O-008 Aberrant Anti-inflammatory Macrophage Function and Differentiation in Wiskott-Aldrich Syndrome Protein-Deficient Mice and Humans

Abstract

BACKGROUND The intestinal mucosal surface is continuously exposed to microbial communities and intestinal homeostasis is dependent on host barrier function and the establishment and persistence of well-regulated immune responses. Innate immune cells play a critical role in the maintenance of mucosal homeostasis by facilitating immune tolerance to luminal antigens. A recent study identified an inflammatory bowel disease (IBD) causal sub-network of genes (e.g., IL10, ARC, NOD2, HCK, WAS), which were highly enriched in bone marrow derived macrophages (Mφ) and predicted to have a role in anti-inflammatory macrophage function. The Wiskott-Aldrich syndrome (WAS) gene was identified within this causal sub-network along with HCK, a protein that interacts with Wiskott-Aldrich syndrome protein (WASP). WASP-deficient mice and up to 10% of WAS patients develop IBD. We have shown that WASP-deficiency in mice, when limited to innate immune cells, is sufficient to render normal wild-type CD4 T cells colitogenic. We hypothesized that WASP-mediated regulation of anti-inflammatory Mφ function is critical for the maintenance of intestinal homeostasis. METHODS We analyzed the expression of Ly6c and MHCII in mucosal macrophages (CD45CD11bCD11cCD64CD103) to distinguish them into pro- and anti-inflammatory Mφ populations. Bone marrow derived Mφ (BMDM) in mice and monocyte derived Mφ in human was polarized into anti-inflammatory M2 Mφ in presence of IL4, TGFb and IL10. RESULTS Analysis of gut resident Mφ population in 5-weeks-old pre-colitic Was mice showed an increase in the percentage of pro-inflammatory Mφ (Ly6c MHCII) and a concomitant decrease in the percentage of anti-inflammatory Mφ (Ly6c MHCII). As predicted, in a bone-marrow chimera experiment, where lethally irradiated Was mice were transferred with a mixture of WT (CD45.1) and Was (CD45.2) bone-marrow in a 1:1 ratio, the Was compartment, harbored an increased percentage of pro-inflammatory Mφ and decreased anti-inflammatory Mφ compared to the WT compartment. Next we examined the role of Mφ-intrinsic WASP in intestinal homeostasis by transferring CD4 T cell into Rag1 mice lacking WASP only in Mφ (Rag1WasLysM). Rag1WasLysM recipient mice, when compared to Rag1 mice, developed more severe weight loss and colonic inflammation with increased pro-inflammatory cytokine expression. In addition, in vitro generation of BMDM M2 Mφ from Was mice expressing classical M2 markers Arg1 and Fizz1, was also markedly reduced when compared with M2 Mφ derived from WT mice. LPS induced a much higher expression of pro-inflammatory cytokines in Was M2 Mφ compared to WT M2 Mφ. Moreover, Was M2 Mφ induced greater proliferation of CD4 T cells, cytokine production, and decreased generation of Tregs compared to WT Mφ. Importantly, the colitis that results from the transfer of CD4 T cells into Rag1Was mice was rescued by the transfer of WT M2 Mφ but not Was M2 Mφ. Similarly, we found that the M2 Mφ generation was also severely impaired in patients with Wiskott-Aldrich syndrome. M2 Mφ from patients expressed higher levels of pro-inflammatory cytokines and was less efficient in the generation Tregs in an in vitro co-culture system. CONCLUSIONS Collectively, these data implicates a critical Mφ-intrinsic functional role for WASP for the generation and maintenance of tolerance in the intestinal mucosa.