DataSheet_7_p38 MAP Kinase Signaling in Microglia Plays a Sex-Specific Protective Role in CNS Autoimmunity and Regulates Microglial Transcriptional States.xlsx

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system, representing the leading cause of non-traumatic neurologic disease in young adults. This disease is three times more common in women, yet more severe in men, but the mechanisms underlying these sex differences remain largely unknown. MS is initiated by autoreactive T helper cells, but CNS-resident and CNS-infiltrating myeloid cells are the key proximal effector cells regulating disease pathology. We have previously shown that genetic ablation of p38α MAP kinase broadly in the myeloid lineage is protective in the autoimmune model of MS, experimental autoimmune encephalomyelitis (EAE), but only in females, and not males. To precisely define the mechanisms responsible, we used multiple genetic approaches and bone marrow chimeras to ablate p38α in microglial cells, peripheral myeloid cells, or both. Deletion of p38α in both cell types recapitulated the previous sex difference, with reduced EAE severity in females. Unexpectedly, deletion of p38α in the periphery was protective in both sexes. In contrast, deletion of p38α in microglia exacerbated EAE in males only, revealing opposing roles of p38α in microglia vs. periphery. Bulk transcriptional profiling revealed that p38α regulated the expression of distinct gene modules in male vs. female microglia. Single-cell transcriptional analysis of WT and p38α-deficient microglia isolated from the inflamed CNS revealed a diversity of complex microglial states, connected by distinct convergent transcriptional trajectories. In males, microglial p38α deficiency resulted in enhanced transition from homeostatic to disease-associated microglial states, with the downregulation of regulatory genes such as Atf3, Rgs1, Socs3, and Btg2, and increased expression of inflammatory genes such as Cd74, Trem2, and MHC class I and II genes. In females, the effect of p38α deficiency was divergent, exhibiting a unique transcriptional profile that included an upregulation of tissue protective genes, and a small subset of inflammatory genes that were also upregulated in males. Taken together, these results reveal a p38α-dependent sex-specific molecular pathway in microglia that is protective in CNS autoimmunity in males, suggesting that autoimmunity in males and females is driven by distinct cellular and molecular pathways, thus suggesting design of future sex-specific therapeutic approaches.

多发性硬化（Multiple sclerosis, MS）是一种中枢神经系统（central nervous system, CNS）自身免疫性脱髓鞘疾病，是青年群体中非创伤性神经系统疾病的首要致病原因。该疾病的女性发病率约为男性的三倍，但男性患者的病情更为严重，而这种性别差异背后的分子机制目前仍未完全阐明。多发性硬化由自身反应性辅助T细胞（T helper cells）启动，但中枢神经系统驻留髓系细胞与浸润髓系细胞才是调控疾病病理进程的关键近端效应细胞。

我们此前的研究表明，在髓系谱系中广泛敲除p38α丝裂原活化蛋白激酶（p38α MAP kinase），可在多发性硬化的自身免疫模型——实验性自身免疫性脑脊髓炎（experimental autoimmune encephalomyelitis, EAE）中发挥保护作用，但该保护作用仅见于雌性个体，雄性个体中未见明显效果。为精准阐明背后的作用机制，我们采用多种遗传学手段及骨髓嵌合体（bone marrow chimeras）模型，分别在小胶质细胞（microglial cells）、外周髓系细胞或二者中同时敲除p38α。

同时敲除两种细胞中的p38α，重现了此前观察到的性别差异：雌性个体的EAE病情得到缓解。意外的是，仅在外周髓系细胞中敲除p38α，可在两种性别个体中均发挥保护作用。相反，仅在小胶质细胞中敲除p38α，仅会加重雄性个体的EAE病情，这揭示了p38α在小胶质细胞与外周髓系细胞中发挥着截然相反的调控作用。

批量转录组测序分析显示，p38α在雄性与雌性个体的小胶质细胞中调控着不同的基因模块。对从炎症性中枢神经系统中分离的野生型（wild type, WT）及p38α缺陷型小胶质细胞进行单细胞转录组测序分析，结果揭示了多种复杂的小胶质细胞状态，这些状态通过不同的趋同转录轨迹相互关联。

在雄性个体中，小胶质细胞p38α缺失会促进小胶质细胞从稳态向疾病相关状态转化，伴随Atf3、Rgs1、Socs3及Btg2等调控基因的表达下调，以及Cd74、Trem2、主要组织相容性复合体（Major Histocompatibility Complex, MHC）I/II类基因等炎症相关基因的表达上调。而在雌性个体中，p38α缺失的作用则截然不同：其转录谱具有独特性，不仅上调了组织保护相关基因的表达，还上调了一小部分在雄性个体中也会上调的炎症相关基因。

综上，本研究揭示了小胶质细胞中一条依赖p38α的性别特异性分子通路，该通路在雄性个体的中枢神经系统自身免疫中发挥保护作用；这一结果提示，雄性与雌性的自身免疫疾病由不同的细胞及分子通路驱动，也为未来开发性别特异性治疗方案提供了理论依据。