NLRP3 inflammasome activation and impaired Treg-mediated immune tolerance are associated with spontaneous preterm birth: A case-control study with in vitro probiotic metabolite intervention.

BACKGROUND

Spontaneous preterm birth (sPTB) is a leading cause of neonatal morbidity and mortality worldwide. Excessive NLRP3 inflammasome activation coupled with impaired regulatory T cell (Treg)-mediated immune tolerance may be associated with sPTB; however, whether probiotic-derived metabolites can modulate this immunological imbalance under in vitro conditions remains unclear.

METHODS

A case-control study enrolled 45 women with sPTB and 50 term controls. Peripheral blood and fetal membrane/decidual tissues were collected to assess NLRP3 pathway activation (NLRP3, caspase-1, and IL-1β) and immune tolerance markers (Treg proportion, IL-10, and TGF-β). Multivariable logistic regression was performed to evaluate independent associations with sPTB. In vitro experiments used paired samples from six donors, and data were analysed with linear mixed-effects models treating donor as a random effect. Fetal membrane explants and peripheral blood mononuclear cells (PBMCs) were cultured in vitro under lipopolysaccharide-stimulated conditions and treated with sodium butyrate or Lactobacillus rhamnosus GG culture supernatant to evaluate effects on NLRP3 pathway markers and the proportion of CD4⁺CD25⁺Foxp3⁺ cells. An uninoculated, identically processed MRS broth control was included.

RESULTS

The sPTB group exhibited significantly elevated NLRP3, caspase-1, and IL-1β expression alongside reduced Treg proportions and decreased IL-10 and TGF-β levels compared with term controls (all P < 0.001). Tissue NLRP3 expression (adjusted OR = 2.74) and Treg proportion (adjusted OR = 0.61) were independently associated with sPTB status. Under in vitro conditions, both interventions attenuated LPS-induced NLRP3 pathway marker expression and increased the proportion of CD4⁺CD25⁺Foxp3⁺ cells with higher anti-inflammatory cytokine concentrations in fetal membrane explants and PBMCs, whereas the MRS broth control produced no significant change.

CONCLUSIONS

NLRP3 pathway overactivation and reduced Treg proportion coexisted and were each independently associated with sPTB. Probiotic-derived metabolites modulated NLRP3-related and Treg-related measurements at the maternal-fetal interface under in vitro conditions. These cross-sectional and in vitro findings describe associations only and do not establish causality or a preventive effect.