ARTICLE IN PRESS

Developmental and Comparative Immunology (2008) 32, 563–574

Available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/devcompimm

Gene expression profiling of chicken lymphoid cells
after treatment with Lactobacillus acidophilus
cellular components
Jennifer T. Brisbina,1, Huaijun Zhoua,b,c,1, Joshua Gongb,, Parviz Sabourb,
Mohammad Reza Akbaria, Hamid Reza Haghighia, Hai Yub, Anthony Clarked,
Aimie Jey Sarsona, Shayan Sharifa,
a

Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada N1G 2W1
Food Research Program, Agriculture and Agri-Food Canada, Guelph, Ontario, Canada N1G 5C9
c
Department of Poultry Science, Texas A&M University, College Station, TX, USA
d
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada
b

Received 19 July 2007; received in revised form 6 September 2007; accepted 18 September 2007
Available online 16 October 2007

KEYWORDS
Gene expression;
Chicken;
Cecal tonsils;
Microarray;
Immune system;
Lactobacillus;
TLR;
STAT pathway

Abstract
Lactobacillus acidophilus has been shown to exert immunostimulating activities in a
number of species, including the chicken. To examine the molecular mechanisms of this
phenomenon, we investigated spatial and temporal expression of immune system genes in
chicken cecal tonsil and spleen mononuclear cells in response to structural constituents of
L. acidophilus. Using a low-density chicken immune system microarray, we found that
cecal tonsil cells responded more rapidly than spleen cells to the bacterial stimuli, with
the most potent stimulus for cecal tonsil cells being DNA and for splenocytes being the
bacterial cell wall components. We also discovered that in both splenocytes and cecal
tonsil cells, STAT2 and STAT4 genes were highly induced. Given the close interactions
between cecal tonsil cells and commensal bacteria, we further examined the involvement
of STAT2 and STAT4 signaling pathways in cellular responses to bacterial DNA. Our results
revealed that the expression of STAT2, STAT4, IL-18, MyD88, IFN-a, and IFN-g genes were
up-regulated in cecal tonsil cells after treatment with L. acidophilus DNA.
& 2007 Elsevier Ltd. All rights reserved. 

Corresponding author. Tel.: +1 519 780 8027; fax: +1 829 2600. 
Also for correspondence. Tel.: +1 519 824 4120x54641;

fax: +1 519 824 5930.
E-mail addresses: GongJ@AGR.GC.CA (J. Gong),
Shayan@uoguelph.ca (S. Sharif).
1
These two authors contributed equally to this work.
0145-305X/$ - see front matter & 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.dci.2007.09.003

1. Introduction
Members of the intestinal microbiota, including commensal
bacteria, play an important role in the development and
regulation of the immune system. Commensal bacteria

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present in the intestine are in close contact with cells of the
gut-associated lymphoid tissues (GALT) and interactions
between the cells and commensal bacteria may modulate
the immune response [1]. The observed modulation of
immune response is thought to be mediated in part by the
interactions between Toll-like receptors (TLRs) and microbial ligands, collectively known as pathogen-associated
molecular patterns (PAMPs) [2,3]. PAMPs are small molecular
motifs of various microorganisms, such as bacteria, viruses,
protozoa, and fungi that are recognized by host TLRs as well
as other pattern recognition receptors. Various bacterial
PAMPs, including peptidoglycan and unmethylated CpG DNA,
from Gram-positive commensal bacteria modulate the
immune response by the alteration of cytokine gene
expression [4,5]. Cytokines are responsible for regulating
the functions of immune system cells through the modification of the transcriptional profile of a cell to increase or
decrease the expression of relevant genes. Alteration of
cytokine gene expression by commensal bacteria is
mediated by a number of pathways, including the activation
of nuclear factor kB (NF-kB) and the signal transducer and
activator of transcription (STAT)-mediated signaling pathways [6]. The STAT pathway is responsible for converting
cytokine signals into gene expression profiles that regulate
the immune response in a number of different cell types.
Lactobacillus spp. are the predominant commensal
bacteria in the intestinal tract of young chicks [7,8] with
Lactobacillus acidophilus being one of the normal members
of the chicken microbiota [7]. L. acidophilus alone or in
combination with other probiotic bacteria has been shown
to stimulate the induction of systemic and mucosal antibody-mediated immune responses as well as to increase the
number of T lymphocytes in the cecal tonsils of chickens
[9–14]. Despite the evidence for immunomodulatory effects
of L. acidophilus, the underlying molecular mechanisms of
immunoregulatory activities of this organism in chickens are
unknown. The objective of the current study was, therefore,
to examine spatial and temporal expression of immune
system genes in chicken lymphoid tissues in response to
various PAMPs of L. acidophilus. We hypothesized that
bacterial DNA and cell wall components stimulate chicken
lymphoid cells and that cells of the GALT respond differently
to these PAMPs than cells of other lymphoid tissues.
Therefore, gene expression profiles in cecal tonsil cells
were compared to those of spleen cells after treatment with
L. acidophilus DNA and cell wall components. Furthermore,
given the fact that cecal tonsils are a major component of
the GALT in the chicken and are in close contact with the
intestinal microbiota, we set out to more specifically
examine the molecular mechanisms of cell activation by
Lactobacillus PAMPs.

2. Materials and methods
2.1. Preparation of spleen and cecal tonsil
mononuclear cells
For the microarray hybridization study, spleens and cecal
tonsils were harvested from a mixed sex population of
mature White Leghorns (Arkell Poultry Research Station,
University of Guelph, ON, Canada). For each of the four

J.T. Brisbin et al.
individual replicates, spleens and cecal tonsils were isolated
from seven birds and then pooled. For the real-time PCR
analysis of the STAT pathway, cecal tonsils were harvested
from mature female Barred Rock chickens (Arkell Poultry
Research Station). Chickens were euthanized by cervical
dislocation according to the University of Guelph Animal
Care Committee guidelines. Tissues were rinsed three times
in 1  Hank’s balanced salt solution (HBSS) and then minced
with sterile scalpels. The tissue was further disrupted with
the flat end of a 10-mL syringe plunger and strained through
a 40-mm nylon cell strainer to obtain a single-cell suspension. The suspension was then overlaid onto a Histopaque1077 (Sigma, Oakville, ON, Canada) density gradient and
mononuclear cells at the interface were collected and
washed twice in 1  HBSS and then suspended in RPMI (RPMI
containing 10% fetal bovine serum, 2% chicken serum,
0.146 g L-glutamine, 1.6 mM 2-mercaptoethanol, 200 U/mL
penicillin, 80 mg/mL streptomycin, 25 mg gentamicin, and
250 mg fungizone). Cells were counted by the trypan blue
dye exclusion assay before being resuspended in RPMI.

2.2. Isolation of cell envelope from L. acidophilus
To isolate the cell envelope fraction, L. acidophilus was
grown overnight at 37 1C in Lactobacilli MRS broth (Becton
Dickinson, Oakville, ON, Canada) under anaerobic conditions
(80% N2, 15% CO2, 5% H2). Bacterial cells were pelleted by
centrifugation (12,000g) for 10 min at 4 1C, washed twice
with PBS, and resuspended in 30 mL of 10 mM piperazineN,N0 -bis-2-ethanesulfonic acid (pH 6.8) with 0.85% (wt/vol)
NaCl. The cells were disrupted by three passages through a
French Press (10,000 p.s.i) at 0 1C followed by centrifugation
at 12,000g for 10 min at 4 1C. The supernatant containing the
cell envelope fraction was recovered by ultracentrifugation
(35,000 rpm) at 4 1C for 2 h in rotor 60 Ti (Beckman Coulter,
Mississauga, ON, Canada), followed by washing and resuspension in 10 mM N-2-hydroxyethylpiperazine-N0 -2-ethanesulfonic acid (pH 7.2).
To isolate the peptidoglycan-enriched cell envelope
extract (PECE), a method for isolation of peptidoglycan
sacculi, similar to other methods used for isolation of
peptidoglycan sacculi, encapsulating glycogen granules from
a rumen bacterium were used [15]. Briefly, an overnight
culture of L. acidophilus was added to an equal volume of
boiling 8% (wt/vol) SDS, incubated with agitation for 30 min,
cooled, and centrifuged. The insoluble fraction was washed
with equal volumes of 2% (wt/vol) SDS, 0.5 M NaCl, and
0.85% (wt/vol) NaCl, and the pellet resuspended in 10 mM
Tris HCl (pH 8.0) containing 5 mM MgCl2, 20 mg/mL bovine
pancreatic deoxyribonuclease I (type IV, Sigma), and
10 mg/mL bovine pancreatic ribonuclease A (type I-AS,
Sigma), and incubated at 37 1C for 30 min. The cell envelope
extract enriched in peptidoglycan sacculi was recovered by
centrifugation, washed, and lyophilized. The Branford assay
was used to verify that no contaminating proteins were
present in the extract.

2.3. Isolation of DNA from L. acidophilus
L. acidophilus cells from an overnight culture were pelleted
by centrifugation (3300g) and resuspended in 1 mL of 50 mM

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Gene expression profiling of chicken lymphoid cells
Tris–HCl (pH 8.0) containing 25% sucrose (wt/vol) and 0.5 mg
of lysozyme. After incubation at room temperature for
5 min, 200 mL of 0.25 M EDTA (pH 8.0) was added. The
bacterial suspension was then treated with 50 mg RNase at
37 1C for 30 min, followed with an overnight incubation with
4 mg proteinase K and 400 mL of 2% (wt/vol) SDS. The DNA
was isolated via a phenol chloroform procedure and was
precipitated with an equal volume of isopropanol. The string
of DNA was added to 100 mL of sterile TE (10 mM Tris–HCL,
1 mM EDTA, pH 8.0), and 1 mL of 95% EtOH was added and
incubated at 80 1C for 15 min. The DNA was pelleted,
washed with 70% ethanol, dried in vacuum dessicator, and
resuspended in 100 mL of sterile nuclease-free water. The
DNA was fragmented by vortexing for 2 min with 0.5 g sterile
0.5 and 0.1 mm zirconium beads.

2.4. In vitro stimulation of spleen and cecal tonsil
cells
In order to determine the optimal concentration of the
various bacterial components needed for stimulation of
spleen and cecal tonsil mononuclear cells, ranges of
concentrations of the structural components (5, 10, and
15 mg/mL of PECE; 5, 20, and 50 mg protein/mL of the cell
envelope fraction; 2.5, 5, and 10 mg/mL of DNA) were tested
by semi-quantitative RT-PCR (data not shown). Two proinflammatory cytokines, IL-6 and IFN-g, were used to
evaluate cell stimulation.
For the microarray hybridizations, each of the four
biological replicates had 1.5 mL of the spleen or cecal tonsil
mononuclear cell suspensions (1  107 cells/mL) isolated
from separate birds seeded into 24-well flat-bottom plates.
Four wells were left unstimulated per time point while two
wells were stimulated with each of the following: 10 mg/mL
DNA, 5 mg/mL PECE, and 20 mg/mL cell envelope fraction.
The cells from various treatments were incubated at 41 1C in
a humidified 5% CO2 environment and harvested for RNA
extraction at 3, 6, and 12 h post-stimulation.
For the real-time PCR analysis of the STAT pathway, each
of the five replicates isolated from separate birds had 200 mL
of cecal tonsil mononuclear cell suspensions (1.5  107
cells/mL) seeded into two wells of a 96-well flat-bottom
plate. One well was left unstimulated per time point while
the other well was stimulated with 10 mg/mL DNA. The cells
were incubated at 41 1C in a humidified 5% CO2 environment
and harvested for RNA extraction at 1, 3, 6, 12, and 18 h
post-stimulation.

2.5. cDNA array
The chicken immune system low-density array used for
hybridization in these studies was an extended version of
the array developed by our group [16]. The array has been
submitted to the National Center for Biotechnology Information Gene Expression Omnibus database under the
accession number GPL4585.

2.6. Array hybridizations
Total RNA was extracted using TRIzol Reagent (Invitrogen,
Burlington, ON, Canada) from stimulated and control cells

565
according to the manufacturer’s recommendations.
Total RNA was then treated with DNase using the DNA-free
Kit (Ambion, Austin, TX, USA) and 5 mg was transcribed
into cDNA using 100 ng of random hexamers and SuperScript
II Reverse Transcriptase (Invitrogen) using a 2:3 ratio of
5-(3-aminoallyl)-dUTP (Ambion) to unlabeled dTTP (0.5 mM
dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.3 mM dTTP, and 0.2 mM
5-(3-aminoallyl)-dUTP) for 2 h at 42 1C. Following reverse
transcription 6 mL of 0.5 M EDTA and 6 mL 1 N NaOH
were added, the mixture incubated at 65 1C for 15 min,
and neutralized with 16 mL of 1 M Tris (pH 7.4). The samples
were concentrated into approximately 5 mL with Microcon
YM-30 filters (Millipore, Mississauga, ON, Canada) and the
cDNA was then labeled with the Cy3 and Cy5 monofunctional
dyes (GE Healthcare Bio-Sciences, Baie d’Urfe, QC, Canada).
The unincorporated dye was removed with the QIAquick PCR Purification kit (Qiagen) and the labeled cDNA
samples were dried in a speed-vac. The pellet was then
dissolved in 12 mL of salt-based hybridization buffer (Ocimum Biosolutions, Indianapolis, IN, USA) and 1 mL of salmon
sperm DNA. The labeled samples were then heated at 95 1C
for 5 min and cooled to room temperature prior to
hybridization.
To account for any bias inherent to the fluorescent
dyes, two of the four replicates had the unstimulated
samples labeled with Cy3 and the stimulated samples
labeled with Cy5, and in the remaining two replicates,
the opposite labeling was performed. The labeled probes
were hybridized to the microarrays for 16 h at 65 1C.
Following hybridization, the slides were washed and
images acquired using a ScanArray Express instrument and
analyzed with the ScanArray Express software, version 3.0
(PerkinElmer, Montreal, QC, Canada). Subsequent to
normalization, the data were analyzed as previously
described [16] using a mixed model, which included
the effects of treatment, time point, dye, replicate, slide
and interactions between treatment and time point.
The criteria for differential expression were established
to include statistical significance reported at Po0.05,
a signal/noise ratio X2 and a fold-change of intensity
4 1.2.

2.7. Real-time RT-PCR validation of microarray
results
Subsets of genes showing differential expression during
microarray analysis were selected for validation by realtime RT-PCR. The cDNA synthesis on two of the
four replicates was performed with 5 mg of total RNA
using 100 ng of random hexamers and SuperScript II Reverse
Transcriptase (Invitrogen), according to the manufacturer’s
recommendations. Expression levels were normalized to
b-actin gene expression, which was used as an internal
housekeeping control. Real-time quantification was
performed in the LightCycler Instrument (Roche Diagnostics,
Laval, QC, Canada) using the SYBR green dye. PCR mixtures
(final volume of 20 mL) contained 2 mL of the LightCycler
FastStart DNA Master SYBR Green I (Roche Diagnostics),
2 mL of a 1:100 dilution of the cDNA, 3 mM MgCl2, and 0.5 mM
of each primer (Table 1). The cycling conditions included
an initial heat-denaturing step at 95 1C for 10 min, 40 cycles

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J.T. Brisbin et al.

Table 1

Real-time PCR primer sequences and annealing temperatures

Target mRNA

50 primer

30 primer

Annealing temperature (1C)

IgA
STAT2a
STAT4b
b-Actin
LFA-1c
K123
Caspase 3
IFN-gd
IFN-ae
MyD88f
IL-18g

50 -AATCCTTTCCACTCCCAACTGCTCG-30
50 -TGCAGGGAACAGAACAAATGAGGG-30
50 -ATGCTGGCAGAGAAACTTATGGGG-30
50 -CAACACAGTGCTGTCTGGTGGTA-30
50 -TGGGGCTTCAGTTTGTGCTGTGG-30
5-GATCTCACTGAGCTGCCTCTGGCT-30
50 -TGCAGAAGTCTAGCAGGGAAACCC-30
50 -CTGAAGAACTGGACAGAGAG-30
50 -ATCCTGCTGCTCACGCTCCTTCT-30
50 -AGAAGGTGTCGGAGGATGGT-30
50 -GAAACGTCAATAGCCAGTTGC-30

50 -CCATTTGTCTCTTGCCTTAC-30
50 -GGAAGCTGGCTCACATTAGA-30
50 -CGTACCCATCAATCCAGAGAGGAA-30
50 -ATCGTACTCCTGCTTGCTGATCC-30
50 -GCTCTATGTCCAGTTCTACC-30
50 -ATCCAGGCTGGGTTCTCTGGCT-30
50 -AAGTTTCCTGGCGTGTTCCTTCAG-30
50 -CACCAGCTTCTGTAAGATGC-30
50 -GGTGTTGCTGGTGTCCAGGATG-30
50 -GCTGGATGCTATTGCCTCGCTG-30
50 -TCCCATGCTCTTTCTCACAACA-30

58
58
58
58
58
58
58
64
64
58
64

a

Signal transducer and activator of transcription 2.
Signal transducer and activator of transcription 4.
c
Lymphocyte function-associated antigen-1.
d
Interferon-g.
e
Interferon-a.
f
Myeloid differentiation primary response 88.
g
Interleukin-18.
b

at 95 1C for 10 s, annealing as described in Table 1 for
each of the primers, and product elongation and signal
acquisition (single mode) at 72 1C for 10 s. Following
amplification, the melting curves were determined
in a three-segment cycle of 95 1C for 0 s, 65 C for 15 s, and
95 C for 0 S at the continuous acquisition mode. The
temperature transition rates were set at 20 1C/s except for
segment three of the melting curve analysis where it was set
to 0.1 1C/s.
The real-time quantitative PCR data were analyzed
using the 2DDCt method of Livak and Schmittgen [17].
Briefly, the relative level of each mRNA normalized to
the b-actin gene was calculated using the following
equation: fold change ¼ 2Ct target (control)–Ct target (treatment)/
2Ct b-actin (control)Ct b-actin (treatment).

2.8. Real-time RT-PCR analysis of the STAT2 and
STAT4 pathways
The cDNA synthesis was performed with 0.5 mg of total
RNA using oligo dT primers and SuperScript II Reverse
Transcriptase (Invitrogen) according to the manufacturer’s
instructions. Expression levels were normalized to
b-actin expression, which was used as an internal housekeeping control. Real-time quantification was performed
in the LightCycler 480 Instrument (Roche Diagnostics)
using the SYBR green dye. PCR mixtures (final volume of
20 mL) contained 10 mL of the LightCycler 480 SYBR Green I
Master Mix (Roche Diagnostics), 5 mL of a 1:25 dilution
of the cDNA, and 0.5 mM of each primer (Table 1). The
cycling conditions and data analysis were as described above. Treatments were considered statistically
significant when at Po0.05 using a paired two-tailed
T-test comparing the treated cells and the untreated
controls.

3. Results
3.1. Gene expression of chicken mononuclear cells
stimulated with L. acidophilus bacterial
components
Ranges of concentrations of the various bacterial components were tested in order to determine the optimal
concentration for cell stimulation. This was done based on
the highest level of cell activation exerted by the various
concentrations (data not shown). Subsequently, chicken
mononuclear cells isolated from spleen and cecal tonsils
were exposed to 10 mg/mL DNA, 5 mg/mL PECE, or 20 mg
protein/mL of the cell envelope fraction for 3, 6, and 12 h.
Of the 122 genes on the microarray, a total of 58 genes
(48%) had expression changes that were statistically
significant (Po0.05), had a signal to noise ratio 42, and
had a fold change over 1.2 in at least one of the
experimental parameters (accession numbers GSM146933–
GSM147004, NCBI Gene Expression Omnibus).
Genes belonging to the following families displayed
differential expression in spleen and cecal tonsil cells after
stimulation with bacterial PAMPs: chemokine and chemokine
receptors, cytokine and cytokine receptors, adhesion
molecules, surface molecules, immunoglobulin and T-cell
receptors, antigen processing, apoptosis, and transcription
and signal transduction.
At each of the three time points, between 0% and 25% of
the genes represented on the microarray showed differential expression (Tables 2 and 3). The least number of
differentially regulated genes in both spleen and cecal tonsil
mononuclear cells was observed at 3 h post-stimulation.
Cecal tonsil cells appeared to respond faster to various
treatments compared to spleen cells. The highest number of
genes that displayed significant changes in expression was

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Table 2 Statistically significant changes in gene expression in spleen mononuclear cells after stimulation with 10 mg/mL DNA,
5 mg/mL peptidoglycan enriched cell envelope, or 20 mg/mL the cell envelope fraction with Po0.05
Accession number

Gene name

P-values
3h

Induction following DNA stimulation
BU422799 est
Putative BAK (pro-apoptotic)
AF083029
Chicken caspase 3

0.002
0.000005
0.0000001
0.0004
0.04

0.000004

0.0000002
0.000000000004
0.0000002
0.000000006
0.0006
0.0000004
0.0000000008
0.00000007
0.004

0.03
0.005

0.000005
0.001
2E-22
0.000009
0.03
0.000000000001
0.000009
0.000002
0.0008

Repression following peptidoglycan enriched cell envelope stimulation
AF039943
Chicken Akt1
Y08823
Chicken CD80-like protein (precursor)
AF029369
Chicken chemokine receptor CRL1
0.01
BG625680 est
Putative E-selectin
0.05
BQ038594
Chicken IL-16
BQ038261 est
Putative LFA-1 alpha
Induction following cell envelope stimulation
Z48921
Chicken B-2-microglobulin
AB015289
Chicken BASH
L14414
Chicken B-F
AF083029
Chicken caspase 3
AJ292037
Chicken CD164 (partial)
AF153205
Chicken CD44
L13285
Chicken CD45
AJ446108 est
Putative CD82
BU141439 est
Putative CDw137
J00889
Chicken C-myc
AF296874
Chicken Fas (partial)
BU409623 est
Putative granzyme-like molecule
J02579
Chicken HSP 70

12 h

0.03

Repression following DNA stimulation
L13285
Chicken CD45
AF294794
Chicken CXCR4
K01458
Chicken GAPDH
BU428135/AF096264
Putative JAK-like molecule (Jak 2, JAK 3
homologue)
BQ038261 est
Putative LFA-1 alpha
AJ447751 est
Putative STAT2
AB092341
Chicken TCR beta chain constant region
AY046915
Chicken VAV3 (GDP/GTP exchange factor)
Induction following peptidoglycan-enriched cell envelope stimulation
Z48921
Chicken B-2-microglobulin
L14414
Chicken B-F
BM488143
Chicken B-L beta chain
AF083029
Chicken caspase 3
AF153205
Chicken CD44
J00889
Chicken C-myc
BX277938 est
Putative ICAM-1
CB017585
Chicken IgM heavy chain constant region
AF143806
Chicken IL-2 alpha receptor (CD25)
AJ621251
Chicken IL-3
AJ309540
Chicken IL-6 precursor
AJ292038
Chicken invariant chain (partial)
Y14970
Chicken K123
AF218784
Chicken Rfp-y (class 1 alpha chain)
AJ447751 est
Putative STAT2
BU417667 est
Putative STAT4
AI981819
Putative TAP-2
AY633575
Chicken TLR3

6h

0.001

0.00000006
0.00000003
0.000004
0.0000000002
0.000001

0.001
0.00008

0.02
0.005
0.00002
0.0003
0.000000008
7E-20
0.0000003
0.00009
0.009
0.00005
0.02
0.000000002

0.0006
0.0001
0.02

0.0003

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J.T. Brisbin et al.

Table 2 (continued )
Accession number

Gene name

P-values
3h

AF082665
X07174
AF143806
AJ292038
Y14970
BG625630
AF218784
AJ447751 est
BU417667 est

6h

Chicken IFN alpha/beta receptor 2 (IFNAR2)
Chicken IgG heavy chain constant region
Chicken IL-2 alpha receptor (CD25)
Chicken Invariant chain (partial)
Chicken K123
Chicken NK-kB p50
Chicken Rfp-y (class 1 alpha chain)
Putative STAT2
Putative STAT4

Repression following cell envelope stimulation
BU128302 est
Putative Calnexin
AJ454899 est
Putative Calreticulin
AF318895
Chicken CBL
Y08823
Chicken CD80-like protein (precursor)
AF294794
Chicken CXCR4
S40610
Chicken IgA heavy chain constant region
BQ038261 est
Putative LFA-1 alpha
AI981598
Chicken myelomonocytic growth factor
AB092341
Chicken TCR beta chain constant region
BG710003
Chicken TGF B receptor 1
AJ632302
Chicken TLR7

12 h

0.02
0.0005
0.002
0.000000001
0.0000000000002
7E-16
0.0002
7E-21
7E-14
0.0003
0.0001
0.02
0.002

0.0001
0.000004
0.00002
0.00003
0.00005
0.000000000001
0.0003
0.04

Table 3 Statistically significant changes in gene expression in cecal tonsil mononuclear cells after stimulation with 10 mg/mL
DNA, 5 mg/mL peptidoglycan enriched cell envelope, or 20 mg/mL the cell envelope fraction with Po0.05
Accession number

Gene name

P-values
3h

Induction following DNA stimulation
AF039943
Chicken Akt1
Z48921
Chicken B-2-microglobulin
AB015289
Chicken BASH
L14414
Chicken B-F
AF083029
Chicken Caspase3
AJ292037
Chicken CD164 (partial)
AF153205
Chicken CD44
L13285
Chicken CD45
BU141439 est
Putative CDw137
J00889
Chicken C-myc
AF296874
Chicken Fas (partial)
J02579
Chicken HSP 70
X07174
Chicken IgG heavy chain constant region
AF143806
Chicken IL-2 alpha receptor (CD25)
AJ292038
Chicken Invariant chain (partial)
BG625630
Chicken NK-kB p50
AJ447751 est
Putative STAT2
BU417667 est
Putative STAT4
Repression following DNA stimulation
BU128302 est
Putative Calnexin
AF294794
Chicken CXCR4

6h

12 h

0.0002
0.001
0.0005
0.000005
2E-18
0.000008
0.0005
0.004
0.01
0.00002

0.000001

0.0009
0.007
0.0001
0.02
0.0000004
0.000000002
2E-15
0.00000000002
0.0007
0.00006

0.003

0.000004
0.005

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Table 3 (continued )
Accession number

Gene name

P-values
3h

S40610
BQ038261 est
AI981598
AB092341
BG710003

Chicken IgA heavy chain constant region
Putative LFA-1 alpha
Chicken Myelomonocytic growth factor
Chicken TCR beta chain constant region
Chicken TGF B receptor 1

12 h

0.00004
0.0001
0.0000008
0.000000000002
0.0003

Induction following peptidoglycan enriched cell envelope stimulation
M59925
Chicken CD3
Z26484
Chicken CD8b
AJ419896
Chicken Common gamma chain receptor (putative a/b)
CB017585
Chicken IgM heavy chain constant region
AI981042
Chicken p53
AI981819
Putative TAP-2
AJ005071
Chicken Tapasin
BG710003
Chicken TGF B receptor 1
AI981542 est
Putative Zap-70
Repression following
L13285
AF294794
AF082664
AF082665
BU428135/AF096264
BQ038261 est
AJ447751 est
BU417667 est
AY046915

peptidoglycan enriched cell envelope stimulation
Chicken CD45
Chicken CXCR4
Chicken IFN alpha/beta receptor 1 (IFNAR1)
Chicken IFN alpha/beta receptor 2 (IFNAR2)
Putative JAK-like molecule (Jak 2, JAK 3 homologue)
Putative LFA-1 alpha
Putative STAT2
Putative STAT4
Chicken VAV3 (GDP/GTP exchange factor)

Repression following
AJ292037
Z26484
AF029369
AF296874
X56931
L19258
J02579
AF082664
S40610
AJ493595
BU428135/AF096264
AJ005071

cell envelope stimulation
Chicken CD164 (partial)
Chicken CD8b
Chicken Chemokine receptor CRL1
Chicken Fas (partial)
Chicken GATA3
Chicken Grb2
Chicken HSP 70
Chicken IFN alpha/beta receptor 1 (IFNAR1)
Chicken IgA heavy chain constant region
Chicken IL-17
Putative JAK-like molecule (Jak 2, JAK 3 homologue)
Chicken Tapasin

at 6 h post-stimulation for all three treatments in cecal
tonsil cells, whereas in the spleen the highest number of
differentially expressed genes for the cell envelope was at
6 and 12 h for DNA and PECE.
In spleen cells, the cell envelope fraction was the most
potent stimulus, as it increased the expression level of 22
genes compared to 18 and 2 genes in the PECE and DNA
treatment groups, respectively. In splenocytes, caspase 3
was the only gene that was induced after treatment of cells
with all three bacterial components. There was, however,
some degree of overlap in the expression pattern of genes in
splenocytes after treatment with cell envelope and PECE
fractions. The genes that were in common between cell
envelope and PECE treatment were: K123, IL-2 alpha

6h

0.000008
0.002
0.00007
0.00000006
0.00001
0.0000008
0.0008
0.00000006
0.003
0.00003
9E-15
0.008
0.0006
0.003
0.00004 0.000002

1E-35

0.00000000005
0.0000000000007
0.00007
0.0002
0.000004

0.04
0.006
0.0004
0.00002
0.02
0.001
0.008
0.000000001
0.01
0.02
0.04

receptor (CD25), CD44, CD45, b2-microglobulin, MHC class
I heavy chain (B-F), invariant chain, Rfp-y (class 1 alpha
chain), caspase 3, C-myc, STAT2 and STAT4.
In cecal tonsil cells, stimulation with DNA altered the
expression of the highest number of genes, as it resulted in
the increased expression of 18 genes compared to nine
genes in PECE-treated cells and no genes in the cell
envelope fraction-treated cells. In cecal tonsil cells, no
overlap was observed in the expression profile of genes that
were induced after DNA or PECE treatment. Treatment of
cecal tonsil cells with DNA induced the expression of genes,
including several genes in common with splenocytes treated
with bacterial cell wall components, such as b2-microglobulin, MHC class I heavy chain (B-F), caspase 3, CD25, CD44,

ARTICLE IN PRESS
570

J.T. Brisbin et al.

CD45, C-myc, IL-2 alpha receptor (CD25), invariant chain,
STAT2 and STAT4.

3, and K123 were induced in the majority of the time points,
being validated by real-time PCR (data not shown).

3.2. Validation of microarray results by real-time
PCR

3.3. Real-time PCR analysis of gene expression of
the STAT2 and STAT4 pathways

Genes that were differentially expressed in multiple
combinations of cell type, time, and treatment and had a
fold change of more than 1.5 were selected for validation
using real-time RT-PCR. These genes included K123, STAT2,
STAT4, IgA, LFA-1 alpha, and caspase 3. Expression levels
were normalized to b-actin expression, which was used as an
internal housekeeping control. Overall, there was 84%
agreement between real-time PCR and microarray gene
expression data with the findings that STAT2, STAT4, caspase

Cecal tonsils are a major component of the GALT in
chickens and given their close associations to the commensal
bacteria and our observation that gene expression in cecal
tonsil cells was most affected by DNA stimulation; the
response of cecal tonsil mononuclear cells to DNA was
examined in more detail. In vitro stimulation was performed
similar to the microarray experiments and analysis of gene
expression was examined with real-time PCR. Given the
observed increase in gene expression of the transcription

STAT 2
25

STAT 4
15

*

*

Fold Change

Fol d Change

20
15
10

10

*

5

5

*
*

*
0

0
1

3

6

12

18

1

3

*

20

*

10

*

0
1

*

*

Fold Change

Fold Change

40

20

10

*
0

3

6

12

1

18

3

6

IL-18
5

*

Fol d Change

4

Fold Change

18

MyD88

30

20

10

*

*

3
2

*

1

*

0
3

12

Hours Post Stimulation

Hours Post Stimulatio n

1

18

*

50

30

12

IFN- α

IFN- γ
60

6

Hours Post Stimulatio n

Hours Post Stimulation

6

12

Hours Post Stimulation

0

18

1

3

6

12

18

Hours Post-stimulation

Fig. 1 Real-time PCR analysis of chicken cecal tonsil mononuclear cells stimulated with 10 mg/mL of L. acidophilus DNA for 1, 3, 6,
12, and 18 h. RNA was isolated, reverse transcribed and used for real-time PCR analysis with primers listed in Table 1. Relative
expression was determined with the EDDCt method using the PCR efficiencies determined with the standard curve included in each
run. (A) STAT2 gene expression, (B) STAT4 gene expression, (C) IFN-g gene expression, (D) IFN-a gene expression, (E) IL-18 gene
expression, and (F) MyD88 gene expression. Expression of target genes is normalized to b-actin and is presented as mean7the
standard error. * indicates that the treated samples are statistically different from the untreated control samples (Po0.05) using a
paired two tailed T-test.

ARTICLE IN PRESS
Gene expression profiling of chicken lymphoid cells
factors, STAT2 and STAT4, we sought to determine whether
the expression of genes associated with activation of the
STAT pathway, including myeloid differentiation primary
response (MyD) 88, IFN-a, IFN-g, and IL-18 were up-regulated
as well.
STAT2 had a similar pattern of gene expression as in the
microarray experiments with increased expression at 6 and
12 h post-stimulation, with a fold change of 10.55 and 15.93,
respectively, in DNA-treated cells compared to untreated
cells (Fig. 1a). STAT4 also had a similar pattern of expression
as in the microarray experiments with an increase in
expression that peaked at 12 h post-stimulation with a fold
change of 8.93 (Fig. 1b).
The highest level of IFN-g gene expression in stimulated
cells compared to untreated cells was observed at 6 h poststimulation (33.82). IFN-g gene expression increased at 3 h
post-stimulation with a fold change of 5.71 and after the
peak at 6 h remained up-regulated throughout the observation period. The difference between treated and untreated
cells was reduced to 15.75 and 8.16 by 12 and 18 h poststimulation, respectively (Fig. 1c).
IFN-a expression was increased at all time points in DNAtreated cells compared to untreated cells. IFN-a expression
peaked at 6 h and remained high through 12 h, but dropped
at 18 h post-stimulation (Fig. 1d). The fold changes were
7.31, 4.81, 14.92, 12.90, and 2.11 for 1, 3, 6, 12, and 18 h
post-stimulation time points, respectively.
The highest level of IL-18 expression was at 1 h with a fold
change of 14.09, which was then reduced to 1.17 and 1.86 at
3 and 6 h post-stimulation, respectively (Fig. 1e). The
expression of IL-18 at 12 and 18 h post-stimulation was
down-regulated in DNA-treated cells compared to untreated
cells.
The expression of MyD88 was increased in treated cells
compared to untreated control cells with fold changes of
1.94, 1.84, 2.00, 3.27, and 1.26 for 1, 3, 6, 12, and 18 h
post-stimulation, respectively (Fig. 1f).

4. Discussion
Lactic acid bacteria (LAB), made up of species from the
genera Bifidobacterium, Lactobacillus, and Lactococcus,
have been extensively used as probiotics for control of
intestinal colonization with enteric pathogens. Moreover,
the potential for use as live mucosal delivery systems and
the immunomodulatory abilities and of these probiotic
bacteria have been examined [18–20]. Various Lactobacillus
spp., including L. acidophilus, have been shown to induce
the activation and maturation of mouse and human DCs and
lymphocytes and the subsequent cytokine production by
these cells [21–24]. LAB are able to induce interferon (IFN)g, interleukin (IL)-6, IL-10, and tumor necrosis factor -a in
human peripheral blood mononuclear cells [4,25], and
activation of the NF-kB and STAT signaling pathways in
macrophages [6]. In order to examine the immunoregulatory
abilities of L. acidophilus in the chicken, we investigated
the spatial and temporal expression of immune system genes
in cecal tonsil and spleen mononuclear cells in response to
the structural constituents of this bacterium. We discovered
that, compared to splenocytes, cecal tonsil cells responded
more rapidly to bacterial PAMPs. This result may be

571
attributed to the fact that cecal tonsils, given their location
in the intestine, are continuously exposed to bacterial
PAMPs. This prior exposure may explain the shorter time
required for triggering gene expression in cecal tonsil cells.
Sasai and co-workers [26] have reported that following
infection of chickens with Salmonella, cecal tonsil cells
undergo rapid changes, whereas there is nearly no change in
spleen cells. The fact that gene expression changes
associated with treatment of spleen cells by bacterial cell
envelope peaked at 6 h may be due to the fact that the cell
envelope faction contains a more complex mixture of
different PAMPs, while DNA and PECE are less complex,
leading to the faster response of cells to these bacterial
components.
After stimulation of splenocytes with the cell envelope
and PECE fractions, there was a unique set of genes, which
displayed statistically significant differential expression in
each group. However, there was also a degree of overlap
between the two treatment groups in genes displaying
expression changes. Given that both cell envelope and PECE
fractions are derived from bacterial cell wall, they are
probably enriched in similar type of PAMPs, most notably
peptidoglycan. Treatment of splenocytes with bacterial
DNA, however, only induced the expression of two genes.
Previous studies have shown that chickens respond to
bacterial DNA, although the ortholog of mammalian TLR9
has not been found in avian species [27,28]. However, it is
conceivable that chickens respond to bacterial DNA in a
tissue- and bacteria-dependent manner.
In contrast to spleen cells, however, DNA was the most
potent stimulus for cecal tonsil cells, while PECE and the cell
envelope fraction altered the expression of very few genes
and, moreover, the ones that were altered in expression were
primarily down-regulated. A minimal or down-regulatory
response of cecal tonsil cells to the cellular components of
L. acidophilus is not surprising given the fact that this bacteria
a normal member of the commensal microbiota. Downregulation of a number of genes may represent the adapted
response to these normal members of the microbiota.
In cecal tonsil cells, there was no overlap in genes that
displayed altered expression among the three treatments,
indicating that cecal tonsil cells respond differently to
different bacterial components. Overall, these findings
suggest a spatial control of gene expression in lymphoid
tissues of the chicken in response to different stimuli. These
tissues differ in their cellular compositions [26,29], which
may partly explain the observed differences in gene
expression profiles. Moreover, it is possible that the cytokine
milieu and the activation state of cells of their prior
exposure in these tissues are different, leading to the
varying responses to bacterial PAMPs.
In spite of the spatial and temporal differences between
spleen and cecal tonsil mononuclear cells, there was some
overlap in gene expression profiles of spleen cells treated
with the two structural components (PECE and cell envelope)
and cecal tonsil cells treated with DNA. There were nine
genes that were in common among the above treatment
groups indicating that different tissues can respond similarly
to different stimuli. These genes belonged to the following
families: cytokine and cytokine receptors (IL-2 receptor alpha
or CD25), surface molecules (CD44 and CD45), antigen
processing and presentation (b2-microglobulin, MHC class I

ARTICLE IN PRESS
572
(B-F), invariant chain), apoptosis (caspase 3), and transcription and signal transduction (C-myc, STAT2, and STAT4).
Induction of genes encoding members of the STAT family is
consistent with the observations made by Sun et al. [30] that
subsequent to in vivo treatment of mice with Lactobacillus
peptidoglycan, the STAT pathway is activated. Miettinen
et al. [6] also observed an increase in STAT signaling after
treatment of human macrophages with Lactobacillus rhamnosus and Streptococcus pyogenes. STAT2 plays an important
role in signal transduction after engagement of type I
interferons (IFN-a and -b) with their receptors [31]. STAT4 is
important in the development of T helper (Th) 1 cells by
acting as a member of the IL-12 signaling pathway and
playing a role in the induction of IFN-g [32,33]. IL-18 is also
able, with either IL-12 or IFN-a, to induce IFN-g in a STAT4dependent manner [34]. The combined effect of IL-12, IL18, and IFN-g provides an appropriate milieu for naı¨ve T cells
to differentiate into committed Th1 cells [33,35,36]. IFN-a is
also capable of activating STAT4 in human T and NK cells,
resulting in an increase in IFN-g expression [34,37]. The
induction of STAT2 and STAT4 genes in our studies may,
therefore, be related to the activation status of cells and
their differentiation to effector cells. Interestingly, in cecal
tonsil cells treated with PECE where STAT2 and STAT4 genes
were repressed, type I interferon receptors were also
repressed, raising the possibility that the type I interferon
pathway is completely or partially down-regulated after
stimulation with peptidoglycan in cells of the chicken GALT.
Given the ability of L. acidophilus DNA to act as a potent
stimulus for cecal tonsil mononuclear cells, the expression
of STAT2 and STAT4 genes along with MyD88, IFN-a, IFN-g,
and IL-18 was examined further in cecal tonsil cells after
treatment with DNA. The same expression pattern of STAT2
and STAT4 was observed as in the microarray experiments.
MyD88 is involved in signal transduction from TLRs, including
those that bind to bacterial peptidoglycan and DNA [38].
Since a TLR responsible for binding to bacterial DNA has not
yet been identified in chickens, MyD88 was used as a marker
for the activation of the TLR signaling machinery. IFN-a, IFNg, and IL-18 were screened since, in mammals, these
cytokines are known to utilize the STAT2/4 pathways for
signaling. After DNA stimulation of cecal tonsil cells, the
expression of MyD88 gene was increased indicating that
bacterial DNA stimulation in chickens, similar to mammals
[39], may operate in an MyD88-dependent manner.
In our experiments, IFN-a expression was increased and
correlated well with the observed increase in STAT2
expression, which is expected as IFN-a is the only known
activator of STAT2 [40]. Others have reported an increase in
IFN-a expression after treatment of macrophages with
another LAB, Lactobacillus gasseri, and have associated this
increase with the production of IL-1 and, subsequently,
enhanced gut immunity [41]. In mammals, type I interferons
have a number of effects on the immune system, including
enhancement of MHC class I on all cell types, up-regulation
of IFN-g, and cytoxicity in NK cells, promoting isotype
switching in B cells, and induction of proliferation,
cytoxicity, and Th1 differentiation in T cells [42]. In our
experiments, the increase in STAT2 and IFN-a expression
correlated with an increase in the expression of genes
involved in apoptosis, T cell activation, and MHC class I
processing and presentation, as determined by microarray

J.T. Brisbin et al.
analysis. These results indicate that in chicken cecal tonsil
cells, IFN-a may signal through STAT2 to generate effector
functions similar to those observed in mammals.
The increase in STAT4 gene expression in cecal tonsil cells
treated with DNA may indicate that L. acidophilus generates
a Th1 phenotype. It has been demonstrated that in vitro
treatment of cells with DNA extracted from Bifidobacterium
and Lactobacillus results in cell activation and the expression of Th1 cytokines, including IL-12, IL-18, and IFN-g [43].
In accordance with these results, we noted a significant
increase in the expression of Th1 cytokines, including IL-18
and IFN-g after stimulation of cecal tonsil cells with
L. acidophilus genomic DNA. Overall, these results suggest
that DNA of probiotic bacteria in chickens possess the ability
to stimulate the development of Th1-like responses. Despite
the increase in STAT4 expression, we did not observe a
significant change in the expression of IL-12 after stimulation of cecal tonsil cells with DNA. Although STAT4 is usually
associated with IL-12 signaling, it has also been demonstrated that type I interferons could activate STAT4 leading
to the production of IFN-g [31,32]. However, there may be a
difference in the level of STAT4 activation induced by type I
interferons and IL-12. Athie-Morales et al. [37] have
described the transient nature of STAT4 activation in cells
stimulated with IFN-a, whereas IL-12 induced a more longlasting STAT4 activation. In our experiments, IL-18 expression was increased in cecal tonsil cells treated with
L. acidophilus DNA. This cytokine has also been shown to
induce to synergistically act with IFN-a or IL-12 to induce
IFN-g in a STAT4-dependent manner [34]. Hence, it is
possible that L. acidophilus induces the expression of IL-18
and IFN-a, but not IL-12, to transiently up-regulate the
expression of IFN-g in chicken immune system cells.
Very little is known about cytokine signaling pathways in
chickens, but our findings indicate that, similar to human
cells, type I interferons may activate both STAT2 and STAT4
pathways, leading to the production of Th1 cytokines
and, perhaps, the development of a Th1 phenotype.
This conclusion is consistent with the previous finding that
a Lactobacillus-based probiotic enhanced local immunity to
Eimeria acervulian in chickens and this immunity was
associated with elevated IL-2 and IFN-g production [44].
Development of the Th1 phenotype may also be associated
with the up-regulation of T cell activation markers, such as
CD44 and CD25, and an increase in the expression of the
genes involved in antigen uptake and processing [45].
Indeed, in our microarray analysis, we noted the induction
of several genes, including CD44, CD25, MHC class I heavy
chain (B-F), b2-microglobulin, and the invariant chain after
stimulation of cecal tonsil cells with DNA. This is the first
report to demonstrate the immunomodulatory effects of
genomic DNA, PECE, and the cell envelope of L. acidophilus,
a frequently used probiotic bacteria, on chicken immune
system cells. The findings of the present study also shed
light on the underlying molecular mechanisms of immunomodulation mediated by probiotic L. acidophilus.

Acknowledgements
This study was supported by the Canadian Poultry Research
Council, Poultry Industry Council, Natural Science and

ARTICLE IN PRESS
Gene expression profiling of chicken lymphoid cells
Engineering Research Council of Canada, and Agriculture
and Agri-Food Canada through the MII program. Dr. H. Zhou
was an NSERC Visiting Fellow to Canadian Federal Government Laboratories.

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