Deletion of Endogenous Neuregulin-4 Limits Adaptive Immunity During Interleukin-10 Receptor-Neutralizing Colitis.

BACKGROUND: Growth factors are essential for maintenance of intestinal health. We previously showed that exogenous neuregulin-4 (NRG4) promotes colonocyte survival during cytokine challenge and is protective against acute models of intestinal inflammation. However, the function(s) of endogenous NRG4 are not well understood. Using NRG4-/- mice, we tested the role of endogenous NRG4 in models of colitis skewed toward either adaptive (interleukin-10 receptor [IL-10R] neutralization) or innate (dextran sulfate sodium [DSS]) immune responses. METHODS: NRG4-/- and wild-type cage mate mice were subjected to chronic IL-10R neutralization colitis and acute DSS colitis. Disease was assessed by histological examination, inflammatory cytokine levels, fecal lipocalin-2 levels, and single cell mass cytometry immune cell profiling. Homeostatic gene alterations were evaluated by RNA sequencing analysis from colonic homogenates, with real-time quantitative polymerase chain reaction confirmation in both tissue and isolated epithelium. RESULTS: During IL-10R neutralization colitis, NRG4-/- mice had reduced colonic inflammatory cytokine expression, histological damage, and colonic CD8+ T cell numbers vs wild-type cage mates. Conversely, in DSS colitis, NRG4-/- mice had elevated cytokine expression, fecal lipocalin-2 levels, and impaired weight recovery. RNA sequencing showed a loss of St3gal4, a sialyltransferase involved in immune cell trafficking, in NRG4-null colons, which was verified in both tissue and isolated epithelium. The regulation of St3gal4 by NRG4 was confirmed with ex vivo epithelial colon organoid cultures from NRG4-/- mice and by induction of St3gal4 in vivo following NRG4 treatment. CONCLUSIONS: NRG4 regulates colonic epithelial ST3GAL4 and thus may allow for robust recruitment of CD8+ T cells during adaptive immune responses in colitis. On the other hand, NRG4 loss exacerbates injury driven by innate immune responses.

Neuregulin-4 (NRG4) is a growth factor that protects the epithelial cells lining the colon from injury and restrains innate (non-specific) immune responses. Here we show that NRG4's role in inflammation is context-specific, and mice that lack NRG4 have impaired adaptive immunity in a model of chronic immune-mediated colitis.

Sprouty2 limits intestinal tuft and goblet cell numbers through GSK3ß-mediated restriction of epithelial IL-33.

Dynamic regulation of intestinal cell differentiation is crucial for both homeostasis and the response to injury or inflammation. Sprouty2, an intracellular signaling regulator, controls pathways including PI3K and MAPKs that are implicated in differentiation and are dysregulated in inflammatory bowel disease. Here, we ask whether Sprouty2 controls secretory cell differentiation and the response to colitis. We report that colonic epithelial Sprouty2 deletion leads to expanded tuft and goblet cell populations. Sprouty2 loss induces PI3K/Akt signaling, leading to GSK3ß inhibition and epithelial interleukin (IL)-33 expression. In vivo, this results in increased stromal IL-13+ cells. IL-13 in turn induces tuft and goblet cell expansion in vitro and in vivo. Sprouty2 is downregulated by acute inflammation; this appears to be a protective response, as VillinCre;Sprouty2F/F mice are resistant to DSS colitis. In contrast, Sprouty2 is elevated in chronic colitis and in colons of inflammatory bowel disease patients, suggesting that this protective epithelial-stromal signaling mechanism is lost in disease.

The ErbB3 receptor tyrosine kinase negatively regulates Paneth cells by PI3K-dependent suppression of Atoh1.

Paneth cells (PCs), a secretory population located at the base of the intestinal crypt, support the intestinal stem cells (ISC) with growth factors and participate in innate immunity by releasing antimicrobial peptides, including lysozyme and defensins. PC dysfunction is associated with disorders such as Crohn's disease and necrotizing enterocolitis, but the specific pathways regulating PC development and function are not fully understood. Here we tested the role of the neuregulin receptor ErbB3 in control of PC differentiation and the ISC niche. Intestinal epithelial ErbB3 knockout caused precocious appearance of PCs as early as postnatal day 7, and substantially increased the number of mature PCs in adult mouse ileum. ErbB3 loss had no effect on other secretory lineages, but increased expression of the ISC marker Lgr5. ErbB3-null intestines had elevated levels of the Atoh1 transcription factor, which is required for secretory fate determination, while Atoh1+ cells had reduced ErbB3, suggesting reciprocal negative regulation. ErbB3-null intestinal progenitor cells showed reduced activation of the PI3K-Akt and ERK MAPK pathways. Inhibiting these pathways in HT29 cells increased levels of ATOH1 and the PC marker LYZ. Conversely, ErbB3 activation suppressed LYZ and ATOH1 in a PI3K-dependent manner. Expansion of the PC compartment in ErbB3-null intestines was accompanied with elevated ER stress and inflammation markers, raising the possibility that negative regulation of PCs by ErbB3 is necessary to maintain homeostasis. Taken together, our data suggest that ErbB3 restricts PC numbers through PI3K-mediated suppression of Atoh1 levels leading to inhibition of PC differentiation, with important implications for regulation of the ISC niche.

ErbB4 signaling stimulates pro-inflammatory macrophage apoptosis and limits colonic inflammation.

Efficient clearance of pro-inflammatory macrophages from tissues after resolution of a challenge is critical to prevent prolonged inflammation. Defects in clearance can contribute to conditions such as inflammatory bowel disease, and thus may be therapeutically targetable. However, the signaling pathways that induce termination of pro-inflammatory macrophages are incompletely defined. We tested whether the ErbB4 receptor tyrosine kinase, previously not known to have role in macrophage biology, is involved in this process. In vitro, pro-inflammatory activation of cultured murine and human macrophages induced ErbB4 expression; in contrast, other ErbB family members were not induced in pro-inflammatory cells, and other innate immune lineages (dendritic cells, neutrophils) did not express detectable ErbB4 levels. Treatment of activated pro-inflammatory macrophages with the ErbB4 ligand neuregulin-4 (NRG4) induced apoptosis. ErbB4 localized to the mitochondria in these cells. Apoptosis was accompanied by loss of mitochondrial membrane potential, and was dependent upon the proteases that generate the cleaved ErbB4 intracellular domain fragment, suggesting a requirement for this fragment and mitochondrial pathway apoptosis. In vivo, ErbB4 was highly expressed on pro-inflammatory macrophages but not neutrophils during experimental DSS colitis in C57Bl/6 mice. Active inflammation in this model suppressed NRG4 expression, which may allow for macrophage persistence and ongoing inflammation. Consistent with this notion, NRG4 levels rebounded during the recovery phase, and administration of exogenous NRG4 during colitis reduced colonic macrophage numbers and ameliorated inflammation. These data define a novel role for ErbB4 in macrophage apoptosis, and outline a mechanism of feedback inhibition that may promote resolution of colitis.