QS207. Intestinal Injury Induced by Ischemia/Reperfusion Requires Activation of Toll-Like Receptor 4

Abstract

Introduction: Intestinal ischemia/reperfusion injury (IRI) is a major clinical problem in surgery and transplantation. Toll-like receptor 4 (TLR4) has been implicated as a potential link between the innate and adaptive immune systems in this complex process, however very little data exists regarding TLR4 in intestinal IRI. We have previously demonstrated in a pilot study that TLR4 is up-regulated after intestinal IRI. Compared to sham controls, wild-type (WT) mice undergoing intestinal IRI had decreased survival, significant histopathological injury, increased myeloperoxidase activity and infiltration of CD3+ cells, as well as increased production of several chemokines and adhesion molecules. Here for the first time we use TLR4 knockout (TLR4KO) mice to further investigate the role of TLR4 in intestinal IRI and its effects on cytokine/chemokine programs. Methods: Under anesthesia, male C57BL10 WT and TLR4KO mice underwent 100 min of warm intestinal IRI. Total jejunoileal ischemia was induced by placing atraumatic clamps on the superior mesenteric and ileocolic arteries and ligating collateral vessels. Separate survival and analysis groups were performed, and intestinal tissue was harvested at 4h and 24h. Analysis included histopathology, immunostaining for CD3, myeloperoxidase activity (MPO), and PCR for cytokines, chemokines, and adhesion molecules. Results: TLR4KO mice had markedly improved survival over WT mice (100% vs. 33%, p < 0.05). On histopathological examination, TLR4KO mice had near normal-appearing villous architecture, while WT mice had significant histopathological injury including mucosal erosions and villous congestion/hemorrhage. Immunostaining for CD3 revealed less infiltration of CD3+ cells in the TLRKO mice as compared to WT (3.9 +/− 0.7 vs. 6.2 +/− 1.4 per hpf at 4h, p < 0.001; and 4.8 +/− 1.1 vs. 7.0 +/− 2.3 per hpf at 24h, p < 0.02). Additionally, ischemic intestinal tissue from TLR4KO mice had decreased neutrophil myeloperoxidase activity (0.11 +/− 0.06 vs. 0.88 +/− 0.5 U/g at 4h, p<0.005). There was also decreased production of mRNA for chemokines IP-10, MCP-1, RANTES and the cytokine IFN-γ. Interestingly, there was increased mRNA production for IL-13, STAT6, and MAdCAM-1 in the TLR4KO mice. Conclusion: This study is the first investigation of intestinal IRI in TLR4KO mice. Our results show that the absence of TLR4 is protective, demonstrating for the first time that TLR4 is required for IRI in the intestine. This TLR4-dependent cascade results in tissue injury, myeloperoxidase activity, expression of cytokines/chemokines, infiltration of T cells, and subsequent animal death. These data support findings in other organ models that TLR4 is a mechanistic link between the innate and adaptive immune systems. These results implicate TLR4 as a potential therapeutic target for the prevention of intestinal IRI.