| The toll-like receptors (TLRs) are a family of pattern recognition receptors (PRR) which provide a sensory mechanism for the detection of infectious threats. TLR9 recognizes bacterial DNA or synthetic CpG oligodeoxynucleotides (ODN). Cells that express TLR9 when stimulated with CpG ODN proliferate and produce Th1-like pro-inflammatory cytokines and upregulate co-stimulatory molecules. Our hypothesis is that innate immune responses to the TLR9 agonist CpG DN in Peyer's patches (PP) are attenuated compared to other tissues such as blood or lymph nodes.;We conducted a number of experiments to test this hypothesis. We initially assessed the immunostimulator activity of three available classes of CpU ODN in lymph nodes (LN), peripheral blood mononuclear cells (PBMC) and PP since this had not been done in ruminants. We found that CpU ODN induced strong IFNalpha, 1FNgamma, IL-12, lymphocyte proliferation and NK-like activity in LN and PBMC. In contrast. these responses were significantly less in PP stimulated with CpG ODN. We wondered whether the reduced responses of PP cells to CpG ODN were unique to the TLR9 agonist. For this reason we tested responses of cells from these tissues to poly (I:C), LPS. and single-stranded RNA, which are agonists for TLR3, TLR4, and TLR7/8 respectively. Additionally, we tested combinations of TLRs since others have reported that multiple TLR agonists may induce synergistic responses. All TLR agonists or their combinations either failed to induce detectable responses or the responses were significantly less in PP compared to other tissues. Thus we concluded that PP cells responses to TLR stimulation were attenuated. In all tissues tested, there were no synergistic responses (IFNalpha, IFNgamma and lymphocyte proliferation) following stimulation with combinations of agonists.;Finally, we examined the capacity of IL-10 secreting B cells (B regs) to respond to CpG ODN. To achieve this, we compared CD21 + B cells from blood and JPP. Unlike blood CD21+ B cells, CD21+ B cells from JPP proliferated poorly in response to CpG ODN. Moreover, PP CD21+ B cells, unlike blood CD21 + B cells, do not secrete IgM or IL-12 in response to CpG stimulation, although both PP and blood CD21+ B cells express similar level of TLR9 mRNA. Neutralization of IL-10 did not enhance CpG-induced proliferative responses in PP CD21+ B cells. Thus IL-10 does not play a direct role in the hyporesponsiveness of PP CD21+ B cells to CpG ODN. To further explore the mechanism by which PP Bregs fail to respond to CpG ODN stimulation, we used a kinome analysis to determine whether the TLR9 pathway was functional in PP Bregs compared to blood CD21 + B cells. We observed that peptides representing critical adaptor molecules downstream of TLR9 such as IRAK1, TAK1, Casp8, p-38 MAPK, JNK, FOS, IKKalpha, NFkappaB-p65 were not phosphorylated in JPP CD21+ B cells following CpG ODN stimulation.;In conclusion, we clearly demonstrated that TLR9-induced responses in cells from PP are significantly attenuated. This is a consequence of PP CD21 + B cells (Bregs) that spontaneously secrete IL-10, which in turn "conditions" an anti-inflammatory environment in this tissue leading to poor cytokine responses to the TLR9 agonist, CpG ODN. Additionally, we show that Bregs are unresponsiveness to TLR9 stimulation. This unresponsiveness is due to regulatory mechanisms in Bregs leading to a dysfunctional TLR9 signaling pathway. These may represent strategies by which PP dampen innate responses to pathogen-associated molecular patterns (PAMPs) in intestinal immune tissues to maintain intestinal immune homeostasis. These conclusions are consistent with our hypothesis that TLR responses in PP cells are attenuated. and this is due to B cell-mediated regulatory mechanisms that are unique to the intestinal microenvironment. (Abstract shortened by UMI.). |
