TLR2 and TLR4 Mediate Differential Responses to Limb Ischemia through MyD88-Dependent and Independent Pathways

INTRODUCTION The danger signal HMGB1 is released from ischemic myocytes, and mediates angiogenesis in the setting of hindlimb ischemia. HMGB1 is a ligand for innate immune receptors TLR2 and TLR4. While both TLR2 and TLR4 signal through myeloid differentiation factor 88 (MyD88), TLR4 also uniquely signals through TIR-domain-containing adapter-inducing interferon-β (TRIF). We hypothesize that TLR2 and TLR4 mediate ischemic myocyte regeneration and angiogenesis in a manner that is dependent on MyD88 signaling. METHODS Mice deficient in TLR2, TLR4, MyD88 and TRIF underwent femoral artery ligation in the right hindlimb. Laser Doppler perfusion imaging was used to assess the initial degree of ischemia and the extent of perfusion recovery. Muscle regeneration, necrosis and fat replacement at 2 weeks post-ligation were assessed histologically and vascular density was quantified by immunostaining. In vitro, endothelial tube formation was evaluated in matrigel in the setting of TLR2 and TLR4 antagonism. RESULTS While control and TLR4 KO mice demonstrated prominent muscle regeneration, both TLR2 KO and TRIF KO mice exhibited marked necrosis with significant inflammatory cell infiltrate. However, MyD88 KO mice had a minimal response to the ischemic insult with little evidence of injury. This observation could not be explained by differences in perfusion recovery which was similar at two weeks in all the strains of mice. TLR2 KO mice demonstrated abnormal vessel morphology compared to other strains and impaired tube formation in vitro. DISCUSSION TLR2 and TRIF signaling are necessary for muscle regeneration after ischemia while MyD88 may instead mediate muscle injury. The absence of TLR4 did not affect muscle responses to ischemia. TLR4 may mediate inflammatory responses through MyD88 that are exaggerated in the absence of TLR2. Additionally, the actions of TLR4 through TRIF may promote regenerative responses that are required for recovery from muscle ischemia.