Loss of γδ intraepithelial lymphocytes and reduced immunosurveillance of the epithelial barrier precedes the onset of Crohn’s disease‐like ileitis

Abstract

Inflammatory bowel disease, including Crohn’s disease (CD), affects over 3 million Americans. Recently, single cell sequencing of resected ileal tissue from CD patients revealed a loss of γδ intraepithelial lymphocytes (IELs). IELs expressing the γδ T cell receptor (γδ IEL) bridge innate and adaptive immunity and migrate dynamically within the intestinal epithelium as a means of immunosurveillance. However, the contribution of γδ IELs to the initiation of ileitis remains unknown. We and others have found that the onset of histopathology in TNFΔARE/+mice, which develop a chronic CD‐like ileitis, occurs at 8 weeks of age. We profiled the IEL populations of TNFΔARE/+mice prior to and during the initial stages of ileal inflammation (4, 6, 8 and 10 weeks). In agreement with CD patient data, we observed about a 2‐fold reduction in the γδ IEL population in ilea of 8‐week‐old TNFΔARE/+mice compared to TNF+/+ (WT) littermates (p=0.05). Seeding of γδ IELs within the epithelial compartment is regulated in part by expression of epithelial butyrophilin‐like genes Btnl1 and Btnl6. We find that the expression of both Btnl1 and Btnl6is reduced in 4‐ and 6‐week‐old TNFΔARE/+mice (p=0.05). Furthermore, the frequency of EdU+ γδ IELs is decreased in TNFΔARE/+mice compared to WT littermates at 6 weeks of age (p=0.05). γδ IEL motility and function contribute to maintenance of an intact epithelial barrier, as we have recently demonstrated that γδ IELs facilitate shedding of apoptotic epithelial cells. Cleaved caspase 3 staining in fixed ileal tissue revealed increased shedding of apoptotic enterocytes in 6‐week‐old TNFΔARE/+mice compared to WTlittermates (p=0.05). Since γδ IEL surveillance behavior is altered during the shedding of apoptotic enterocyte, we next assessed γδ IEL motility in 6‐week‐old WT and TNFΔARE/+ mice using intravital microscopy. γδ IEL migratory behavior is significantly impaired in TNFΔARE/+ mice, as reflected by reduced track speed (7.9 vs 2.8 μm/min, p=0.005) and increased arrest coefficient (p=0.001) relative to WT mice. To determine the contribution of γδ IELs in the development of CD‐like ileitis, we generated TNFΔARE/+ mice expressing diphtheria toxin (DT) receptor driven by a γδ T‐cell‐specific promoter (TNFΔARE/+ TcrdGDL). Our preliminary findings indicate that DT‐treated 3‐week‐old TNFΔARE/+ TcrdGDL mice exhibit reduced survival compared to TNFΔARE/+ littermates treated with vehicle control (Mantel‐Cox log‐rank test, p=0.155). In summary, we report that the onset of ileal inflammation coincides with the reduction of γδ IELs, which may be attributed to decreased epithelial Btnl expression and impaired γδ IEL proliferation. Reduced survival of TNFΔARE/+ mice following γδ T cell depletion suggests that γδ IEL‐mediated maintenance and surveillance of the barrier is critical to maintain mucosal tolerance prior to disease onset. These studies suggest that restoring γδ IEL number or migratory behavior may be effective therapeutic strategies to maintain remission in CD patients.