PROJECT SUMMARY Tuberculosis (TB) occurs more frequently in males (~70% of cases) than females. While reduced access to care for females and undercounting of female cases may contribute modestly to this longstanding epidemiologic observation in some countries, both human studies (the Lűbeck disaster and eunuch studies) as well as animal models underscore the fact that biological factors play a dominant role in female resistance to TB. Despite the importance of this sex-bias in TB, it has gone largely neglected by basic science researchers. The central scientific premise of this application is that defining the biological mechanisms of the male bias for TB will enlighten our mechanistic understanding of TB pathogenesis and protective human immune responses in TB. A multidisciplinary team with decades of expertise on sex differences (Klein), TB pathogenesis (Bishai), and sex differences in HIV (Scully) will conduct the project. This proposal will investigate the impact of genetics (X chromosome complement) and sex steroid hormones on TB pathogenesis using cellular tools, animal models, and human samples. A key tool used in Aim 1 will be the novel four core genotype (FCG) mouse model, in which animals with XX genotypes have male gonads/hormone levels and those with XY genotypes have female gonads/hormone levels. This model will enable us to differentiate the impact of genetics from those of the sex steroids on murine control of TB. The X chromosome encodes numerous genes of immunologic importance including the genes that encode for TLR7, TLR8, CYBB, NEMO, CD40L, and FOXP3. The process of X-chromosome inactivation (XCI)--in which one female X chromosome is epigenetically silenced--is designed to provide balanced gene dosing between females and males. Certain genes, however, can escape XCI, leading to a double gene dose in females. The process of gene escape from XCI has not been studied in the context of TB, and advanced molecular tools will be used in Aim 2 to investigate gene escape from XCI as a cause of the male bias in severity of TB. While it is widely known that sex steroid signaling modulates immune function, the impact of sex steroids as a basis for the male bias in TB has not been thoroughly investigated. Testosterone has an immunotolerizing effect, reducing levels of IFN-γ and elevating levels of IL-4. In contrast, estrogen promotes higher levels of macrophage activation and increases in TNF-α levels. In Aim 2, we will carefully dissect the role of sex steroids using adoptive transfer methods as well as gonadectomized mice with selective hormone replacement. Lastly, in Aim 3 we will extend these studies to assess the intersection of HIV infection, a critical risk for TB, and sex differences in immunity. We will assess immune responses in samples from people living with HIV (PLWH) on ART versus healthy controls (HCs), specifically balancing groups for sex. We will evaluate the ability of whole blood and hMDM from PLWH and HCs to contain Mtb growth both alone and in the presence of DHEA. Lastly, we will assess transcriptomic responses in PBMCs from male and female PLWH and HCs following Mtb infection.

PROJECT NARRATIVE Despite the fact that TB occurs more frequently in males (~70% of cases) than in females, very little is known about the biological factors that lead to this difference. In this study, a team with expertise on sex differences, TB pathogenesis, and HIV will apply cutting edge tools to evaluate genetic and hormonal explanations for sex differences in TB outcomes. Because HIV-associated TB is an important contributor to the global TB epidemic, the team will also evaluate sex differences in immune responses among human males and females living with HIV.