S1762 TLR-3 Mediated Regulation of B7 Negative Co-Stimulators on Stromal Cells: A Potential Mechanism of Colonic Immune Evasion During Intestinal Viral Infection

BACKGROUND: Activation of TLR3 triggers specific signaling pathways leading to the activation of NF-kB and/or IRF3 transcription factors, enabling the host to mount innate immune responses against different dsRNA viruses and serve as a “danger” receptor in nonviral diseases. However, TLR3-mediated organ specific activation of the B7-coinhibitory pathway PD-L1/PD-1 can be exploited locally leading to the impairment in antiviral T effector cell responses promoting successful viral invasion of the host. Despite significant advances in understanding of the process of TLR3 signaling, the mechanisms by which TLR3 influence homeostasis and contribute to the B7 negative molecules-mediated viral invasion in the gut remain obscure. Human colonic stromal cells, A.k.a. myofibroblasts/fibroblasts (CMFs), are one of the important cell types taking part in innate immunity. CMFs express TLR3, and represent a major cell population expressing B7 negative costimulators PD-L1 and PD-L2 in normal colon. CMFs suppress activated CD4+ effector T cell responses in a PD-L1/2 dependent manner. We hypothesized that in human colonic mucosa, CMFs might be among the first cells to respond to TLR3 mediated stimuli via modulation of PD-L1/2 expression and, thus, may contribute to the impaired T effector cell responses associated with viral invasion. METHODS: TLR3, PD-L1/2 expression on human CMFs isolated from normal mucosa in response to the synthetic analog of TLR3 ligand, poly(I:C), was quantified using real-time RT-PCR, Western blot and FACS analysis. RESULTS: TLR3 stimulation on CMFs by poly(I:C) resulted in robust upregulation of PD-L1 and PD-L2 mRNA and cell surface protein. The expression of IFN-β precedes the upregulation of PD-L1 and PD-L2 mRNA. Stimulation of CMFs with IFN-β leads to the upregulation of the PD-L1 and PD-L2 protein expression (Westen blot analysis) suggesting involvement of IFN-β in the TLR3-mediated PD-L1/2 upregulation. Blocking of the MyD88 adaptor expression in CMFs by using specific siRNA did not affect PD-L2 upregulation in response to the TLR3 stimulation, but reduced the induction of the PD-L1. It is possible that TLR3mediated upregulation of PD-L1 on CMFs may be synergized by activation of heat shock proteins that are known to signal through the TLR4MyD88 adaptor pathway. CONCLUSION: The presented results indicate that CMFs are among the key players in innate immune responses in colonic mucosa. Our data suggest that TLR3 mediated PD-L1/2 upregulation on CMFs may favor suppression of T effector cells responses and this may allow the progression of host invasion during intestinal viral infection. Supported by AGA, CCFA foundations and NIDDK.