Heme activates TLR4-mediated inflammatory injury via MyD88/TRIF signaling pathway in intracerebral hemorrhage

Abstract

BackgroundInflammatory injury plays a critical role in intracerebral hemorrhage (ICH)-induced neurological deficits; however, the signaling pathways are not apparent by which the upstream cellular events trigger innate immune and inflammatory responses that contribute to neurological impairments. Toll-like receptor 4 (TLR4) plays a role in inflammatory damage caused by brain disorders.MethodsIn this study, we investigate the role of TLR4 signaling in ICH-induced inflammation. In the ICH model, a significant upregulation of TLR4 expression in reactive microglia has been demonstrated using real-time RT-PCR. Activation of microglia was detected by immunohistochemistry, cytokines were measured by ELISA, MyD88, TRIF and NF-κB were measured by Western blot and EMSA, animal behavior was evaluated by animal behavioristics.ResultsCompared to WT mice, TLR4−/− mice had restrained ICH-induced brain damage showing in reduced cerebral edema and lower neurological deficit scores. Quantification of cytokines including IL-6, TNF-α and IL-1β and assessment of macrophage infiltration in perihematoma tissues from TLR4−/−, MyD88−/− and TRIF−/− mice showed attenuated inflammatory damage after ICH. TLR4−/− mice also exhibited reduced MyD88 and TRIF expression which was accompanied by decreased NF-κB activity. This suggests that after ICH both MyD88 and TRIF pathways might be involved in TLR4-mediated inflammatory injury possibly via NF-κB activation. Exogenous hemin administration significantly increased TLR4 expression and microglial activation in cultures and also exacerbated brain injury in WT mice but not in TLR4−/− mice. Anti-TLR4 antibody administration suppressed hemin-induced microglial activation in cultures and in the mice model of ICH.ConclusionsOur findings suggest that heme potentiates microglial activation via TLR4, in turn inducing NF-κB activation via the MyD88/TRIF signaling pathway, and ultimately increasing cytokine expression and inflammatory injury in ICH. Targeting TLR4 signaling may be a promising therapeutic strategy for ICH.

Hemolysis and free hemoglobin revisited: exploring hemoglobin and hemin scavengers as a novel class of therapeutic proteins

“…In addition, activation of TLRs and downstream inflammatory signaling, particularly of the TLR-4 pathway, can be triggered by free hemin in some models. [22][23][24] In summary, the definitive pathophysiology of extracellular Hb is dependent on timing, quantity, and tissue localization of Hb/ hemin exposure in a specific clinical condition and may result from cumulative effects largely described by the 4 mechanisms discussed in the sections above. For example, systemic and, to some extent, pulmonary hypertension is the most apparent and readily measurable effect of free Hb after intravascular hemolysis.…”

A20 Ameliorates Intracerebral Hemorrhage–Induced Inflammatory Injury by Regulating TRAF6 Polyubiquitination

“…We previously showed that inflammatory injury can be induced by the activation of TLR2/TLR4 dimers, which subsequently activated NF-kB through MyD88 signaling (30,31). Recent studies have indicated that A20 can disturb ubiquitin enzyme complexes that are important for inhibiting NF-kB activation in TLR4 signaling.…”

Cerebral Neovascularization in Diabetes: Implications for Stroke Recovery and beyond

“…Evidence from intracranial hemorrhage models suggests that hemoglobin and heme released from red blood cells enter the brain parenchyma and free iron from further degradation of the heme molecule, disrupts cellular integrity and function via increased oxidative stress. [106][107][108][109] It is also intriguing that heme upregulates TLR4, 110 a gate keeper of the innate immune system and TLR4 mediates disruption of endothelial barrier function. 111 These observations collectively raise the possibility of bleeding and TLR4 being additional mechanisms involved in the vasoregression and impaired repair process after diabetic stroke.…”