Transcriptomic profiling of the ovarian immune landscape reveals distinct macrophage subsets and activation of the NLRP3 inflammasome likely contributing to accelerated follicular atresia in classic galactosemia

BACKGROUND: Premature ovarian insufficiency (POI) is a complication that affects 80% of females with classic galactosemia (CG), an autosomal recessive metabolic disorder caused by mutations in the GALT gene. The immune system plays a critical role in normal ovarian physiology and in the development of pathological conditions, including POI. While previous animal studies have shown that accumulation of toxic galactose metabolites trigger cellular stress pathways, and may contribute to the disease's complications, it remains unclear how these influence the ovarian immune landscape in CG patients. METHODS: Here, we performed single-nucleus and spatial transcriptomic analyses on ovary biopsies from pre-pubertal girls with CG and compared with control ovary samples to explore the changes in ovarian immune milieu. We specifically investigated the gene expression profiles and altered signaling pathways in macrophages and endothelial cells. Mouse ovary cultures were treated with 50mM D-galactose for 48 h and immunohistochemical analysis was performed to evaluate the role of oxidative stress and activation of the NLRP3 inflammasome. RESULTS: Our transcriptomic analysis reveals diverse subpopulations of macrophages inside the ovarian stroma. We noticed a marked increase in both monocyte-derived and tissue-resident macrophages, and expression of proinflammatory cytokine genes (TNF, IL1B, CCL2, CXCL10, CXCL8) in the CG ovary. Transcriptomic profiles of endothelial cells reveal upregulation of cell-adhesion molecules (VCAM1, ICAM1, PECAM1, SELP, SELE) and chemokines (CCL21, CX3CL1) which can modulate immune cell extravasation at the site of inflammation in the CG. IHC data revealed increased expression of NLRP3, CASP-1 and TNF-α expression in the ovaries of CG. Furthermore, D-galactose toxicity in mouse ovaries resulted in oxidative stress evident by increased expression of 8-OHdG and 4-HNE, and increased atresia of ovarian follicles indicated by cleaved-CASP3 expression. We found an increased expression of NLRP3, cleaved CASP1, activated GSDMD, cleaved IL-1β and TNF-α in the D-galactose-treated mouse ovaries, suggesting inflammasome-mediated pyroptotic cell-death and inflammation, which can eventually contribute to augmented follicular atresia. CONCLUSIONS: Our findings provide molecular insights into the proinflammatory immune response driven by oxidative stress-induced activation of the NLRP3 inflammasome pathway in the CG ovary. This study elucidates the plausible role of ovarian pyroptotic macrophages in the exacerbating the inflammatory microenvironment, which may explain the early onset of POI in CG patients.