The Effects And Mechanisms Of HMGB1on Cerebral Inflammation And Peripheral Immunodepression Caused By Stroke

Background—stroke is one of the leading causes of death and disability worldwide. Cerebral ischemia/reperfusion injury (CIRI) is induced by recanalization of blood flow in the ischemic area. After CIRI, two different responses ocurred including central inflammation and peripheral stroke-induced immunodepression (SIID), characterized by increased neutrophils and macrophages in injuried brain tissue and lymphopenia in peripheral system, respectively. However, the mechanisms of both above responses remian unclear. High-mobility group box1protein (HMGB1), a novel cytokine-like protein, is released by necrotic neurons and mediates cerebral inflammation and brain injury after CIRI. Previous researches showed that CIRI induced infiltration of micaroglia/macrophages, neutrophils and lymphocytes in the ischemic brain tissues and contributed cerebral inflammatory response. It is known that HMGB1release results in activation of micaroglia/macrophages. Because of the crosstalk between macrophages and T lymphocytes and low concentration of HMGB1promotes T-cell immuno-function, it is possible that HMGB1may mediate activation of T cells directly or act on micaroglia/macrophages through activating T cells. However, the precise mechanisms are unclear. In addition, HMGB1is released in the peripheral blood due to damaged blood-brain barrier after CIRI. Previous studies showed that in vitro, high concentration of HMGB1induced T cell immunosuppression directly or indirectly through inhibiting dendritic cells (DCs) activities. However, the association between high level HMGB1in the peripheral blood and lymphopenia caused by CIRI remains unkonwn.Objectives—in this study, we will investigate the sub-location and expression of HMGB1in the ischemic brain, the degree of cerebral inflamation, as well as absolute numbers of T lymphocytes and their subsites in the peripheral organs after CIRI by using Glycyrrhizin, a HMGB1antagonist to inhibit HMGB1in focal cerebral ischemia/reperfusion injury models. We also will detect the effect of HMGB1on brain injury post CIRI in vivo or neuronal death in vitro splenocytes-neuronal coculture system in T cell dificient animals. Our purpose is to clarify the effects and mecahnisms of HMGB1on central inflammation and peripheral immunodepression induced by stroke. Our findings will provide new drug target for stroke treatment.Methods—In vivo, two ischemic models were used in this study including transient focal ischemic model and permernant distal MCA occlusion ischemic model. Focal ischemia was induced by intraluminal MCA suture occlusion (MCAO) for100min in rats and60min in mice followed by reperfusion; in the distal MCA occlusion (dMCAO) model, the distal MCA was permanently occluded and the bilateral common carotid arteries (CCAs) were transiently occluded for60min. Animals were IP-injected with Glycyrrhizin (200mg/kg) or vehicle once immediately post-reperfusion and once3.5h after reperfusion (2injections total). TTC-staining was used for measuring infarct sizes, immunofluorescent and confocal microscopy to detect HMGB1cytosolic release and microglia/macrophages (anti-CD68antibody labeled) and neutrophil activation (anti-MPO) in brain tissues, as well as Western blot and ELISA to assess HMGB1release in CSF or serum, respectively. Flow cytometry was used to measure lymphocyte subpopulations in the blood, spleen and ischemic brain.Results—1. HMGB1was widely expressed in the NeuN+neurons in the sham group, and was localized in the nuclei. However, HMGB1translocation was observed in the peri-infarction area12h post stroke and, only few NeuN+cells were detected in the ischemic core48h post stroke. This suggests that a large number of neurons died post stroke and that HMGB1was released from the dying neurons. Correspondingly, increased HMGB1levels were found in the cerebralspinal fluid (CSF) and serum of rats with stroke.2. At the same time, the number of CD68+/HMGB1+macrophages/microglia and MPO+neutrophils increased in the ischemic area72h after stroke. This implies that macrophages/microglia and neutrophils had accumulated in the ischemic core and there was an inflammatory cellular response in the ischemic brain.3. Injection of Glycyrrhizin post stroke significantly reduced infarction volume, inhibited HMGB1translocation and release from neurons and improved neuronal survival, as well as inhibited the infiltration of CD68+macrophages/microglia and MPO+neutrophils in the ischemic core, compared with the vehicle group, suggesting that the drug robustly suppresses brain inflammation and cerebral injury following stroke.4. Glycyrrhizin attenuated the total number of brain mononuclear cells, inhibited T cells and monocytes infiltration.5. Glycyrrhizin only protects WT rats or mice from ischemic injury but not in nude rats or SCID mice, also Glycyrrhizin can inhibit the increase effects of infarct in SCID mice by splenocytes transferred from WT mice; in in vitro splenocytes and neurons coculture system, Glycyrrhizin protects damaged effect of splenocytes derived from WT not SCID mice on neuronal death.6. Glycyrrhizin inhibited HMGB1release in the cytosolic space and CSF, and remarkably reduced HMGB1levels in the serum.7. With the decreased level of HMGB1in the blood, Glycyrrhizin attenuated reduction in both peripheral blood mononuclear cells (PBMCs) and the total number of splenocytes, also inhibited stroke-induced reduction in the absolute number of T cells in the blood and spleen.Conclusion1. HMGB1is localized in the nuclei of neurons in normal condition. It transfers from nuclei to cytosol then releases to exralcellular space, even to CSF and blood post CIRI;2. HMGB1might contribute to the cerebral inflammation by activating infiltrated T cell in ischemic area and induce SIID through mediating T cell immunosuppression because of its dual-effects on T cell immunity;3. Glycyrrhizin, targeting on HMGB1, protects against ischemic injury by inhibiting cerebral inflammation and modulating the immune system.