Analysis of the CCR3 promoter reveals a regulatory region in exon 1 that binds GATA-1

BACKGROUND: CC Chemokine Receptor 3 (CCR3), the major chemokine receptor expressed on eosinophils, binds promiscuously to several ligands including eotaxins 1, 2, and 3. Even though the only cells that consistently accumulate following eotaxin administration in vivo are myeloid cells (primarily eosinophils), other cell types have recently been shown to express CCR3. It is therefore important to elucidate the molecular mechanisms regulating receptor expression. RESULTS: In order to define regions responsible for CCR3 transcription, a DNAse hypersensitive site was identified in the vicinity of exon 1. Coupled with our previous data implicating exon 1 in CCR3 transcription, we hypothesized that transcription factors bind to exon-1. Electrophoretic mobility shift analysis revealed that nuclear proteins in eosinophilic cells bound to exon 1. Furthermore, antibody interference and mutation studies demonstrated GATA-1 binding to exon 1. In order to test the 1.6-kb CCR3 promoter element (that includes exon 1) for in vivo function, this region was used to generate transgenic mice that expressed a reporter protein. Strong transgene expression was achieved, with the pattern of expression suggesting a broad acting promoter. CONCLUSION: The transcription factor GATA-1 binds to CCR3 exon 1. The 1.6-kb CCR3 promoter element, that includes exon 1, is a strong promoter in vivo

Development and Application of a Functional Human Esophageal Mucosa Explant Platform to Eosinophilic Esophagitis.

There is an increasing prevalence of esophageal diseases but intact human tissue platforms to study esophageal function, disease mechanisms, and the interactions between cell types in situ are lacking. To address this, we utilized full thickness human donor esophagi to create and validate the ex vivo function of mucosa and smooth muscle (n = 25). Explanted tissue was tested for contractile responses to carbachol and histamine. We then treated ex vivo human esophageal mucosa with a cytokine cocktail to closely mimic the Th2 and inflammatory milieu of eosinophilic esophagitis (EoE) and assessed alterations in smooth muscle and extracellular matrix function and stiffening. We found that full thickness human esophagus as well as the individual layers of circular and longitudinal muscularis propria developed tension in response to carbachol ex vivo and that mucosa demonstrated squamous cell differentiation. Treatment of mucosa with Th2 and fibrotic cytokines recapitulated the majority of the clinical Eosinophilic Esophagitis Diagnostic Profile (EDP) on fluidic transcriptional microarray. Transforming growth factor-beta-1 (TGFβ1) increased gene expression of fibronectin, smooth muscle actin, and phospholamban (p < 0.001). The EoE cocktail also increased stiffness and decreased mucosal compliance, akin to the functional alterations in EoE (p = 0.001). This work establishes a new, transcriptionally intact and physiologically functional human platform to model esophageal tissue responses in EoE

Regulation of Carcinogenesis by IL-5 and CCL11: A Potential Role for Eosinophils in Tumor Immune Surveillance

The role of the immune system in the surveillance of transformed cells has seen a resurgence of interest in the last 10 years, with a substantial body of data in mice and humans supporting a role for the immune system in host protection from tumor development and in shaping tumor immunogenicity. A number of earlier studies have demonstrated that eosinophils, when recruited into tumors, can very effectively eradicate transplantable tumors. In this study, we investigated whether eosinophils also play a role in tumor immune surveillance by determining the incidence of methylcholanthrene (MCA)-induced flbrosarcomas in IL-5 transgenic mice that have greatly enhanced levels of circulating eosinophils, CCL11 (eotaxin-l)-deficient mice that lack a key chemokine that recruits eosinophils into tissues, and the eosinophil-deficient mouse strains, IL-5/CCL11-/- and ΔdblGATA. It was found that MCA-induced tumor incidence and growth were significantly attenuated in IL-5 transgenic mice of both the BALB/c and C57BL/6 backgrounds. Histological examination revealed that the protective effect of IL-5 was associated with massively enhanced numbers of eosinophils within and surrounding tumors. Conversely, there was a higher tumor incidence in CCL11-/- BALB/c mice, which was associated with a reduced eosinophil influx into tumors. This correlation was confirmed in the eosinephil-deficient IL-5/CCL11-/- and ΔdblGATA mouse strains, where tumor incidence was greatly increased in the total absence of eosinophils. In addition, subsequent in vitro studies found that eosinophils could directly kill MCA-induced fibrosarcoma cells. Collectively, our data support a potential role for the eosinophil as an effector cell in tumor immune surveillance

Bone marrow cell derived arginase I is the major source of allergen-induced lung arginase but is not required for airway hyperresponsiveness, remodeling and lung inflammatory responses in mice

<p>Abstract</p> <p>Background</p> <p>Arginase is significantly upregulated in the lungs in murine models of asthma, as well as in human asthma, but its role in allergic airway inflammation has not been fully elucidated in mice.</p> <p>Results</p> <p>In order to test the hypothesis that arginase has a role in allergic airway inflammation we generated arginase I-deficient bone marrow (BM) chimeric mice. Following transfer of arginase I-deficient BM into irradiated recipient mice, arginase I expression was not required for hematopoietic reconstitution and baseline immunity. Arginase I deficiency in bone marrow-derived cells decreased allergen-induced lung arginase by 85.8 ± 5.6%. In contrast, arginase II-deficient mice had increased lung arginase activity following allergen challenge to a similar level to wild type mice. BM-derived arginase I was not required for allergen-elicited sensitization, recruitment of inflammatory cells in the lung, and proliferation of cells. Furthermore, allergen-induced airway hyperresponsiveness and collagen deposition were similar in arginase-deficient and wild type mice. Additionally, arginase II-deficient mice respond similarly to their control wild type mice with allergen-induced inflammation, airway hyperresponsiveness, proliferation and collagen deposition.</p> <p>Conclusion</p> <p>Bone marrow cell derived arginase I is the predominant source of allergen-induced lung arginase but is not required for allergen-induced inflammation, airway hyperresponsiveness or collagen deposition.</p

Leveraging Multilayered “Omics” Data for Atopic Dermatitis: A Road Map to Precision Medicine

Atopic dermatitis (AD) is a complex multifactorial inflammatory skin disease that affects ~280 million people worldwide. About 85% of AD cases begin in childhood, a significant portion of which can persist into adulthood. Moreover, a typical progression of children with AD to food allergy, asthma or allergic rhinitis has been reported (“allergic march” or “atopic march”). AD comprises highly heterogeneous sub-phenotypes/endotypes resulting from complex interplay between intrinsic and extrinsic factors, such as environmental stimuli, and genetic factors regulating cutaneous functions (impaired barrier function, epidermal lipid, and protease abnormalities), immune functions and the microbiome. Though the roles of high-throughput “omics” integrations in defining endotypes are recognized, current analyses are primarily based on individual omics data and using binary clinical outcomes. Although individual omics analysis, such as genome-wide association studies (GWAS), can effectively map variants correlated with AD, the majority of the heritability and the functional relevance of discovered variants are not explained or known by the identified variants. The limited success of singular approaches underscores the need for holistic and integrated approaches to investigate complex phenotypes using trans-omics data integration strategies. Integrating omics layers (e.g., genome, epigenome, transcriptome, proteome, metabolome, lipidome, exposome, microbiome), which often have complementary and synergistic effects, might provide the opportunity to capture the flow of information underlying AD disease manifestation. Overlapping genes/candidates derived from multiple omics types include FLG, SPINK5, S100A8, and SERPINB3 in AD pathogenesis. Overlapping pathways include macrophage, endothelial cell and fibroblast activation pathways, in addition to well-known Th1/Th2 and NFkB activation pathways. Interestingly, there was more multi-omics overlap at the pathway level than gene level. Further analysis of multi-omics overlap at the tissue level showed that among 30 tissue types from the GTEx database, skin and esophagus were significantly enriched, indicating the biological interconnection between AD and food allergy. The present work explores multi-omics integration and provides new biological insights to better define the biological basis of AD etiology and confirm previously reported AD genes/pathways. In this context, we also discuss opportunities and challenges introduced by “big omics data” and their integration

Newly Developed and Validated Eosinophilic Esophagitis Histology Scoring System and Evidence that it Outperforms Peak Eosinophil Count for Disease Diagnosis and Monitoring

Eosinophilic esophagitis is diagnosed by symptoms, and at least 15 intraepithelial eosinophils per high power field in an esophageal biopsy. Other pathologic features have not been emphasized. We developed a histology scoring system for esophageal biopsies that evaluates eight features: eosinophil density, basal zone hyperplasia, eosinophil abscesses, eosinophil surface layering, dilated intercellular spaces, surface epithelial alteration, dyskeratotic epithelial cells and lamina propria fibrosis. Severity (grade) and extent (stage) of abnormalities were scored using a 4 point scale (0 normal; 3 maximum change). Reliability was demonstrated by strong to moderate agreement among 3 pathologists who scored biopsies independently (p≤0.008). Several features were often abnormal in 201 biopsies (101 distal, 100 proximal) from 104 subjects (34 untreated, 167 treated). Median grade and stage scores were significantly higher in untreated compared to treated subjects (p≤0.0062). Grade scores for features independent of eosinophil counts were significantly higher in biopsies from untreated compared to treated subjects (basal zone hyperplasia p≤0.024 and dilated intercellular spaces p≤0.005), and were strongly correlated (r-square\u3e0.67). Principal components analysis identified 3 principal components that explained 78.2% of the variation in the features. In logistic regression models, 2 principal components more closely associated with treatment status than log distal peak eosinophil count (r-square 17, area under the curve 77.8 vs r-square 9, area under the curve 69.8). In summary, the eosinophilic esophagitis histology scoring system provides a method to objectively assess histologic changes in the esophagus beyond eosinophil number. Importantly, it discriminates treated from untreated patients, uses features commonly found in such biopsies, and is utilizable by pathologists after minimal training. These data provide rationales and a method to evaluate esophageal biopsies for features in addition to peak eosinophil count