Interleukin-33 (IL-33) is a pleiotropic cytokine that can promote type 2 inflammation but also drives immunoregulation through Foxp3+Treg expansion. How IL-33 is exported from cells to serve this dual role in immunosuppression and inflammation remains unclear. Here, we demonstrate that the biological consequences of IL-33 activity are dictated by its cellular source. Whereas IL-33 derived from epithelial cells stimulates group 2 innate lymphoid cell (ILC2)–driven type 2 immunity and parasite clearance, we report that IL-33 derived from myeloid antigen-presenting cells (APCs) suppresses host-protective inflammatory responses. Conditional deletion of IL-33 in CD11c-expressing cells resulted in lowered numbers of intestinal Foxp3+Treg cells that express the transcription factor GATA3 and the IL-33 receptor ST2, causing elevated IL-5 and IL-13 production and accelerated anti-helminth immunity. We demonstrate that cell-intrinsic IL-33 promoted mouse dendritic cells (DCs) to express the pore-forming protein perforin-2, which may function as a conduit on the plasma membrane facilitating IL-33 export. Lack of perforin-2 in DCs blocked the proliferative expansion of the ST2+Foxp3+Treg subset. We propose that perforin-2 can provide a plasma membrane conduit in DCs that promotes the export of IL-33, contributing to mucosal immunoregulation under steady-state and infectious conditions.

Intestinal epithelial cells (IEC) have important functions in nutrient absorption, barrier integrity, regeneration, pathogen-sensing, and mucus secretion. Goblet cells are a specialized cell type of IEC that secrete Trefoil factor 3 (TFF3) to regulate mucus viscosity and wound healing, but whether TFF3-responsiveness requires a receptor is unclear. Here, we show that leucine rich repeat receptor and nogo-interacting protein 2 (LINGO2) is essential for TFF3-mediated functions. LINGO2 immunoprecipitates with TFF3, co-localizes with TFF3 on the cell membrane of IEC, and allows TFF3 to block apoptosis. We further show that TFF3-LINGO2 interactions disrupt EGFR-LINGO2 complexes resulting in enhanced EGFR signaling. Excessive basal EGFR activation in Lingo2 deficient mice increases disease severity during colitis and augments immunity against helminth infection. Conversely, TFF3 deficiency reduces helminth immunity. Thus, TFF3-LINGO2 interactions de-repress inhibitory LINGO2-EGFR complexes, allowing TFF3 to drive wound healing and immunity.

Whether conventional dendritic cells (cDC) acquire subset identity under direction of Wnt family glycoproteins is unknown. We demonstrate that Wnt4, a b-catenin-independent Wnt ligand, is produced by both hematopoietic and nonhematopoietic cells and is both necessary and sufficient for preconventional DC1/cDC1 maintenance. Whereas bone marrow cDC precursors undergo phosphoJNK/c-Jun activation upon Wnt4 treatment, loss of cDC Wnt4 in CD11c Cre Wnt4 flox/flox mice impaired differentiation of CD24 + , Clec9A + , CD103 + cDC1 compared with CD11c Cre controls. Conversely, single-cell RNA sequencing analysis of bone marrow revealed a 2-fold increase in cDC2 gene signature genes, and flow cytometry demonstrated increased numbers of SIRP-a + cDC2 amid lack of Wnt4. Increased cDC2 numbers due to CD11c-restricted Wnt4 deficiency increased IL-5 production, group 2 innate lymphoid cell expansion, and host resistance to the hookworm parasite Nippostrongylus brasiliensis. Collectively, these data uncover a novel and unexpected role for Wnt4 in cDC subset differentiation and type 2 immunity.

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Intestinal epithelial cells (IEC) comprise diverse lineages that serve distinct roles necessary for regulation of nutrient absorption, regeneration, immunity, and homeostasis 1,2 . Goblet cells secrete Trefoil factor 3 (TFF3) to maintain mucus viscosity and drive mucosal healing by inhibiting cell death and influencing tight junction protein expression 3 . However, whether TFF3 signaling relies upon conventional ligand-receptor interactions has been unclear for decades. This study demonstrates that the orphan transmembrane protein leucine rich repeat receptor and nogo-interacting protein 2 (LINGO2) immunoprecipitates with TFF3, that LINGO2 and TFF3 co-localize at the IEC cell surface, and that TFF3/LINGO2 interactions block IEC apoptosis. Loss of function studies show that TFF3-driven STAT3 and EGFR activation are both LINGO2 dependent. Importantly, we demonstrate that TFF3 disrupts LINGO2/EGFR interactions that normally restrict EGFR activity, resulting in enhanced EGFR signaling. Excessive EGFR activation in Lingo2 gene deficient mice exacerbates colitic disease and accelerates host resistance to parasitic nematodes, whereas TFF3 deficiency results in host susceptibility. Thus, our data demonstrating that TFF3 functions through a previously unrecognized ligandreceptor interaction with LINGO2 to de-repress LINGO2-dependent inhibition of EGFR activation provides a novel conceptual framework explaining how TFF3-mediates mucosal wound healing through enhanced activation of the EGFR pathway.3 Although Trefoil factor 3 (TFF3) is well known to drive reparative pathways at respiratory, ocular, genitourinary and gastrointestinal mucosa, the identity of a potential TFF3 receptor has remained elusive for several decades 4-8 . In addition to enhancing mucous viscosity 9 , TFF3 induces epithelial cell survival and proliferation, activates EGFR, β-catenin, and STAT3 signaling pathways, and controls tight junction protein expression through ill-defined mechanisms to protect against gastrointestinal (GI) tissue injury and inflammation 3,10 . Since the anti-inflammatory activity of TFF3 suggested that leukocytes may be directly responsive to TFF3, we utilized a human macrophage/monocyte cell line (U937) to first ask whether cytokine release could be regulated in response to rTFF3 exposure in order to isolate the TFF3 receptor. We found that rhTFF3 caused a dose-dependent reduction in endotoxin-mediated TNF release over a range of 1-100 ng/ml and that TFF3 suppression was lost at a higher range of rhTFF3 (100-1000 ng/ml) ( Figure S1A). TFF3-induced interleukin 10 (IL-10) production was found over several orders of magnitude, but was lost at higher doses of rhTFF3 ( Figure 1A). As the dose response is similar to the established range of activity for most cytokine and growth factor receptors 11 , these observations suggested that TFF3 was interacting with a receptor in a conventional manner.TFF3 was likely to interact with its receptor through low-affinity interactions because glycosylation has been shown critical for biological...