Monocyte-Derived ADA2 Regulates Myeloid—T Cell Crosstalk and Drives Pro-Inflammatory Responses Linked with Intestinal Inflammation in Crohn’s Disease 2307789

Abstract Introduction Crohn’s disease (CD) is a chronic inflammatory disorder marked by dysregulated immune responses. Purinergic signaling contributes to mucosal injury, yet the role of adenosine-metabolizing enzymes remains unclear. Adenosine deaminase 2 (ADA2), a secreted myeloid-derived enzyme that converts adenosine to inosine, has been insufficiently studied in CD. We investigated whether monocyte-derived ADA2 is linked to intestinal inflammation. Methods Twenty-nine patients with CD undergoing endoscopy at the University Hospital Heidelberg were enrolled. Paired inflamed and non-inflamed intestinal tissue samples were collected. ADA2 activity and cytokine/chemokine levels were measured in tissue digestion medium. Tissue-derived cells were analyzed by flow cytometry to characterize monocyte populations and their intracellular ADA2 expression. Results ADA2 activity was increased in inflamed versus non-inflamed tissue, along with pro-inflammatory mediators IL-6, IL-1β and IFN-γ. Intracellular ADA2 was reduced in CD14+ monocytes from inflamed regions, suggesting that the protein was actively secreted into the extracellular space. Notably, ADA2 activity was significantly associated with CXCL10 levels, a chemokine for CXCR3-dependent cell recruitment. Accordingly, the CXCR3+ cells were increased specifically within ADA2+CD14+ monocytes in inflamed tissues, while no difference was noted in ADA2-CD14+ population. In general, ADA2+CD14+ monocytes exhibited a pro-inflammatory signature enriched in TNFa, IL-23, and IFN-γ, compared to ADA2-CD14+ monocytes. Subsequent analysis of bulk RNA-seq data of ADA2-stimulated macrophages confirmed upregulation of IL23, IL1B, and IL6 -key drivers of pathogenic Th17 cell differentiation. Consistently, ADA2 stimulation of peripheral blood mononuclear cells increased the frequency of CCR6+ CD4+ Th17-like cells. Conclusion These findings suggest ADA2 as a previously unrecognized regulator of myeloid—T cell crosstalk in CD, highlighting a potential pathogenic axis and therapeutic target. Funding Source German Research Foundation (DFG), Heidelberg University Faculty of Medicine Topic Categories Immune Mechanisms of Human Disease (HUM)