Sex-specific gut microbial and metabolic responses to inhaled diesel exhaust particle exposure are modified by probiotic treatment in C57BL/6 mice

Background Epidemiological and experimental studies indicate that susceptibility to traffic-generated particulate matter (PM)-induced metabolic dysfunction varies by sex, with females frequently exhibiting greater vulnerability. Recent evidence suggests that disruption of the lung-gut axis and subsequent gut-derived inflammatory signaling may contribute to these outcomes. However, the effects of inhaled PM on gut microbiome signaling and the metabolic milieu across sexes remain inadequately characterized. This study examined whether diesel exhaust particulate (DEP) exposure induces sex-specific metabolic and inflammatory responses and whether probiotic supplementation differentially modifies these effects in males and females. Methods Male and female C57BL/6 mice were exposed to 35 µg DEP (1 mg/mL, SRM-2975) or saline control via oropharyngeal aspiration twice weekly for 50 days, with or without probiotic supplementation (Winclove Ecologic® Barrier probiotics). Systemic metabolic outcomes in plasma were assessed using a multiplex hormone panel. Gut microbiome composition was characterized using 16S rRNA sequencing, and gut-derived signaling was evaluated through plasma lipopolysaccharide (LPS) quantification and short-chain fatty acid (SCFA) analysis. Three-way ANOVAs with sex as a biological variable were conducted to determine differential responses to DEP exposure and probiotic intervention. Results DEP exposure induced sex-dependent alterations in gut microbial composition, circulating metabolites, and endotoxemia. In females, DEP exposure resulted in taxonomic shifts and reduced microbial diversity, whereas probiotic treatment produced the most pronounced community-level expansion in DEP-exposed males. Beta-diversity analyses confirmed significant treatment-associated differences in community composition, identifying probiotic treatment as the primary driver, with stronger community-level effects in females and more limited effects in males. These changes corresponded with alterations in circulating SCFA and LPS levels, with more pronounced effects observed in females. Conclusions Biological sex is a major determinant of susceptibility to DEP-induced metabolic and inflammatory dysregulation. Females demonstrated heightened systemic and gut-derived responses to DEP exposure. Probiotic supplementation modified several of these functions, with effects varying by sex. Notably, probiotic supplementation produced opposing effects on glucagon in DEP-exposed animals depending on sex, underscoring the importance of biological sex in determining both the direction and magnitude of microbiome-targeted intervention outcomes. These findings support the inclusion of sex as a biological variable in environmental health research and suggest that microbiome-targeted interventions may provide sex-specific protective effects against PM-induced metabolic dysfunction.