Cytokine Signaling in Splenic Leukocytes from
Vaccinated and Non-Vaccinated Chickens after
Intravenous Infection with Salmonella Enteritidis
Marta Matulova, Hana Stepanova, Frantisek Sisak, Hana Havlickova, Marcela Faldynova, Kamila Kyrova,
Jiri Volf, Ivan Rychlik*
Veterinary Research Institute, Brno, Czech Republic
Abstract
In order to design a new Salmonella enterica vaccine, one needs to understand how naive and immune chickens interact
differently when exposed to S. enterica. In this study we therefore determined the immune response of vaccinated and nonvaccinated
chickens after intravenous infection with Salmonella enterica serovar Enteritidis (S. Enteritidis). Using flow
cytometry we showed that 4 days post infection (DPI), counts of CD4 and B-lymphocytes did not change, CD8 and cd Tlymphocytes
decreased and macrophages and heterophils increased in the spleen. When vaccinated and non-vaccinated
chickens were compared, only macrophages and heterophils were found in significantly higher counts in the spleens of the
non-vaccinated chickens. The non-vaccinated chickens also expressed higher anti-LPS antibodies than the vaccinated
chickens. The expression of interleukin (IL)1b, IL6, IL8, IL18, LITAF, IFNc and iNOS did not exhibit any clear pattern in the cells
sorted from the spleens of vaccinated or non-vaccinated chickens. Only IL17 and IL22 showed a differential expression in
the CD4 T-lymphocytes of the vaccinated and non-vaccinated chickens at 4 DPI, both being expressed at a higher level in
the non-vaccinated chickens. Due to a similar IFNc expression in the CD4 T-lymphocytes in both the vaccinated and nonvaccinated
chickens, and a variable IL17 expression oscillating around IFNc expression levels, the IL17:IFNc ratio in CD4 Tlymphocytes
was found to be central for the outcome of the immune response. When IL17 was expressed at higher levels
than IFNc in the non-vaccinated chickens, the Th17 immune response with a higher macrophage and heterophil infiltration
in the spleen dominated. However, when the expression of IL17 was lower than that of IFNc as in the vaccinated chickens,
the Th1 response with a higher resistance to S. Enteritidis infection dominated.
Citation: Matulova M, Stepanova H, Sisak F, Havlickova H, Faldynova M, et al. (2012) Cytokine Signaling in Splenic Leukocytes from Vaccinated and NonVaccinated
Chickens after Intravenous Infection with Salmonella Enteritidis. PLoS ONE 7(2): e32346. doi:10.1371/journal.pone.0032346
Editor: Dipshikha Chakravortty, Indian Institute of Science, India
Received December 16, 2011; Accepted January 27, 2012; Published February 24, 2012
Copyright: ß 2012 Matulova et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work has been supported by the projects MZE0002716202 of the Czech Ministry of Agriculture, AdmireVet project CZ.1.05/2.1.00/01.0006 –
ED0006/01/01 from the Czech Ministry of Education and project 524/09/0215 from the Czech Science Foundation. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: rychlik@vri.cz
Introduction
Non-typhoid salmonellosis together with campylobacteriosis
belong among the two most important causes of human
gastrointestinal disorders in developed countries. The most
important reservoirs of Salmonella enterica for humans are found in
farm animals, poultry and pigs in particular. Since it is believed
that a decrease in the prevalence of S. enterica in farm animals will
result in a lower incidence of human salmonellosis, measures on
how to decrease S. enterica prevalence in farm animals are
continuously being sought.
One of the possible measures targeted at the decrease of S.
enterica prevalence in poultry is vaccination. However, in order to
design new a Salmonella vaccine for chickens with significantly
improved performance over the current ones, one needs to
understand how both naive and immune chickens interact when
infected with S. enterica. Earlier studies were focused either on the
characterization of cellular infiltrates or cytokine signaling in the
infected tissues, mostly in the cecal wall, liver or spleen of chickens
orally infected with Salmonella. It is therefore known that
heterophils form the initial cellular infiltrate [1,2] followed by
the infiltration of macrophages and T-lymphocytes [3]. Recently,
significant changes in cd T-lymphocytes have been described in
chickens after S. Enteritidis infection [4].
Leukocytes infiltrating the site of infection communicate in
a controlled fashion by cytokine release. The cytokines produced
after S. enterica infection include proinflammatory cytokines
and chemotactic chemokines such as IL1b, IL6 or IL8, Th1
cytokines such as IFNc and Th17 cytokines such as IL17 or IL22
[5]. A lower level of cellular infiltrate and a lower level of
cytokine expression were commonly observed in the tissues of
chickens that had been vaccinated prior to the infection than
in those exposed to the infection for the first time [6–8].
However, since cytokine signaling is usually determined by realtime
PCR using RNA/cDNA isolated from whole target tissue,
information about the contribution of individual cellular
subpopulations in chickens is essentially unavailable. And if
there were attempts to determine cytokine signaling in particular
cell population of chickens, e.g. cd T-lymphocytes [9], this was
performed alone not providing sufficiently general overview on
the immunological processes occurring in chickens after S.
enterica infection.
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The whole effort towards understanding the chicken immune
response to vaccination and (re)infection is also adversely affected
by the fact that although newly-hatched chickens are highly
sensitive to S. enterica, older, non-vaccinated chickens are quite
resistant to the infection [6]. However, if a new vaccine is being
tested, this commonly requires a primary vaccination on the day of
hatching, revaccination 2–3 weeks later and challenge an
additional 2–3 weeks later. Following the above vaccination
scheme, the challenge at around 6 weeks of age becomes an issue
because this is time when even the non-vaccinated birds are
already relatively resistant to oral challenge [6]. This is why in this
study we focused on the immune response of vaccinated and nonvaccinated
chickens after intravenous infection with S. Enteritidis.
Using flow cytometry we first characterized the dynamics of
leukocyte (macrophages, heterophils, B-lymphocytes, CD8, CD4
and cd T-lymphocytes) infiltrates in the spleen and in FACS sorted
leukocyte subpopulations we next determined Th1 and Th17
cytokine expression. This allowed us to characterize roles of
individual leukocyte subpopulations during primary and secondary
exposure of chickens to S. Enteritidis infection.
Results
S. Enteritidis challenge
Over 106
CFU of S. Enteritidis per gram of spleen was recorded
in the non-vaccinated chickens at 4 days post infection (DPI).
Counts of S. Enteritidis more than 106 lower were observed in
chickens which had been vaccinated before the challenge.
Fourteen days after infection, S. Enteritidis counts decreased in
the spleens of both vaccinated and non-vaccinated chickens to
104
CFU/g (Fig. 1A).
The intravenous mode of administration of the S. Enteritidis
used for challenge resulted in a high antibody production. Four
days post intravenous infection, higher antibody levels were
recorded in the non-vaccinated birds when compared with the
vaccinated birds. Fourteen days post infection, a further increase
in antibody production was recorded in all infected birds with the
non-vaccinated birds exhibiting significantly higher antibody titers
than the vaccinated birds (Fig. 1B).
Cellular infiltrates after i.v. challenge determined by flow
cytometry
CD4 lymphocytes, i.e. CD4+ CD82 TCR12, and Blymphocytes
represented the subpopulation counts which did not
change significantly in the spleen of chickens after the challenge
(see Figs. 2 and 3).
Double positive CD4 and CD8 lymphocytes, i.e. CD4+ CD8+
TCR12, were represented at relatively low levels in the spleen.
Similar to CD4+ lymphocytes, this subpopulation only weakly
responded to the infection at 4 DPI. However, this subpopulation
significantly increased in the non-vaccinated chicken at 14 DPI
(Fig. 3).
CD8 T-lymphocytes (CD8+ CD42 TCR12) and cd Tlymphocytes
(TCR1+, CD42 and CD8 either positive or negative)
decreased in response to S. Enteritidis infection. CD8 T-lymphocytes
decreased in counts 4 DPI whilst at 14 DPI their counts returned to
those before the challenge. Unlike CD8, cd T-lymphocytes
decreased in number at both time points after the challenge.
However, the response characteristics of CD8 and cd T-lymphocytes
did not differ in the vaccinated or non-vaccinated chickens (Fig. 3).
Macrophages and heterophils were the two subpopulations
which increased in response to the infection. Monocytes/
macrophages increased 4 DPI and decreased nearly to the levels
present in the non-infected chickens at 14 DPI. On the other
hand, heterophils remained at a high level both at 4 and 14 DPI.
Four days post infection, the infiltration of macrophages and
heterophils was significantly higher in the non-vaccinated chickens
than in vaccinated chickens (Fig. 3).
Cytokine expression in the spleen, purified leukocytes
and sorted cellular subpopulations
There were 3 different groups formed by 9 cytokines tested in
this study. The first group comprised of IL1b, IL6, IL8 and IL18
Figure 1. Intravenous infection of chickens with S. Enteritidis. Panel A, S. Enteritidis counts in the spleens of vaccinated and non-vaccinated
chickens, 4 and 14 days after intravenous challenge, respectively. Panel B, serological response to the infection. nv-ni, non-vaccinated and noninfected
chickens; v-i-4, vaccinated and infected and 4 days post challenge; nv-i-4, non-vaccinated and infected and 4 days post challenge etc. Table
below – t-test comparison of biological relevant groups, ni, non-infected chickens; vi4, vaccinated and infected and 4 days post challenge; ni4, nonvaccinated
and infected and 4 days post challenge etc. ns – non-significant difference, * P,0.05, ** P,0.01, *** P,0.001.
doi:10.1371/journal.pone.0032346.g001
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which were produced predominantly by macrophages. Especially
for IL1b and IL8, the contribution of any other cell type was
marginal. The second group was formed by iNOS and LITAF
which were expressed in all the leukocyte subpopulations at similar
levels. The last group comprised IFNc, IL17 and IL22, production
of which was dependent on T-lymphocytes (Fig. 4–6).
The expression of IL1b, IL6, IL8 and IL18 was dependent
mainly on macrophages followed by B-lymphocytes. These
cytokines remained expressed at the same level in macrophages
after S. Enteritidis infection although their expression significantly
increased in the spleen. Except for IL1b at 4 DPI, expression
profiles of these cytokines did not differ significantly between the
vaccinated and non-vaccinated chickens. However, the data for
IL1b and IL8 must be taken with care because when comparing
their expression in the spleen, total leukocytes and macrophages
(considering also the size of macrophage population) it is clear that
these two cytokines must have been induced in macrophages
during labeling and cell sorting (Fig. 4).
Expression of LITAF and iNOS was not restricted to any
leukocyte subpopulation although LITAF was slightly more
transcribed in macrophages and B-lymphocytes and iNOS was
slightly more expressed in all T-lymphocyte subpopulations.
Furthermore, LITAF was not inducible in the sorted leukocytes
whilst iNOS was induced in macrophages in response to the
infection at 4 DPI. When the vaccinated and non-vaccinated
chickens were compared, iNOS was expressed at a significantly
higher level in the spleen and the total number of leukocytes of
non-vaccinated chickens at 4 DPI. However, there were no
significant differences in iNOS expression among any of the sorted
subpopulations originating from the vaccinated or non-vaccinated
chickens (Fig. 5).
The expression of IL17, IL22 and IFNc was dependent on the
T-lymphocytes and all these cytokines were clearly induced after S.
Enteritidis challenge. IL17 and IL22 were induced mainly at 4
DPI and decreased at 14 DPI whilst IFNc remained at an
increased transcription rate both at 4 and 14 DPI (Fig. 6). The
expression of all these 3 cytokines in CD8 and cd T-lymphocytes
did not significantly differ between the vaccinated and nonvaccinated
chickens. Even the expression of IFNc in CD4 Tlymphocytes
did not differ between the vaccinated and nonvaccinated
chickens. The only difference in cytokine signaling
between the vaccinated and non-vaccinated chicken was the
expression of IL17 and IL22 in CD4 T-lymphocytes at 4 DPI.
These two cytokines were expressed in significantly higher levels in
CD4 T-lymphocytes originating from the non-vaccinated chickens
than in those from the vaccinated chickens (Fig. 6).
Figure 2. Gating strategy for the characterization of cellular infiltrates in the spleen and for the sorting of leukocyte
subpopulations. The leukocytes were gated based on the CD45 expression (P1) and only CD45+ cells were included in the sorting and analyses.
Sorted populations were followed: CD4 T-lymphocytes (P4+P3), CD8+ T-lymphocytes (P5), cd T-lymphocytes (P6), monocytes/macrophages (P7), Blymphocytes
(P8) and heterophils (P9).
doi:10.1371/journal.pone.0032346.g002
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Discussion
In this study we were interested in the identification of
differences in the immune response between vaccinated and
non-vaccinated chickens to S. Enteritidis infection. Unlike the
majority of previous studies, for the characterization of immune
response we used sorted leukocyte subpopulations from the spleens
of intravenously infected chickens. Perhaps by adopting the lessfrequent,
intravenous mode of infection, which resulted in a severe
systemic infection in both the vaccinated and non-vaccinated
chickens, the differences in cytokine signaling between the
vaccinated and non-vaccinated chickens were not great despite
the fact that the vaccinated chickens were 10 times more resistant
than the non-vaccinated ones, although this was only evident at 4
DPI (Fig. 2A). The i.v. infection itself can be understood as a
certain limit of this study. However, a more relevant oral infection
of 42-day-old chickens followed by an analysis of splenic
subpopulations would likely result in insignificant differences since
splenomegaly as an indicator of cellular infiltrates into the spleen is
obvious after i.v. infection but is essentially absent after oral
infection of 42-day-old chickens [10]. In addition, a similar
immune response can be expected also in the spleens of very
young chickens infected in hatcheries, in which colonization of the
spleen and liver is quite common.
Four days after the infection, an increase in macrophages and
heterophils was observed in the spleens of the infected chickens.
The changes in the cellular composition in the spleens of the
infected chickens were therefore in clear contradiction to the
response of the Balb/C mice to the infection with the same S.
Enteritidis strain [11]. As all the flow cytometry calculations were
performed within CD45 positive cells considered as 100%, the
increase of macrophages and heterophils should automatically
lead to a decrease in all other subpopulations. This may explain
the decrease of CD8 and cd T-lymphocytes. However, as Blymphocytes
and CD4 T-lymphocytes did not change, counts of
these two subpopulations had to increase in their absolute counts
albeit at lower rate than the macrophages and heterophils. In the
case of B-lymphocytes and CD4 T-lymphocytes, the increase in
their absolute counts in the spleen due to the infiltration from
circulation is as likely as their clonal expansion after antigen
stimulation. The clonal expansion of B-lymphocytes is supported
also by a high antibody production (Fig. 2B). We also noticed that
double positive CD4 and CD8 lymphocytes increased in the
spleens of non-vaccinated chickens at 14 DPI. Certain authors
proposed that such subpopulation might represent the memory T
cells [12]. Their increase at 14 DPI but not at 4 DPI would
support this although the absence of their increase in already
vaccinated chickens cannot be explained by any current model.
Cytokines with a clear response to the infection and/or a
different response in the vaccinated and non-vaccinated chickens
included iNOS, IFNc, IL17 and IL22. The difference in iNOS
expression between the vaccinated and non-vaccinated chickens
was exhibited more in the spleen and total leukocytes whilst none
of the sorted subpopulations exhibited the same expression profile.
This apparent contradiction can be explained by a higher
infiltration of macrophages into the spleen of the non-vaccinated
chickens than in the vaccinated chickens (Fig. 3). This result also
shows that the increase in iNOS expression in different tissues after
Figure 3. Relative representation of leukocyte subpopulations in the spleens of infected chickens 4 and 14 days post infection. Light
blue columns - vaccinated and infected chickens 14 DPI, blue columns vaccinated and infected chickens 4 DPI, green columns – non-infected
chickens, red columns non-vaccinated and infected chickens 4 DPI, pink columns - non-vaccinated and infected chickens 14 DPI. Table below – t-test
comparison of biologically relevant groups, ni, non-infected chickens; vi4, vaccinated and infected and 4 days post challenge; ni4, non-vaccinated and
infected and 4 days post challenge etc. ns – non-significant difference, * P,0.05, ** P,0.01, *** P,0.001.
doi:10.1371/journal.pone.0032346.g003
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Figure 4. Expression of IL1, IL6, IL8 and IL18 in sorted splenic leukocytes after intravenous S. Enteritidis challenge. Light blue columns
- vaccinated and infected chickens 14 DPI, blue columns vaccinated and infected chickens 4 DPI, green columns – non-infected chickens, red columns
non-vaccinated and infected chickens 4 DPI, pink columns - non-vaccinated and infected chickens 14 DPI. Table below – t-test comparison of
biologically relevant groups, ni, non-infected chickens; vi4, vaccinated and infected and 4 days post challenge; ni4, non-vaccinated and infected and 4
days post challenge etc. ns – non-significant difference, * P,0.05, ** P,0.01, *** P,0.001.
doi:10.1371/journal.pone.0032346.g004
Figure 5. Cytokine expression of LITAF and iNOS in sorted splenic leukocytes after intravenous S. Enteritidis challenge. Light blue
columns - vaccinated and infected chickens 14 DPI, blue columns vaccinated and infected chickens 4 DPI, green columns – non-infected chickens, red
columns non-vaccinated and infected chickens 4 DPI, pink columns - non-vaccinated and infected chickens 14 DPI. Table below – t-test comparison
of biologically relevant groups, ni, non-infected chickens; vi4, vaccinated and infected and 4 days post challenge; ni4, non-vaccinated and infected
and 4 days post challenge etc. ns – non-significant difference, * P,0.05, ** P,0.01, *** P,0.001.
doi:10.1371/journal.pone.0032346.g005
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Salmonella infection [13–15] can be caused both by its induction in
macrophages (Fig. 3) and by an influx of leukocytes from
circulation to the site of inflammation, as we proposed earlier
based on a high iNOS expression in the blood of healthy adult
hens [16].
The only significantly different response in any of the sorted
leukocytes of the vaccinated or non-vaccinated chickens was the
expression of two cytokines characteristic of the Th17 immune
response, namely IL17 and IL22. For IL17 in CD4 Tlymphocytes
we noticed that its expression levels oscillated
around the expression levels of IFNc, a central cytokine of the
Th1 branch of the immune response. In the CD4 T-lymphocyte
populations from each of the non-vaccinated chickens, the
expression of IL17 was always higher than the expression of
IFNc whilst in the CD4 T-lymphocyte populations from each of
the vaccinated chickens, the expression of IL17 always dropped
below the expression of IFNc (Fig. 7). At 4 DPI, CD4 Tlymphocytes
of the vaccinated chickens therefore produced
nearly 3 times more IFNc than IL17 while CD4 T-lymphocytes
from the non-vaccinated chickens produced 1.76 more IL17
than IFNc. Nothing like this was observed in the CD8 or cd Tlymphocytes
which also induced IFNc, IL17 and IL22 expression
in response to S. Enteritidis infection (Figs. 4 and 5). The Th1
immune response with IFNc as its central cytokine is generally
considered as the most important for resistance to Salmonella
infection and consistent with this, there were lower counts of S.
Enteritidis in the spleens of vaccinated chickens (Fig. 2A). On the
other hand, the characteristics of IL17 signaling and the Th17
branch of immune response include inflammation and leukocyte
infiltration. This is in agreement with higher heterophil counts,
higher macrophage counts and also higher IL17, IL22 and iNOS
expression observed in the spleen of the non-vaccinated chickens
(Figs. 3 and 6).
The fact that the IFNc:IL17 ratio and that the Th1 branch of
immune response is central for resistance to S. Enteritidis infection
comes also from data at 14 DPI. At this time point, the expression
of IL17 and IL22 dropped significantly when compared with 4
DPI and the differences in their expression between CD4 Tlymphocytes
from the vaccinated and non-vaccinated chickens
disappeared. As the expression of IFNc did not decline that
rapidly, the IFNc:IL17 ratio increased to values between 12 and
35 in the CD4 T-lymphocytes from the non-vaccinated and
Figure 6. Expression of IFNc, IL17 and IL22 in sorted splenic leukocytes after intravenous S. Enteritidis challenge. Light blue columns vaccinated
and infected chickens 14 DPI, blue columns vaccinated and infected chickens 4 DPI, green columns – non-infected chickens, red columns
non-vaccinated and infected chickens 4 DPI, pink columns - non-vaccinated and infected chickens 14 DPI. Table below – t-test comparison of
biologically relevant groups, ni, non-infected chickens; vi4, vaccinated and infected and 4 days post challenge; ni4, non-vaccinated and infected & 4
days post challenge etc. ns – non-significant difference, * P,0.05, ** P,0.01, *** P,0.001.
doi:10.1371/journal.pone.0032346.g006
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vaccinated chickens, respectively. In both the cases, this polarized
the immune response towards a Th1 response and increased the
resistance to S. Enteritidis infection as documented by the decrease
in S. Enteritidis counts in the spleen and a reduction in
macrophage infiltration of the spleen in both the vaccinated and
non-vaccinated chickens. These results are also in total agreement
with previous experimental data [7,15]. Although the results
presented in this study were obtained with a rather small number
of chickens, in conclusion, we have shown that i) the vaccinated
chickens responded to S. Enteritidis infection by the Th1 branch of
immune response ii) the non-vaccinated chickens responded by the
Th17 branch of immune response at 4 DPI, however, at 14 DPI
they re-oriented their immune response towards the Th1 branch,
iii) the only cellular subpopulation controlling the polarization was
formed by CD4 T-lymphocytes and iv) the polarization in CD4 Tlymphocytes
was achieved by the regulation in expression of IL17
and IFNc. Finally, it would be interesting to compare the
IFNc:IL17 ratio in chickens vaccinated with different live,
attenuated Salmonella vaccines, both to confirm or exclude whether
the observed phenomenon is specific for the vaccination with the
SPI1 mutant or whether it is common to all attenuated vaccine
strains. In the latter case it would be interesting to correlate the
IFNc:IL17 ratio with a real protection determined by bacterial
counts in the spleen of vaccinated and non-vaccinated chickens
after the challenge.
Materials and Methods
Ethics Statement
The handling of animals in the study was performed in
accordance with current Czech legislation (Animal protection and
welfare Act No. 246/1992 Coll. of the Government of the Czech
Republic). The specific experiments were approved by the Ethic
Committee of the Veterinary Research Institute (permit number
48/2010) followed the Committee for Animal Welfare of the
Ministry of Agriculture of the Czech Republic (permit number
MZe 1226).
Bacterial strain and chicken line
S. Enteritidis strain 147, a clone spontaneously resistant to
nalidixic acid, was used in this study. The construction of isogenic
mutant with completely removed Salmonella Pathogenicity Island
1 (SPI-1) used as a live attenuated vaccine for the vaccination of
egg laying ISA Brown chickens (Hendrix Genetics, Boxmeer,
Netherlands) was described earlier [1]. Immediately after transport,
3 chickens were sacrificed straight away and proved
Salmonella negative by culture.
Vaccination and infection
An eighteen-hour-old culture of the SPI-1 mutant grown
statically at 37uC in LB broth was used for oral vaccination of 6
one-day-old chickens. The chickens were vaccinated using an oral
gavage into the crop with 106
CFU in 0.1 ml of inoculum and
revaccinated with the same amount of vaccine on day 21 of life.
The challenge strain was prepared by growth in LB broth at
37uC for 18 hours, pelleting bacteria at 10 000 g for 1 min and
resuspending the pellet in the same volume of PBS as was the
original volume of LB broth. Three weeks after the revaccination,
i.e. on day 42 of chicken’s life, the chickens were infected
intravenously into the wing vein with 107
CFU in 0.1 ml of wild
type S. Enteritidis. In addition to the vaccinated chickens, 6 nonvaccinated
chickens were intravenously challenged on day 42 of
life. Finally 3 non-vaccinated and non-infected chickens sacrificed
on day 46 of life were used as controls.
Intravenously infected chickens were sacrificed 4 and 14 days
post infection (DPI). At the end of the experiment, blood from
each bird was collected for serological tests. During necropsy,
approx. 100 mg of spleen was taken for bacteriological analysis,
30 mg was taken into RNALater (Qiagen) for subsequent RNA
purification and the rest of the spleen was used for the isolation of
splenic leukocytes.
Cell sorting by flow cytometry
The spleens were collected into ice cold RPMI 1640 medium
(Sigma) and during all additional steps the cells were washed or
kept on ice. The cell suspensions were prepared by pressing the
tissue through a fine nylon mesh. The erythrocytes were removed
by cold hemolytic solution (8.26 g of NH4Cl, 1 g of KHCO3 and
0.037 g of EDTA per liter of distilled water) and the cells were
washed twice in 30 ml of cold PBS. After the last washing step, the
splenic leukocytes were resuspended in PBS and a small aliquot
(26106
cells) was transferred into 500 ml of Tri Reagent (MRC) to
purify the RNA from the total leukocytes. The remaining
leukocytes were used for surface marker staining. In total 46107
of cells were stained in each sample. The first panel of primary
antibodies (all Southern Biotech, Alabama, USA) consisted of antiCD45:APC
(clone LT40), anti-CD4:FITC (clone CT-4), antiCD8a:SPRD
(clone CT-8) and anti-TCR1:PE (clone TCR-1).
The second panel of antibodies consisted of anti-CD45:APC (clone
LT40), anti-monocyte/macrophage:FITC (clone KUL01) and
anti-Bu-1:PE (clone AV20). A mouse IgG1 isotype control for
each fluorochrome was also used. After 20 min of incubation and
subsequent washing in PBS, the cells were subjected to sorting
using a FACSAria instrument (BD Biosciences) with a 4 channel
sorter. The cells were collected in PBS containing 20% of bovine
Figure 7. Transcription of IFNc and IL17 in T-lymphocytes of vaccinated or non-vaccinated chickens 4 days post intravenous
challenge. A, B, C - individual chickens vaccinated or non-vaccinated chickens. The most right panel shows polarization towards IFNc or IL17
transcription ratio in CD4 T-lymphocytes of vaccinated and non-vaccinated chickens, respectively. Squares, transcription of IL17 and triangles,
transcription of IFNc.
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serum, pelleted by centrifugation and lysed in 500 ml of Tri
Reagent. A small aliquot from each sample was left for purity
analysis. The purity of sorted populations was: CD8+, 96.761.4
(mean%6SD); CD4+, 94.162.1; cdTCR+, 93.562.6; B-lymphocytes
92.463.1; Monocytes/Macrophages, 89.963.0. Heterophils
were sorted as well, however this population lost its typical FSC/
SSC properties and purity of this population was under 60%.
Because of this, heterophils were not included in the real-time
PCR quantification of the cytokine response. The relative
representation of each population was analyzed using FACSDiva
software (BD Biosciences) with only CD45+ positive cells included
in the analysis. A general gating strategy is shown in Fig. 2.
RNA purification and reverse transcription
Fifty ml of bromoanisol was added to the sorted cells in 500 ml of
Tri Reagent and the samples were vigorously shaken for 10 s. The
samples were centrifuged at 4uC for 10 min at 10 000 g, 500 ml of
the upper aqueous phase was collected and mixed with an equal
volume of 70% ethanol and this mixture was applied to RNeasy
purification columns (Qiagen). Washing and RNA elution was
performed exactly as recommended by the manufacturer. The
concentration and purity of RNA was determined spectrophotometrically
(Nanodrop, Agilent) and the RNA was immediately
reverse transcribed into cDNA using MuMLV reverse transcriptase
(Invitrogen) and oligo dT primers. After reverse transcription,
the cDNA was diluted 10 times with sterile water and saved at
220uC prior real-time PCR.
Real-time PCR
Real-time PCR was performed in 3 ml volumes in 384-well
microplates using QuantiTect SYBR Green PCR Master Mix
(Qiagen) and a Nanodrop pipetting station from Inovadyne for
PCR mix dispensing. Amplification of PCR products and signal
detection were performed using a LightCycler II (Roche) with an
initial denaturation at 95uC for 15 min followed by 40 cycles of
95uC for 20 s, 60uC for 30 s and 72uC for 30 s. Each sample was
subjected to real-time PCR in triplicate and the mean values of the
triplicates were used for subsequent analysis. The Ct values of
genes of interest were normalized (DCt) to an average Ct value of
three house-keeping genes (GAPDH, TBP and UB) and the
relative expression of each gene of interest was calculated as 22DCt
.
These expression levels were used for data analysis and are
presented in the figures as average 6 SD. All the primers
sequences have been described earlier [13].
ELISA detection of anti-Salmonella LPS antibodies
A commercial FLOCKSCREENTM
Salmonella Enteritidis Antibody
ELISA kit (x-OvO Limited) was used for the detection of
anti-LPS serum antibodies. ELISA was performed exactly as
recommended by the manufacturer except for the fact that the
sera were diluted from 1:10 up to 1:4000 using sample dilution
buffer to reach the absorbance which could be measured by
spectrophotometer, i.e. ranging from 0.2 to 1.8. Real absorbance
was then calculated knowing the read absorbance and particular
dilution.
Statistics
The data were analyzed by both parametric (t-test) and nonparametric
tests (Mann-Whitney U test) using Prism Graph Pad
Software (La Jolla, USA). Although the results of all statistical tests
were very similar (though not identical), the results of the t-test are
presented in the results section and in all the figures. In addition,
we show the statistics only for biologically relevant data - for
example, we do not show the results of the statistical comparison
between vaccinated&infected chickens 4 DPI and non-vaccinated&infected
chickens 14 DPI, etc. Similarly, since the expression of
some of the cytokines was clearly associated with particular cellular
subpopulations, we only present the statistical results for the
appropriate subpopulations.
Acknowledgments
The technical assistance of Klara Mollova from Babak Myeloma Group,
Department of Pathological Physiology, Faculty of Medicine, Masaryk
University, who assisted us with flow cytometry and cell sorting, is highly
appreciated. Authors also would like to thank Peter Eggenhuizen for his
English language corrections.
Author Contributions
Conceived and designed the experiments: MM IR. Performed the
experiments: HS FS HH MF KK JV. Analyzed the data: IR JV. Wrote
the paper: MM IR.
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