Physiological microbial exposure transiently inhibits mouse lung ILC2 responses to allergens.

Lung group 2 innate lymphoid cells (ILC2s) control the nature of immune responses to airway allergens. Some microbial products, including those that stimulate interferons, block ILC2 activation, but whether this occurs after natural infections or causes durable ILC2 inhibition is unclear. In the present study, we cohoused laboratory and pet store mice as a model of physiological microbial exposure. Laboratory mice cohoused for 2 weeks had impaired ILC2 responses and reduced lung eosinophilia to intranasal allergens, whereas these responses were restored in mice cohoused for ≥2 months. ILC2 inhibition at 2 weeks correlated with increased interferon receptor signaling, which waned by 2 months of cohousing. Reinduction of interferons in 2-month cohoused mice blocked ILC2 activation. These findings suggest that ILC2s respond dynamically to environmental cues and that microbial exposures do not control long-term desensitization of innate type 2 responses to allergens.

Oral mucosa effectively protects against peanut allergy in mice.

BACKGROUND: Oral consumption of peanut products early in life reduces the incidence of peanut allergy in children. However, little is known about whether exposure via the oral mucosa alone is sufficient or whether the gastrointestinal tract must be engaged to protect against peanut allergy. OBJECTIVE: We used a mouse model and examined the effects of peanut allergen administration to only the oral cavity on allergy development induced by environmental exposure. METHODS: Naive BALB/c mice were administered peanut flour (PNF) sublingually, followed by epicutaneous exposure to PNF to mimic a human condition. The sublingual volume was adjusted to engage only the oral cavity and prevent it from reaching the esophagus or gastrointestinal tract. The efficacy was evaluated by examining the anaphylactic response, antibody titers, and T follicular helper cells. RESULTS: The mice exposed epicutaneously to PNF developed peanut allergy, as demonstrated by increased plasma levels of peanut-specific IgE and the manifestation of acute systemic anaphylaxis following intraperitoneal challenge with peanut extract. The development of peanut allergy was suppressed when mice had been given PNF sublingually before epicutaneous exposure. There were fewer T follicular helper cells in the skin-draining lymph nodes of mice that received sublingual PNF than in the mice that received PBS. Suppression of IgE production was observed with sublingual PNF at 1/10 of the intragastric PNF dose. CONCLUSION: Administration of peanut allergens only to the oral cavity effectively prevents the development of peanut allergy. The capacity of the oral mucosa to promote immunologic tolerance needs to be evaluated further to prevent food allergy.

Cholinergic sensing of allergen exposure by airway epithelium promotes type 2 immunity in the lungs.

BACKGROUND: Nonneuronal cells, including epithelial cells, can produce acetylcholine (ACh). Muscarinic ACh receptor antagonists are used clinically to treat asthma and other medical conditions; however, knowledge regarding the roles of ACh in type 2 immunity is limited. OBJECTIVE: Our aim was to investigate the roles of epithelial ACh in allergic immune responses. METHODS: Human bronchial epithelial (HBE) cells were cultured with allergen extracts, and their ACh production and IL-33 secretion were studied in vitro. To investigate immune responses in vivo, naive BALB/c mice were treated intranasally with different muscarinic ACh receptor antagonists and then exposed intranasally to allergens. RESULTS: At steady state, HBE cells expressed cellular components necessary for ACh production, including choline acetyltransferase and organic cation transporters. Exposure to allergens caused HBE cells to rapidly release ACh into the extracellular medium. Pharmacologic or small-interfering RNA-based blocking of ACh production or autocrine action through the M3 muscarinic ACh receptors in HBE cells suppressed allergen-induced ATP release, calcium mobilization, and extracellular secretion of IL-33. When naive mice were exposed to allergens, ACh was quickly released into the airway lumen. A series of clinical M3 muscarinic ACh receptor antagonists inhibited allergen-induced IL-33 secretion and innate type 2 immune response in the mouse airways. In a preclinical murine model of asthma, an ACh receptor antagonist suppressed allergen-induced airway inflammation and airway hyperreactivity. CONCLUSIONS: ACh is released quickly by airway epithelial cells on allergen exposure, and it plays an important role in type 2 immunity. The epithelial ACh system can be considered a therapeutic target in allergic airway diseases.

Epithelial overexpression of IL-33 induces eosinophilic esophagitis dependent on IL-13.

BACKGROUND: Eosinophilic esophagitis (EoE) is an increasingly common inflammatory condition of the esophagus; however, the underlying immunologic mechanisms remain poorly understood. The epithelium-derived cytokine IL-33 is associated with type 2 immune responses and elevated in esophageal biopsy specimens from patients with EoE. OBJECTIVE: We hypothesized that overexpression of IL-33 by the esophageal epithelium would promote the immunopathology of EoE. METHODS: We evaluated the functional consequences of esophageal epithelial overexpression of a secreted and active form of IL-33 in a novel transgenic mouse, EoE33. EoE33 mice were analyzed for clinical and immunologic phenotypes. Esophageal contractility was assessed. Epithelial cytokine responses were analyzed in three-dimensional organoids. EoE33 phenotypes were further characterized in ST2-/-, eosinophil-deficient, and IL-13-/- mice. Finally, EoE33 mice were treated with dexamethasone. RESULTS: EoE33 mice displayed ST2-dependent, EoE-like pathology and failed to thrive. Esophageal tissue remodeling and inflammation included basal zone hyperplasia, eosinophilia, mast cells, and TH2 cells. Marked increases in levels of type 2 cytokines, including IL-13, and molecules associated with immune responses and tissue remodeling were observed. Esophageal organoids suggested reactive epithelial changes. Genetic deletion of IL-13 in EoE33 mice abrogated pathologic changes in vivo. EoE33 mice were responsive to steroids. CONCLUSIONS: IL-33 overexpression by the esophageal epithelium generated immunopathology and clinical phenotypes resembling human EoE. IL-33 may play a pivotal role in the etiology of EoE by activating the IL-13 pathway. EoE33 mice are a robust experimental platform for mechanistic investigation and translational discovery.

Lung-resident CD69+ST2+ TH2 cells mediate long-term type 2 memory to inhaled antigen in mice.

BACKGROUND: Chronic airway diseases such as asthma are characterized by persistent type 2 immunity in the airways. We know little about the mechanisms that explain why type 2 inflammation continues in these diseases. OBJECTIVE: We used mouse models to investigate the mechanisms involved in long-lasting immune memory. METHODS: Naive mice were exposed intranasally to ovalbumin (OVA) antigen with Alternaria extract as an adjuvant. Type 2 memory was analyzed by parabiosis model, flow cytometry with in vivo antibody labeling, and intranasal OVA recall challenge. Gene-deficient mice were used to analyze the mechanisms. RESULTS: In the parabiosis model, mice previously exposed intranasally to OVA with Alternaria showed more robust antigen-specific immune responses and airway inflammation than mice with circulating OVA-specific T cells. After a single airway exposure to OVA with Alternaria, CD69+ST2+ TH2-type T cells, which highly express type 2 cytokine messenger RNA and lack CD62L expression, appeared in lung tissue within 5 days and persisted for at least 84 days. When exposed again to OVA in vivo, these cells produced type 2 cytokines quickly without involving circulating T cells. Development of tissue-resident CD69+ST2+ TH2 cells and long-term memory to an inhaled antigen were abrogated in mice deficient in ST2 or IL-33, but not TSLP receptor. CONCLUSION: CD69+ST2+ TH2 memory cells develop quickly in lung tissue after initial allergen exposure and persist for a prolonged period. The ST2/IL-33 pathway may play a role in the development of immune memory in lung to certain allergens.

Allergen-induced DNA release by the airway epithelium amplifies type 2 immunity.

BACKGROUND: Alternaria alternata and house dust mite exposure evokes IL-33 secretion from the airway epithelium, which functions as an alarmin to stimulate type 2 immunity. Extracellular DNA (eDNA) is also an alarmin that intensifies inflammation in cystic fibrosis, chronic obstructive pulmonary disease, and asthma. OBJECTIVE: We investigated the mechanisms underlying allergen-evoked DNA mobilization and release from the airway epithelium and determined the role of eDNA in type 2 immunity. METHODS: Human bronchial epithelial (hBE) cells were used to characterize allergen-induced DNA mobilization and extracellular release using comet assays to measure DNA fragmentation, Qubit double-stranded DNA assays to measure DNA release, and DNA sequencing to determine eDNA composition. Mice were used to investigate the role of eDNA in type 2 immunity. RESULTS: Alternaria extract rapidly induces mitochondrial and nuclear DNA release from human bronchial epithelial cells, whereas house dust mite extract induces mitochondrial DNA release. Caspase-3 is responsible for nuclear DNA fragmentation and becomes activated after cleavage by furin. Analysis of secreted nuclear DNA showed disproportionally higher amounts of promotor and exon sequences and lower intron and intergenic regions compared to predictions of random DNA fragmentation. In mice, Alternaria-induced type 2 immune responses were blocked by pretreatment with a DNA scavenger. In caspase-3-deficient mice, Alternaria-induced DNA release was suppressed. Furthermore, intranasal administration of mouse genomic DNA with Alternaria amplified secretion of IL-5 and IL-13 into bronchoalveolar lavage fluid while DNA alone had no effect. CONCLUSION: These findings highlight a novel, allergen-induced mechanism of rapid DNA release that amplifies type 2 immunity in airways.

Eosinophils promote effector functions of lung group 2 innate lymphoid cells in allergic airway inflammation in mice.

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) are critical mediators of type 2 respiratory inflammation, releasing IL-5 and IL-13 and promoting the pulmonary eosinophilia associated with allergen provocation. Although ILC2s have been shown to promote eosinophil activities, the role of eosinophils in group 2 innate lymphoid cell (ILC2) responses is less well defined. OBJECTIVE: We sought to investigate the role of eosinophils in activation of ILC2s in models of allergic asthma and in vitro. METHODS: Inducible eosinophil-deficient mice were exposed to allergic respiratory inflammation models of asthma, such as ovalbumin or house dust mite challenge, or to innate models of type 2 airway inflammation, such as inhalation of IL-33. Eosinophil-specific IL-4/13-deficient mice were used to address the specific roles for eosinophil-derived cytokines. Direct cell interactions between ILC2s and eosinophils were assessed by in vitro culture experiments. RESULTS: Targeted depletion of eosinophils resulted in significant reductions of total and IL-5+ and IL-13+ lung ILC2s in all models of respiratory inflammation. This correlated with reductions in IL-13 levels and mucus in the airway. Eosinophil-derived IL-4/13 was necessary for both eosinophil and ILC2 accumulation in lung in allergen models. In vitro, eosinophils released soluble mediators that induced ILC2 proliferation and G protein-coupled receptor-dependent chemotaxis of ILC2s. Coculture of ILC2s and IL-33-activated eosinophils resulted in transcriptome changes in both ILC2s and eosinophils, suggesting potential novel reciprocal interactions. CONCLUSION: These studies demonstrate that eosinophils play a reciprocal role in ILC2 effector functions as part of both adaptive and innate type 2 pulmonary inflammatory events.

ATP functions as a primary alarmin in allergen-induced type 2 immunity.

Environmental allergens that interact with the airway epithelium can activate cellular stress pathways that lead to the release of danger signals known as alarmins. The mechanisms of alarmin release are distinct from damage-associated molecular patterns (DAMPs), which typically escape from cells after loss of plasma membrane integrity. Oxidative stress represents a form of allergen-induced cellular stress that stimulates oxidant-sensing mechanisms coupled to pathways, which facilitate alarmin mobilization and efflux across the plasma membrane. In this review, we highlight examples of alarmin release and discuss their roles in the initiation of type 2 immunity and allergic airway inflammation. In addition, we discuss the concept of alarmin amplification, where "primary" alarmins, which are directly released in response to a specific cellular stress, stimulate additional signaling pathways that lead to secretion of "secondary" alarmins that include proinflammatory cytokines, such as IL-33, as well as genomic and mitochondrial DNA that coordinate or amplify type 2 immunity. Accordingly, allergen-evoked cellular stress can elicit a hierarchy of alarmin signaling responses from the airway epithelium that trigger local innate immune reactions, impact adaptive immunity, and exacerbate diseases including asthma and other chronic inflammatory conditions that affect airway function.

Detergent exposure induces epithelial barrier dysfunction and eosinophilic inflammation in the esophagus.

BACKGROUND: Eosinophilic esophagitis (EoE) is a chronic allergic disease associated with type 2 inflammation and epithelial barrier dysfunction. The etiology is unknown, however, genetic heritability studies suggest environmental factors play a key role in pathogenesis. Detergents, such as sodium dodecyl sulfate (SDS), are common ingredients in household products such as dish soap and toothpaste. We hypothesized detergent exposure decreases epithelial barrier function and induces esophageal inflammation. METHODS: Immortalized esophageal epithelial cells (EPC2) were cultured in air-liquid interface (ALI) and exposed to SDS. Barrier function/activity was assessed by transepithelial electrical resistance (TEER), FITC-dextran flux, and RT-PCR. Additionally, SDS-treated mouse esophageal organoids were evaluated for morphology. To investigate the effects of SDS in vivo, mice were treated with 0.5% SDS in drinking water for 14 days. Esophagi were assessed by gross morphology, histopathology, protein expression, and bulk RNA sequencing. RESULTS: When EPC2 cells were exposed to SDS (5 µg/ml) for 96 h, TEER decreased (p = 0.03), and FITC-dextran flux increased (p = 0.0002). mRNA expression of IL-33 increased 4.5-fold (p = 0.02) at 6 h and DSG1 decreased (p < 0.0001) by 72 h. Disrupted epithelial integrity was noted in SDS-treated esophageal organoids. When mice were exposed to SDS, they showed increased esophageal width, chemokine, and metalloprotease levels. Mice treated with SDS also showed increased IL-33 protein expression, basal zone hyperplasia, CD4+ cell infiltration, and esophageal eosinophilia. RNA sequencing revealed upregulation of immune response pathway genes. CONCLUSION: Exposure to SDS decreases esophageal barrier integrity, stimulates IL-33 production, and promotes epithelial hyperplasia and tissue eosinophilia. Detergents may be a key environmental trigger in EoE pathogenesis.

Allergies Come Clean: The Role of Detergents in Epithelial Barrier Dysfunction.

PURPOSE OF REVIEW: The prevalence and incidence of allergic disease have been rising in Westernized countries since the twentieth century. Increasingly, evidence suggests that damage to the epithelium initiates and shapes innate and adaptive immune responses to external antigens. The objective of this review is to examine the role of detergents as a potential risk factor for developing allergic disease. RECENT FINDINGS: Herein, we identify key sources of human detergent exposure. We summarize the evidence suggesting a possible role for detergents and related chemicals in initiating epithelial barrier dysfunction and allergic inflammation. We primarily focus on experimental models of atopic dermatitis, asthma, and eosinophilic esophagitis, which show compelling associations between allergic disease and detergent exposure. Mechanistic studies suggest that detergents disrupt epithelial barrier integrity through their effects on tight junction or adhesion molecules and promote inflammation through epithelial alarmin release. Environmental exposures that disrupt or damage the epithelium may account for the increasing rates of allergic disease in genetically susceptible individuals. Detergents and related chemical compounds represent possible modifiable risk factors for the development or exacerbation of atopy.

Blocking the inhibitory receptor programmed cell death 1 prevents allergic immune response and anaphylaxis in mice.

BACKGROUND: Food allergy and acute anaphylaxis can be life-threatening. While T follicular helper (Tfh) cells play a pivotal role in the allergic immune responses, the immunologic mechanisms that regulate the production of antibodies (Abs) that mediate anaphylaxis are not fully understood. OBJECTIVE: The aim of this study was to investigate the role of the inhibitory receptor programmed cell death protein 1 (PD-1), which is highly expressed on Tfh cells, in allergic immune responses using an animal model of peanut allergy and anaphylaxis. METHODS: Naive wild-type mice were exposed to peanut flour intranasally and then challenged with peanut extract to induce systemic anaphylaxis. The roles of PD-1 were examined by blocking Abs and using gene-deficient animals. A hapten model and passive cutaneous anaphylaxis were used to characterize allergen-specific Abs. RESULTS: Treatment with anti-PD-1 enhanced development of Tfh cells and germinal center B cells in mice exposed to peanut flour. Nonetheless, anti-PD-1 or its ligand fully protected mice from developing anaphylaxis. Anti-PD-1 treatment or genetic deficiency of PD-1 in CD4+ T cells inhibited production of peanut-specific IgE and increased the levels of IgG. The passive cutaneous anaphylaxis showed that peanut-specific Abs generated in anti-PD-1-treated animals prevented, rather than provoked, anaphylaxis when transferred to naive animals. Anti-PD-1 promoted production of Abs with low affinity for an antigen in the hapten model. CONCLUSION: Blockade of the pathway between PD-1 and its ligand is protective against allergic immune responses. The direct interaction between Tfh cells and B cells may play a pivotal role in controlling Ab quality and clinical manifestation of allergic diseases.

TLR3-driven IFN-ß antagonizes STAT5-activating cytokines and suppresses innate type 2 response in the lung.

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) are involved in type 2 immune responses in mucosal organs and are associated with various allergic diseases in humans. Studies are needed to understand the molecules and pathways that control ILC2s. OBJECTIVE: The aims of this study were to develop a mouse model that limits the innate type 2 immune response in the lung and to investigate the immunologic mechanisms involved in regulation of lung ILC2s. METHODS: Naive BALB/c mice were administered various Toll-like receptor agonists and exposed intranasally to the fungal allergen Alternaria alternata. The mechanisms were investigated using gene knockout mice as well as cultures of lung cells and isolated lung ILC2s. RESULTS: Polyinosinic-polycytidylic acid, or poly (I:C), effectively inhibited innate type 2 response to A alternata. Poly (I:C) promoted production of IFNα, -ß, and -γ, and its inhibitory effects were dependent on the IFN-α/ß receptor pathway. IFN-ß was 100 times more potent than IFN-α at inhibiting type 2 cytokine production by lung ILC2s. Signal transducer and activator of transcription 5 (STAT5)-activating cytokines, including IL-2, IL-7, and thymic stromal lymphopoietin, but not IL-33, promoted survival and proliferation of lung ILC2s in vitro, while IFN-ß blocked these effects. Expression of the transcription factor GATA3, which is critical for differentiation and maintenance of ILC2s, was inhibited by IFN-ß. CONCLUSIONS: IFN-ß blocks the effects of STAT5-activating cytokines on lung ILC2s and inhibits their survival and effector functions. Administration of IFN-ß may provide a new strategy to treat diseases involving ILC2s.

A mouse model of the LEAP study reveals a role for CTLA-4 in preventing peanut allergy induced by environmental peanut exposure.

BACKGROUND: A human study, Learning Early About Peanut Allergy (LEAP), showed that early introduction of peanut products decreases the prevalence of peanut allergy among children. However, the immunologic mechanisms mediating the protective effects of consuming peanut products are not well understood. OBJECTIVE: The objective was to develop a mouse model that simulates the LEAP study and investigate the underlying mechanisms for the study observations. METHODS: Adult naive BALB/c mice were fed a commercial peanut butter product (Skippy) or buffer control and concomitantly exposed to peanut flour through the airway or skin to mimic environmental exposure. The animals were analyzed for anaphylactic reaction and by molecular and immunologic approaches. RESULTS: After exposure to peanut flour through the airway or skin, naive mice developed peanut allergy, as demonstrated by acute and systemic anaphylaxis in response to challenge with peanut extract. Ingestion of Skippy, however, nearly abolished the increase in peanut-specific IgE and IgG and protected animals from developing anaphylaxis. Skippy-fed mice showed reduced numbers of T follicular helper (Tfh) cells and germinal center B cells in their draining lymph nodes, and single-cell RNA sequencing revealed a CD4+ T-cell population expressing cytotoxic T lymphocyte-associated protein 4 (CTLA-4) in these animals. Critically, blocking CTLA-4 with antibody increased levels of peanut-specific antibodies and reversed the protective effects of Skippy. CONCLUSION: Ingestion of a peanut product protects mice from peanut allergy induced by environmental exposure to peanuts, and the CTLA-4 pathway, which regulates Tfh cell responses, likely plays a pivotal role in this protection.

Group 2 Innate Lymphoid Cells Promote Development of T Follicular Helper Cells and Initiate Allergic Sensitization to Peanuts.

Peanut allergy is a growing public concern; however, little is known about the immunological mechanism(s) that initiate the disease process. Our knowledge is also limited regarding the role of group 2 innate lymphoid cells (ILC2s) in regulating humoral immunity. To fill these major gaps in our knowledge, we investigated the immunological mechanisms involved in peanut allergen sensitization by using mouse models. To mimic environmental exposure in humans, naive BALB/c mice were exposed to peanut flour by inhalation without any exogenous adjuvants. When exposed to peanut flour, naive mice developed T follicular helper (Tfh) cells in their lung draining lymph nodes and produced IgE Abs to peanuts. Mice deficient in IL-13 showed decreased numbers of Tfh cells and germinal center B cells and produced significantly fewer IgE Abs. IL-13 was necessary and sufficient for induction of CD11c+ MHC class IIhi dendritic cells that are implicated in Tfh cell development. Importantly, lung ILC2s served as a predominant early source of IL-13 when naive mice were exposed to peanut flour. Furthermore, mice that are deficient in lung ILC2s by bone marrow transfer from Rora sg/sg mice or by genetic manipulation produced significantly fewer IgE Abs to peanuts compared with control mice. These findings suggest lung ILC2s that serve as a rapid source of IL-13 upon allergen exposure play a major role in Tfh cell development, IgE Ab production, and initiation of peanut allergy.

COX Inhibition Increases Alternaria-Induced Pulmonary Group 2 Innate Lymphoid Cell Responses and IL-33 Release in Mice.

The cyclooxygenase (COX) metabolic pathway regulates immune responses and inflammation. The effect of the COX pathway on innate pulmonary inflammation induced by protease-containing fungal allergens, such as Alternaria alternata, is not fully defined. In this study, we tested the hypothesis that COX inhibition augments Alternaria-induced pulmonary group 2 innate lymphoid cell (ILC2) responses and IL-33 release. Mice were treated with the COX inhibitors indomethacin, flurbiprofen, or vehicle and challenged intranasally with Alternaria extract for four consecutive days to induce innate lung inflammation. We found that indomethacin and flurbiprofen significantly increased the numbers of ILC2 and IL-5 and IL-13 expression by ILC2 in the lung. Indomethacin also increased ILC2 proliferation, the percentages of eosinophils, and mucus production in the lung. Both indomethacin and flurbiprofen augmented the release of IL-33 in bronchoalveolar lavage fluid after Alternaria challenge, suggesting that more IL-33 was available for ILC2 activation and that a COX product(s) inhibited IL-33 release. This is supported by the in vitro finding that the COX product PGE2 and the PGI2 analogs cicaprost decreased Alternaria extract-induced IL-33 release by human bronchial epithelial cells. Although contrasting effects of PGD2, PGE2, and PGI2 on ILC2 responses have been previously reported, the overall effect of the COX pathway on ILC2 function is inhibitory in Alternaria-induced innate airway inflammation.

BCL6 modulates tissue neutrophil survival and exacerbates pulmonary inflammation following influenza virus infection.

Neutrophils are vital for antimicrobial defense; however, their role during viral infection is less clear. Furthermore, the molecular regulation of neutrophil fate and function at the viral infected sites is largely elusive. Here we report that BCL6 deficiency in myeloid cells exhibited drastically enhanced host resistance to severe influenza A virus (IAV) infection. In contrast to the notion that BCL6 functions to suppress innate inflammation, we find that myeloid BCL6 deficiency diminished lung inflammation without affecting viral loads. Using a series of Cre-transgenic, reporter, and knockout mouse lines, we demonstrate that BCL6 deficiency in neutrophils, but not in monocytes or lung macrophages, attenuated host inflammation and morbidity following IAV infection. Mechanistically, BCL6 bound to the neutrophil gene loci involved in cellular apoptosis in cells specifically at the site of infection. As such, BCL6 disruption resulted in increased expression of apoptotic genes in neutrophils in the respiratory tract, but not in the circulation or bone marrow. Consequently, BCL6 deficiency promoted tissue neutrophil apoptosis. Partial neutrophil depletion led to diminished pulmonary inflammation and decreased host morbidity. Our results reveal a previously unappreciated role of BCL6 in modulating neutrophil apoptosis at the site of infection for the regulation of host disease development following viral infection. Furthermore, our studies indicate that tissue-specific regulation of neutrophil survival modulates host inflammation and tissue immunopathology during acute respiratory viral infection.

Roles of innate lymphoid cells (ILCs) in allergic diseases: The 10-year anniversary for ILC2s.

In the 12 years since the discovery of innate lymphoid cells (ILCs), our knowledge of their immunobiology has expanded rapidly. Group 2 ILCs (ILC2s) respond rapidly to allergen exposure and environmental insults in mucosal organs, producing type 2 cytokines. Early studies showed that epithelium-derived cytokines activate ILC2s, resulting in eosinophilia, mucus hypersecretion, and remodeling of mucosal tissues. We now know that ILC2s are regulated by other cytokines, eicosanoids, and neuropeptides as well, and interact with both immune and stromal cells. Furthermore, ILC2s exhibit plasticity by adjusting their functions depending on their tissue environment and may consist of several heterogeneous subpopulations. Clinical studies show that ILC2s are involved in asthma, allergic rhinitis, chronic rhinosinusitis, food allergy, and eosinophilic esophagitis. However, much remains unknown about the immunologic mechanisms involved. Beneficial functions of ILCs in maintenance or restoration of tissue well-being and human health also need to be clarified. As our understanding of the crucial functions ILCs play in both homeostasis and disease pathology expands, we are poised to make tremendous strides in diagnostic and therapeutic options for patients with allergic diseases. This review summarizes discoveries in immunobiology of ILCs and their roles in allergic diseases in the past 5 years, discusses controversies and gaps in our knowledge, and suggests future research directions.

Gastrointestinal Eosinophil Responses in a Longitudinal, Randomized Trial of Peanut Oral Immunotherapy.

BACKGROUND & AIMS: Gastrointestinal side effects are common during oral immunotherapy (OIT) and eosinophilic esophagitis (EoE) is a potential complication. We aimed to characterize eosinophilic gastrointestinal responses to peanut OIT, in which peanut protein is given orally, with incremental increases in dose over time. METHODS: Twenty adults with IgE-mediated peanut allergy were randomly assigned to groups given peanut OIT (n = 15) or placebo (n = 5); 1 additional subject withdrew before randomization. Serial gastrointestinal biopsies were collected at baseline (n = 21, 0 weeks), following dose escalation (n = 10, 52 weeks), and during the maintenance phase (n = 11, 104 weeks). Endoscopic findings were characterized using the EoE endoscopic reference score. Biopsies were assessed for eosinophils per high-power field (eos/hpf) and other pathology features using EoE histologic scoring system scores. We performed immunohistochemical analyses of eosinophil peroxidase deposition, quantified using automated image analysis. RESULTS: At baseline, no subjects reported current gastrointestinal symptoms. However, 3 of the 21 subjects (14%) had esophageal peak eosinophil counts ≥15 eos/hpf and all subjects had dilated intercellular spaces (DIS). OIT induced or exacerbated esophageal eosinophilia (EE) at 52 weeks in most subjects (peak eosinophil counts >5 eos/hpf in 6 of 7 patients [86%]; peak eosinophil counts ≥15 eos/hpf in 4 of 7 patients [57%]). One subject met clinicopathologic criteria for EoE and withdrew; no significant changes in esophageal peak eosinophil counts were observed in the placebo group. EE in the OIT group corresponded with significant increases in EoE histologic scoring system scores and deposition of eosinophil peroxidase. In 4 of 6 participants (67%), OIT-induced EE and gastrointestinal eosinophilia resolved by the end of the maintenance phase. Gastrointestinal symptoms were not clearly associated with EE or gastrointestinal eosinophilia. CONCLUSIONS: In this pilot study, we found that peanut OIT-induced EE and gastrointestinal eosinophilia are usually transient and are not always associated with gastrointestinal symptoms. Clinicaltrials.gov no: NCT02103270.

Estrogen receptor-α signaling increases allergen-induced IL-33 release and airway inflammation.

BACKGROUND: Group 2 innate lymphoid cells (ILC2) are stimulated by IL-33 to increase IL-5 and IL-13 production and airway inflammation. While sex hormones regulate airway inflammation, it remained unclear whether estrogen signaling through estrogen receptor-α (ER-α, Esr1) or ER-ß (Esr2) increased ILC2-mediated airway inflammation. We hypothesize that estrogen signaling increases allergen-induced IL-33 release, ILC2 cytokine production, and airway inflammation. METHODS: Female Esr1-/- , Esr2-/- , wild-type (WT), and IL33fl/fl eGFP mice were challenged with Alternaria extract (Alt Ext) or vehicle for 4 days. In select experiments, mice were administered tamoxifen or vehicle pellets for 21 days prior to challenge. Lung ILC2, IL-5 and IL-13 production, and BAL inflammatory cells were measured on day 5 of Alt Ext challenge model. Bone marrow from WT and Esr1-/- female mice was transferred (1:1 ratio) into WT female recipients for 6 weeks followed by Alt Ext challenge. hBE33 cells and normal human bronchial epithelial cells (NHBE) were pretreated with 17ß-estradiol (E2), propyl-pyrazole-triol (PPT, ER-α agonist), or diarylpropionitrile (DPN, ER-ß agonist) before allergen challenge to determine IL-33 gene expression and release, extracellular ATP release, DUOX-1 production, and necrosis. RESULTS: Alt Ext challenged Esr1-/- , but not Esr2-/- , mice had decreased IL-5 and IL-13 production, BAL eosinophils, and IL-33 release compared to WT mice. Tamoxifen decreased IL-5 and IL-13 production and BAL eosinophils. IL-33eGFP + epithelial cells were decreased in Alt Ext challenged Esr1-/- mice compared to WT mice. 17ß-E2 or PPT, but not DPN, increased IL-33 gene expression, release, and DUOX-1 production in hBE33 or NHBE cells. CONCLUSION: Estrogen receptor -α signaling increased IL-33 release and ILC2-mediated airway inflammation.