Toll-like receptor ligands have previously been shown to modulate the production of cytokines in several chicken cell subsets, including in macrophages, heterophils and B cells [10–12]. Here, we show that a similar phenomenon may be extended to chicken CD4+ T cells.
In mammals, CD4+ T cells may be classified into several different subsets such as TH1, TH2, TH17 and regulatory T cells (TREG) [13]. In addition to producing a distinct profile of cytokines and performing different effector functions, each cell subset also expresses a different repertoire of TLRs. For example, TLR10 has been detected in human regulatory T cells, but not in non-regulatory CD4+ T cells [14]. Nevertheless, CD4+ T cells, in general, express transcripts for TLRs 2, 3, 4, 5, 7/8 and 9 in both mice and humans [9]. In chicken CD4+ T cells, it was shown that they also express TLRs transcripts including those for TLRs 2, 3 and 4 [3]. However, the study by Iqbal et al., (2005) used semi-quantitative PCR and as such, we employed real-time PCR to provide a more accurate quantification of TLR transcript levels (Figure 1). Our results suggest chicken CD4+ T cells express TLRs 2, 3, 4 and 21 at the transcript level, and not TLRs 5 and 7. Moreover, transcripts for TLR2 were the most abundant, followed by transcripts for TLR3 and lastly by TLRs 4 and 21. This therefore raises the possibility that chicken CD4+ T cells may have the potential to respond directly to PAMPs derived from both viral and bacterial pathogens.
Different mammalian CD4+ cell subsets produce a distinct profile of cytokines upon stimulation. These cytokines include IFN-γ, which is produced by TH1 cells and IL-4/13 which are produced by TH2 cells, as well as IL-17 and IL-10, which are produced by TH17 cells and TREGS, respectively. Although in chickens it is not yet known if such CD4+ T cell subsets exist, evidence accumulated over the last few years raises the possibility that at least some of these subsets might [15–17]. As such, in the present study, we examined the above cytokines to determine how TLR ligands modulate their expression (Figures 2 and 3).
In mammals, TLR2 ligands such as Pam3CSK4 have been shown to directly activate CD4+ T cells and induce their production of IFN-γ, in the absence of T cell receptor (TCR) signaling [5, 7]. Our results suggest that this may also be the case in chicken CD4+ T cells, as treatment with Pam3CSK4 significantly up-regulated IFN-γ transcripts at 3 (p ≤0.01) and 8 (p ≤0.01) hours post-treatment (Figure 2). This effect was not limited only to Pam3CSK4, as both poly I:C (p ≤0.01) and LPS (p ≤0.05) significantly enhanced IFN-γ transcripts levels as well (Figure 2). This is in contrast to what occurs in mammals, as poly I:C and LPS fail to directly up-regulate IFN-γ production in TH1 cells [7]. In fact, when combined with TCR stimulation, LPS inhibits IFN-γ production in mammalian T cells, which was shown to be mediated by the TIR-domain-containing adapter-inducing interferon-β (TRIF) pathway [18]. In chickens, emerging evidence suggests that both TLRs 3 and 4 signal through the TRIF pathway as indicated by a robust type I IFN response following treatment with these ligands [19–21]. However, as these ligands both up-regulated IFN-γ transcripts in the present study, this raises the possibility that there might be some differences between the chicken and mammalian TRIF pathways, potentially with respect to the accessory and signaling molecules involved. As such, future studies should be aimed at further elucidating the mechanisms involved in the TRIF signaling pathway in chickens.
We discovered that there was a significant down-regulation (controls were set to 1) of IFN-γ transcripts following treatment CpG ODN at 1 (p ≤0.01) and 8 (p ≤0.05) hours post-treatment (Figure 2). This is in contrast with what has been shown in mammalian CD4+ T cells, because CpG ODN enhances production of cytokines such as IL-2 and IFN-γ by these cells [22]. However, this enhanced cytokine production occurs only in conjunction with TCR signaling, and as such, future studies may consider exploring whether adding anti-chicken CD3 may alter the responses to CpG ODN and other TLR ligands.
In addition to enhancing IFN-γ production, TLR ligands may also modulate responses of other T cell subsets, such as TH17 cells. For example, treatment of naïve CD4+ T cells with Pam3CSK4 promotes their differentiation into TH17 cells [8]. Importantly, when fully differentiated TH17 cells are treated with TLR ligands including Pam3CSK4 and LPS, but not poly I:C, a significant increase in IL-17 production is observed [8]. However, this does not appear to be the case in chickens, as suggested by our results (Figure 3). We found that both poly I:C, and the high dose of CpG ODN (p ≤0.01) significantly down-regulated IL-17 transcripts at 1 hour and 8 hours post-treatment, respectively. Moreover, we found that treatment with the low dose of LPS significantly down-regulated IL-17 transcripts at 1 hour post-treatment (p ≤0.01), while the high dose significantly up-regulated IL-17 at 1 hour post-treatment (p ≤0.05). Although the reason behind this observation is not known, we have observed a similar down-regulation of murine natural killer T (NKT) cell activities in response to some TLR ligands, including CpG ODN, which we have attributed to a TLR-mediated increase in dual specific protein phosphatases (DUSPs) (Villanueva et al., unpublished data). As such, future studies may consider employing additional assays in order to examine the role of DUSPs in chicken TLR mediated responses.
In mammals, evidence suggests that TH2 cells are non-responsive to TLR ligands and fail to become activated and up-regulate the production of IL-4 [7]. This seems to also be the case in chickens, as we did not detect any significant up-regulation of IL-4 or IL-13 in response to any of the TLR ligand treatments (data not shown). In addition, we also did not detect any significant up-regulation of IL-10 either in response to Pam3CSK4 or any of the other TLR ligands (data not shown). Although in mammals IL-10 may be produced by TH2 cells as well as TREGS, in chickens, evidence suggests that stimulated CD4+ CD25+ regulatory T cells are the predominant source of IL-10, as they produce more than 30 times the amount of IL-10 when compared against stimulated CD4+ CD25- T cells [15]. Nevertheless, TLR ligands have been shown to directly activate mammalian TREGS and promote their proliferation and survival, however this occurred only in conjunction with TCR stimulation [4]. Therefore, we speculate that this lack of up-regulation in the chicken CD4+ T cells may be due to lack of TCR stimulation. However, there are a few other possible explanations that could be considered. For example, i) chicken TREGS may not respond to TLR ligands or ii) our T cell population may potentially be limited in its diversity and may have an oligoclonal or monoclonal nature. As a result, the population of T cells that we have used in the present study might have been devoid of TH2 and TREG populations.