P142 Genetic ablation of DNA methyltransferase 3A in myeloid cells impairs M1/M2 polarization and leads to pro-inflammatory macrophages

• Published in: Journal of Crohn's and colitis 2024-01, Vol.18 (Supplement_1), p.i440-i440
• Main Authors: Bordoni, D, Escudero-Hernández, C, Kimmig, F, Mishra, N, Fazio, A, Tran, F, Imm, S, Hinrichsen, F, Weber-Stiehl, S, Stengel, S, Sommer, F, Falk-Paulsen, M, Rosenstiel, P
• Format: Article
• Language: English
• ISSN: 1873-9946; 1876-4479
• DOI: 10.1093/ecco-jcc/jjad212.0272

Abstract Background Genetic variants in the de novo DNA methyltransferase DNMT3A associate with increased risk for inflammatory bowel disease (IBD). While DNMT3A loss-of-function somatic mutations have been attributed to myelodysplasia and acute myeloid leukemia, the exact mechanisms how defective DNMT3A is linked to IBD remain elusive. Since macrophages are key gatekeepers for immune homeostasis, we aimed to characterize how DNMT3A loss affects monocytic lineage differentiation and inflammatory properties. Methods We generated mice lacking Dnmt3a expression in myeloid cells (Dnmt3aLysM) using a LysM-driven cre-lox system and confirmed gene deletion by PCR and western blot of gut lamina propria CD11b+ enriched cells and 5-day in vitro differentiated bone marrow-derived macrophages (BMDM, 20ng/mL M-CSF). Four-day polarized BMDMs (resting: 20ng/mL M-CSF; M1: 50ng/mL LPS/IFNγ; M2: 50ng/mL IL-4/IL-13) were functionally characterized using RNA-seq, flow cytometry, and arginase and nitric oxide synthase (NOS) activities. FITC-labelled zymosan served to quantify phagocytosis, and co-culture with intestinal epithelial ModeK wounded monolayers to assess healing. To evaluate Dnmt3a loss during inflammation in vivo, colitis was induced using dextran sodium sulfate (DSS). Results M2 polarization induces Dnmt3a expression in wild-type (WT) BMDMs while inflammatory stimuli decrease it. Dnmt3a-deficient BMDMs differentiated with IL4/IL13 in vitro increase ERK1/2 and PI3K signaling, decrease M2 markers and upregulate inflammatory transcripts, e.g. Irg1,Tnf compared to WT cells. IL4/IL13 treated BMDMs confirmed M2 polarization defects as they acquire morphological and functional M1-like features in the absence of Dnmt3a, especially those from aged (>34 weeks old) mice, e.g. failed spindle M2 morphology, increased phagocytosis, decreased arginase and increased NOS activities. In co-cultures, IL4/IL13 treated Dnmt3aLysM BMDMs inhibited intestinal epithelial regeneration after a scratch lesion compared to M2 WT BMDMs. Increased NOS activity was also detected in M1-polarized BMDMs from aged Dnmt3aLysM mice. In vivo, young Dnmt3aLysM mice display mildly increased splenic T-cell and decreased monocyte numbers. Aged Dnmt3aLysM mice (>34 weeks) exhibit altered bone marrow morphology and hepatosplenomegaly linked to increased myeloproliferation. Dnmt3aLysM mice already at young age displayed increased sensitivity to both acute and chronic DSS colitis. Conclusion Normal Dnmt3a funct