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.

No abstract

High-mobility group protein box-1 (HMGB1) has recently been recognized as a novel candidate in a specific upstream pathway promoting inflammation after brain ischemia. However, its downstream pathway and underlying mechanism have yet to be elucidated. The HMGB1 level in the acute cerebral infarct (ACI) group was significantly increased compared with that of control group, and correlated with the severity of neurologic impairment of ACI patients. Further, recombinant human HMGB1 (rhHMGB1) had no effect on microglia derived from mice lacking the Toll-like receptor 4 (TLR4 À/À ). Intracerebroventricular injection of rhHMGB1 in TLR4 + / + mice cause significantly more injury after cerebral ischemia-reperfusion than control group. But, TLR4À/À mice administered with rhHMGB1 showed moderate impairment after ischemia-reperfusion than TLR4 + / + mice. To determine the potential downstream signaling of HMGB1/TLR4 in cerebral ischemic injury, we used the ischemic-reperfusion model with Toll/interleukin-1 receptor domain-containing adaptorinducing interferon-b knockout mice (TRIF À/À ) and evaluated the activity and expression of TRIF pathway-related kinases. The results suggest that the TRIF pathway is not likely to be involved in TLR4-mediated ischemia brain injury. Finally, we found that TLR4 expressed by immigrant macrophages was involved in the development of ischemic brain damage. These results suggest that HMBG1 mediates ischemia-reperfusion injury by TRIF-adaptor independent Toll-like receptor 4 signaling. The TLR4 expressed by immigrant macrophages may be involved in the development of ischemic brain damage.

In the present study, we observed the expression of toll-like receptor 4 (TLR4) and its downstream signal pathway in peripheral blood monocytes (PBMs) from patients with acute cerebral infarct (ACI). The expression of TLR4 and MyD88 by PBMs was determined by flow cytometry and reverse transcriptase-polymerase chain reaction, and nuclear factor-jB (NF-jB) activity was detected by electrophoretic mobility shift assay. Ischemia/reperfusion injury-induced cerebral edema, infarction area, and neurologic impairment scores were determined in MyD88 gene knockout mice. The results indicated a significant increase in circulating TLR4+ monocytes in ACI patients as compared with the control group and the transient ischemia attack (TIA) group. This change paralleled an elevation in TLR4mRNA transcription and serum tumor necrosis factor-a (TNF-a) and interleukin (IL)-6 in the ACI and TIA groups. Correlation analysis showed TLR4 expression to significantly correlate with cytokine levels and stroke severity. MyD88mRNA differed insignificantly among the three groups. Compared with wild-type mice, 6 h of cerebral ischemia followed by 24 h of reperfusion did not significantly change cerebral edema, cerebral infarction area, and neurologic impairment scores in MyD88 gene knockout mice. Compared with the control group, serum heat shock protein (HSP) 60 increased significantly in the ACI and TIA groups, leading to NF-jB activation in TLR4/CD14-transfected HEK293 cells. It is suggested that upregulated TLR4 expression on PMBs may act as one of the peripheral mechanisms of inflammatory injury after ACI. Moreover, circulating HSP60 may be a ligand for TLR4, which is involved in the peripheral mechanism of inflammatory injury after ACI, possibly through an MyD88-independent signal pathway.

High-mobility group box-1 (HMGB1) was originally identified as a ubiquitously expressed, abundant, nonhistone DNA-binding protein. It has well-established functions in the maintenance of nuclear homeostasis. The HMGB1 can either be passively released into the extracellular milieu in response to necrotic signals or actively secreted in response to inflammatory signals. Extracellular HMGB1 interacts with receptors, including those for advanced glycation endproducts (RAGEs) as well as Toll-like receptor 2 (TLR2) and TLR4. The HMGB1 functions in a synergistic manner with other proinflammatory mediators and acts as a potent proinflammatory cytokine-like factor that contributes to the pathogenesis of diverse inflammatory and infectious disorders. Numerous reports point to HMGB1 as a novel player in the ischemic brain. This review provides an appraisal of the emerging roles of HMGB1 in cerebral ischemia injury, highlighting the relevance of HMGB1-blocking agents as potent therapeutic tools for neuroprotection.