Background Early-life respiratory viral infection is a risk factor for asthma development. Rhinovirus (RV) infection of six-day old but not mature mice causes mucous metaplasia and airway hyperresponsiveness which is associated with expansion of lung type 2 innate lymphoid cells (ILC2s) and dependent on IL-13 and the innate cytokine IL-25. However, contributions of the other innate cytokines, IL-33 and thymic stromal lymphopoietin (TSLP), to the observed asthma-like phenotype have not been examined. Objective We reasoned that IL-33 and TSLP expression are also induced by RV infection in immature mice and required for maximum ILC2 expansion and mucous metaplasia. Methods We inoculated six day-old BALB/c (wild-type) and TSLP receptor knockout (TSLPR KO) mice with sham HeLa cell lysate or RV. Selected mice were treated with neutralizing antibodies to IL-33 or recombinant IL-33, IL-25 or TSLP. ILC2s were isolated from RV-infected immature mice and treated with innate cytokines ex vivo. Results RV infection of six-day old mice increased IL-33 and TSLP protein abundance. TSLP expression was localized to the airway epithelium, whereas IL-33 was expressed in both epithelial and subepithelial cells. RV-induced mucous metaplasia, ILC2 expansion, airway hyperresponsiveness and epithelial cell IL-25 expression were attenuated by anti-IL-33 treatment and in TSLPR KO mice. Administration of intranasal IL-33, but not TSLP, was sufficient for mucous metaplasia. Finally, TSLP was required for maximal ILC2 gene expression in response to IL-25 and IL-33. Conclusion The generation of mucous metaplasia in immature, RV-infected mice involves a complex interplay between the innate cytokines IL-25, IL-33 and TSLP.

Analyses of frequency profiles of markers on disease or drug-response related genes in diverse populations are important for the dissection of common diseases. We report the results of analyses of data on 405 SNPs from 75 such genes and a 5.2 Mb chromosome, 22 genomic region in 1871 individuals from diverse 55 endogamous Indian populations. These include 32 large (>10 million individuals) and 23 isolated populations, representing a large fraction of the people of India. We observe high levels of genetic divergence between groups of populations that cluster largely on the basis of ethnicity and language. Indian populations not only overlap with the diversity of HapMap populations, but also contain population groups that are genetically distinct. These data and results are useful for addressing stratification and study design issues in complex traits especially for heterogeneous populations.

Background: Mechanisms linking cortical actin with adherens junctions (AJs) required for forming restrictive endothelial barrier remain unclear. Results: We show that neural Wiskott-Aldrich syndrome protein (N-WASP) binds AJ constituent, p120-catenin, forms cortical actin, and links AJs with actin. Conclusion: N-WASP stabilizes AJs and thereby maintains endothelial barrier function. Significance: N-WASP represents a novel target for preventing leaky endothelial barrier syndrome.

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Early-life wheezing-associated infections with rhinovirus (RV) have been associated with asthma development in children. We have shown that RV infection of 6-day-old mice induces mucous metaplasia and airways hyperresponsiveness, which is dependent on IL-13, IL-25, and type 2 innate lymphoid cells (ILC2s). Infection of immature mice fails to induce lung IFN-g expression, in contrast to mature 8-week-old mice with a robust IFN-g response, consistent with the notion that deficient IFN-g production in immature mice permits RV-induced type 2 immune responses. We therefore examined the effects of intranasal IFN-g administration on RVinduced ILC2 expansion and IL-13 expression in 6-day-old BALB/c and IL-13 reporter mice. Airway responses were assessed by histology, immunofluorescence microscopy, quantitative polymerase chain reaction, ELISA, and flow cytometry. Lung ILC2s were also treated with IFN-g ex vivo. We found that, compared with untreated RV-infected immature mice, IFN-g treatment attenuated RV-induced IL-13 and Muc5ac mRNA expression and mucous metaplasia. IFN-g also reduced ILC2 expansion and the percentage of IL-13-secreting ILC2s. IFN-g had no effect on the mRNA or protein expression of IL-25, IL-33, or thymic stromal lymphoprotein. Finally, IFN-g treatment of sorted ILC2s reduced IL-5, IL-13, IL-17RB, ST2, and GATA-3 mRNA expression. We conclude that, in immature mice, IFN-g inhibits ILC2 expansion and IL-13 expression in vivo and ex vivo, thereby attenuating RV-induced mucous metaplasia. These findings demonstrate the antagonistic function of IFN-g on ILC2 expansion and gene expression, the absence of which may contribute to the development of an asthma-like phenotype after early-life RV infection.

Background We have shown that rhinovirus (RV), a cause of asthma exacerbations, colocalizes with CD68- and CD11b-positive airway macrophages following experimental infection in humans. We have also shown that RV-induced cytokine expression is abolished in TLR2−/− bone marrow-derived macrophages. Objective We hypothesize that TLR2+ macrophages are required and sufficient for RV-induced airway inflammation in vivo. Methods To determine the requirement and sufficiency of TLR2 for RV-induced airway responses, naïve and ovalbumin-sensitized and challenged C57BL/6 wild-type and TLR2−/− mice were infected with RV1B followed by IgG or anti-TLR2. Bone marrow chimera experiments using OVA-treated C57BL/6 and TLR2−/− mice were also performed. Finally, naïve TLR2−/− mice underwent intranasal transfer of bone marrow-derived wild type macrophages. Results RV1B infection of naïve wild-type mice induced an influx of airway neutrophils and CD11b+ exudative macrophages which was reduced in TLR2−/− mice. In allergen-exposed mice, RV-induced neutrophilic and eosinophilic airway inflammation and hyperresponsiveness were reduced in TLR2−/− and anti-TLR2-treated mice. Transfer of TLR2−/− bone marrow into wild type ovalbumin-treated, C57BL/6 mice blocked RV-induced airway responses, whereas transfer of wild type marrow to TLR2−/− mice restored them. Finally, transfer of wild-type macrophages to naïve TLR2−/− mice was sufficient for neutrophilic inflammation after RV infection, whereas macrophages treated with IL-4 (to induce M2 polarization) were sufficient for eosinophilic inflammation, mucous metaplasia and airways hyperresponsiveness. Conclusions TLR2 is required for early inflammatory responses induced by RV, and TLR2+ macrophages are sufficient to confer airway inflammation to TLR2−/− mice, with the pattern of inflammation depending on macrophage activation state.

BSTRACTActivation of sphingosine-1-phosphate receptor 1 (S1PR1) plays a key role in repairing endothelial barrier function. We addressed the role of phosphorylation of the three intracellular tyrosine residues of S1PR1 in endothelial cells in regulating the receptor responsiveness and endothelial barrier function regulated by sphingosine 1-phosphate (S1P)-mediated activation of S1PR1. We demonstrated that phosphorylation of only Y143 site was required for S1PR1 internalization in response to S1P. Maximal S1PR1 internalization was seen in 20 min but S1PR1 returned to the cell surface within 1 h accompanied by Y143-dephosphorylation. Cell surface S1PR1 loss paralleled defective endothelial barrier enhancement induced by S1P. Expression of phospho-defective (Y143F) or phosphomimicking (Y143D) mutants, respectively, failed to internalize or showed unusually high receptor internalization, consistent with the requirement of Y143 in regulating cell surface S1PR1 expression. Phosphorylation of the five S1PR1 C-terminal serine residues did not affect the role of Y143 phosphorylation in signaling S1PR1 internalization. Thus, rapid reduction of endothelial cell surface expression of S1PR1 subsequent to Y143 phosphorylation is a crucial mechanism of modulating S1PR1 signaling, and hence the endothelial barrier repair function of S1P.

Objectives Increased vascular permeability is a hallmark of sepsis and acute respiratory distress syndrome. Angiopoietin (Ang2) induces vascular leak, and excess Ang2 generation is associated with patient mortality from these diseases. However, mechanisms dampening Ang2 generation during injury remain unclear. Interestingly, microRNA-150 levels were decreased in septic patients. MicoRNAs (miR) regulate signaling networks by silencing mRNAs containing complementary sequences. Thus, we hypothesized that miR-150 suppresses Ang2 generation and thereby resolves vascular injury. Approach and Results Wild-type or miR-150−/− mice or endothelial cells were exposed to lipopolysaccharide or sepsis and Ang2 levels, adherens junction re-annealing, endothelial barrier function and mortality were determined. While Ang2 transiently increased during lipopolysaccharide-induced injury in wild-type endothelial cells and lungs, miR-150 expression was elevated only during recovery from injury. Deletion of miR-150 caused a persistent increase in Ang2 levels and impaired adherens junctions re-annealing after injury, resulting thereby in an irreversible increase in vascular permeability. Also, miR-150−/− mice died rapidly after sepsis. Rescuing miR-150 expression in endothelial cells prevented Ang2 generation, thereby restoring vascular barrier function in miR-150−/− mice. miR-150 terminated Ang2 generation by targeting the transcription factor, early growth response 2. Thus, early growth response 2 or Ang2 depletion in miR-150−/− endothelial cells restored junctional re-annealing and reinstated barrier function. Importantly, upregulating miR-150 expression by injecting a chemically synthesized miR-150 mimic into WT-mice vasculature decreased EGR2 and Ang2 levels and hence mortality from sepsis. Conclusions miR-150 is a novel suppressor of Ang2 generation with a key role in resolving vascular injury and reducing mortality resulting from sepsis.