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.

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:PURPOSE: Estrogen receptor alpha (ERα, encoded by ESR1) is a well-characterized transcription factor expressed in more than 75% of breast tumors and is the key biomarker to direct endocrine therapies. On the other hand, much less is known about estrogen receptor beta (ERβ, encoded by ESR2) and its importance in cancer. Previous studies had some disagreement, however most reports suggested a more favorable prognosis for patients with high ESR2 expression. METHODS: To add further clarity to ESR2 in breast cancer, we interrogated a large population-based cohort of primary breast tumors (n=3207) from the SCAN-B study (Sweden Cancerome Analysis Network—Breast [SCAN-B], ClinicalTrials.gov identifier: NCT02306096). Spearman rank correlation was used to determine correlations between expression of ESR1 and ESR2. Kruskal-Wallis non-parametric test and Wilcoxon rank sum test were used to compare and plot expression of the ESR1 and ESR2 genes in various clinical groups such as PAM50 subtype and age groups in both the SCAN-B and TCGA cohorts. Transformed ESR2 expression data was divided into tertiles, with the first tertile defined as ESR2-high, and the bottom two tertiles at ESR2-low. Mann Whitney U test and Fisher’s exact test were used to evaluate significant differences in the clinicopathological variables for the ESR2-high and ESR2-low groups. Survival analysis was performed by Kaplan-Meier and Cox regression survival analyses that included a median follow-up of 6.2 years (IQR = 2.2). RESULTS: Using RNA-seq data, we found that ESR2 is expressed at low levels overall with a slight inverse correlation to ESR1 expression (Spearman R = -0.18, p = 2.2e-16), and highest ESR2 expression in the basal- and normal-like PAM50 subtypes. ESR2-high tumors had favorable overall survival (p=0.006), particularly in subgroups receiving endocrine therapy (p=0.03) and in triple-negative breast cancer (p=0.01). These results were generally robust in multivariable analyses accounting for patient age, size, node status, and grade. Gene modules consistent with immune response were associated to ESR2-high tumors. CONCLUSIONS: Taken together, our results indicate that ESR2 is generally expressed at low levels in breast cancer but associated with improved overall survival and may be related to immune response modulation.

Project description:E2 exposure significantly decreased peak viral titer in hNECs from female donors. We used microarray analyses to identify global gene expression patterns between E2 and vehicle exposed hNECs from female donors Influenza causes an acute infection characterized by virus replication in respiratory epithelial cells. The severity of influenza and other respiratory diseases changes over the life course and during pregnancy in women, suggesting that sex steroid hormones, such as estrogens, may be involved. Using primary, differentiated human nasal epithelial cell (hNEC) cultures from adult male and female donors, we exposed cultures to the endogenous 17Î²-estradiol (E2) or select estrogen receptor modulators (SERMs), then infected cultures with a seasonal influenza A virus (IAV) to determine whether estrogenic signaling could affect the outcome of IAV infection and whether these effects where sex-dependent. Estradiol, raloxifene, and bisphenol A decreased IAV titers in hNECs from female, but not male, donors. The estrogenic decrease in viral titer was dependent on the genomic estrogen receptor- 2 (ESR2) as neither genomic ESR1 nor non- genomic GPR30 were expressed in hNEC cultures and addition of the genomic ER antagonist ICI 182,780 reversed the antiviral effects of E2. Treatment of hNECs with E2 had no effect on interferon or chemokine secretion, but significantly downregulated cell metabolic processes, including genes that encode for zinc finger proteins, many of which contain estrogen response elements in their promoters. These data provide novel insights into the cellular and molecular mechanisms of how natural and synthetic estrogens impact IAV infection in respiratory epithelial cells derived from humans. Primary human nasal epithelial cells from females were exposed to E2 for 24h prior to infection, then infected with an H3N2 strain of influenza a virus for 2 hours. At 24 and 48h post infection, hNECs were collected in Trizol for RNA extraction and hybridization on Affymetrix Human Gene ST 2.0 microarrays.

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Expression data from BMDM

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