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.

Eosinophil Peroxidase: A Biomarker for Eosinophilic Chronic Rhinosinusitis Agnostic of Polyp Status.

OBJECTIVE: To evaluate eosinophil peroxidase (EPX) as a biomarker for tissue levels of eosinophilia, cytokines, and chemokines within chronic rhinosinusitis (CRS). METHODS: Twenty-eight subjects undergoing sinonasal surgery were prospectively enrolled. Ethmoid tissue was analyzed with an in-house EPX immunoassay and a 48-plex cytokine-chemokine array. Clinical severity was assessed using SNOT-22 and Lund-Mackay scores. Subjects were grouped as follows: controls, polyp status (CRS with [CRSwNP] and without nasal polyps [CRSsNP]), tissue eosinophilia (eosinophilic CRS [eCRS], non-eosinophilic CRS [neCRS]), or combinations thereof (eCRSwNP, eCRSsNP, neCRSsNP). eCRS was defined as >10 eosinophils per high power field (HPF). Subjects without CRS or asthma were enrolled as controls. RESULTS: EPX was elevated in CRSwNP compared to control (p = 0.007), in eCRS compared to neCRS (p = 0.002), and in eCRSwNP along with eCRSsNP compared to neCRSsNP (p = 0.023, p = 0.015, respectively). eCRS displayed elevated IL-5 compared to neCRS (p = 0.005). No significant differences in EPX or IL-5 were observed between eCRSwNP and eCRSsNP. IL-5 was elevated in eCRSwNP (p = 0.019) compared neCRSsNP. Area under the receiver operator characteristic curve was 0.938 (95% CI, 0.835-1.00) for EPX and tissue eosinophilia, with an optimal cut-point of 470 ng/mL being 100% specific and 81.25% sensitive for tissue eosinophilia. Linear regression revealed a strong correlation between EPX and IL-5 (R2 = 0.64, p < 0.001). Comparing EPX and IL-5, only EPX displayed significant correlation with SNOT-22 (p = 0.04) and Lund-Mackay score (p = 0.004). CONCLUSION: EPX is associated with tissue eosinophilia in CRS patients regardless of polyp status. EPX correlates with IL-5 and could be potentially considered a biomarker for anti-IL-5 therapies. LEVEL OF EVIDENCE: 3 Laryngoscope, 134:69-78, 2024.

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.

Viruses in chronic rhinosinusitis: a systematic review.

Background: Unlike acute rhinosinusitis (ARS) which is mostly viral in etiology, the role of viruses in chronic rhinosinusitis (CRS) remains unclear. Viruses may play a role in initiation, exacerbations or perpetuate chronic inflammatory responses in the sinonasal mucosa. Research needs to characterize whether viruses are part of the normal sinonasal microbiome, colonizers or pathogenic. Methods: Systematic review of the English literature was conducted. Following databases were searched with an initial search conducted in November 2021 and then updated through June 2023: Ovid Medline (1946 to present), Ovid Embase (1988 to present), Scopus (2004 to present) and Web of Science (1975 to present). MeSH (Medical Subject Headings) terms included: viruses, virus diseases, sinusitis, and rhinovirus. Keywords: virus, viral infection*, sinusitis, rhinovirus, chronic rhinosinusitis, CRS, respiratory virus, respiratory infection*, and exacerbat*. A supplementary search was conducted through September 2023: Ovid Medline (1946 to present), Epub Ahead of Print, In-Process & Other Non-Indexed Citations and Ovid MEDLINE(R) Daily. Keywords used were: virus, viral infection*, sinusitis, chronic rhinosinusitis, CRS, respiratory virus, respiratory infection*, and exacerbat*. Results: Thirty studies on viruses in CRS met inclusion criteria for full review. These included 17 studies on prevalence of virus in CRS, 5 examining probable causes of host susceptibility to viral infections in CRS, and 8 studies examining pathological pathways in viral association of CRS. The prevalence of viruses in nasal specimens of CRS subjects was higher as compared to controls in most studies, though a few studies showed otherwise. Rhinovirus was the most common virus detected. Studies showed that viruses may be associated with persistent hyper-responsiveness in the sinonasal mucosa, susceptibility to bacterial infections, upregulation of genes involved in the immune response and airway remodeling as well as CRS exacerbations. Presence of viruses was also associated with worse symptom severity scores in CRS subjects. Conclusion: Most data show higher presence of viruses in nasal and serum samples of CRS subjects as compared to controls but their exact role in CRS pathophysiology in unclear. Large studies with longitudinal sampling at all disease phases (i.e., prior to disease initiation, during disease initiation, during disease persistence, and during exacerbations) using standardized sampling techniques are needed to definitively elucidate the role of virus in CRS.

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.

Genome-wide Epigenetic Study of Chronic Rhinosinusitis Tissues Reveals Dysregulated Inflammatory, Immunologic and Remodeling Pathways.

BACKGROUND: Epigenetics studies mechanisms such as DNA methylation, histone modifications, non-coding RNAs, and alternative polyadenylation that can modify gene activity without changing the underlying DNA nucleotide base-pair structure. Because these changes are reversible, they have potential in developing novel therapeutics. Currently, seven pharmaceutical agents targeting epigenetic changes are FDA approved and commercially available for treatment of certain cancers. However, studies investigating epigenetics in chronic rhinosinusitis (CRS) have not been undertaken previously in the United States. OBJECTIVES: The goal of this study was to investigate sinonasal DNA methylation patterns in CRS versus controls, to discern environmentally-induced epigenetic changes impacting CRS subjects. METHODS AND RESULTS: Ethmoidal samples from CRS and inferior turbinate mucosal tissue samples from controls without CRS were studied. DNA methylation was studied by Reduced Representation Bisulfite Sequencing. RADMeth® biostatistical package was used to identify differentially methylated regions (DMRs) between CRS and controls. Ingenuity Pathway analysis of DMRs was performed to identify top upstream regulators and canonical pathways. Ninety-three samples from 64 CRS subjects (36 CRSwNP; 28 CRSsNP) and 29 controls were studied. CRS and control samples differed in 13 662 CpGs sites and 1381 DMRs. Top upstream regulators identified included: 1. CRS versus controls: TGFB1, TNF, TP53, DGCR8, and beta-estradiol. 2. CRSwNP versus controls: TGFB1, CTNNB1, lipopolysaccharide, ID2, and TCF7L2. 3. CRSsNP versus controls: MYOD1, acetone, ID2, ST8SIA4, and LEPR. CONCLUSIONS: Differential patterns of methylation were identified between controls and CRS, CRSwNP, and CRSsNP. Epigenetic, environmentally-induced changes related to novel, inflammatory, immunologic, and remodeling pathways appear to affect epithelial integrity, cell proliferation, homeostasis, vascular permeability, and other yet uncharacterized pathways and genes.

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.

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.

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.

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.

Rethinking blood eosinophil counts: Epidemiology, associated chronic diseases, and increased risks of cardiovascular disease.

Background: The distribution and determinants of blood eosinophil counts in the general population are unclear. Furthermore, whether elevated blood eosinophil counts increase risk for cardiovascular disease (CVD) and other chronic diseases, other than atopic conditions, remains uncertain. Objective: We sought to describe the distribution of eosinophil counts in the general population and determine the association of eosinophil count with prevalent chronic disease and incident CVD. Methods: A population-based adult cohort was followed from January 1, 2006, to December 31, 2020. Electronic health record data regarding demographic characteristics, prevalent clinical characteristics, and incident CVD were extracted. Associations between blood eosinophil counts and demographic characteristics, chronic diseases, laboratory values, and risks of incident CVD were assessed using chi-square test, ANOVA, and Cox proportional hazards regression. Results: Blood eosinophil counts increased with age, body mass index, and reported smoking and tobacco use. The prevalence of chronic obstructive pulmonary disease, hypertension, cardiac arrhythmias, hyperlipidemia, diabetes mellitus, chronic kidney disease, and cancer increased as eosinophil counts increased. Eosinophil counts were significantly associated with coronary heart disease (hazard ratio [HR], 1.44; 95% CI, 1.12-1.84) and heart failure (HR, 1.62; 95% CI, 1.30-2.01) in fully adjusted models and with stroke/transient ischemic attack (HR, 1.37; 95% CI, 1.16-1.61) and CVD death (HR, 1.49; 95% CI, 1.10-2.00) in a model adjusting for age, sex, race, and ethnicity. Conclusions: Blood eosinophil counts differ by demographic and clinical characteristics as well as by prevalent chronic disease. Moreover, elevated eosinophil counts are associated with risk of CVD. Further prospective investigations are needed to determine the utility of eosinophil counts as a biomarker for CVD risk.

White blood cells and chronic rhinosinusitis: a Mendelian randomization study.

BACKGROUND: Risk factors for the pathogenesis of chronic rhinosinusitis (CRS) remain largely undetermined, which is likely due to the heterogeneity of the disease. White blood cell counts have been largely unexplored as a risk factor for CRS even though different types of white blood cells are involved in the inflammatory process of CRS. OBJECTIVE: To investigate causal associations between different types of white blood cells on risk of CRS utilizing a Mendelian randomization (MR) analysis. METHODS: A two-sample MR analysis was performed using respective GWAS summary statistics for the exposure traits (neutrophil count, eosinophil count, basophil count, lymphocyte count, and monocyte count) and outcome trait (CRS). For the exposure traits, the European Bioinformatics Institute database of complete GWAS summary data was used. For the outcome trait, summary statistics for CRS GWAS were obtained from FinnGen. Primary analysis for MR was performed using inverse-variance weighted two-sample MR. Sensitivity analyses included weighted median, MR-Egger, and MR-PRESSO (raw and outlier-corrected). RESULTS: Eosinophils were associated with CRS (OR = 1.55 [95% CI 1.38, 1.73]; p = 4.3E-14). Eosinophil results were similar across additional MR methods. MR results did not demonstrate significant causal relationships between neutrophils, lymphocytes, monocytes, or basophils with CRS. No significant pleiotropic bias was observed. CONCLUSIONS: In a two-sample MR analysis, a potential causal link between blood eosinophil counts and CRS has been demonstrated. In addition, causal relationships between blood counts among other white blood cell types and CRS were not found. Further studies involving genetic variation in CRS are needed to corroborate genetic causal effects for CRS.

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.

Eosinophil Peroxidase Staining Enhances the Diagnostic Utility of the Cytosponge in Eosinophilic Esophagitis.

INTRODUCTION: We aimed to assess the diagnostic utility of eosinophil peroxidase (EPX) staining on Cytosponge (CS) samples in eosinophilic esophagitis (EoE). METHODS: Esophageal biopsy (BX) samples from adult subjects with EoE were assessed using peak eosinophils per high-power field (eos/hpf), EPX, and the EoE histologic scoring system. EPX staining and eos/hpf were compared (BX vs CS). RESULTS: CS EPX positivity correlated with eos/hpf (CS [ r = 0.82, P < 0.0001]; BX [ r = 0.65, P < 0.0001]) and EoE histologic scoring system (grade [ r = 0.62, P < 0.00001]; stage [ r = 0.61, P < 0.0001]). CS EPX identified subjects with active EoE (area under the curve = 0.86, P < 0.0001). DISCUSSION: The correlation of CS EPX with eosinophilic inflammation and histologic disease severity supports its diagnostic utility in EoE.

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.