| Backgroud1.Multiple sclerosis (MS) is a chronic autoimmune disease with complex clinical, genetic and pathological backgrounds, characterized by inflammatory demyelination in the central nervous system (CNS). It has been shown that MS can be influenced by multiple factors, including several endogenous regulatory factors and pathways. Gr-1+/CD11b+ myeloid cells constitute a population of immature myeloid cells with potent immunosuppressive functions. These cells have been shown to expand markedly during multiple diseases and regulate the progression and severity of disease. Unfortunately, there are few reports on the cell population during experimental autoimmune encephalomyelitis (EAE), a classical model for human MS. It has been generally accepted that programmed death ligand-1/programmed death-1 (PD-L1/PD-1) are the principal molecules belonging to the B7/CD28 costimulatory superfamily and the PD-L1/PD-1 pathway can induce immuno- suppression during EAE. Whether Gr-1+/CD11b+ myeloid cells from EAE mice induce immunosuppression via the expression of PD-L1 is still not well established and needs further investigation.2.It is now well established that the brain, ranging from mammalian to human, has the capacity to produce new neurons into adulthood. The phenomenon might offer a possible implication for the repair of lesions in the CNS during multiple diseases. Multipotent neural stem/progenitor cells (NSC/NPC) have been demonstrated to be produced by two discrete regions, namely, the subgranular zone (SGZ) of dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ). Several studies on the adult neurogenesis during EAE have been reported, however, the conflicting results are obtained. Moreover, all the studies have focused on the adult neurogenesis in the SVZ and the changes in the DG of hippocampus are not addressed. Thus, further studies are needed to clarify the above questions.Objective1.To observe the changes of Gr-1+/CD11b+ myeloid cells in the spleen and the CNS of mice with MOG35-55 peptide-induced EAE as well as the changes of PD-L1 expressed on the cell population; to investigate the contribution of PD-L1 to the inhibition of in vitro T cell proliferation by the Gr-1+/CD11b+ myeloid cells from EAE mice.2.To explore the effects of MOG-induced chronic/persistent EAE on the adult neurogenesis, especially on the NSC/NPC proliferation in the DG and SVZ regions. Methods1. Experimental autoimmune encephalomyelitis (EAE) was induced in female C57BL/6 mice by the subcutaneous immunization with MOG35-55 peptide. The histopathological changes were demonstrated by hematoxylin&eosin (HE) staining and Luxol fast blue (LFB) myelin staining. The presence of Gr-1+/CD11b+ myeloid cells in the spleen and the CNS was determined and the quantitive change of the cell population were then examined. The expression of PD-L1 on Gr-1+/CD11b+ myeloid cells were measured by flow cytometric analysis. By in vitro cell coculture, we tested the possible suppressive effect of Gr-1+/CD11b+ myeloid cells from the CNS tissue of EAE mice on the proliferation of activated CD4+ and CD8+ T cells from control mice.2. A model of chronic/persistent EAE was established by the immunization with MOG35-55 peptide. A 5-bromo-2′-deoxyuridine (BrdU) incorporation was used and brain sections from EAE or control mice were then stained with anti-BrdU antibody. The proliferation of NSC/NPC was determined by counting the number of BrdU-labeled cells in the SGZ of the DG of the hippocampus and in the SVZ from EAE or control mice.Results1.The immunization with MOG35-55 peptide led to limb paralysis in female C57BL/6 mice, with the morbility of 80 %. HE staining showed a large quantity of inflammatory cells infiltrated in the CNS of EAE mice and the LFB myelin staining showed many demyelinated lesions among the regions with inflammatory infiltration. In contrast, no clinical symptoms and histopathological changes were observed in the control mice. In EAE mice, many Gr-1+/CD11b+ myeloid cells were present in the CNS, while no positive cells were found in control mice. Compared with the control group, the weight of spleen in EAE group was prominently increased (146.5±12.4 mg vs. 67.2±8.7 mg, P<0.001). Flow cytometric analysis showed an extensive expansion of splenic Gr-1+/CD11b+ myeloid cells in EAE mice (P < 0.001 vs. control group). Consistent with the results of immunofluorescent staining, there was an increased number of Gr-1+/CD11b+ myeloid cells in the CNS of EAE mice, while no positive cells were seen in the control group. In the further study, we found that the expression of PD-L1 on the Gr-1+/ CD11b+ myeloid cells from EAE mice was upregulated. In vitro functional analysis showed that these cells could significantly suppress the proliferation of activated CD4+ T and CD8+ T cells from control mice, and the suppressive effect was alleviated after the blockade of PD-L1.2.MOG35-55 peptide-immunized C57BL/6 mice developed chronic/persistent EAE, an EAE subtype believed to be analogous to the progressive forms of MS. EAE mice reached the peak of disease rapidly after the onset, followed by a brief remission and then a stable chronic phase, while no clinical symptoms were developed in the control mice. Double immunofluorescent staining with anti-CD4 and anti-BrdU showed that no proliferating CD4+ T cells (BrdU+/CD4+) were observed in the SGZ of DG of the hippocampus. There were BrdU+/CD4+ T cells distributed among the stem cells in the SVZ, although these cells occupied only 0.3 % of all dividing cells in this region and were thus negligible in our statistical analysis of the number of BrdU+ cells in the region. Compared with the control group, the number of BrdU+ cells in the SGZ of DG of the hippocampus was significantly decreased (reduction of 15.7±4.0 %, mean±SEM; P < 0.05). The quantification of BrdU+ cells in the SVZ showed similar results (reduction of 10.0±2.9 %; P < 0.05). All these results suggest that there is an impaired proliferation of NSC/NPC in EAE mice.Conclusions1.EAE induces an extensive expansion of Gr-1+/CD11b+ myeloid cells with an increased expression of PD-L1 in the spleen and the CNS. Furthermore, the Gr-1+/CD11b+ myeloid cells can suppress in vitro T cell proliferation via the PD-L1 signaling, which may provide a new immunoregulatory pathway for EAE.2.MOG35-55 peptide-induced Chronic/persistent EAE lead to a significantly decreased NSC/NPC proliferation in the SVZ. Furthermore, the SGZ of DG of the hippocampus reveals a reduction of proliferation of the NSC/NPC as well, which may provide a possible explanation for the extensive cognitive impairment in human chronic MS. |
