Priya Rajavelu

Significance of para-esophageal lymph nodes in food or aeroallergen-induced iNKT cell-mediated experimental eosinophilic esophagitis

Eosinophilic esophagitis (EoE) is a recently recognized inflammatory disorder driven by food hypersensitivity; however, the specific foods and mechanisms involved are unclear. In patients with EoE, we have found that hypersensitivities to corn and peanuts are the most common. Accordingly, we sensitized and exposed mice either intranasally or intragastrically with corn or peanut extract or saline. Esophageal eosinophilia, the genes of eosinophil-directed cytokines, and allergen-induced antibodies were examined in mice challenged with corn or peanut extract or saline. A high number of esophageal lamina propria eosinophils as well as eosinophilic microabscesses, intraepithelial eosinophils, extracellular eosinophilic granules, thickened and disrupted epithelial mucosa, and mast cell hyperplasia were observed in the esophagus of peanut or corn allergen-challenged mice. Mechanistic analysis indicated that para-esophageal lymph nodes might be critical in the trafficking of eosinophils to the esophagus and in EoE association to airway eosinophilia. Furthermore, experimentation with gene-targeted mice revealed that peanut allergen-induced EoE was dependent on eotaxin and invariant natural killer T (iNKT) cells, as CD1d and eotaxin-1/2 gene-deficient mice were protected from disease induction. Thus we provide evidence that para-esophageal lymph nodes are involved in food- or aeroallergen-induced eosinophilia and patchy EoE pathogenesis, likely a mechanism dependent on eotaxins and iNKT cells.

Esophageal functional impairments in experimental eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is an emerging chronic esophageal disease. Despite the increasing diagnosis of EoE globally, the causes of EoE and other esophageal eosinophilic disorders are not clearly understood. EoE pathology includes accumulation of inflammatory cells (e.g., eosinophils, mast cells), characteristic endoscopic features (e.g., furrows, the formation of fine concentric mucosal rings, exudates), and functional impairments (e.g., esophageal stricture, dysmotility). We hypothesized that the esophageal structural pathology and functional impairments of EoE develop as a consequence of the effector functions of the accumulated inflammatory cells. We analyzed eosinophils (anti-major basic protein immunostaining), esophageal stricture (X-ray barium swallowing), and esophageal motility (isometric force) in two established transgenic murine models of EoE (CD2-IL-5 and rtTA-CC10-IL-13) and a novel eosinophil-deficient model (ΔdblGATA/CD2-IL-5). Herein, we show the following: 1) CD2-IL-5 and doxycycline (DOX)-induced rtTA-CC10-IL-13 mice have chronic eosinophilic and mast cell esophageal inflammation; 2) eosinophilic esophageal inflammation promotes esophageal stricture in both transgenic murine models; 3) the eosinophil-deficient ΔdblGATA/CD-2-IL-5 mice were protected from the induction of stricture, whereas the eosinophil-competent CD2-IL-5 mice develop esophageal stricture; 4) esophageal stricture is not reversible in DOX-induced rtTA-CC10-IL-13 mice (8 wk DOX followed by 8 wk no-DOX); and 5) IL-5 transgene-induced (CD2-IL-5) EoE evidences esophageal dysmotility (relaxation and contraction) that is independent of the eosinophilic esophageal inflammation: CD2-IL-5 and ΔdblGATA/CD2-IL-5 mice have comparable esophageal dysmotility. Collectively, our present study directly implicates chronic eosinophilic inflammation in the development of the esophageal structural impairments of experimental EoE.

Airway epithelial SPDEF integrates goblet cell differentiation and pulmonary Th2 inflammation

Epithelial cells that line the conducting airways provide the initial barrier and innate immune responses to the abundant particles, microbes, and allergens that are inhaled throughout life. The transcription factors SPDEF and FOXA3 are both selectively expressed in epithelial cells lining the conducting airways, where they regulate goblet cell differentiation and mucus production. Moreover, these transcription factors are upregulated in chronic lung disorders, including asthma. Here, we show that expression of SPDEF or FOXA3 in airway epithelial cells in neonatal mice caused goblet cell differentiation, spontaneous eosinophilic inflammation, and airway hyperresponsiveness to methacholine. SPDEF expression promoted DC recruitment and activation in association with induction of Il33, Csf2, thymic stromal lymphopoietin (Tslp), and Ccl20 transcripts. Increased Il4, Il13, Ccl17, and Il25 expression was accompanied by recruitment of Th2 lymphocytes, group 2 innate lymphoid cells, and eosinophils to the lung. SPDEF was required for goblet cell differentiation and pulmonary Th2 inflammation in response to house dust mite (HDM) extract, as both were decreased in neonatal and adult Spdef(-/-) mice compared with control animals. Together, our results indicate that SPDEF causes goblet cell differentiation and Th2 inflammation during postnatal development and is required for goblet cell metaplasia and normal Th2 inflammatory responses to HDM aeroallergen.

Invariant natural killer T-cell neutralization is a possible novel therapy for human eosinophilic esophagitis

Eosinophilic esophagitis (EoE) is a recently recognized inflammatory disorder that needs a potential therapeutic strategy. We earlier showed that iNKT cell‐deficient mice are protected from allergen‐induced EoE. Therefore, we now tested the hypothesis that iNKT cells are induced in the human EoE and is a novel possible target for the treatment of human EoE. Accordingly, we examine number of iNKT cells and eosinophils and expression of iNKT‐associated cell surface receptors and chemokines by performing immunofluorescence, qPCR and ELISA in the esophageal biopsies and blood samples of normal subjects (comparison control) and EoE patients. Herein, we show that iNKT cell number, their receptor subcomponents Vα24 and Vβ11 expression, and associated chemokine CXCL16 levels (or expression) are induced significantly in EoE patients compared with normal individuals. In addition, we show that CXCL16 levels (or expression) correlate with the mRNA levels of Vα24 receptor but not well with esophageal eosinophilia in human EoE. Of note, we show that in vivo activation of iNKT cells is sufficient to induce EoE in mice. Furthermore, we show that anti‐mCD1d‐ and anti‐hVα24Jα18‐neutralizing antibody treatment protects allergen‐induced experimental EoE. Taken together, we have shown first time that iNKT cells have a critical pathogenic role in human and experimental EoE. iNKT cell neutralization by humanized anti‐CD1d and anti‐Vα24Jα18 antibodies might be a novel and potential therapy for human EoE.

Involvement of interleukin-18 in the pathogenesis of human eosinophilic esophagitis

IL-18 is induced in food allergy and EoE is food allergen-induced disease. Therefore, we tested the hypothesis whether IL-18 is involved in food allergen-induced EoE pathogenesis. Accordingly, we examined normal SPT+ and SPT- EoE patient blood and biopsy samples for IL-18, IL-18Rα, ICAM and VCAM expression. Herein, we show increased IL-18 level is highly significant in food allergen SPT+ compared to SPT- EoE patients. We also report that IL-18Rα+ cells and mRNA levels are induced in the esophageal biopsies of EoE patients and blood IL-18 levels correlate with esophageal eosinophilia (P<0.01). Additionally, we report that the levels of esophageal eosinophil and mast cells correlate with ICAM expression in human EoE. Mechanistically, we show that IL-18 in vitro stimulates iNKT cells and endothelial cells and induce eosinophil active cytokines IL-5 and IL-13. We provide the evidence that IL-18 is critical cytokine involved in activation of iNKT cells and ICAM in promoting human EoE.

Regulatory effects of IL-15 on allergen-induced airway obstruction

Background: Airway obstruction is a physiologic feature of asthma, and IL‐15 might have an important role in asthma pathogenesis. Objective: We tested the hypothesis that regulation of IL‐15 is critical for preservation of allergen‐induced airway hyperresponsiveness (AHR), airway resistance, and compliance in response to methacholine. Methods: Airway inflammation, AHR, resistance, and compliance were assessed in Il15 gene–deficient mice and IL‐15–overexpressing mice in an allergen‐induced murine model of asthma. We assessed eosinophil numbers by using anti–major basic protein immunostaining, goblet cell hyperplasia by using periodic acid–Schiff staining, and cytokine and chemokine levels by performing quantitative PCR and ELISA. Results: We made a novel observation that IL‐15 deficiency promotes baseline airway resistance in naive mice. Moreover, rIL‐15 delivery to the lung downregulates expression of proinflammatory cytokines and improves allergen‐induced AHR, airway resistance, and compliance. These observations were further validated in doxycycline‐inducible CC10–IL‐15 bitransgenic mice. Doxycycline‐exposed, Aspergillus species extract–challenged CC10–IL‐15 bitransgenic mice exhibited significantly reduced levels of proinflammatory cytokines (IL‐4, IL‐5, and IL‐13) and decreased goblet cell hyperplasia. Airway obstruction, including AHR and airway resistance, was diminished in allergen‐challenged doxycycline‐exposed compared with non–doxycycline‐exposed CC10–IL‐15 bitransgenic mice. Mechanistically, we observed that IL‐15–mediated protection of airway obstruction is associated with induced IFN‐&ggr;– and IL‐10–producing regulatory CD4+CD25+ forkhead box p3 (Foxp3)+ T cells. Additionally, we found that a human IL‐15 agonist (ALT‐803) improved airway resistance and compliance in an experimental asthma model. Conclusion: We report our novel finding that IL‐15 has a potent inhibitory effect on the airway obstruction that occurs in response to environmental allergens.

Airway Epithelial KIF3A Regulates Th2 Responses to Aeroallergens

KIF3A, the gene encoding kinesin family member 3A, is a susceptibility gene locus associated with asthma; however, mechanisms by which KIF3A might influence the pathogenesis of the disorder are unknown. In this study, we deleted the mouse Kif3a gene in airway epithelial cells. Both homozygous and heterozygous Kif3a gene–deleted mice were highly susceptible to aeroallergens from Aspergillus fumigatus and the house dust mite, resulting in an asthma-like pathology characterized by increased goblet cell metaplasia, airway hyperresponsiveness, and Th2-mediated inflammation. Deletion of the Kif3a gene increased the severity of pulmonary eosinophilic inflammation and expression of cytokines (Il-4, Il-13, and Il-17a) and chemokine (Ccl11) RNAs following pulmonary exposure to Aspergillus extract. Inhibition of Kif3a disrupted the structure of motile cilia and impaired mucociliary clearance, barrier function, and epithelial repair, demonstrating additional mechanisms by which deficiency of KIF3A in respiratory epithelial cells contributes to pulmonary pathology. Airway epithelial KIF3A suppresses Th2 pulmonary inflammation and airway hyperresponsiveness following aeroallergen exposure, implicating epithelial microtubular functions in the pathogenesis of Th2-mediated lung pathology.

A critical role for IL-18 in transformation and maturation of naive eosinophils to pathogenic eosinophils

The current studies demonstrate a critical role of IL-18 in transforming IL-5 generated naïve eosinophils into the distinct inflammatory CD101+CD274+ expressing mature and activated tissue eosinophils that promote disease pathogenesis.