MASARYK
UNIVERSITY
F A C U L T Y O F S C I E N C E
Mechanism of
cyanobacterial
lipopolysaccharide
effects on epithelial and
immune cells
Doctoral thesis
P E T R A R A P T O V A
Supervisor: RNDr. Lenka Šindlerové, Ph.D.
Department of Experimental Biology of Experimental
Biology
Programme Animal Physiology, Immunology and
Developmental Biology
Brno 2024
MUNI
SCI
MECHANISMUS ÚČINKU SINICOVÉHO LIPOPOLYSACHARIDU NA EPITELIÁLNÍ A IMUNITNÍ
BUŇKY
Bibliografický záznam
Autor:
Název práce:
Studijní program:
Vedoucí práce:
Rok:
Počet stran:
Klíčová slova:
Petra Raptová
Přírodovědecká fakulta
Masarykova univerzita
Ústav experimentální biologie
Mechanismus účinku sinicového lipopolysacharidu na
epiteliální a imunitní buňky
Fyziologie, imunologie a vývojová biologie živočichů
RNDr. Lenka Šindlerová, Ph.D.
2024
130
vodní květ, keratinocyty, imunitní buňky, zánět, toll-like
receptor, TLR, lipopolysacharid, LPS
2
MECHANISM OF CYANOBACTERIAL LIPOPOLYSACCHARIDE EFFECTS ON EPITHELIAL AND
IMMUNE CELLS
Bibliographic record
Author: Petra Raptova
Faculty of Science
Masaiyk University
Title of Thesis: Mechanism of cyanobacterial lipopolysaccharide effects on
epithelial and immune cells
Degree Programme: Animal Physiology, Immunology and Developmental Biology
Supervisor: RNDr. Lenka Sindlerova, Ph.D.
Year: 2024
Number of Pages: 130
Keywords: harmful algal bloom, keratinocytes immune cells,
inflammation, toll-like receptor, TLR, lipopolysaccharides, LPS
3
MECHANISMUS ÚČINKU SINICOVÉHO LIPOPOLYSACHARIDU NA EPITELIÁLNÍ A IMUNITNÍ
BUŇKY
Abstrakt
Sinicové vodní květy (cyanoHABs) představují závažný problém na celém světě. Nejenže
vyčerpávají živiny v životním prostředí, ale jsou také zdrojem různých toxinů a
biologicky aktivních molekul. Povrch sinic, podobně jako u G-bakterií, obsahuje lipopolysacharid
(LPS). Několik studií, včetně naší, prokázalo imunomodulační účinnost
sinicového LPS (cyanoLPS).
Protože kůže je prvním orgánem, který se při rekreačních aktivitách dostává do
kontaktu s vodou kontaminovanou cyanoHAB, studovali jsme účinky LPS pocházejícího
z cyanoHAB na keratinocyty. Sledovali jsme produkci cytokinů, expresi keratinů a
změny v proliferaci a migraci keratinocytů in vitro. Abychom zjistili, zda hlavní účinek
způsobuje cyanoLPS nebo bakteriální LPS z G- heterotrofních bakterií přítomných
v cyanoHAB, použili jsme axenické sinicové kultury druhů, které dominovaly studované
vodní květy.
Přestože nejsilněji spouštěly prozánětlivou odpověď různých buněčných typů
LPS izolované z environmentálních směsí, i cyanoLPS Aphanizomenon flos-aquae
PCC7905 (Ap-1) působil podobně. Zatímco Toll-like receptor (TLR) 4 je kanonickým
receptorem pro bakteriální LPS především z čeledi Enterobacteríaceae (např.
Escheríchia colí), LPS jiných typů bakterií může působit také jako antagonista TLR4
nebo signalizovat prostřednictvím TLR2. U cyanoLPS však není konkrétní receptor
znám. Proto jsme se pokusili objasnit mechanismus účinku Ap-1. Inhibice TLR2, stejně
jako knock-out mutace TLR2 vedly ke snížení buněčné odpovědi. Naopak TLR2
knock-in ji vyvolal. Při up/down regulaci TLR4 se odpověď buněk nezměnila.
NGS analýza mikrobiálního složení cyanoHAB ukázala, že nejaktivnější LPSs pocházely
z biomas s nejvyšším výskytem heterotrofních bakterií, především kmene Pro-
teobacteria.
Naše výsledky naznačují, že LPS pocházející z biomasy cyanoHAB může mít prozánětlivé
účinky na buňky cílových orgánů, které jsou pravděpodobně způsobeny především
LPS heterotrofních bakterií. Minimálně cyanoLPS Aphanizomenon flos-aquae
však také působí prozánětlivě, a to prostřednictvím TLR2.
5
MECHANISMUS UCINKU SINICOVEHO LIPOPOLYSACHARIDU NA EPITELlALNl A IMUNITNi
BUNKY
Abstract
Cyanobacterial water blooms (cyanoHABs) are a serious problem worldwide. They not
only deplete nutrients in the environment but are also a source of various toxins and
biologically active molecules. The surface of cyanobacteria, like that of G-bacteria, contains
lipopolysaccharide (LPS). Several studies, including ours, have demonstrated the
immunomodulatory effects of cyanobacterial LPS (cyanoLPS).
Because the skin is the first organ to come into contact with cyanoHAB-contaminated
water during recreational activities, we studied the effects of cyanoHAB-derived
LPS on keratinocytes. We determined cytokine production, keratin expression, and
changes in keratinocyte proliferation and migration in vitro. To determine whether the
main effect is caused by cyanoLPS or bacterial LPS from G-heterotrophic bacteria present
in cyanoHAB, we used axenic cyanobacterial cultures of the species that dominated
the water blooms studied.
Although LPS isolated from environmental mixtures showed the most potent
pro-inflammatory response of different cell types, the cyanoLPS Aphanizomenon
flos-aquae PCC7905 (Ap-1) also exerted similar effects. While Toll-like receptor (TLR)
4 is the canonical receptor for bacterial LPS primarily from the Enterobactehaceae
family (e.g. Escherichia coli], LPS from other bacterial types can also act as an antagonist
of TLR4 or signal through TLR2. However, the specific receptor for cyanoLPS is
unknown. Therefore, we attempted to elucidate the mechanism of action of Ap-1. Inhibition
of TLR2 as well as knock-out mutations of TLR2 led to a reduced cellular response.
In contrast, TLR2 knock-in induced it. When TLR4 was up/downregulated, the
cell response was not altered.
NGS analysis of the microbial composition of cyanoHABs showed that the most
active LPSs isolated from the biomasses with the highest abundance of heterotrophic
bacteria, especially the Proteobacteria phylum.
Our results suggest that LPS derived from cyanoHAB biomass may have proinflammatory
effects on target organ cells, which are likely to be mainly caused by LPS
from heterotrophic bacteria. However, atleastthe cyanoLPS ofAphanizomenon flos-aquae
also exerts pro-inflammatory effects through TLR2.
7
MECHANISMUS UCINKU SINICOVEHO LIPOPOLYSACHARIDU NA EPITELlALNl A IMUNITNi
BUNKY
Declaration
I declare that I have prepared my thesis independently under the mentorship of the
thesis supervisor and using the information sources provided in the thesis.
9
MECHANISMUS ÚČINKU SINICOVÉHO LIPOPOLYSACHARIDU NA EPITELIÁLNÍ A IMUNITNÍ
BUŇKY
Acknowledgements
I would like to thank my supervisor RNDr. Lenka Šindlerová, Ph.D. for her guidance,
patience and helping hand with anything I needed. To our colleagues at RECETOX without
whom we would have the cyanoLPS samples. And DBIS and OFIŽ for a very friendly
and professional work environment.
The results of the project were created with the financial support of the provider Czech
Science Foundation within project No. GA19-09980S and of the provider Institute of
Biophysics of the Czech Academy of Sciences within the Internal Support of Research
program 2022. This work was supported by the European Union's Horizon 2020 research
and innovation programme under grant agreement No. 857560—CETOCOEN
Excellence We thank the Research Infrastructure RECETOX RI (No. LM2018121) financed
by the Ministry of Education, Youth and Sports, and Operational Programme
Research, Development, and Education—project CETOCOEN EX-CELLENCE (No.
CZ.02.1.01/0.0/0.0/17_043/0009632) for supportive background and the Institute of
Botany for the long-term research development project (RVO 67985939).
QfDBIS IBPDepartment of Biophysics of Immune system
Institute of Biophysics
Šablona DP 3.4.5-SCI-programova-english (2023-05-26) © 2014, 2016, 2018-2023 Masarykova univerzita 11
TABLE OF CONTENTS
Table of Contents
List of Publications 15
Abbreviations 16
1 Preface 19
2 Overview 20
2.1 Cyanobacteria and Lipopolysaccharides 20
2.1.1 Cyanobacterial Harmful Algal Blooms 20
2.1.2 LPS 20
2.2 Skin 23
2.2.1 Skin response to LPS 24
2.3 Macrophage response to LPS 28
2.4 Toll-like receptors 29
2.4.1 TLR4 30
2.4.2 TLR2 31
2.4.3 N F K B 31
2.4.4 MAPK 32
2.4.5 Type I interferons 32
3 Motivation and hypothesis 34
4 Aims 35
5 Results and discussion 36
5.1 CyanoLPS pro-inflammatory processes in keratinocytes 38
5.2 Differences in O-antigen structures of cyanoLPS 48
5.3 The main contributor to the pro-inflammatory activation of CyanoHABderived
LPS is G- bacteria 50
5.4 Effect of axenic cyanoLPS can on macrophages 54
5.5 The activity of cyanoLPS is dependent on TLR2, but not TLR4 59
6 Conclusion 64
Bibliography 65
13
TABLE OF CONTENTS
Appendix A 84
Appendix B 85
14
LIST OF PUBLICATIONS
List of Publications
List of publications included in thesis (Appendices A, B]
I. P. Raptová, V. Skočková, P. Babica, I. Sovadinová, E. Sychrová, P. Vídeňská,
P. Šplíchalová, 0. Vašíček, L. Šindlerová, Cyanobacterial bloom-associated lipopoly'saccharides
induce pro-inflammatory processes in keratinocytes in vitro. Environmental
Toxicology and Pharmacology, doi: 10.1016/j.etap.2023.104342.
IF 4.3; Q l (Toxicology)
Personal contribution (40%): design and implementation of experiments, analysis,
and interpretation of data, writing
II. V. Skočková, P. Raptová, K. Pospíchalová, I. Sovadinová, E. Sychrová, M. Smutná,
K. Hilscherová, P. Babica, L. Šindlerová, Cyanobacterial harmful bloom lipopolysaccharides:
pro-inflammatory effects on epithelial and immune cells in vitro. Archives
of Toxicology, 2023, doi: 10.1007/s00204-023-03644-8. IF 6.1;
Q l (93.1% in Toxicology)
Personal contribution (20%): design and implementation of experiments, analysis,
and interpretation of data, writing
15
ABBREVIATIONS
Abbreviations
Ap - Aphanizomenon klebahnii/floss-aquae
BMDM - bone marrow derived macrophage
CCL - C-C motif ligand
CD - cluster of differentiation
COX-II - cyclooxygenase II
CyanoHAB - cyanobacterial harmful algal bloom
cyanoLPS - cyanobacterial lipopolysaccharide
CyP - cyanobacterial product
DAMP - danger-associated molecular pattern
DNA - deoxyribonucleic acid
Do - Dolichospermum curvum
EC - Escherichia coli
ERK - extracellular signal-regulated kinase
G- - Gram-negative
HKLM - heat-killed Listeria monocytogenes
IFN - interferon
IKK - IKB kinase
IL - interleukin
iNOS - inducible nitric oxide synthase
IRF - interferon regulatory factor
JNK/SAPK - c Jun NH(2)-terminal kinase/stress-activated protein kinase
K - keratin
LPS - lipopolysaccharide
MAPK - mitogen-activated protein kinase
MD-2 - myeloid differentiation factor 2
Mi - Microcystis aeruginosa
miRNA - micro RNA
MyD88 - myeloid differentiation primary response 88
N F K B - Nuclear factor kappa B
NGS - next generation sequencing
NLR - nucleotide-binding domain and leucine-rich repeat containing
gene receptor
NO - nitric oxide
NOD - nucleotide-binding oligomerization domain
PAMP - pathogen-associated molecular pattern
PI - Planktothrix agardhii
poly(LC) - polyinosinic:polycytidylic acid
PRR - pattern recognition receptor
16
ABBREVIATIONS
RNA - ribonucleic acid
ROS - reactive oxygen species
SDS-PAGE - sodium dodecyl sulfatepolyacrylamide gel electrophoresis
Th - T helper lymphocyte
TLC - thin layer chromatography
TLR - Toll-like receptor
TNFa - tumor necrosis factor a
TRIF - TIR-domain-containing adapter-inducing INF-p
UV - ultraviolet
17
PREFACE
1 Preface
The aim of this thesis is to elucidate the pro-inflammatory potential and mechanism of
action of lipopolysaccharides (LPS) isolated from cyanobacterial water blooms as well
as laboratory cultures. Microcystis, Aphanizomenon, Dolichospermum, and Planktothhx
discussed in this thesis are common bloom-forming genera. We focused on the response
of human keratinocytes as skin is the first organ exposed to cyanobacterial LPS
(cyanoLPS) contaminated water. Macrophages were used to study the mechanism behind
cyanoLPS activation.
The first part of the thesis describes current knowledge of inflammatory response
of epithelial and immune cells. Furthermore, the recognition and signalling pathways
connected with bacterial LPS is discussed. The second part describes our findings
about pro-inflammatory activation of keratinocytes after exposure to cyanoLPS. It presents
my contribution and key results from our publications (attached in Appen-dices
A, B). Moreover, the potential mechanism of cyanoLPS signalling is discussed. These
data have not yet been published and are included only in this thesis.
19
OVERVIEW
2 Overview
2.1 Cyanobacteria and Lipopolysaccharides
2.1.1 Cyanobacterial Harmful Algal Blooms
Cyanobacteria are highly diverse photosynthetic procaryotic organisms. They can be
unicellular or form colonies and filaments1
. They inhabit aquatic as well as terrestrial
environments. Some even survive extreme conditions in the arctic2
or deserts3
. Spirulina
spp. and Aphanizomenon flos-aquae are currently used as food supplements or
food colourings4
'5
. In aquatic ecosystems, cyanobacteria can form harmful algal blooms
(CyanoHABs), a process intensified by human activities such as nutrient over-enrichment
or climate change6
. They are a source of toxins and biologically active molecules
and thus are not only an ecological but also an immunological threat7
. Furthermore, in
last decades there is an increasing occurrence of CyanoHABs in the temperate zone8
.
The best-known CyanoHAB-derived toxins are microcystins, most commonly produced
by Microcystis sp. They show hepatotoxicity9
, can cause DNA damage1 0
'1 1
and
activate neutrophiles1 2
. Cylindrospermopsin, another cyanobacterial toxin, production
was first observed in Cylindrospermopsis raciborskii13
and can also be produced by
some strains of Aphanizomenon14
'15
. It also has negative effects on the liver and kidneys1
6
'1 7
and can activate innate immunity1 8
. Skin irritation is commonly caused by
lyngbyatoxin and aplysiatoxin1 9
.
2.1.2 LPS
LPS generally consist of three main domains - lipid A, core polysaccharide and O-antigen
polysaccharide. 75% of the Gram-negative (G-) bacterial outer cell wall of consists
of LPS (Fig. 1). It contributes to structural integrity of the cell wall but can also help the
bacterium to evade environmental threats and the eucaryotic immune system. Lipid A
is extremely conserved, but the core polysaccharide is variable, and the O-antigen is
highly variable. Lipid A is considered to be the crucial pro-inflammatory activator interacting
with the typical LPS receptor Toll-like receptor 4 (TLR4). However,
20
OVERVIEW
differences in the O-antigen can modulate the pro-inflammatory response and interact
with the complement system. The variability in the O-antigen is commonly used to differentiate
bacterial serotypes2 0
.
S LPS
Fig. 1 Bacterial lipopolysaccharide221
Similarly to G- bacteria, the cyanobacterial cell wall also contains LPS2 1
. It is known
that the structure and chemical composition of cyanoLPS differ from the typical LPS of
G- bacteria2 2
. This can result in different affinity and/or specificity of cyanoLPS.
Our previous studies show that CyanoHAB-derived LPS can activate epithelial and
immune cells in vitro23
-27
. Others have shown pro-inflammatory activation of innate
immunity after exposure to cyanoLPS of a specific cyanobacterial species2 8 - 3 1
. Furthermore,
we observed that even when not active by itself, cyanoLPS could potentiate the
pro-inflammatory effect of cylindrospermopsin1 8
. On the other hand, cyanoLPS of Oscillatoha
[Planktothrix] FP1 exhibits anti-inflammatory properties antagonizing the
pro-inflammatory effects of Escherichia coli LPS3 2
'3 3
.
The effects of cyanoLPS from the cyanobacterium Geitlerinema sp. strain HCC1097
have been intensively studied and the cyanoLPS was reported to induce elevation of
pro-inflammatory mediators in rat microglia3 1
, human monocytes2 9
, as well as murine
and human airway epithelium3 4
. This cyanoLPS also activated murine
21
OVERVIEW
B-lymphocytes3 5
. However, it was active only in higher concentrations and not as potent
as the E. coli 055:B5 LPS.
Other studies report that cyanoLPS from Scytonema javanicum UHM GB-9-9, but
notS. ocellatum UHM HX-22-2 was able to induce pro-inflammatory cytokines in rat
microglia. Again, it was less potent than E. coli 026:B6 LPS3 6
. Similar trend was observed
after exposure to cyanoLPS from M. aeruginosa UTCC 2993 0
. We also observed
a pro-inflammatory activation of immune cells following exposure to cyanoLPS from
non-axenic and axenic M. aeruginosa PCC 7806, and axenic M. aeruginosa
HAMBI/UHCC 130. The inflammatory response occurred only at a higher concentration
compared to E. coli 026:B6 LPS2 7
. On the other hand, one study showed M. aeruginosa
NIES-44-derived cyanoLPS to be more potent than E. coli NBRC 3301 LPS2 8
.
22
OVERVIEW
2.2 Skin
Skin is the third biggest organ of the body3 7
after the intestine3 8
and lungs3 9
. It is composed
of two main parts, epidermis, and dermis. The epidermis is mainly composed of
keratinocytes, but melanocytes, Merkel, and Langerhans cells are also found here
(Fig. 2). Its main functions are prevention from pathogens, water retention, and protection
from UV light. The dermis located beneath acts as mechanical protection. It is
formed from extracellular matrix, mainly collagen and elastin fibres produced by fibroblasts.
It also contains blood and lymphatic vessels, sweat and exocrine glands, and
hair follicles.
Skin is the first organ that gets in contact with CyanoHAB-contaminated water
during recreational activities. Skin irritation after the contact with cyanobacteria is
usually associated with cyanotoxins. For example, most "swimmers itch" cases are
caused by alkaloid lyngbyatoxin1 9
'4 0
. Cylindrospermopsin induces hypersensitivity on
skin4 1
as well as inflammation of airway epithelium4 2
. Debromoaplysiatoxin was observed
to have caused skin irritation in a dog4 3
as well as rabbits, mice, and humans4 4
.
Langerhans cell Keratlnocyte DETC
OY ö T c e l l dermal D C
C D 8 * T c e l l
Mast cell
C D 4 * T c e l l
Blood vessel
Macrophage
Fig. 2 Anatomy of skin2 2 2
Stratum
corneum
Epidermis
Dermis
23
OVERVIEW
However, skin irritation was also observed upon contact with cyanobacterial strains
that do not produce cyanotoxins4 5
. Since the bacterial (usually E. coli) LPS is routinely
used to induce inflammation in model human keratinocyte cell line HaCaT used in this
thesis4 6 - 5 0
, as well as in other primary keratinocyte models5 1
or primary animal
keratinocytes5 2
. Since cyanoLPS is always present in cyanobacterial sample, there is a
possibility, that it could have caused the pro-inflammatory response. This hypothesis
is supported by our previous results, where we have shown the pro-inflammatory activation
of mammalian cells after the axenic cyanoLPS challenge2 6
'2 7
. Furthermore, Torokne
et a/.53
hypothesized that G- heterotrophic bacteria present in environmental
CyanoHABs can cause inflammatory activation because none of their axenic cyanobacterial
strains caused skin irritation compared to environmental samples.
2.2.1 Skin response to LPS
The first defence of skin against invading pathogens is the top layer of epidermis consisting
of cornified keratinocytes. Breaching this layer by external stimuli allows the
direct contact of live skin cells with bacteria from the environment. Recognition of invading
pathogens is important for cutaneous immune response (Fig. 3). Keratinocytes
express various pattern recognition receptors (PRRs) like TLRs5 4
or nucleotide-binding
domain and leucine-rich repeat containing gene family receptors (NLR) (formerly
known as NOD-like receptors)5 5
. Recognition of pathogen- or danger-associated molecular
patterns (PAMPs, DAMPs) by PRRs leads to production of inflammatory mediators.
Production of cytokines and chemokines in turn leads to recruitment and activation
of innate and adaptive immunity5 6
.
Exposure to bacterial LPS elevates pro-inflammatory cytokines tumor necrosis
factor a (TNFa), interleukin (IL)-l, and IL-65 7
'5 8
. IL-8 is also produced5 9
'6 0
serving as a
chemoattractant and is also important for promotion of acute phase of inflammation6 1
.
Another important mediator involved is the inducible cyclooxygenase COX-II62
. It regulates
levels of prostaglandin E26 3
modulating the migration of immune cells into
24
OVERVIEW
tissue6 4
'6 5
. Exposure to LPS can also modulate the proliferative and migratory potential
of keratinocytes. Its effect depends on the concentration and origin of LPS5 8
'6 6
-6 7
.
ANTIMICROBIAL PEPTIDES
IL1A
1MB
« IL18
[S IL18BP
- IL36A
j£ IL36B
O IL36G
r IL36RN
O I L 3 3
U
IL37
IL38
IL-1 family
cytokines
IL19
IL20
IL24
IL12A
IL12B
IL23A
EBI3
IFNK
TGFB1
IL-20 family TGFB2
cytokines TGFB3
IL-12/IL-23
cytokines
Type I
IFN
TNF
TSLP
IL6
IL7
IL15
TGFB
cytokine
CO
a.
o
i-
o
<
5
o
a.
o
Fig. 3 Skin inflammation223
AREG
BTC
EGF
HBEGF
Epidermal
Growth
factors
VEGFA I Vascular
CTGF
FGF1
FGF2
Connective
Tissue
Growth Factors
NGF I Nerve
Cytokines
IL-6 is produced by immune cells, mesenchymal cells, endothelial cells, fibroblasts, and
many others6 8
'6 9
. Exposure to bacterial LPS increases keratinocyte IL-6 production5
8
'7 0
"7 3
. Other stimuli, for example UVB, can also cause IL-6 production in skin tissue7
4
'7 5
. Furthermore, IL-6 is robustly produced after the integrity of skin is disturb
ed5 7
'7 6
'7 7
IL-8 is a chemokine well-known for its role in neutrophil migration7 8
'7 9
. Similarly
to IL-6, it is produced during the initial inflammatory phase after a skin injury5 7
.
Keratinocytes respond to bacterial LPS by producing IL-84 9
'5 1
'7 0
'8 0
'8 1
. The expression of
IL-8 is not limited to keratinocytes, but in vivo the whole skin tissue contributes, too8 2
.
25
OVERVIEW
C-C motif ligand 2 (CCL2) also known as monocyte chemoattractant protein 1 is also a
chemokine. It attracts macrophages and T-lymphocytes but not neutrophils or eosinophils
into the tissue8 3 - 8 6
. Porphyromonas gingivalis LPS is able to increase CCL2 production
in keratinocytes8 7
. Conversely, E. coli LPS was shown to be unable to do so8 8
.
On the other hand, it could elevate CCL2 in other epithelial and immune cells8 4
'8 8 - 1 0 0
.
Another important chemokine attracting macrophages and T-lymphocytes is
CCL201 0 1 - 1 0 3
. There is a positive loop between cytokines produced by Thl7 lymphocytes
and CCL20 produced by keratinocytes1 0 4
. Bacterial LPS can stimulate CCL20 production
in various epithelial cells1 0 5 - 1 0 7
. However, this activation is dependent on the
bacterial strain1 0 6 - 1 0 9
.
Keratins
Keratins (K) are one of the intermediary filaments in epithelial cells. Keratin heterodimers
form filaments providing mechanical as well as structural stability of the cell1 1 0
.
Their expression is spatiotemporally regulated1 1 1
'1 1 2
.
Some keratins are only expressed as a response to external stimuli. K6 and K16
are elevated 6 h after skin injury followed by cytoskeletal reorganisation. After 12 h
K17 can be detected1 1 3
. In some tissues, K6 is naturally present1 1 4
. It is important for
regulation of keratinocyte migration1 1 5
. The key initiators of K6 and K16 expression
are IL-1 and TNF-a1 1 6
.
K17 is not present in unstimulated keratinocytes in vivo. It can be detected after
injury1 1 3
and in hyperproliferating cells1 1 7
. For example, it is expressed in cells infected
by human papilloma virus1 1 8
or in carcinomas8 3
'1 1 9
. Elevation of K17 can also be affected
by pro-inflammatory cytokines1 2 0 - 1 2 2
. Interestingly, it contains nuclear localisation
signal1 2 3
. In the nucleus it interacts with several proteins and binds to DNA resulting
in expression of pro-inflammatory mediators1 2 4
.
26
OVERVIEW
Proliferation and migration
Part of the pro-inflammatory response of keratinocytes is elevation of proliferation
and migration of cells. Confluent keratinocytes proliferate very little because of contact
inhibition1 2 5
. However, disruption of skin integrity results in pro-inflammatory response,
elevated proliferation, and remodelling of the tissue1 2 6
. Several studies note
decrease of keratinocyte wound healing after LPS challenge in vitro as well as
in v/vo6 6
'1 2 7
'1 2 8
. On the other hand, is seems that LPS can also elevate proliferation and
migration potential of keratinocytes depending on the concentration and origin of
LPS5 8
'1 2 9
.
27
OVERVIEW
2.4 Toll-like receptors
TLRs are transmembrane glycoprotein PRRs responsible for recognition of PAMPs and
DAMPs1 3 8
. They are found on professional immune cells like lymphocytes and macrophages
as well as epithelial and endothelial cells. Their compartmentalization is
strictly regulated. TLR1,2,4, 5,6 and 10 are localised on the cell surface1 3 9
'1 4 0
and upon
ligand binding some can be internalised in endosomes1 4 1
'1 4 2
. They are responsible for
detecting surface-associated components of pathogens, for example LPS, lipopeptides,
lipoteichoic acid or flagellin1 4 3
"1 4 6
. TLR3, 7, 8, 9, and 11 are strictly located in endosomes1
3 9
'1 4 0
as they recognise bacterial and viral nucleic acids like dsRNA or poly(LC)
(polyinosinic:polycytidylic acid)1 4 4
(Fig. 4).
Triacyl lipopeptides Diacyl lipopeptides Flagellin LPS
I 1 1 1
TLR2-TLR1 TLR2-TLR6 TLR5 TLR4
Fig. 4 TLR signalling pathways224
29
OVERVIEW
2.3 Macrophage response to LPS
Macrophages are an integral part of innate immunity. Tissue-resident macrophages
have the ability to self-renew and are of embryonic origin1 3 0
. During pathological processes,
macrophages can also origin from tissue-infiltrating monocytes1 3 1
. Macrophages
clean up pathogens, damaged cells and participate in tissue repair1 3 2
. They can
be stimulated by endogenously produced mediators or exogenous stimuli like LPS.
LPS recognition leads to production of pro-inflammatory mediators. The production
of pro-inflammatory cytokines TNFa, IL-ip, IL-6, and IL-8 is typical for the early
response. Among them, TNFa is one of the main parameters measured in experiments
modelling the inflammation1 3 3
'1 3 4
. At a later stage, a negative regulation loop is involved,
and anti-inflammatory cytokines are produced.
TNFa is a pleiotropic pro-inflammatory cytokine playing a role during inflammation,
cell proliferation, differentiation, and apoptosis. LPS has long been known to be a
very potent inducer of TNFa1 3 5
. Even now, E. coli LPS is still used to model the inflammation
in macrophages.
Additionally, inducible nitric oxide synthase (iNOS) is activated, catalysing the
pro-duction of nitric oxide (NO). NO is an important messenger molecule. When produced
by the endothelium, it mediates vasodilatation, and its deficiencies are associated
with several pathologies1 3 6
. During the inflammatory activation of macrophages
caused by E. coli LPS, iNOS is activated resulting in local elevation of NO. This local and
temporal elevation of NO1 3 3
'1 3 4
acts as an antimicrobial agent1 3 7
.
28
OVERVIEW
Ligand binding to TLRs activates MyD88-dependent and/or TRIF-dependent pathways.
Nuclear factor kappa B (NFKB), mitogen-activated protein kinase (MAPK) and
interferon regulatory factor (IRF) pathways1 4 7 - 1 4 9
are triggered downstream. They
lead to phosphorylation of transcription factors p65, Ap-1, IRF3 and IRF7 respectively
resulting in production of pro-inflammatory mediators1 5 0
'1 5 1
.
In keratinocytes, LPS challenge causes phosphorylation of the N F K B subunits
and MAPK4 7
'4 9
'5 2
and is connected with production of cytokines IL-1(3, IL-6, IL-8, IL-10,
IL-12, IL-31 or TNF-a4 9 - 5 2
. Jeong et al.47
even observed expression of iNOS. Elevation
of IL-6 and IL-8 after LPS exposure is also observed in intestinal epithelium2 5
'5 0
. Macrophages
exhibit activation of these pathways, too. Compared to the epithelial cells,
macrophages are more sensitive to lower concentration of LPS. Apart from above mentioned
cytokines, there is a robust induction of iNOS4 8
'1 3 3
'1 3 4
.
2.4.1 TLR4
Activated TLR4 forms a homodimer and is considered the canonical LPS receptor1 5 2
.
However, for proper LPS binding and the signalling cascade activation, co-receptors
cluster of differentiation (CD) 14 and myeloid differentiation factor 2 (MD-2) are necessary1
5 3 - 1 5 6
.
CyP (cyanobacterial product) from Oscillatoha planktothhx is the most studied
cyanoLPS interacting with TLR43 2
. On its own, it does not affect production of cytokines3
2
'3 3
. However, it can decrease the cytokine response of monocytes and dendritic
cells exposed to bacterial LPS3 2
'1 5 7
. The same effect was observed on whole blood3 3
'1 5 8
.
It is suggested that CyP interacts with MD-2 antagonising the bacterial LPS3 2
'3 3
. Moreover,
it induces production of miRNA146a silencing synthesis of downstream kinases
of the TLR signalling pathway1 5 7
'1 5 9
'1 6 0
. Furthermore, cyanoLPS from Spirulina sp. was
shown to reduce tumor growth in mice in TLR4 dependent manner1 6 1
.
30
OVERVIEW
2.4.2 T L R 2
Lipopeptide, peptidoglycan, lipoteichoic acids or zymosan are typical ligands for
TLR21 6 2
. It usually forms a heterodimer with TLR1 or TLR6 which changes the ligand
specificity1 6 3
. For bacterial LPS, TLR4 is recognised as a canonical receptor, but it is to
note that the studies mostly refer to enterobacterial-like LPS. Interestingly, some studies
show that TLR2 does not interact with E. coli LPS1 6 4
-1 6 5
. On the other hand, others
suggest that non-enterobacterial-like LPS (also referred to as atypical LPS) binds to
TLR2 resulting in pro-inflammatory activation of cells.
This hypothesis was recently supported by some publications. In one of them, Helicobacter
pylori LPS was shown to activate human gastric adenocarcinoma cell line via
TLR21 6 6
. Moreover, Ochrobactrum intermedium LPS used both TLR2 and TLR4 in
mouse peritoneal macrophages and activated human embryonic kidney cell line
HEK293 expressing respective receptors. Furthermore, this group proposed a hypothetical
TLR2/4 heterodimer formed only after LPS-binding1 6 7
. However, there is no
knowledge about the ability of cyanoLPS to interact with TLR2.
The responsiveness of TLR2 to LPS can also be specific for host species. For example,
in mice, Legionella pneumophila LPS requires TLR2 and does not interact with
TLR41 6 8
'1 6 9
. On the other hand, human cells detect L. pneumophila LPS via TLR4 but not
TLR21 7 0
. Ovine luteal endothelium detects E. coli LPS using both TLR2 and TLR41 7 1
.
Interestingly, Rhodobacter sphaeroides LPS has agonistic effect in horses or hamsters
and antagonistic effect in humans and mice1 7 2
.
2.4.3 N F K B
N F K B family consists of 5 proteins p65/RelA, RelB, cRel, p50, and p52. NFkB is kept
inactive in the cytoplasm by the IKB kinase (IKK) complex1 7 3
. Upon E. coli LPS binding
TLR4 associates with adaptor molecules MyD88 and TRIF1 7 4
. This initiates downstream
kinases leading to phosphorylation of IKB resulting in its release from N F K B 1 5 0
.
TLR signalling typically leads to activation of the p65 subunit which is in turn translocated
to the nucleus1 3 4
'1 7 5
'1 7 6
. Here it acts as a transcription factor inducing expression
31
OVERVIEW
of cytokines, chemokines, adhesion molecules or cell cycle regulators1 4 7
. Immune cells
recognising LPS via TLRs also generate reactive oxygen species (ROS). Increase in ROS
further triggers N F K B activation amplifying the cellular response1 7 7
'1 7 8
. Furthermore,
N F K B activation was also shown to be connected with proliferation and migration of
keratinocytes1 7 9
.
2.4.4 MAPK
MAPK interact with numerous signalling pathways. MAPK family includes p38, and
cjun NH(2)-terminal kinase also known as stress-activated protein kinase QNK/SAPK),
and extracellular signal-regulated kinase (ERK)1 8 0
. MAPK respond to various stimuli
ranging from growth factors and cytokines to UV or oxidative stress1 8 1
. Phosphorylation
of p38, JNK/SAPK and ERK1/2 after LPS exposure has been known for a long
time1 8 2
.
E. coli LPS (usually strain 0111:B4) is currently used to model inflammatory processes
in different cells in vitro. Mouse macrophage cell line RAW264.7 as well as primary
bone marrow derived macrophages (BMDMs) produce TNFa, IL-6 and NO after
exposure to E. coli LPS. Furthermore, phosphorylation of p38, JNK/SAPK and ERK1/2
is observed1 8 3 - 1 8 7
. In the case of RAW264.7 inhibition of p38 reduces NO production
quite significantly and there is a decrease of TNFa and IL-6 production. However, the
strongest down-regulation is observed by inhibiting N F K B 1 8 5
' 1 8 7
. In contrast, inhibition
of p38 in BMDMs does not affect IL-6 production at all1 8 3
-1 8 5
. Macrophages of the central
nervous system also respond to LPS by activating the MAPK pathways. They start
to produce TNFa and IL-6 and express iNOS necessary for NO synthesis9 8
'1 8 8
'1 8 9
. MAPK
signalization after LPS exposure is not restricted to immune cells, LPS can for example
induce p38 in mammary epithelial cells as well1 9 0
.
2.4.5 Type I interferons
LPS can trigger several signalling pathways. Some lead to production of type I IFN,
namely iFNfB1 4 2
'1 9 1
'1 9 2
. E. coli LPS used as a model TLR4 ligand was shown to induce
32
OVERVIEW
IFNp in murine peritoneal macrophage and bone marrow derived macrophages
(BMDMs)1 9 3
'1 9 4
Murine BMDMs also respond with IFNp production after exposure to various TLR2
ligands9 7
'1 4 1
'1 9 4
. This seems dependent on internalisation of the receptor after ligand
binding1 4 1
'1 4 2
. Some, however, report that TLR2 ligands do not induce IFNp in murine
peritoneal macrophages1 9 3
. Additionally, a mutant Listeria monocytogenes unable to
modify its peptidoglycan to escape lysosomal degradation induced higher IFNp response
in peritoneal macrophages and BMDMs. However, this response was TLR2-dependent
only in peritoneal macrophages not in BMDMs1 4 2
.
33
MOTIVATION AND HYPOTHESIS
3 Motivation and hypothesis
Several groups including ours have shown pro-inflammatory activation of various cells
after cyanoLPS exposure. Apart from us, they have mostly focused on immune cells.
Furthermore, the mechanism of this activation is not known. On the other hand, binding
of bacterial LPS with TLRs has been well-established. Therefore, we aim to elucidate
the effects of cyanoLPS from environmental samples on keratinocytes and focus
on the potential mechanism of cyanoLPS from axenic cyanobacterial cultures.
Hypothesis: CyanoLPS triggers pro-inflammatory responses in keratinocytes and
macrophages via TLRs.
Keratinocytes
Cyanobacteria
• • • • • " •
C C L 2 0 . • > . * . • * • IL-8
••••
INFLAMMATORY RESPONSE
Fig. 5 A schematic illustration of the aims of the thesis; created with BioRender.com
34
AIMS
4 Aims
• To compare the effects of cyanoLPS isolated from harmful algal blooms dominated
by certain cyanobacterial species and corresponding axenic cyanobacterial
laboratory culture on keratinocytes in vitro.
• To investigate this activation after exposure to cyanoLPS isolated from environmental
samples containing a mixture of cyanobacterial species.
• To verify the pro-inflammatory activation of cyanoLPS isolated from axenic cyanobacterial
laboratory cultures and elucidate the mechanism of signalling.
35
RESULTS AND DISCUSSION
5 Results and discussion
First, to get a general understanding of the effects of cyanoLPS we selected four cyanobacterial
water bloom samples from the RECETOX archive, each dominated by a different
cyanobacterial species. For this purpose, we selected blooms containing Microcystis
aeruginosa, Planktothrix agardhii, Aphanizomenon klebahnii, and Dolichospermum
curvum, because they are common freshwater bloom-forming species worldwide.
Moreover, six corresponding axenic cyanobacterial laboratory cultures were obtained
from Pasteur Culture Collection of Cyanobacteria, UTEX Culture Collection of Algae and
HAMBI Culture Collection of the University of Helsinki (Tab. 1). Secondly, to see the
effects of complex environmental samples of cyanobacterial water blooms were collected
from water bodies in the Czech Republic (Tab. 1).
As skin is the first organ getting in contact with CyanoHAB-contaminated water
during recreational activities HaCaT cell line was chosen to model keratinocytes
in vitro. To study the immune cell activation and mechanism of cyanoLPS, murine macrophage
cell line RAW264.7 and human monocytic cell line THP-1 were used. To verify
the mechanism of action, knock-in human embryonic kidney cell lines HEK293 were
employed.
36
RESULTS AND DISCUSSION
Tab. 1 Sampling localities, dates, and dominants of CyanoHABs and axenic cell
cultures used for LPS isolation.
Sample
Source of LPS Dominating species
code
EC Purchased Escherichia coli 0 1 1 1 : B 4
HAB-Ap Ruda Pond Jul 25, 2013 Aphanizomenon klebahnii
HAB-PI K a m e n s k ý S u t r á k Pond Aug 2 8 , 2 0 1 4 Ptanktothrix agardhii
HAB-Do S t a ň k o v s k ý r y b n í k Pond Sep 1 1 , 2 0 1 3 Dolichospermum curvum
HAB-Mi N o v é M l ý n y Reservoir Aug 1 4 , 2 0 1 2 Microcystis aeruginosa
Ap-1
Laboratory culture
Aphanizomenon fios-aquae P C C 7 9 0 5
Pl-1
Laboratory culture
Ptanktothrix aghardii P C C 7 8 0 5
Do-1
Laboratory culture
Dolichospermum U H C C / H A M B I 0 3 5 2
Do-2
Laboratory culture
Dolichospermum flos-aquae U T E X 1 4 4 4
Mi-1
Laboratory culture
Microcystis aeruginosa U H C C / H A M B I 130
Mi-2
Laboratory culture
Microcystis aeruginosa P C C 7 8 0 6
A D v o r s k ý r y b n í k Pond Jun 19, 2019 Ptanktothrix agardhii, Planktolyngbya limnetica .Aphanizomenon so.
B H o r n í š t í t a r s k ý r y b n í k Pond Jul 10, 2019
Dolichospermum flos-aquae, Chroococcus limneticus, Aphanocapsa
holstatica
C Hejtman Pond Aug 0 7 , 2 0 1 9 Woronichinia nacgcliana, Microcystis wesenbergii
D S t a r ý K a n c l í ř Pond Aug 0 7 , 2 0 1 9
Sphaerospermopsis aphanizomenoides , Aphanizomenon klebahnii,
Microcystis sp.{M. aeruginosa, Raphidiopsisraciborskii),
Dolichospermum sp. (D. crassum ), Planktolyngbya sp.
E V ý t o p a Pond Aug 07, 2019
Dolichospermum sp. (D. planctonicum , D. crassum , D. ucrainicum ,
D. flos-aquae )
F N o v ý K a n c l í ř Pond Aug 0 7 , 2 0 1 9
Microcystis sp. [M. flos-aquae , M. viridis, M. aeruginosa,
M. wesenbergii)
G Ruda Pond A u g 0 7 , 2 0 1 9
Dolichospermum flos-aquae, Sphaerospermopsis sp.
(S. aphanizomenoides, S. reniforme ), Aphanizomenon issatchenkoi
H K a m e n s k ý Š u t r á k Pond Aug 2 7 , 2 0 1 9 Planktothrix agardhii
J F r a n t i š k ů v r y b n í k Pond Aug 2 7 , 2 0 1 9 Microcystis aeruginosa
K N o v é M l ý n y - s t ř e d Reservoir Aug 27, 2019 Microcystis sp. (M. botrys, M. aeruginosa)
L Vranov Reservoir, Bítov bay Aug 27, 2019 Microcystis sp. (M. flos-aquae , M. aeruginosa )
M Letovice Reservoir Sep 1 7 , 2 0 1 9 Microcystis sp. (M. flos-aquae , M. viridis, M. wesenbergii)
N O l š o v c c Pond Sep 1 7 , 2 0 1 9 Planktothrix agardhii, Microcystis novacekii, Aphanizomenon sp.
0 M l ý n s k ý r y b n í k Pond Sep 1 7 , 2 0 1 9 Raphidiopsis raciborskii
P Brno Reservoir Sep 2 4 , 2 0 1 9 Microcystis sp. (M. aeruginosa , M. novacekii), Woronichinia nacgcliana
R Letovice Reservoir Aug 2 7 , 2 0 2 0 Microcystis sp. [M. aeruginosa, M. viridis), Woronichinia naegeliana
S K o n v e n t s k ý r y b n í k Pond Aug 2 7 , 2 0 2 0
Planktothrix agardhii, Raphidiopsis raciborskii, Microcystis so.,
Pseudanabaena sp.
T K a c h l i č k a Pond Aug 2 7 , 2 0 2 0
Dolichospermum sp. (D. crassum ), Aphanizomenon klebahnii,
Woronichinia sp.
U T r n á v k a Reservoir Aug 27, 2020
Microcystis sp. (M. flos-aquae , M. aeruginosa, M. viridis),
Aphanizomenon klebahnii
37
RESULTS AND DISCUSSION
5.1 CyanoLPS pro-inflammatory processes in keratinocytes
Skin is the first organ that gets in contact with CyanoHAB contaminated water. Cyanotoxins
are usually associated with skin irritation and/or hypersensitivity1 9
'4 0
'4 1
-4 3
but
the contribution of cyanoLPS to skin irritation is not well explored. However, mild skin
erythema was observed upon contact with cyanobacterial strains that do not produce
cyanotoxins4 5
. This would suggest that not only toxins, but other cyanobacterial components
could contribute to the overall skin irritation.
CyanoLPS as a component of the cyanobacterial cell wall is found in all cyanoHABs
and is always present in the contaminated water. To study effects on keratinocytes,
the most abundant cells in skin, HaCaT cell l i n e 4 6 - 5 0
or primary keratinocytes
models of human or animal origin5 1
'5 2
are used. To model the inflammatory state of the
keratinocytes, the bacterial (usually E. coli) LPS is routinely employed.
To have an in vitro keratinocyte model HaCaT cells were grown to form a confluent
layer. Because we studied inflammatory processes, we used non-toxic concentrations
(20 and 50 |ig/mL) of cyanoLPS (Fig. 6). These concentrations are environmentally
relevant, as the endotoxin activity of studied cyanoLPS in the cell culture medium
was in the range of endotoxin activity found in the water sample from respective
cyanoHABs (Appendix B Tab. S3)2 5
.
Fig. 6 Cytoxicity assays. Total cell protein BCA quantification assay (A, B), mitochondrial activity (MTT) (C, D),
and LDH activity in medium (E, F) were measured after 24 h exposure to all used LPS (50 ng/ml and 20 ng/ml].
Untreated HaCaT cells were used as a negative control. HaCaT cells lysed by supplier-provided lysis buffer were
used as a positive control in LDH assay (E, F). n = 3-5. Data were converted to a percentage of the control and
expressed as the mean ± SEM. Data were statistically analyzed using one sample t-test (** p < 0.01]. EC - Escherichia
coli 0111:B4, HAB-Ap - Aphanizomenon bloom, HAB-P1 - Planktothrix bloom, HAB-Do - Dolichospermum
bloom, HAB-Mi - Microcystis bloom, Ap-1 - Aphanizomenon PCC7905, Pl-1 - Planktothrix PCC7805, Do-1 Dolichospermum
UHCC 0352, Do-2 - Dolichospermum UTEX 1444, Mi-1 - Microcystis UHCC 130 and Mi-2 - Microcystis
PCC 7806. (figure on the next page)
38
RESULTS AND DISCUSSION
A 150
§ 1 0 0
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o 50-
Q.
B
I I X
V
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§ 100
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control EC HAB-Ap HAB-PI HAB-Do HAB-Mi control E C Ap-1 PI-1 Do-1 Do-2 Mi-1 Mi-2
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39
RESULTS AND DISCUSSION
We observed that cyanoLPSs isolated from water bloom dominated by Microcystis aeruginosa
(HAB-Mi) and Dolichospermum curvum (HAB-Do) were able to induce the increase
in the production of IL-6 and IL-8 (Fig. 7A, B). Both concentrations induced production
of CCL20 but only the higher one lead to its significant elevation (Fig. 7C). Exposure
to the higher concentration also resulted in significant increase of CCL2 levels
released into the culture medium (Fig. 7D). The effects of the higher concentrations of
cyanoLPSs on cytokines production were comparable or even greater than the effects
of same concentration of E. coli 0111:B4 LPS. In contrast, cyanoLPS isolated from water
bloom dominated by Aphanizomenon flos-aquae (HAB-Ap) and Planktothrix
agardhii (HAB-P1) did not show any effect on the studied cytokines (Fig. 7A-D). On the
other hand, cyanoLPS isolated from axenic culture of Aphanizomenon flos-aquae
PCC 7905 (Ap-1) was the only axenic sample able to elevate the production of cytokines
(IL-6, IL-8 and CCL20). We have observed statistically significant, but probably
not physiologically relevant increase in IL-6 after exposure to cyanoLPS isolated from
axenic culture of Dolichospermum UHCC/HAMBI 0352 (Do-1) (Fig. 7E-H).
Fig. 7 Production of pro-inflammatory cytokines and chemokines by HaCaT cells after exposure
to LPS isolated from water blooms (A, C, E, G) and axenic cyanobacterial cultures (B, D, F, H).
Levels of IL-6 [A, B), IL-8 (C, D), CCL2 (E, F) and CCL20 (G, H) in the medium after 24 h exposure to
studied LPS (20 ng/ml and 50 ng/ml] were measured using ELISA. E. coli LPS was used as a positive
control. Data from five independent experiments are expressed as a mean ± SEM. Data were statistically
analyzed using ANOVA followed by Dunnett's multiple comparison test (** p < 0.01 and * p < 0.05].
EC - Escherichia coli 0111:B4, HAB-Ap - Aphanizomenon bloom, HAB-P1 - Planktothrix bloom,
HAB-Do - Dolichospermum bloom, HAB-Mi - Microcystis bloom, Ap-1 - Aphanizomenon PCC7905,
Pl-1 - Planktothrix PCC7805, Do-1 Dolichospermum UHCC 0352, Do-2 - Dolichospermum UTEX 1444,
Mi-1 - Microcystis UHCC 130 and Mi-2 - Microcystis PCC 7806. (figure on the next page)
40
RESULTS AND DISCUSSION
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41
RESULTS AND DISCUSSION
For the evaluation of environmental samples, only 50 [ig/mL of cyanoLPS was used.
Samples G, S and T behaved similarly to HAB-Mi and HAB-Do elevating all the studied
cytokines. Samples A and H were also able to increase CCL2 production but were not
as potent as E. coli LPS (Fig. 8A-D).
ctrl. EC A B C D E F G H J K L M N O P R S T U
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I * mm
c -eb e e e H
T 3 T 3 J
a T J a
T 3 T 3 T 3 T D
Ctrl. EC A B C D E F G H J K L M N O P R S T U
Fig. 8 Production of pro-inflammatory cytokines and chemokines by HaCaT cells after exposure to LPS
isolated from 19 CyanoHAB-LPS from 18 recreational water bodies at a final concentration of 50 ng/ml for
24 h. Negative control was untreated, and LPS from E. coli was used as a positive control. The concentration of
IL-6 (A), IL-8 (B), CCL2 (C), and CCL20 (D] in the culture medium was determined by ELISA. n = 4. Data are expressed
as the mean ± SD. Data were statistically analyzed by unpaired t-test (*p < 0.05, **p < 0.01]. EC - Escherichia
coli 0111:B4.
Similarly to us, other studies show IL-6 production in in vitro keratinocytes after the
exposure to bacterial LPS5 8
'7 0
'7 1
'7 3
. Furthermore, keratinocytes located at the edge of a
cutaneous wound produce it right after skin injury7 6
'7 7
, and the levels of IL-6 are kept
high during the initial inflammatory phase5 7
.
IL-8 is a strong inducer of cell migration and proliferation7 8
'7 9
and is, similarly
to IL-6, produced during initial inflammatory phase after skin injury5 7
. Its production
42
RESULTS AND DISCUSSION
can be also increased after E. coli LPS exposure of in vitro keratinocytes like HaCaT cell
line4 9
'8 0
'8 1
or primary keratinocyte cell line HEKa5 1
. However, also other bacterial LPS
(e.g. P. gingivalis] activate keratinocytes or even skin explants to produce IL-87 0
'8 2
.
CCL2 is a chemoattractant for macrophages and T-lymphocytes. Elevation of its
production was observed in gingival keratinocytes after P. gingivalis LPS exposure8 7
.
Interestingly, Svensson et al. published E. coli LPS not to be able to induce CCL2 in HaCaT
keratinocytes8 8
, which is in contrast to our results. On the other hand, it has been
shown to elevate CCL2 production in monocytes and macrophages8 4
'9 3 - 9 8
, fibroblasts8
8
'8 9
'9 9
'1 0 0
'1 9 5
, alveolar epithelium and macrophages9 0
'9 1
or microglia9 2
.
CCL20 is a chemoattractant mainly for lymphocytes. In skin tissue, it is known
to be elevated by bacterial LPS, however, this activation is dependent on the bacterial
species/strain. E. coli LPS is a stabile activator of CCL20 production1 0 6 - 1 0 8
in contrast
to P. gingivalis and S. typhymurium LPSs1 0 8
'1 0 9
. Keratinocytes can also produce CCL20
after the exposure to other bacterial components like flagellin and poly I:C1 9 6
or pro-inflammatory
mediators1 9 7
. Interestingly, several studies including ours have observed
CCL20 to be strictly inducible with no basal production1 0 6
'1 0 8
'1 9 6
'1 9 7
contrary to others
who detected basal production of CCL20 between 20-100 pg/ml in keratinocytes1
0 7
'1 9 8
'1 9 9
.
Changes in proliferation, migration, and keratins expression are other hallmarks
of activated keratinocytes. Elevation of K6, K16 and K17 is typical for wounded
skin or hyperproliferating keratinocytes1 1 3
'2 0 0
. Their expression can also be affected by
pro-inflammatory cytokines1 1 6
. We have not observed any significant changes in K6
nor K17 levels apart from a slight increase of K17 in the case of 50 [ig/mL Mi-2 (LPS
isolated from axenic culture of Microcystis aeruginosa PCC 7806) (Fig. 9A-D). Although
healthy cells should have a very low expression of K178 3
, HaCaT cells seem to have very
high basal expression of K6 and K17 and as a result we don't see any changes in their
expression after cyanoLPS challenge or cyanoLPS does not affect their expression.
43
RESULTS AND DISCUSSION
B
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2.0-
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5
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20 |jg/ml 50 [jg/rnl
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60 kD
48 kD
124 kD
Fig. 9 Keratin expression by HaCaT cell line after LPS exposure. K6 (A, B] and K17 (C, D] expression after 24 h
exposure to tested cyanoLPS (50 ng/ml and 20 ng/ml] w a s
detected by western blot. Relative protein levels were
detected by densitometry and the ratio of protein of interest and vinculin was calculated. Data from five independent
experiments were integrated and expressed as the mean ± SEM. Data were statistically analyzed using ANOVA
followed by Dunnett's multiple comparison test (*p < 0.05]. Representative blots are shown in E. EC - Escherichia
coli 0111:B4, HAB-Ap - Aphanizomenon bloom, HAB-PI - Planktothrix bloom, HAB-Do - Dolichospermum bloom,
HAB-Mi - Microcystis bloom, Ap-1 - Aphanizomenon PCC7905, Pl-1 - Planktothrix PCC7805, Do-1 Dolichospermum
UHCC 0352, Do-2 - Dolichospermum UTEX1444, Mi-1 - Microcystis UHCC 130 and Mi-2 - Microcystis PCC 7806.
RESULTS AND DISCUSSION
Since it is known that keratinocytes, when reaching confluence, stop proliferating because
of contact inhibition1 2 5
and that disruption of skin integrity triggers proliferation
and remodelling of the tissue1 2 6
, we used scratch/wound-healing assay to evaluate the
effect of cyanoLPS on proliferation and migration of HaCaT cells. Even though changes
in keratins expression were not detectable in our model, we observed an increase in
wound closure speed after the exposure to LPS isolated from archived HABs and axenic
cultures apart from both strains of Microcystis aeruginosa (Fig. 10).
Similarly to our study, Lee et a/.58
showed elevated proliferation and migration
of keratinocytes when using subtoxic concentrations of a bacterial LPS. However, the
role of bacterial LPS in activation of keratinocytes and other epithelial cell types seems
to be ambivalent. For example, P. aeruginosa LPS was shown to induce increased proliferation
and migration in the airway epithelium1 2 9
, on the other hand, it was observed
to cause a decline in wound healing in epidermal keratinocytes being even more effective
in inhibiting proliferation and migration compared to E. coli LPS6 7
. Decreased proliferation
of in vitro keratinocytes after E. coli LPS exposure was also published by Bassino
et a/.66
. Moreover, lower proliferation was observed with human gingival epithelial
cell line after exposure to P. gingivalis LPS1 2 8
. Also in vivo in mice, lower cutaneous
wound repair after Klebsiella pneumoniae LPS injection was observed1 2 7
.
When studying the environmental samples, we aimed to distinguish proliferation
and migration by stopping the proliferation using mitomycin c. Because of the
number of tested samples, we switched to 96-well plate format, but we did not see any
changes with or without mitomycin c treatment (Fig. 11).
45
RESULTS AND DISCUSSION
Ctrl. EC HAB-Ap
Fig. 10 Changes in proliferation and migration of HaCaT cells after LPS exposure were measured using a
wound-healing assay. Wound healing was observed after 48 h exposure to tested LPS (50 ng/ml) isolated from
water blooms (A) or axenic cyanobacterial cultures (B). Data from seven independent experiments were integrated
and expressed as a percentage of wound closure after 48 h and as the mean ± SEM. Representative pictures
of wound healing after exposure to CyanoHAB LPS (C) and LPS from axenic cyanobacterial cultures (D) are shown.
Data were statistically analyzed using an unpaired t-test (** p < 0.01 and * p < 0.05). EC - Escherichia coli 0111:B4,
HAB-Ap - Aphanizomenon bloom, HAB-P1 - Planktothrix bloom, HAB-Do - Dolichospermum bloom, HAB-Mi - Microcystis
bloom, Ap-1 - Aphanizomenon PCC7905, Pl-1 - Planktothrix PCC7805, Do-1 Dolichospermum UHCC 0352,
Do-2 - Dolichospermum UTEX1444, Mi-1 - Microcystis UHCC 130 and Mi-2 - Microcystis PCC 7806.
46
RESULTS AND DISCUSSION
100-
80
3 60
o
1
without mitomycin-c
l l l l l l l l l l l l l l l l . i l
B
10(h
Ctrl. E C A B C D E F G H J K L M N O P R S T U
with mitomycin-c
Ctrl. EC A M N
Fig. 11 Changes in migration of HaCaT cells after LPS exposure were measured using a wound-healing
assay. Confluent HaCaT cells were treated with 19 CyanoHAB-LPS from 18 recreational water bodies
at a final concentration of 50 ug/ml for 4 days. Negative control was untreated, and LPS from £ coli was used
as a positive control. Wound closure of the scratched monolayer was assessed after 12 h. Cell proliferation
was either unaffected (A] or stopped by mitomycin c pre-treatment (1 ug/ml, 2 h) (B). Data are expressed as
the mean ± SD. Data were statistically analyzed by unpaired t-test. EC - Escherichia coli 0111:B4.
47
RESULTS AND DISCUSSION
5.2 Differences in O-antigen structures of cyanoLPS
The differences in activity of cyanoLPS among each other and in comparison to E. coli
can be due to their structural differences. There are not many studies focusing on the
structure of cyanoLPS. In general, cyanoLPS has different ratio, type and distribution
of monosaccharides compared to the bacterial one and it also differs in the fatty acids
content2 9
. It is known that the number of fatty acid chains and their length have a direct
influence on the endotoxic activity of LPS2 2
.
Here we characterised cyanoLPS using thin layer chromatography (TLC) and
sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) followed by silver
or Pro-Q™ Emerald 300 staining. These methods inform about the general characteristics
e.g. length and charge of the O-antigen. LPS containing the O-antigen is described
as "smooth" while LPS lacking it is called "rough". The primary purpose of the
O-antigen variability is to evade environmental threats and interact with the eucaryotic
immune system2 0 1
. In general, smooth LPS helps the bacteria to evade the complement
system while rough LPS induces the immune system2 0
-2 0 2
.
HAB-Do and HAB-Mi contain smooth LPS ranging from long to very long O-antigen.
Both axenic M. aueruginosa and Do-1 LPS mirror this trend, while Do-2 exhibits
rough LPS as well as smooth LPS. HAB-Ap and Ap-1 show mostly intermediate length
of O-antigen (Fig. 12). Both P. aghardii samples produce faint or no signal, similarly to
our findings on other P/an/ctot/jr/x-dominated blooms and non-axenic P. aghardii culture2
5
-2 0 3
. Notably more laddering and signal intensity was found in CyanoHAB-derived
LPS compared to the axenic LPS. This could be a result of the presence of G- heterotrophic
bacteria in the CyanoHAB.
48
RESULTS AND DISCUSSION
Fig. 12 SDS-PAGE of LPS followed by Pro-Q™ Emerald 300 staining. LPSs were isolated from CyanoHABs or
axenic cyanobacterial cultures. SR—semi-rough LPS. Core OS—core oligosaccharide, EC - Escherichia coli
0111:B4, HAB-Ap - Aphanizomenon bloom, HAB-P1 - Planktothrix bloom, HAB-Do - Dolichospermum bloom,
HAB-Mi - Microcystis bloom, Ap-1 - Aphanizomenon PCC7905, Pl-1 - Planktothrix PCC7805, Do-1 Dolichospermum
UHCC 0352, Do-2 - Dolichospermum UTEX1444, Mi-1 - Microcystis UHCC 130 and Mi-2 - Microcystis PCC 7806.
49
RESULTS AND DISCUSSION
5.3 The main contributor to the pro-inflammatory activation of
CyanoHAB-derived LPS is G- bacteria
Allergenic effects of cyanobacterial water bloom have previously been sho wn to be associated
with the aqueo us extract o f the bio mass rather than the to xins5 3
. Mo reo ver,
the extracts fro m axenic cultures were not active in contrast to the enviro nmental and
non-axenic samples. As LPS had to be in the extracts, it indicates, that LPS from heterotrophic
bacteria co uld have important impact on the bioactivity of the samples. Therefore,
we focused o n composition of the biomasses to study possible co nnectio n o f specific
bacterial genera presence with the bioactivity of HAB.
The co mpo sitio n of CyanoHABs HAB-Ap, HAB-P1, HAB-Do and HAB-Mi was determined
using NGS analysis (Fig. 13). Co nsiderable variatio n was fo und amo ng the
samples. The most prevalent amo ng the G- bacteria was the Proteobacteha phylum
with minimal representation in HAB-P1 (1.80%) and the maximal representatio n in
HAB-Do (56.99%). Other pro minent phylla were Bacteroidetes (HAB-Do 14.74%,
HAB-Mi 10.02%), Verrucomicrobia (HAB-Do 9.47%, HAB-P1 1.38%), and Chloroflexi
(HAB-Do 3.57%, HAB-P11.11%). In HAB-Mi, Gemmatimonadetes phylum was also detected
(1.85%).
A
100-,
£ so
0
1 60-
C
_Q
ra<d 40-
>
a. 20-
0 -
1.11%1.8Ů°/.1.80B
/«
B
100,
• Unassigned
^1 Firmicutes (£+)
_ l AclinoSacteria (G+j
• Verrucamicrcbia (C-j
• S a ixh a ri bacteria (G-]
• Planctomyceies (G-]
^1 Pralennacteria [G-;
^ Gemmatimonacctcs \.G-\
M Chlorotlesj (Q -)
• Bacteroidetes (G-[
=1 Cyanobacteria
H A B - A p H A B - P I H A B - D o H A B - M i Ap-1 PI-1 Do-1 Do-2 Mi-1 Mi-2
Fig. 13 Taxonomie abundance at the phylum level in the water bloom and axenic cyanobacterial culture
biomasses. Phyla with abundance belo w 0.01% are grouped as rare in the graph, and only phyla with abundance
> 1% are indicated. HAB-Ap - Aphanizomenon blo o m, HAB-PI - Planktothrix blo o m, HAB-Do - Dolichospermum
bloom, HAB-Mi - Microcystis blo o m, Ap-1 - Aphanizomenon PCC7905, Pl-1 - Planktothrix PCC7805, Do-1 Dolichospermum
UHCC 0352, Do -2 - Dolichospermum UTEX 1444, Mi-1 - Microcystis UHCC 130 and Mi-2 - Microcystis
PCC 7806.
50
RESULTS AND DISCUSSION
Differences in taxonomic representation are also evident when comparing the ten
most abundant G- bacterial genera in respective water bloom biomasses (Fig. 14). In
all samples, Rhodobacter, Roseomonas, Halomonas, and Vibrio genera were present.
Moreover, unspecified genera from Nitrosomonadaceae and Comamonadaceae (labeled
as 1) were also found in each HAB. Nevertheless, the abundance of these bacteria
varied among the samples.
On the other hand, there were 11 bacterial genera unique to only one of the
studied water blooms - four of them were unique to HAB-P1 [Caldilineaceae, Thiotrix,
Rhodopirellula, and Chtoniobacterales 1), three to HAB-Do [Anaerolineaceae, Lautropia,
and Variovorax], and two to HAB-Ap [Emticicia, Rubrivivax) and to HAB-Mi [Gemmatimonas
and Azohydromonas). Similarly to the common genera, their abundances varied
among the biomasses.
As our results show, the active HAB-Do and HAB-Mi LPS were isolated from biomasses
containing the highest proportion of G- bacteria, primarily from the Proteobacteria
phylum. The most interesting genera found were Vibrio, Roseomonas, and Lautropia
including some pathogenic species, similarly to the Alcaligenaceae family.
V. cholerae is known for its cholera toxin causing cholera disease. However, LPS
is also bioactive in some strains. The structure of the LPS serves as a classifier for different
V. cholerae strains, with strains other than 01 and 0139 (which were proved to
cause several cholera outbreaks) have been shown to cause gastrointestinal and extraintestinal
infections2 0 4
. Moreover, the Vibrio O-antigen influences intestinal colonization
by the bacteria2 0 5
. V. vulnificus, which is also pathogenic to humans, depends on
an LPS-containing capsid that exhibits pro-inflammatory activity for successful infection2
0 6
.
Roseomonas genera contains two species, R. mucosa and R.gilardii. The first one
is an occasional immunocompromised patients' pathogen, commonly found as a part
of skin microbiome2 0 7
.
Lautropia is a genera containing the species L. mirabilis, an opportunistic pathogen
in humans with underlying conditions2 0 8
'2 0 9
. Family Alcaligenaceae contains the
51
RESULTS AND DISCUSSION
pathogenic Bordetella genus. There are three species, B. pertussis, B. parapertussis, and
B. bronchiseptica, which are causative agents of human respiratory tract infections including
whooping cough2 1 0
. Their LPS is an important virulent factor of these bacteria.
On the other hand, in M/crocvst/s-dominated HAB, the most prevalent genus was
Rhodobacter, known as a non-pathogenic one. It even includes the species
R. sphaeroides with TLR4 antagonizing LPS2 1 1
'2 1 2
.
Based on our results, we cannot confirm pathogenic species/strains presence
in the studied biomasses, but the abundance of genera or families including pathogens
in active LPS samples supports the hypothesis that these bacteria may contribute significantly
to the overall bioactivity of LPS of environmental mixtures.
52
RESULTS AND DISCUSSION
Cytophagaceae 1
Emticicia
Flavobacterium
Gemmatimonas
Anaerolineaceae 1
Caldilineaceae 1
Rhodopirellula
Megan ema
Methylomsula
Phenylobacterium
Rhizobiales 1
Rhizobiales 2
Rhizobiales 3
Rhodobacter
Roseomonas
Sphingopyxis
Alcaligenaceae 1
Azohydromonas
Ccmamonadaceae 1
Ccmamonadaceae 2
Lautropia
Nitrosomonadaceae 1
Oxalobacteraceae 1
Rubriviyax
Variovorax
Acidibacter
Halomonas
Silanimonas
Thiothrix
Vibrio
Proteobacteria 1
Chthoniobacterales 1
Chthoniobacterales 2
6
67
110
1903
1122
272
22
• 1148
\ 943
34
1464
43
15
406
39
49
20
47
158
14
40
40
29
4
68
8
355
61
8038
2318
162
21
•
8725
5900
882
833
167
4441
671
3133
4398
t
2579
1624
6065
2942
146
482
848
t
578
9
62
28
3239
1211
24
1469
499
443
443
\ 1549
204
75
544
36
HAB-Ap HAB-PI HAB-Do HAB-Mi
Fig. 14 Representation of the ten most abundant bacterial taxa in each CyanoHAB. The graph illustrates NGS
data for each water bloom biomass, displaying the ten most abundant Gram-negative (G-] bacterial taxa (cyanobacteria
were excluded from the dataset). Each bacterium is labeled by its genus, determined at the Taxonomy 5
level of NGS analysis. If the genus was unknown, the nearest established level of taxonomy was utilized. The results
were categorized by phylum, with genera belonging to Bacteroidetes in yellow, Gemmatimonadetes in orange,
Chloroflexi in blue, Planctomycetes in pink, Proteobacteria in green (ranging from lighter to darker shades representing
the classes Alpha-, Beta-, Gammaproteobacteria, and TA18], and Verrucomicrobia in grey. The numbers
indicate the respective counts of each taxon. HAB-Ap - Aphanizomenon bloom, HAB-PI - Planktothrix bloom,
HAB-Do - Dolichospermum bloom, HAB-Mi - Microcystis bloom.
RESULTS AND DISCUSSION
5.4 Effect of axenic cyanoLPS can on macrophages
In recent years, the pro-inflammatory effect of cyanoLPS on human immune cells has
been shown2 3
-2 7
-2 9
'3 5
. The endotoxic activity of LPS is usually tested using Limulus amebocyte
lysate or PyroGene™. However, this test does not reliably reflect the biological
activity of LPS as we have shown previously2 3
-2 7
-2 0 3
. All used axenic strains show little
to no endotoxin activity by PyroGene™rFC assay, similarly to HAB-Ap, HAB-P1, and
HAB-Do. HAB-Mi was the only cyanoLPS sample exhibiting significant endotoxin activity
(Tab.2).
Tab.2 Endotoxin activity of LPS isolated from CyanoHAB biomasses, axenic cyanobacterial cultures,
and positive controls measured by PyroGene™ assay. EC - Escherichia coli 0111:B4,
HAB-Ap - Aphanizomenon bloom, HAB-P1 - Planktothrix bloom, HAB-Do - Dolichospermum bloom,
HAB-Mi - Microcystis bloom, Ap-1 - Aphanizomenon PCC7905, Pl-1 - Planktothrix PCC7805, Do-1 Dolichospermum
UHCC 0352, Do-2 - Dolichospermum UTEX 1444, Mi-1 - Microcystis UHCC 130 and
Mi-2 - Microcystis PCC 7806.
PyroGene™ PyroGene'"
sample ,
[*103
EU/mgLPS] [*103EU/mgd.w.]
EC 3 689.95 HAB-Ap
0.43 6.9
HAB-Pl 0.17 1.3
HAB-Do 0.58 7.1
HAB-Mi 37 202.67 265 092.9
Ap-1 0.07 1.7
Pl-1 0.24 1.9
Do-1 0.09 0.8
Do-2 0.05 0.8
Mi-1 2.95 46.4
Mi-2 9.40 117.2
Because we observed slight pro-inflammatory response of epithelial cells to some of
the axenic cyanoLPS we wanted to confirm, it is also able to activate innate immunity.
For this purpose, mouse macrophage cell line RAW264.7 was used. Again, we used
non-toxic concentrations of cyanoLPS (Fig. 15A). After exposure to Ap-1, we observed
high production of TNFa, a typical pro-inflammatory cytokine. Its production was significantly
increased also upon the Pl-1 and Do-1 challenge (Fig. 15B). Nevertheless,
A p l was the only one able to trigger the production of NO as well (Fig. 15C). On the
other hand, none of the axenic cyanoLPS elevated IL-6 levels (Fig. 15D). Exposure to
54
RESULTS AND DISCUSSION
E. coli 0111:B4 LPS, the positive control, resulted in elevation of all measured parameters
and was more potent compared to cyanoLPS (Fig. 15).
300
2
c
O
o
'S 200
X
B 100
E 6 000
Ol
s
I
pos, drl. EC Ap-1 PI-1 Do-1 Do-2 Mi-1 Mi-2 Ctrl. EC Ap-1 PI-1 Do-1 Do-2 Mi-1 Mi-2
Ctrl. EC Ap-1 PI-1 Do-1 Do-2 Mi-1 Mi-2 Ctrl. EC Ap-1 PI-1 Do-1 Do-2 Mi-1 Mi-2
Fig. 15 LDH activity in medium and production of pro-inflammatory mediators by RAW264.7 cells. Levels
of LDH [A], TNFa (B), IL-6 (C), and NO (D) produced by RAW 264.7 cells after exposure to LPS isolated from axenic
cyanobacterial cultures. LDH was measure using Cytotoxicity Detection Kit. Cells were lysed by supplier-provided
lysis buffer were used as a positive control. Cytokines were measured by ELISA, concentrations of nitrites as an
indirect way to determine NO production were measured using Griess reaction. All were measured after 24 h
exposure to 10, 50,100, and 1 000 ng/ml of studied LPS and positive control E. coli LPS. Data are expressed as the
mean ± SEM; n > 3. Data were statistically analyzed using ANOVA followed by Dunnett's multiple comparison test
or Kruskal-Wallis test for non-homogenous data variation (** p < 0.01 and * p < 0.05]. EC - Escherichia coli
0111:B4, Ap-1 - Aphanizomenon PCC7905, Pl-1 - Planktothrix PCC7805, Do-1 Dolichospermum UHCC 0352,
Do-2 - Dolichospermum UTEX1444, Mi-1 - Microcystis UHCC 130 and Mi-2 - Microcystis PCC 7806.
These results are consistent with the findings of previous reports that utilized cyanoLPS
isolated from non-axenic cyanobacterial cultures2 0 3
. Similarly, we observed
that cyanoLPS from axenic M. aeruginosa laboratory culture required concentrations
55
RESULTS AND DISCUSSION
10-100 times higher than E. coli 0111:B4 and CyanoHAB LPS to activate neutrophils
and monocytes2 7
. Furthermore HAB-Ap (labeled APH LPS in the publication) was also
able to potentiate the effects of non-toxic concentration of cylindrospermopsin (an
A. flos-aquae toxin) on immune cells1 8
.
It is evident that bacterial LPS and cyanoLPS exhibit varying degrees of potency,
and exposed cells respond to these LPS differently in terms of the produced inflammatory
mediators. We hypothesized that the observed difference in cellular responses to
cyanoLPS compared to bacterial LPS could be attributed to the activation of different
receptors or signaling pathways. Upon LPS treatment, several signaling pathways are
activated. Some lead to the production of type IIFN1 4 2
'1 9 1
'1 9 2
. Other result in the phosphorylation
of the transcription factor N F K B 2 1 3 - 2 1 7
or activate MAP kinase signal-
jng214,215
Depending on the strain, in murine macrophage cell line RAW264.7 cyanoLPS
samples were found to activate only certain signaling pathways that are typically activated
by bacterial LPS. We detected elevated expression of IFNfB after exposure to all
studied cyanoLPS (Fig. 16A). Furthermore, the transcription factor N F K B was activated
by Ap-1, Pl-1, and Mi-1 (Fig. 16B). The phosphorylation of p38 was only triggered by
Mi-1, while there was a slight, but not statistically significant, elevation observed after
exposure to Ap-1 (Fig. 16C). Only Ap-1 was able to activate SAPK/JNK pathway
(Fig. 16D). To evaluate the human relevance of our findings, we employed human monocytic
cell line THP-1 containing N F K B reporter gene (THP-l.luc cell line). All cyanoLPS
samples slightly elevated the activity of NFKB, but only the effect of Ap-1 was statistically
significant (Fig. 17).
56
RESULTS AND DISCUSSION
B 3n
3.5i
2.5
* 1.5
< 0.5
I 0.2-
0.1
0.0 r-i n
Ctrl. EC Ap-1 PI-1 Do-1 Do-2 Mi-1 Mi-2
COy 2-
T
P-NFkB
NFkB
Ctrl. E C Ap-1 PI-1 Do-1 Do-2 Mi-1 Mi-2
mm—mm *• 65 kD
65 kD
1
I
p-p38
p3S
Ctrl. E C Ap-1 PI-1 Do-1 Do-2 Mi-1 Mi-2
• M t
m
38 kD
Z
a.
<
in
X
X
J H
x
Ctrl. E C Ap-1 PI-1 Do-1 Do-2 Mi-1 Mi-2
38 kD p-SAPK,JNK
SAPK/JNK
54 kD
46 kD
54 kD
46 kD
Fig. 16 Activation of signaling pathways by cyanoLPS. Expression of IFNß mRNA 4 h after exposure to cyanoLPS
and positive control was detected by qPCR (A). The results are presented as phosphorylation of selected signaling
pathways - NFKB (B), p38 (C), and SAPK/JNK (D] - was detected using western blotting 30 min. after the exposure.
The results are expressed as a mean ratio of phosphorylated vs. total protein optical density; ± SEM; n > 3. Data
were statistically analyzed using an unpaired t-test (** p < 0.01 and * p < 0.05]. EC - Escherichia coli 0111:B4,
Ap-1 - Aphanizomenon PCC7905, Pl-1 - Planktothrix PCC7805, Do-1 Dolichospermum UHCC 0352, Do-2 - Dolichospermum
UTEX1444, Mi-1 - Microcystis UHCC 130 and Mi-2 - Microcystis PCC 7806.
57
RESULTS AND DISCUSSION
2 000-|
1 500-
1 000
200
150-
100-
50-
I
X X
EC Ap-1 PI-1 Do-1 Do-2 Mi-1 Mi-2
Fig. 17 Activation of NFKB in THP-l.luc cells. The cells were exposed to cyanoLPS and EC for 10 h and then the
luminescence was measured. The data are expressed as a mean percentage of negative control (i.e. untreated cells)
± SEM; n > 3. Data were statistically analyzed using a one-sample t-test with the negative control = 100% (** p <
0.01 and *p < 0.05). The red line denotes the level of 100% of control. EC - Escherichia coli 0111:B4, Ap-1 -Aphanizomenon
PCC7905, Pl-1 - Planktothrix PCC7805, Do-1 Dolichospermum UHCC 0352, Do-2 - Dolichospermum UTEX
1444, Mi-1 - Microcystis UHCC 130 and Mi-2 - Microcystis PCC 7806.
58
RESULTS AND DISCUSSION
5.5 The activity of cyanoLPS is dependent on TLR2, but not TLR4
Based on these findings and our previous data, which revealed a distinct pattern of
cytokine production by murine macrophages after exposure to cyanoLPS compared to
E. coli 0111:B4 and P. aeruginosa LPS2 0 3
, we hypothesized that the activation of immune
cells by cyanoLPS is not mediated via the typical LPS receptor TLR4, but rather
through TLR2. It is known that the structure and chemical composition of cyanoLPS
differ from the typical LPS of G- bacteria2 2
, which can result in different affinity and/or
specificity of cyanoLPS for binding to receptors. This difference could explain the specific
patterns in the activation of signaling pathways by cyanoLPS we observed.
Although TLR4 is widely recognized as a canonical receptor for bacterial LPS
signaling2 1 8
, it is important to note that other TLRs, such as TLR2, as well as other PRRs,
may also play a role in cellular responses to different types of LPS, including cyanoLPS.
To investigate whether cyanoLPS also activates TLR4 in murine macrophages,
we pre-treated the cells with TLR4 inhibitor. The E. coli 0111:B6 LPS elevated the production
of TNFa, but this effect was significantly reduced when TLR4 inhibitor was
used (Fig. 18A). However, the effects of HKLM (heat-killed L. monocytogenes), a model
TLR2 agonist, and the studied cyanoLPS were not affected by TLR4 inhibition. On the
other hand, a co-treatment with TLR2 neutralizing antibody was able to significantly
decrease the TNFa production of Ap-1 as well as E. coli LPS (Fig. 18B). The reduction
of TNFa production by other cyanoLPS in the presence of neutralizing antibody was
not statistically significant, but a decreasing trend was observed in most of them.
59
RESULTS AND DISCUSSION
=• 10 000'
£
• without inhibitor
• with inhibitor
fill ["ill - ni-, r,.
Ctrl. EC Ap-1 PI-1 Do-1 Do-2 Mi-1 Mi-2
• without neutralizing antibody
• with neutralizing antibody
J k i l i
Ctrl. EC HKLM Ap-1 PI-1 Do-1 Do-2 Mi-1 Mi-2
Fig. 18 Inhibition of TLR2 and TLR4. RAW 264.7 cells were pre-treated with TLR4 inhibitor for 6 h (A] or cotreated
with TLR2 neutralizing antibody (B] and exposed to cyanoLPS and positive controls for 24 h. Then the
medium was collected and the concentration of TNFa was determined by ELISA. Results are expressed as a mean
± SEM; n > 3. Data were statistically analyzed using two-way ANOVA (** p < 0.01 and * p < 0.05]. EC - Escherichia
coli 0111:B4, Ap-1 - Aphanizomenon PCC7905, Pl-1 - Planktothrix PCC7805, Do-1 Dolichospermum UHCC 0352,
Do-2 - Dolichospermum UTEX1444, Mi-1 - Microcystis UHCC 130 and Mi-2 - Microcystis PCC 7806.
To further validate these findings, we purchased a HEK293 cell line overexpressing
TLR4/CD14/MD2 or TLR2/CD14. Cells containing an empty plasmid (null) were used
as a negative control. E. coli LPS was able to elevate the production of IL-8 in both
TLR-overexpressing cell lines (Fig. 19). Exposure to HKLM, Ap-1, and Do-1 induced an
increase in IL-8 production only in TLR2/CD14 overexpressing cell line. In addition,
exposure to Pl-1 and Mi-2 led to a slight but not statistically significant increase in IL-8
production by TLR2/CD14 overexpressing cells. In contrast, none of the cyanoLPS had
an impact on the cytokine production by TLR4/CD14/MD2 overexpressing cells.
60
RESULTS AND DISCUSSION
3 000n
2 500>
1 500 <
D)
£ 1 OOOH
CO
500•
null
• TLR4
• TLR2
i
I
**
i
£L
Ctrl.
J L J L
EC HKLM Ap-1 PI-1 Do-1 Do-2 Mi-1
J L
Mi-2
Fig. 19 Production ofIL-8 by HEK293 cell overexpressing TLR4/CD14/MD2 or TLR2/CD14. The cell lines,
including cells transfected by the empty vector (null], were exposed to 50 |ig/ml of E. coli 0111:B4 LPS and cyanoLPS
and 10"8/ml HKLM for 24 h. Then, the concentration of IL-8 was determined by ELISA. The results are
presented as a mean ± SEM; n > 3. Data were statistically analyzed using two-way ANOVA (** p < 0.01 and * p <
0.05]. EC - Escherichia coli 0111:B4, HKLM - heat-killed Listeria monocytogenes, Ap-1 - Aphanizomenon PCC7905,
Pl-1 - Planktothrix PCC7805, Do-1 Dolichospermum UHCC 0352, Do-2 - Dolichospermum UTEX1444, Mi-1 - Microcystis
UHCC 130 and Mi-2 - Microcystis PCC 7806.
Lastly, we utilized three different THP-1 TLR2V- clones, prepared using
pSpCas9(BB)-2A-GFP (Px458) plasmid. The gRNA was targeted in the 4t h
exon of the
t/r2gene (sequences 5'-ACCGCATGGTATCTGCAA-3'; 5'-TTGCAGATACCATTGCGGT-3').
The successful mutation was verified using Sanger sequencing. Exposure to Ap-1 induced
the production of TNFa in naive cells and in the scrambled control, but this effect
was completely abolished in all TLR2V- clones (Fig. 20). The other cyanoLPS samples
triggered only minimal TNFa production in the control cell lines, while the TLR2Vclones
did not exhibit any response at all.
61
RESULTS AND DISCUSSION
2 000
1 500
a. 1 000 >
S 200 f
****** ******
• naive cells
• scrambled Ctrl.
• clone A
• clone B
• clone C
100
J L J L J L
Ctrl. EC HKLM Ap-1 PI-1 Do-1
J L
Do-2
J L J L
Mi-1 Mi-2
Fig. 20 Activation of TNFa production by cyanoLPS dependent on TLR2 or TLR4 expression. TLR2-/- clones
as well as naive cells and scrambled control cells were exposed to cyanoLPS and positive controls and production
of TNFa into the medium was determined using ELISA. The results are expressed as a mean ± SEM; n = 4. Data
were statistically analyzed using two-way ANOVA (** p < 0.01 and * p < 0.05]. EC - Escherichia coli 0111:B4,
HKLM - heat-killed Listeria monocytogenes, Ap-1 - Aphanizomenon PCC7905, Pl-1 - Planktothrix PCC7805, Do-
1 - Dolichospermum UHCC 0352, Do-2 - Dolichospermum UTEX 1444, Mi-1 - Microcystis UHCC 130 and Mi-2 -Microcystis
PCC 7806.
These findings suggest that in human cells, cyanoLPS derived from A. flos-aquae
PCC7905 predominantly signals via TLR2. It is in accordance with other studies suggesting
that atypical LPS can signal using TLR2. For example, Legionella interrogans
and L. pneumophila LPS utilize TLR2 in several cell types1 6 8
'2 1 9
. Similarly, Helicobacter
pylori LPS activated human gastric adenocarcinoma cell line via TLR21 6 6
and Ochrobactrum
intermedium LPS signaled through both TLR2 and TLR4 in mouse peritoneal macrophages
or HEK293 cells expressing the respective receptors1 6 7
.
Interestingly, our study also demonstrated that E. coli 0111:B4 LPS can signal
through both TLR4 and TLR2. Although TLR2 activation is described for
62
RESULTS AND DISCUSSION
non-enterobacterial-like LPS, evidence of E. co//-induced TLR2 activation can be found
in the literature1 7 1
. Still, this is a very rare observation.
Murine BMDMs and chicken fibroblasts were shown to produce IFNfB in response
to activation of TLR4 by G- bacteria LPS, as well as by activation of TLR2 by various
specific ligands1 4 1
'1 9 4
'2 2 0
. In contrast, murine peritoneal macrophages were reported to
produce IFN(3 after TLR4 but not TLR2 activation1 9 3
.
63
CONCLUSION
6 Conclusion
The work presented in this thesis has led to the following conclusions:
• CyanoHAB-derived LPS can have a pro-inflammatory effect on keratinocytes in
vitro.
• Most of the pro-inflammatory effect stems from heterotrophic G- bacteria.
• The activity of the CyanoHAB-derived LPS depends on the specific composition
of bacterial genera.
• CyanoLPS is not as potent as E. coli 0111:B4 but some can induce inflammatory
processes in vitro.
• A. flos-aquae PCC7905 cyanoLPS induces pro-inflammatory processes in vitro
via TLR2, but not TLR4.
Fig. 21 Mechanism of Aphanizomenonflos-aquaePCC7905 cyanoLPS signalling; created
with BioRender.com
64
BIBLIOGRAPHY
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Appendix A
Cyanobacterial bloom-associated lipopolysaccharides induce pro-inflammatory
processes in keratinocytes in vitro
P. Raptová, V. Skočková, P. Babica, I. Sovadinová, E. Sychrová, P. Vídeňská,
P. Šplíchalová, 0. Vašíček, L. Šindlerová
Environmental Toxicology and Pharmacology, 2023, doi: 10.1016/j.etap.2023.104342
84
Environmental Toxicology and Pharmacology 105 (2024) 104342
ELSEVIER
Contents lists available at ScienceDirect
Environmental Toxicology and Pharmacology
journal homepage: www.elsevier.com/locate/etap
Cyanobacterial bloom-associated lipopolysaccharides induce
pro-inflammatory processes in keratinocytes in vitro
P. R a p t o v á a , b
, V. S k o č k o v á a , b
, P. B a b i c a c , d
, I. Sovadinovác
, E. Sychrová', P. Vídeňská'
P. Šplíchalová', O. Vašíčeka
, L. Šindlerováa
'
a
Department ofBiophysics of Immune System, Institute of Biophysics of the Czech Academy of Sciences, Brno 61200, Czech Republic
b
Department of Experimental Biology, Faculty of Science, Masaryk University, Brno 62500, Czech Republic
c
RECETOX, Faculty of Science, Masaryk University, Brno 62500, Czech Republic
d
Department of Experimental Phycology and Ecotoxicology, Institute of Botany of the Czech Academy of Sciences, Brno 60200, Czech Republic
A R T I C L E I N F O A B S T R A C T
Keywords:
Cyanobacterial harmful algae bloom
lipopolysaccharide
Axenic cyanobacteria
Inflammation
Human HaCaT keratinocytes
Genomic analyses
Our previous studies have shown that CyanoHAB LPS (lipopolysaccharides) and LPS from cyanobacterial cultures
induce pro-inflammatory effects on intestinal epithelial and immune cells in vitro. To expand our understanding,
we investigated their impact on human keratinocytes, which are targeted during water recreational activities.
LPS samples were isolated from CyanoHAB biomasses dominated by Microcystis, Aphanizomenon, Planktothrix,
and Dolichospermum, or from axenic cultures of these genera. We identified two CyanoHAB biomasses containing
a high proportion of Gram-negative bacteria, including potentially pathogenic genera. These biomasses showed
the highest induction of interleukin (IL) 8, IL-6, C-C motif chemokine ligand (CCL) 2 (also known as MCP-1), and
CCL20 production by HaCaT cells. Interestingly, all CyanoHAB-derived LPS and LPS from axenic cultures (except
for Microcystis) accelerated cell proliferation and migration. Our findings highlight the role of G- bacteria
composition and LPS structural disparities in influencing these effects, with implications for skin health during
recreational activities.
1. I n t r o d u c t i o n
Cyanobacterial harmful blooms (CyanoHABs) pose a serious
ecological concern, and their occurrence is projected to rise in the future
(Huisman et al., 2018). These blooms can generate toxins, leading to
adverse health issues, including fatalities. Furthermore, CyanoHABs can
produce other bioactive molecules which, when combined with the
toxins, can potentiate their effects. Our previous studies have demonstrated
that CyanoHAB-derived lipopolysaccharides (LPS) alone can
activate intestinal and immune cells in vitro (Moosova et al., 2019b;
Skockova et al., 2023a), as well as enhance the pro-inflammatory effect
of non-toxic concentrations of cylindrospermopsin in vitro (Moosova
et al., 2019a).
Dermal exposure to CyanoHAB LPS is a potential route of exposure
during recreational activities, alongside the more commonly recognized
oral exposure. While skin irritation from contact with CyanoHABs has
primarily been associated with cyanotoxins like cylindrospermopsin or
aplysiatoxins (Osborne et al., 2001; Stewart et al., 2006), skin effects
have also been observed upon contact with cyanobacterial strains that
do not produce cyanotoxins (Pilotto et al., 2004). Moreover, allergic skin
reactions have been reported when exposed to cyanotoxin-free components
of cyanobacteria (Bernstein et al., 2011; Torokne et al., 2001). In
these cases, the cyanobacteria samples were detoxified by deproteinization,
suggesting that LPS may have contributed to the observed
effects.
Importantly, it should be noted that LPS in CyanoHABs does not
originate solely from cyanobacteria. A significant number of various
bacteria inhabit cyanobacterial colonies or the surrounding water,
including Gram-negative species capable of producing LPS (Cai et al.,
2014; Mankiewicz-Boczek and Font-Najera, 2022; Seymour et al., 2017;
Woodhouse et al., 2018). Previous research has shown that bacterial LPS
can stimulate skin tissue explants to produce pro- and anti-inflammatory
cytokines and chemokines (Gvirtz et al., 2020). The predominant cell
type in the skin is keratinocytes, which can secrete cytokines such as
* Corresponding author.
E-mail addresses: raptova@ibp.cz (P. Raptová), skockova@ibp.cz (V. Skočková), pavel.babica@recetox.muni.cz (P. Babica), sovadinova@recetox.muni.cz
(I. Sovadinová), eliska.sychrova@recetox.muni.cz (E. Sychrová), petra.videnska@recetox.muni.cz (P. Vídeňská), petra.splichalova@recetox.muni.cz
(P. Splíchalová), ondrej.vasicek@ibp.cz (O. Vašíček), sindler@ibp.cz (L. Sindlerová).
https://doi.Org/10.1016/j.etap.2023.104342
Received 26 June 2023; Received in revised form 23 October 2023; Accepted 8 December 2023
Available online 12 December 2023
1382-6689/© 2023 Elsevier B.V. All rights reserved.
P. Raptovä et al. Environmental Toxicology and Pharmacology 105 (2024) 104342
interleukin (IL) 1, tumor necrosis factor-alpha (TNF-a), or IL-6, thus
activating skin-resident immune cells and regulating their response.
Keratinocytes also release chemokines, such as IL-8, C-C motif chemokine
ligand (CCL) 2 (also known as MCP-1), and CCL20, which attract
immune cells from the circulation to the site of infection (Behfar et al.,
2018; Nestle et al., 2009). Additionally, activated keratinocytes undergo
cytoskeletal remodeling, replacing keratin (K) dimers K1/K10 in intermediate
filaments with K6/K16 and K6/K17 dimers. This substitution is
crucial for intensive proliferation and collective migration, facilitating
the replacement of damaged cells and the closure of the wound (Zhang
et al., 2019).
In this study, we aimed to investigate the pro-inflammatory effects of
LPS isolated from CyanoHAB biomasses and axenic cyanobacteria cultures
(single-strain cyanobacteria grown without heterotrophic bacteria)
on the human keratinocyte cell line HaCaT. Specifically, our focus
was on LPS derived from Microcystis, Planktothrix, Aphanizomenon, and
Dolichospermum, which represent common bloom-forming cyanobacterial
genera (Huisman et al., 2018). The HaCaT cell line is widely used for
pro- and anti-inflammatory studies using various LPS as well as for
protective, pro-healing, anti-inflammatory, or anti-aging effects of
cyanobacteria-derived compounds in cosmetics and dermatology (Cai
et al., 2018; Favas et al., 2022; Jang and Kim, 2021; Lopes et al., 2020;
Olaru and Jensen, 2010).
2. M a t e r i a l s a n d m e t h o d s
2.1. Cyanobacterial biomass preparation
The preparation of the water bloom biomasses collected from water
reservoirs in the Czech Republic is described in (Skočková et al., 2023a).
Briefly, the biomasses were concentrated using a plankton net with a 20
um mesh, transported on ice to the laboratory, and then frozen and
lyophilized, as previously published (Javurek et al., 2015; Pipal et al.,
2020; Smutná et al., 2023; Szmucová, 2019). Species composition was
determined by light microscopy in the samples fixed with 2% formaldehyde.
The cell concentration of each species (cell/ml) was transformed
to biovolume (um3
/ml) using established methods (Hillebrand
et al., 1999; Skácelova and Leps, 2014). Subsequently, the biovolumes of
individual species were expressed as a percentage of the total phytoplankton
biovolume of the sample. The sample codes, dominant species,
biovolume percentages for each sample, and other characteristics of the
biomasses are summarized in Table 1.
Axenic cultures of cyanobacteria were obtained from the Pasteur
Culture Collection of Cyanobacteria (PCC, Paris, France), UTEX Culture
Collection of Algae (UTEX, Austin, TX, USA), and HAMBI Culture
Collection of University of Helsinki (HAMBI/UHCC, Helsinki, Finland).
Specifically, Aphanizomenonflos-aquaePCC7905 (abbreviated as Ap-1),
Planktothrix agardhü PCC7805 (Pl-1), Dolichospermum UHCC/HAMBI
0352 (alias 1TU35S12, Do-1), Dolichospermumfios-aquaeUTEX1444
(Do-2), Microcystis aeruginosa UHCC/HAMBI 130 (Mi-1), and Microcystis
aeruginosa PCC7806 (Mi-2) were used. All cultures were grown in a
mixture of Zehnder medium (Staub, 1961), Bristol (modified Bold)
medium (Bold, 1949), and distilled water in a 1:1:2 (v/v/v) ratio
(Moosova et al., 2019b), except the strains obtained from UHCC/HAMBI,
which were cultured in the Zehnder medium. The cultures
were grown under continuous illumination from the cool white fluorescent
tubes (1300-3000 lux) at 22 °C ± 2 °C The cultures were
handled aseptically, and grown in filter-cap tissue culture flasks (TPP,
Trasadingen, Switzerland) without active aeration. To ensure the axenic
status of the batch cultures before the biomass collection, an aliquot of
the culture was spread over a 100 mm Petri dish with 1.8% (w/v) R2A
agar (Sigma-Aldrich, St. Louis, MO, USA) and the dishes were evaluated
for bacterial growth after 5-7 days at 30 ° C The absence of heterotrophic
bacteria was further controlled by fluorescence microscopy after
staining the culture sample with 5 ug/ml Hoechst 33342 (Thermo Fisher
Scientific, Waltham, MA, USA). Cyanobacterial biomass was collected
from the cultures approaching the stationary phase of the growth, harvested
by centrifugation, and freeze-dried.
2.2. LPS preparation
The extraction of a fraction of cyanobacterial LPS was performed
using a hot phenol-water extraction method, as previously described
(Bernardova et al., 2008; Blahova et al., 2013; Moosova et al., 2019b;
Skockovä et al., 2023a). In brief, 1 g of lyophilized cyanobacterial
biomass was suspended in 50 ml MilliQ water and sonicated using an
ultrasonic bath (10 min). The suspension was then heated to 68 °C,
mixed with 50 ml of pre-warmed 90% phenol, and stirred for 20 min at
68 ° C After cooling to 4 °C, the mixture was centrifuged (5,630g, 30
min, 4 °C), and the supernatant (aqueous phase with LPS) was collected.
The phenol layer was re-extracted with 50 ml of MilliQ water, and
pooled supernatants were purified by 48-h dialysis using cellulose
membranes (33 x 21 mm, Sigma-Aldrich, D9652) against MilliQ water
(1 1) containing toluene (10 n.1/1) to prevent bacterial contamination.
After 24 h of dialysis, the water was changed and dialysis continued for
another 24 h. The dialyzed extracts were centrifuged (5,630g, 30 min,
4 °C), and collected supernatants were lyophilized. The resulting
semi-purified freeze-dried extract of LPS was resuspended in 3.75 ml of
Table 1
Endotoxin activity of LPS isolated from CyanoHAB biomasses, axenic cyanobacterial cultures, and positive controls measured by PyroGene™ assay. Cyanobacterial
compositions of the CyanoHAB biomasses are indicated. The values for biomasses and positive controls were previously published (Skockova et al., 2023a).
Sample code Locality Dominating species
(% biovolume)
mg LPS/g
biomass d.\
PyroGene™
(xl03
EU/mgLPS)
PyroGene™
(EU/mg d.w.)
CyanoHABs (Skockova et al., 2023a)
HAB-Ap Ruda
25.7.2013
Kamenický Šutrák
28.08.2014
Staňkov
11.9.2013
Nové Mlýny, 14.8.2012HAB-Mi
Axenic cultures
Ap-1
Pl-1
Do-1
Do-2
Mi-1
Mi-2
Gram-negative bacteria (positive controls) (Skockovä et al., 2023a)
EC Escherichia cofi 0111:B4
PA Pseudomonas aeruginosa
Aphanizomenon klebahnU (92.4%) (Pipal et al., 2020) 15.9
Planktothrix agardhii (92%) (Smutná et al., 2023) 7.8
Dolichospermum curvum (73.5%) (Szmucová, 2019) 12.4
Microcystis aeruginosa (99.8%) (Javurek et al., 2015) 7.1
Aphanizomenon flos-aquae PCC 7905 25.7
Planktothrix aghardii PCC7805 7.9
Dolichospermum UHCC/HAMBI 0352 8.9
Dolichospermumflos-aquae UTEX 1444 16.5
Microcystis aeruginosa UHCC/HAMBI 130 15.7
Microcystis aeruginosa PCC 7806 12.5
0.43
0.17
0.58
37,202. 67
0.07
0.24
0.09
0.05
2.95
9.40
3689.95
387.66
6.9
1.3
7.1
265,092.9
1.7
1.9
0.8
0.8
46.4
117.2
2
P. Raptova et al.
0.1 M Tris-HCl buffer (pH 7.4) containing 25 ug/ml ribonuclease A
(RNase, Sigma-Aldrich, R4642) and incubated for 16 h at 37 °C. Next,
3.75 ml of 90% phenol in 0.1 M Tris-HCl was added, vortexed, and
incubated for 4 min at room temperature (RT). The solution was
centrifuged (18,41Og, 15 min, RT), the aqueous phase was separated and
purified for 48 h by dialysis for a second time (see above) and then
lyophilized. The purified LPS (freeze-dried powder) was weighed to
assess the content of LPS in the biomass and kept at — 20 °C. Isolated LPS
were dissolved in MilliQ water for characterization by SDS-PAGE, or in
PBS with 0.1% BSA (v/v, Sigma-Aldrich) for PyroGene™ rFC or in vitro
cell assays at a concentration of 10 mg/ml.
2.3. Characterization of LPS by TLC
Isolated LPS were separated using a thin-layer chromatography
(TLC) method adapted from a previously reported protocol (Caroff et al.,
1990). LPS samples (LPS isolated from Escherichia coli OHl:B4, Pseudomonas
aeruginosa, and cyanobacterial samples) were loaded (50 ug)
onto Alugram Sil G 5 x 10 cm TLC plates (Macherey-Nagel, Diiren,
Germany). The mobile phase consisted of a mixture of isobutyric
acid-1.5 M ammonium hydroxide (32:23, v/v). After the solvent front
reached the end of the plate, the plate was dried at 60 °C for 10 min,
covered with 10% (v/v) sulfuric acid in ethanol, and spots were visualized
by charring at 145 °C for 10 min. Finally, the developed plates
were photodocumented under white light (Alliance Q9 Advanced
system).
2.4. Characterization of LPS by SDS-PAGE
The isolated LPS were analyzed by sodium dodecyl sulfatepolyacrylamide
gel electrophoresis (SDS-PAGE) according to
(Skockova et al., 2023a). Briefly, 10 ug of LPS were mixed with MilliQ
water, 4 x Laemmli loading buffer (Bio-Rad, Hercules, CA, USA;
161-0747), and 2-mercaptoethanol (final concentration 357 mM), and
heated for 10 min at 65 °C. Stacking gels were prepared with 4% (w/v)
acrylamide and separating gels with a 10-20% (w/v) gradient (1 mm
thickness) (Serva, Heidelberg, Germany). Electrophoresis was conducted
at 130 V. LPS were stained with Pro-Q™ Emerald 300 Lipopolysaccharide
Gel Stain Kit (Thermo Fisher Scientific) according to the
manufacturers instructions. Briefly, the washed gels were fixed with
50% (v/v) methanol-5% (v/v) acetic acid for 45 min at RT, washed with
3% (v/v) acetic acid for 2 x 1 5 min, and then oxidized for 30 min with
the periodic acid-based oxidizing solution supplied with the kit. After
washing with 3% (v/v) acetic acid (3x15 min), the gels were incubated
for 90 min with 1 x Pro-Q Emerald 300 staining buffer. Finally, the gels
were washed with 3% acetic acid for 2 x 20 min and immediately
visualized under UV light and documented (Alliance Q9 Advanced,
Uvitec Cambridge, UK).
Silver staining of LPS was conducted according to previously published
protocols (Fomsgaard et al., 1990; Tsai and Frasch, 1982). First,
the washed gels were fixed with 40% (v/v) ethanol-5% (v/v) acetic acid
overnight at RT. Then, they were oxidized with 0.7% (w/v) periodic acid
in 40% (v/v) ethanol-5% (v/v) acetic acid for 5 min, and washed with
deionized water (dH20; 2 x 5 min and 1 x 1 0 min). The silver staining
solution was freshly prepared by mixing 28 ml of 0.1 M sodium hydroxide,
2.3 ml of 24% (w/w) ammonium hydroxide, 5 ml of 20% (w/v)
silver nitrate (Sigma-Aldrich) and filled up to 150 ml with dH20. The
gels were incubated in the staining solution for 10 min, and then washed
with dH20 ( 2 x 5 min and 1 x 10 min). The developing process was
carried out with 50 ug/ml citric acid in 0.05% (v/v) formalin for
approximately 2-3 min, followed by washing with dH20 to achieve
suitable levels of background staining. Finally, the gels were fixed with
5% (v/v) acetic acid for 1 min, washed, and documented under white
light (Alliance Q9 Advanced system).
To detect any protein contaminations, the gels containing LPS samples
were stained with Coomassie blue. Washed gels were fixed with
Environmental Toxicology and Pharmacology 105 (2024) 104342
40% (v/v) ethanol-20% (v/v) acetic acid for at least 1 h at RT, rinsed 5
min with dH20, and stained overnight with colloidal 0.1% (w/v) Coomassie
Brilliant Blue G250 (Serva, Heidelberg, Germany) dissolved in
17% (w/v) ammonium sulfate-34% (v/v) methanol-3% (v/v) phosphoric
acid. Then they were washed twice with dH20 for 5 min and destained
with 10% (v/v) methanol-10% (v/v) acetic acid for 3 x 1 0 min.
The gels were then washed with water until the background was sufficiently
low. Finally, the de-stained gels were photodocumented under
white light (Alliance Q9 Advanced system).
2.5. DNA extraction
The extraction of DNA from cyanobacterial and cyanobacterial water
bloom biomass was conducted based on previous studies (Sehnal et al.,
2022; Skockova et al., 2023a). Briefly, freeze-dried biomass was resuspended
in 0.15 M NaCl/0.1 M EDTA solution and homogenized using
three freeze-thaw cycles with liquid nitrogen. Samples were then
centrifuged (7,200g, 10 min) and the pellet was resuspended in 0.5 ml of
TE buffer. Then, 1 ul of RNase (10 mg/ml) was added and the samples
were incubated at 37 °C for 1 h. Afterward, 100 ul of lysozyme (50
mg/ml) was added, and the samples were incubated at 37 °C for 30 min.
Then, 10 ul of protein kinase K (50 mg/ml) and 2% (final concentration)
of sodium dodecyl sulfate (SDS) were added, and samples were incubated
at 55 °C for 1 h. Selective precipitation was performed by adding
150 ul of 5 M NaCl to the tubes followed by 0.1 volumes (of total volume)
of 10% cetyltrimethylammonium bromide stock solution, mixed
by inversion, and incubated at 65 °C for 10 min. For purification, 1 vol of
chloroform was added, and tubes were incubated on ice for 30 min.
Then, samples were centrifuged (10 min, 7,200g, 4 °C), and the supernatant
was transferred to fresh tubes, mixed with 0.6 volumes of isopropanol,
and incubated overnight at 4 °C. Following this, samples were
centrifuged (30 min, 16,000g, 4 °C) and the pellet was washed twice
with 1 ml of 70% ethanol. Finally, samples were centrifuged (30 min, 16,
OOOg, 4 °C), the pellet air-dried, and then resuspended in 30 ul TE buffer.
The concentration and quality of the extracted DNA were checked via
NanoDrop (Thermo Fisher Scientific) and agarose gel electrophoresis.
The isolated DNA underwent further processing through qPCR and 16 S
Amplicon sequencing.
2.6. qPCR detection of cyanobacteria, heterotrophic bacteria, and gramnegative
heterotrophic bacteria
DNA quantification was performed using qPCR and specific FAMBHQl-labeled
Taqman probes and primers that targeted 16 S rRNA
genes as reported previously (Skockova et al., 2023a). The primers and
probes were designed to detect the gene sequences specific for three
groups of microorganisms: (1) cyanobacteria, (2) total heterotrophic
bacteria, and (3) a subset of G- heterotrophic bacteria. qPCR was conducted
on a LightCycler® 96 Real-time PCR System under the same
conditions as reported in previous studies (Laboha et al., 2023;
Lang-Yona et al., 2014). Calibration curves were derived using standard
DNA extracted from E. coli CCM 3954 (for total and gram-negative
bacteria), and Microcystis aeruginosa PCC 7806 (for cyanobacteria).
The results were expressed as a percentage share of each group's DNA
sequences (i.e., cyanobacteria, heterotrophic bacteria, G- bacteria) in
the total quantified DNA sequences in the sample, which was the sum of
cyanobacteria and total heterotrophic bacteria (equal to 100%). The
share of heterotrophic bacteria, other than G- bacteria, was determined
by subtracting the share of G- bacteria DNA sequences from the total
heterotrophic bacteria DNA sequences.
2.7. 16 S Amplicon sequencing
To target the hypervariable region of the bacterial 16 S rRNA gene,
the extracted DNA was used as a template in PCR as reported previously
(Sehnal et al., 2022). The Illumina 16 S Metagenomic Sequencing
3
P. Koptová et al.
Library Preparation protocol was followed to prepare the metagenomic
library, with small adaptations as described in (Sehnal et al., 2022).
Sequencing was carried out using the MiSeq instrument and the MiSeq
reagent kit V2, following the manufacturer's instructions (Illumina, San
Diego, CA, USA). Initially, paired reads obtained from the 16 S rRNA
sequencing were processed using a Python 3-based in-house pipeline.
This involved trimming the low-quality 3' ends of the reads, discarding
read pairs that contained unspecified base N, and eliminating pairs that
consisted of very short reads. Further details on the preparation of the
library, sequencing, and data processing are presented in (Sehnal et al.,
2022).
2.8. Endotoxin activity by PyroGene™rFC assay
The endotoxin activity of isolated LPS was determined using the
PyroGene™ Recombinant Factor C Endpoint Fluorescent Assay (Lonza,
Basel, Switzerland). 100 ul of LPS samples were mixed in 96-well
microplate wells with 100 ul of reaction solution (rFC enzyme solution,
rFC assay buffer, and fluorogenic substrate in the ratio of 1:4:5) and
incubated for 1 h at 37 °C. Fluorescence was measured using a microplate
reader (Infinite M200, Tecan, Mánnendorf, Switzerland; excitation
390 nm, emission 440 nm), and endotoxin activity was calculated according
to the standard curve. Data were expressed as endotoxin activity
in endotoxin units (EU) per mg of dry weight (d. w.) biomass and per mg
of the respective LPS (Table 1).
2.9. Cell culture
Human immortalized keratinocyte cell line HaCaT (CLS, Eppelheim,
Germany) was cultured in Dulbecco's Modified Eagle Medium (DMEM;
Gibco - Thermo Fisher Scientific) supplemented with 10% heatinactivated
low endotoxin fetal bovine serum (LE FBS; v/v, PAA, GE
Healthcare, Chicago, IL, USA), 2 mM L-glutamine (Gibco, New York, NY,
USA) and 100 U/ml of penicillin and 100 ug/ml of streptomycin (Pen/
Strep; Gibco) in stationary T-flasks (TPP, Trasadingen, Switzerland) at
37 °C, in a 5% CC>2/95% air atmosphere. HaCaT cells were used in
passages ranging from the 30t h
to the 80t h
.
2.10. Cell exposure studies
For all experiments, cells were seeded in 24-well or 96-well tissue
culture plates (TPP) at the density of 4.0 x 104
cell/cm2
in 0.5 ml or 0.2
ml cultivation media, respectively, and cultured for 72 h to form a
confluent monolayer. After confluent layer formation, the culture medium
was changed, and the cells were exposed to LPS (50 ug/ml or 20
ug/ml) for 24 h.
2.11. Determination of cell viability
Cell viability assessment was based on the determination of the total
protein concentration in whole cell lysates, as described previously
(Moosova et al., 2019b). Protein concentrations were determined using
BCA™ protein assay (Pierce, Waltham, MA, USA); bovine serum albumin
(Sigma-Aldrich) was used as a standard for quantification.
The potential cytotoxic effect of cyanobacterial LPS was determined
by measurement of lactate dehydrogenase (LDH) activity in cell culture
media using the Cytotoxicity Detection KitPLUS (Roche, Pleasanton, CA,
USA), as described previously (Bino et al., 2016). Briefly, 80 ul of
samples were mixed with the reaction mixture in a ratio of 1:1 and
incubated RT. The absorbance was measured at 492 nm using a
SPECTRA Sunrise microplate reader (Tecan). Untreated HaCaT cells
lysed by supplier-provided lysis buffer were used as a positive control.
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)
labeling was applied to evaluate metabolically active cells
reflecting the number of viable cells as described previously (Moosova
et al., 2019a; Šafaříková et al., 2018). Briefly, the culture medium was
Environmental Toxicology and Pharmacology 105 (2024) 104342
changed to 200 ul of MTT solution (0.125 mg/ml, diluted in culture
medium) and the cells were incubated with MTT for 15 min at 37 °C
Formed formazan crystals were dissolved by 50 ul 0.1 M HCl-10% (v/v)
Triton X-100 and the absorbance was measured at 570 nm using a
SPECTRA Sunrise microplate reader.
2.12. Measurement of cytokines and chemokines concentration by ELISA
The concentration of IL-6, IL-8, CCL2, and CCL20 in the cultivation
medium was determined by commercially available ELISA immunoassays
(Invitrogen, Waltham, MA, USA - human IL-6 Uncoated ELISA kit
REF 88-7066-88, human IL-8 Uncoated ELISA kit REF 88-8086-88,
human CCL2 Uncoated ELISA kit REF 88-7399-88; R&D Systems,
Minneapolis, MN, USA - human CCL20/MIP-3a DuoSet DY360). The
assays were performed as described previously (Vasicek et al., 2020).
Results were expressed as pg/ml. Minimum detectable concentrations
for cytokines and chemokines were the following: IL-8 = 2 pg/ml, IL-6
= 2 pg/ml, and CCL2 = 7 pg/ml according to manufacturer information.
The minimal detectable concentration of CCL20 kit was not provided by
the manufacturer.
2.13. Western blot analysis of K6 and Kl 7
HaCaT cells were lysed using 200 ul of lysis buffer (1% SDS); 100 mM
Tris, pH 7.4; 10% glycerol; 1 mM phenylmethylsulfonyl fluoride). Protein
concentrations were determined using BCA™ protein assay
(Pierce). The protein expression was detected as described previously
(Pekarova et al., 2015). Briefly, equal protein amounts from each cell
lysate were suplemented with Laemmli buffer (200 mM Tris, pH 6.8; 3%
SDS, 30% glycerol, 0.03% bromphenol blue, 3% p-mercaptoethanol, and
200 mM dithiothreitol) and subjected to SDS-PAGE on a 10% polyacrylamide
gel. Proteins were transferred to polyvinylidene difluoride
membrane (Immobilon-P, Millipore, Burlington, MA, USA) and then
incubated with antibodies against K6 (1:200; Invitrogen; 14-9788-82)
or K17 (1:1000; Invitrogen; MA5-15534), overnight, 4 ° C Membranes
were further incubated with HRP-linked anti-mouse (1:2000; Cell
signaling technology, Danvers, MA, USA, #7076) or anti-rabbit (1:
2000; Cell signaling technology, #7074) IgG antibodies for 1 h. The
blots were visualized using SuperSignal West PicoChemiluminescent
Substrate (Pierce) and exposed to CP-B X-ray films (Agfa, Mortsel,
Belgium). The relative levels of proteins were quantified by scanning
densitometry using the ImageJ™ program (Schneider et al., 2012), with
the individual band density value expressed in arbitrary units. Equal
protein loading was verified by vinculin immunoblotting (1:1000; Cell
signaling technology, #13901). The data were expressed as a ratio of the
optical density of the target protein and the respective vinculin band
optical density.
2.14. Wound healing assay
To determine changes in the proliferation and migration of HaCaT
cells after LPS exposure, the wound healing assay was performed. A
scratched wound line was created in the centre of the confluent layer
using a 200 ul micropipette tip. The scratched cells were washed with
tempered PBS. Cells were exposed to LPS (50 ug/ml) for 48 h.
Images of scratched areas were taken using a light microscope (Zeiss,
Jena, Germany; 100 x magnification) at time points of 0 h and 48 h. The
scratched areas were measured using the ImageJ™ program. The Sobel
method was used to enhance edges (Process-Find Edges). The image was
sharpened (Process-Sharpen) and an automatically determined
threshold was applied (Image-Adjust-Threshold-B&W-Apply). After
applying the Sobel method again (Process-Find Edges) the image was
inverted without changing the pixel values (Image-Lookup Tables-Invert
LUT). The area of the wound was calculated (Analyse-Analyse particles).
The resolution of the taken images was 2,048 x 2,048 so the size for
calculation was set to 105
-107
-pixel units. The percentage of wound area
4
P. Raptovä et al. Environmental Toxicology and Pharmacology 105 (2024) 104342
in the image was determined at every time point. The change was
expressed as a change in the area ("A") after 48 h using the formula M(t)
= [[A(0)-A(f)]/A(0)]* 100 (Treloar and Simpson, 2013).
2.15. Statistical analysis
Data are reported as means ± SEM; the number of independent
replicates is indicated in each figure. Data were analyzed using one-way
ANOVA followed by Dunnett's test, unpaired t-test, or one sample t-test,
respectively, following the nature of the experiment (GraphPad Prism
software, version 8.4.2, Science Plus Group bv, Groningen, Netherlands,
2012). Statistical analysis used for the particular data is indicated in
each figure. Ap-value < 0.05 was considered significant (*).
3. R e s u l t s
3.1. Endotoxin activity of the LPS
The endotoxin activity of the LPS isolated from CyanoHAB biomasses
and positive controls was previously characterized (Skockova et al.,
2023a). Among tested samples, the LPS from Microcystis-dominated
CyanoHAB exhibited the highest activity, being ten times more active
than E. coli LPS and a hundred times more active than Pseudomonas LPS
(Table 1). The endotoxin activity was also determined for the LPS isolated
from axenic cultures. As shown in Table 1, LPS from Aphanizomenon,
Planktothrix, and Dolichospermum axenic cultures showed very
low endotoxin activity. Microcystis LPS displayed endotoxin activity one
or two orders of magnitude higher than the aforementioned strains.
However, it was still two to four orders of magnitude lower than the
positive controls and the Microcystis-dominated CyanoHAB LPS. Interestingly,
Aphanizomenon axenic culture had the highest amount of LPS in
the biomass, accounting for 2.5% (Table 1).
3.2. Thin layer chromatography and SDS-PAGE of cyanobacterial LPS
Isolated LPS from G- heterotrophic bacteria, CyanoHABs, and axenic
cyanobacterial cultures exhibited distinct migration patterns in TLC.
The TLC analysis revealed sample-specific migration patterns for each
LPS type (Fig. 1). Bacterial LPS from£. coli and P. aeruginosa displayed a
rapidly migrating spot closest to the solvent front, with LPS from
P. aeruginosa exhibiting a more intense spot compared to the faint spot
observed for E. coli LPS. Additionally, a smearing spot was observed in
the middle section of the TLC plate for both bacterial LPS. In contrast,
only cyanobacterial LPS from HAB-Mi and Pl-1 samples displayed
similar rapidly migrating spots closer to the solvent front. The TLC
pattern for other cyanobacterial LPS samples exhibited varying patterns,
with one or multiple spots located in the lower or middle part of the TLC
plate. Notably, LPS from HAB-Do and Mi-1 showed no or very faint spots
in the TLC analysis.
Silver staining of LPS resolved by SDS-PAGE exhibited a characteristic
laddering pattern for E. coli LPS. The lower part of the gel displayed
bands corresponding to the lipid A, core oligosaccharide, and LPS with
short O-antigen, indicating rough and semi-rough LPS (Fig. 2A). The
middle and upper parts of the gel exhibited larger molecular weight
bands representing smooth LPS with intermediate, long and very long Oantigens
(Sarkar et al., 2014; Werts et al., 2001). P. aeruginosa LPS
appeared as a single band in the silver staining, likely corresponding to
lipid A. However, Pro-Q staining revealed laddering bands corresponding
to rough, semi-rough, and smooth LPS types (Gupta et al.,
1997; Hao et al., 2015; Pourcel et al., 2020; Zhang et al., 2018). Silver
staining of LPS from Microcystis-dominated samples displayed bands
corresponding to smooth LPS with molecular weights ranging from intermediate
up to very long O-chain antigen. Rough LPS-type bands were
only observed in LPS isolated from Do-2 {Dolichospermum UTEX1444).
The bands detected by silver staining were among the most intense ones
visualized by Pro-Q Emerald 300, but also additional bands were
observed. Pro-Q staining of Microcystis LPS samples showed a consistent
laddering pattern across the region of smooth LPS. The Do-2 sample
exhibited a laddering pattern from the lowest molecular weights up to
the long O-antigen region. However, no major bands were detected in
the LPS isolated from Do-1, the other Dolichospermum culture (UHCC/HAMBI
0352), while the LPS migration pattern of HAB-Do resembled
HAB-Mi. Interestingly, LPS from CyanoHAB dominated by Aphanizomenon
displayed a clear laddering pattern with highly intense bands in
the intermediate region (Fig. 2B), which were not observed in the silver
staining (Fig. 2A). The most intense band in HAB-Ap LPS corresponded
with a major band in the intermediate region of LPS from Ap-1 culture
(Fig. 2B). As we reported previously (Skockova et al., 2023a), LPS from
Planktothrix water bloom (HAB-Pl) showed only faint bands in the very
long O-antigen region, while no bands were detected in the Pl-1 sample
from axenic culture. Coomassie blue staining confirmed the absence of
protein contaminations in samples (Fig. SI).
' i f
....
- Front
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Fig. 1. Thin-layer chromatography (TLC) of LPS samples isolated from CyanoHABs (A) and axenic cyanobacterial cultures (B). EC - Escherichia coli OHl:B4, PA Pseudomonas
aeruginosa, HAB-Ap - Aphanizomenon bloom, HAB-Pl - Planktothrix bloom, HAB-Do - Dolichospermum bloom, HAB-Mi - Microcystis bloom, Ap-1 Aphanizomenon
PCC7905, Pl-1 - Planktothirx PCC7805, Do-1 Dolichospermum UHCC 0352, Do-2 - Dolichospermum UTEX 1444, Mi-1 - Microcystis UHCC 130 and Mi-2 Microcystis
PCC 7806.
5
P. Raptovä. et al Environmental Toxicology and Pharmacology 105 (2024) 104342
O CA
O Q_
E - 1
B
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Core O S
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+ Lipid A
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Fig. 2. SDS-PAGE of LPS followed by silver staining (A) or Pro-Q™ Emerald 300 staining (B). LPSs were isolatedfromCyanoHABs or axenic cyanobacterial cultures.
SR—semi-rough LPS. Core OS—core oligosaccharide. Descriptions of LPS structures and types correspond to the published SDS-PAGE separation patterns of LPS
isolated horn Escherichia coli and Pseudomonas aeruginosa (Gupta et al., 1997; Hao et al., 2015; Pourcel et al., 2020; Sarkar et al., 2014; Werts et al., 2001; Zhang et al.,
2018). Pro-Q staining images of CyanoHAB samples, E. coli and P. aeruginosa were adapted from our previous article (Skockova et al., 2023a). EC - Escherichia coli
0111:B4, PA - Pseudomonas aeruginosa, HAB-Ap - Aphanizomenon bloom, HAB-P1 - Planktothrix bloom, HAB-Do - Dolichospermum bloom, HAB-Mi - Microcystis
bloom, Ap-1 - Aphanizomenon PCC7905, Pl-1 - Planktothirx PCC7805, Do-1 Dolichospermum UHCC 0352, Do-2 - Dolichospermum UTEX 1444, Mi-1 - Microcystis UHCC
130 and Mi-2 - Microcystis PCC 7806.
3.3. Non-toxic concentrations of several cyanobacterial LPS induce proinflammatory
cytokines production
The potential cytotoxicity of studied LPS was determined by MTT,
LDH, and BCA assays. 24-hour exposure to LPS (20 ug/ml or 50 ug/ml)
did not lead to changes in the whole protein content (Fig. S2A, D) and
mitochondrial activity (Fig. S2B, E). Also, the LDH activity in treated
samples was comparable to the untreated cells (Fig. S2C, F). Thus, the
used concentrations of LPS were considered non-toxic.
HAB-Do and HAB-Mi were the most potent LPS in triggering the
release of pro-inflammatory cytokines. Both experimental concentrations
of these two samples led to a significant increase in IL-6 and IL-8
(Fig. 3A, C). The higher concentration resulted also in elevated production
of chemokines CCL2 and CCL20 (Fig. 3E, G). In contrast, HABAp
and HAB-Pl did not induce any significant increase in the concentration
of studied cytokines in the media. On the other hand, Ap-1 was
the only axenic LPS able to induce the production of CCL20 (Fig. 3H).
The higher Ap-1 concentration also caused an increase in IL-6 level
(Fig. 3B). The increase of IL-6 caused by the lower concentration of Do-1
was statistically significant, but very slight and probably not physiologically
relevant, and it was not confirmed by the effect of higher LPS
concentration (Fig. 3B).
3.4. Most of the cyanobacterial LPS induce wound healing
A wound-healing assay was used to study changes in cell proliferation
and migration. Interestingly, treatment with all LPS isolated from
water blooms resulted in faster wound closure (Fig. AA). Similarly, LPS
from axenic cultures Ap-1, Pl-1, Do-1, Do-2 had the same effect (Fig. 4C).
However, Mi-1 and Mi-2 had no significant effect on wound healing
(Fig. 4C). These changes were not accompanied by changes in K6 and
K17 expression studied by western blot (Fig. S3).
3.5. Biomass DNA composition
qPCR analysis was conducted using specific TaqMan probes to
determine the relative abundance of total heterotrophic bacteria, Gbacteria,
and cyanobacteria in the water bloom biomasses utilized for
LPS isolation. The results of the analysis for water bloom biomasses were
previously published (Skockova et al., 2023a) (Fig. 5A). The findings
revealed that in HAB-Do biomass, the G- bacteria accounted for 50% of
all quantified DNA sequences, while cyanobacteria constituted only
26%. On the other hand, cyanobacteria represented 99% of the DNA
sequences in HAB-Pl DNA.
Biomasses of P. aghardii PCC7805 (Pl-1), Dolichospermum UHCC/
P. Raptovä et al. Environmental Toxicology and Pharmacology 105 (2024) 104342
= • 1 0 0 -
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7
P. Raptová et ah Environmental Toxicology and Pharmacology 105 (2024) 104342
Fig. 3. Production of pro-inflammatory cytokines and chemokines by HaCaT cells after exposure to LPS isolated from water blooms (A, C, E, F) and axenic cyanobacterial
cultures (B, D, F, H). Levels of IL-6 (A, B), IL-8 (C, D), CCL2 (E, F) and CCL20 (G, H) in the medium after 24-h exposure to studied LPS (20 |ig/ml and
50 |ig/ml) were measured using ELISA. E. coli LPS was used as a positive control. Data from five independent experiments are expressed as a mean ± SEM. Data were
statistically analyzed using ANOVA followed by Dunnett's multiple comparison test (** p < 0.01 and * p < 0.05). EC - Escherichia coli O H l : B 4 , PA - Pseudomonas
aeruginosa, HAB-Ap - Aphanizomenon bloom, HAB-Pl - Planktothrix bloom, HAB-Do - Dolichospermum bloom, HAB-Mi - Microcystis bloom, Ap-1 - Aphanizomenon
PCC7905, Pl-1 - Planktothirx PCC7805, Do-1 Dolichospermum UHCC 0352, Do-2 - Dolichospermum UTEX 1444, Mi-1 - Microcystis UHCC 130 and Mi-2 - Microcystis
PCC 7806.
EC HAB-Ap HAB-Pl HAB-Do HAB-Mi
SB S S S
control EC HAB-Ap HAB-Pl HAB-Do HAB-Mi
control EC
Fig. 4. Changes in proliferation and migration of HaCaT cells after LPS exposure were measured using a wound-healing assay. Wound healing was observed after 48h
exposure to tested LPS (50 |ig/ml) isolated from water blooms (A) or axenic cyanobacterial cultures (C). Data from seven independent experiments were integrated
and expressed as a percentage of wound closure after 48 h and as the mean ± SEM. Representative pictures of wound healing after exposure to CyanoHAB LPS (B)
and LPS from axenic cyanobacterial cultures (D) are shown. Data were statistically analyzed using an unpaired t-test (** p < 0.01 and * p < 0.05). EC - Escherichia coli
0111:B4, PA - Pseudomonas aeruginosa, HAB-Ap - Aphanizomenon bloom, HAB-Pl - Planktothrix bloom, HAB-Do - Dolichospermum bloom, HAB-Mi - Microcystis
bloom, Ap-1 - Aphanizomenon PCC7905, Pl-1 - Planktothirx PCC7805, Do-1 Dolichospermum UHCC 0352, Do-2 - Dolichospermum UTEX 1444, Mi-1 - Microcystis UHCC
130 and Mi-2 - Microcystis PCC 7806.
100
80
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20
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H A B - D o HAB-Mi o-L
Ü. I?
I • cyanobacteria
• G - bacteria
• Other bacteria
Ap-1 Pl-1 Do-1 Do-2 Mi-1 Mi-2
Fig. 5. Quantification of DNA sequences in water bloom and axenic cyanobacterial culture biomasses using qPCR. TaqMan Probes specifically targeting total
heterotrophic bacteria, Gram-negative bacteria (G-) and cyanobacteria were employed to quantify their respective DNA shares. The results are presented as percentage
of cyanobacteria, G- and the remaining bacterial population (i.e., total heterotrophic bacteria excluding G-) among the total quantified DNA. EC - Escherichia
coli O l l i : B 4 , PA - Pseudomonas aeruginosa, HAB-Ap - Aphanizomenon bloom, HAB-Pl - Planktothrix bloom, HAB-Do - Dolichospermum bloom, HAB-Mi - Microcystis
bloom, Ap-1 - Aphanizomenon PCC7905, Pl-1 - Planktothirx PCC7805, Do-1 Dolichospermum UHCC 0352, Do-2 - Dolichospermum UTEX 1444, Mi-1 - Microcystis UHCC
130 and Mi-2 - Microcystis PCC 7806.
HAMBI 0352 (Do-1), D.flos-aquae UTEX 1444 (Do-2), andM. aeruginosa
PCC 7806 (Mi-2) exhibited 100% of cyanobacterial DNA composition
(Fig. 5B). The only biomass in which G- bacterial DNA was detected was
M. aeruginosa UHCC/HAMBI 130 (Mi-1), but it constituted only 0.08%
of the DNA composition. Additionally, DNA sequences belonging to
other bacteria were found in the biomasses Mi-1, accounting for 0.02%,
and in A.flos-aquaePCC 7905 (Ap-1) biomass, constituting 0.08% of the
DNA composition.
3.6. Taxonomie diversity of the biomasses
The composition of water blooms determined using NGS analysis
exhibited considerable variation, with cyanobacteria ranging from
94.84% in HAB-Pl to 13.60% in HAB-Do, reflecting their relative
8
P. Raptová et al.
abundance (Fig. 6A). Among the Gram-negative (G-) bacterial phyla,
Proteobacteria was the most prevalent in the water bloom biomasses,
ranging from 1.80% in the PianíctotfirĎc-dominated bloom to 56.99% in
Doiic/iospermum-dominated bloom. Another prominent G- phylum was
Bacteroidetes, primarily found in HAB-Do (14.74%) and HAB-Mi
(10.02%). In the other two HABs, Bacteroidetes constituted less than
0.5% (Tab. SI). Verrucomicrobia (HAB-Do 9.47%, HAB-Pl 1.38%) and
Chloroflexi (HAB-Do 3.57%, HAB-Pl 1.11%) also contributed significantly
to the G- bacterial population. Gemmatimonadetes was detected in
HAB-Mi (1.85%). The biomasses also exhibited the presence of
G+ bacterial phyla, namely Actinobacteria, Deinococcus-Thermus, and
Firmicutes, but in relatively small proportions, up to 0.34% (Tab. SI).
In the laboratory cultures, Proteobacteria also emerged as the dominant
G- bacterial phylum; however, it was present in very low quantities,
ranging from 0.12% in Do-2 to 3.59% in Mi-1 (Fig. 6B). Do-1 and Mi-2
exclusively contained Proteobacteria among the G- bacteria (Fig. 6B, Tab.
SI). The remaining biomasses showed minimal amounts (0.004 -
0.024%; Tab.Sl) of the G- phylum Bacteroidetes. Traces of the
G+ bacterial phylum Firmicutes (up to 0.031%) were detected in Ap-1,
Do-1, and both M. aeruginosa cultures. G+ Actinobacteria was only
found in the Mi-1 biomass (0.004%).
Differences in taxonomie representation are also evident when
comparing the ten most abundant G- bacterial genera in water bloom
biomasses. Several bacterial genera were present in all samples,
including Rhodobacter, Roseomonas, Halomonas, Vibrio, and unspecified
genera from Nitrosomonadaceae and Comamonadaceae (labeled as 1)
(Fig. 7). However, their abundance varied among the samples. For
example, the counts of Rhodobacter were twelve times higher in HAB-Mi
than in HAB-Ap and 67 times and 162 times higher than in HAB-Do and
HAB-Pl, respectively. Halomonas counts ranged from 15 to 75 counts in
HAB-Ap, HAB-Pl, and HAB-Mi, but reached 4,398 in HAB-Do. A similar
pattern was observed for Vibrio, with counts ranging from 8 to 36 in
HAB-Ap, HAB-Pl, and HAB-Mi, but reaching 2,579 counts in HAB-Do.
Roseomonas was relatively rare in HAB-Ap and HAB-Pl (22 and 47,
respectively), but abundant in HAB-Mi (1,211) and especially in HAB-Do
(5,900). The counts of Nitrosomonadaceae were similar in HAB-Ap and
HAB-Mi (1,464 and 1,549, resp.), while in the other biomasses, the
abundance of Nitrosomonadaceae was lower (671 counts in HAB-Do and
14 in HAB-Pl). The counts of bacteria Comamonadaceae 1 were relatively
consistent, ranging from 158 to 943 counts.
On the other hand, 11 bacterial genera were unique to only one of the
studied water blooms. Two genera were unique to HAB-Ap {Emticicia,
Rubrivivax), four to HAB-Pl (Caldilineaceae, Thiotrix, Rhodopirelkda, and
Chtoniobacterales 1), three to HAB-Do {Anaerolineaceae, Lautropia, and
Variovorax), and two to HAB-Mi {Gemmatimonas and Azohydromonas).
However, their abundance varied among the biomasses. The combined
count of these unique bacteria reached 110 counts in HAB-Ap, 868 in
HAB-Pl, 981 in HAB-Mi, and even 9,892 counts in HAB-Do.
Environmental Toxicology and Pharmacology 105 (2024) 104342
4. D i s c u s s i o n
CyanoHABs are occurring worldwide producing various toxins and
bioactive compounds including LPS. Our previous results, as well as
results of other studies, have shown that LPS from CyanoHABs and also
from cyanobacterial cultures can have pro-inflammatory effects in intestinal,
airway epithelial, and immune cells (Blahova et al., 2013;
Laboha et al., 2023; Moosova et al., 2019b; Skockova et al., 2023a,b;
Swartzendruber et al., 2022). Our study focused on human keratinocytes,
which represent other potential target cells exposed to cyanobacterial
LPS during recreational activities.
The LPS used in this study were obtained from environmental water
bloom samples dominated by the most common bloom-forming cyanobacteria,
namely M. aeruginosa, D. curvum, P. agardhii, and A. klebahnii,
along with their respective axenic cultures. These cyanobacterial bloomderived
LPSs, also known as cyanoHAB LPSs, have previously been
investigated by our research group on intestinal and immune cells in
vitro. We have shown that HAB-Do and HAB-Mi induce proinflammatory
effects in human enterocyte-like cells and murine RAW
264.7 macrophages in vitro (Skockova et al., 2023a). In this study, we
show that these LPS induce the production of pro-inflammatory cytokines
such as IL-6, IL-8, CCL2, and CCL20 in keratinocytes as well.
Although, the effects of HAB-Do and HAB-Mi were observed in various
cell types, HAB-Ap and HAB-Pl LPS had minimal effects. The differences
in the observed effects may be attributed to the composition of the
biomasses. qPCR and NGS data indicated that the active HAB-Do and
HAB-Mi LPS were isolated from biomasses containing a high proportion
of G- bacteria, primarily from the Proteobacteria phylum. This phylum
includes genera known to possess bioactive LPS, such as Escherichia,
Salmonella, and Vibrio. An increased abundance of Proteobacteria in the
intestinal microbiome has been associated with inflammatory bowel
diseases (Morgan et al., 2012).
Although our CyanoHAB samples did not contain representatives of
the Escherichia or Salmonella genera, they did contain varying amounts
of bacteria from the Vibrio genus. Vibrio species are predominantly
aquatic and include numerous pathogens affecting humans and fish.
Vibrio cholerae, the well-known causative agent of cholera, primarily
relies on its cholera toxin as a virulence factor; however, LPS also plays a
role. The structure of the LPS serves as a classifier for different
V. cholerae strains, with the O l serotype being responsible for the majority
of cholera cases (Baker-Austin et al., 2018). Another serogroup
associated with cholera outbreaks is 0139. However, strains other than
Ol and 0139 have been shown to cause gastrointestinal and extraintestinal
infections (Deshayes et al., 2015). Additionally, the O-antigen
of Vibrio species appears to influence intestinal colonization (BakerAustin
et al., 2018). V. vulnificus, another human pathogen within this
genus, relies on the bacterial capsule containing LPS, which exhibits
pro-inflammatory activity, for successful infection (Jones and Oliver,
1.11%1.80%1.&
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• Planctomycetes (G-)
• Proteobacteria (G-)
• Gemmatimonadetes (G-)
• Chloroflexi (G-)
• Bacteroidetes (G-)
• Cya nobacteria
HAB-Ap HAB-Pl HAB-Do HAB-Mi Ap-1 PI-1 Do-1 Do-2 Mi-1 Mi-2
Fig. 6. Taxonomie abundance at the phylum level in the water bloom and axenic cyanobacterial culture biomasses. Phyla with abundance below 0.01% are grouped
as rare in the graph, and only phyla with abundance > 1% are indicated. Detailed information regarding taxonomie abundance can be found in the Supplementary
Material (Table SI). HAB-Ap - Aphanizomenon bloom, HAB-Pl - Planktothrix bloom, HAB-Do - Dolichospermum bloom, HAB-Mi - Microcystis bloom, Ap-1 - Aphamzomenon
PCC7905, Pl-1 - Planktothirx PCC7805, Do-1 Dolichospermum UHCC 0352, Do-2 - Dolichospermum UTEX 1444, Mi-1 - Microcystis UHCC 130 and Mi-2 Microcystis
PCC 7806.
9
P. Raptovä et at Environmental Toxicology and Pharmacology 105 (2024) 104342
Cytophagaceae 1
Emticicia
Flavobacterium
Gemmatimonas
Anaerolineaceae 1
Caldilineaceae 1
Rhodopirellula
Meganema
Methylorosula
Phenylobacterium
Rhizobiales 1
Rhizobiales 2
Rhizobiales 3
Rhodobacter
Roseomonas
Sphingopyxis
Alcaligenaceae 1
Azohydromonas
Comamonadaceae 1
Comamonadaceae 2
Lautropia
Nitrosomonadaceae 1
Oxalobacteraceae 1
Rubrivivax
Variovorax
Acidibacter
Halomonas
Silanimonas
Thiothrix
Vibrio
Proteobacteria 1
Chthoniobacterales 1
Chthoniobacterales 2
1903
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HAB-Ap HAB-PI HAB-Do HAB-Mi
Fig. 7. Representation of the ten most abundant bacterial taxa in each CyanoHAB. The graph illustrates NGS data for each water bloom biomass, displaying the ten
most abundant Gram-negative (G-) bacterial taxa (cyanobacteria were excluded from the dataset). Each bacterium is labeled by its genus, determined at the Taxonomy
5 level of NGS analysis. If the genus was unknown, the nearest established level of taxonomy was utilized. The results were categorized by phylum, with
genera belonging to Bacteroidetes in yellow, Gemmatimonadetes in orange, Chloroflexi in blue, Planctomycetes in pink, Proteobacteria in green (ranging from lighter to
darker shades representing the classes Alpha-, Beta-, Gammaproteobacteria, and TA18), and Vermcomicrobia in grey. The numbers indicate the respective counts of
each taxon. Detailed data can be found in Tab. S2. HAB-Ap - Aphanizomenon bloom, HAB-PI - Planktothrix bloom, HAB-Do - Dolichospermiim bloom, HAB-Mi Microcystis
bloom.
2009). Other Vibrio species pathogenic to humans include V. parahaemolyticus,
V. alginolyticus, and V.fluvialis,while others like V. harveyi
and V. angwllarum affect fish and other animals. As HAB-Mi has two to
three orders of magnitude higher abundance of Vibrio genera in comparison
with the other bloom samples (2,579 vs. 8, 9, and 36 counts), it
could be an important contribution to the enormous endotoxin activity
of this LPS. Another genus present in all CyanoHAB samples was
Roseomonas, containing two species (R. mucosa and R. gilardu). The
first-mentioned is a part of the human skin microbiome and was
described as an occasional human pathogen in immunocompromised
patients (Romano-Bertrand et al., 2016).
In addition to the aforementioned genera, Comamonadaceae, Rhodobacter,
and several other taxa were identified in the CyanoHAB samples.
Comamonadaceae, which were found in all samples in relatively
10
P. Raptova et al.
similar amounts, comprise the genera Comamonas, Acidovorax, and
Delftia. Some species within these genera have been associated with
opportunistic human infections, particularly in individuals with underlying
conditions (Bilgin et al., 2015; Ryan et al., 2022). Rhodobacter, a
non-pathogenic genus, includes the well-known representative
R. sphaeroides, whose LPS antagonizes Toll-like receptor 4. Notably, its
lipid A has shown a protective effect against the toxic effects of E. coli
D31m4 LPS in mice (Qureshi et al., 1991) and also in human keratinocytes
(Shi et al., 2014). Furthermore, certain bacterial families and
genera were present only in the active LPS of HAB-Do and HAB-Mi.
These include Cytophagaceae, Alcaligenaceae (which contains the pathogenic
BordeteUa genus), a genus of Rhizobkdes, Azohydromonas (found
only in HAB-Mi), and Anaerolineaceae, Variovorax, and Lautropia (found
only in HAB-Do). The last-mentioned genus contains species L. mirabilis,
an opportunistic pathogen in humans with underlying conditions (Ben
Dekhil et al., 1997; Calheiros Cruz et al., 2022). While our results do not
confirm the presence of these pathogenic species in the biomasses,
similarities in LPS structure within genera can be inferred. Overall, our
findings suggest that the biological activity of LPS isolated from water
bloom environmental mixtures in human in vitro cells is influenced by
the presence and abundance of heterotrophic G- bacteria.
This conclusion is supported also by the results obtained from assays
with axenic cultures of the respective dominant cyanobacterial species.
A. flos-aquae-derived LPS (Ap-1) was the only axenic LPS inducing
CCL20 production by HaCaT cells, accompanied also by IL-6 production
at the higher LPS concentration. A slight increase in the production of IL-
6 was observed in response to the lower concentration of LPS from Do-1.
However, this effect was not confirmed at the higher concentration and
the overall changes were relatively minor, leading to the conclusion that
they cannot be considered (patho)physiologically relevant. On the other
hand, our previously published study showed the pro-inflammatory
activity of LPS from D. fhs-aquae (strain CCALA007) culture in enterocytes
and macrophages in vitro. Although the CCALA007 culture originates
from UTEX1444 (Do-2), CCALA007 strain was not fully axenic
and its biomass contained 1.7% of G- bacterial DNA share according to
qPCR quantification (Skockova et al., 2023a). Probably, G- bacterial LPS
contributed to the pro-inflammatory effects induced in vitro by LPS from
Dolichospermum CCALA007 in the previous study in contrast to axenic
Dolichospermum UHCC/HAMBI 0352 and UTEX 1444 in this study.
Nevertheless, the differences in the observed effects could also depend
on the target cell type and the number of specific receptors expressed on
the cell surface.
Differences in the LPS structure among individual samples were
observed through TLC, SDS-PAGE, and subsequent silver and Pro-Q
staining. Distinctive bands were corresponding to rough (i.e. truncated
LPS without O-antigen) LPS in the lower part of the polyacrylamide gel
were only observed in E. coli, P. aeruginosa, and the axenic D. flos-aquae
UTEX 1444 strain. In contrast, LPS horn Dolichospermum UHCC/HAMBI
0352 did not exhibit reactivity with silver staining, and only very faint
bands were visible after Pro-Q staining in the region associated with
long and very long O-antigen, indicating smooth LPS (i.e. wild type LPS
with O-antigen which is highly variable in length and composition).
Furthermore, the migration pattern of the two Dolichospermum strains
differed in TLC, suggesting structural dissimilarities in their LPS.
Moreover, variations in the staining intensity between silver and Pro-Q
Emerald 300 dye could be attributed to differences in the chemical
composition of Do-1 and Do-2 LPS molecules. Pro-Q™ Emerald 300 dye
is known to be more sensitive to hydrophobic and poorly charged LPS
(Snyder et al., 2009), while silver staining has been reported to have less
reactivity for LPS containing acidic sugars (Fujii et al., 2012).
On the other hand, LPS derived from both M. aeruginosa cultures and
Microcystis-dominated water bloom LPS exhibited similar migration
patterns in SDS-PAGE with prominent bands at molecular weights in the
regions associated with long and very long O-antigen of smooth LPS. The
prevalence of smooth LPS bands over rough LPS bands has been previously
reported for M. aeruginosa NIES-87 (Fujii et al., 2012). Although
Environmental Toxicology and Pharmacology 105 (2024) 104342
HAB-Ap and Ap-1 shared a strong band in the intermediate O-antigen
region, the HAB-Ap displayed a much higher intensity of Pro-Q staining
and more numerous bands than Ap-1. These distinct patterns could be
attributed to the presence of several G- bacteria genera, which were
much more abundant in HAB-Ap than in other biomasses. Notably,
genera such as Methylorosula, Sphingopyxis, and Rhizobkdes 1 were present
in HAB-Ap biomass in counts ranging from one to two thousand,
while their counts in other biomasses were up to thirty. These genera
were also among the three most abundant G- genera in the HAB-Ap
biomass. In contrast, LPS isolated from water bloom and cultures of
Planktothrix showed minimal or no reactivity with silver or Pro-Q
Emerald 300. These findings are consistent with our previously published
results using the non-axenic Planktotrhix CYA126/8 culture
(Skockova et al., 2023a) and our recent observations from other
Planktothrix-dominated blooms (Skockova et al., 2023b).
Although the aforementioned results indicate differences in LPS
structure and their pro-inflammatory activity in keratinocytes, the
wound-healing assay revealed a contrasting pattern. Nearly all LPS
samples, including those from HAB and laboratory cultures, induced
comparable rates of wound closure by stimulating the proliferation and/
or migration of HaCaT keratinocytes. This finding aligns with the
observation by Lee et al. (2017), who reported elevated keratinocyte
proliferation and migration after exposure to subtoxic concentrations of
LPS (Lee et al., 2017). However, both axenic samples of M. aeruginosa
(Mi-1 and Mi-2) exerted a distinct effect comparable with the negative
control. This divergent response pattern suggests that the impact of LPS
on wound healing in keratinocytes is likely mediated through receptors
or mechanisms that differ from the pro-inflammatory activities typically
associated with LPS.
5. C o n c l u s i o n
Previous studies have established that LPS derived from CyanoHABs
and cyanobacterial cultures can trigger pro-inflammatory effects in intestinal,
airway epithelial, and immune cells. In our investigation, we
discovered that specific CyanoHAB LPS, along with LPS from cultured
Aphanizomenon PCC7905 stimulated the production of proinflammatory
cytokines, such as IL-6, IL-8, CCL2, and CCL20, in
human keratinocytes HaCaT, while also enhancing wound healing. The
observed variation in the effects among different LPS samples, with
greater activity observed for LPS isolated from natural water blooms
than those for axenic cultures, suggest that the composition of the
biomass and the presence of specific bacteria, particularly G- bacteria,
influenced the biological activity of the LPS. Furthermore, variations in
LPS structure were identified among the samples, as evidenced by
distinct staining patterns in TLC, SDS-PAGE silver, and Pro-Q Emerald
300 staining, indicating differences in the chemical composition of the
LPS molecules. Notably, most cyanobacterial LPS exhibited bands
associated with long and very long O-antigens, indicative of smooth LPS.
In summary, this study sheds light on the pro-inflammatory properties
of CyanoHAB LPS on the human keratinocyte cell line HaCaT. The
composition and abundance of G- bacteria, along with structural disparities
in the LPS, emerged as crucial factors influencing the observed
effects. A deeper understanding of the specific interactions between LPS
and target cells holds significant value in clarification of the health risks
associated with CyanoHAB exposure.
D e c l a r a t i o n o f g e n e r a t i v e A l a n d A l - a s s i s t e d t e c h n o l o g i e s i n t h e
w r i t i n g p r o c e s s
During the preparation of this work, the author(s) used DeepL and
Grammarly to check and improve English and ChatGPT to rewrite the
abstract and highlights from our own text to the required number of
words and characters. After using this tool/service, the author(s)
reviewed and edited the content as needed and take(s) full responsibility
for the content of the publication.
11
P. Raptová et ah
C R e d i T a u t h o r s h i p c o n t r i b u t i o n s t a t e m e n t
P e t r a R a p t o v á : Investigation, Formal analysis, Writing - original
draft, Writing - review & editing. V e r o n i k a S k o č k o v á : Investigation,
Writing - review & editing. P a v e l B a b i c a : Conceptualization, Methodology,
Writing - original draft, Writing - review & editing, Project
administration, Funding acquisition. I v a S o v a d i n o v á : Methodology,
Writing - original draft, Writing - review & editing. E l i š k a S y c h r o v á :
Investigation. P e t r a V í d e ň s k á : Investigation. O n d ř e j V a š í č e k : Investigation,
Writing - review & editing. L e n k a S i n d l e r o v á : Conceptualization,
Methodology, Formal analysis, Writing - original Draft, Writing
- review & editing, Project administration, Funding acquisition,
Supervision.
D e c l a r a t i o n o f C o m p e t i n g I n t e r e s t
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
D a t a A v a i l a b i l i t y
Data will be made available on request.
A c k n o w l e d g e m e n t s
The results of the project were created with the financial support of
the provider Czech Science Foundation within project No. GA19-
09980S. Authors thank the Research Infrastructure RECETOX RI (No.
LM2023069) financed by the Ministry of Education, Youth and Sports,
and Operational Programme Research, Development, and Education—project
CETOCOEN EXCELLENCE (No. CZ.02.1.01/0.0/0.0/
17_043/0009632) for supportive background. The research was also
supported by the long-term research development project of the Institute
of Botany (RVO 67985939). We also thank to Ms. Hana Klímová for her
technical assistance and Lukáš Kubala for preparing the graphical
abstract.
A p p e n d i x A . S u p p o r t i n g i n f o r m a t i o n
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.etap.2023.104342.
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13
SUPPLEMENTARY DATA
Title: Cyanobacterial bloom-associated lipopolysaccharides induce pro-inflammatory processes in
keratinocytes in vitro
Authors: Raptová P. 1,2, Skočková V. 1,2, Babica P. 3,4, Sovadinová 1.3, Sychrová E. 3, Vídeňská P. 3,
Splíchalová P. 3, Vašíček 0.1, Sindlerová L. 1*
Affiliation:
1
Department of Biophysics of Immune System, Institute of Biophysics of the Czech Academy of
Sciences, Brno 61200, Czech Republic; raptova@ibp.cz (R.P.), skockova@ibp.cz (V.S.);
ondrej.vasicek@ibp.cz (O.V.)
2
Department of Experimental Biology, Faculty of Science, Masaryk University, Brno 62500, Czech
Republic
3
RECETOX, Faculty ofScience, Masaryk University, Brno 62500, Czech Republic;
pavel.babica@recetox.muni.cz (P.B.); sovadinova@recetox.muni.cz (I.S.);
eliska.sychrova@recetox.muni.cz (E.S.); petra.videnska@recetox.muni.cz (P.V.);
petra.splichalova@recetox.muni.cz (P.S.)
4
Department of Experimental Phycology and Ecotoxicology, Institute of Botany, Brno 60200, Czech
Republic
* Correspondence: sindler@ibp.cz
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and 20 u.g/ml). Untreated HaCaT cells were used as a negative control. HaCaT cells lysed by supplierprovided
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Fig. S3: Keratin expression by HaCaT cell line after LPS exposure. K6 (A, B) and K17 (C, D) expression
after 24 h exposure to tested cyanoLPS (50 ug/ml and 20 ug/ml) was detected by western blot. Relative
protein levels were detected by densitometry and the ratio of protein of interest and vinculin was
calculated. Data from five independent experiments were integrated and expressed as the mean ±
SEM. Data were statistically analyzed using ANOVA followed by Dunnett's multiple comparison test.
Representative blots are shown in E.
BIBLIOGRAPHY
Appendix B
Cyanobacterial harmful bloom lipopolysaccharides: pro-inflammatory
effects on epithelial and immune cells in vitro
V. Skočková, P. Raptová, K. Pospíchalová, I. Sovadinová, E. Sychrová, M. Smutná, K.
Hilscherová, P. Babica, L. Šindlerová
Archives of Toxicology, 2023, doi: 10.1007/s00204-023-03644-8
85
Archives of Toxicology (2024) 98:481 -491
https://doi.org/! 0.1007/s00204-023-03644-8
IMMUNOTOXICOLOGY
®Check for
updates
Cyanobacterial harmful bloom lipopolysaccharides: pro-inflammatory
effects on epithelial and immune cells in vitro
V. Skockovä1
'2
• P. Raptovä1
'2
• K. Pospfchalovä1
• I. Sovadinovä3
• E. Sychrovä3
• M. Smutnä3
• K. Hilscherovä3
•
P. Babica3
'4
• L. Sindlerovä1
©
Received: 31 July 2023 / Accepted: 16 November 2023 / Published online: 8 December 2023
©The Author(s) 2023
Cyanobacterial harmful blooms (CyanoHABs) pose a global ecological problem, and their lipopolysaccharides (LPS) are
among the bioactive compounds they release. Previous studies on CyanoHAB-LPS from single cyanobacterial species have
shown varying bioactivities in different in vitro cell models. In this study, we isolated LPS from 19 CyanoHAB samples
collected at 18 water bodies in the Czech Republic over two consecutive seasons. The proportions of cyanobacteria, Gramnegative
bacteria (G-), and other bacteria in the biomass were determined by qPCR, while the cyanobacterial genera were
identified using light microscopy. In vitro models of keratinocytes (HaCaT), the intestinal epithelium (co-culture of differentiated
Caco-2 cells and peripheral blood mononuclear cells — PBMC), and PBMC alone were treated with isolated LPS
at concentrations of 50, 100, and 1 pg/ml, respectively. The endotoxin activities of these concentrations were within the
range measured in the aquatic environment. Approximately 85-90% of the samples displayed biological activity. However,
the potency of individual LPS effects and response patterns varied across the different in vitro models. Furthermore, the
observed activities did not exhibit a clear correlation with the taxonomic composition of the phytoplankton community, the
relative share of microbial groups in the biomass, endotoxin activity of the LPS, or LPS migration and staining pattern in
SDS-PAGE. These findings suggest that the effects of CyanoHAB-LPS depend on the specific composition and abundance
of various LPS structures within the complex environmental sample and their interactions with cellular receptors.
Keywords Lipopolysaccharide • Cyanobacterial harmful blooms • Inflammation • Keratinocytes • Enterocytes • Immune
cells
S3 L. Sindlerovä K . Hilscherovä
sindler@ibp.cz klara.hilscherova@recetox.muni.cz
V. Skockovä P. Babica
skockova@ibp.cz pavel.babica@recetox.muni.cz
Abstract
P. Raptovä
raptova@ibp.cz
Department of Biophysics of Immune System, Institute
of Biophysics of the Czech Academy of Sciences,
Brno 61200, Czech RepublicK. Pospfchalovä
katerina.pospichalova@ibp.cz
Department of Experimental Biology, Faculty of Science,
Masaryk University, Brno 62500, Czech RepublicI. Sovadinovä
sovadinova@recetox.muni.cz
R E C E T O X , Faculty of Science, Masaryk University,
Brno 62500, Czech RepublicE. Sychrovä
eliska.sychrova@recetox.muni.cz i
Department of Experimental Phycology and Ecotoxicology,
Institute of Botany of the Czech Academy of Sciences,
Brno 60200, Czech Republic
M . Smutnä
marie.smutna@recetox.muni.cz
Ö Springer
482 Archives of Toxicology (2024) 98:481-491
Introduction
The occurrence of cyanobacterial harmful blooms (CyanoHABs)
in freshwater reservoirs is a global environmental issue
(Huisman et al. 2018). Cyanobacteria are capable of producing
a wide range of hazardous toxins, mostly secondary metabolites,
some of which, such as microcystin or cylindrospermopsin,
are well-studied and characterized. Their production
can lead to adverse health effects, including fatalities (Buratti
et al. 2017). However, there are also bioactive and potentially
toxic cyanobacterial secondary metabolites, like puwainaphycins
and minutissamides, for which limited information exists
regarding their environmental occurrence, fate, effects, and
associated risks (Huisman et al. 2018; Kubíčkova et al. 2019;
Vašíček et al. 2020). CyanoHABs also represent a source of
endotoxins/lipopolysaccharides (LPS). LPS form an integral
part of the outer membrane of the cell wall in cyanobacteria
and heterotrophic Gram-negative (G-) bacteria which associate
with CyanoHABs (Caroff and Novikov 2020; Gemma et al.
2016). Consequently, LPS is inherently present in any location
with CyanoHAB occurrence, where it may be released
into the water during cell division and lysis (Durai et al. 2015;
Sulcetal. 2017).
Some cases of recreational exposure to CyanoHABs leading
to skin irritation, gastroenteritis, and other adverse inflammation-based
effects were previously published (Kubíčkova
et al. 2019), and also our previous results indicate the proinflammatory
potency of several CyanoHAB-derived LPS
and ability to potentiate the effect of cylindrospermopsin.
However, these studies were primarily focused on Microcystis
aeruginosa (Bláhová et al. 2013) or on a limited number
of CyanoHAB biomasses heavily dominated by a selected
cyanobacterial species of interest (Moosova et al. 2019; Moosová
et al. 2019; Raptová et al. 2023; Skočková et al. 2023).
Consequently, these studies may not sufficiently represent the
variability of potencies and activities of CyanoHAB-derived
LPS across a broader spectrum of naturally occurring water
blooms with different dominants and species composition.
Therefore, in the present study, we collected 19 CyanoHAB
biomasses from 18 recreational water bodies in the Czech
Republic during two consecutive seasons to more comprehensively
characterize the diversity of CyanoHAB-LPS, their
endotoxin activities, and biological effects on cellular in vitro
models of keratinocytes (HaCaT cells), enterocytes (differentiated
Caco-2 cells) co-cultured with human peripheral blood
mononuclear cells (PBMC), and PBMC themselves.
Methods
Detailed specifications of used buffers, media, antibodies,
and kits please find in the Supplementary Material.
CyanoHAB samples
The preparation of the CyanoHAB biomasses collected
from water reservoirs in the Czech Republic is described
in Skočková et al. (2023). The phytoplankton cell counts
and species composition were determined using microscopic
evaluation of sample aliquots fixed with 2% formaldehyde.
The cell counts were converted to biovolumes (Hillebrand
et al. 1999; Skácelová and Lepš 2014) and expressed as a
percentage share of biovolume in a particular sample. The
sample codes, dominant groups and species, biovolume
percentages for each sample, and identification of sampled
localities are summarized in Supplementary Table SI.
LPS preparation and electrophoretic separation
The extraction of CyanoHAB-LPS was performed using
a hot phenol-water extraction method, as previously
described (Skočková et al. 2023). Isolated and purified LPS
were separated by SDS-PAGE followed by silver or Pro-Q™
Emerald 300 staining. Protein contamination was evaluated
using SDS-PAGE and Sypro Ruby staining, nucleic acid
contamination by agarose gel electrophoresis and ethidium
bromide staining. For procedure details see the Supplementary
Materials.
DNA extraction
The extraction of DNA from CyanoHAB biomasses was
conducted based on previous studies (Sehnal et al. 2022;
Skočková et al. 2023). The details of the procedure are
described in Supplementary Materials.
qPCR detection of cyanobacteria, heterotrophic
bacteria, and Gram-negative heterotrophic bacteria
DNA quantification was performed using qPCR and specific
FAM-BHQ1-labeled Taqman probes and primers that
targeted 16S rRNA genes as reported previously (Skočková
et al. 2023). The primers and probes (Supplementary
Table S2) were designed to detect the gene sequences specific
for three groups of microorganisms: cyanobacteria, total
heterotrophic bacteria, and a subset of G- heterotrophic bacteria.
The qPCR was conducted on a LightCycler® 96 Realtime
PCR System under the same conditions as reported
in a previous study (Lang-Yona et al. 2014). qPCR values
(copies of respective 16S rRNA genes) were converted to
cell equivalents using a calibration curve constructed using
a cultured E. coli C C M 3954 (for total and G- bacteria) or
Microcystis aeruginosa PCC7806 (for cyanobacteria). The
obtained cell count equivalents were normalized per total
isolated DNA, and expressed as a percentage share of each
Springer
Archives of Toxicology (2024) 98:481 -491 483
group, with the sum of cyanobacteria and total heterotrophic
bacteria considered as 100%. The share of heterotrophic bacteria
other than G- bacteria was determined by subtracting
G- bacteria from the total heterotrophic bacteria.
Endotoxin activity by PyroGene™ rFC assay
The endotoxin activity of isolated LPS was determined using
the PyroGene™ Recombinant Factor C Endpoint Fluorescent
Assay (Lonza, Switzerland) following the manufacturer's
instructions, as described previously (Skockova et al. 2023).
Fluorescence was measured using a microplate reader (Infinite
M200, Tecan, Switzerland; excitation 380 nm, emission
440 nm), and endotoxin activity was calculated according to
the standard curve. Data were expressed as endotoxin activity
in endotoxin units (EU) per g of biomass dry weight (d.
w.) and per mg of the respective LPS, endotoxin activities of
CyanoHAB-LPS concentrations used in in vitro experiments
were calculated (Table S3).
Cell lines
Caco-2 cells (human colorectal adenocarcinoma cell line;
CLS, Germany) were maintained in the Caco-2 medium.
The cells were seeded at a density of 2 x 104
cells/cm2
on
24-well cell culture inserts (Corning, USA) and maintained
for 21 days to reach a differentiated monolayer. The apical
(300 ul) and basolateral (800 ul) culture media were changed
three times per week.
HaCaT cells (human keratinocyte cell line; CLS, Germany)
were maintained in HaCaT medium. The cells were
seeded at a density of 4 x 104
cells/cm2
on a 24-well or
96-well plate (TPP, Switzerland) and maintained for 3 days
to reach a confluence.
PBMC isolation
Peripheral blood from healthy donors was obtained based on
the collection protocol approved by The Ethics Committee
for Research at Masaryk University, Brno, Czech Republic
(number EKV-2018-083). The PBMC isolation procedure
was based on the work of Meital et al. (2019). The detailed
procedure is provided in the Supplementary Materials.
Treatment
HaCaT cells were treated with 50 ug/ml of CyanoHAB-LPS,
Caco-2/PBMC 100 ug/ml, and PBMC 1 ug/ml, the non-toxic
concentrations (Fig. SI). A detailed description of the procedure
is in Supplementary Materials.
Lactate dehydrogenase assay
Cytotoxicity of LPS treatments was evaluated by lactate
dehydrogenase (LDH) activity in cell culture medium
using the Cytotoxicity Detection Kit PLUS (Roche, USA)
as described previously (Bind et al. 2016).
TEER
Trans-epithelial electric resistance (TEER) measures
changes in the permeability of the monolayer which
increases during inflammation. TEER of the Caco-2 monolayer
was measured by ECIS® TEER24 (Applied BioPhysics,
USA) at two time points: before the assembly
of the co-culture and after 4 days of the exposure to LPS.
For the measurement at the end of the experiment, the
co-culture was disassembled and only inserts with Caco-2
cell monolayer were put in the TEER measuring plate. As
Fig. 1 Quantification of
DNA sequences in CyanoHAB
biomasses using qPCR.
TaqMan Probes specifically
targeting total heterotrophic
bacteria, Gram-negative bacteria
(G-), and cyanobacteria
were employed to quantify
their respective DNA shares.
The results are presented as a
percentage of cyanobacteria,
G- and the remaining bacterial
population (i.e., total heterotrophic
bacteria excluding G-)
among the total quantified DNA
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Archives ofToxicology (2024) 98:481 -491 485
•^Fig. 2 Response of keratinocytes to L P S exposure. Confluent HaCaT
cells were treated with 19 CyanoHAB-LPS from 18 recreational
water bodies (A-U) at a final concentration of 50 ug/ml for 1 day.
Negative control was untreated, and L P S from E. coli (EC) was used
as a positive control. The concentration of a IL-6, b IL-8, c C C L 2 ,
and d C C L 2 0 in the culture medium was determined by ELISA. Protein
expression of involucrin and filaggrin by the cells: e representative
blots and relative concentration of f involucrin and g filaggrin
expressed as the optical density of the bands. Data are expressed as
the m e a n ± S D . Data were statistically analyzed by unpaired i-test.
n=4. *p<0.05, **p<0.01
a negative control, two empty inserts with a cultivation
medium were used.
Wound healing assay
Wound healing assay studies changes in cell migration and
proliferation under pro-inflammatory conditions. Confluent
HaCaT cells grown on the 96-well plate were used for
the assay. Half of the plates was treated with mitomycin at
a final concentration of 1 ug/ml for 2 h to stop the proliferation
and observe only migration. After PBS wash, the
wounds were made by BioTek AutoScratch Wound Making
Tool (Agilent, USA), the cells were washed again, supplemented
with HaCaT culture medium, and treated with LPS
as mentioned above. The cells were maintained in BioTek
BioSpa 8 Automated Incubator (Agilent) and photos of
the wounds were acquired by BioTek Cytation 5 Imaging
Reader (Agilent) each 6 h for 24 h. The area of the wounds
was calculated in the Gen5 program (Agilent).
Flow cytometry
Under pro-inflammatory conditions, the monocytes differentiate
into macrophages and change the expression of
specific surface molecules CD 16 and CD 14. After 4 days
of exposure, PBMC were collected and the expression of
the markers was measured by flow cytometry. Detailed
procedure is provided in the Supplementary Materials.
The data were analyzed in the FlowJo program (BD Life
Sciences, USA). The gating strategy is described in Fig.
S2. tSNE analysis included four parameters (FSC, SSC,
CD14, CD16).
ELISA
Upon pro-inflammatory stimuli, cells produce cytokines
to drive inflammation and regeneration. Concentrations of
selected cytokines were measured in the media by commercially
available ELISA kits according to the manufacturer's
instructions.
Western blot
Pro-inflammatory activation can change the expression of
various proteins by the cell. To study these changes, the
cells were lysed, sonicated (Sonopuls, Bandelin, Germany),
and boiled (10 min). Protein concentration was measured by
BCA assay (Pierce™ BCA ProteinAssay kit, ThermoFisher
Scientific, USA) according to the manufacturer's instructions,
the samples were adjusted to the same concentration
and supplemented with 5 xLaemli buffer. The SDS-PAGE
electrophoresis and western blotting were done as described
previously (Pekarova et al. 2015). The detailed procedures
can be found in Supplementary Materials.
Statistical analysis
Data are presented as a mean ± SD. The number of independently
repeated experiments («) is stated in the figure legend.
Statistical analysis was performed using GraphPad Prism
version 9.5.1 for Windows (GraphPad Software, USA).
Outliers were identified by the ROUT method and filtered
out. Significant difference from negative control (named as
"control" throughout the text and figures) was determined
by unpaired f-test. In case of disagreement of variances, the
Mann-Whitney test was used. Data normalized to control
were analyzed by one-sample f-test. *p<0.05, **p<0.01.
Results
Taxonomy evaluation, endotoxin activity
of CyanoHAB-LPS, and smooth vs. rough LPS
composition
Cyanobacteria accounted for at least 47% biovolume of the
phytoplankton biomass (Table SI and Fig. S3). The exception
was the sample B, where picocyanobacterium Aphanocapsa
holstatica constituted ~ 50% of the cell count, but its
share in the biovolume was < 0.1% due to its small cell size
compared to the larger taxa. The LPS yield (Table S3) significantly
positively correlated with cyanobacterial biovolume
in the biomass, in contrast to the eukaryotic algae biovolume,
while the correlation with the endotoxin activity of
the biomass was weaker and not significant (Fig. S4). There
were only weak and nonsignificant correlations between LPS
yield and qPCR-based estimate of cyanobacterial and bacterial
biomass in the sample, while the endotoxin activity did
not correlate with any of the parameters (Fig. S5). Endotoxin
activity of the majority of LPS samples fell within the range
of tens of EU/mg LPS (Table S3). The highest activity was
shown by sample G with almost 8 000 EU/mg LPS. Three
other samples (A, K, and N) reached more than 1 000 EU/
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Archives of Toxicology (2024) 98:481 -491 487
•4Fig. 3 Response of Caco-2/PBMC co-culture to LPS exposure. Coculture
of differentiated Caco-2 cells and human PBMC were treated
with 19 CyanoHAB-LPS from 18 recreational water bodies (A-U) at
a final concentration of 100 ug/ml for 4 days. Negative control was
untreated, and LPS from E. coli (EC) was used as a positive control.
Single cultures of differentiated Caco-2 cells or PBMC were used
as controls of the model. The concentration of a IL-8 in the apical
medium and b IL-8, c CCL2, d CCL20, e IL-6, and f IL-10 in the
basolateral medium was determined by ELISA (« = 8). g Expression
of CD16 by monocytes in PBMC fraction was determined by flow
cytometry (n = 6, dashed line represents control), h Representative
tSNE visualization of monocytes from samples with significantly
increased CD16 expression. Data are expressed as the mean±SD.
Data were statistically analyzed by unpaired /-test (a-f) or one sample
Mest (g). *p<0.05, **p <0.01. MFI median fluorescence index
mg. In contrast, samples B and O exhibited relatively low
endotoxin activity with less than 10 EU/mg LPS. However,
even the highest endotoxin activity was not comparable to E.
coli LPS with more than 3.5 X 106
EU/mg LPS. The migration
patterns of isolated LPS in SDS-PAGE showed variation
among individual samples (Fig. S6). Pro-Q Emerald 300
staining exhibited higher sensitivity for CyanoHAB-LPS
than silver staining, although certain samples (e.g., H and
N) exhibited only faint staining even with Pro-Q (Fig. S6).
Common characteristics among cyanoHAB-LPS included
dominant presence of smooth LPS with larger molecular
weight (bands > 20 kDa), while rough or semi-rough LPS
observed in E. coli LPS (bands < 15 kDa) were absent,
except the samples O, S and J (Pro-Q only). Contamination
with protein or DNA (Fig. S7 or S8) was not detected, while
RNA contamination in the isolated CyanoHAB-LPS was
below < 2% w/w (Fig. S8).
Biomass DNA composition highly differs
qPCR analysis showed four samples (A, D, E, and P) to
contain only DNA of G- bacteria and cyanobacteria (Fig. 1).
Seven samples (N, H, L, P, K, J, and F) showed cyanobacterial
DNA share higher than 75% (94.2%, 89.98%, 89.0%,
86.8%, 85.7%, 79.0%, 76.1%, resp.), other five samples were
in the range 50-75% (E = 74.1%, T =58.3%, U =54.8%,
M = 52.7%, and R = 51.3%). In contrast, two biomasses contained
less than 10% of cyanobacterial DNA (G = 6.6% and
A =5.1%). Similarly, the share of G- bacteria D N A varied
among the samples with six biomasses above 50%
(A =94.9%, B =82.6%, C =63.2%, D =62.0%, S =54.1%,
and G =52.6%) and four biomasses under 10% (H =7.2%,
L = 5.1%, K = 4.3%, and N =4.2%).
Three CyanoHAB-LPS induced pro-inflammatory
cytokine production in keratinocytes
Three LPS samples (G, S, and T) induced substantial
elevation of all four detected pro-inflammatory cytokines
(Fig. 2a-d). The other two samples (A and H) caused just a
slight increase in CCL2 production. The cytokine response
after exposure to the remaining samples was comparable
to the negative control. Although filaggrin and involucrin
expression decreases during inflammation, we did not
observe any down-regulation of these proteins (Fig. 2e-g).
On the contrary, the level of involucrin was elevated upon
exposure to some otherwise inactive samples (C, H, J, and
L) and even to one cytokine release-inducing LPS (T). In the
wound healing assay, no significant effects on HaCaT cell
migration and proliferation were observed (Fig. S9).
One CyanoHAB-LPS induced pro-inflammatory
response in the intestinal model
Only one LPS sample (G) was highly active throughout
most of the measured parameters, inducing the production
of all pro-inflammatory cytokines and expression of CD 16
(Fig. 3a-g). The tSNE analysis confirmed the results showing
similar clustering of the cells in sample G compared
to the positive control, indicating changes in cell size and
expression of markers typical for activated monocytes/macrophages
(Fig. 3h). Few other samples (A, D, E, H, and S)
elevated level of one or two cytokines but never to the same
extent as sample G. Despite the pro-inflammatory responses
to several CyanoHAB-LPS evident from cytokine levels and
monocyte activation, the integrity of the Caco-2 monolayer
did not appear to be compromised. No drop in TEER or
change in the tight junction protein expression was observed
(Fig. S10).
Almost all CyanoHAB-LPS induce some
pro-inflammatory response in PBMC
The P B M C response dynamics were completely different
from that of epithelial cells. Most LPS samples caused
an elevation of one cytokine level at least (Fig. 4a-e). Some
samples significantly changed all studied parameters (e.g.,
A and G), while some significantly influenced few and
seemed to be inactive in others (e.g., C and L). Three LPS
samples (P, R, and U) caused no or very weak pro-inflammatory
response. CD 16 expression patterns differed from the
results of the co-culture model (Fig. 4f). Sample G and E.
coli LPS caused a decrease in CD 16 expression which was
opposite to the effect of indirect PBMC activation. Sample
A worked similarly. On the other hand, three other samples
(L, N, and T) elevated CD16 expression. Samples L and N
acted similarly enhancing some cytokine production to a
comparable extent, while sample T significantly increased
the levels of all measured cytokines. In accordance with
this observation, clustering of the cell subpopulations was
_) Springer
488 Archives ofToxicology (2024) 98:481-491
(a)
14 000
12 000_
. 10 000-
E
S 8 000
6 000
4 000
2 000
0
(b)
A A
(c)
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concst_l
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concst_l
L T
similar in samples G and A together with the positive control Clustering of sample T cells was quite unique among the
on the one side and L with N on the other side (Fig. 4g). PBMC-activating LPS.
Springer
Archives of Toxicology (2024) 98:481 -491 489
•<Fig. 4 Response of P B M C to LPS exposure. Human P B M C were
treated with 19 CyanoHAB-LPS from 18 recreational water bodies
(A-U) at a final concentration of 1 ug/ml for 4 days. Negative control
was untreated, and LPS from E. coli (EC) was used as a positive
control. The concentration of a IL-6, b IL-8, c C C L 2 , d CCL20,
and e TNFot in the culture medium was determined by E L I S A (n = 6).
f Expression of CD16 by monocytes in P B M C fraction was determined
by flow cytometry (n = 5, dashed line represents control), g
Representative tSNE visualization of monocytes from samples with
significantly changed CD16 expression. Data are expressed as the
mean ± SD. Data were statistically analyzed by unpaired /-test (a-e)
or one sample /-test (f).*p<0.05, **p<0.01. MFI median fluorescence
index
Discussion
In this study, 19 CyanoHAB biomasses from various recreational
water bodies in the Czech Republic were collected
during two consecutive seasons to investigate the
bioactivity of their LPS. To study this issue, we used three
different in vitro cell models and a commercial endotoxin
activity assay. Among the individual samples, only sample
G exhibited pro-inflammatory responses in all three
in vitro models, and its response was often comparable
to E. coli LPS. Sample G also had the highest endotoxin
activity from all studied CyanoHAB-LPS. Similar patterns
of effects in PBMC responses were observed in sample A
but its pro-inflammatory effect on epithelial models was
weaker. The dominant cyanobacteria in sample A were
Planktothrix agardhii, accompanied by Aphanizomenon
sp., and Planktolyngbya sp., while sample G was dominated
by Dolichospermum flos-aquae, Sphaerospermopsis
sp., and Aphanizomenon issatchenkoi. Our previous results
show CyanoHAB-LPS isolated from Dolichospermum flosagi/ae-dominated
water bloom to be more bioactive than
the ones isolated from Planktothrix agardhii- and Aphanizomenon
flos-aquae-dominated blooms (Raptova et al.
2023; Skockova et al. 2023). Other samples with outstanding
profile of effects in our CyanoHAB-LPS collection were
samples S and T. They were (together with sample G) highly
active in HaCaT keratinocytes. The more bioactive sample
S was dominated by cyanobacteria Planktothrix agardhii,
Raphidiopsis (ex. Cylindrospermopsis) raciborskii, Microcystis
sp., and Pseudanabaena sp., sample T by Dolichospermum
sp., Aphanizomenon klebahnii, and Woronichinia
sp. The other samples dominated by Planktothrix agardhii
(H, N) or Dolichospermum sp. (E) showed much lower bioactivity
in the epithelial cell models. The only exception
was the induction of CD 16 expression in PBMC by sample
N, although its other effects on these cells were either much
lower than those of others or were not even detectable. With
these results it seems that dominance of cyanobacteria Dolichospermum
or Planktothrix might serve as some kind of a
predictor of potential biological activity of the LPS produced
by the water bloom but for sure it does not stand alone.
Microcystis is one of the most common bloom forming
cyanobacterial genera in the fresh waters around the
world (Huisman et al. 2018). Samples dominated by this
genus showed no pro-inflammatory effects in epithelial
cells, in contrast to our other previous results, where one
particular M/crocysf/s-dominated CyanoHAB-LPS induced
strong pro-inflammatory effects in HaCaT as well as Caco-2
cells (Raptova et al. 2023; Skockova et al. 2023). In PBMC,
the effect differed. Samples L (Microcystis) and N (dominated
with Planktothrix but more than 20% of biomass made
up of Microcystis) showed similar pattern in cytokine activation
and above all significant induction of CD 16 expression
and similar clustering of PBMC subpopulations. The other
Mz'crocysfK-dominated samples (F, J, K, and M) activated
the PBMC to produce cytokines but they did not change
their differentiation status. Finally, samples P and U were
completely inactive. Previously, Microcystis aeruginosadominated
CyanoHAB-LPS were shown to activate human
whole blood cells as well as isolated neutrophils (Blahova
et al. 2013; Moosova et al. 2019) but our results show that
not all the environmental mixtures have this ability.
The other LPS-producing organisms in the water blooms
are G- bacteria. Their share highly varied among the samples
from approximately 4% (sample N) to 94% (sample A).
Samples with the highest in vitro bioactivity had G- bacteria
share from low (T = 22%) to average (G = 53%, S = 54%)
and high (A = 95%). The share of G- bacteria also was not
reflected by the endotoxin activity of the CyanoHAB-LPS,
where samples S and T showed less than one hundred EU/
mg LPS, sample A displayed 1 227 EU/mg LPS, and sample
G exhibited almost eight thousand EU/mg LPS. Our analysis
also showed that endotoxin activity of the CyanoHAB-LPS
did not significantly correlate with quantities of G- bacteria,
cyanobacteria or other bacteria in the biomass. None of the
above mentioned parameters could clearly eludcidate the
bioactivity of the CyanoHAB-LPS.
No relationships between endotoxin activity, in vitro
activity and SDS-PAGE migration pattern were observed.
In general, CyanoHAB-LPS samples were dominated by
bands > 20 kDa, indicating dominance of smooth LPS, which
is in agreement with previous studies (Fujii et al. 2012; Raptova
et al. 2023; Skockova et al. 2023; Swanson-Mungerson
et al. 2019). In contrast to E. coli LPS, CyanoHAB-LPS
were stained more intensively with Pro-Q Emerald 300 compared
to silver staining, which suggests differences in their
chemistry, such as presence of more hydrophobic and poorly
charged LPS that are preferentially stained with Pro-Q Emerald
300 (Snyder et al. 2009), or presence of acidic sugars for
which silver staining has a low affinity (Fujii et al. 2012).
Very faint LPS staining was observed for samples H and
N dominated by Planktothrix agardhii, which corresponds
to other reports with LPS from CyanoHABs or cultures of
Planktothrix (Raptova et al. 2023; Skockova et al. 2023).
Springer
490 Archives of Toxicology (2024) 98:481-491
Overall, our results demonstrate that certain environmental
LPS associated with CyanoHABs can indeed
induce pro-inflammatory effects in human skin and intestinal
epithelial cells, as well as in immune cells in vitro.
Although a majority of the tested CyanoHAB-LPS in our
study did not exhibit significant effects in the epithelial
cells, about 15-20% of samples were found to be active
in at least one cell model. Moreover, these effects were
induced in vitro at endotoxin concentrations that can be
found in natural waters with CyanoHABs. The literature
reports significant variability in the endotoxin activity of
CyanoHAB-LPS, with values ranging from 1500 times
lower to 5000 times higher than those observed for the
most active sample G in our study (Bernardova et al. 2008;
Bláhová et al. 2013; Moosová et al. 2019; Skočková et al.
2023). However, the potency of individual LPS effects and
response patterns across the different in vitro models and
endpoints varied significantly in this study, mostly regardless
of endotoxin activity. The main driver behind these
differences remains to be identified. So far, the observed
activities did not exhibit a clear correlation with the taxonomie
composition of the phytoplankton community, the
relative share of microbial groups in the biomass, endotoxin
activity of the LPS, or LPS migration pattern or
band intensity in the SDS-PAGE. The resulting effects
of CyanoHAB-LPS likely depend on the specific composition
and abundance of various LPS structures in the
complex environmental sample and their mixture interactions
with cellular receptors. Predicting these effects
from other parameters is challenging. Further research is
needed to better understand the complexities involved in
CyanoHAB-LPS effects.
Supplementary Information The online version contains supplementary
material available at https://doi.org/10.1007/s00204-023-03644-8.
Acknowledgements The authors want to thank Dr. Olga LepšováSkácelová
for her help with taxonomie analyses, Ms. Michaela Pešková
for human serum preparation, M s . Vanda Oravcová for co-culture
model optimization, M s . Hana Klímová and Mr. Robert Stanko for
LPS sample preparation and electrophoresis, and Ms. Iva Zaplatilová
for technical support.
Author contributions Conceptualization: L Š , V S ; Methodology: V S ,
PR, L Š , P B , IS, K H ; Formal analysis and investigation: V S , PR, K P ,
IS, E S , M S ; Writing — original draft preparation: V S , L S ; Writing —
review and editing: PR, P B , IS, ES, K H ; Funding acquisition: L S , PB;
Project administration: L S ; Resources: L S , P B , K H ; Supervision: L S .
Funding Open access publishing supported by the National Technical
Library in Prague. The research was supported by Czech Science
Foundation grant No. GA19-09980S. Authors thank the Research Infrastructure
R E C E T O X RI (No. LM2023069) financed by the Ministry of
Education, Youth and Sports, and Operational Programme Research,
Development, and Education—project C E T O C O E N E X C E L L E N C E
(No. CZ.02.1.01/0.0/0.0/17_043/0009632) for supportive background.
This work was supported by the European Union's Horizon
2020 research and innovation programme under grant agreement No.
857560—CETOCOEN Excellence. This publication reflects only the
author's view and the European Commission is not responsible for
any use that may be made of the information it contains. This study
was also supported by long-term research development project of the
Institute of Botany (RVO 67985939).
Availability of data and materials The datasets generated during and/or
analyzed during the current study are available from the corresponding
author on a reasonable request.
Declarations
Conflict of interest The authors have no relevant financial or non-financial
interests to disclose.
Ethical approval Not applicable.
Open Access This article is licensed under a Creative Commons Attribution
4.0 International License, which permits use, sharing, adaptation,
distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
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otherwise in a credit line to the material. If material is not included in
the article's Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creativecommons.Org/licenses/by/4.0/.
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Cyanobacterial harmful bloom lipopolysaccharides: pro-inflammatory effects on epithelial and immune
cells in vitro
Skočková V.1 , 2
, Raptová P.1 , 2
, Pospíchalová K.1
, Sovadinová I.3
, Sychrová E.3
, Smutná, M . 3
, Hilscherová, K.3
,
Babica P.3
-4
, ŠindlerováL.1
*
'Department of Biophysics of Immune System, Institute of Biophysics of the Czech Academy of Sciences, Brno
61200, Czech Republic; skockova@ibp.cz (V.S.); raptova@ibp.cz (P.R.); katerina.pospichalova@ibp.cz (K.P.)
2
Department of Experimental Biology, Faculty of Science, Masaryk University, Brno 62500, Czech Republic
3
RECETOX, Faculty of Science, Masaryk University, Brno 62500, Czech Republic; sovadinova@recetox.muni.ez
(I.S.); eliska.sychrova@recetox.muni.ez (E.S.); marie.smutna@recetox.muni.ez (M.S.);
ldara.hilscherova@recetox.muni.ez (K.H.); pavel.babica@recetox.muni.ez (P.B.)
4
Department of Experimental Phycology and Ecotoxicology, Institute of Botany of the Czech Academy
of Sciences, Brno 60200, Czech Republic
correspondence: sindler@ibp.ez (L.S.)
Tab. SI Sampling localities, dates and dominants of CyanoHABs used for LPS isolation.
Sample
code
Locality GPS
Sampling
date
Dominant groups and species (based on %biovolume)
A Dvorský rybník Pond
48°51'32.960"N,
17°4'21.948"E
Jun 19,
2019
Planktothrix agardhii (26.1%), Planktolyngbya limnetica
(10.5%), Aphanizomenon sp. (7.4%), algae (52.7%)
B
Horní štítarský rybník 48°55'50.545"N,
Jul 10, 2019
Dolichospermum flos-aquae (0.5%), Chroococcus limneticus
B
Pond 15°51'4.550"E
Jul 10, 2019
(0.2%), Aphanocapsa holstatica (<0.1%), algae (99.3%)
fj Hejtman Pond
48°57'52.352"N, Aug 07, Woronichinia naegeliana (82.3%), Microcystis wesenbergii
Hejtman Pond
14°56'28.401"E 2019 (10.4%), algae (1.9%)
Sphaerospermopsis aphanizomenoides (26%), Aphanizomenon
48°58'18.773"N,
14°53'55.219"E
Aug 07,
2019
klebahnii (19.2%), Microcystis sp. (16.1%) [mainly M. aeruginosa
D Starý Kanclíř Pond
48°58'18.773"N,
14°53'55.219"E
Aug 07,
2019
(10.4%)], Raphidiopsis raciborskii (8.7%), Dolichospermum sp.
48°58'18.773"N,
14°53'55.219"E
Aug 07,
2019
(6.1%) [mainly D. crassum (5.8%)], Planktolyngbya sp. (6%),
algae (16.4%)
E Výtopa Pond
48°59'3.797"N,
14°52'47.859"E
Aug 07,
2019
Dolichospermum sp. (95.6%) [mainly D. planctonicum (38.4%),
D. crassum (23.7%), D. ucrainicum (20.9%), D. flos-aquae
(10.5%)], algae (0.2%)
F Nový Kanclíř Pond
48°57'50.984"N,
14°53'50.893"E
Aug 07,
2019
Microcystis sp. (91.7%) [mainly M. flos-aquae (45.8%), M. viridis
(23%), M. aeruginosa (10.7%), M. wesenbergii (8.7%)], algae
(0.8%)
Dolichospermum flos-aquae (43.2%),
G Ruda Pond
48°56'51.739"N, Aug 07, Sphaerospermopsis sp. (16.1%) [mainly S. aphanizomenoides
G Ruda Pond
14°46'24.711"E 2019 (9.9%), S. reniforme (6.2%)], Aphanizomenon issatchenkoi
(16.1%), algae (21.2%)
H
Kamenský Šutrák
Pond
48°50'47.773"N,
16°48'34.583"E
Aug 27,
2019
Planktothrix agardhii (90.2%), algae (1.5%)
J
Františkův rybník
Pond
48°43'5.907"N,
16°51'48.166"E
Aug 27,
2019
Microcystis aeruginosa (94.8%), algae (<0.1%)
K
Nové Mlýny - střed 48°53'43.067"N, Aug 27, Microcystis sp. (96.9%) [mainly M. botrys (80.4%), M. aeruginosa
K
Reservoir 16°36'57.501"E 2019 (16.5%), ] algae n.d.
T
Vranov Reservoir, 48°56'13.533"N, Aug 27, Microcystis sp. (97.5%) [mainly M. flos-aquae (88.1%), M.
Bítov bay 15°43'25.236"E 2019 aeruginosa (1.1%)}, algae (0.5%)
M Letovice Reservoir
49°33'25.999"N, Sep 17, Microcystis sp. (99.8%), [mainly M. flos-aquae (42.2%), M. viridis
M Letovice Reservoir
16°32'12.920"E 2019 (33.5%), M. wesenbergii (20.3%)], algae n.d.
N Olšovec Pond
49o
20'11.679"N, Sep 17, Planktothrix agardhii (68.5%), Microcystis novacekii (22.5%),
N Olšovec Pond
16°45'36.605"E 2019 Aphanizomenon sp. (6%), algae (3%)
0 Mlýnský rybník Pond
48o
47'6.200"N,
16°49'18.614"E
Sep 17,
2019
Raphidiopsis raciborskii (72.2%), algae (25.7%)
p Brno Reservoir
49°14'20.086"N, Sep 24, Microcystis sp. (69.2%) [mainly M. aeruginosa (45.7%), M.
Brno Reservoir
16°30'31.262"E 2019 novacekii (20.8%)], Woronichinia naegeliana (25.4%), algae (2%)
R Letovice Reservoir
49°33'13.753"N, Aug 27, Microcystis sp. (65.5%) [mainly M. aeruginosa (49.3%), M. viridis
R Letovice Reservoir
16°33'20.420"E 2020 (16.1%)], Woronichinia naegeliana (34.5%), algae n.d.
c Konventský rybník 49o
34'49.600"N, Aug 27, Planktothrix agardhii (22.8%), Raphidiopsis raciborskii (8%),
o Pond 15°56'17.721"E 2020 Microcystis sp. (7%), Pseudanabaena sp. (7.1%), algae (43.1%)
T Kachlička Pond
49°33'8.816"N,
15°25'57.236"E
Aug 27,
2020
Dolichospermum sp. (30.6%) [mainly D. crassum (23.6%)],
Aphanizomenon klebahnii (15.9%), Woronichinia sp. (5.8%),
algae (40.2%)
U Trnávka Reservoir
49°31'22.777"N,
15°13'1.081"E
Aug 27,
2020
Microcystis sp. (76%) [mainly M. flos-aquae (57.7%), M.
aeruginosa (10.2%), M. viridis (7%)], Aphanizomenon klebahnii
(14.8%), algae (6.8%)
Cyanobacterial genera and/or species with more than 5% biovolume share in the concentrated biomass are listed. The only exception
is the sample B, where all the cyanobacterial species are listed due to a very low biovolume share of cyanobacteria in this mixture.
The sum of biovolume share of all eukaryotic algae is also provided. n.d.= not detected.
Tab. S2 List of specific primers and FAM-BHQ1-labeled probes used for Taqman qPCR
Target Primer Sets and Probe Sequences (5'->3')
Forward: A C G G G T G A G T A A C R C G T R A
Cyanobacteria Reverse: C C A T G G C G G A A A A T T C C C C
Probe: C T C A G T C C C A G T G T G G C T G N T C
Forward: T C C T A C G G G A G G C A G C A G T
Total heterotrophic bacteria Reverse: G G A C T A C C A G G G T A T C T A A T C C T G T T
Probe: C G T A T T A C C G C G G C T G C T G G C A C - 3 '
Forward: A A C T G G A G G A A G G T G G G G A T
Gram-negative bacteria Reverse: A G G A G G T G A T C C A A C C G C A
Probe: G A C G T A A G G G C C A T G A G G A C T T G A C G T C
Primers and probes are based on the publication: Lang-Yona, N.; Lehahn, Y.; Herut, B.; Burshtein, N.; Rudich, Y. Marine aerosol as a possible
source for endotoxins in coastal areas. Sci. Total. Environ. 2014, 499, 311-318. https://doi.Org/10.1016/i.scitotenv.2014.08.054.
Tab. S3 LPS yield from the CyanoHAB biomasses and endotoxin activity. The endotoxin activity
of the isolated CyanoHAB-LPS and the water from the site of the collection was assessed by Pyrogene™ assay.
The endotoxin activity of the final concentration used for the treatment in the in vitro experiments was calculated.
The green color denotes biomasses, blue water in situ, and yellow culture media.
Sample
code
LPS yield
(mgLPS/g
biomass
d.w.)
PyroGene™
(EU/mg LPS)
PyroGene™
(EU/g
biomass
d.w.)
PyroGene™
(EU/ml water)
Caco-2/PBMC
(EU/ml medium)
HaCaT
(EU/ml medium)
PBMC (EU/ml
medium)
A 6.2 1 226.8 7 619 884 122.68 61.34 1.23
B 5.1 5.2 27 901 0.52 0.26 0.01
C 49.6 17.4 865 368 1.74 0.87 0.02
D 23.6 20.3 478 647 2.03 1.02 0.02
E 66.9 30.1 2011 935 3.01 1.51 0.03
F 20.5 35.9 735 174 3.59 1.80 0.04
G 11.4 7 870.7 89 726 317 787.07 393.54 7.87
H 3.3 12.3 41 898 1.23 0.62 0.01
J 23.1 435.0 10 047 623 43.50 21.75 0.44
K 8.3 1 864.0 15 546 51 186.40 93.20 1.86
L 20.1 45.5 913 147 4.55 2.28 0.05
M 33.9 81.2 2 754 7 438 8.12 4.06 0.08
N 13.7 1 617.7 22 195 135 161.77 80.89 1.62
0 9.5 7.9 74 230 0.79 0.40 0.01
P 15.2 170.4 2 586 44 17.04 8.52 0.17
R 47.0 38.4 1 807 695 3.84 1.92 0.04
S 20.0 70.8 1 416 141 7.08 3.54 0.07
T 24.0 21.6 518 164 2.16 1.08 0.02
U 47.0 73.9 3 473 51 7.39 3.70 0.07
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Fig. SI Cytotoxicity of used LPS concentrations, (a) The concentration of LDH in culture medium after
treatment of HaCaT cells with LPS in concentration 50 tig/ml for 1 day, n = 3. Cells lysed by supplier-provided
lysis buffer were used as a positive control, (b) Percentage of viable (Pi-negative) PBMC after treatment
of Caco-2/PBMC co-culture with LPS in concentration 100 tig/ml for 4 days, n = 6. (c) Percentage of viable
(Pl-negafive) cells after treatment of P B M C with LPS in concentration 1 tig/ml for 4 days, n = 5. Data were
converted to a percentage of the control (untreated cells) and expressed as the mean ± SD. Data were statistically
analyzed by one-sample f-test. * p < 0.05
Fig. S2 Flow cytometry gating strategy, (a) Monocytes, based on FSC-A/SSC-A. (b) Single cells, based
on FSC-A/FSC-H. (c) Single cells, based on SSC-A/SSC-W. (d) Live cells, based on propidium iodide negativity,
(e) Determination of CD14/CD16 double negative cells (quartile Q4). (f) CD16 expression in quartiles Q l - Q3.
(a)
100%
~ 75%
I 50%
cu
>
™ 25%cu
0%
A B C D E F G H J K L M N O P R S T U
Cyanobacteria Euglenophyta Chrysophyta Xanthophyceae
Bacillariophyceae • Dinophyceae l Cryptophyceae l Chlorophyta
(b)
100%
~ 75%
o
>
o
cu
>
cu
50%
i5 25%
I
0%
A B C
Aphanizomenon sp.
Dolichospermum sp.
Planktothrix agardhii
I Other cyanobacteria
E F G H J K L
Sphaerospermopsis sp.
• Pseudanabaena sp.
• Microcystis sp.
Eukaryotic Algae
M N O P R S T U
Raphidiopsis raciborskii
• Planktolyngbya sp.
Woronichinia sp.
Fig. S3 Composition of CyanoHABs used for LPS isolation as a percentage of biovolume. (a) Biovolume share
of the main phytoplankton groups, (b) Biovolume of dominant cyanobacterial genera or species.
(a) 100
in
in
>- .2
on - Q
0 _ CiO
—I \
IS)
Q -
aa
E
1 0
IW)
D L S j T
IV
G
••
Ps = 0.610, p=0.006
ID
O
+
O
+
00
o
+
a i o
o <H
+ +
(b) 100 000
>. 7 10 000
!> -a
U g 1000
.£ E
x o
2 3
•g -55
1 0 0
w 10
i
C
D
H
C
B
H
Ps = 0.174, p = 0.475
1 0 ro
o
+ +
00
o
+
en
o
+
o
+
Cyanobacteria biovolume density
(u.m3
/g biomass d.w.)
Cyanobacteria biovolume density
(um3
/g biomass d.w.)
1 0 0
>- °
oo - Q
0 - OJO
_ l \
IS)
o_
—I
aa
E
1 0
•
• • J
A
1 . . . s T
„ G
0
H
= -0.058, p=0.813
o o
+ +
LTl
o
+
ID
O
+
o
+
00 0 1
o o
+ +
100 000
>. -7 10 000
> -a
1 g 1000
.S E
2 l
•g -55
1 0 0
B 10
• N
•
E P \E P \
H
-0.271, p = 0.262
m ^ - in
0 0 0
+ + +
to r~ 0 0 CTI
0 0 0 0
+ + + +
Eukaryotic algae biovolume density
(um3
/g biomass d.w.)
Eukaryotic algae biovolume density
(um3
/g biomass d.w.)
(c)
LE+15
u
ro
I 1,E+14
OJO
"> 1,E+13
J3
g i
5- g
U
.«1
U £
or o
£E+12
1,E+11
1,E+10
Ps =
p =
0.34
0.159
H
URPs =
p =
0.34
0.159
D
L
\
E
UR
.•*
f
G
0
#
B
N
LD I D
O O
r~ 0 0 ai
0 0 0
+ + +
o <H
+ +
Cyanobacteria biovolume density
(um3
/g biomass d.w.)
Fig. S4 Relationship between phytoplankton biovolume and LPS yield or endotoxin activity in CyanoHABs.
(a) Cyanobacteria or (b) eukaryotic algae biovolumes normalized per dry weight (d.w.) of biomass and plotted
against LPS yield (left) or endotoxin activity (right) per CyanoHAB biomass d.w. (c) Biovolume of cyanobacteria
compared to qPCR-based cyanobacterial cell count estimates (equivalents of Microcystis) per CyanoHAB biomass
d.w. Letters (A-U) represent the sample codes, p = Spearman's rank correlation coefficient and two-tailed p value
(a)
2 c
o_ aa_ i \
IS)
CL.
—I
on
E
100
10
M
0
ps = 0.199, p = 0.414
o o a i o < H ( N p n ^ - L n
+ + + + + + + +
100 000
— 10 000
>• >
> -a
o
ono
T3
c Z>
1000
100
~ 10
N
6
ftft
E
o H
Ps =0.077, p = 0.96 0
0 0 C T l O < H ( N P 0 ^ - L n
+ + + + + + + +
(b)
2 coi c
Q _
—I
on
E
(c)
in
to
2 c
>- °
is, -°
0 _ BD
_ l \
IS)
CL.
—I
aa
E
qPCR Cyanobacteria
(Microcystis cells eq./g biomass d.w.)
100
10
S L PjT
N
0
G
K
ps = 0.271, p = 0.261
100
M
10 G
JT
K
ps = 0.289, p = 0.231
o o c n o < H ( N i r i ^ - L n
O O * - H * - H < - H T - < T - < * H
+ + + + + + + +
qPCR Gram negative bacteria
(E. coli cells eq./g biomass d.w.)
o o c n o < H ( N i r i ^ - L n
O O * - H * - H < - H T - < T - < * H
+ + + + + + + +
qPCR Cyanobacteria
(Microcystis cells eq./g biomass d.w.]
>• >
> -6
u %
.£ E
•g -55
,q =>
100 ooo
10 ooo
1000
100
~ 10
N
G
K ,
_ |
"H*
P F.
L C
•
1P F.
L C
o
B
•
Ps =0.118, p = 0.74 1
oo oi o <H I N
O O < H < H T H
+ + + + +
^t
+
qPCR Gram negative bacteria
(E. coli cells eq./g biomass d.w.)
>
ro
c
100 000
- 10 000
1000
100
10
N
=•
A
E P
S
M L
1,
D C
B
0
H
0.098, p = 0.86 9
o o c n o < H ( N i r i ^ - L n
+ + + + + + + +
qPCR Other bacteria
(E. coli cells eq./g biomass d.w.)
qPCR Other bacteria
(E. coli cells eq./g biomass d.w.)
Fig. S5 Relationship between qPCR-based estimates of microorganism quantity of microorganisms
in CyanoHABs and LPS yield or endotoxin activity, (a) Cyanobacteria, (b) Gram-negative bacterial, and
(c) other bacterial (total bacteria without Gram-negative) cell counts (equivalents of Microcystis or E. coli)
as estimated by qPCR per CyanoHAB biomass dry weight (d.w.), and plotted against either LPS yield (left)
or endotoxin activity (right) per CyanoHAB biomass d.w. Letters (A-U) represent the sample codes,
ps= Spearman's rank correlation coefficient, and two-tailed /?-value.
(a) (b)
103 •
EC A B C D E EC MWM
10 ug 1 (jg
EC F
10 ug
2R
,3 -
130 •
LOO •
111 -
100 •
MWM EC R
1 My
T U EC
10 ug
: •MWM EC A
10 ug
E MWM
I'M •
] 10 •
ICO •
7 0 •
1 "0 -
1 JO .
ICO •
MWM EC L M N
10 ug
j no •
130 -
100 ft
-
MWM EC R S
10 ug
U EC MWM
10 ug
Fig. S6 Separation of LPS by SDS-PAGE followed with (a) silver or (b) Pro-Q™ Emerald 300 staining. LPS
isolated from CyanoHAB biomasses (samples A-U) were loaded at 10 ug per lane. EC - purified LPS from E. coli
011LB4 (Sigma-Aldrich L2630), M W M - molecular weight marker (PageRuler™ Plus Prestained Protein
Ladder, Thermo 26619).
MWM EC A B C D E MWM
10 |jg
2W •
no •
too •
70 .
55 -
1.30 •
100 •
100 •
70 •
3b '
75 '
E C MWM
10 \ig
Fig. S7 Separation of LPS by SDS-PAGE followed with S Y P R O ™ Ruby Protein staining. LPS isolated from
CyanoHAB biomasses (samples A-U) were loaded at 10 ug per lane. EC - purified LPS from E. coli 011LB4
(Sigma-Aldrich L2630), M W M - molecular weight marker (PageRuler™ Plus Prestained Protein Ladder, Thermo
26619).
(a) (b)
0 1 2 A B C D E F G H J K L M N O P R S T U
Ug RNA + 10 ng EC LPS LPS sample #
Fig. S8 Agarose gel electrophoresis of LPS and nucleic acids followed by ethidium bromide staining, (a) Model D N A or R N A (0.5 ug) was loaded alone or in the mix
with 10 ugLPS fromis. coli O l 11:B4 (EC LPS). The presence of LPS was found to affect the migration pattern of RNA bands and cause their smearing, (b) Detection of nucleic
acids in the LPS samples isolated from CyanoHABs (samples A-U) loaded at 10 ug per lane. As a control, 10 ug LPS from E. coli O l 11:B4 (EC LPS) was mixed with 0-2 ug
of semipurified RNA. (c) Densitometric evaluation of 0-2 ug R N A + 10 ug E. coli O H L B 4 (EC LPS), (d) Estimated amounts of RNA in the LPS samples isolated from
CyanoHABs (samples A-U) based on densitometric analysis of the agarose gels, expressed as % of sample weight (w/w). Model DNA and RNA were isolated from a fish cell
line NKA-1.
(a) (b)
80S
b U - T _
I Ininri
I
80-
3 60
40
1 I k 1 I
Fig. S9 Migration potential of keratinocytes. Confluent HaCaT cells were treated with 19 CyanoHAB-LPS
from 18 recreational water bodies (A-U) at a final concentration of 50 ug/ml for 4 days. Negative control was
untreated, and LPS from E. coli (EC) was used as a positive control. Wound closure of the scratched monolayer
was assessed after 12 h. Cell proliferation was either (a) unaffected or (b) stopped by mitomycin c pre-treatment
(1 ug/ml, 2 h). (c) Selection of representative images. Data are expressed as the mean ± SD. Data were statistically
analyzed by unpaired /-test.
(a)
* I Aril A
A.C f- <b O 0 <o << G ^ ^ -t- v >}• ^ O <> <?- <b ^
(b)
ZO-1
OcclurJJn
Claudin 4
Claudin 2
mM • M M
Vincijlin — —
H-actin
control EC G A D S B E F
195 kDa
• 59 kDa
(e)
| 4 -
c
tII
'I0 i
^ 1
(f)
o
Q
o
0.2
0.6
0.4"JO
. O - 1
- ^ " -
T
control E C G A B E F
Fig. S10 Caco-2 monolayer integrity. Co-culture of differentiated Caco-2 cells and human PBMC were treated
with 19 CyanoHAB-LPS from 18 recreational water bodies (A-U) at a final concentration of 100 ug/mlfor4 days.
Negative control was untreated, and LPS from E. coli (EC) was used as a positive control. Simple cultures
of differentiated Caco-2 cells were used as a control of the model (control Caco). (a) Trans-epithelial electric
resistance (TEER) of the Caco-2 monolayer, n = 4. Expression of tight junction proteins: (b) representative blots
and relative concentration of (c) claudin 2, (d) claudin 4, (e) occludin, and (f) zonula occludens 1 expressed
as the optical density of the bands, n = 4 - 8. Data are expressed as the mean ± SD. Data were statistically analyzed
by unpaired f-test.
DETAILED METHODOLOGY
LPS isolation
1 g of lyophilized cyanobacterial biomass was mixed with 50 ml of MilliQ water and sonicated using an ultrasonic
bath (10 min). The suspension was then heated to 68 °C, mixed with 50 ml of pre-warmed 90% phenol, and stirred
for 20 min at 68 °C. After cooling to 4 °C, the mixture was centrifuged (5 630 g, 30 min, 4 °C), and the supernatant
(aqueous phase with LPS) was collected. The phenol layer was re-extracted with 50 ml of MilliQ water, and pooled
supernatants were purified by dialysis (48 h) using cellulose membranes (33x21 mm, Sigma-Aldrich, USA,
D9652) against MilliQ water (11) containing toluene (10 ul/1) to prevent bacterial contamination. After 24 h of
dialysis, the water was changed and dialysis continued for another 24 h. The dialyzed extracts were centrifuged
(5 630 g, 30 min, 4 °C), collected as supernatants and lyophilized. The resulting semi-purified freeze-dried extract
of LPS was resuspended in 3.75 ml of 0.1M Tris-HCl buffer (pH 7.4) containing 25 ug/ml ribonuclease A (RNase,
Sigma-Aldrich, R4642) and incubated for 16 h at 37 °C. Next, 3.75 ml of 90% phenol in 0.1M Tris-HCl was added,
vortexed, and incubated for 4 min at room temperature (RT). The solution was centrifuged (18 410 g, 15 min, RT),
the aqueous phase was separated and purified for 48 h by dialysis for a second time (see above) and then
lyophilized. The purified LPS (freeze-dried powder) was weighed to assess the content of LPS in the biomass and
kept at -20 °C. Isolated LPS were dissolved (at a concentration of 10 mg/ml) in MilliQ water for characterization
by SDS-PAGE or agarose electrophoresis, or in PBS with 0.1% BSA (v/v, Sigma-Aldrich) for PyroGene™ rFC
or in vitro cell assays.
SDS-PAGE and agarose electrophoresis of CyanoHAB-LPS
Isolated LPS samples (10 ug) were diluted with MilliQ water and 4x Laemmli loading buffer (Bio-Rad, Hercules,
CA, USA, 161-0747) containing 2-mercaptoethanol (final concentration 357 mM), and heated for 10 min at 65 °C.
Commercially available LPS purified from Escherichia coli serotype O l 11:B4 (Sigma-Aldrich, L2630) was used
as a positive control (1 or 10 ug per lane). The 4% (w/v) acrylamide stacking gels were used for SDS-PAGE
in combination with 10-20% (w/v) gradient separating gels (1 mm thickness) prepared from 40% (w/v)
acrylamide/bis-acrylamide solution, 37.5:1 (Serva, Heidelberg, Germany) using a Mini-Protean gel casting and
electrophoresis system (Bio-Rad). Electrophoresis was conducted at 130 V, and the gels were then washed
with deionized water, and stacking gels were removed.
For the silver staining of LPS, the washed gels were fixed with 40% (v/v) ethanol-5% (v/v) acetic acid overnight
at RT. Then, the fixed gels were oxidized with 0.7% (w/v) periodic acid in 40% (v/v) ethanol-5% (v/v) acetic acid
for 5 min, and washed with deionized water (2x5 min and 1x10 min). The silver staining solution was freshly
prepared by mixing 28 ml of 0.1 M sodium hydroxide, 2.3 ml of 24% (w/w) ammonium hydroxide, 5 ml of 20%
(w/v) silver nitrate (Sigma-Aldrich) and filled up to 150 ml with deionized water. The gels were incubated in the
staining solution for 10 min and then washed with deionized water (2x5 min and 1x10 min). The developing
process was carried out with 50 ug/ml citric acid in 0.05% (v/v) formalin for approximately 2-3 min, followed by
washing with deionized water to achieve suitable levels of background staining. Finally, the gels were fixed with
5% (v/v) acetic acid for 1 min, washed, and documented under white light (Alliance Q9 Advanced system, Uvitec
Cambridge, UK).
LPS were stained with the Pro-Q™ Emerald 300 Lipopolysaccharide Gel Stain Kit (ThermoFisher Scientific,
Waltham, M A , USA) according to the manufacturer's protocol. Washed gels were fixed with 50% (v/v) methanol-
5% (v/v) acetic acid for 45 min at RT and then washed with 3% (v/v) acetic acid for 2x15 min. LPS were oxidized
for 30 min with a periodic acid-based oxidizing solution supplied with the kit. The gels were washed again with
3% (v/v) acetic acid (3x15 min) and then incubated for 90 min with lx Pro-Q™ Emerald 300 staining buffer.
Before UV-light visualization and documentation (Alliance Q9 Advanced), the gels were washed with 3% acetic
acid for 2x20 min. To visualize protein contamination of LPS, the gels were restained overnight with SYPRO®
Ruby (ThermoFisher Scientific), washed with 10% methanol with 7% acetic acid (v/v) for 30 min and finally with
deionized water for 2x5 min, prior documentation (Alliance Q9 Advanced).
Agarose gel electrophoresis of LPS and nucleic acids was conducted as reported previously (Sychrová et al., 2022,
https://doi.Org/10.1016/j.etap.2022.103869), using 1% gel, TAE buffer, and Bioline HyperLadder™ 50 bp
molecular weight marker (MWM). The gels were stained using a 1% solution of ethidium bromide and documented
with the Alliance Q9 Advanced system (Cambridge, UK). LPS from Escherichia coli serotype O l 11:B4 (SigmaAldrich)
and its mixture with DNA or RNA from a fish cell line NKA-1 (isolated as reported previously in
Součková et al., 2023, https://doi.org/10.1016/j.aquatox.2023.106517) were used as positive controls and to create
a calibration curve for the RNA amount in the mixture with LPS. Densitometrie analysis was conducted using
ImageJ software (Schneider et al., 2012, https://doi.org/10.1038/nmeth.2089).
DNA extraction
Briefly, freeze-dried biomass (4-10 mg) was resuspended in 0.5 ml of 0.15 M NaCl/0.1 M EDTA solution
and homogenized using three freeze-thaw cycles with liquid nitrogen. Samples were then centrifuged (7 200 g,
10 min), the supernatant was discarded, and the pellet was resuspended in 0.5 ml of TE buffer. Then, 1 ul of RNase
(10 mg/ml) was added and the samples were incubated at 37 °C for 1 h. Afterwards, 100 ul of lysozyme (50 mg/ml)
was added, and the samples were incubated at 37 °C for 30 min. After incubation, 10 ul of protein kinase K
(50 mg/ml) and 2% (final concentration) sodium dodecyl sulfate (SDS) were added, and samples were incubated
at 55 °C for 1 h. Then, selective precipitation was performed by adding 150 ul of 5M NaCl to the tubes followed
by 0.1 volumes (of total volume) of 10% cetyltrimethylammonium bromide stock solution, mixed by inversion,
and incubated at 65 °C for 10 min. For purification (to allow for protein precipitation), 1 volume of chloroform
was added, and tubes were incubated on ice for 30 min. Then, samples were centrifuged (7 200 g, 10 min, 4 °C),
and the supernatant was transferred to fresh tubes, mixed with 0.6 volumes of isopropanol, and incubated overnight
at 4 °C. Following this, samples were centrifuged (16 000 g, 30 min, 4 °C), the isopropanol supernatant was
discarded, and the pellet was washed twice with 1 ml of 70% ethanol. Finally, samples were centrifuged (16 000
g, 30 min, 4 °C), the supernatant discarded, the pellet air-dried, and then resuspended in 30 ul of TE buffer.
The concentration and quality of the extracted DNA were checked via NanoDrop (Thermo Scientific) and agarose
gel electrophoresis. The isolated DNA underwent further processing through qPCR.
Cell lines media
Caco-2 medium: high-glucose Dulbecco's Modified Eagle's Medium (DMEM; Gibco) supplemented
with 10% heat-inactivated low endotoxin fetal bovine serum (FBS; v/v, PAA), 1% non-essential amino acids (v/v,
Gibco), ImM sodium pyruvate (Gibco), 2mM L-glutamine (Gibco), and 100 U/ml of penicillin and 100 jig/ml
of streptomycin (Pen/Strep; Gibco)
HaCaT medium: high-glucose D M E M supplemented with 10% FBS, 2 mM L-glutamine, and Pen/Strep
PBMC isolation
30 ml of blood was collected to 300 ul of 0.5M EDTA. 6ml aliquots of blood were centrifuged (400 g, 25 min,
20 °C, the lowest acceleration, no brake; Rotina 420, Hettich Zentrifugen, Germany), buffy coat was collected,
pooled into 3 tubes, and washed twice with 8 ml of PBS+lmM EDTA (150 g, 10 min, 20 °C, the lowest
acceleration, no brake). The washed buffy coat was diluted by ImM EDTA/PBS up to 8 ml. Each 2 ml
of suspension was carefully layered on 2 ml of Histopaque 1077 and centrifuged (400 g, 30 min, 18 °C, the lowest
acceleration, no brake). PBMC layers were collected, pooled, and washed with 5 ml of ImM EDTA/PBS (500 g,
10 min, 20 °C). Washed PBMC were resuspended in PBS and counted (CASY Cell Counter, Innovatis). PBMC
were diluted in RPMI (Gibco) with 20% FBS up to concentration 2*106
cells/ml, seeded in 24 plates, 500 ul per
well, and placed in the incubator. After 2 h, cells were extremely gently washed with pre-warmed PBS and 800 ul
of fresh RPMI with 20% FBS was added. Cells prepared in this way were used either for the assembly of a coculture
or for direct LPS treatment.
Treatment
Confluent HaCaT cells were treated with LPS at a final concentration of 50 ug/ml. Positive control was treated
with£. coli O H L B 4 LPS (Sigma-Aldrich) diluted in 0.1% BSA in PBS, while the negative control cells received
no treatment. After 1 day of exposure, the medium was collected, and the cells were washed with ice-cold PBS
and prepared for western blot.
Co-culture was assembled by putting an insert with differentiated Caco-2 cells into the well with PBMC. LPS was
applied to the apical part of the insert (on Caco-2 cells) at a final concentration of 100 ug/ml. The positive control
consisted of a co-culture treated with E. coli LPS, while the negative control group was a co-culture without any
treatment. Additional controls included an insert with Caco-2 cells without PBMC, either untreated or treated with
E. coli LPS, and untreated PBMC without the insert. After 4 days of exposure, the media were collected for further
analyses, the Caco-2 cells were washed with ice-cold PBS and prepared for western blot, PBMC were collected
and prepared for flow cytometry.
PBMC were treated with LPS at a final concentration of 1 ug/ml. Positive control was treated with E. coli LPS,
and negative control was untreated. After 4 days of exposure, the medium was collected for further analyses,
the cells were collected and prepared for flow cytometry.
Lactate dehydrogenase assay
80 ul of the sample was mixed with the reaction mixture in a ratio of 1:1 and incubated RT. The absorbance was
measured at 492 nm using a SPECTRA Sunrise microplate reader (Tecan, Mannedorf, Switzerland). Untreated
cells lysed by supplier-provided lysis buffer were used as a positive control.
Flow cytometry
The medium from PBMC was collected and centrifuged to capture all floating cells. PBMC in wells were incubated
with 500 ul of 10 mM EDTA/PBS (10 min, 37 °C). Detached cells were moved to a flow cytometry tube. Another
500 ul of 10 mM EDTA/PBS was added to the wells, and any remaining cells were scraped with a cell scraper and
moved to the tube with the rest of the respective sample. Cells were washed with PBS and blocked with 150 ul
of PBS with 10% human serum (15 min, 4 °C). Human serum was a heat-inactivated pool of sera from 4 healthy
donors prepared in our lab. Blocked cells were stained with antibodies against CD14 (APC anti-human CD14,
2109040) and CD16 (FITC anti-human CD16, 2110030, both SONY Biotechnology), 3 ul of each per sample
(20 min, 4 °C). Stained cells were washed with cold PBS, the cells were re-suspended in 150 ul of cold PBS and
kept on ice until measured on a flow cytometer (BD FACSVerse, BD Biosciences, USA). Immediately before
measurement, 2 ul of propidium iodide (1 mg/ml) was added to the samples as a viability marker.
ELISA
Kits from Invitrogen and RaD Systems were used for the analyses. Invitrogen: Human CCL2 Uncoated ELISA
Kit, Human IL-6 Uncoated ELISA Kit, Human IL-8 Uncoated ELISA Kit, Human IL-10 Uncoated ELISA Kit,
Human TNFa Uncoated ELISA Kit; RaD Systems: Human CCL20 DuoSet ELISA. The absorbance was measured
using a SPECTRA Sunrise microplate reader.
Western blot
The proteins were separated by SDS polyacrylamide gel electrophoresis using 7.5% gel (ZO-1), 10% gel (filaggrin,
involucrin), 12.5% gel (claudin 4, occludin) or 15% gel (claudin 2). Primary antibodies against human ZO-1
(1:1000; 33-9100, Thermo Fisher Scientific), occludin (1:1000, sc-133256), claudin 2 (1:1000, sc-293233),
claudin 4 (1:1000, sc-376643), filaggrin (1:500, sc-66192) and involucrin (1:200, sc-53361, all Santa Cruz
Biotechnology, USA) were used. The total level of vinculin (1:1000, #13901), GAPDH (1:1000, #2118S, both
Cell signalling technology), or |3-actin (1:5000, sc-47778, Santa Cruz Biotechnology) was detected as a loading
control. The secondary antibodies conjugated with HRP were diluted 1:2000, anti-mouse IgG (#7076) or antirabbit
IgG (#7074, both Cell signalling technology), respectively. The immunoreactive bands were detected by an
ECL detection reagent kit (Pierce, USA) using Amersham Imager 680 (GE HealthCare, USA). Relative protein
levels were quantified by ImageJ software (Schneider et al., 2012, https://doi.org/10.1038/nmeth.2089), and the
level of the protein of interest was normalized to the respective loading control.