P0006Ikaros family transcription factors drive monocyte fate into the tissue to define treatment outcomes in IBD.

Abstract Background Response to advanced therapies in IBD is variable, highlighting the need for biomarkers to guide therapy. Circulating monocytes, precursors of intestinal macrophages shape inflammation and repair, yet the transcriptional programs directing their fate in IBD remain unclear1. Although Ikaros family transcription factors (TFs) regulate lymphocyte differentiation2, their roles in monocyte-macrophage biology in IBD are unknown. We investigated whether Ikaros TFs shape monocyte function and determine response to advanced therapies. Methods Peripheral blood was collected at baseline (W0) and post-induction (W8/12) from IBD patients initiating or switching advanced therapies, and from healthy controls (HC, n = 10). Patients were classified as Super-Responders (clinical remission at W8/12; SR, n = 20) or Non-Responders (NR, n = 20). Monocyte subsets and TFs were analysed by multiparametric flow cytometry. Plasma proteomics with gene-ontology analysis was performed. Imaging Mass Cytometry (IMC, CyTOF) characterised myeloid populations and Ikaros expression in intestinal biopsies. In vitro functional studies were performed on monocyte-derived macrophages polarized into inflammatory, alternative, or regulatory states; Ikaros or Helios were silenced using siRNA, followed by phenotypic, cytokines, and transcriptional profiling. Results At W0, IBD patients had elevated circulating monocytes (vs HC; p 0.001). SR showed a significant decline at W8/12, whereas NR remained elevated. Non-classical monocytes (CD14-CD16+) were increased (vs HC p 0.05) and persisted in NR (vs SR; p 0.05) at W8/12. Helios was absent in IBD monocytes at W0 but detectable in HC (mean expression:47.3%). By W8/12, Helios re-emerged in SR (34.1%) but not in NR. Proteomics identified 49 differentially abundant proteins in NR revealing dysregulated monocyte chemotaxis, immune activation, and macrophage reprogramming. IMC showed increased lamina propria macrophages with high Ikaros expression and enriched monocytes infiltration. In vitro, inflammatory stimuli induced Ikaros, while IL-10 upregulated Helios. Ikaros silencing reduced IL6/IL1β secretion, whereas Helios silencing enhanced inflammatory activation of macrophages. Conclusion Monocyte subset distribution, Ikaros, and Helios dynamics clearly distinguished SR from NR. Persistent Helios loss and expansion of non-classical monocytes in NR indicate defective regulatory reprogramming with consequences for monocytes infiltration and macrophage behaviour. Functional silencing revealed for the first time opposing roles of Ikaros and Helios in macrophage activation, identifying these transcription factors as promising biomarkers and potential targets to improve treatment response in IBD. References: 1.Amamou A, O’Mahony C, Leboutte M, Savoye G, Ghosh S, Marion-Letellier R. Gut Microbiota, Macrophages and Diet: An Intriguing New Triangle in Intestinal Fibrosis. Microorganisms. 2022;10(3):490. doi:10.3390/microorganisms10030490 2.Ueno A, Jeffery L, Kobayashi T, Hibi T, Ghosh S, Jijon H. Th17 plasticity and its relevance to inflammatory bowel disease. J Autoimmun. 2018;87:38-49. doi:10.1016/j.jaut.2017.12.004 Conflict of interest: Amamou, Asma: None O’Mahony, Cian: No conflict of interest Toman, Jan: No conflict of interest Lopez Espinar, Aida: No conflict of interest Deery, Alan: No conflict of interest Kowalski, Piotr S.: No conflict of interest Iacucci, Marietta: Grant: Pentax, Olympus, Eli lilly, Helmsley Personal Fees: Pentax, Pfitzer, Janssen,EliLilly, J & J Ghosh, Subrata: None None None