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Aim: Recovery of damaged mucosal surfaces following inflammatory insult requires diverse regenerative mechanisms that remain poorly defined. Previously, we demonstrated that the reparative actions of Trefoil Factor 3 (TFF3) depend upon the enigmatic receptor, leucine rich repeat and immunoglobulin-like domain containing nogo receptor 2 (LINGO2). This study examined the related orphan receptor LINGO3 in the context of intestinal tissue damage to determine whether LINGO family members are generally important for mucosal wound healing and maintenance of the intestinal stem cell (ISC) compartment needed for turnover of mucosal epithelium.Methods and Results: We find that LINGO3 is broadly expressed on human enterocytes and sparsely on discrete cells within the crypt niche, that contains ISCs. Loss of function studies indicate that LINGO3 is involved in recovery of normal intestinal architecture following dextran sodium sulfate (DSS)-induced colitis, and that LINGO3 is needed for therapeutic action of the long acting TFF2 fusion protein (TFF2-Fc), including a number of signaling pathways critical for cell proliferation and wound repair. LINGO3-TFF2 protein-protein interactions were relatively weak however and LINGO3 was only partially responsible for TFF2 induced MAPK signaling suggesting additional un-identified components of a receptor complex. However, deficiency in either TFF2 or LINGO3 abrogated budding/growth of intestinal organoids and reduced expression of the intestinal ISC gene leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), indicating homologous roles for these proteins in tissue regeneration, possibly via regulation of ISCs in the crypt niche.Conclusion: We propose that LINGO3 serves a previously unappreciated role in promoting mucosal wound healing.

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.

BackgroundSolitary chemosensory cells (SCCs) are rare epithelial cells enriched in nasal polyps and are the primary source of interleukin-25 (IL-25), an innate cytokine eliciting T-helper 2 (Th2) immune response. Although it is proposed that SCCs are stimulated by antigens released by upper airway pathogens, the exogenous triggers of human SCCs remain elusive. We studied patients with noninvasive fungal rhinosinusitis to determine whether extracts of Aspergillus fumigatus and Alternaria alternata stimulate SCC proliferation as an early event in type 2 inflammation.MethodsMulticolor flow cytometry, immunofluorescence, and enzyme-linked immunoassay were used to interrogate mucosa from patients with mycetomas and allergic fungal rhinosinusitis (AFRS) for SCCs and IL-25. Primary sinonasal epithelial cells from AFRS patients and noninflamed inferior turbinates were stimulated with fungal extracts for 72 hours, and SCC population frequency as well as mitotic activity were quantified using flow cytometry.ResultsSCCs producing IL-25 are enriched in inflamed mucosa compared with intrapatient noninflamed control tissue (38.6% vs 6.5%, p = 0.029). In cultured sinonasal epithelial cells from AFRS nasal polyps, Aspergillus fumigatus and Alternaria alternata stimulated higher SCC frequency compared with controls (27.4% vs 10.6%, p = 0.002; 18.1% vs 10.6%, p = 0.046), which led to increased IL-25 secretion in culture media (75.5 vs 3.3 pg/mL, p < 0.001; 32.3 vs 3.3 pg/mL, p = 0.007). Ki-67 expression was higher in SCCs grown in fungal stimulation conditions compared with controls.ConclusionAlthough fungal antigens are known to potentiate immune response through innate cytokines, including IL-25, the early expansion of SCCs in the presence of fungus has not been described. This early event in the pathogenesis of noninvasive fungal rhinosinusitis may represent a target for intervention.

Coordinated efforts between macrophages and epithelia are considered essential for wound healing, but the macrophage-derived molecules responsible for repair are poorly defined. This work demonstrates that lung macrophages rely upon Trefoil factor 2 to promote epithelial proliferation following damage caused by sterile wounding, Nippostrongylus brasiliensis or Bleomycin sulfate. Unexpectedly, the presence of T, B, or ILC populations was not essential for macrophage-driven repair. Instead, conditional deletion of TFF2 in myeloid-restricted CD11cCre TFF2 flox mice exacerbated lung pathology and reduced the proliferative expansion of CD45- EpCAM+ pro-SPC+ alveolar type 2 cells. TFF2 deficient macrophages had reduced expression of the Wnt genes Wnt4 and Wnt16 and reconstitution of hookworm-infected CD11cCre TFF2flox mice with rWnt4 and rWnt16 restored the proliferative defect in lung epithelia post-injury. These data reveal a previously unrecognized mechanism wherein lung myeloid phagocytes utilize a TFF2/Wnt axis as a mechanism that drives epithelial proliferation following lung injury.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is commonly characterized by type-2 inflammation. It is established that group-2 innate lymphoid cells (ILC2s) are a subset of immune cells important in orchestrating mucosal type-2 response. IL-25 is an epithelial-derived cytokine that is a critical activator of ILC2s. Recent evidence demonstrates that specialized taster epithelial cells, such as solitary chemosensory cells (SCCs), may be producers of IL-25. To elucidate the relationship between SCCs and ILC2s in CRSwNP, we sought to quantify ILC2s and SCCs to determine if these cell types are enriched in nasal polyps compared to healthy sinonasal mucosa. We quantified SCCs and ILC2s using multicolor flow cytometry in nasal polyps and non-inflamed turbinate mucosa from seven patients and investigated the role of IL-13 and dexamethasone on SCC frequency using tissue explants of nasal polyps and turbinate mucosa. SCCs were found to be the primary source of IL-25. Nasal polyps demonstrated higher populations of SCCs (33.0% vs 5.6%, p < 0.001) and ILC2s (2.40% vs 0.19%, p = 0.008) compared to patient-matched nonpolypoid turbinates. In cultured polyp explants, exogenous IL-13 increased the proportion of epithelial SCCs (40.2% IL-13 condition vs 28.9% untreated, p = 0.012), and this effect was reversed by addition of dexamethasone (40.2% vs 8.9%, p < 0.0005). These data support SCC and ILC2 expansion as well as increased IL-25 production in nasal polyps and may represent early events in the pathogenesis of CRSwNP. IL-13 stimulates proliferation of SCC in a feed-forward loop, a process that is steroid-sensitive.

Co‐evolutionary adaptation of hookworms with their mammalian hosts has been selected for immunoregulatory excretory/secretory (E/S) products. However, it is not known whether, or if so, how host immunological status impacts the secreted profile of hematophagous adult worms. This study interrogated the impact of host Signal transducer and activator of transcription 6 (STAT6) expression during the experimental evolution of hookworms through the sequential passage of the life cycle in either STAT6 deficient or WT C57BL/6 mice. Proteomic analysis of E/S products by LC–MS showed increased abundance of 15 proteins, including myosin‐3, related to muscle function, and aconitate hydratase, related to iron homeostasis. However, most E/S proteins (174 of 337 unique identities) were decreased, including those in the Ancylostoma‐secreted protein (ASP) category, and metallopeptidases. Several identified proteins are established immune‐modulators such as fatty acid‐binding protein homologue, cystatin, and acetylcholinesterase. Enrichment analysis of InterPro functional categories showed down‐regulation of Cysteine‐rich secretory proteins, Antigen 5, and Pathogenesis‐related 1 proteins (CAP), Astacin‐like metallopeptidase, Glycoside hydrolase, and Transthyretin‐like protein groups in STAT6 KO‐adapted worms. Taken together, these data indicate that in an environment lacking Type 2 immunity, hookworms alter their secretome by reducing immune evasion proteins‐ and increasing locomotor‐ and feeding‐associated proteins. [ABSTRACT FROM AUTHOR]

Schistosoma haematobium is the etiologic agent for urogenital schistosomiasis, a major source of morbidity and mortality for more than 112 million people worldwide. Infection with S. haematobium results in a variety of immunopathologic sequelae caused by parasite oviposition within the urinary tract, which drives inflammation, hematuria, fibrosis, bladder dysfunction, and increased susceptibility to urothelial carcinoma. While humans readily develop urogenital schistosomiasis, the lack of an experimentally-tractable model has greatly impaired our understanding of the mechanisms that underlie this important disease. We have developed an improved mouse model of S. haematobium urinary tract infection that recapitulates several aspects of human urogenital schistosomiasis. Following microinjection of purified S. haematobium eggs into the bladder wall, mice consistently develop macrophage-rich granulomata that persist for at least 3 months and pass eggs in their urine. Importantly, egg-injected mice also develop urinary tract fibrosis, bladder dysfunction, and various urothelial changes morphologically reminiscent of human urogenital schistosomiasis. As expected, S. haematobium egg-induced immune responses in the immediate microenvironment, draining lymph nodes, and systemic circulation are associated with a Type 2-dominant inflammatory response, characterized by high levels of interleukin-4, eosinophils, and IgE. Taken together, our novel mouse model may help facilitate a better understanding of the unique pathophysiological mechanisms of epithelial dysfunction, tissue fibrosis, and oncogenesis associated with urogenital schistosomiasis.

Th2 cells drive protective immunity against most parasitic helminths, but few mechanisms have been demonstrated that facilitate pathogen clearance. We show that IL-4 and IL-13 protect against intestinal lumen-dwelling worms primarily by inducing intestinal epithelial cells (IECs) to differentiate into goblet cells that secrete resistin-like molecule (RELM) beta. RELM-beta is essential for normal spontaneous expulsion and IL-4-induced expulsion of Nippostrongylus brasiliensis and Heligmosomoides polygyrus, which both live in the intestinal lumen, but it does not contribute to immunity against Trichinella spiralis, which lives within IEC. RELM-beta is nontoxic for H. polygyrus in vitro but directly inhibits the ability of worms to feed on host tissues during infection. This decreases H. polygyrus adenosine triphosphate content and fecundity. Importantly, RELM-beta-driven immunity does not require T or B cells, alternative macrophage activation, or increased gut permeability. Thus, we demonstrate a novel mechanism for host protection at the mucosal interface that explains how stimulation of epithelial cells by IL-4 and IL-13 contributes to protection against parasitic helminthes that dwell in the intestinal lumen.

Th2 cells drive protective immunity against most parasitic helminths, but few mechanisms have been demonstrated that facilitate pathogen clearance. We show that IL-4 and IL-13 protect against intestinal lumen-dwelling worms primarily by inducing intestinal epithelial cells (IECs) to differentiate into goblet cells that secrete resistin-like molecule (RELM) beta. RELM-beta is essential for normal spontaneous expulsion and IL-4-induced expulsion of Nippostrongylus brasiliensis and Heligmosomoides polygyrus, which both live in the intestinal lumen, but it does not contribute to immunity against Trichinella spiralis, which lives within IEC. RELM-beta is nontoxic for H. polygyrus in vitro but directly inhibits the ability of worms to feed on host tissues during infection. This decreases H. polygyrus adenosine triphosphate content and fecundity. Importantly, RELM-beta-driven immunity does not require T or B cells, alternative macrophage activation, or increased gut permeability. Thus, we demonstrate a novel mechanism for host protection at the mucosal interface that explains how stimulation of epithelial cells by IL-4 and IL-13 contributes to protection against parasitic helminthes that dwell in the intestinal lumen.

[D4.sup.+] type 1 T regulatory (Tr1) cells are induced in the periphery and have a pivotal role in promoting and maintaining tolerance. The absence of surface markers that uniquely identify [...]

Antigen encounter directs CD4+ T cells to differentiate into T helper or regulatory cells. This process focuses the immune response on the invading pathogen and limits tissue damage. Mechanisms that govern T helper cell versus T regulatory cell fate remain poorly understood. Here, we show that the E3 ubiquitin ligase Cul5 determines fate selection in CD4+ T cells by regulating IL-4 receptor signaling. Mice lacking Cul5 in T cells develop Th2 and Th9 inflammation and show pathophysiological features of atopic asthma. Following T cell activation, Cul5 forms a complex with CIS and pJak1. Cul5 deletion reduces ubiquitination and subsequent degradation of pJak1, leading to an increase in pJak1 and pSTAT6 levels and reducing the threshold of IL-4 receptor signaling. As a consequence, Cul5 deficient CD4+ T cells deviate from Treg to Th9 differentiation in low IL-4 conditions. These data support the notion that Cul5 promotes a tolerogenic T cell fate choice and reduces susceptibility to allergic asthma. Cytokine signaling influences the differentiation of CD4+ T cells into varying functional subsets. Here the authors show that an E3 ubiquitin ligase Cul5 alters TH2 and TH9 development and absence of Cul5 in T cells results in higher levels of allergy-associated IL-4 and IL-9 secreting T cells. [ABSTRACT FROM AUTHOR]

Tissue damage in the upper and lower airways caused by mechanical abrasion, noxious chemicals, or pathogenic organisms must be followed by rapid restorative processes; otherwise, persistent immunopathology and disease may ensue. This review will discuss evidence for the important role served by trefoil factor (TFF) family members in healthy and diseased airways of humans and rodents. Collectively, these peptides serve to both maintain and restore homeostasis through their regulation of the mucous layer and their control of cell motility, cell differentiation, and immune function in the upper and lower airways. We will also discuss important differences in which trefoil member tracks with homeostasis and disease between humans and mice, which poses a challenge for research in this area. Moreover, we discuss new evidence supporting newly identified receptor binding partners in the leucine-rich repeat and immunoglobulin-like domain–containing NoGo (LINGO) family in mediating the biological effects of TFF proteins in mouse models of epithelial repair and infection. Recent advances in our knowledge regarding TFF peptides suggest that they may be reasonable therapeutic targets in the treatment of upper and lower airway diseases of diverse etiologies. Further work understanding their role in airway homeostasis, repair, and inflammation will benefit from these newly uncovered receptor–ligand interactions. [ABSTRACT FROM AUTHOR]

Communication between the nervous and immune systems serves a key role in host‐protective immunity at mucosal barrier sites including the respiratory tract. In these tissues, neuroimmune interactions operate in bidirectional circuits that can sense and respond to mechanical, chemical, and biologic stimuli. Allergen‐ or helminth‐induced products can produce airway inflammation by direct action on nociceptive afferents and adjacent tissues. The activity of nociceptive afferents can regulate innate and adaptive immune responses via neuropeptides and neurotransmitter signaling. This review will summarize recent work investigating the role of neuropeptides CGRP, VIP, neuromedins, substance P, and neurotransmitters dopamine and the B2‐adrenoceptor agonists epinepherine/norepinepherine, each of which influence type 2 immunity by instructing mast cell, innate lymphoid cell type 2, dendritic cell, and T cell responses, both in the airway and the draining lymph node. Afferents in the airway also contain receptors for alarmins and cytokines, allowing their activity to be modulated by immune cell secreted products, particularly those secreted by mast cells. Taken together, we propose that further investigation of how immunoregulatory neuropeptides shape respiratory inflammation in experimental systems may reveal novel therapeutic targets for addressing the increasing prevalence of chronic airway disease in humans. [ABSTRACT FROM AUTHOR]