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Comprehensive analysis of gene expression induced by IFN-γ in the human oral mucosal epithelial cell line HSC-2

ABSTRACT: Gram-negative bacterial lipopolysaccharide (LPS) is recognized at the host cell surface by TLR4 and intracellularly by inflammatory caspases such as caspase-4 (CASP4). While LPS from Porphyromonas gingivalis (PgLPS) is known to engage TLR4, it remains unclear whether it is also sensed intracellularly. Here, we examined the intracellular recognition of PgLPS. PgLPS alone did not stimulate HSC-2 oral epithelial cells, but IFN-γ priming followed by lipofection of PgLPS induced IL-18 maturation, GSDMD activation, and pyroptotic cell death. These responses were unaffected by TLR4 inhibition, but were strongly suppressed by CASP4 inhibition. Furthermore, recombinant CASP4 was directly activated by PgLPS in vitro. These findings indicate that PgLPS is recognized intracellularly by CASP4 independently of TLR4, leading to IL-18 maturation, secretion, and pyroptotic cell death. Since pyroptosis induced by lipofection of PgLPS required IFN-γ priming, we performed RNA-seq analysis of genes induced by IFN-γ and identified 974 upregulated genes. Consistent with previous reports, increased expression of GBP1–7 was observed, suggesting that GBPs are also involved in PgLPS-induced pyroptosis in HSC-2 cells. Indeed, knockdown of GBP1 strongly suppressed IL-18 secretion and cell death induced by PgLPS. Taken together, these results indicate that intracellular PgLPS induces pyroptosis in HSC-2 cells, in which GBPs play a critical role.

ORGANISM(S): Homo sapiens

PROVIDER: GSE306855 | GEO | 2025/08/31

REPOSITORIES: GEO

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Project description:Background: In previous studies we showed that IL-27 shares several effects with IFN-γ in human cancer cells. To identify novel extracellular mediators, potentially involved in epithelial ovarian cancer (EOC) biology, we analyzed the effect of IL-27 or IFN-γ on the secretome of EOC cells in culture. Methods: The secretome of cytokine-treated or untreated SKOV3 cells was analyzed by nano-UHPLC-MS/MS and eluting peptides by an Orbitrap Fusion Tribrid mass spectrometer. Extracellular GBP1 was studied by ELISA, immunoprecipitation, GTP-agarose pull-down, and Western blotting. GBP and STAT proteins were analysed on cell lysates by Western blot. GBP1 was transfected using lipofectamine reagent and cell viability measured by MTT assay. Results: IL-27 and IFN-γ modulate the extracellular release of a limited fraction of proteins that were also induced in the whole cell. Among these proteins we focused our attention on GBP1, a guanylate-binding protein and GTPase, which is a mediator of several biological activities of IFNs. Interestingly GBP1, 2, and 5 were induced by cytokine treatment in EOC cells, but only GBP1 was secreted. ELISA and immuno-blotting analyses showed that cytokine-stimulated EOC cells release full-length GBP1 molecule in vitro, through non-classical pathways, not involving microvesicles. Moreover, soluble, full-length GBP1 accumulates in the ascites of most EOC patients, suggesting a potential role of extracellular GBP1 in the tumor environment. EOC cells enriched from ascites showed constitutive tyrosine-phosphorylated STAT1 and STAT3 proteins and GBP1 expression, supporting a role of STAT-activating cytokines in GBP1 induction in vivo. Data from the TCGA dataset of EOC indicate that high GBP1 gene expression correlates with a better overall survival. Accordingly GBP1 transfection reduced SKOV3 cell proliferation, suggesting a potential anti-tumor activity of GBP1. Conclusions: Our data show for the first time that soluble GBP1 is released by cytokine-stimulated EOC cells in vitro and accumulates in EOC patients’ ascites. GBP1 expression may have anti-tumor effects, in EOC, as suggested by TCGA dataset analysis and in vitro transfection experiments. Further studies to identify the biological role of soluble GBP1 in the tumor environment of EOC are warranted.

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Comprehensive analysis of gene expression induced by IFN-γ in the human oral mucosal epithelial cell line HSC-2

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