Streptococcus anginosus supernatant promotes macrophage pyroptosis-associated activation and phenotypes associated with the gastric inflammation-to-cancer transition via NLRP3-related signaling

Streptococcus anginosus (SA) is enriched in the gastric cancer (GC) microenvironment and has been associated with GC risk. However, its potential role in the gastric inflammation-to-cancer transition remains unclear. This study aimed to determine whether soluble factors derived from SA drive macrophage pyroptosis-associated activation and thereby contribute to the formation of a pro-inflammatory and pro-tumorigenic microenvironment, with a particular focus on the involvement of NLRP3-related signaling. Gastric tissue samples from non-Helicobacter pylori-infected patients with chronic superficial gastritis (CSG), intestinal metaplasia (IM), and GC were analyzed for SA abundance, macrophage infiltration, and pyroptosis-related marker expression. In vitro, filter-sterilized SA culture supernatant (SA-SN) was used to evaluate its effects on gastric cancer cell viability and migration. To explore the underlying molecular mechanisms, RNA sequencing (RNA-seq) was performed on SA-SN-treated macrophages, followed by analysis of differentially expressed genes and KEGG pathway enrichment. Based on the enrichment of pyroptosis-related signalingpathways and changes in key molecules identified by transcriptomic analysis, we further validated the regulatory effects of SA-SN on macrophage pyroptosis-associated responses and its influence on intestinal metaplasia-associated markers in gastric epithelial cells. The NLRP3 inhibitor MCC950 was used to assess the involvement of NLRP3-related signaling. Untargeted metabolomics was performed to identify SA-SN-associated metabolites, followed by functional validation of 15(S)-HPETE. SA abundance, macrophage infiltration, and NLRP3/IL-1β pathway-related marker expression were higher in IM and GC tissues than in CSG tissues, and these parameters were positively correlated. In vitro experiments showed that SA-SN enhanced the viability and migratory ability of gastric cancer cells. RNA-seq analysis identified 1,175 differentially expressed genes in SA-SN-treated macrophages, including 535 upregulated and 640 downregulated genes. Among these, NLRP3 and IL-1β were significantly upregulated, whereas Caspase-1 and GSDMD showed no significant changes. KEGG pathway enrichment analysis revealed significant enrichment of the NOD -like receptor, TNF , IL-17 , NF-κB , and Toll-like receptor signaling pathways. Based on these findings, further functional validation showed that SA-SN promoted pyroptosis-associated responses in macrophages, including increased inflammatory cytokine release, enhanced ASC speck formation, and upregulated expression of Cleaved Caspase-1 , GSDMD-N , and IL-1β . Conditioned medium from SA-SN-treated macrophages increased EMT -associated markers in gastric cancer cells and intestinal metaplasia-associated markers in gastric epithelial cells. These effects were partially attenuated by MCC950，supporting the involvement of NLRP3-related signaling.However, the specific causal contribution of IL-1β to these downstream phenotypic changes was not directly assessed. Metabolomic analysis identified 15(S)-HPETE as a differentially abundant metabolite. Functional assays showed that 15(S)-HPETE enhanced malignant phenotype-associated responses in AGS cells, promoted pyroptosis-associated activation in macrophages, and increased intestinal metaplasia-associated marker expression in gastric epithelial cells. SA-SN may contribute to a pro-inflammatory and pro-tumorigenic microenvironment by promoting macrophage pyroptosis-associated activation involving NLRP3-related signaling. Increased IL-1β expression and release accompanied these responses; however, the direct causal role of IL-1β in the induction of EMT-associated and intestinal metaplasia-associated markers remains to be established. Furthermore, 15(S)-HPETE may be a candidate metabolic mediator involved in this process. Further validation in larger clinical cohorts and in vivo models is required.