Disrupted Tuzzerella abundance and impaired L-glutamine levels induce Treg accumulation in ovarian endometriosis: a comprehensive multi-omics analysis

Abstract

Introduction: The microbial community plays a crucial role in the pathological microenvironment. However, the structure of the microbial community within endometriotic lesions and its impact on the microenvironment is still limited.

Methods: All 55 tissue samples, including ovarian ectopic (OEMs) and normal (NE) endometrium, were subjected to 16S rRNA sequencing, metabolomic and proteomic analysis.

Results: We found the abundance of Tuzzerella is significantly lower in OEMs compared to NE tissue (p < 0.01). We selected samples from these two groups that exhibited the most pronounced difference in Tuzzerella abundance for further metabolomic and proteomic analysis. Our findings indicated that endometriotic lesions were associated with a decrease in L-Glutamine levels. However, proteomic analysis revealed a significant upregulation of proteins related to the complement pathway, including C3, C7, C1S, CLU, and A2M. Subsequent metabolic and protein correlation predictions demonstrated a negative regulation between L-Glutamine and C7. In vitro experiments further confirmed that high concentrations of Glutamine significantly inhibit C7 protein expression. Additionally, immune cell infiltration analysis, multiplex immunofluorescence, and multifactorial testing demonstrated a positive correlation between C7 expression and the infiltration of regulatory T cells (Tregs) in ectopic lesions, while L-Glutamine was found to negatively regulate the expression of chemotactic factors for Tregs.

Conclusion: In this study, we found a clear multi-omics pathway alteration, "Tuzzerella (microbe)-L-Glutamine (metabolite)-C7 (protein)," which affects the infiltration of Tregs in endometriotic lesions. Our findings provide insights into endometriosis classification and personalized treatment strategies based on microbial structures.

Keywords: Endometriosis; Immune infiltration; Metabolomics; Microbiome; Proteomics.

Fig. 1

Overall microbiota composition in ectopic endometrial tissue and normal endometrial tissue. A Venn diagram. B The Chao1 and Shannon indices. C The principal component analysis (PCA). D The barplot of phylum community structure in NE group and OEMs group. E The barplot of phylum community structure in NE group and OEMs group. F The LEfe table of Meta Statistic. G Differential microbial genera in NE group and OEMs group. NE normal endometrium group, OEMs ovarian endometriosis. **p < 0.01; *p < 0.05

Fig. 2

Overall analysis of differential metabolites in ectopic endometrium with low abundance of Tuzzerella and normal endometrium. A The principal component analysis (PCA). B Volcano plot of differential metabolites. C The unit-less Z-score values in two groups. D Top 20 upregulated and downregulated KEGG pathways (OEMs vs NE). E A Sankey diagram depicting the top 100 bacteria and metabolites. F Metabolites associated with Tuzzerella. VIP variable important in projection, NE normal endometrium group, OEMs ovarian endometriosis

Fig. 3

Complement-related proteins exhibit abnormally high expression in ectopic endometrial tissue with low abundance of Tuzzerella. A The principal component analysis (PCA). B Volcano plot of differential proteins. C Display of significantly upregulated KEGG pathways (OEMs group vs NE group). D The chord diagram generated from KEGG enrichment analysis. E qPCR validation of C3, C7, C1S, CLU, and A2M mRNA expression in ectopic and normal endometrium. The mRNA expression levels were calculated using 2^−∆CT formula. **p < 0.01, ***p < 0.001, ****p < 0.0001. G Heatmap representation of complement proteins expressed in tissues with different biological functions. F Protein–protein interaction (PPI) network visualization of the correlation between complement proteins and other proteins. NE normal endometrium group, OEMs ovarian endometriosis

Fig. 4

Glutamine negatively regulates the expression of C7 in ectopic endometrial tissue with low abundance of Tuzzerella. A Significant matrix plot depicting the connections between differential proteins and differential metabolites. B Cornetwork plot illustrating the relationships between differential proteins and differential metabolites. C Heatmap showing the downstream related metabolites of l-Glutamine, and the negative correlation between l-Glutamine and GFPT2. F Western blot analysis demonstrating the expression of C7 after treatment with high concentrations of glutamine. ####, p < 0.0001, vs EC; *, p < 0.05; A-446: glutaminase inhibitor

Fig. 5

The levels of glutamine and high expression of C7 within ectopic lesions are correlated with the infiltration quantity of Tregs. A Analysis of immune cell infiltration. B Multiple fluorescence detection of FoxP3 + CD3 + Tregs, with C7 protein labeled with 647 fluorescence. The expression level of C7 protein is positively correlated with the infiltration quantity of Tregs. C Expression profile of chemokines. **, p < 0.01. ***, p < 0.001