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

Abstract

IntroductionThe 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.MethodsMice 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.ResultsWhile 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.DiscussionTLR2 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.

TLR4 Deters Perfusion Recovery and Upregulates Toll-like Receptor 2 (TLR2) in Ischemic Skeletal Muscle and Endothelial Cells

“…In TLR2KO mice, we have shown that this recovery was not observed with persistence of necrotic myocytes and grossly abnormal vascular structures supporting the role of TLR2 in angiogenesis in vivo (9). This result was confirmed in TLR4KO mice, where perfusion and vascularity in the ischemic hindlimb were increased compared with WT mice.…”

“…*p < 0.03, TLR4KO to TLR2KO mice; 3-5 animals/group, ANOVA. (C) PMNs were identified on H and E sections by using characteristic nuclear morphology as described (9). Percent PMN nuclei relative to total nuclei is shown from tibialis anterior muscle obtained 3 d after FAL in control, TLR2/4 double KO, TLR2KO and TLR4KO mice (p value nonsignificant among the groups).…”

“…Whereas most have focused on the identification and testing of proangiogenic compounds or cellular therapies, there has been much less emphasis on evaluating the role of modulating inflammation to improve tissue recovery from the ischemic insult. We and others have investigated the role of toll-like receptor (TLR) activation in promoting angiogenesis and muscle recovery in a murine model of hindlimb ischemia (7)(8)(9)(10)(11)(12)(13)(14). TLRs mediate innate immune responses to bacterial components, including gram-negative bacterial lipopolysaccharide (LPS) (TLR4) and gram-positive bacterial lipoproteins (TLR2) (15)(16)(17)(18).…”

“…TLRs also sense endogenously released damageassociated molecular patterns (DAMPS), such as high-mobility group box-1 (HMGB1) to initiate an immune response (19). We have previously demonstrated that HMGB1, TLR4 and TLR2 modulate inflammation and muscle recovery after ischemia (8)(9)(10). TLR2 knockout (TLR2KO) mice develop abnormal vasculature, possibly influencing the regenerative process, whereas TLR4KO mice exhibit a robust regenerative phenotype.…”

Dynamic localization and the associated translocation mechanism of HMGBs in response to GCRV challenge in CIK cells

“…Extracellular HMGB1 interacts with cell surfaceexpressed receptors: RAGE, TLR2 and TLR4, which promote the activation of the MyD88-mediated NF-kB pathway. 7,[32][33][34] HMGB1 or HMGB2 released from necrotic cells can exert a proinflammatory effect by transmitting a damage signal to neighboring cells. 35,36 In the cytoplasm, HMGBs bind immunogenic nucleotides and deliver them to the cytosolic nucleic acid sensors retinoic acid-inducible gene I, MDA5, AIM2 and DAI and to the endosome nucleic acid-sensing TLRs (TLR3, TLR7 and TLR9).…”