| Background and objective:Intracerebral hemorrhage(ICH) accounts for 10-15% of all stroke cases worldwide and is associated with high morbidity and mortality. The proportion of deaths caused by ICH increased to 61.7% of all deaths with cerebrovascular disease, which is leading cause of death in china. Significant progress has been made in increasing understanding of pathophysiology of ICH, but therapeutic methods to treat ICH remain limited. Recent studies indicated that inflammation plays an important role in ICH-induced second injury. After ICH, the inflammatory factors released by activated microglia initiated brain injury, meanwhile, the infiltration of inflammatory cells from the periphery further increases the inflammatory injury. Inhibiting peripheral inflammatory cell infiltration using fingolimod can reduce inflammatory damage after ICH. However, another study showed that fingolimod therapy is associated with increased neuroinflammation. These results indicated that specific inhibit the inflammatory cells infiltration or increase the regulatory cells(Tregs) infiltration could be a new method for ICH therapy.Foxp3+Tregs sustain immune homeostasis and self-tolerance by restricting the activation and release of cytokines from a wide range of inflammatory cells. Impairment of the regulatory capacity of Tregs could result in many autoimmune diseases(experimental autoimmune encephalomyelitis, thyroiditis and type I diabetes). In ischemic stroke, Tregs can accumulate and proliferate in the ischemic hemisphere for up to 30 days after middle cerebral arterial occlusion(MCAO), and depleting Tregs using a CD25 antibody increased neuroinflammation. Previous study showed that infusion of Tregs was also protective after ICH or ischemic stroke. This indicated that the similar function of Tregs was shared by ischemic and hemorrhagic stroke, but the potential mechanisms involved in Tregs-mediated central nervous system(CNS) protection remain to be elucidated. Furthermore, transfusing of Tregs is technically demanding and time consuming, and it yields a limited number of cells. Thus, in this study, we used Foxp3 DTR mice, CD25 antibodies and CD28-SA to further explore the functions of Tregs in ICH and to provide a new target for ICH therapy.Meanwhile, microglia as an important CNS resident macrophage also participate in the pathogenesis of ICH induced inflammation. Increasing evidences indicated that the microglia could be divided into 2 different phenotypes according to their surface marker and the cytokines they release. M1 phenotype can release large amount of pro-inflammatory factors, increase the inflammatory reaction and damage the surrounding brain tissue. M2 phenotype can release protective factors to decrease the inflammatory reaction. The enhanced phagocytic ability could eliminate the damaged tissue and accelerate brain self-repair. By now, there is little evidence about the temporal change of M1&M2 in peri-hematoma tissues after ICH. Previous studies in ischemic stroke indicated that the activated M2 phenotype can shift toward M1 phenotype and aggravate the inflammation in ischemic brain tissue. Revert this pathogenesis process may improve the neurological functions and ameliorate the inflammation.Previous studies showed that two main pathways were involved in Tregs mediated inhibition of inflammation. One pathway was mediated by the surface markers expressed on Tregs such as Cytotoxic T lymphocyte antigen(CTLA-4) and lymphocyte activation gene 3(LAG-3), which directly inhibit the activation of antigen presenting cells(APCs) and other lymphocytes by cells-to-cells interactions. The other pathway was mediated by the cytokines released by Tregs such as IL-10, TGF-β, which also can ameliorate the inflammation.The studies about the crosstalks between the microglia /macrophage and Tregs hint that Tregs could be an effect way to revert the microglia/macrophage polarization and IL-10, TGF-β are both involved in this transition. Furthermore, studies in traumaticbrain injury models showed that GSK3β/PTEN/PI3K/Akt axis may be one of important pathways involved in microglia polarization.Thus, we speculate that the cytokines released by Tregs can be one of important pathway to shift the microglia/macrophage polarization towards M2 phenotype through GSK3β/PTEN axis. In this study, we intend to identify the exact role of Tregs in ICH and further, explore the potential mechanisms involved in Tregs mediated protection.Methods:Part I. Tregs ameliorate the inflammatory injury induced by ICH1. ICH mice models were constructed, the hemorrhagic hemispheres were discarded 1, 4, 7 and 14 days after ICH. The hemispheres were digested and pipetted into single cell suspension. Then gradient centrifugation was performed to purify the suspension. After stained with fluorescently-labeled antibodies, the temporal changes of total total inflammatory cells, total T cells, Th cells and Treg cells were analysed by FACS. The expression changes of Foxp3 in peri-hematoma tissue after ICH was evaluated by Western-Blots.2. Diphtheria toxin(DTX) were peritoneally injected in Foxp3 DTR mice or WT mice to deplete Tregs, 48 hours later, ICH mice models were made. The NDS were evaluated at day 1, 3, 5 and 7 after ICH. 4 days after ICH,the brain tissues were discarded, the water content, hematoma volumes were compared with the results in WT mice respectively. The neuro-degenerations and the pro-inflammatory factors(IL-1β, IL-6, TNF-α, IL-10 and TGF-β) in Foxp3 DTR mice were analysed by FJB-Staining and RT-PCR.3. CD25 antibodies and the control IgG were peritoneally injected in WT mice, 48 hours later, ICH mice models were constructed. The NDS were evaluated at day 1, 3, 5 and 7 after ICH. Four days after ICH,the brain tissues were removed, the water content, hematoma volumes were compared with the results in IgG treated mice respectively. The neuro-degenerations and the pro-inflammatory factors(IL-1β, IL-6, TNF-α, IL-10 and TGF-β) in CD25 antibodies treated mice were analysed by FJB-staining and RT-PCR.4. ICH mice models were constructed. 3 hours after ICH, CD28 superagonists(CD28-SA) and the control IgG were peritoneally injected in WT mice. The NDS were evaluated at day 1, 3, 5 and 7 after ICH. Four days after ICH,the brain tissues were removed, the water content, hematoma volumes were compared with the results in IgG treated mice respectively. The neuro-degenerations and the pro-inflammatory factors(IL-1β, IL-6, TNF-α, IL-10 and TGF-β) in CD28-SA antibodies treated mice were analysed by FJB-staining and RT-PCR.Part II. Tregs shift the hemoglobin activated microglia/macrophage towards M2 phenotype1. Microglia primary culture were supplemented with different concentration of hemoglobin(Hb, 5μM, 10μM, 20μM and 40μM) and cultured for 6 hours. Then, the TNF-α expression was compared among the groups by RT-PCR. Further, after digested, the microglia were harvested and stained with annexin V-FITC and propidium iodide(PI). Then the apoptosis rate were analysed by FACS among the groups.2. DTX was injected in Foxp3 DTR mice to deplete Tregs 2 days before ICH induction. CD28-SA was injected in WT mice to boost Tregs 3 hours after ICH induction. Then, the mice were sacrificed 4 days after ICH and the hemorrhagic hemisphere were removed and digested to single cells suspension. Then, CD45-APC, CD11b-Percp cy5.5, MHC-II-FITC and CD206-PE were stained and the numbers of microglia /macrophage were calculated by FACS. Meanwhile, the MFI of MHC-II and CD206 on CD45+CD11b+ cells were also analysed3. Microglia primary cultures were supplemented with 10μM Hb and cultured for 6 hours. Then the freshly isolated tregs from the spleen were added on a transwell on the top of microglia. 40 hours later, the microglia was harvested. The MHC-II & CD206 expression were analysed by FACS and the expression of TNF-α and IL-6 were analysed by RT-PCR.Part III Treg shift microglia/macrophage polarization toward M2 phenotype by IL-10/GSK3β/PTEN axis1. Microglia primary cultures were supplemented with 10μM Hb and cultured for 6 hours. Then the freshly isolated tregs from the spleen were added on a transwell on the top of microglia. IL-10 antibodies or TGF-β antibodies were used to block the Tregs associated cytokines. 40 hours later, the microglia was harvested. The MHC-II & CD206 expression were analysed by FACS and the expression of TNF-α and IL-6 were analysed by RT-PCR among the groups.2. Microglia primary cultures were supplemented with 10μM Hb and cultured for 6 hours. Then the freshly isolated tregs from the spleen were added on a transwell on the top of microglia. IL-10 antibodies or TGF-β antibodies were used to block the Tregs associated cytokines. 40 hours later, the microglia was harvested. The expression of GSK3β,P-GSK3β,PTEN and P-PTEN were analysed by Western-blots.3. Microglia primary cultures were infected by GSK3β-ShRNA,PTEN-ShRNA, contr-ShRNA or GFP containing particles for 48 hours. The successful rates for lentivirus infection were confirmed by cell immunofluorescence. Then, the four groups’ microglia were activated by 10μM Hb and cultured for 6 hours. Then the freshly isolated tregs from the spleen were added on a transwell on the top of microglia. IL-10 antibodies or TGF-β antibodies were used to block the Tregs associated cytokines. 40 hours later, the microglia was harvested. The expression of P-GSK3β, and P-PTEN were analysed by Western-blots.Results:Part I Tregs ameliorate the inflammatory injury induced by ICH1. The number of microglia/macrophage remained unchanged after ICH. The total T cells infiltration increased after ICH, peaked at 4 days and gradually decreased. 14 days after ICH, the numbers of total T cells were still higher than the sham-operated mice. Numbers of Th cells shared the same variation trend with total T cells. But Tregs began to increase 4 days after ICH and sustained until 14 days after ICH. The foxp3 expression was significantly increased in Th cells both in brain and spleen.2. Compared to the DTX-treated WT mice, the Foxp3 DTR mice showed a significant increase in NDS that peaked at 3 days. Tregs depletion also resulted in an increase in brain water content, hematoma volume. Furthermore, the FJB-positive cells were significantly increased in Foxp3 DTR mice. The expression of inflammatory factors in peri-hematomal tissues indicated that an increase in the expression of IL-1β, IL-6, and TNF-αwith the changes in IL-1βand TNF-αbeing the most significant, while the expression of IL-10 and TGF-βremainedunchanged.3. Compared to the IgG-ICH control group, injection of CD25 antibodies resulted in an increase in NDS after ICH. At 4 days after ICH, Treg depletion resulted in an increase in brain water content, hematoma volume, the number of FJB-positive cellsand the expression of IL-1β, IL-6, TNF-α(IL-6 was the most significant), while the expression of TGF-βand IL-10 remainedunchanged.4. Compared to the IgG-ICH group, CD28-SA reduced NDS after ICH. At 4 days after ICH, compared to IgG-ICH group, CD28-SA resulted in decrease in brain water content, hematoma volume,FJB-positive cellsand the expression of IL-1β,IL-6 and TNF-α, while the TGF-βand IL-10 increased significantly(IL-10 was most clearly increased).Part II. Tregs shift the polarization of microglia/macrophages toward the M2 phenotype after ICH1. RT-PCR results indicated that the expression of TNF-α in microglia increased with 5μM Hb stimulation. 20μM Hb was the most effective concentration which was associated with the highest level of TNF-α expression. The apoptosis rate of cultured microglia rose significantly with the stimulatory concentration increased.2. Compared to the sham-operated WT mice, the numbers of microglia/macrophages remaind stable at 4 days after ICH, but the expression of both MHC-II and CD206 increased in this population. Compared to the WT-ICH mice, Treg depletion induced an increase in the numbers of microglia/macrophages, and also an increase in the expression of MHC-II on microglia/macrophages, but the expression of CD206 on microglia/macrophages decreased. On the contrary, compared to the WT-ICH group, the number of microglia/macrophages and the MFI of MHC-II in the cells remained stable after CD28-SA was injected, but CD206 was significantly increased.3. To validate the results of in vivo tests, an in vitro Tregs/microglia transwell co-culture model was used to simulate ICH. Compared to the vehicle group, the MFI of CD206 and MHC-II wassignificantly increased in microglia, and these changes were accompanied by an increase in the mRNA expression of TNF-αand IL-6. Compared to the hemoglobin-stimulated group, Tregs decreased the MFI of MHC-II and the mRNA levels of TNF-αand IL-6 and, increased the MFI of CD206 in microglia.Part III The IL-10/GSK3β/PTEN axis mediates the Tregs-induced polarization of microglia1. Compared tocells treated with the vehicle, only the IL-10 antibody reversed the Tregs-induced polarization toward the M2 phenotype in microglia. This result was indicated by the increased the MFI of MHC-II, decreased the MFI of CD206, and increased the mRNA expression of TNF-αand IL-6. However, the TGF-βantibody had little effect on the Tregs-induced microglia polarization.2. After stimulationwith hemoglobin, GSK3β, P-PTEN and P-GSK3β were down regulated compared to stimulation with vehicle,but PTEN remainedunchanged. Tregs preventedthese decreases in the levels of GSK3β,P-GSK3βand P-PTEN. An IL-10 antibody, but not TGF-β, abolished the influence of Tregs on microglia(decreased GSK3β,P-GSK3β and P-PTEN).3. Western-blot showed the P-GSK3β and P-PTEN was knockdown by correspondingShRNA.After co-cultured with Tregs, GSK3β knockdown resulted in a decrease in P-PTEN in the Hb-activated microglia, but knockdown of PTEN had little effect on P-GSK3β levels. This indicates that GSK3β is in the upstream of PTEN. Thus, Tregs modulate microglia/macrophage polarization towards the M2 phenotype through the IL-10/GSK3β/PTEN axis.Conclusions:1. Depleting Tregs usingCD25 antibodies or in Foxp3 DTR mice exacerbated ICH induced brain inflammatory injury, while therapeutically boosting Tregs using CD28-SAameliorated these injuries.2. Tregs depletion shifted the Hb-activated microglia polarization towards the M1 phenotype, while boosting Tregs shifted the Hb-activated microglia polarization toward the M2 phenotype.3. IL-10 originating from Tregs can convert Hb-activated microglia/macrophages towardsthe M2 phenotype through GSK3β/PTEN axis.This study indicatesthat Tregs protect against ICH-induced inflammatory injury by modulating microglia/macrophage polarization through the IL-10/GSK3β/PTEN axis. Boosting Treg may be a new target for ICH therapy. |
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