Establishment of humanized gut and vaginal microbiomes in lupus-prone mice

Objective: We aimed to establish humanized fecal and vaginal microbiomes in lupus-prone mice to enable mechanistic studies of the microbiome contribution to systemic lupus erythematosus (SLE). Lupus features gut dysbiosis and altered estrogen metabolism, increasing 16α-hydroxyestrone (16α-OHE), a metabolite linked to immune activation. Unlike estradiol, 16α-OHE may fail to maintain vaginal Lactobacillus dominance, promoting dysbiosis and systemic inflammation. Vaginal microbiota are critical for women’s health, yet their role in autoimmunity remains poorly understood due to limited models. Humanized microbiome techniques now allow translational approaches in chronic disease models. Applying these methods in lupus-prone mice offers a unique opportunity to test how gut dysbiosis, estrogen metabolism, and vaginal microbiome disruption interact to exacerbate autoimmunity. Hypothesis: Broad-spectrum antibiotic depletion followed by human fecal microbiota transplant (HFMT) would humanize fecal and vaginal microbial communities in two murine lupus models. Methods: Female mice from four groups (NZBWF1, NZW/LacJ, pristane-treated C57BL/6, and vehicle-treated C57BL/6 controls) received oral antibiotics for two weeks to deplete endogenous microbiota. Fecal pellets and vaginal washes were collected pre- and post-treatment for DNA quantification and cultivability was assessed on non-differential TSA and Lactobacillus-selective MRS agar. After depletion, mice received weekly oral gavage of human fecal microbiota from a healthy reproductive-age human female donor for three weeks. Reconstitution was assessed by reassessing DNA concentration and by PCR using primers specific for human-associated bacterial strains, including N. timonensis, E. aerofaciens, B. vulgatus, and the vaginal Lactobacillus species L. crispatus, L. iners, L. jensenii, and L. gasserii. Results: Antibiotics markedly reduced fecal and vaginal DNA (16.95 ± 5.22 ng/µL to 4.92 ± 2.22 ng/µL and 27.74 ± 5.17 ng/µL to 13.32 ± 3.01 ng/µL, respectively), and eliminated cultivability, confirming effective microbial depletion. After three weeks of human fecal microbiota gavage, fecal and vaginal DNA concentration increased toward baseline and PCR confirmed human-associated gut and vaginal taxa in fecal and vaginal samples across all antibiotic-treated groups. 16S rRNA gene analyses further characterized engraftment of a humanized microbiome. Conclusions: Antibiotic pretreatment followed by HFMT effectively established human-derived gut and vaginal signatures in lupus-prone mice. This platform enables investigation of gut and vaginal dysbiosis in SLE and future studies on how altered estrogen disrupts Lactobacillus dominance, impairs mucosal immune tolerance, and exacerbate systemic autoimmunity, clarifying the mechanistic links between sex hormones, microbiome composition, and lupus progression, and addressing a critical gap in understanding hormonal–microbial crosstalk in autoimmune disease. Funding: Supported by U54HL16191, P30GM149404, and T32HL105324. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.