Project description: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. Microglial p38α deficiency in males 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. The effect of p38α deficiency was divergent in female microglia, with an upregulation of a small subset of inflammatory genes that overlapped with males, but mostly a unique transcriptional profile that also included an upregulation of potentially tissue reparative genes. Taken together, these results reveal the presence of 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 informing the design of novel future sex-specific therapeutic approaches.

Project description:sex-specific role of p38-MAPK in EAE is regulated by estrogen receptor alpha; Biological sex is a critical factor in regulating immune function. A striking example of this is the higher prevalence of autoimmune diseases such as multiple sclerosis (MS) and lupus in females compared to males. While many studies have implicated the role of sex hormones such as estrogens and androgens in these sex differences, surprisingly little is known about other molecular pathways that underlie sex differences or interact with sex hormones. We have previously shown that conditional ablation of p38α MAP kinase signaling in myeloid cells (p38αCKO) was protective in a mouse model of MS, experimental autoimmune encephalomyelitis (EAE), in female but not male mice. This sex difference was dependent on the presence of sex hormones, leading us to hypothesize that the pathogenic function of p38α in EAE depends on estrogen signaling via one of the two nuclear estrogen receptors, encoded by Esr1 and Esr2. To test this hypothesis, we performed experiments with p38αCKO macrophages, which demonstrated that the effects of estradiol and p38α were independent of one another in vitro. Since many sex hormone effects are lost in vitro, we generated p38αCKO mice lacking either Esr1 or Esr2, and evaluated their EAE susceptibility in vivo. Myeloid-specific deletion of Esr1 abrogated protection in p38αCKO females, although global deletion of Esr1 and Esr2 did not. Moreover, global or myeloid-specific disruption of Esr1 unexpectedly promoted protection from EAE in p38αCKO males. Mechanistically, Esr1 deletion resulted in partial reprogramming of p38α-dependent transcriptional modules in male macrophages, in particular those regulated by TGFβ, BRD4, and SMARCA4. These results demonstrate that estrogen signaling in myeloid cells plays an important sex-specific role in programming their dependence on specific intracellular signaling pathways in the context of autoimmune disease pathogenesis, suggesting potential avenues for sex-specific therapeutics or combinatorial approaches for the treatment of such diseases.

Project description:SPEACC-seq is a novel high-throughput method which enables forward genetic screens to identify cell-cell interaction mechanisms that uncovered an astrocyte-microglia regulatory circuit mediated by amphiregulin and IL33-ST2. signaling. Cell-cell interactions in the central nervous system (CNS) play central roles in neurologic diseases. However, little is known about the specific molecular pathways involved, and methods for their systematic identification are limited. For example, several factors mediate microglia-astrocyte interactions that promote CNS pathology, but less is known about regulatory interactions that limit tissue pathology. Here we report the development of SPEACC-seq (Stimulation, Perturbation, and Encapsulation of interACting Cells followed by Sequencing), a forward genetic screening platform which combines genome-wide CRISPR/Cas9 perturbations, cell co-culture in picoliter droplets, and microfluidic-based fluorescence activated droplet sorting to identify mechanisms of cell-cell communication. Using SPEACC-seq in combination with an in vivo perturb-seq screen, we identified microglia-produced amphiregulin as a suppressor of disease promoting astrocyte responses in experimental autoimmune encephalomyelitis (EAE), a pre-clinical model of multiple sclerosis (MS). The production of microglial amphiregulin was induced via ST2 signaling by IL-33 released from astrocytes during EAE. Indeed, the genetic inactivation of ST2 or amphiregulin in microglia, or IL-33 or amphiregulin signaling in astrocytes resulted in the worsening of EAE, suggesting that IL-33-induced microglial amphiregulin limits disease-promoting astrocyte responses associated with CNS pathology. This regulatory loop was also detected in human astrocytes and microglia both in vitro and in MS patient CNS samples. In summary, we developed SPEACC-seq, a high-throughput, droplet-based forward genetic screening platform for the identification of cell-cell interaction mechanisms, which identified a novel microglia-astrocyte negative feedback loop that limits CNS pathology.

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P38 MAP kinase signaling in microglia plays a sex-specific protective role in CNS autoimmunity and regulates microglial transcriptional states

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