p38 MAP Kinase Signaling in Microglia Plays a Sex-Specific Protective Role in CNS Autoimmunity and Regulates Microglial Transcriptional States

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.

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Single-cell transcriptome analysis of CD34+stem cell-derived myeloid cells identifies a CFU-GEMM-like population permissive to human cytomegalovirus infection

10.1101/438457 ◽

2018 ◽

Cited By ~ 2

Author(s):

Melissa Galinato ◽

Kristen Shimoda ◽

Alexis Aguiar ◽

Fiona Hennig ◽

Dario Boffelli ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Viral Entry ◽

Single Cells ◽

Myeloid Cells ◽

Therapeutic Interventions ◽

Transcriptional Analysis ◽

Viral Gene ◽

Small Subset ◽

Cmv Infection ◽

Hematopoietic Stem

ABSTRACTMyeloid cells are important sites of lytic and latent infection by human cytomegalovirus (CMV). We previously showed that only a small subset of myeloid cells differentiated from CD34+hematopoietic stem cells is permissive to CMV replication, underscoring the heterogeneous nature of these populations. The exact identity of susceptible and resistant cell types, and the cellular features characterizing permissive cells, however, could not be dissected using averaging transcriptional analysis tools such as microarrays and, hence, remained enigmatic. Here, we profile the transcriptomes of ∼ 7000 individual cells at day one post-infection using the 10X genomics platform. We show that viral transcripts are detectable in the majority of the cells, suggesting that virion entry is unlikely to be the main target of cellular restriction mechanisms. We further show that viral replication occurs in a small but specific sub-group of cells transcriptionally related to, and likely derived from, a cluster of cells expressing markers of Colony Forming Unit – Granulocyte, Erythrocyte, Monocyte, Megakaryocyte (CFU-GEMM) oligopotent progenitors. Compared to the remainder of the population, CFU-GEMM cells are enriched in transcripts with functions in mitochondrial energy production, cell proliferation, RNA processing and protein synthesis, and express similar or higher levels of interferon-related genes. While expression levels of the former are maintained in infected cells, the latter are strongly down-regulated. We thus propose that the preferential infection of CFU-GEMM cells may be due to the presence of a pre-established pro-viral environment, requiring minimal optimization efforts from viral effectors, rather than to the absence of specific restriction factors. Together, these findings identify a potentially new population of myeloid cells susceptible to CMV replication, and provide a possible rationale for their preferential infection.AUTHOR SUMMARYMyeloid cells such as monocytes and dendritic cells are critical targets of CMV infection. To identify the cellular factors that confer susceptibility or resistance to infection, we profiled the transcriptomes of ∼ 7,000 single cells from a population of semi-permissive myeloid cells infected with CMV. We found that viral RNAs are detectable in the majority of the cells, but that marked expression of CMV lytic genes occurs in only a small subset of cells transcriptionally related to a cluster of CFU-GEMM progenitors that express similar amounts of transcripts encoding interferon-related anti-viral factors as the rest of the population but higher levels of transcripts encoding proteins required for energy, RNA, and protein production. We thus conclude that the preferential infection of CFU-GEMM cells might be due to the pre-existing presence of an intracellular environment conducive to infection onset, rather than to the absence of anti-viral factors restricting viral entry or initial gene expression. Together, these findings uncover a new type of myeloid cells potentially permissive to CMV infection, expand our understanding of the cellular requirements for successful initiation of CMV infection, and provide new pro- and anti-viral gene candidates for future analyses and therapeutic interventions.

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