Synergistic activation of gingival fibroblasts by Fusobacterium nucleatum and IFNγ is driven by autocrine mechanisms and enhances inflammatory responses of immune cells

Gingival fibroblasts (GFs) are important sentinel cells which modulate immune responses within inflamed gingival tissue in response to oral bacteria and inflammatory mediators during periodontitis. Yet, the mechanisms governing GF-immune cell cross-talk remain poorly defined. Here, we investigated how the interaction between the oral pathobiont Fusobacterium nucleatum and the proinflammatory cytokine interferon-γ (IFNγ) modulates primary human GF inflammatory responses and interactions with immune cells. Transcriptomic analysis using RNA-seq revealed a unique transcriptional program induced in GFs infected with F. nucleatum in the presence of IFNγ. This program was characterized by synergistic upregulation of multiple genes involved in immune cells recruitment and activation, including CXC and CC family chemokines, colony stimulating factors, and components of the TNF pathway. The synergistic effect was specific to GFs and was not observed in keratinocytes or macrophages. However, it was not restricted to F. nucleatum, as Porphyromonas gingivalis and Filifactor alocis also synergized with IFNγ to induce CXCL9, CXCL10, and CXCL11 expression in GFs. These transcriptional changes had pronounced functional consequences: conditioned media from GFs exposed to both F. nucleatum and IFNγ enhanced neutrophil viability and activation, as shown by increased CD11b surface expression, and promoted a proinflammatory macrophage phenotype, characterized by reduced CD163 and increased CD80 expression. Mechanistically, the synergistic activation of GFs by F. nucleatum and IFNγ was driven by an autocrine TNF-dependent loop that promoted cross-talk between STAT1, c-Jun N-terminal kinase (JNK), and NFκB signaling pathways, leading to amplification of inflammatory gene expression. Collectively, our findings identify an autocrine TNF-dependent signaling circuit that integrates bacterial and IFNγ-derived signals in GFs to orchestrate stromal–immune cell communication, which may contribute to excessive activation of neutrophils and macrophages during periodontitis.