Background: Asthma is a chronic respiratory disease with marked clinical and pathophysiological heterogeneity. Specific pathways are thought to be involved in the pathomechanisms of different inflammatory phenotypes of asthma; however, direct in vivo comparison has not been performed. Methods:We developed mouse models representing three different phenotypes of allergic airway inflammation-eosinophilic, mixed, and neutrophilic asthma via different methods of house dust mite sensitization and challenge. Transcriptomic analysis of the lungs, followed by the RT-PCR, western blot, and confocal microscopy, was performed. Primary human bronchial epithelial cells cultured in air-liquid interface were used to study the mechanisms revealed in the in vivo models.Results: By whole-genome transcriptome profiling of the lung, we found that airway tight junction (TJ), mucin, and inflammasome-related genes are differentially expressed in these distinct phenotypes. Further analysis of proteins from these families revealed that Zo-1 and Cldn18 were downregulated in all phenotypes, while increased Cldn4 expression was characteristic for neutrophilic airway inflammation.Mucins Clca1 (Gob5) and Muc5ac were upregulated in eosinophilic and even more in neutrophilic phenotype. Increased expression of inflammasome-related molecules such as Nlrp3, Nlrc4, Casp-1, and IL-1β was characteristic for neutrophilic asthma. In addition, we showed that inflammasome/Th17/neutrophilic axis cytokine-IL-1βmay transiently impair epithelial barrier function, while IL-1β and IL-17 increase mucin expressions in primary human bronchial epithelial cells.Conclusion: Our findings suggest that differential expression of TJ, mucin, and inflammasome-related molecules in distinct inflammatory phenotypes of asthma may be linked to pathophysiology and might reflect the differences observed in the clinic. K E Y W O R D Sendotype, epithelial barrier, house dust mite, phenotype, precision medicine Tan and Hagner equal first-author contribution.
1 The calcitonin receptor-like receptor (CRLR) and speci®c receptor activity modifying proteins (RAMPs) together form receptors for calcitonin gene-related peptide (CGRP) and/or adrenomedullin in transfected cells. 2 There is less evidence that innate CGRP and adrenomedullin receptors are formed by CRLR/ RAMP combinations. We therefore examined whether CGRP and/or adrenomedullin binding correlated with CRLR and RAMP mRNA expression in human and rat cell lines known to express these receptors. Speci®c human or rat CRLR antibodies were used to examine the presence of CRLR in these cells. 3 We con®rmed CGRP subtype 1 receptor (CGRP 1 ) pharmacology in SK-N-MC neuroblastoma cells. L6 myoblast cells expressed both CGRP 1 and adrenomedullin receptors whereas Rat-2 ®broblasts expressed only adrenomedullin receptors. In contrast we could not con®rm CGRP 2 receptor pharmacology for Col-29 colonic epithelial cells, which, instead were CGRP 1 -like in this study.
We report here on the characterization and immunohistochemical localization in human tissues of calcitonin receptor-like receptor (CRLR) which was recently found to mediate the effects of both calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM). Western blot analysis using antibodies raised against the first extracellular loop and the carboxy-terminal part of hCRLR, respectively, detected two major bands corresponding to about 70 and 60 kDa in membrane preparations of cultured endothelial cells and numerous organs including lung, heart ventricle and kidney. Immunohistochemical analysis of the cardiovascular system revealed CRLR-like immunoreactivity (CRLR-LI) in the endothelium of all blood vessels including large and small arteries, veins and capillaries, and in heart muscle cells and endocardium. The lung showed intense staining over the alveolar capillaries. Within the digestive tract, staining was observed over the cells lining the excretory ducts of the parotid gland, over the epithelium of the fundic glands of stomach, endocrine cells of the duodenum and ileum and some myenteric ganglia. The kidney presented staining of the juxtaglomerular arteries, the glomerular capillaries and chief cells of the collecting duct. Within the endocrine organs, a strong CRLR-LI signal was observed over the Langerhans islets, and weak immunoreactivity in the Leydig cells of testis. Spleen showed intense staining in trabecular veins and sinuses. Macrophages displayed a variable immunoreactivity. Our data demonstrate a wide distribution of CRLR throughout the human body and suggest CRLR to be involved in the mediation of a variety of actions in addition to vascular control.
Similar to T-helper (Th) cells, CD8+ T cells also differentiate into distinct subpopulations.However, the existence of IL-9-producing CD8 + T (Tc9) cells has not been elucidated so far. We show that murine CD8 + T cells activated in the presence of IL-4 plus TGF-β develop into transient IL-9 producers characterized by specific IFN-γ and IL-10 expression patterns as well as by low cytotoxic function along with diminished expression of the CTL-associated transcription factors T-bet and Eomesodermin. Similarly to the CD4 + counterpart, Tc9 cells required for their differentiation STAT6 and IRF4. Tc9 cells deficient for these master regulators displayed increased levels of Foxp3 that in turn suppressed IL-9 production. In an allergic airway disease model, Tc9 cells promoted the onset of airway inflammation, mediated by subpathogenic numbers of Th2 cells. This support was specific for Tc9 cells because CTLs failed to exert this function. We detected increased Tc9 frequency in the periphery in mice and humans with atopic dermatitis, a Th2-associated skin disease that often precedes asthma. Thus, our data point to the existence of Tc9 cells and to their supportive function in Th2-dependent airway inflammation, suggesting that these cells might be a therapeutic target in allergic disorders.Keywords: Allergic airway inflammation r Atopic dermatitis r CD8 + T cells r IL-9 r Tc9 cells Additional supporting information may be found in the online version of this article at the publisher's web-site 607Introduction IL-9 is a pleiotropic cytokine produced by different cell types such as T cells, innate lymphoid cells, eosinophils, and mast cells [1,2]. IL-9 secretion by T cells was originally linked to Th2-mediated (where Th is T-helper) diseases such as allergic airway inflammation [3,4] and parasite infection [5,6] However, in vitro IL-9 is differently regulated from other classical Th2-cytokines such as IL-4, IL-5, and IL-13. IL-9 is induced by the combination of the Th2-skewing cytokine IL-4 with TGF-β [7] and, under these conditions Th2-cytokines are strongly inhibited [8,9]. Therefore, besides Th1, Th2, Th17, and Treg cells, IL-9-producing cells have been established as an additional Th-cell subset, termed Th9 cells. The differentiation of Th9 cells is governed by the transcription factors IRF4, PU.1, and STAT6 [10,11]. IRF4, also important for Th2-, Th17-, and Tfh-fate decisions [12][13][14][15] regulates IL-9 production directly by binding to the Il9 promoter [10]. Likewise, PU.1 enhances IL-9 production, at least partly by binding to the Il9 promoter [11]. In contrast, STAT6, activated by IL-4, contributes to IL-9 production indirectly. It represses the expression of two transcription factors, Treg-specific Foxp3 and Th1-associated T-bet that inhibit IL-9 production [8,9,16].Similarly to Th cells, CD8 + T cells differentiate into at least four effector subsets with different phenotype: CTLs, Tc2, Tc17, or CD8 + Treg cells [17]. The best characterized CD8 + T-cell subpopulation, CTLs kill infected or tumorogenic cells ...
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