| Intracerebral hemorrhage(ICH) represents the acute manifestation of progressive cerebral small-vessel disease. ICH is associated with high rate of mortality and morbidity. It accounts for about 10% to 15% of all strokes worldwide. Currently, there is no effective therapy to improve survival rate or treatment to improve the quality of life for survivors.ICH is mainly caused by mechanical injury that results in compression because of the mass effect of hematoma. But the clinical trials for surgical hematoma evacuation had an unfavourable outcome versus conservative treatment. Increasing evidence indicates that secondary injury following ICH plays an important role in the neurological deterioration experienced by patients with ICH. The secondary brain injury after ICH is including hematoma toxicity, high metabolic injury, excitotoxicity, oxidative stress, and inflammatory injury. Innate immunity and inflammatory responses have been found to play critical roles in ICH-induced secondary brain injury.Toll-like receptors(TLRs) that recognize distinct pathogen-associated and damage-associated molecular patterns play a key role in innate immunity and inflammatory responses. Recently studies suggested that TLR4 may be the upstream signal that triggers inflammatory injury in ICH. Our previous study suggested that Hb potentiates microglial activation via TLR4, in turn inducing NF-kappa B activation via the My D88/TRIF signaling pathway, and ultimately increasing cytokine expression and inflammatory injury after ICH. However, the inflammation injury was not completely inhibited when TLR4 signaling was blocked.The recent identification of co-receptors that enhance TLRs function suggests an emerging paradigm in which TLRs act together with other cell surface molecules to bridge microbe recognition and initiation of signaling responses, for example, TLR1/TLR2 and TLR2/TLR6 heterodimers in ischemic brain injury, TLR4/TLR6 heterodimer in arteriosclerosis and Alzheimer’s disease. These findings led to the speculation that combinatorial signaling among the TLRs might account for the breadth of this repertoire.Rodriguez-Yanez et al reported that expression levels of TLR2 and TLR4 in monocytes at admission were independently associated with poor functional outcome in patients with ICH, suggesting that both TLR2 and TLR4 participate in ICH-induced brain injury. However, it remains unclear how TLR2 functions in ICH-induced inflammatory injuries, and whether TLR2 interacts with TLR4 in ICH. Thus, this study was aimed to investigate the role of TLR2 and TLR2/TLR4 heterodimerization in ICH-induced inflammatory injury.In the present study, we generated a mouse model of ICH by injection of autologous blood. The expression of TLR2 and its role in inflammatory injury after ICH were described. Formation of TLR2 and TLR4 heterodimer was detected both in in vitro and in vivo ICH models. Inhibition of TLR2/TLR4 heterodimer formation via the administration of Sparstolonin B(Ssn B) reduced ICH-induced inflammatory injury and neurological deficits.Part one: Activation of TLR2 by hemoglobin(Hb) Mediates ICH-Induced Inflammatory InjuryObjective: Explore the expression of TLR2 after ICH and the role of TLR2 in inflammatory injury after ICH.Methods: Firstly, the mice autologous blood ICH models were established. Total RNA and protein were collected from perihematomal tissues to detect the expression of TLR2. Expression of TLR2 by microglia, astrocytes, and neurons present in perihematomal tissues obtained 3 days after ICH were analyzed by flow cytometry and fluorescence microscopy. Neurological deficit score(NDS), brain water content(BWC) and inflammation factors were tested in TLR1-/-, TLR6-/-, WT and TLR2-/- mice. Western blot were conducted to identify the blood components that activate TLR2.Results:1. Both real-time PCR and western blot assays detected significantly higher levels of TLR2 in ICH tissues compared with sham controls. The highest levels of TLR2 expression were detected in microglia, whereas levels were unchanged in neurons. Similar results were obtained in fluorescence microscopy studies of microglia, astrocytes, and neurons.2. TLR2-/- mice exhibited significantly less brain edema up to 5 days after ICH compared with TLR1-/-, TLR6-/-, and wild-type(WT) mice. They had significantly lower levels of proinflammatory cytokines and lower neurologic deficit scores(NDS) but higher levels of anti-inflammatory cytokine. The NDS of TLR2-/- mice were still lower than those of WT mice in collagenase models. TLR2-/- mice had less neuronal apoptosis and degeneration, whereas this was not the case in TLR1-/- or TLR6-/- mice. In TLR2-/- mice, all of the blood-derived inflammatory cells present in perihematomal tissues were present at significantly lower levels compared with control mice.3. In Western blot assays, higher levels of TLR2 expression were detected for microglia treated with whole blood lysate and RBC lysate solution, suggesting that RBC lysate are responsible for upregulation of TLR2 expression. Among the RBC components, only Hb was found to significantly increase TLR2 expression in a dosedependent manner. In addition, Hb promoted microglial activation, resulting in a significant increase in the production of inflammatory cytokines. These effects were blocked in TLR2-/- mice, and were also blocked in WT mice treated with an anti-TLR2 antibody.4. In transwell experiments, when cultured microglia were treated with Hb then placed in transwells above neuron-enriched cultures(WT), a significant decrease in neuron survival was observed and quantitated, whereas microglia from TLR2-/- mice had significantly improved neuronal survival.5. Three days after Hb or saline was stereotactically injected into the striatum of WT and TLR2-/- mice, compared with the corresponding sham controls or TLR2-/- mice, WT mice had a significant increase in brain edema, accompanied by an increase in neurologic deficits, a greater number of apoptosis neurons, increased neuronal degeneration, and higher concentrations of TNF-a, IL-1β, and IL-6.Conclusions: All these results suggest that TLR2 may contribute to ICH-induced brain injury, suggesting that TLR2 is important for the induction of inflammatory cell infiltration into perihematomal tissues following ICH. The expression of TLR2 by microglia mediates Hb-induced neuronal death.Part two: Hb Promotes TLR2/TLR4 Heterodimer formation on microglia to Induce Inflammatory InjuryObjective: To examine the association between TLR2 and TLR4 in in vivo and in vitro ICH models.Methods: Immunoprecipitation assays were performed to detected TLR2/TLR4 heterodimer formation after ICH. The BWC, NDS, and proinflammatory cytokines were calculated in TLR2-/-/TLR4-/- mice, TLR2-/- and TLR4-/- mice 3 days after ICH. HEK293 cells and mammalian cells, expressing TLR2 or TLR4 or both TLR2 and TLR4, were stimulated with various conditions to identify the interaction of TLR2 and TLR4. Immunoprecipitation assays were conducted to detected TLR2/TLR4 heterodimer formation in WT, TRIF-/- and My D88-/- mice. Microglia was treated with My D88 si RNA to find out the critical bind site for TLR2/TLR4 heterodimer formation. Finally, The BWC, NDS, and proinflammatory cytokines were conducted in WT and My D88-/- mice.Results:1. Immunoprecipitation assays detected TLR2/TLR4 heterodimer after ICH, with the highest levels 1 day after ICH. Immunofluorescence assays detected overlay of TLR2 and TLR4 distribution in perihematomal tissues. The immunoprecipitation results indicated that only Hb(the component of RBC lysate) induced heterodimer formation in cell culture. The double-hybrid results indicated that there is no auto-transcriptional activity of TLR2 or TLR4. However, the luciferase activity was significantly increased only when TLR2 and TLR4 co-transfected into cells, especially when the cells were treated with Hb.2. For HEK293 cells expressing both TLR2 and TLR4, treatment with Hb significantly increased NF-kappa B activation. The BWC, NDS, and proinflammatory cytokines decreased significantly in TLR2-/-/TLR4-/- mice 3 days after ICH, compared with TLR2-/-or TLR4-/- mice.3. Twenty-four hours after ICH, TLR2/TLR4 heterodimer formation was detected in WT and TRIF-/- mice, yet not in My D88-/- mice. My D88R196 A, but not My D88R288 A, impaired the ability of My D88 to induce NF-kappa B–dependent luciferase gene expression in HEK293 cells co-expressing TLR2 and TLR4. Moreover, Hb induced co-precipitation of TLR2 and TLR4 in HEK293 cells co-expressing WT My D88 or My D88R288 A, not My D88R196 A. My D88-si RNA demonstrated a significant decrease in the production of inflammatory cytokines and NO in microglia treated with My D88-si RNA. Additionally, NDS, BWC, and inflammatory cytokines decreased significantly in My D88-/- mice 3 days after ICH.Conclusion: Hb, but not other blood components, triggered inflammatory injury in ICH via assembly of TLR2/TLR4 heterodimers. My D88, but not TRIF, was required for ICH-induced TLR2/TLR4 heterodimerization.Part three: Ssn B attenuates ICH-induced inflammatory injury by disrupting TLR2/TLR4 heterodimer formation.Objective: In this part, we explored the treatment of ICH by disrupting TLR2/TLR4 heterodimer formation.Methods: Co-immunoprecipitation was performed to screen for compounds able to disrupt TLR2/TLR4 heterodimer formation. NF-κB activity was measured using a reporter gene assay system during cell transfection and mutation experiments. A liquid chromatography-mass spectrometry(LC-MS) experiment was conducted to test whether the screened compound can cross the blood-brain barrier. Animal ICH models were used to explore the therapeutic effect of sparstolonin B(Ssn B) on ICH-induced inflammatory injury.Results: Among the screened compounds, Ssn B abolished the formation of TLR2 and TLR4 heterodimers, exhibiting potential as a therapeutic compound. Ssn B significantly inhibited Hb-induced NF-κB expression in HEK293 cells that expressed both TLR2 and TLR4, whereas the mutation of residue Arg196 in My D88 abolished this inhibition. Moreover, Ssn B alleviated NDS, brain water content and inflammation factor expression in perihematomal tissue samples from ICH mice.Conclusion: These results suggest that the inhibition of TLR2/TLR4 heterodimer formation via the administration of Ssn B reduced ICH-induced inflammatory injury and neurological deficits. Ssn B may be a novel drug candidate for ICH treatment. |
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