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Characterization of responses elicited by Toll-like receptor agonists in cells of the bursa of Fabricius in chickens
Veterinary Immunology and Immunopathology 149 (2012) 237–244
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Research paper
Characterization of responses elicited by Toll-like receptor agonists in cells of the bursa of Fabricius in chickens Michael St. Paul, Sarah Paolucci, Leah R. Read, Shayan Sharif ∗ Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada N1G 2W1
a r t i c l e
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Article history: Received 15 June 2012 Received in revised form 12 July 2012 Accepted 17 July 2012 Keywords: B cells Bursa of Fabricius Chicken Innate response Toll-like receptor TLR3 TLR4 TLR21
a b s t r a c t Toll-like receptors (TLRs) are an evolutionarily conserved group of pattern recognition receptors that play an important role in mediating host-responses to pathogens. Several TLRs have been identified in chickens and their expression has been detected in many immune cell subsets including in B cells. However, the mechanisms through which TLRs modulate B cell responses have not been well characterized in chickens. The aim of the present study was to elucidate the responses mounted by cells of the bursa of Fabricius to treatment with the TLR 3, 4 and 21 ligands, poly I:C, lipopolysaccharide (LPS) and CpG oligodeoxynucleotides (ODN), respectively. The relative expression of several immune system genes was quantified at 1, 3, 8 and 18 h post-treatment. The results show that all three ligands induced the up-regulation of interferon (IFN)-␥ and interleukin (IL)-10 transcripts and promoted the up-regulation of transcripts associated with antigen presentation, namely CD80 and major histocompatibility complex (MHC) class II. Furthermore, the results indicated that LPS and poly I:C induced the greatest IFN-␥ and IL-10 responses, respectively, while CpG ODN was the most efficacious inducer of CD80 and MHC-II expression. Future studies may be aimed at elucidating the mechanisms of TLR-mediated activation of chicken B cells. These mechanisms may be then exploited for the development of adjuvants with enhanced ability to stimulate B cell responses. © 2012 Elsevier B.V. All rights reserved.
1. Background Toll-like receptors (TLRs) are an evolutionarily conserved group of pattern recognition receptors that play an important role in mediating host-responses to pathogens (Akira et al., 2001; Medzhitov, 2001). The ligands for TLRs are typically conserved structural motifs expressed by microbes, termed pathogen-associated molecular patterns (PAMPs). To date, several TLRs have been identified in chickens, each responding to different PAMPs. For example, TLR3 binds double-stranded RNA, a product of some viruses during their replication cycle, while TLR4 binds lipopolysaccharide (LPS), which is present in the
∗ Corresponding author. Tel.: +1 519 824 4120x54641; fax: +1 519 824 5930. E-mail address: [email protected] (S. Sharif). 0165-2427/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetimm.2012.07.008
cell wall of Gram-negative bacteria, and TLR21 binds unmethylated CpG DNA motifs found in the nucleic acids of certain bacteria and viruses (Leveque et al., 2003; Temperley et al., 2008; Keestra et al., 2010). TLRs have been identified in many cell subsets of both the innate and adaptive immune system including macrophages, heterophils, T cells and B cells (Iqbal et al., 2005). Responses mediated by interactions between TLRs and their ligands typically include the production of cytokines and cellular activation (Hopkins and Sriskandan, 2005). In the case of mammalian B cells, treatment with TLR ligands induces the production of immunoglobulins and cytokines, such as interferon (IFN)-␥ and interleukin (IL)-10 (Barr et al., 2007; Giordani et al., 2009). Moreover, TLR stimulation facilitates antibody isotype switching (Jegerlehner et al., 2007) and promotes the proliferation of B cells and enhances their antigen presentation capabilities (Jiang
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et al., 2007). To date, several TLRs have been identified in mammalian B cells as reviewed by Booth et al. (2010). Although extensively studied in mammals, the TLRmediated responses of B cells have not been well characterized in chickens. It has been previously shown that chicken B cells express TLRs 2, 3, 4, 5, 7 and 21 at the transcript level (Iqbal et al., 2005; Han et al., 2010), and they proliferate in response to CpG oligodeoxynucleotides (ODN) (Wattrang, 2009). Additionally, our group has previously found that chicken B cells up-regulate certain immune system genes in response to LPS (Sarson et al., 2007). Although these studies suggest that chicken B cells may express functional TLRs 4 and 21, the responses of chicken B cells to stimulation with these TLR ligands and others have not been well examined. The bursa of Fabricius in chickens is the primary site for development and differentiation of B cells and approximately 98% of bursal lymphocytes are B cells (Davison et al., 2008). Therefore, the present study was an attempt to elucidate and characterize the responses of bursal cells to treatment with the TLR3, 4 and 21 ligands, poly I:C, LPS and CpG ODN, respectively. The results show that all three ligands induced the transcriptional up-regulation of IFN-␥ and IL-10, and also promoted the up-regulation of transcripts associated with antigen presentation, namely CD80 and major histocompatibility complex (MHC) class II. 2. Methods 2.1. Chickens Four-week-old broiler chickens (n = 6) were procured from the Arkell Poultry Research Center, University of Guelph (Guelph, ON). This research was approved by the University of Guelph Animal Care Committee and complied with the guidelines of the Canadian Council on Animal Care. 2.2. B cell isolation The bursa of Fabricius was collected from 6 chickens, minced and filtered through a 40 m nylon cell strainer to obtain a single cell suspension. The suspension was overlaid onto a Histopaque-1077 (Sigma–Aldrich, Oakville, ON) gradient and centrifuged at 400 × g for 30 min, and the cells were harvested from the plasma-Histopaque interface and washed 3× in RPMI-1640 (Invitrogen, Burlington, ON) supplemented with 10% heat-inactivated fetal bovine serum, 200 U/mL penicillin, 80 g/mL streptomycin, 25 mg gentamicin, 10 mM HEPES buffer, 50 M -mercaptoethanol, and 2 mM l-glutamine. Bursal cells were seeded into 48well plates at 1 × 107 cells/mL for in vitro stimulation with TLR ligands. As indicated before, 98% of lymphocytes in the chicken bursa of Fabricius are B cells (Davison et al., 2008), therefore, the cell cultures used in this study were highly enriched for B cells. 2.3. TLR ligands Poly I:C and LPS from Escherichia coli 0111:B4 were (Oakville, purchased from Sigma–Aldrich-Canada ON), while synthetic class B CpG ODN 2007
[5 -TCGTCGTTGTCGTTTTGTCGTT-3 ] and non-CpG ODN [5 -TGCTGCTTGTGCTTTTGTGCTT-3 ] were purchased from Eurofins MWG Operon (Ebersberg, GER). All of the ligands used were re-suspended in sterile phosphate buffered saline (PBS, pH 7.4) and diluted to working concentrations in RPMI medium of the same formulation used to culture bursal cells. 2.4. Experimental design Bursal cells from 6 chickens were stimulated with either a low or high dose of the TLR3 ligand poly I:C (5 g/mL and 50 g/mL), the TLR4 ligand LPS (0.5 g/mL and 5 g/mL) and the TLR21 ligand CpG ODN (0.5 g/mL and 5 g/mL), while control groups received either nonCpG ODN (5 g/mL) or medium. These doses were selected as they have been shown to be immunostimulatory for several different chicken cell populations (Kogut et al., 2005; Keestra and van Putten, 2008; Villanueva et al., 2011; He et al., 2012). At 1, 3, 8 and 18 h post-stimulation, cells were harvested for RNA extraction. 2.5. RNA extraction and cDNA synthesis Total RNA was extracted from the bursal cells using TRIzol® (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol and treated with DNA Free® (Ambion, Austin, TX) DNAse. Subsequently, 500 ng of purified RNA was reverse transcribed to cDNA using Superscript® II First Strand Synthesis kit (Invitrogen, Carlsbad, CA) and oligodT primers, according to the manufacturers recommended protocol. The resulting cDNA was subsequently diluted 1:10 in DEPC treated water. 2.6. Quantitative real-time PCR Quantitative real-time PCR using SYBR Green was performed on diluted cDNA using the LightCycler® 480 II (Roche Diagnostics GmbH, Mannheim, GER) as previously described (Villanueva et al., 2011). Briefly, each reaction involved a pre-incubation at 95 ◦ C for 10 min, followed by 45 cycles of 95 ◦ C for 10 min, 55–64 ◦ C (TA as per primer) for 5 s, and elongation at 72 ◦ C for 10 s. Subsequent melt curve analysis was performed by heating to 95 ◦ C for 10 s, cooling to 65 ◦ C for 1 min, and heating to 97 ◦ C. Many of these primers have been used in previous studies, and the new ones were designed using the NCBI Primer-Blast (St. Paul et al., 2011, 2012). Primers were synthesized by Sigma–Aldrich-Canada (Oakville, ON), and their specific sequences and accession numbers are outlined in Table 1. 2.7. Data analysis Relative expression levels of all genes was calculated relative to the housekeeping gene -actin using the LightCycler® 480 Software (Roche Diagnostics GmbH, Mannheim, GER), based on the formula developed by Pfaffl (2001). Data represent mean fold change from medium treated controls ± standard error. Statistical significance
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Table 1 Primer sequences and accession numbers used for quantitative real-time PCR. Target gene
Primer sequence
GenBank accession number
TLR3
F: 5 -TCAGTACATTTGTAACACCCCGCC-3 R: 5 -GGCGTCATAATCAAACACTCC-3 F: 5 -TGCCATCCCAACCCAACCACAG -3 R: 5 -ACACCCACTGAGCAGCACCAA -3 F: 5 -CCTGCGCAAGTGTCCGCTCA-3 R: 5 -GCCCCAGGTCCAGGAAGCAG-3 F: 5 -ACACTGACAAGTCAAAGCCGCACA-3 R: 5 -AGTCGTTCATCGGGAGCTTGGC-3 F: 5 -TGTGCCCACGCTGTGCTTACA-3 R: 5 -CTTGTGGCAGTGCTGGCTCTCC-3 F: 5 -AGCAGATCAAGGAGACGTTC-3 R: 5 -ATCAGCAGGTACTCCTCGAT-3 F: 5 -CCTGGTGATGCTGTGAATTG-3 R: 5 -CTTCTGTGTCGTTGCATTCAG-3 F: 5 -CTGTTCCTTCACATCCTGAGAG -3 R: 5 -CTTCAACACCATCTATTTGCCAG-3 F: 5 -CCACGGACGTGATGCAGAAC-3 R: 5 -ACCGCGCAGGAACACGAAGA-3 F: 5 -CAACACAGTGCTGTCTGGTGGTA-3 R: 5 -ATCGTACTCCTGCTTGCTGATCC-3
DQ780341
TLR4 TLR21 IFN-␥ IL-4 IL-10 CD40 CD80 MHC II -Actin
between treatment groups and control groups was calculated using a paired student’s t-test. 3. Results To examine the effects of TLR ligand stimulation on the expression of TLRs, we quantified the transcript levels of TLRs 3, 4 and 21 (Fig. 1). TLR3 transcripts were up-regulated at 3 h (p ≤ 0.05) and 8 h (p ≤ 0.05) postlow dose poly I:C treatment (5 g/mL), while high dose poly I:C (50 g/mL) up-regulated TLR3 transcripts at 3 h post-treatment (p ≤ 0.01) (Fig. 1A). TLR4 transcripts were down-regulated at 1 h post-low dose (0.5 g/mL) LPS treatment (p ≤ 0.05), while subsequently up-regulated at 3 h post-treatment (p ≤ 0.01) (Fig. 1B). Treatment with the high dose of LPS (5 g/mL), however, did not lead to any downregulation in gene expression, and only up-regulated TLR4 transcripts at 3 h post-treatment (p ≤ 0.05). Treatment with the low dose of CpG ODN (0.5 g/mL) up-regulated TLR21 transcripts at 1 h (p ≤ 0.05), 3 h (p ≤ 0.05) and 18 h (p ≤ 0.05) post-stimulation, while high dose CpG ODN (5 g/mL) treatment up-regulated TLR21 transcripts at all the time points (Fig. 1C). As TLR stimulation is known to up-regulate IFN-␥ and IL-10 in mammalian B cells (Barr et al., 2007; Giordani et al., 2009), we decided to see if this was the case in chicken bursal cells (Fig. 2). Treatment with the low dose of poly I:C significantly up-regulated IFN-␥ transcripts at 1 h post-treatment (p ≤ 0.01), while transcripts were upregulated in response to treatment with the high dose of poly I:C at 1 h (p ≤ 0.01), 3 h (p ≤ 0.05) and 8 h (p ≤ 0.05) post-treatment (Fig. 2A). Treatment with the low dose of LPS up-regulated IFN-␥ transcripts at 1 h (p ≤ 0.01) and 18 h (p ≤ 0.05) post-treatment, and treatment with the high dose of LPS up-regulated transcripts at 1 h (p ≤ 0.01), 8 h (p ≤ 0.05) and 18 h post-treatment (p ≤ 0.01) (Fig. 2B). IFN-␥ transcripts were up-regulated at 1 h post-treatment with both the low dose (p ≤ 0.01) and high dose (p ≤ 0.05) of CpG ODN (Fig. 2C). Treatment with only the high
AY064697 AJ720600.1 X99774 AJ621249.1 AJ621614 AJ293700 Y08823 113206149 X00182
dose of poly I:C significantly up-regulated IL-10, and this occurred at both 1 h post-treatment (p ≤ 0.01) and 18 h post-treatment (p ≤ 0.05) (Fig. 2D). Similar results were observed in response to LPS, as only the high dose of LPS upregulated IL-10 at 1 h post-treatment (p ≤ 0.05) (Fig. 2E). In response to CpG ODN treatment, the low dose up-regulated IL-10 at 1 h post-treatment (p ≤ 0.05) (Fig. 2F). Additionally, as mammalian B cells are known to produce IL-4 (Harris et al., 2000), we attempted to quantify IL-4 expression in the present study, however, there was very little or no IL4 expression in bursal cells cultured with or without TLR stimulation (data not shown). In addition to cytokines, TLR stimulation in mammalian B cells has been shown to up-regulate surface proteins, namely CD40, CD80 and MHC-II (Barr et al., 2007). As demonstrated in Fig. 3, this also occurs in chicken bursal cells. Treatment with the low dose of poly I:C up-regulated CD40 transcripts at 1 h post-treatment (p ≤ 0.01) while down-regulating them at 8 h (p ≤ 0.05) post-treatment (Fig. 3A). Similar results were observed in response to the high dose poly I:C treatment, as transcripts were up-regulated at 1 h (p ≤ 0.01) and 3 h (p ≤ 0.01) post-treatment, while down-regulated at 8 h post-treatment (p ≤ 0.05). Treatment with the low dose of LPS up-regulated CD40 transcripts at 1 h post-treatment (p ≤ 0.05), while treatment with the high dose of LPS upregulated transcripts at 1 h (p ≤ 0.01) and 3 h (p ≤ 0.01) post-treatment (Fig. 3B). Treatment with the low dose of CpG ODN up-regulated CD40 at 1 h post-treatment (p ≤ 0.05) (Fig. 3C). However, treatment with non-CpG ODN significantly down-regulated CD40 transcripts at 3 h post-treatment (p ≤ 0.05). Moreover, treatment with only the low dose of poly I:C up-regulated CD80, and this occurred at 1 h post-treatment (p ≤ 0.01) (Fig. 3D). Similar results were obtained in the case of bursal cell responses to LPS, as only the low dose of LPS upregulated CD80 and this also occurred at 1 h post-treatment (p ≤ 0.05) (Fig. 3E). However, treatment with the low dose of CpG ODN significantly up-regulated CD80 at all
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Fig. 1. Relative gene expression of TLR3, 4 and 21 transcripts in chicken bursal cells. Bursal cells were collected at 1, 3, 8 and 18 h post-treatment with the TLR ligands poly I:C (5 g/mL and 50 g/mL), LPS (0.5 g/mL and 5 g/mL) and CpG ODN (0.5 g/mL and 5 g/mL). The control groups received either non-CpG ODN (5 g/mL) or medium. Data represent mean fold change from medium treated controls ± standard error. Statistical significance between treatment groups and control groups was calculated using a paired student’s t-test and was considered statistically significant from the medium control group if p ≤ 0.05 (*) or p ≤ 0.01 (**), and in the case of CpG ODN, considered statistically significant from the non-CpG ODN control group if p ≤ 0.05 (#).
sampling time points (p ≤ 0.05), while treatment with the high dose of CpG ODN significantly up-regulated CD80 solely at 18 h post-treatment (p ≤ 0.05) (Fig. 3F). Treatment with non-CpG ODN significantly down-regulated CD80 at 3 h post-treatment (p ≤ 0.05). Lastly, treatment with the low dose of poly I:C significantly up-regulated MHC II
transcripts at 3 h (p ≤ 0.05) and 18 h (p ≤ 0.05) posttreatment (Fig. 3G). Treatment with the low dose of LPS significantly up-regulated MHC II transcripts at 1 h posttreatment (p ≤ 0.01), while the high dose LPS treatment up-regulated transcripts at 1 h (p ≤ 0.05), 3 h (p ≤ 0.05) and 18 h (p ≤ 0.05) post-treatment (Fig. 3H). Treatment with
Fig. 2. Relative gene expression of IFN-␥ and IL-10 transcripts in chicken bursal cells. Bursal cells were collected at 1, 3, 8 and 18 h post-treatment with the TLR ligands poly I:C (5 g/mL and 50 g/mL), LPS (0.5 g/mL and 5 g/mL) and CpG ODN (0.5 g/mL and 5 g/mL). The control groups received either non-CpG ODN (5 g/mL) or medium. Data represent mean fold change from medium treated controls ± standard error. Statistical significance between treatment groups and control groups was calculated using a paired student’s t-test and was considered statistically significant from the medium control group if p ≤ 0.05 (*) or p ≤ 0.01 (**), and in the case of CpG ODN, considered statistically significant from the non-CpG ODN control group if p ≤ 0.05 (#).
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Fig. 3. Relative gene expression of CD40, CD80 and MHC II transcripts in chicken bursal cells. Bursal cells were collected at 1, 3, 8 and 18 h post-treatment with the TLR ligands poly I:C (5 g/mL and 50 g/mL), LPS (0.5 g/mL and 5 g/mL) and CpG ODN (0.5 g/mL and 5 g/mL). The control groups received either non-CpG ODN (5 g/mL) or medium. Data represent mean fold change from medium treated controls ± standard error. Statistical significance between treatment groups and control groups was calculated using a paired student’s t-test and was considered statistically significant from the medium control group if p ≤ 0.05 (*) or p ≤ 0.01 (**), and in the case of CpG ODN, considered statistically significant from the non-CpG ODN control group if p ≤ 0.05 (#).
the low dose of CpG ODN significantly up-regulated MHC II transcripts at all sampling time points (p ≤ 0.05), while treatment with the high dose of CpG ODN up-regulated MHC II transcripts at 1 h (p ≤ 0.05) and 3 h (p ≤ 0.05) posttreatment, and treatment with non-CpG ODN significantly up-regulated MHC II transcripts at 1 h post-treatment (p ≤ 0.01) (Fig. 3I). 4. Discussion TLR stimulation of mammalian B cells promotes the production of cytokines and enhances their antigen presentation capabilities (Barr et al., 2007; Jiang et al., 2007). Indeed, as the present study suggests, similar responses may also occur in chicken bursal cells. It has been established that chicken B cells express several TLRs at the transcript level including TLRs 3, 4 and 21 (Iqbal et al., 2005; Han et al., 2010). These findings are similar to what is found in mammals, except with respect to TLR3. For the most part, mammalian B cells express little TLR3 and typically do not respond to poly I:C, as suggested by their lack of proliferation and up-regulation of co-stimulatory molecules (Genestier et al., 2007; Gururajan et al., 2007; Douagi et al., 2009). This is in contrast to our findings in chicken bursal cells, as poly I:C treatment induced a large (nearly 10-fold) up-regulation of
TLR3 transcripts, in addition to up-regulating several other genes, such as IL-10. In mammals, B cells produce a variety of cytokines which function to modulate several aspects of the immune system, including T cell and inflammatory responses (Harris et al., 2000). Of note are the cytokines IFN-␥, IL-4 and IL-10. Interferon-␥ is a cytokine with several functions, including activating anti-viral cell subsets, namely CD8+ T cells and natural killer cells, in addition to promoting the up-regulation of several interferon inducible genes and modulating the immune response towards a T-helper (TH )-1 bias (Romagnani, 1997; Samuel, 2001). This is in contrast to IL-4, which promotes a TH 2 biased response. In mammals, TLR stimulation in B cells largely drives the up-regulation of IFN-␥, while having little effect on IL-4 (Agrawal and Gupta, 2011). We observed a similar result in chicken bursal cells, as IFN-␥ transcripts were up-regulated in response to poly I:C, LPS and CpG ODN treatment, while none of the ligands up-regulated IL-4 transcripts. Furthermore, it appears that of the TLR ligands tested, LPS induced the greatest up-regulation of IFN-␥. This finding raises the possibility that B cells might be one of the contributing sources to IFN-␥ expression in the spleens of LPS treated chickens (St. Paul et al., 2011). As such, future studies may consider employing LPS as an adjuvant to elicit a TH 1biased response, as studies in mice have highlighted the
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importance of IFN-␥ producing B cells in promoting the differentiation of naive CD4+ T into TH 1 cells during antigen presentation (Harris et al., 2000). Being an anti-inflammatory and regulatory cytokine, one function of IL-10 is to modulate the immune response through regulating the function of T cells, macrophages and dendritic cells (Saraiva and O’Garra, 2010). To this end, IL-10 producing B cells contribute to the downregulation of the deleterious immune responses associated with several autoimmune diseases including experimental autoimmune encephalomyelitis, inflammatory bowel disease and ulcerative colitis (Fillatreau et al., 2008). In mammals, TLR signaling is one stimulus that induces B cells to produce IL-10, as observed in murine B cells following treatment with LPS and CpG ODN (Lampropoulou et al., 2008; Agrawal and Gupta, 2011). Our results suggest that this may also be the case in chicken bursal cells, as we observed an up-regulation of IL-10 transcripts in response to all three TLR ligands. Although only a modest up-regulation was observed in response to LPS and CpG ODN, treatment with poly I:C induced a large up-regulation (over fivefold) of IL-10 transcripts. Furthermore, it appears that the production of IL-10 in response to a TLR ligand treatment is also dependent on the dose of the ligand. For example, it is clear from our results that both doses of poly I:C are immunostimulatory for B cells, yet only the high dose significantly up-regulated IL-10 transcripts. Similarly, only the low dose of CpG ODN significantly upregulated IL-10, while the high dose did not. Indeed, a similar phenomenon occurs in mammalian B cells, as in some instances the lower doses of CpG ODN and other TLR ligands induce a greater production of IL-10 compared to a higher dose (Agrawal and Gupta, 2011). Although the mechanisms behind this finding is not known, it highlights the importance of selecting an appropriate dose of TLR ligands when administering them as vaccine as adjuvants in order to achieve the desired result. In addition to the dose of the TLR ligand, the IL-10 responses of mammalian B cells may also be dependent on the tissue of origin of the B cells. As indicated by Booth and colleagues, B cells isolated from the peripheral blood do not produce IL-10, even after stimulation with TLR ligands or CD40 ligand, which is in contrast to B cells isolated from Peyer’s patches, as the latter cells constitutively produce IL-10 (Booth et al., 2009). This phenomenom may be attributed to the phenotype of each tissue’s resident B cells. For instance, unlike in the peripheral blood, Peyer’s patches contain many regulatory B cells (Booth et al., 2009). Furthermore, the activation state of B cells is known to influence the IL-10 response as well. In support of this notion, Agrawal and colleagues found that naïve human B cells produce significantly less IL-10 compared to that of memory B cells in response to TLR ligands (Agrawal and Gupta, 2011). In the present study, the B cells found in the bursal cell population must have included a significant number of developing and naïve B cells. Considering that, future studies may explore whether the IL-10 responses of chicken B cells to TLR ligands varies depending on the type of B cells (regulatory or effector) in addition to their activation state (naïve, memory or plasma cell).
In general, B cells are capable of presenting antigens via MHC-II to CD4+ T cells (Rivera et al., 2001). However, naïve B cells do not constitutively express high levels of MHC-II or the co-stimulatory molecule CD80, and require a degree of activation and maturation in order to obtain their antigen presenting capabilities (Good et al., 2009). In mammals, it has been shown that TLR ligands provide a sufficient stimulus to activate and induce the maturation of B cells, as evident in murine B cells which up-regulate MHC-II, CD80 and CD86 in response to LPS and CpG ODN (Barr et al., 2007). It appears that a similar response occurs in chicken bursal cells. All three of the TLR ligands induced the up-regulation of both MHC-II and CD80. Moreover, the results suggested that CpG induced the highest degree of alteration in the expression of MHC-II and CD80, as evident through the sustained up-regulation of these genes across all sampling time points. Generally, CpG ODN are categorized as class A, B or C depending on the sequence flanking the ODN (Vollmer et al., 2004). Of the three classes, class B CpG ODN are the most immunostimulatory for B cells, as evident through their increased cytokine production and enhanced antigen presenting capabilities (Krug et al., 2001). Interestingly, our findings indicate that the lower dose of CpG ODN induced a greater up-regulation of MHC II and CD80 compared to that of the higher dose, a phenomenon which has been previously observed in mammalian B cells (Lampropoulou et al., 2008; Agrawal and Gupta, 2011). Indeed, the effects of CpG ODN on promoting the up-regulation of MHC-II and CD80 in bursal cells may be one explanation as to why CpG ODN induces a robust antibody-mediated response in chickens when administered as a vaccine adjuvant (Vleugels et al., 2002; Mallick et al., 2011). In addition to providing activation signals to CD4+ T cells via antigen presentation and cytokines, B cells also receive help from T cells through CD40/CD40 ligand interactions. Studies in mice have demonstrated the importance of CD40 ligand during the formation of germinal centers, and CD40 ligand also sustains the production of antibodies and promotes isotype switching (Lee et al., 2003). Moreover, CD40 activated B cells have proven to be efficacious antigen presenting cells, inducing the clonal expansion of T cells (von Bergwelt-Baildon, 2002). As our results suggest, treatment with TLR ligands up-regulates CD40 transcripts, thereby raising the possibility that TLR activated bursal cells are more responsive to CD40 ligand, although further studies are needed to confirm this. These findings are similar to those in mammals, as an up-regulation of CD40 has previously been reported following treatment of B cells with TLR ligands (Jiang et al., 2007). However, we also noted that poly I:C has a down-regulatory activity for CD40 expression. Although the reason for this is unknown, it may be attributed in part to the large up-regulation of IL-10 in response to poly I:C treatment, as IL-10 has been shown to down-regulate CD40 expression in mice (Qin et al., 2006). In conclusion, we have demonstrated that chicken bursal cells respond to treatment with ligands for TLRs 3, 4 and 21 by up-regulating IFN-␥ and IL-10 transcripts, in addition to up-regulating transcripts of genes associated with antigen presentation, namely CD80 and MHC II. Future studies may consider employing these ligands as adjuvants
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in chickens to enhance the immunogenicity of vaccines. Furthermore, detailed molecular mechanisms of chicken B cell responses to TLR ligands needs to be elucidated. Funding Michael St. Paul was a recipient of the Ontario Ministry of Agriculture and Rural Affairs (OMAFRA) HQP Scholarship and an Ontario Graduate Scholarship in Science and Technology. This work was funded by Natural Sciences and Engineering Research Council of Canada, Poultry Industry Council, and OMAFRA. Conflict of interest statement The authors declare that there is no conflict of interest. Acknowledgments The authors would like to thank Hamid Haghighi and Jennifer Brisbin for their assistance. References Agrawal, S., Gupta, S., 2011. TLR1/2, TLR7, and TLR9 signals directly activate human peripheral blood naive and memory B cell subsets to produce cytokines, chemokines, and hematopoietic growth factors. J. Clin. Immunol. 31, 89–98. Akira, S., Takeda, K., Kaisho, T., 2001. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat. Immunol. 2, 675–680. Barr, T.A., Brown, S., Ryan, G., Zhao, J., Gray, D., 2007. TLR-mediated stimulation of APC: distinct cytokine responses of B cells and dendritic cells. Eur. J. Immunol. 37, 3040–3053. von Bergwelt-Baildon, M.S., 2002. Human primary and memory cytotoxic T lymphocyte responses are efficiently induced by means of CD40activated B cells as antigen-presenting cells: potential for clinical application. Blood 99, 3319–3325. Booth, J., Wilson, H., Jimbo, S., Mutwiri, G., 2010. Modulation of B cell responses by Toll-like receptors. Cell Tissue Res., 131–140. Booth, J.S., Griebel, P.J., Babiuk, L.A., Mutwiri, G.K., 2009. A novel regulatory B-cell population in sheep Peyer’s patches spontaneously secretes IL-10 and downregulates TLR9-induced IFNalpha responses. Mucosal Immunol. 2, 265–275. Davison, F., Kaspers, B., Schat, K.A., 2008. Avian Immunology. Elsevier. Douagi, I., Gujer, C., Sundling, C., Adams, W.C., Smed-Sörensen, A., Seder, R.A., Karlsson Hedestam, G.B., Loré, K., 2009. Human B cell responses to TLR ligands are differentially modulated by myeloid and plasmacytoid dendritic cells. J. Immunol. 182, 1991–2001. Fillatreau, S., Gray, D., Anderton, S.M., 2008. Not always the bad guys: B cells as regulators of autoimmune pathology. Nat. Rev. Immunol. 8, 391–397. Genestier, L., Taillardet, M., Mondiere, P., Gheit, H., Bella, C., Defrance, T., 2007. TLR agonists selectively promote terminal plasma cell differentiation of B cell subsets specialized in thymus-independent responses. J. Immunol. 178, 7779–7786. Giordani, L., Sanchez, M., Libri, I., Quaranta, M.G., Mattioli, B., Viora, M., 2009. IFN-alpha amplifies human naive B cell TLR-9-mediated activation and Ig production. J. Leuk. Biol. 86, 261–271. Good, K.L., Avery, D.T., Tangye, S.G., 2009. Resting human memory B cells are intrinsically programmed for enhanced survival and responsiveness to diverse stimuli compared to naive B cells. J. Immunol. 182, 890–901. Gururajan, M., Jacob, J., Pulendran, B., 2007. Toll-like receptor expression and responsiveness of distinct murine splenic and mucosal B-cell subsets. PloS One 2, e863. Han, Y., Niu, M., An, L., Li, W., 2010. Involvement of TLR21 in baculovirusinduced interleukin-12 gene expression in avian macrophage-like cell line HD11. Vet. Microbiol. 144, 75–81. Harris, D.P., Haynes, L., Sayles, P.C., Duso, D.K., Eaton, S.M., Lepak, N.M., Johnson, L.L., Swain, S.L., Lund, F.E., 2000. Reciprocal regulation of
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Research paper
Characterization of responses elicited by Toll-like receptor agonists in cells of the bursa of Fabricius in chickens Michael St. Paul, Sarah Paolucci, Leah R. Read, Shayan Sharif ∗ Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada N1G 2W1
a r t i c l e
i n f o
Article history: Received 15 June 2012 Received in revised form 12 July 2012 Accepted 17 July 2012 Keywords: B cells Bursa of Fabricius Chicken Innate response Toll-like receptor TLR3 TLR4 TLR21
a b s t r a c t Toll-like receptors (TLRs) are an evolutionarily conserved group of pattern recognition receptors that play an important role in mediating host-responses to pathogens. Several TLRs have been identified in chickens and their expression has been detected in many immune cell subsets including in B cells. However, the mechanisms through which TLRs modulate B cell responses have not been well characterized in chickens. The aim of the present study was to elucidate the responses mounted by cells of the bursa of Fabricius to treatment with the TLR 3, 4 and 21 ligands, poly I:C, lipopolysaccharide (LPS) and CpG oligodeoxynucleotides (ODN), respectively. The relative expression of several immune system genes was quantified at 1, 3, 8 and 18 h post-treatment. The results show that all three ligands induced the up-regulation of interferon (IFN)-␥ and interleukin (IL)-10 transcripts and promoted the up-regulation of transcripts associated with antigen presentation, namely CD80 and major histocompatibility complex (MHC) class II. Furthermore, the results indicated that LPS and poly I:C induced the greatest IFN-␥ and IL-10 responses, respectively, while CpG ODN was the most efficacious inducer of CD80 and MHC-II expression. Future studies may be aimed at elucidating the mechanisms of TLR-mediated activation of chicken B cells. These mechanisms may be then exploited for the development of adjuvants with enhanced ability to stimulate B cell responses. © 2012 Elsevier B.V. All rights reserved.
1. Background Toll-like receptors (TLRs) are an evolutionarily conserved group of pattern recognition receptors that play an important role in mediating host-responses to pathogens (Akira et al., 2001; Medzhitov, 2001). The ligands for TLRs are typically conserved structural motifs expressed by microbes, termed pathogen-associated molecular patterns (PAMPs). To date, several TLRs have been identified in chickens, each responding to different PAMPs. For example, TLR3 binds double-stranded RNA, a product of some viruses during their replication cycle, while TLR4 binds lipopolysaccharide (LPS), which is present in the
∗ Corresponding author. Tel.: +1 519 824 4120x54641; fax: +1 519 824 5930. E-mail address: [email protected] (S. Sharif). 0165-2427/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetimm.2012.07.008
cell wall of Gram-negative bacteria, and TLR21 binds unmethylated CpG DNA motifs found in the nucleic acids of certain bacteria and viruses (Leveque et al., 2003; Temperley et al., 2008; Keestra et al., 2010). TLRs have been identified in many cell subsets of both the innate and adaptive immune system including macrophages, heterophils, T cells and B cells (Iqbal et al., 2005). Responses mediated by interactions between TLRs and their ligands typically include the production of cytokines and cellular activation (Hopkins and Sriskandan, 2005). In the case of mammalian B cells, treatment with TLR ligands induces the production of immunoglobulins and cytokines, such as interferon (IFN)-␥ and interleukin (IL)-10 (Barr et al., 2007; Giordani et al., 2009). Moreover, TLR stimulation facilitates antibody isotype switching (Jegerlehner et al., 2007) and promotes the proliferation of B cells and enhances their antigen presentation capabilities (Jiang
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et al., 2007). To date, several TLRs have been identified in mammalian B cells as reviewed by Booth et al. (2010). Although extensively studied in mammals, the TLRmediated responses of B cells have not been well characterized in chickens. It has been previously shown that chicken B cells express TLRs 2, 3, 4, 5, 7 and 21 at the transcript level (Iqbal et al., 2005; Han et al., 2010), and they proliferate in response to CpG oligodeoxynucleotides (ODN) (Wattrang, 2009). Additionally, our group has previously found that chicken B cells up-regulate certain immune system genes in response to LPS (Sarson et al., 2007). Although these studies suggest that chicken B cells may express functional TLRs 4 and 21, the responses of chicken B cells to stimulation with these TLR ligands and others have not been well examined. The bursa of Fabricius in chickens is the primary site for development and differentiation of B cells and approximately 98% of bursal lymphocytes are B cells (Davison et al., 2008). Therefore, the present study was an attempt to elucidate and characterize the responses of bursal cells to treatment with the TLR3, 4 and 21 ligands, poly I:C, LPS and CpG ODN, respectively. The results show that all three ligands induced the transcriptional up-regulation of IFN-␥ and IL-10, and also promoted the up-regulation of transcripts associated with antigen presentation, namely CD80 and major histocompatibility complex (MHC) class II. 2. Methods 2.1. Chickens Four-week-old broiler chickens (n = 6) were procured from the Arkell Poultry Research Center, University of Guelph (Guelph, ON). This research was approved by the University of Guelph Animal Care Committee and complied with the guidelines of the Canadian Council on Animal Care. 2.2. B cell isolation The bursa of Fabricius was collected from 6 chickens, minced and filtered through a 40 m nylon cell strainer to obtain a single cell suspension. The suspension was overlaid onto a Histopaque-1077 (Sigma–Aldrich, Oakville, ON) gradient and centrifuged at 400 × g for 30 min, and the cells were harvested from the plasma-Histopaque interface and washed 3× in RPMI-1640 (Invitrogen, Burlington, ON) supplemented with 10% heat-inactivated fetal bovine serum, 200 U/mL penicillin, 80 g/mL streptomycin, 25 mg gentamicin, 10 mM HEPES buffer, 50 M -mercaptoethanol, and 2 mM l-glutamine. Bursal cells were seeded into 48well plates at 1 × 107 cells/mL for in vitro stimulation with TLR ligands. As indicated before, 98% of lymphocytes in the chicken bursa of Fabricius are B cells (Davison et al., 2008), therefore, the cell cultures used in this study were highly enriched for B cells. 2.3. TLR ligands Poly I:C and LPS from Escherichia coli 0111:B4 were (Oakville, purchased from Sigma–Aldrich-Canada ON), while synthetic class B CpG ODN 2007
[5 -TCGTCGTTGTCGTTTTGTCGTT-3 ] and non-CpG ODN [5 -TGCTGCTTGTGCTTTTGTGCTT-3 ] were purchased from Eurofins MWG Operon (Ebersberg, GER). All of the ligands used were re-suspended in sterile phosphate buffered saline (PBS, pH 7.4) and diluted to working concentrations in RPMI medium of the same formulation used to culture bursal cells. 2.4. Experimental design Bursal cells from 6 chickens were stimulated with either a low or high dose of the TLR3 ligand poly I:C (5 g/mL and 50 g/mL), the TLR4 ligand LPS (0.5 g/mL and 5 g/mL) and the TLR21 ligand CpG ODN (0.5 g/mL and 5 g/mL), while control groups received either nonCpG ODN (5 g/mL) or medium. These doses were selected as they have been shown to be immunostimulatory for several different chicken cell populations (Kogut et al., 2005; Keestra and van Putten, 2008; Villanueva et al., 2011; He et al., 2012). At 1, 3, 8 and 18 h post-stimulation, cells were harvested for RNA extraction. 2.5. RNA extraction and cDNA synthesis Total RNA was extracted from the bursal cells using TRIzol® (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol and treated with DNA Free® (Ambion, Austin, TX) DNAse. Subsequently, 500 ng of purified RNA was reverse transcribed to cDNA using Superscript® II First Strand Synthesis kit (Invitrogen, Carlsbad, CA) and oligodT primers, according to the manufacturers recommended protocol. The resulting cDNA was subsequently diluted 1:10 in DEPC treated water. 2.6. Quantitative real-time PCR Quantitative real-time PCR using SYBR Green was performed on diluted cDNA using the LightCycler® 480 II (Roche Diagnostics GmbH, Mannheim, GER) as previously described (Villanueva et al., 2011). Briefly, each reaction involved a pre-incubation at 95 ◦ C for 10 min, followed by 45 cycles of 95 ◦ C for 10 min, 55–64 ◦ C (TA as per primer) for 5 s, and elongation at 72 ◦ C for 10 s. Subsequent melt curve analysis was performed by heating to 95 ◦ C for 10 s, cooling to 65 ◦ C for 1 min, and heating to 97 ◦ C. Many of these primers have been used in previous studies, and the new ones were designed using the NCBI Primer-Blast (St. Paul et al., 2011, 2012). Primers were synthesized by Sigma–Aldrich-Canada (Oakville, ON), and their specific sequences and accession numbers are outlined in Table 1. 2.7. Data analysis Relative expression levels of all genes was calculated relative to the housekeeping gene -actin using the LightCycler® 480 Software (Roche Diagnostics GmbH, Mannheim, GER), based on the formula developed by Pfaffl (2001). Data represent mean fold change from medium treated controls ± standard error. Statistical significance
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Table 1 Primer sequences and accession numbers used for quantitative real-time PCR. Target gene
Primer sequence
GenBank accession number
TLR3
F: 5 -TCAGTACATTTGTAACACCCCGCC-3 R: 5 -GGCGTCATAATCAAACACTCC-3 F: 5 -TGCCATCCCAACCCAACCACAG -3 R: 5 -ACACCCACTGAGCAGCACCAA -3 F: 5 -CCTGCGCAAGTGTCCGCTCA-3 R: 5 -GCCCCAGGTCCAGGAAGCAG-3 F: 5 -ACACTGACAAGTCAAAGCCGCACA-3 R: 5 -AGTCGTTCATCGGGAGCTTGGC-3 F: 5 -TGTGCCCACGCTGTGCTTACA-3 R: 5 -CTTGTGGCAGTGCTGGCTCTCC-3 F: 5 -AGCAGATCAAGGAGACGTTC-3 R: 5 -ATCAGCAGGTACTCCTCGAT-3 F: 5 -CCTGGTGATGCTGTGAATTG-3 R: 5 -CTTCTGTGTCGTTGCATTCAG-3 F: 5 -CTGTTCCTTCACATCCTGAGAG -3 R: 5 -CTTCAACACCATCTATTTGCCAG-3 F: 5 -CCACGGACGTGATGCAGAAC-3 R: 5 -ACCGCGCAGGAACACGAAGA-3 F: 5 -CAACACAGTGCTGTCTGGTGGTA-3 R: 5 -ATCGTACTCCTGCTTGCTGATCC-3
DQ780341
TLR4 TLR21 IFN-␥ IL-4 IL-10 CD40 CD80 MHC II -Actin
between treatment groups and control groups was calculated using a paired student’s t-test. 3. Results To examine the effects of TLR ligand stimulation on the expression of TLRs, we quantified the transcript levels of TLRs 3, 4 and 21 (Fig. 1). TLR3 transcripts were up-regulated at 3 h (p ≤ 0.05) and 8 h (p ≤ 0.05) postlow dose poly I:C treatment (5 g/mL), while high dose poly I:C (50 g/mL) up-regulated TLR3 transcripts at 3 h post-treatment (p ≤ 0.01) (Fig. 1A). TLR4 transcripts were down-regulated at 1 h post-low dose (0.5 g/mL) LPS treatment (p ≤ 0.05), while subsequently up-regulated at 3 h post-treatment (p ≤ 0.01) (Fig. 1B). Treatment with the high dose of LPS (5 g/mL), however, did not lead to any downregulation in gene expression, and only up-regulated TLR4 transcripts at 3 h post-treatment (p ≤ 0.05). Treatment with the low dose of CpG ODN (0.5 g/mL) up-regulated TLR21 transcripts at 1 h (p ≤ 0.05), 3 h (p ≤ 0.05) and 18 h (p ≤ 0.05) post-stimulation, while high dose CpG ODN (5 g/mL) treatment up-regulated TLR21 transcripts at all the time points (Fig. 1C). As TLR stimulation is known to up-regulate IFN-␥ and IL-10 in mammalian B cells (Barr et al., 2007; Giordani et al., 2009), we decided to see if this was the case in chicken bursal cells (Fig. 2). Treatment with the low dose of poly I:C significantly up-regulated IFN-␥ transcripts at 1 h post-treatment (p ≤ 0.01), while transcripts were upregulated in response to treatment with the high dose of poly I:C at 1 h (p ≤ 0.01), 3 h (p ≤ 0.05) and 8 h (p ≤ 0.05) post-treatment (Fig. 2A). Treatment with the low dose of LPS up-regulated IFN-␥ transcripts at 1 h (p ≤ 0.01) and 18 h (p ≤ 0.05) post-treatment, and treatment with the high dose of LPS up-regulated transcripts at 1 h (p ≤ 0.01), 8 h (p ≤ 0.05) and 18 h post-treatment (p ≤ 0.01) (Fig. 2B). IFN-␥ transcripts were up-regulated at 1 h post-treatment with both the low dose (p ≤ 0.01) and high dose (p ≤ 0.05) of CpG ODN (Fig. 2C). Treatment with only the high
AY064697 AJ720600.1 X99774 AJ621249.1 AJ621614 AJ293700 Y08823 113206149 X00182
dose of poly I:C significantly up-regulated IL-10, and this occurred at both 1 h post-treatment (p ≤ 0.01) and 18 h post-treatment (p ≤ 0.05) (Fig. 2D). Similar results were observed in response to LPS, as only the high dose of LPS upregulated IL-10 at 1 h post-treatment (p ≤ 0.05) (Fig. 2E). In response to CpG ODN treatment, the low dose up-regulated IL-10 at 1 h post-treatment (p ≤ 0.05) (Fig. 2F). Additionally, as mammalian B cells are known to produce IL-4 (Harris et al., 2000), we attempted to quantify IL-4 expression in the present study, however, there was very little or no IL4 expression in bursal cells cultured with or without TLR stimulation (data not shown). In addition to cytokines, TLR stimulation in mammalian B cells has been shown to up-regulate surface proteins, namely CD40, CD80 and MHC-II (Barr et al., 2007). As demonstrated in Fig. 3, this also occurs in chicken bursal cells. Treatment with the low dose of poly I:C up-regulated CD40 transcripts at 1 h post-treatment (p ≤ 0.01) while down-regulating them at 8 h (p ≤ 0.05) post-treatment (Fig. 3A). Similar results were observed in response to the high dose poly I:C treatment, as transcripts were up-regulated at 1 h (p ≤ 0.01) and 3 h (p ≤ 0.01) post-treatment, while down-regulated at 8 h post-treatment (p ≤ 0.05). Treatment with the low dose of LPS up-regulated CD40 transcripts at 1 h post-treatment (p ≤ 0.05), while treatment with the high dose of LPS upregulated transcripts at 1 h (p ≤ 0.01) and 3 h (p ≤ 0.01) post-treatment (Fig. 3B). Treatment with the low dose of CpG ODN up-regulated CD40 at 1 h post-treatment (p ≤ 0.05) (Fig. 3C). However, treatment with non-CpG ODN significantly down-regulated CD40 transcripts at 3 h post-treatment (p ≤ 0.05). Moreover, treatment with only the low dose of poly I:C up-regulated CD80, and this occurred at 1 h post-treatment (p ≤ 0.01) (Fig. 3D). Similar results were obtained in the case of bursal cell responses to LPS, as only the low dose of LPS upregulated CD80 and this also occurred at 1 h post-treatment (p ≤ 0.05) (Fig. 3E). However, treatment with the low dose of CpG ODN significantly up-regulated CD80 at all
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Fig. 1. Relative gene expression of TLR3, 4 and 21 transcripts in chicken bursal cells. Bursal cells were collected at 1, 3, 8 and 18 h post-treatment with the TLR ligands poly I:C (5 g/mL and 50 g/mL), LPS (0.5 g/mL and 5 g/mL) and CpG ODN (0.5 g/mL and 5 g/mL). The control groups received either non-CpG ODN (5 g/mL) or medium. Data represent mean fold change from medium treated controls ± standard error. Statistical significance between treatment groups and control groups was calculated using a paired student’s t-test and was considered statistically significant from the medium control group if p ≤ 0.05 (*) or p ≤ 0.01 (**), and in the case of CpG ODN, considered statistically significant from the non-CpG ODN control group if p ≤ 0.05 (#).
sampling time points (p ≤ 0.05), while treatment with the high dose of CpG ODN significantly up-regulated CD80 solely at 18 h post-treatment (p ≤ 0.05) (Fig. 3F). Treatment with non-CpG ODN significantly down-regulated CD80 at 3 h post-treatment (p ≤ 0.05). Lastly, treatment with the low dose of poly I:C significantly up-regulated MHC II
transcripts at 3 h (p ≤ 0.05) and 18 h (p ≤ 0.05) posttreatment (Fig. 3G). Treatment with the low dose of LPS significantly up-regulated MHC II transcripts at 1 h posttreatment (p ≤ 0.01), while the high dose LPS treatment up-regulated transcripts at 1 h (p ≤ 0.05), 3 h (p ≤ 0.05) and 18 h (p ≤ 0.05) post-treatment (Fig. 3H). Treatment with
Fig. 2. Relative gene expression of IFN-␥ and IL-10 transcripts in chicken bursal cells. Bursal cells were collected at 1, 3, 8 and 18 h post-treatment with the TLR ligands poly I:C (5 g/mL and 50 g/mL), LPS (0.5 g/mL and 5 g/mL) and CpG ODN (0.5 g/mL and 5 g/mL). The control groups received either non-CpG ODN (5 g/mL) or medium. Data represent mean fold change from medium treated controls ± standard error. Statistical significance between treatment groups and control groups was calculated using a paired student’s t-test and was considered statistically significant from the medium control group if p ≤ 0.05 (*) or p ≤ 0.01 (**), and in the case of CpG ODN, considered statistically significant from the non-CpG ODN control group if p ≤ 0.05 (#).
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Fig. 3. Relative gene expression of CD40, CD80 and MHC II transcripts in chicken bursal cells. Bursal cells were collected at 1, 3, 8 and 18 h post-treatment with the TLR ligands poly I:C (5 g/mL and 50 g/mL), LPS (0.5 g/mL and 5 g/mL) and CpG ODN (0.5 g/mL and 5 g/mL). The control groups received either non-CpG ODN (5 g/mL) or medium. Data represent mean fold change from medium treated controls ± standard error. Statistical significance between treatment groups and control groups was calculated using a paired student’s t-test and was considered statistically significant from the medium control group if p ≤ 0.05 (*) or p ≤ 0.01 (**), and in the case of CpG ODN, considered statistically significant from the non-CpG ODN control group if p ≤ 0.05 (#).
the low dose of CpG ODN significantly up-regulated MHC II transcripts at all sampling time points (p ≤ 0.05), while treatment with the high dose of CpG ODN up-regulated MHC II transcripts at 1 h (p ≤ 0.05) and 3 h (p ≤ 0.05) posttreatment, and treatment with non-CpG ODN significantly up-regulated MHC II transcripts at 1 h post-treatment (p ≤ 0.01) (Fig. 3I). 4. Discussion TLR stimulation of mammalian B cells promotes the production of cytokines and enhances their antigen presentation capabilities (Barr et al., 2007; Jiang et al., 2007). Indeed, as the present study suggests, similar responses may also occur in chicken bursal cells. It has been established that chicken B cells express several TLRs at the transcript level including TLRs 3, 4 and 21 (Iqbal et al., 2005; Han et al., 2010). These findings are similar to what is found in mammals, except with respect to TLR3. For the most part, mammalian B cells express little TLR3 and typically do not respond to poly I:C, as suggested by their lack of proliferation and up-regulation of co-stimulatory molecules (Genestier et al., 2007; Gururajan et al., 2007; Douagi et al., 2009). This is in contrast to our findings in chicken bursal cells, as poly I:C treatment induced a large (nearly 10-fold) up-regulation of
TLR3 transcripts, in addition to up-regulating several other genes, such as IL-10. In mammals, B cells produce a variety of cytokines which function to modulate several aspects of the immune system, including T cell and inflammatory responses (Harris et al., 2000). Of note are the cytokines IFN-␥, IL-4 and IL-10. Interferon-␥ is a cytokine with several functions, including activating anti-viral cell subsets, namely CD8+ T cells and natural killer cells, in addition to promoting the up-regulation of several interferon inducible genes and modulating the immune response towards a T-helper (TH )-1 bias (Romagnani, 1997; Samuel, 2001). This is in contrast to IL-4, which promotes a TH 2 biased response. In mammals, TLR stimulation in B cells largely drives the up-regulation of IFN-␥, while having little effect on IL-4 (Agrawal and Gupta, 2011). We observed a similar result in chicken bursal cells, as IFN-␥ transcripts were up-regulated in response to poly I:C, LPS and CpG ODN treatment, while none of the ligands up-regulated IL-4 transcripts. Furthermore, it appears that of the TLR ligands tested, LPS induced the greatest up-regulation of IFN-␥. This finding raises the possibility that B cells might be one of the contributing sources to IFN-␥ expression in the spleens of LPS treated chickens (St. Paul et al., 2011). As such, future studies may consider employing LPS as an adjuvant to elicit a TH 1biased response, as studies in mice have highlighted the
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importance of IFN-␥ producing B cells in promoting the differentiation of naive CD4+ T into TH 1 cells during antigen presentation (Harris et al., 2000). Being an anti-inflammatory and regulatory cytokine, one function of IL-10 is to modulate the immune response through regulating the function of T cells, macrophages and dendritic cells (Saraiva and O’Garra, 2010). To this end, IL-10 producing B cells contribute to the downregulation of the deleterious immune responses associated with several autoimmune diseases including experimental autoimmune encephalomyelitis, inflammatory bowel disease and ulcerative colitis (Fillatreau et al., 2008). In mammals, TLR signaling is one stimulus that induces B cells to produce IL-10, as observed in murine B cells following treatment with LPS and CpG ODN (Lampropoulou et al., 2008; Agrawal and Gupta, 2011). Our results suggest that this may also be the case in chicken bursal cells, as we observed an up-regulation of IL-10 transcripts in response to all three TLR ligands. Although only a modest up-regulation was observed in response to LPS and CpG ODN, treatment with poly I:C induced a large up-regulation (over fivefold) of IL-10 transcripts. Furthermore, it appears that the production of IL-10 in response to a TLR ligand treatment is also dependent on the dose of the ligand. For example, it is clear from our results that both doses of poly I:C are immunostimulatory for B cells, yet only the high dose significantly up-regulated IL-10 transcripts. Similarly, only the low dose of CpG ODN significantly upregulated IL-10, while the high dose did not. Indeed, a similar phenomenon occurs in mammalian B cells, as in some instances the lower doses of CpG ODN and other TLR ligands induce a greater production of IL-10 compared to a higher dose (Agrawal and Gupta, 2011). Although the mechanisms behind this finding is not known, it highlights the importance of selecting an appropriate dose of TLR ligands when administering them as vaccine as adjuvants in order to achieve the desired result. In addition to the dose of the TLR ligand, the IL-10 responses of mammalian B cells may also be dependent on the tissue of origin of the B cells. As indicated by Booth and colleagues, B cells isolated from the peripheral blood do not produce IL-10, even after stimulation with TLR ligands or CD40 ligand, which is in contrast to B cells isolated from Peyer’s patches, as the latter cells constitutively produce IL-10 (Booth et al., 2009). This phenomenom may be attributed to the phenotype of each tissue’s resident B cells. For instance, unlike in the peripheral blood, Peyer’s patches contain many regulatory B cells (Booth et al., 2009). Furthermore, the activation state of B cells is known to influence the IL-10 response as well. In support of this notion, Agrawal and colleagues found that naïve human B cells produce significantly less IL-10 compared to that of memory B cells in response to TLR ligands (Agrawal and Gupta, 2011). In the present study, the B cells found in the bursal cell population must have included a significant number of developing and naïve B cells. Considering that, future studies may explore whether the IL-10 responses of chicken B cells to TLR ligands varies depending on the type of B cells (regulatory or effector) in addition to their activation state (naïve, memory or plasma cell).
In general, B cells are capable of presenting antigens via MHC-II to CD4+ T cells (Rivera et al., 2001). However, naïve B cells do not constitutively express high levels of MHC-II or the co-stimulatory molecule CD80, and require a degree of activation and maturation in order to obtain their antigen presenting capabilities (Good et al., 2009). In mammals, it has been shown that TLR ligands provide a sufficient stimulus to activate and induce the maturation of B cells, as evident in murine B cells which up-regulate MHC-II, CD80 and CD86 in response to LPS and CpG ODN (Barr et al., 2007). It appears that a similar response occurs in chicken bursal cells. All three of the TLR ligands induced the up-regulation of both MHC-II and CD80. Moreover, the results suggested that CpG induced the highest degree of alteration in the expression of MHC-II and CD80, as evident through the sustained up-regulation of these genes across all sampling time points. Generally, CpG ODN are categorized as class A, B or C depending on the sequence flanking the ODN (Vollmer et al., 2004). Of the three classes, class B CpG ODN are the most immunostimulatory for B cells, as evident through their increased cytokine production and enhanced antigen presenting capabilities (Krug et al., 2001). Interestingly, our findings indicate that the lower dose of CpG ODN induced a greater up-regulation of MHC II and CD80 compared to that of the higher dose, a phenomenon which has been previously observed in mammalian B cells (Lampropoulou et al., 2008; Agrawal and Gupta, 2011). Indeed, the effects of CpG ODN on promoting the up-regulation of MHC-II and CD80 in bursal cells may be one explanation as to why CpG ODN induces a robust antibody-mediated response in chickens when administered as a vaccine adjuvant (Vleugels et al., 2002; Mallick et al., 2011). In addition to providing activation signals to CD4+ T cells via antigen presentation and cytokines, B cells also receive help from T cells through CD40/CD40 ligand interactions. Studies in mice have demonstrated the importance of CD40 ligand during the formation of germinal centers, and CD40 ligand also sustains the production of antibodies and promotes isotype switching (Lee et al., 2003). Moreover, CD40 activated B cells have proven to be efficacious antigen presenting cells, inducing the clonal expansion of T cells (von Bergwelt-Baildon, 2002). As our results suggest, treatment with TLR ligands up-regulates CD40 transcripts, thereby raising the possibility that TLR activated bursal cells are more responsive to CD40 ligand, although further studies are needed to confirm this. These findings are similar to those in mammals, as an up-regulation of CD40 has previously been reported following treatment of B cells with TLR ligands (Jiang et al., 2007). However, we also noted that poly I:C has a down-regulatory activity for CD40 expression. Although the reason for this is unknown, it may be attributed in part to the large up-regulation of IL-10 in response to poly I:C treatment, as IL-10 has been shown to down-regulate CD40 expression in mice (Qin et al., 2006). In conclusion, we have demonstrated that chicken bursal cells respond to treatment with ligands for TLRs 3, 4 and 21 by up-regulating IFN-␥ and IL-10 transcripts, in addition to up-regulating transcripts of genes associated with antigen presentation, namely CD80 and MHC II. Future studies may consider employing these ligands as adjuvants
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