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Bulk RNAseq of human Tregs transduced with a wild type HDAC7 or a version carrying the MS-protective R166H variant

ABSTRACT: Genome-wide association studies identifying hundreds of susceptibility loci for autoimmune diseases indicate that genes active in immune cells predominantly mediate risk. Identification and functional characterization of causal variants remains challenging. Here, we focused on the immunomodulatory role of a protective variant in HDAC7 (rs148755202, HDAC7.p.R166H), identified in a study of low-frequency coding variation in multiple sclerosis (MS). Through transcriptomic analysis, we demonstrate that HDAC7 regulates genes essential for the function of FoxP3+ regulatory T cells (Tregs), an immunosuppressive subset of CD4 T cells that is dysfunctional in patients with MS. Moreover, Treg cell-specific conditional hemizygous deletion of HDAC7 increases severity of experimental autoimmune encephalitis (EAE), a mouse model of MS. Strikingly, Tregs transduced with the protective HDAC7 R166H variant exhibit higher suppressive capacity in an in vitro functional assay, mirroring functional defects previously observed in patients. In vivo modelling of the human HDAC7 R166H variant, by generation of a knock-in mouse model bearing the orthologous R150H substitution, showed decreased EAE severity linked to transcriptomic alterations of brain infiltrating Tregs as assessed by single cell RNA-seq. Our data suggest that dysregulation of epigenetic modifiers, a novel molecular class associated with disease risk, can influence disease onset. Finally, our approach provides a template for translation of genetic susceptibility loci to detailed functional characterization, using human in vitro and mouse in vivo modelling.

ORGANISM(S): Homo sapiens

PROVIDER: GSE215818 | GEO | 2022/10/17

REPOSITORIES: GEO

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Project description:Vitamin D3 (VitD) insufficiency is postulated to represent a major modifiable risk factor for multiple sclerosis (MS). While low VitD levels strongly correlate with higher MS risk in white populations, this is not the case for other ethnic groups, suggesting the existence of a genetic component. Moreover, VitD supplementation studies in MS so far have not shown a consistent benefit. We sought to determine whether direct manipulation of VitD levels modulates central nervous system (CNS) autoimmune disease in a sex-by-genotype-dependent manner. To this end, we used a dietary model of VitD modulation, together with the autoimmune animal model of MS, experimental autoimmune encephalomyelitis (EAE). To assess the impact of genotype-by-VitD interactions on EAE susceptibility, we utilized a chromosome substitution (consomic) mouse model that incorporates the genetic diversity of wild-derived PWD/PhJ mice. High VitD was protective in EAE in female, but not male C57BL/6J (B6) mice, and had no effect in EAE-resistant PWD/PhJ (PWD) mice. EAE protection was accompanied by sex- and genotype-specific suppression of proinflammatory transcriptional programs in effector CD4 T effector cells, but not CD4 Treg cells. Decreased expression of proinflammatory genes was observed with high VitD in female CD4 T cells, specifically implicating a key role of MHC class II genes, interferon gamma, and Th1 cell-mediated neuroinflammation. In consomic strains, effects of VitD on EAE were also sex- and genotype-dependent, whereby high VitD: 1) was protective, 2) had no effect, and, 3) unexpectedly had disease-exacerbating effects. Systemic levels of 25(OH)D differed across consomic strains, with higher levels associated with EAE protection only in females. Analysis of expression of key known VitD metabolism genes between B6 and PWD mice revealed that their expression is genetically determined and sex-specific, and implicated Cyp27b1 and Vdr as candidate genes responsible for differential EAE responses to VitD modulation. Taken together, our results support the observation that the association between VitD status and MS susceptibility is genotype-dependent, and suggest that the outcome of VitD status in MS is determined by gene-by-sex interactions.

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. Taken together, these results reveal the presence of a p38α-dependent male-specific molecular pathway in microglia that is protective in CNS autoimmunity, 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.

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Bulk RNAseq of human Tregs transduced with a wild type HDAC7 or a version carrying the MS-protective R166H variant

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