Endogenous NRG4 promotes ST3GAL4 expression, leukocyte infiltration, and inflammation in IL‐10R neutralization colitis

Abstract

BackgroundThe ErbB family of receptor tyrosine kinases are essential for the regulation of colonic epithelial cell proliferation, differentiation, survival, and wound healing. We previously showed that ErbB4 is induced as a compensatory response by intestinal inflammation; treatment with exogenous NRG4 (an ErbB4 ligand) protects colonocytes against cytokine‐induced apoptosis and ameliorates murine experimental colitis. Exogenous NRG4 can further alleviate intestinal inflammation by suppressing the activity of pro‐inflammatory macrophages. However, the role of endogenous NRG4 in the intestine has not been described.HypothesisBased on our previous studies, we hypothesized that NRG4 deletion would exacerbate intestinal inflammation.MethodsColitis was induced in 5‐week‐old wild‐type (WT) and NRG4 knockout (KO) mice by i.p. injection with 1 mg/kg IL‐10R neutralizing antibody, weekly for four weeks. Inflammation was assessed by fecal lipocalin‐2 ELISA, plasma myeloperoxidase ELISA, and total spleen weight. Disease was assessed by histological scoring of paraffin embedded colon rolls. Cytokine (TNF, IL‐1b, IFNg, and IL‐6) expression was determined by qPCR on RNA from full thickness distal colon homogenates. ST3GAL4 expression was assessed by qPCR and immunohistochemistry. Intestinal immune cell populations were identified and analyzed by mass cytometry (CyTof) using FlowJo software.ResultsFecal lipocalin expression was elevated in all mice after IL‐10R antibody treatment. Surprisingly, WT mice displayed significantly more fecal lipocalin than NRG4 KO cage mates. Similarly, after 4 weeks, WT mice had significantly higher plasma myeloperoxidase (2‐fold, p<0.001), increased spleen weight (57% more, p<0.01), and more histological injury and inflammation (72% higher score, p<0.05) versus their KO cage mates. Furthermore, mRNA levels for IFNg (3.7‐fold difference, p<0.01) and IL‐6 (1.6‐fold difference, p<0.05) were significantly higher in WT versus KO cage mates. CyTof analysis identified a reduction in CD8+ T cell populations, as well as alterations in overall T cell populations. Expression of ST3GAL4, a sialyltransferase that contributes to inflammatory leukocyte recruitment, was significantly reduced in NRG4‐null colons and epithelia (>90% reduction, p<0.01 and p<0.001 respectively), suggesting a mechanism for altered T cell populations.ConclusionIn contrast to the protective effects of exogenous NRG4, endogenous NRG4 appears to participate in recruitment of CD8+ T cells and intestinal inflammation, possibly through regulation of ST3GAL4.