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Background This study group has previously identified IL-9–producing mucosal mast cell (MMC9) as the primary source of IL-9 to drive intestinal mastocytosis and experimental IgE-mediated food allergy. However, the molecular mechanisms that regulate the expansion of MMC9s remain unknown. Objectives This study hypothesized that IL-4 regulates MMC9 development and MMC9-dependent experimental IgE-mediated food allergy. Methods An epicutaneous sensitization model was used and bone marrow reconstitution experiments were performed to test the requirement of IL-4 receptor α (IL-4Rα) signaling on MMC9s in experimental IgE-mediated food allergy. Flow cytometric, bulk, and single-cell RNA-sequencing analyses on small intestine (SI) MMC9s were performed to illuminate MMC9 transcriptional signature and the effect of IL-4Rα signaling on MMC9 function. A bone marrow–derived MMC9 culture system was used to define IL-4–BATF signaling in MMC9 development. Results Epicutaneous sensitization– and bone marrow reconstitution–based models of IgE-mediated food allergy revealed an IL-4 signaling-dependent cell-intrinsic effect on SI MMC9 accumulation and food allergy severity. RNA-sequencing analysis of SI-MMC9s identified 410 gene transcripts reciprocally regulated by IL-4 signaling, including Il9 and Batf. In silico analyses identified a 3491-gene MMC9 transcriptional signature and identified 2 transcriptionally distinct SI MMC9 populations enriched for metabolic or inflammatory programs. Employing an in vitro MMC9-culture model system showed that generation of MMC9-like cells was induced by IL-4 and this was in part dependent on BATF. Conclusions IL-4Rα signaling directly modulates MMC9 function and exacerbation of experimental IgE-mediated food allergic reactions. IL-4Rα regulation of MMC9s is in part BATF-dependent and occurs via modulation of metabolic transcriptional programs.

BACKGROUND: There is no widely adopted severity grading system for acute allergic reactions, including anaphylactic and nonanaphylactic reactions, thus limiting the ability to optimize and standardize management practices and advance research. OBJECTIVE: The aim of this study was to develop a severity grading system for acute allergic reactions for use in clinical care and research. METHODS: From May to September 2020, we convened a 21-member multidisciplinary panel of allergy and emergency care experts; 9 members formed a writing group to critically appraise and assess the strengths and limitations of prior severity grading systems and develop the structure and content for an optimal severity grading system. The entire study panel then revised the grading system and sought consensus by utilizing Delphi methodology. RESULTS: The writing group recommended that an optimal grading system encompass the severity of acute allergic reactions on a continuum from mild allergic reactions to anaphylactic shock. Additionally, the severity grading system must be able to discriminate between clinically important differences in reaction severity to be relevant in research while also being intuitive and straightforward to apply in clinical care. Consensus was reached for all elements of the proposed severity grading system. CONCLUSION: We developed a consensus severity grading system for acute allergic reactions, including anaphylactic and nonanaphylactic reactions. Successful international validation, refinement, dissemination, and application of the grading system will improve communication among providers and patients about the severity of allergic reactions and will help advance future research.

BACKGROUND: The incidence of eosinophilic esophagitis (EoE) is greater in male than female subjects, and the underlying molecular basis for this sex bias remains unclear. OBJECTIVE: We sought to delineate the contribution of the sex hormone estrogen to the EoE phenotype and esophageal epithelial barrier function and remodeling. METHODS: We performed demographic and incidence analyses of EoE in male and female subjects from a single-center pediatric cohort. Estrogen-responsive gene expression analyses and estrogen receptor (ESR) immunofluorescence staining of esophageal biopsy specimens from patients with EoE and control subjects were performed. The effect of 17β-estradiol (E2) on IL-13–induced signaling pathways, gene expression, and esophageal epithelial architecture and barrier function in a primary human esophageal keratinocyte cell (EPC2) culture system (EPC2–air-liquid interface) was examined. RESULTS: We observed a male predominance in patients with EoE. Analyses of RNA sequencing data sets revealed a significant dysregulation of the estrogen-responsive gene network and expression of ESR1 and ESR2 in esophageal biopsy specimens from patients with EoE compared with control subjects. IL-13 stimulation of EPC2–air-liquid interface cells led to altered cellular architecture with induced dilation of intercellular spaces and barrier dysfunction. Pretreatment of EPC2s with E2 prior to IL-13 exposure abrogated IL-13– induced architectural changes and esophageal barrier dysfunction. Mechanistically, E2-protective effects were dependent on ESR2 and associated with diminishing of IL-13– induced tyrosine kinase 2 and signal transducer and activator of transcription 6 phosphorylation and EoE-dysregulated gene expression. CONCLUSIONS: Estrogen-responsive genes are modified in patients with EoE compared with control subjects. E2 attenuated IL-13– induced architectural changes and esophageal epithelial barrier dysfunction through inhibition of the IL-13/tyrosine kinase 2/ signal transducer and activator of transcription 6 pathway via ESR2-dependent process. Estrogen hormone signaling may protect against development of EoE in female subjects.

Background Eosinophilic esophagitis (EoE) is characterized by histopathologic modifications of esophageal tissue, including eosinophil-rich inflammation, basal zone hyperplasia, and dilated intercellular spaces (DIS). The underlying molecular processes that drive the histopathologic features of EoE remain largely unexplored. Objective We sought to investigate the involvement of solute carrier family 9, subfamily A, member 3 (SLC9A3) in esophageal epithelial intracellular pH (pHi) and DIS formation and the histopathologic features of EoE. Methods We examined expression of esophageal epithelial gene networks associated with regulation of pHi in the EoE transcriptome of primary esophageal epithelial cells and an in vitro esophageal epithelial 3-dimensional model system (EPC2-ALI). Molecular and cellular analyses and ion transport assays were used to evaluate the expression and function of SLC9A3. Results We identified altered expression of gene networks associated with regulation of pHi and acid-protective mechanisms in esophageal biopsy specimens from pediatric patients with EoE (healthy subjects, n = 6; patients with EoE, n = 10). The most dysregulated gene central to regulating pHi was SLC9A3. SLC9A3 expression was increased within the basal layer of esophageal biopsy specimens from patients with EoE, and expression positively correlated with disease severity (eosinophils/high-power field) and DIS (healthy subjects, n = 10; patients with EoE, n = 10). Analyses of esophageal epithelial cells revealed IL-13–induced, signal transducer and activator of transcription 6–dependent SLC9A3 expression and Na+-dependent proton secretion and that SLC9A3 activity correlated positively with DIS formation. Finally, we showed that IL-13–mediated, Na+-dependent proton secretion was the primary intracellular acid-protective mechanism within the esophageal epithelium and that blockade of SLC9A3 transport abrogated IL-13–induced DIS formation. Conclusions SLC9A3 plays a functional role in DIS formation, and pharmacologic interventions targeting SLC9A3 function may suppress the histopathologic manifestations in patients with EoE.

Food allergy naturally resolves in a proportion of food-allergic children without intervention; however the underlying mechanisms governing the persistence or resolution of food allergy in childhood are not understood.This study aimed to define the innate immune profiles associated with egg allergy at age 1 year, determine the phenotypic changes that occur with the development of natural tolerance in childhood, and explore the relationship between early life innate immune function and serum vitamin D.This study used longitudinally collected PBMC samples from a population-based cohort of challenge-confirmed egg-allergic infants with either persistent or transient egg allergy outcomes in childhood to phenotype and quantify the functional innate immune response associated with clinical phenotypes of egg allergy.We show that infants with persistent egg allergy exhibit a unique innate immune signature, characterized by increased numbers of circulating monocytes and dendritic cells that produce more inflammatory cytokines both at baseline and following endotoxin exposure when compared with infants with transient egg allergy. Follow-up analysis revealed that this unique innate immune signature continues into childhood in those with persistent egg allergy and that increased serum vitamin D levels correlate with changes in innate immune profiles observed in children who developed natural tolerance to egg.Early life innate immune dysfunction may represent a key immunological driver and predictor of persistent food allergy in childhood. Serum vitamin D may play an immune-modulatory role in the development of natural tolerance.

Background GTPase of immunity-associated protein 5 (GIMAP5) is essential for lymphocyte homeostasis and survival. Recently, human GIMAP5 single nucleotide polymorphisms have been linked to an increased risk for asthma, whereas loss of Gimap5 in mice has been associated with severe CD4+ T cell–driven immune pathology. Objective We sought to identify the molecular and cellular mechanisms by which Gimap5 deficiency predisposes to allergic airway disease. Methods CD4+ T-cell polarization and development of pathogenic CD4+ T cells were assessed in Gimap5-deficient mice and a human patient with a GIMAP5 loss-of-function (LOF) mutation. House dust mite–induced airway inflammation was assessed by using a complete Gimap5 LOF (Gimap5sph/sph) and conditional Gimap5fl/flCd4Cre/ert2 mice. Results GIMAP5 LOF mutations in both mice and human subjects are associated with spontaneous polarization toward pathogenic TH17 and TH2 cells in vivo. Mechanistic studies in vitro reveal that impairment of Gimap5-deficient TH cell differentiation is associated with increased DNA damage, particularly during TH1-polarizing conditions. DNA damage in Gimap5-deficient CD4+ T cells could be controlled by TGF-β, thereby promoting TH17 polarization. When challenged with house dust mite in vivo, Gimap5-deficient mice displayed an exacerbated asthma phenotype (inflammation and airway hyperresponsiveness), with increased development of TH2, TH17, and pathogenic TH17/TH2 cells. Conclusion Activation of Gimap5-deficient CD4+ T cells is associated with increased DNA damage and reduced survival that can be overcome by TGF-β. This leads to selective survival of pathogenic TH17 cells but also TH2 cells in human subjects and mice, ultimately promoting allergic airway disease.

Background Severe IgE-mediated, food-induced anaphylactic reactions are characterized by pulmonary venous vasodilatation and fluid extravasation, which are thought to lead to the life-threatening anaphylactic phenotype. The underlying immunologic and cellular processes involved in driving fluid extravasation and the severe anaphylactic phenotype are not fully elucidated. Objective We sought to define the interaction and requirement of IL-4 and vascular endothelial (VE) IL-4 receptor α chain (IL-4Rα) signaling in histamine-abelson murine leukemia viral oncogene homology 1 (ABL1)–mediated VE dysfunction and fluid extravasation in the severity of IgE-mediated anaphylactic reactions in mice. Methods Mice deficient in VE IL-4Rα and models of passive and active oral antigen– and IgE-induced anaphylaxis were used to define the requirements of the VE IL-4Rα and ABL1 pathway in severe anaphylactic reactions. The human VE cell line (EA.hy926 cells) and pharmacologic (imatinib) and genetic (short hairpin RNA knockdown of IL4RA and ABL1 ) approaches were used to define the requirement of this pathway in VE barrier dysfunction. Results IL-4 exacerbation of histamine-induced hypovolemic shock in mice was dependent on VE expression of IL-4Rα. IL-4– and histamine-induced ABL1 activation in human VE cells and VE barrier dysfunction was ABL1-dependent. Development of severe IgE-mediated hypovolemia and shock required VE-restricted ABL1 expression. Treatment of mice with a history of food-induced anaphylaxis with the ABL kinase inhibitor imatinib protected the mice from severe IgE-mediated anaphylaxis. Conclusion IL-4 amplifies IgE- and histamine-induced VE dysfunction, fluid extravasation, and the severity of anaphylaxis through a VE IL-4Rα/ABL1–dependent mechanism. These studies implicate an important contribution by the VE compartment in the severity of anaphylaxis and identify a new pathway for therapeutic intervention of IgE-mediated reactions.

The intestinal epithelium is a single-cell layer that constitutes the largest and most important barrier against the external environment. It acts as a selectively permeable barrier permitting the absorption of nutrients, electrolytes and water, while maintaining an effective defense against intraluminal toxins, antigens and enteric flora. The epithelium maintains its selective barrier function through the formation of complex protein-protein networks that mechanically link adjacent cells and seal the intercellular space. The protein networks connecting epithelial cells form three adhesive complexes: desmosomes, adherens junctions and tight junctions. These complexes consist of transmembrane proteins that interact extracellularly with adjacent cells and intracellularly with adaptor proteins that link to the cytoskeleton. Over the past decade, there has been increasing recognition of an association between disrupted intestinal barrier function and the development of autoimmune and inflammatory diseases. In this review, we summarize the evolving understanding of the molecular composition and regulation of intestinal barrier function. We discuss the interactions between innate and adaptive immunity and intestinal epithelial barrier function, as well as the impact of exogenous factors on intestinal barrier function. Finally, we summarize clinical and experimental evidence demonstrating intestinal epithelial barrier dysfunction as a major factor contributing to the predisposition to inflammatory diseases including food allergy, inflammatory bowel diseases and celiac disease.

Background Resistin-like molecule (RELM) β is a cysteine-rich cytokine expressed in the gastrointestinal tract and implicated in insulin resistance and gastrointestinal nematode immunity; however, its function primarily remains an enigma. Objective We sought to elucidate the function of RELM-β in the gastrointestinal tract. Methods We generated RELM-β gene–targeted mice and examined colonic epithelial barrier function, gene expression profiles, and susceptibility to acute colonic inflammation. Results We show that RELM-β is constitutively expressed in the colon by goblet cells and enterocytes and has a role in homeostasis, as assessed by alterations in colon mRNA transcripts and epithelial barrier function in the absence of RELM-β. Using acute colonic inflammatory models, we demonstrate that RELM-β has a central role in the regulation of susceptibility to colonic inflammation. Mechanistic studies identify that RELM-β regulates expression of type III regenerating gene (REG) (REG3β and γ), molecules known to influence nuclear factor κB signaling. Conclusions These data define a critical role for RELM-β in the maintenance of colonic barrier function and gastrointestinal innate immunity. Clinical implications These findings identify RELM-β as an important molecule in homeostatic gastrointestinal function and colonic inflammation, and as such, these results have implications for a variety of human inflammatory gastrointestinal conditions, including allergic gastroenteropathies.

Background Eotaxin-2 is a member of the eotaxin subfamily of CC chemokines that display eosinophil-specific, chemotactic properties and has been associated with allergic disorders. However, the contribution of eotaxin-2 to the development of defined pathogenic features of allergic disease remains to be defined. Objective We sought to determine whether eotaxin-2 was a cofactor with IL-5 for the regulation of pulmonary eosinophilia and to identify the combined role of these molecules in the induction of phenotypic characteristics of allergic lung disease. Methods We instilled recombinant eotaxin-2 into the airways of wild-type mice that had been treated systemically with IL-5 or into IL-5-transgenic mice and characterized pulmonary eosinophil numbers, IL-13 production, and airway hyperreactivity (AHR) to methacholine. Mice deficient in the IL-4 receptor α-chain, IL-13, and signal transducers and activators of transcription 6 or mice treated with anti-CCR3 monoclonal antibody were also used. Results Eotaxin-2 and IL-5 cooperatively promoted eosinophil accumulation, IL-13 production, and AHR to methacholine. Neither eotaxin-2 nor IL-5 alone induced these features of allergic disease. IL-13 production was critically dependent on eotaxin-2- and IL-5-regulated eosinophilia, which predisposed to the development of AHR. AHR was dependent on IL-13 and signaling through the IL-4R α-chain and signal transducers and activators of transcription 6 pathways and the presence of eosinophils in the lung. Conclusion These investigations demonstrate important cooperativity between eotaxin-2, IL-5, and IL-13 signaling systems and eosinophils for the development of hallmark features of allergic disease of the lung.

Background Food-mediated allergic reactions have emerged as a major health problem. The underlying mechanisms that promote uncontrolled type 2 immune responses to dietary allergens in the gastrointestinal tract remain elusive. Objective We investigated whether altering IL-25 signaling enhances or attenuates allergic responses to food allergens. Methods Mice of an IL-25 transgenic mouse line (iIL-25Tg mice), which constitutively overexpress intestinal IL-25, and Il17rb −/− mice, in which Il17rb gene expression is disrupted, were sensitized and gavage fed with ovalbumin (OVA). We assessed symptomatic characteristics of experimental food allergy, including incidence of diarrhea, incidence of hypothermia, intestinal T H 2 immune response, and serum OVA-specific IgE and mast cell protease 1 production. Results Rapid induction of Il25 expression in the intestinal epithelium preceded onset of the anaphylactic response to ingested OVA antigen. iIL-25Tg mice were more prone and Il17rb −/− mice were more resistant to experimental food allergy. Resident intestinal type 2 innate lymphoid cells (ILC2s) were identified as the major producers of IL-5 and IL-13 in response to IL-25. Reconstituting irradiated wild-type mice with Rora −/− or Il17rb −/− bone marrow resulted in a deficiency or dysfunction of the ILC2 compartment, respectively, and resistance to experimental food allergy. Repeated intragastric antigen challenge induced a significant increase in numbers of CD4 + T H 2 cells, which enhance IL-25–stimulated IL-13 production by ILC2s ex vivo and in vivo . Finally, reconstituted IL-13–deficient ILC2s had reduced capability to promote allergic inflammation, resulting in increased resistance to experimental food allergy. Conclusion IL-25 and CD4 + T H 2 cells induced by ingested antigens enhance ILC2-derived IL-13 production, thereby promoting IgE-mediated experimental food allergy.

The cytokine IL-9 has been implicated in allergic reactions, including food allergy, but its contribution to parenteral versus oral antigen-induced anaphylaxis remains unclear.We sought to delineate the contribution of the IL-9/IL-9 receptor alpha-chain (IL-9R) pathway to parenteral and oral antigen-induced anaphylaxis.Wild-type, IL-9-deficient (Il9(-/-)), and IL-9R-deficient (Il9R(-/-)) mice were subjected to passive and active parenteral and oral antigen (ovalbumin [OVA])-induced anaphylaxis. Severity of systemic anaphylaxis was gauged by decreased body temperature; intestinal anaphylaxis was assessed based on secretory diarrhea, intestinal mastocytosis, and serum murine mast cell protease 1 level. Specific immunoglobulin isotypes or immunoglobulin receptor-blocking antibodies were administered before challenge to define the role of the IgE and IgG pathways.Repeated oral antigen challenge of OVA-sensitized wild-type mice induced anaphylaxis with both systemic and intestinal involvement; both were IgE dependent and attenuated in Il9(-/-) and Il9R(-/-) mice. In contrast, parenteral OVA challenge of OVA-sensitized wild-type mice induced systemic anaphylaxis, which was independent of the IL-9/IL-9R pathway. Strikingly, the IL-9/IL-9R pathway had no role in either the IgG or IgE component of parenteral antigen-induced or anti-IgE and anti-FcgammaRII/III mAb-induced systemic anaphylaxis.Parenteral antigen-induced murine systemic anaphylaxis is mediated by both IgG- and IgE-dependent pathways, and both can occur independently of IL-9/IL-9R signaling. In contrast, oral antigen-induced intestinal and systemic anaphylaxis is strictly IgE mediated and requires IL-9/IL-9R signaling. These studies indicate differential involvement of the IL-9/IL-9R pathway in systemic and oral antigen-induced anaphylaxis.

Background Resistin-like molecule (Relm) α is a secreted protein and a hallmark signature gene for alternatively activated macrophages. Relm-α is highly induced by allergic inflammatory triggers and perceived to promote tissue repair. Yet the function of Relm-α remains unknown. Objective We sough to determine the role of Relm-α in dextran sodium sulfate (DSS)–induced colonic injury. Methods The cellular source of Relm-α was determined after oral DSS-induced colitis. Retnla −/− mice were generated, subjected to DSS treatment, and monitored for disease progression (clinical and histopathologic features). Cytokine production in the supernatants of ex vivo colon cultures, and of LPS-stimulated macrophages incubated with Relm-α was assessed. Relm-α was administered intraperitoneally, and the cellular recruitment to the peritoneum was assessed. Results After innate intestinal stimulation with DSS, Relm-α was highly expressed by eosinophils and epithelial cells. Retnla gene–targeted mice were protected from DSS-induced colitis (eg, decreased diarrhea, rectal bleeding, colon shortening, disease score, and histopathologic changes). Relm-α coactivated IL-6 and TNF-α release and inhibited IL-10 release from LPS-activated bone marrow–derived macrophages. Consistent with these finding, colon cultures of DSS-treated Retnla −/− mice produced decreased IL-6 and increased IL-10 ex vivo . Furthermore, Retnla −/− mice had substantially decreased c-Jun N-terminal kinase phosphorylation in vivo . In vivo administration of Relm-α initiated cellular recruitment to the peritoneum, and Relm-α was able to induce eosinophil chemotaxis in vitro . Conclusions These findings demonstrate a central proinflammatory role for Relm-α in colonic innate immune responses, identifying a novel pathway for regulation of macrophage activation.

Rationale Eosinophil-associated gastrointestinal diseases (EGID) are frequently associated with extraintestinal features including bronchopulmonary manifestations. The factors predisposing to bronchial hyperresponsiveness (BHR) in EGID are unknown. To elucidate the mechanistic link between EGID and BHR we employed murine models of EGID and eotaxin-1 (eotaxin)-transgene induced EGID. Methods Mice were sensitized and orally challenged with ovalbumin coated encapsulated particles to induce EGID and bronchial responsiveness was examined. Furthermore, transgenic mice expressing eotaxin in the intestine (using the rat fatty acid-binding promoter) were used to specifically elucidate the contribution of this chemokine in EGID-associated BHR. Results We show induction of allergen-induced EGID directly correlated with the development of BHR. The development of BHR in mice with allergen-induced EGID was dependent on eotaxin expression in the GI tract. Expression of eotaxin in the gastrointestinal tract of transgenic mice was sufficient to promote BHR. BHR was shown to be directly linked to the aberrant CD4 + Th 2 -type T-cell (Th 2 ) production of interleukin-(IL)-13. Interestingly, transgenic expression of eotaxin was linked with enhanced Th 2 -cytokine synthesis (IL-4, -5 and -13) and the production of mucosal IgG 1 in the gastrointestinal lumen. We also show that eotaxin treatment of CD4 + T-cells enhances IL-13 production in vitro. Conclusions These studies suggest that elevated expression of eotaxin in the gastrointestinal compartment can lead to increased CD4 + T-cell derived Th 2 -cytokine production that drives aberrant immunophysiological responses in distant non-inflamed mucosal tissue (the lung). These results provide a possible explanation for altered lung function seen in some patients with inflammatory gastrointestinal disorders.