Cell, Inflammation,
Wound healing
Cell
Mitochondrial function/dysfunction
ROS
Hypoxia
Lysosomal function/dysfunction
Cell death
2
Mitochondria
̶ production of ATP for cellular energy needs
̶ metabolism of amino acids
̶ regulation of the redox state of cells
̶ heme synthesis
̶ differentiation and activation processes of immune cells
̶ crucial functions in the cell death program
3
Mitochondrial network
Mitochondrial fusion and fission
̶ processes occur in response
to various extra- or
intracellular changes
̶ changes in nutrient supply,
energy or redox status,
during cell differentiation in a
cell-type dependent manner
4
Front. Cell Dev. Biol., 2017
Mitochondrial fusion and fission
̶ response to metabolic/pathogenic conditions
̶ FUSION = autonomously integrate
̶ 1. fusion of the outer membrane between 2
adjacent mitochondria
̶ mediated by mitofusin 1 and 2
̶ 2. fusion of the inner membrane
̶ cardiolipin, dynamin-like GRPase optic atrophy (OPA)
̶ important for maintenance of mitochondrial DNA
integrity and cellular respiration5
Research Reports in Clinical Cardiology 2014(default):111
Mitochondrial fusion and fission
̶ FISSION
̶ important to allow inheritance of
mitochondria by daughter cells during cell
division
̶ when damaged and deleted - damaged
mitochondria facilitates their removal by
mitophagy
6
Research Reports in Clinical Cardiology 2014(default):111
Mitochondria and ROS
̶ production of reactive oxygen species
̶ generated by mitochondria via the electron transport chain
̶ byproduct during mitochondrial energy production,
consequence of fatty acid β-oxidation, exposure to
radiation, light, metals, and redox drugs
̶ ROS function:
̶ second messengers in various signaling pathways
in immune cells: Ca2+-NFAT signaling pathway, which is critical in T cell
activation.
̶ ROS can also damage bacterial pathogens, but
̶ if produced excessively - damage the
producing cell or neighboring cells.
7
Int. J. Mol. Sci. 2019, 20(18), 4407
Sites of ROS production
̶ mitochondrial ROS (mROS)
are basically produced as
byproducts of this bioenergetic
metabolism
̶ Cyt c, cytochrome c; MAO, monoamine
oxidase; NOX-4, NADPH oxidase 4;
VDAC, voltage-dependent anion channel
8
Oxidative stress
̶ result of imbalance between
ROS production and antioxidation
̶ pathological defects in living organisms
̶ cancer, atherosclerosis, neurological diseases, aging, and
diabetes, damage of cellular components (DNA, RNA, lipids, and proteins)
̶ non-enzymatic defense:
̶ flavonoids, vitamins (A, C, and E), and glutathione
̶ enzymatic antioxidants:
̶ Superoxide dismutase (SOD), superoxide reductase, catalase, glutathione peroxidase,
glutathione reductase, peroxiredoxins (Prdxs), and thioredoxins (Trx)
9
Int. J. Mol. Sci. 2019, 20(18), 4407
Effect of mitochondria in immune reaction
̶ mitochondrial DAMPs
̶ in extracellular space and circulation.
̶ mitochondrial proteins
̶ FRP receptors - production of chemoattractants.
̶ mitochondrial ROS
̶ intracellular signaling, damage cells.
̶ mitochondrial ATP and cardiolipin
̶ activate the NLRP3 inflammasome or TLR4 production
of pro-inflammatory cytokines.
̶ mitochondrial DNA
̶ activate TLR9, NLRP3 inflammasomes or the
cGAS pathway - production of pro-inflammatory
cytokines.10
Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease
Volume 1866, Issue 10, 1 October 2020, 165845
Mitochondria – induction of immune response
̶ mitochondrial danger associated molecules (DAMPs)
that resemble structures of bacterial derived pathogen
associated molecular patterns (PAMPs)
̶ mitochondrial DAMPS - mitochondrial DNA with hypomethylated CpG
motifs, specific lipid present in prokaryotic bacteria and mitochondria, i.e.
cardiolipin.
̶ via DAMPs mitochondria guide the immune response
̶ mitochondrial DAMPs - negative impact- released by
damaged cells, without the presence of an infection undesired
inflammatory response, resulting in tissue
damage and organ dysfunction
̶ after a trauma11
Nature Education 3(9):15
PRRs as "microbial sensors"
̶ to detect a set of evolutionarily conserved
molecules found in a variety of pathogens PAMPs,
expressed in a wide variety of
microorganisms, including those that do not
cause disease.
̶ In patients with severe infections such as sepsis, PAMPs are the
major external "stimulators" of the inflammatory response.
̶ DAMPs are capable of initiating an
inflammatory response similar to that produced
by PAMPs, no microbial infections present.
̶ DAMPs in SIRS (internal or endogenous "stimulators")
Definujte zápatí – název prezentace nebo pracoviště12
Mitochondria originated following
endocytosis of a proteobacteria by
another prokaryotic cell.
Nature Reviews Genetics 5, 123–135
Mitochondrial dysfunction
13
Nature Reviews Molecular Cell Biology volume 17, pages308–321(2016)
Mitochondrial dysfunction
14
Mitochondrial dysfunction and heart
15
European Society of Cardiology Journals 23. 10. 2018
Mitochondrial dysfunction
̶ mutations in mitochondrial DNA and
oxidative stress – risk factor for
neurodegenerative diseases
̶ strong evidence that mitochondrial dysfunction
occurs early and acts causally in disease
pathogenesis
̶ disease-specific proteins interact with
mitochondria
16
Nature volume 443, pages787–795(2006)
Mitochondrial dysfunction
17
Oxidative Medicine and
Cellular Longevity 2019
Mitochondrial dysfunction
18
Oxidative Medicine and Cellular Longevity 2019
Mitochondria and hypoxia
19
Nature Reviews Cancer volume 2, pages38–47(2002)
Mitochondria and hypoxia
̶ Acute/chronic
̶ decreased flux through the
tricarboxylic acid (TCA) cycle
̶ activity of the electron transport
chain (ETC)
̶ hypoxia-induced ROS, …
̶ low vs. no oxigen
20
Cell death
Definujte zápatí – název prezentace nebo pracoviště21
Current Aging Science Volume 10 , Issue 1 , 2017
Cell death
Signals from the lysosome: a control centre
for cellular clearance and energy metabolism
̶ degradation and recycling of cellular
waste
̶ via endocytosis and autophagy
̶ Lysosomal and autophagy dysfunction in
̶ lysosomal storage diseases (LSDs) and
̶ common neurodegenerative diseases
̶ defective cellular clearance and
accumulation of toxic material
23
Nature Reviews Molecular Cell Biology volume 14, pages283–296(2013)
Defective cellular clearance in
neurodegenerative diseases
̶ loss-of-function mutations of
genes involved in the
lysosomal–autophagic pathway
̶ gain-of-function mutations of
aggregate-prone proteins
̶ enhanced protein aggregation and
impairment of lysosomal–autophagic
pathways
24
Nature Reviews Molecular Cell Biology volume 14, pages283–296(2013)
Lysosoms and starvation
̶ limited nutrient availability
and mediates the starvation
response by regulating lipid
catabolism
̶ used also by tumor cells
25
Nature Reviews Molecular Cell Biology volume 14, pages283–296(2013)
Inflammation
Inflammation
Acute fase reaction
Cytokines, chemokines
26
Inflammation
 Inflammation is the response of living tissue to damage.
The acute inflammatory response has 3 main functions:
The affected area is occupied by a transient material called
the acute inflammatory exudate. The exudate carries proteins,
fluid and cells from local blood vessels into the damaged area
to mediate local defences.
If an infective causative agent (e.g. bacteria) is present in the
damaged area, it can be destroyed and eliminated by
components of the exudate.
The damaged tissue can be broken down and partially
liquefied, and the debris removed from the site of damage.
Inflammation
̶ In all these situations, the inflammatory stimulus will be met by a series of
changes in the human body; it will induce production of certain cytokines and
hormones, which in turn will regulate haematopoiesis, protein synthesis and
metabolism.
̶ Most inflammatory stimuli are controlled by a normal immune system. The
human immune system is divided into two parts which constantly and closely
collaborate - the innate and the adaptive immune system.
Inflammation – innate system
̶ The innate system reacts promptly without specificity and memory.
Phagocytic cells are important contributors in innate reactivity together with
enzymes, complement activation and acute phase proteins.
̶ When phagocytic cells are activated, the synthesis of different cytokines is
triggered. These cytokines are not only important in regulation of the innate
reaction, but also for induction of the adaptive immune system. There,
specificity and memory are the two main characteristics.
Inflammation – adaptive immune response
̶ In order to induce a strong adaptive immune response, some lymphocytes
must have been educated to recognize the specific antigen on the antigenpresenting
cell (APC) in context of self major histocompatibility molecules.
The initial recognition will mediate a cellular immune reaction, production of
antigen-specific antibodies or a combination of both. Some of the cells, which
have been educated to recognize a specific antigen will survive for a long
time with the memory of the specific antigen intact, rendering the host
"immune" to the antigen.
Non-specific Immunity
Differences between innate (non-specific)
and specific (adaptive) immunologic
reaction of organism
Specific Immunity
̶ Response is antigen-independent
̶ There is immediate maximal response
̶ Non-antigen-specific
̶ Exposure results in no immunologic memory
̶ Response is antigen-dependent
̶ There is a lag time between exposure and
maximal response
̶ Antigen-specific
̶ Exposure results in immunologic memory
Causes of Inflammation
̶ infectious inflammatory stimuli (viruses, bacteria, fungi and parasites)
̶ by non-infectious inflammatory stimuli, as in rheumatoid arthritis and graftversus
host disease
̶ by tissue necrosis as in cancer
̶ by burns and toxic influences caused by drugs or radiation
Clin Microbiol Rev. 2012 Oct; 25(4): 609–634.
Danger recognition by the innate immune
system
̶ PRRs such as TLR, NLR, and
RAGE sense danger associated
with infection via recognition of
evolutionarily conserved PAMPs
on pathogens or sterile injury via
recognition of DAMPs.
̶ Activation of cell surface or
intracellular PRRs leads to
intracellular signaling and
inflammatory responses
Definujte zápatí – název prezentace nebo pracoviště34
Mucosal Immunology volume 9, pages567–582 (2016)
Contribution of DAMPs to inflammatory
response in IBD
̶ nonapoptotic cell death,
mucosal oxidative
stress, and deregulation
of homeostatic
pathways lead to
overwhelming release
of DAMPs, creating a
proinflammatory milieu
Definujte zápatí – název prezentace nebo pracoviště35
Mucosal Immunology volume 9, pages567–582 (2016)
Types of inflammation
̶ Acute
̶ Chronic
̶ Local
̶ Systemic
General and local clinical symptoms of the
acute phase reaction
General symptoms
fever
tachycardia
hyperventilation
tiredness
Loss of appetite
Local symptoms
calor
rubor
dolor
tumor
functio laesa
Local Inflammation
The acute inflammatory response
involves three processes:
̶ changes in vessel caliber (=
vasodilation) and, consequently, slower
blood flow
̶ increased vascular permeability and
formation of the fluid exudate
̶ formation of the cellular exudate by
emigration of the neutrophil polymorphs
into the extravascular space.
Early Stages
The steps involved in the acute inflammatory response are:
• Small blood vessels adjacent to the area of tissue damage initially become dilated with
increased blood flow, then flow along them slows down.
• Endothelial cells swell and partially retract so that they no longer form a completely intact
internal lining.
• The vessels become leaky, permitting the passage of water, salts, and some small proteins
from the plasma into the damaged area (exudation). One of the main proteins to leak out is
fibrinogen.
• Circulating neutrophil polymorphs initially adhere to the swollen endothelial cells
(margination), then actively migrate through the vessel basement membrane (emigration),
passing into the area of tissue damage.
• Later, small number of blood monocytes (macrophages) migrate in a similar way, as do
Iymphocytes.
Systemic manifestation of inflammation
• Increase of body temperature (fever)
• Acute phase reaction
Justin Root
Acute phase reaction
The acute phase reaction is the body's first-line inflammatory defense system,
functioning without specificity and memory, and in front of, and in parallel with,
the adaptive immune system.
In the acute phase reaction, several biochemical, metabolic, hormonal and cellular
changes take place in order to fight the stimulus and re-establish a normal functional
state in the body.
An increase in the number of granulocytes will increase the phagocytotic capacity,
an increase in scavengers will potentiate the capability to neutralize free oxygen
radicals, and an increase in metabolic rate will increase the energy available for
cellular activities, despite a reduced food intake.
Some of these changes can explain the symptoms of an acute phase reaction, which
are typically fever, tiredness, loss of appetite and general sickness, in addition to local
symptoms from the inducer of the acute phase.
Canadian Medical Association Journal 182(18):E834-8
Systemic effects of acute/chronic
inflammation
 Pyrexia
Polymorphs and macrophages produce compounds known as endogenous
pyrogens, which act on the hypothalamus to set the thermoregulatory mechanisms
at a higher temperature. Release of endogenous pyrogen is stimulated by
phagocytosis, endotoxins and immune complexes.
 Constitutional symptoms
Constitutional symptoms include malaise, anorexia and nausea. Weight loss is
common when there is extensive chronic inflammation.
 Local or systemic Iymph node enlargement commonly accompanies inflammation,
while splenomegaly is found in certain specific infections (e.g. malaria, infectious
mononucleosis).
Mol Cancer Res; 11(9); 967–72. ©2013 AACR.
Systemic effects of inflammation
Haematological changes
̶ Increased erythrocyte sedimentation rate. An increased erythrocyte sedimentation rate is a non-specific
finding in many types of inflammation.
̶ Leukocytosis.
̶ Neutrophilia occurs in pyogenic infections and tissue destruction;
̶ eosinophilia in allergic disorders and parasitic infection;
̶ Iymphocytosis in chronic infection (e .g. tuberculosis), many viral infections and in whooping cough; and
̶ monocytosis occurs in infectious mononucleosis and certain bacterial infections (e.g. tuberculosis, typhoid).
̶ Anaemia.
̶ blood-loss in the inflammatory exudate (e.g. in ulcerative colitis),
̶ haemolysis (due to bacterial toxins), and
̶ 'the anemia of chronic disorders' due to toxic depression of the bone marrow.
Amyloidosis
̶ Longstanding chronic inflammation (for example, in rheumatoid arthritis, tuberculosis and
bronchiectasis), by elevating serum amyloid A protein (SAA), may cause amyloid to be deposited in
various tissues resulting in secondary (reactive) amyloidosis.
Difference between anaemia
of chronic disease and irondeficiency
anaemia
Anemia of Chronic Diseases Iron Deficiency Anemia
Seru Iron Reduced Reduced
Transferrin Reduced to normal Increased
Transferrin Saturation Reduced Reduced
Ferritin Normal to increased Reduced
Soluble transferrin receptor Normal Increased
Cytokine level Increased Normal
Hepcidin Increased Reduced
Bone marrow iron stores Normal to increased Reduced
Ery Normal, microcytes Microcytes
J Clin Invest. 2007;117(7):1755-1758. https://doi.org/10.1172/JCI32701.
Acute phase proteins
̶ Induction of the acute phase reaction - changes in
synthesis of many proteins in the liver
̶ measured in plasma.
̶ Regulation of protein synthesis - at the level of both
transcription (DNA, RNA) and translation to protein.
̶ The cells have intricate systems for up- and down-regulation of protein synthesis,
initiated by a complex system of signals induced in the acute phase reaction.
Biotechnology Advances Volume 34, Issue 3, May–June 2016
Acute phase proteins
Function related to
̶ limiting the negative effects of the acute phase stimulus
or
̶ repair of inflammatory induced damage.
Examples are enzyme inhibitors limiting the negative effect of enzymes released
from neutrophils, scavengers of free oxygen radicals, increase in some transport
proteins and increased synthesis and activity of the cascade proteins such as
coagulation and complement factors.
The protein synthesis may be upregulated even if plasma levels are normal, due
to increased consumption of acute phase proteins. N Engl J Med 1999; 340:448-454
DOI: 10.1056/NEJM199902113400607
Function Positive acute phase protein Increase up to
Protease inhibitors Alfa 1-antitrypsin
Alfa 1-antichymotrypsin
4 x
6 x
Coagulation proteins (serin
proteinases)
fibrinogen
prothrombin
factor VIII
plasminogen
8 x
Complement factors C1s
C2b
C3, C4, C5
C9
C5b
2 x
Transport proteins haptoglobin
hemopexin
ferritin
8 x
2 x
4 x
Scavenger proteins ceruloplasmin 4 x
Others alfa1-acid glycoprotein (orosomukoid)
serum amyloid A protein
C-reactive protein
4 x
1000 x
1000 x
47
Biochemistry and physiology of the acute
phase reaction
̶ CRP is a major acute phase protein acting mainly
through Ca2+-dependent binding to, and
clearance of, different target molecules in
proteins, having evolved almost unchanged from
primitive to advanced species.
microbes, cell debris and cell nuclear material.
In an acute phase reaction there may be a more than 1000-fold increase in the
serum concentration of CRP. CRP is regarded as an important member of the family
of acute phase, having evolved almost unchanged from primitive to advanced species.
Nature Reviews Rheumatology volume 7, pages282–289 (2011)
C-reactive protein
Most functions of CRP are easily understood in the context of the
body's defenses against infective agents.
• The bacteria are opsonized by CRP and increased phagocytosis
is induced.
• CRP activates complement with the split product being
chemotactic, increasing the number of phagocytes at the site of
infection. Enzyme inhibitors protect surrounding tissue from the
damage of enzymes released from the phagocytes.
• CRP binds to chromatin from dead cells and to cell debris which
are cleared from the circulation by phagocytosis, either directly
or by binding to Fc-, C3b- or CRP-specific receptors. Platelet
aggregation is inhibited, decreasing the possibility of thrombosis.
• CRP binds to low density lipoprotein (LDL) and may clear LDL
from the site of atherosclerotic plaques by binding to cell surface
receptors on phagocytic cells. N Engl J Med 1999; 340:448-454
DOI: 10.1056/NEJM199902113400607
Biologically active products of complement
activation
Chemotactic factors
C5a and MAC (membrane attack complex C5b67) are both chemotactic. C5a is also a potent
activator of neutrophils, basophils and macrophages and causes induction of adhesion molecules
on vascular endothelial cells.
Opsonins
C3b and C4b in the surface of microorganisms attach to C-receptor (CR1) on phagocytic cells and
promote phagocytosis.
Other biologically active products of C activation
Degradation products of C3 (iC3b, C3d and C3e) also bind to different cells by distinct receptors
and modulate their function.
Biologically active products of complement
activation
Activation of complement results in the production of several biologically active
molecules which contribute to resistance, anaphylaxis and inflammation.
Kinin production
C2b generated during the classical pathway of C activation is a prokinin which
becomes biologically active following enzymatic alteration by plasmin.
Anaphylotoxins
C4a, C3a and C5a (in increasing order of activity) are all anaphylatoxins, which
cause basophil/mast cell degranulation and smooth muscle contraction.
Negative proteins of acute phase
̶ Decreases in albumin, transferrin, cortisol-binding globulin, transthyretin and vitamin A
binding protein temporarily lead to an increased supply of free hormones, which usually bind
to these proteins.
̶ Transthyretin (pre-albumin binding thyroxine, transports thyroid hormones from the plexus
choroideus to the cerebrospinal fluid) inhibits the production of IL-1β by monocytes and
endothelial cells. Its decline can thus be considered as a pro-inflammatory mechanism.
These changes in blood protein profiles appear to be partly related to muscle starvation and
catabolism. It is also an offer of amino acids for the production of positive acute phase
proteins.
52
Cytokines
̶ generic name for a diverse group of soluble
proteins and peptides
̶ act as humoral regulators at nano- to
picomolar concentrations under normal or
pathological conditions
̶ modulate the functional activities of individual
cells and tissues.
̶ These proteins also mediate interactions
between cells directly and regulate processes
taking place in the extracellular environment.
Nature Reviews Immunology volume 19, pages205–217(2019)
Cytokine network
This term essentially refers to the extremely complex interactions of cytokines
by which they induce or suppress their own synthesis or that of other
cytokines or their receptors, and antagonize or synergies with each other in
many different and often redundant ways.
These interactions often resemble Cytokine cascades with one cytokine
initially triggering the expression of one or more other cytokines that, in turn,
trigger the expression of further factors and create complicated feedback
regulatory circuits.
Mutually interdependent pleiotropic cytokines usually interact with a variety of
cells, tissues and organs and produce various regulatory effects, both local
and systemic.
International Journal of Oral Science volume 11, Article number: 30 (2019)
“cytokine storm”
̶ extreme increase in inflammatory
cytokines, including IL-1β, IL-2, IL-
6, IL-7, IL-8, IL-10, granulocytecolony
stimulating factor (G-CSF),
granulocyte macrophage-colony
stimulating factor (GM-CSF),
interferon-inducible protein-10
(IP10), monocyte chemotactic
protein 1 (MCP1), macrophage
inflammation protein-1α, IFN-γ, and
TNF-α.
56
Signal Transduction and Targeted Therapy volume 5, Article number: 128 (2020)
57
Journal of Infection 80 (2020) 607-613
Chemokines
Generic name given to a family of pro-inflammatory activationinducible
cytokines. These proteins are mainly chemotactic for
different cell types.
All chemokines possess a number of conserved cysteine
residues involved in intramolecular disulfide bond formation,
which allows chemokines to be grouped into families according
to the presence or absence of one or more conserved cysteine
residues.
Chemokines
̶ According to their mode of expression and function, chemokines
have been categorized as inflammatory chemokines and
homeostatic chemokines.
̶ Inflammatory chemokines are expressed usually by leukocytes or related cells only upon cell
activation. These factors mediate emigration of leukocytes.
̶ Homeostatic chemokines are expressed constitutively and are involved usually in relocation
of lymphocytes or other cell types.
̶ Dual-function chemokines can act as inflammatory cytokines or homeostatic cytokines.
Wound healing
61
Wound healing
̶ Wound healing is the process of repair that follows injury to the
skin and other soft tissues.
̶ Healing is the interaction of a complex cascade of cellular events
that generates resurfacing, reconstitution, and restoration of the
tensile strength of injured tissue.
̶ Under the most ideal circumstances, healing is a systematic
process, traditionally explained in terms of 3 classic phases:
inflammation, proliferation, and maturation.
Wound healing
̶ The inflammatory phase:
̶ a clot forms and cells of inflammation
debride injured tissue.
̶ The proliferative phase:
̶ epithelialization, fibroplasia, and
angiogenesis occur; additionally, granulation
tissue forms and the wound begins to
contract.
̶ The maturation phase:
̶ Collagen forms tight cross-links to other
collagen and with protein molecules,
increasing the tensile strength of the scar.
I. Inflammatory Phase
Immediate to 2-5 days
̶ Hemostasis
̶ Vasoconstriction
̶ Platelet aggregation
̶ Clot formation
̶ Inflammation
̶ Vasodilation
̶ Phagocytosis
̶ Fibrin products
̶ essential to wound healing and
̶ primary component of the wound matrix into which inflammatory cells, platelets,
and plasma proteins migrate.
̶ Removal of the fibrin matrix impedes wound healing.
II. Proliferative Phase
2 days to 3 weeks
̶ B) Granulation
̶ Fibroblasts lay bed of collagen - scaffold for migration
and further fibroblast proliferation
̶ C) Contraction
̶ Wound edges pull together to reduce defect
̶ D) Epithelialization
̶ Crosses moist surface
̶ Cell travel about 3 cm from point of origin in all
directions
vascular network is also re-established
through angiogenesis
- main regulator of angiogenesis is the vascular
endothelial growth factor (VEGF) family, which
includes VEGF-A, VEGF-B, VEGF-C, VEGF-D
and placental growth factor (PlGF)
Journal of Theoretical Biology 459, 2018, 1-17
III. Maturation Phase
̶ During the maturation phase, fibroblasts leave the wound and
collagen is remodeled into a more organized matrix.
̶ Tensile strength increases for up to one year following the injury.
While healed wounds never regain the full strength of uninjured
skin, they can regain up to 70 to 80% of its original strength.
Acute vs. chronic wound
̶ Acute wounds
̶ adequate angiogenesis promotes
re-epithelialization, fibroblasts'
proliferation, and neutrophils' antiinfection
activities.
̶ Chronic wounds
̶ persistent local bacterial infections
hinder the formation of novel blood
vessels. The restricted
angiogenesis hampers fibroblasts'
proliferation and the neutrophils'
anti-infection activities.
68
Front. Bioeng. Biotechnol., 11 June 2020
69
Wound healing in DM
International Journal of Research in Medical Sciences 2017: 5(10):4206
Thank you for attention
Regenerative medicine
72 Nature volume 453, pages314–321(2008)