D28-03 Sex-linked Lung Estrobolome May Promote Pulmonary Arterial Hypertension Penetrance Of Bone Morphogenetic Protein Receptor 2 R899X Mutation Via An Endothelin-1high Endoregulatory Macrophage Phenotype

Abstract Rationale Pulmonary Arterial Hypertension (PAH) is about three times more prevalent in women, with men often experiencing a worse disease prognosis, attributed in part to a deficiency in estrogen-mediated cardioprotection. Despite its protective effects, estrogen and its metabolites, including 16α-hydroxyestrogen (16α-OHE), also exert paradoxical effects in PAH, including disruption of endoprotective bone morphogenetic protein receptor 2 (BMPR2) signaling. Over 300 BMPR2 mutations have already been identified in PAH, but fully restoring lung BMPR2 function remains an unmet need. In mice, introducing the loss-of-function Bmpr2 R899X mutation found in humans, induces pulmonary hypertension (PH) with a higher effect in female animals. Hypothesis Our group hypothesized that this heightened response is partly attributed to a sex-linked increase in potent vasoactive molecules, such as endothelin-1 (ET-1), as a response to a dysfunctional lung microbiome. Methods To test this hypothesis, lung tissue from 12-month-old male and female wild-type and BMPR2+/R899X mutant mice was used for western blot, ELISA, flow cytometry, metagenomic analysis, and immunohistochemistry (IHC). In addition, bone marrow-derived macrophages (BMDMs) treated with vehicle control, 100 ng/mL Lipopolysaccharide (LPS), and/or 16α-OHE (24 hours) were subsequently exposed or not to fluorescent E. coli for a phagocytosis assay. Cell lysates and supernatants were used for immunocytochemistry, Western, and Dot Blot Analysis. Results/Discussion IHC revealed a significant accumulation of CD45+; ET-1high macrophages in the microvasculature of female mutants. In vitro analysis of LPS-treated BMDMs confirmed sex-linked overexpression of ET-1. Metagenomic analysis revealed sex-specific dysbiosis of the lung microbiome, which can disturb the biodistribution of estrogen and its metabolites, via a specific subgroup of β-glucuronidase+ bacteria known as the estrobolome. Indeed, our data indicate that female PH lungs exhibit elevated β-glucuronidase expression, increased lung LPS, and an abundance of estrobolome antibiotic-sensitive bacteria, which could amplify vascular inflammation through the accumulation of 16α-OHE and infiltration of BMDM-producing ET-1 in the lungs. Macrophages play a pivotal role in recognizing and eliminating potential threats; however, opportunistic microbes can trigger an inflammatory cascade when their function is compromised. In general, estrogen limits this effect by modulating the expression pattern of pathogen recognition receptors, but its metabolites also have toxic effects. Conclusion Therefore, lung microbiome dysbiosis may drive the accumulation of damaging estrogen metabolites, contributing to recruitment and differentiation of an endoregulatory ET-1high macrophage phenotype and the development of sex-linked PAH. This abstract is funded by: NIH, HEPP