The role of senescent macrophages in inducing a pro-inflammatory state in regulatory T cells in Crohn‘s disease

Previous studies have identified a pro-inflammatory subpopulation of regulatory T cells (Tregs) in Crohn’s disease, characterized by the upregulation of ten TReg-specific genes induced in Crohn’s (TRICs). These Tregs exhibit high incoming TNF-α signaling and a reduced suppressive capacity. Preliminary scRNA-seq analyses further suggested that senescent M1 macrophages represent a major source of TNFα in this context. Based on these findings, we hypothesized that senescent M1 macrophages may induce TRICs expression via TNFα signaling, thereby impairing Treg function. To address this, we aimed to establish a robust in vitro protocol for inducing senescence in PBMC-derived macrophages to generate Treg-macrophage co-cultures, and to perform functional assays to evaluate Treg activity. We found that H2O2 treatment induces CDKN1A, a marker for cellular senescence, expression in PBMC-derived M1 macrophages without changing phagocytotic activity and cell migration. Importantly, we were able to detect higher secreted TNF-α levels in the medium of H2O2-treated M1 macrophages. Various Treg-macrophage co-culture approaches, including direct, indirect, and macrophage medium setups, were tested, and exposure to macrophage medium resulted in the strongest TRICs induction in Tregs. TRICs induction was stronger with M1 macrophage medium than H2O2-treated M1 macrophage medium and the upregulation of TRICs was found not to be exclusively TNF-α-dependent. Finally, establishment of functional assays was performed to assess Treg suppressive activity and effects on epithelial permeability, providing the basis for future analyses of macrophage-conditioned Tregs. In addition, follow-up experiments will focus on analyzing further features of senescence, considering compatibility with macrophage biology. Together, in this study senescence-associated features were induced in M1 macrophages. However, M1 macrophage medium subsequently co-cultured with Tregs was observed to induce the expression of inflammation-associated genes.