Microglia Confers Strong Immune Inhibition Through PD-L1 In Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is a common central nervous system (CNS) associated inflam-matory demyelinating autoimmune disease. The etiology and pathogenesis of MS is very complex. Considering the difficulty of acquiring pathologic sampling from human, the analog animal model of MS, experimental autoimmune encephalomyelitis (EAE) is established. MS patients and EAE animals have a common characteristic, that is, remission followed by the peak of disease. This display a protection from strong immune response in brain, indicating there occurs negative immune regulation to limit injury to the nerve tissue. What is key cell to mediate CNS-associated immune regulation? What is the mechanism under immune privilege in the brain?There is a specialized phagocytic cells called microglia in the CNS, As the most immune cells, microglia monitor tissue damage or pathological changes. Due to low expression of antigen presenting molecules such as MHC-Ⅱ, B7 and CD40, microglia is a potential antigen-presenting cells(APC)in the CNS. Previous study confirmed that resting microglia not only lack the ability of antigen presentation, but also inhibit activated-CD4+T cell proliferation, suggesting involvement of microglia in the formation of immune privilege under physiological conditions. What is the role of activated microglia in the CNS under pathological conditions?The programmed cell death ligand-1/programmed death-1 (PD-L1/PD-1) is an important member of the co-stimulatory molecule superfamily. PD-L1/PD-1 associated cell signaling play a role in negative regulation, helping to induce and maintain the immune peripheral tolerance by regulating T cell activity. Privelous study showed that PD-L1 expression in the CNS gradually increased during EAE, while blocking PD-L1/PD-1 signaling pathway caused early EAE and aggravated the symptoms of the disease. Given the large number of microglial in the brain as well as involvement in neuroinflammation, whether they take part in immune reguation during EAE by express PD-L1 is need to investigate. We sought to clarify the role of microglia in the negative immune regulation occurred in CNS as well as PD-L1 associated molecular mechanisms. This study helps to provide a theoretical and experimental basis for finding the therapeutic targets in CNS pathological disease. Our study consists of five sections:(1) Induction of EAE in C57BL/6 miceWe first have successfully established the classic animal model of EAE. In brief, myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide companied with Mycobacterium tuber-culosis and pertussis toxin were used to subcutaneously immune sensitive animal strains C57BL/6 mice. After 10 days, mice displayed typical EAE clinical neurological symptoms, a progressive chronic single phase. At about 20 days, EAE reached the peak, accompanied by pathological changes of the CNS. During the peak of EAE, HE staining showed peri vascular lymphocytes infiltration in the white matter of the spinal cord, displaying the sleeve-like inflammatory structural changes. While the control group of animals did not show any symptoms of the nervous system, and any pathological changes in the CNS.(2) Dynamic changes in cell number, activation and PD-L1 expression of microglial during EAEFlow cytometry results showed that the percentage of microglia (CD45low CD11b+) in CD45+cells was 92.9% in the CNS of untreated mice, whereas microglia proportion continued to decline and reach the lowest valley (46.2%) in the peak of EAE, followed by recovery. Absolute count results showed that the number of microglia continue to increase in EAE mice. When compared with control group, there is significantly increased microglia in the onset, increase and peak-onset in CNS during EAE (p<0.05). The number of microglial was correlated with disease progression.We detected the dynamic changes in the expression of antigen-presentation associated membrane molecules in microglia. The results showed that expression of MHC-Ⅱ molecules progressive increased, whereas CD86 expression significantly upregulated in onset phase (p< 0.05), followed by decrease to normal level in increase phase during EAE. The pattern of PD-L1 expression was similar to MHC-II molecules, displaying an increase in pre-onset, onset, increase and peak phase. We also found there was no significant difference in PD-L2 and B7-DC expression in EAE when compared with control mice. Upregulation of MHC-Ⅱ and CD86 molecules in microglia in pre-onset and onset phase reflected potential immune stimultion, whereas increased expression of MHC-Ⅱ and PD-L1 in increase and peak phase suggested potential negative immune regulation.In this study, we also detected the Iba-1 expression in lumbar spinal cord tissue by immunofluorescence to determine the distribution and activation of microglia. In treated group, microglia express low levels of Iba-1, mainly displaying branch-like shape and scattered distribution. In the peak of EAE, Iba-1 expression increased in the lumbar spinal cord tissue. Meanwhile, microglia from outside and posterior median fissure in the white matter showed larger cell body, disappeared branch retraction, stout shape, dendritic or amoebic-like shape, and overlaped each other. In gray matter area, increased expression of Iba-1 expression are also visible, companied with enhanced cell body, the shorter branches, and increased cell density. In the median sulcus white matter, microglia slightly upregulated Iba-1, but cell mophology did not significantly changed.(3) The changes in the number of CD4+ T cells, PD-1 expression, and CD4+ T subset during EAEThis study examined the CD4+ T cell infiltration in the CNS during EAE, showed that CD4+ T cells entered the CNS prior to the clinical symptoms. After onset, the percentage of CD4+T cells in CD45+ cells gradually increased and reached the peak (20.8%) at EAE peak phase, then followed by decline. This data suggest that certain factors trigger a large number of T cell apoptosis, thereby promoting the recovery of EAE. Flow cytometry results showed that infiltrated CD4+T cells in CNS progressively upregulated PD-1, and reached the highest level at peak phase.We also examined the PD-1 expression on CD4+T cells infiltrated to CNS by immun-ohistochemistry fluorescence. The results showed that many CD4+/PD-1+T cells distributed in spinal cord at the peak of EAE. These cells located in the region of microglia aggregation, suggesting microglia and CD4+T cells interact in CNS during EAE.In this study, we detected the dynamic changes in Thl/Th17 subset in the brain and spinal cord during different period of EAE. The results showed that IFN-gamma and IL-17 expression on CD4+T cells in CNS gradually increased and reached the highest level at increase phase of EAE, followed by subsequent decline, suggesting that apoptosis of effector CD4+T cells in the CNS may occur. Accumulated evidence showed that EAE were characterized by self-recovery, during which CD4+Foxp3+T cells play an immportant role. We thus detected the dynamic changes of CD4+Foxp3+Treg, and showed that CD4+Foxp3+ Treg in the brain continued to increase and reached a peak (34.6%) in the peak of EAE, followed by a decline in remission. The finding that down-regulation of IL-17 and IFN-gamma companied with up-regulation of Treg, indicated that the ratio of effector to Treg may affect neuroinflammation. Combined with the previous results, we speculated that microglia may through PD-L1/PD-1-mediated signaling to regulate the differentiation of CD4+T cells to play a negative role in immune regulation.(4) Verification of immune supression by microglia though co-culture of microglia and OVA323-339 specific CD4+T cells in vitroMicroglia significantly upregulated PD-L1 in the peak phase during EAE. Considering the decreased proporsion of Thl/Th17 subset at the same time, we suggested that microglia negatively regulated inflammation in the CNS. We thus coculture sorted-microglia from EAE mice and OVA323-339 specific CD4+T cells in vitro, take the system to determine the immunosuppressive function of microglia.First, coculture of microglial and OTⅡ naive CD4+T cells were used to observe the ability of microglia to present antigen. The results showed that microglia having a certain ability to stimulate T cell proliferation, but the capacity was significantly lower than spleen DC. Having the capability of induction of Thl, microglia tend to induce Thl other than Th17.Second, microglial in vitro was added to spleen DC/CD4+T cell co-culture system to observe whether it can inhibit DC-stimulated CD4+T cell proliferation. The result showed that the number of CD4+T cells significantly decreased after addion of microgla, indicating that microglial from EAE mice can inhibited spDC-stimulated CD4+T cell proliferation. Meanwile, we found that there was an decrease in the precentage of INF-y+CD4+T cells and no change in the the precentage of IL-17+CD4+T cells in microglia/spDC/CD4+T coculture system as compared to spDC/CD4+T coculture system.(5) Explore the immunosuppression mechanism of microglial cellsIn order to discuss the immunosuppression mechanism of microglial cells, we detected the changes of Treg in co-culture system, the results show that the ratio of Treg in EAE-MG/T groups were higher than that in CD4+T group, with statistical difference (P<0.05); joining microglia of EAE peak to spDC/T group,Treg was significantly increased (P<0.05). Results indicate that microglia may play a role in immune suppression induced by Treg.Some experiments showed that NO has the ability to inhibit T cell proliferation. In this study, real-time quantitative PCR were used to detect the expression of nitric oxide synthase in microglia during EAE. Our results showed that microglia from EAE mice expressed highly oxide synthase when compared with untreated group, suggesting that microglia may inhibit T cell proliferation by releasing NO. Addition of nitric oxide synthase inhibitor L-NAME into co-culture system led to a significant recovery of CD4+T cell proliferation.(6) Investigate whether microglia exert suppressive activity by PD-L1/PD-1 signaling pathway in vitroMG/T training system, and blocking anti-PD-L1 antibody CD4+T cells increased and statistically significant (p<0.05), the same results appear in MG/spDC/T Totalculture system. However, compared with the positive control group, i.e. spDC/T, CD4+T cell proliferation recovery is not complete, only 1/4.These described PD-L1 indeed involved in the inhibition mediated by microglia, but not all effects by its cause, there must have been other factors play a role, to be further studied.To investigate whether PD-L1 can affect microglia-mediated T cell differentiation, PD-L1 were blocked in vitro. Our results showed that blocking PD-L1 in microglia/T and microglia/spDC/T system lead to an significant increase in Th1 (p<0.05), indicating that PD-L1 have the ability to inhibit Thl differentiation.We also investigate the role of PD-L1 in microglia-induced Treg, and found that Treg differentiation were not affected after blocking PD-L1 in microglia/T and microglia/spDC/T system (P>0.05). This data indicated that PD-L1 are not involved in the induction of Treg cells by microglia, suggesting that T cell increase by blocking PD-L1 in coculture system is not caused by reduction of Treg cells.To investigate whether PD-L1 affect microglia to secrete NO, we detected the NO concentration of coculture system. We found that blocking PD-L1 lead to lower NO than that of isotype control antibody group, indicating that PD-L1 may regulate T cell proliferation and Thl differentiation by affecting microglia secrete NO.In summary, this research shows that the peak of EAE microglia is a kind of inhibitory cells with immunosuppressive phenotype and inhibit immune response by inducing Treg, the secretion of NO. The PD-L1 expresssed high on the microglial of peak of EAE was associated with the immune regulation by affecting secreted NO of microglial cells.These results confirmed the negative immune regulation played by microglia in the EAE, helping understand the mechanism of EAE and MS, providing a theoretical and experimental basis for treat MS.