Date on Master's Thesis/Doctoral Dissertation

8-2025

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Microbiology and Immunology

Degree Program

Microbiology and Immunology, PhD

Committee Chair

Kosiewicz, Michele

Committee Member

Alard, Pascale

Committee Member

Jala, Venkatakrishna

Committee Member

Powell, David

Author's Keywords

SLE; sex bias; microbiota; androgen; CD103DC; LP stromal cells

Abstract

Systemic lupus erythematosus (SLE) is a complex autoimmune disease involving broad immune dysregulation. Despite decades of research, most lupus treatments still have limited effectiveness and can cause serious side effects. Both genetic and environmental factors contribute to lupus susceptibility, and a better understanding of how environmental factors influence disease could guide future therapeutic strategies. SLE shows a strong female predominance. Growing evidence implicates the gut microbiota and sex hormones as key environmental contributors to some autoimmune diseases, including lupus. The gut microbiota can influence host immune function, and interestingly, recent studies have revealed sex-bias in microbiota composition and function. Moreover, the microbiota and sex hormones interact bidirectionally: androgens can influence microbial communities, while microbiota can produce or metabolize sex steroids. Regulatory T cells (Tregs), especially peripherally-induced pTregs, are key to immune tolerance, and impaired Tregs, particularly their frequencies, is closely associated with lupus. A distinct population of CD103⁺ dendritic cells in the gut excels at inducing pTregs, primarily through the production of retinoic acid (RA) and TGF-β. Lamina propria stromal cells (LPSCs) are essential for CD103⁺ DC development by providing key signals such as retinoic acid (RA) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Previous work from our lab showed that male BWF1 microbiota differs significantly from that of females, and that transferring male microbiota into female recipients delays lupus onset and improves survival. In addition, intact male BWF1 mice are more resistant to disease, but this protection is lost upon castration, supporting a protective role for androgens. However, the precise mechanisms underlying protection mediated by androgens and male-imprinted microbiota remain poorly understood. The overall goal of this dissertation was to identify the key immunological components and elucidate the mechanisms by which androgens and male-associated microbiota suppress lupus-like disease. To address this, we proposed two specific aims. Aim 1 focused on defining the mechanisms by which male microbiota exerts protection in female BWF1 mice. Aim 2 investigated the mechanisms by which androgens contribute to immune regulation and protection from disease observed in lupus-resistant male BWF1 mice. As we sought to understand how male-associated microbiota and androgens mediate protection in lupus-prone BWF1 mice, we first examined regulatory T cell populations. Both female and castrated male mice exhibited significantly reduced frequencies of Foxp3⁺ regulatory T cells (Tregs), particularly the peripherally induced Tregs (pTregs). To identify the source of this defect, we turned to CD103⁺ dendritic cells (CD103⁺DCs), a tolerogenic DC known to promote pTreg differentiation via TGF-β and retinoic acid (RA). CD103⁺DCs from female and castrated male BWF1 mice showed impaired ability to induce pTregs, which was linked to reduced expression of RALDH2, the key enzyme required for RA synthesis. We determined that the defect in female CD103⁺DCs was due to insufficient availability of exogenous RA, as RA supplementation restored their function. We next investigated the role of lamina propria stromal cells (LPSCs), which are known to support CD103⁺DC development and function by producing RA and GM-CSF. LPSCs from female and castrated male mice exhibited reduced expression of RA pathway genes and diminished RALDH enzymatic activity. This implicated LPSC dysfunction as a contributor to defective CD103⁺DC function and pTreg generation by failing to provide a sufficient exogenous source of RA that is required for normal CD103⁺DC development. To determine whether the previously observed protective effect of male microbiota transfer is mediated through restoration of the tolerogenic LPSC–RA–CD103⁺DC–pTreg axis, we transferred intact male microbiota into female and castrated male BWF1 mice. This transfer restored RA pathway gene expression in LPSCs, improved CD103⁺DC function, and restored Foxp3⁺ Treg frequencies. Because gut microbes influence immunity in part through secreted metabolites, we examined phytanic acid (PA), a microbial metabolite enriched in intact male (compared to female or castrated male) mice and a known RXR agonist, as a potential mediator. Administration of PA to female and castrated male mice restored RA-associated gene expression in LPSCs, enhanced CD103⁺DC development and function, increased pTreg frequencies, and ameliorated disease. Our data support a role for PA in modulating LPSCs function and facilitating the tolerogenic CD103⁺ DC–pTreg pathway, which may contribute to the observed lupus protection. In Aim 2, we further examined the mechanisms of androgens in the protection of lupus. We found that androgens are essential for maintaining a protective microbial community in males. Castration increased disease susceptibility that was associated with a shift in microbiota composition and reduced PA levels, and similar to females, decreased pTregs and CD103⁺DC function. Importantly, LPSCs express high levels of androgen receptor and respond robustly to androgen stimulation both in vitro and in vivo. Androgen treatment increased the expression of genes involved in RA synthesis in LPSCs, suggesting that these LPSCs may serve as key mediators of androgen-driven immune tolerance. In this study, we investigated two environmental factors that contribute to lupus protection in male BWF1 mice, sex hormones and gut microbiota. Together, our findings suggest both androgens and male-associated microbiota could modulate lamina propria stromal cells to enhance RA-associated LPSC–CD103⁺ DC–pTreg pathways, contributing to immune tolerance in male BWF1 mice. This work adds to our understanding of how sex-based immune differences arise, and highlights LPSCs as a key piece of the puzzle. Although more work is needed to fully define these mechanisms, targeting stromal metabolism or using microbial metabolites like phytanic acid could provide a more targeted approach to promote tolerance in lupus, potentially with fewer side effects than current lupus treatments.

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