Project description:We detected BALF exosome-derived proteins from adult Scnn1b transgenic (Scnn1b-Tg+) and wild type (WT) mice. A total of 3144 and 3119 proteins were identified in BALF exosomes from Scnn1b-Tg+ and WT mice, respectively.
Project description:The opportunistic pathogen Pseudomonas aeruginosa is responsible for much of the morbidity and mortality associated with cystic fibrosis (CF), a condition that predisposes patients to chronic lung infections. P. aeruginosa lung infections are difficult to treat because P. aeruginosa adapts to the CF lung, can develop multidrug resistance, and can form biofilms. Despite the clinical significance of P. aeruginosa, modeling P. aeruginosa infections in CF has been challenging. Here, we characterize Scnn1b-transgenic (Tg) BALB/c mice as P. aeruginosa lung infection models. Scnn1b-Tg mice overexpress the epithelial Na+ channel (ENaC) in their lungs, driving increased sodium absorption that causes lung pathology similar to CF. We intranasally infected Scnn1b-Tg mice and wild-type littermates with the laboratory P. aeruginosa strain PAO1 and CF clinical isolates and then assessed differences in bacterial clearance, cytokine responses, and histological features up to 12 days postinfection. Scnn1b-Tg mice carried higher bacterial burdens when infected with biofilm-grown rather than planktonic PAO1; Scnn1b-Tg mice also cleared infections more slowly than their wild-type littermates. Infection with PAO1 elicited significant increases in proinflammatory and Th17-linked cytokines on day 3. Scnn1b-Tg mice infected with nonmucoid early CF isolates maintained bacterial burdens and mounted immune responses similar to those of PAO1-infected Scnn1b-Tg mice. In contrast, Scnn1b-Tg mice infected with a mucoid CF isolate carried high bacterial burdens, produced significantly more interleukin 1β (IL-1β), IL-13, IL-17, IL-22, and KC, and showed severe immune cell infiltration into the bronchioles. Taken together, these results show the promise of Scnn1b-Tg mice as models of early P. aeruginosa colonization in the CF lung.
Project description:When animals are infected with helminthic parasites, resistant hosts show type II helper T immune responses to expel worms. Recently, natural helper (NH) cells or nuocytes, newly identified type II innate lymphoid cells, are shown to express ST2 (IL-33 receptor) and produce IL-5 and IL-13 when stimulated with IL-33. Here we show the relevant roles of endogenous IL-33 for Strongyloides venezuelensis infection-induced lung eosinophilic inflammation by using Il33(-/-) mice. Alveolar epithelial type II cells (ATII) express IL-33 in their nucleus. Infection with S. venezuelensis or intranasal administration of chitin increases in the number of ATII cells and the level of IL-33. S. venezuelensis infection induces pulmonary accumulation of NH cells, which, after being stimulated with IL-33, proliferate and produce IL-5 and IL-13. Furthermore, S. venezuelensis infected Rag2(-/-) mice increase the number of ATII cells, NH cells, and eosinophils and the expression of IL-33 in their lungs. Finally, IL-33-stimulated NH cells induce lung eosinophilic inflammation and might aid to expel infected worms in the lungs.
Project description:BackgroundEosinophilic esophagitis (EoE) is a chronic, food-driven allergic disease, characterized by eosinophil-rich inflammation in the esophagus. The histopathological and clinical features of EoE have been attributed to overproduction of the type 2 cytokines IL-4 and IL-13, which mediate profound alterations in the esophageal epithelium and neutralizing of their shared receptor component (IL-4Rα) with a human antibody drug (dupilumab) demonstrates clinical efficacy. Yet, the relative contribution of IL-4 and IL-13 and whether the type II IL-4 receptor (comprised of the IL-4Rα chain in association with IL-13Rα1) mediates this effect has not been determined.MethodsExperimental EoE was induced in WT, Il13ra1-/- , and Krt14Cre /Il13ra1fl/fl mice by skin-sensitized using 4-ethoxymethylene-2-phenyl-2-oxazolin (OXA) followed by intraesophageal challenges. Esophageal histopathology was determined histologically. RNA was extracted and sequenced for transcriptome analysis and compared with human EoE RNAseq data.ResultsInduction of experimental EoE in mice lacking Il13ra1 and in vivo IL-13 antibody-based neutralization experiments blocked antigen-induced esophageal epithelial and lamina propria thickening, basal cell proliferation, eosinophilia, and tissue remodeling. In vivo targeted deletion of Il13ra1 in esophageal epithelial cells rendered mice protected from experimental EoE. Single-cell RNA sequencing analysis of human EoE biopsies revealed predominant expression of IL-13Rα1 in epithelial cells and that EoE signature genes correlated with IL-13 expression compared with IL-4.ConclusionsWe demonstrate a definitive role for IL-13 signaling via IL-13Rα1 in EoE. These data provide mechanistic insights into the mode of action of current therapies in EoE and highlight the type II IL-4R as a future therapeutic target.
Project description:Airway mucus obstruction triggers macrophage activation and MMP12-dependent emphysema microarray expression profiling of lungs from Scnn1b-Tg mice to search for emphysema candidate genes
Project description:BackgroundIL-5 plays a central role in the development and maintenance of eosinophilia (EO) and eosinophil activation in a wide variety of eosinophilic disorders. Although IL-5, IL-3, and GM-CSF can modulate the expression of IL-5 receptor ? (IL-5R?) on eosinophils in vitro, little is known about soluble and surface IL-5R? levels in vivo.ObjectiveTo assess soluble and surface IL-5R? levels in patients with EO and/or mastocytosis.MethodsSurface IL-5R? expression was assessed by flow cytometry in blood and/or bone marrow from subjects with EO (n = 39) and systemic mastocytosis (n = 8) and from normal volunteers (n = 28). Soluble IL-5R? (sIL-5R?) level was measured in a cohort of 177 untreated subjects and correlated with EO, eosinophil activation, and serum tryptase and cytokine levels.ResultsIL-5R? expression on eosinophils inversely correlated with EO (r = -0.48; P < .0001), whereas serum levels of sIL-5R? increased with the eosinophil count (r = 0.56; P < .0001) and serum IL-5 (r = 0.40; P < .0001) and IL-13 (r = 0.29; P = .004) levels. Of interest, sIL-5R? level was significantly elevated in patients with systemic mastocytosis without EO. Although sIL-5R? levels correlated with serum tryptase levels in these patients, eosinophil activation, assessed by CD69 expression on eosinophils and serum eosinophil-derived neurotoxin levels, was increased compared with that in normal subjects.ConclusionsThese data are consistent with an in vivo IL-5R? regulatory pathway in human eosinophils similar to that described in vitro and involving a balance between soluble and surface receptor levels. This may have implications with respect to the use of novel therapeutic agents targeting IL-5 and its receptor in patients with EO and/or mastocytosis.
Project description:CONTEXT:Excessive adipose glucocorticoid action is associated with insulin resistance, but the mechanisms linking adipose glucocorticoid action to insulin resistance are still debated. We hypothesized that insulin resistance from excess glucocorticoid action may be attributed in part to increased ectopic lipid deposition in liver. METHODS:We tested this hypothesis in the adipose specific 11?-hydroxysteroid dehydrogenase-1 (HSD11B1) transgenic mouse, an established model of adipose glucocorticoid excess. Tissue specific insulin action was assessed by hyperinsulinemic-euglycemic clamps, hepatic lipid content was measured, hepatic insulin signaling was assessed by immunoblotting. The role of hepatic lipid content was further probed by administration of the functionally liver-targeted mitochondrial uncoupler, Controlled Release Mitochondrial Protonophore (CRMP). FINDINGS:High fat diet fed HSD11B1 transgenic mice developed more severe hepatic insulin resistance than littermate controls (endogenous suppression of hepatic glucose production was reduced by 3.8-fold, P?<?0.05); this was reflected by decreased insulin-stimulated hepatic insulin receptor kinase tyrosine phosphorylation and AKT serine phosphorylation. Hepatic insulin resistance was associated with a 53% increase (P?<?0.05) in hepatic triglyceride content, a 73% increase in diacylglycerol content (P?<?0.01), and a 66% increase in PKC? translocation (P?<?0.05). Hepatic insulin resistance was prevented with administration of CRMP by reversal of hepatic steatosis and prevention of hepatic diacylglycerol accumulation and PKC? activation. CONCLUSIONS:These findings are consistent with excess adipose glucocorticoid activity being a predisposing factor for the development of lipid (diacylglycerol-PKC?)-induced hepatic insulin resistance.
Project description:Cystic fibrosis is characterized by dehydration of the airway surface liquid layer with persistent mucus obstruction. Th2 immune responses are often manifested as increased mucous cell density (mucous cell metaplasia) associated with mucus obstruction. IL-33 is a known inducer of Th2 immune responses, but its roles in mucus obstruction and related phenotypes in a cystic fibrosis-like lung disease model (i.e., Scnn1b-Tg-positive [Tg+]) mouse, remain unclear. Accordingly, IL-33 knockout (IL-33KO) Tg+ mice were examined and compared with IL-33 heterozygous (IL-33HET) Tg+ mice. As compared with IL-33HET/Tg+ mice, IL-33KO/Tg+ mice had complete absence of bronchoalveolar lavage fluid eosinophilia, accompanied with significant reduction in bronchoalveolar lavage fluid concentration of IL-5, a cytokine associated with eosinophil differentiation and recruitment, and IL-4, a major Th2 cytokine. As compared with IL-33HET/Tg+ mice, IL-33KO/Tg+ mice had significantly reduced levels of Th2-associated gene signatures (Slc26a4, Clca1, Retnla, and Chi3l4), along with complete loss of intracellular mucopolysaccharide staining in the airway epithelium. As compared with IL-33HET/Tg+ mice, although the IL-33KO/Tg+ mice had significantly reduced levels of MUC5AC protein expression, they showed no reduction in the degree of mucus obstruction, MUC5B protein expression, bacterial burden, and neonatal mortality. Interestingly, the histological features, including subepithelial airway inflammation and alveolar space enlargement, were somewhat exaggerated in IL-33KO/Tg+ mice compared with IL-33HET/Tg+ mice. Taken together, our data indicate that although IL-33 modulates Th2 inflammatory responses and MUC5AC protein production, mucus obstruction is not dependent on IL-33.