The Role And Mechanism Of IL-23/IL-17 Axis In Inflammatory Injury Induced By ICH

Intracerebral hemorrhage(ICH)accounts for 20% to 30% of all cases of cerebrovascular disease in China and remains the leading cause of mortality,largely because there currently is no specific treatment.ICH is associated with both primary and secondary injury.The physical effects of the hematoma(mass effect)and disturbance of the adjacent tissue that occur within hours of the onset of bleeding represent the primary insult.The potential benefit of clot removal has been investigated in multiple clinical trials but not confirmed.Instead,most recent attempts to identify novel therapeutic targets have focused on the mechanisms responsible for ICH induced secondary injury,which is caused by the development of edema,free radicals,inflammation,and direct cellular toxicity of the degradation by products of the hematoma.Increasing evidence indicates that inflammation plays a critical role in ICH-induced secondary injury.Within minutes after ICH,microglias are activated and begin to release large amounts of cytokines and chemokines to destroy the blood-brain barrier(BBB).A number of,but not all,studies have shown a benefit of inhibiting microglia activation after ICH with tuftsin or minocycline,which may be due to the effects of microglia in ICH-induced brain injury be both detrimental and beneficial(e.g.clot resolution)?In the later phase of ICH,peripheral inflammatory cells such as macrophages and lymphocytes are mobilized to the perihematoma brain tissue through the damaged BBB.The infiltrating macrophages and T lymphocytes further produce proinflammatory factors to exacerbate brain edema and neurologic deficits after ICH.However,little is known about the mechanisms by which macrophages and lymphocytes are activated after ICH as well as the proinflammatory factors they release to induce the later brain injury.Among the(9)National Natural Science Fund for Distinguished Young Scholars(81525008),National Science Foundation of China(81400974),National Basic Research Program of China(973 Program,2014CB541605)proinflammatory factors released within the injured brain,interleukin(IL)-17 generated by IL-17–expressing helper T(Th17)cells has been shown to induce inflammation in experimental autoimmune encephalomyelitis(EAE),which is a mouse model of multiple sclerosis.Research has also shown that IL-23,a heterodimer of the IL-23p19 and Il-12p40 subunits,released by activated macrophages and dendritic cells(DCs)can induce differentiation of CD4+ T cells into Th17 cells to generate a major inflammatory reaction.This IL-23/IL-17 inflammatory axis has been shown to be essential for the onset and progression of autoimmune inflammatory diseases in multiple mouse models,including EAE and collagen-induced arthritis.Moreover,the T-lymphocyte production of IL-17 in response to macrophage-derived IL-23 was found to play acritical role in the later stage of brain infarction.These data highlight an important role of the IL-23/IL-17 inflammatory axis in the acute neuroinflammatory response.However,the roles of macrophages and T lymphocytes as well as the IL-23/IL-17 inflammatory axis within the neuroinflammation that occurs after ICH remain to be determined.The purpose of this study was to explore the roles of macrophages,T lymphocytes,and the cytokines they secreted in the secondary brain injury caused by ICH.ICH was induced using an established model in various lines of knockout mice,and the molecular events underlying the resulting neuroinflammatory responses were studied.The results of these experiments provide insight into the mechanisms of the neuroinflammation responsible for secondary brain injury after ICH and provide new targets for the development of therapies for ICH patients.Part one: The role of infiltrating of peripheral inflammatory cells and their in the secondary inflammatory injury induced by ICHObjective: Explore the change in value of macrophages and T lymphocytes in ICH brain,and their roles in the secondary inflammatory injury induced by ICH.Methods: Firstly,the WT mice autologous blood ICH models were established.The temporal changes in the numbers of different types of inflammatory cells in the hemorrhagic hemisphere were analyzed by FACS and immunofluorescent staining.Neurological deficit score(NDS),brain water content(BWC)tested in CLPs treated,Fingolimod treated and WTCD3?Gag1-/-mice after ICH.At the same time,the temporal changes in the numbers of macrophages and T lymphocytes in hemorrhagic hemisphere of these three groups of mice were analyzed by FACS.Results:1.Absolute numbers of CD45+ cells,including T lymphocytes(CD3+ cells),CD4+ T lymphocytes,and ??T lymphocytes were increased with the peak on day 4 after ICH,while the number of macrophages peaked at day 1;no changes in the numbers of microglia were observed after ICH.Immunofluorescence staining showed that F4/80+(macrophages/microglia)and CD3+ cells(T lymphocyte)were located along the perihe matoma area after ICH.2.CLPs injection significantly reduced the number of infiltrating macrophages in the brain at 1 day and 4 days after ICH,without influencing the infiltration of T lymphocytes.CLP injection also significantly reduced the NDS and BWC of WT mice after ICH.3.Fingolimod treated significantly reduced the number of infiltrating T lymphocytes in the brain at 1 day and 4 days after ICH,without influencing the infiltration of macrophages.Fingolimod treated also significantly reduced the NDS and BWC of WT mice after ICH.4.NDS and BWC were increased when WT T lymphocytes were transferred into Rag1-/-mice.Conclusions: All these results suggest that infiltrating peripheral macrophages and T lymphocytes can aggravate the the secondary inflammatory injury in mice after ICH.Part two: IL-23 produced by infiltrating macrophages exacerbated the secondary inflammatory injury following ICH.Objective: To examine the role and the source of IL-23 in the secondary inflammatory injury after ICH.Methods: Western blot and real-time PCR analyses were performed to detect IL-23 expression in WT mice at days 1,4,and 7d after ICH.NDS and BWC tested in WT and IL-23-/-mice.Immunofluorescence staining and TUNEL detected the ?III tubulin and TUNEL double-positive cells in the perihematoma area of WT and IL-23-/-mice.Proinflammatory cytokines were calculated in WT and IL-23-/-mice.Real-time PCR analyses were performed to detect IL-23 expression in infiltrating macrophages,microglia and other cells separated by FACs from the brain of WT ICH mice.Results:1.The protein and mRNA levels of IL-23 were significantly higher in the perihematoma tissues of ICH mice,both peaking on day 1 after ICH.2.IL-23-/-mice showed lower NDS and BWC and fewer apoptotic neurons after ICH than WT mice.In addition,IL-1? and TNF-a gene expression in infiltrating CD45+ cells was significantly lower in IL-23-/-mice than in WT mice at day 4 after ICH.3.IL-23 mRNA expression on infiltrating inflammatory cells was highest at day 1 after ICH.Upon sorting of the infiltrating inflammatory cells into macrophages,microglia,and other cells,IL-23 mRNA was found to be expressed dominantly in macrophages,with little expression in microglia and other cells.Conclusion: All these results suggest that IL-23 produced by infiltrating macrophages exacerbated the secondary inflammatory injury following ICHPart three: ??T cell production of IL-17 drived by IL-23 played a critical role in ICH-Induced secondary inflammatory brain injuryObjective: In this part,we explored the the role and the source of IL-23 in the secondary inflammatory injury after ICH,and confirmed the relationship between IL-23 expression and IL-17 production in the ICH brain.Methods: IL-17 expression in WT mice at days 1,4,and 7d after ICH were detected by Western blot and real-time PCR analyses.NDS and BWC tested in WT,IL-17-/-and ??TCR-/-mice.Immunofluorescence staining and TUNEL detected ?III tubulin and TUNEL double-positive cells in the perihematoma area of WT and IL-17-/-mice.Proinflammatory cytokines were calculated in WT and IL-17-/-mice.Immunofluorescence staining detected CD3 and IL-17 double-positive cells in the perihematoma area of WT mice.Results:1.The protein and mRNA levels of IL-17 were significantly higher in the perihematoma tissues of ICH mice,both peaking on day 4 after ICH.2.IL-17-/-mice showed lower NDS and BWC and fewer apoptotic neurons after ICH than WT mice.In addition,IL-1? and TNF-a gene expression in injury brain and infiltrating CD45+ cells were significantly lower in IL-17-/-mice than in WT mice at day 4 after ICH.While the number of infiltration inflammatory cells had not significantly decrease.3.Immunofluorescence staining showed that IL-17 was colocalized with CD3 in the perihematoma area.Flow cytometric analysis various surface markesr expression on IL-17+ cells showed that IL-17+ cells almost all expressed CD45,most expressed CD3,barely expressed CD11 b.Further FACs analysis of ??T and CD4 expression on IL-17+CD3+T cells indicated that most IL-17–producing T lymphocytes were CD4-??TCR+cells at 4 days after ICH.The NDS,BWC and mRNA expression of IL-1? and TNF-a were significantly lower in ??TCR-/-mice than in WT mice after ICH4.The percentage of IL-17+ cells among infiltrating CD3+ T lymphocytes in IL-23-/-mice declined sharply,IL-17+ cells almost can not be detected in IL-23-/-mice.Conclusion: These results suggest that IL-17 played a critical role in ICH-Induced Secondary inflammatory Injury,CD3+CD4-??TCR+ cells rather than CD3+CD4+ cells were the main resource of IL-17.IL-23 was essential for production of IL-17 after ICH.Part four: Hb-Induced TLR2/TLR4 heterodimer formation on infiltrating macrophages intiated IL-23/IL-17 axisObjective: To confirm that Hb induced TLR2/4 heterodimer formation on macrophages,and its role in ICH secondary inflammatory brain injuryMethods: Immunoprecipitation assays were performed to detected TLR2/TLR4 heterodimer formation on brain-derived macrophages and spleen-derived macrophages after ICH,and on BMM stimulated by various red blood cell components.Immunofluorescence staining performed to detecte the expression of TLR2 and TLR4 on F4/80+ cells around perihematomal in ICH mouse brain and BMM stimulated by Hb.Bone marrow chimeric mice were established to verify the critical role of TLR2/TLR4 heterodimer on the peripheral cells in ICH-induced brain injury.The mRNA of IL-23 was detected in BMM from different genotype mice stimulated by Hb.The effects of SsnB were evaluated by NDS,BWC,the number of ?III tubulin and TUNEL double-positive cells and the mRNA of proinflammatory cytokines.Results:1.We observed the colocalization of TLR2 and TLR4 on F4/80+ cells perihematomal in ICH mouse brain tissue by immunohistochemical staining,and the same colocalization on BMM stimulated by Hb.2.Immunoprecipitation assays revealed that Hb induced coprecipitation of TLR2 and TLR4 on brain-derived macrophages of ICH mice,but not on spleen-derived macrophages.3.Among the red blood cell components including Hb,hemin,bilirubin,Fe2+,and Fe3+,only Hb was found to induce coprecipitation of TLR2 and TLR44.Hb-mediated induction of IL-23p19 expression was lower in TLR2-/-and TLR4-/-BMM than in WT BMM and lower still in TLR2-/-/TLR4-/-BMM.5.SsnB treated mice showed lower NDS,BWC,number of ?III tubulin and TUNEL double-positive cells and mRNA of proinflammatory cytokines after ICH.Conclusion: These results suggest that Hb-Induced TLR2/TLR4 heterodimer formation on infiltrating macrophages intiated IL-23/IL-17 axis,SsnB disturbed the formation of TLR2/TLR4 heterodimer,and inhibited the role of IL-23/IL-17 axis in ICH-induced inflammatory injury.