O-022 YI Innate Immune IL10 Receptor Signaling Regulates Mucosal Homeostasis and the Function of Anti-inflammatory Macrophages

IL10 receptor (IL10R) mutations cause severe very early onset IBD in humans, and similarly, IL10Rβ deficient mice develop spontaneous colitis. While recent data show that IL10R signaling is important on T cell populations to prevent IBD, the role of IL10R signaling on innate immune cells in maintaining mucosal homeostasis is unknown. We hypothesized that innate IL10R signaling is a key regulator in the intestine of immune responses and prevention of colitis in mice and humans. We crossed IL10Rβ KO and RAG KO mice to generate IL10Rβ-RAG DKO (10RDKO) mice. RAG KO and 10RDKO mice were adoptively transferred with unfractionated WT CD4+ T cells or fractionated as CD45RBhigh, CD45RBlow alone or in combination. Colitis was assessed based on clinical, endoscopic and histological scores. FACS analysis to assess different innate and adaptive immune cell populations was performed. In addition, bone marrow derived macrophages were generated from WT and IL10Rβ KO mice, and subjected to different pro-inflammatory (M1) or anti-inflammatory (M2) stimuli. Cells were analyzed by FACS and RT-PCR for surface markers and cytokine expression. Similar analysis was performed on monocyte derived macrophages that were generated from 3 IL10R deficient patients and healthy controls. In some experiments murine and human macrophages were used as antigen presenting cells and generation of Tregs from murine WT or healthy donor CD4+ CD25− T naïve cells was evaluated. 10RDKO mice were viable and did not develop spontaneous colitis. However, transfer of total WT CD4+ T cells into 10RDKO mice caused severe intestinal inflammation after 3–4 weeks. Similarly, when CD4+ T cells were transferred into RAG KO mice reconstituted with 10RDKO bone marrow, severe colitis resulted, indicating that the loss of IL10R signaling on innate immune cells is sufficient to cause inflammation. CD4+CD45RBhigh transfer also elicited severe colitis in 10RDKO mice, was not ameliorated by co-transfer of CD45RBlow cells, and was associated with a significant decrease in generation of inducible Tregs. Innate immune cells analysis in young non-colitic IL10Rβ KO mice showed increase in pro-inflammatory LP macrophages and decrease in the tolerogenic subset, suggesting that IL10R-dependent signals modulate generation of different macrophage subsets. Similarly, generation of anti-inflammatory bone marrow derived macrophages was impaired in IL10Rβ deficient mice. These macrophages also expressed significantly higher levels of pro-inflammatory cytokines following LPS stimulation and promoted less generation of Tregs, perhaps due to decreased surface expression of PD-L1 and PD-L2. Finally, anti-inflammatory monocyte-derived macrophages from IL10R deficient patients also expressed lower levels of typical M2 markers, secreted high levels of TNFα and IL6 following LPS stimulation and promoted less generation of Tregs. IL10R-dependent signals play a critical role in generation and function of anti-inflammatory macrophages in mice and humans. Loss of this signaling impairs the cross talk with adaptive immune cells, and can lead to severe colitis. Our findings establish innate immune IL10R signaling as a key regulator of intestinal immune responses. Understanding the down-stream signaling of IL10R in innate immune cells can aid in developing new therapies entailing targeted delivery of IL10 to these cell populations.

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