Inflammatorybowel disease (IBD) is a chronic inflammatory condition that affects theintestine. Environmental factors, infectious microbes,genetic susceptibility,and a dysregulated immune system can result in mucosal inflammation. The mechanismsbehind establishing and maintaining intestinal tolerance are complex, involvinganatomy, cells, and humoral factors. Intestinal macrophages (Mφ) play a key role in controlling gutimmune responses and maintaining homeostasis.Substitution of Mφ with healthy ones improves IBD symptoms in a genetic mouse model ofIBD (Il10rb deficiency). Depletion of endogenous Mφ wasnecessary to achieve therapeutic benefit. We surmised that this step wasnecessary to reset the Mφ niche and to prevent thepolarization of incoming cells by the hyper-inflammatory environment. Wehypothesized that locking Mφ before injection into an anti-inflammatory (M2)phenotype could improve therapeutic efficacy and eliminate the need forconditioning. In this study, we used CRISPR/Cas mediated knock-out (KO) of 12genes or lentiviral addition of one gene to enforce an M2 phenotype in Mφ.Based on cell surface marker expression and cytokine secretion, we selected KOof Stat1, Ecm1, Gsk3a, Dnmt1 and Trim24, or overexpression of constitutivelyactive Stat6 as promising candidates for promoting tolerance in IBD (A). Allthese candidates except Stat1 KO led to an up to 3-fold upregulation of cellsurface markers associated with M2 polarization. In contrast, KO of Stat1 ledto impaired acquisition of the inflammatory (M1) markers CD80 and CD86 inresponse to LPS and IFNγ (B). All candidates led to reduced expression ofpro-inflammatory cytokines and chemokines upon LPS and IFNγ stimulationcompared to control Mφ. Next, we evaluated the capability of geneticallyengineered Mφ to modulate naïve T-cell activation and polarization in vitro.For this,OVA-peptide stimulated macrophages were mixed with OT-II naïve CD4+ Tcells (C). Stat1 KO did not change the proliferation of OVAspecific T cellscompared to control Mφ but drastically increased the percentage of Gata3+ Tcells, indicating increased differentiation towards Th2. While no significantchanges were observed in T-reg induction, expression of mutant Stat6 or KO ofGsk3a and Dnmt1 led reduction of T cell proliferation. Finally, we evaluatedthe therapeutic potential of genetically engineered Mφin Il10rb-deficient IBD mice. Transplantation of Mφengineered to express mutated Stat6 or KO of Stat1 or Gsk3a led to improvedclinical symptoms of IBD. We observed body weight gain (D), improved colonhistopathology, and altered T cell activation in the mesenteric lymph nodes.This study highlights the potential of targeting Mφpolarization as a therapeutic strategy for IBD and possibly other inflammatorydisorders, particularly those that are unresponsive to conventionalanti-inflammatory therapy.