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Wheezing-associated rhinovirus (RV) infections are associated with asthma development. We have shown that infection of immature mice with RV induces type 2 cytokine production and mucous metaplasia which is dependent on IL-33 and type 2 innate lymphoid cells (ILC2s) and intensified by a second heterologous RV infection. We hypothesize that M2a macrophages are required for the exaggerated inflammation and mucous metaplasia in response to heterologous RV infection. Wild-type C57Bl/6J mice and LysMCre IL4Rα KO mice lacking M2a macrophages were treated as follows: (1) sham infection on day 6 of life plus sham on day 13 of life, (2) RV-A1B on day 6 plus sham on day 13, (3) sham on day 6 and RV-A2 on day 13, or (4) RV-A1B on day 6 and RV-A2 on day 13. Lungs were harvested one or seven days after the second infection. Wild-type mice infected with RV-A1B at day 6 showed an increased number of Arg1- and Retnla-expressing lung macrophages, indicative of M2a polarization. Compared to wild-type mice infected with RV on day 6 and 13 of life, the lungs of LysMCre IL4Rα KO mice undergoing heterologous RV infection showed deCreased protein abundance of the epithelial-derived innate cytokines IL-33, IL-25 and TSLP, decreased ILC2s, decreased mRNA expression of IL-13 and IL-5, and decreased PAS staining. Finally, mRNA analysis and immunofluorescence microscopy of doubleinfected LysMCre IL4Rα KO mice showed reduced airway epithelial cell IL-33 expression, and treatment with IL-33 restored the exaggerated mucoinflammatory phenotype. Conclusion: Early-life RV infection alters the macrophage response to subsequent heterologous infection, permitting enhanced IL-33 expression, ILC2 expansion and intensified airway inflammation and mucous metaplasia. [ABSTRACT FROM AUTHOR]

Increased medial arterial thickness is a structural change in pulmonary arterial hypertension (PAH). The role of smooth muscle hypertrophy in this process has not been well studied. Bone morphogenetic proteins (BMPs), transforming growth factor (TGF)-[beta]1, serotonin (or 5-hydroxytryptamine; 5-HT), and endothelin (ET)-l have been implicated in PAH pathogenesis. We examined the effect of these mediators on human pulmonary artery smooth muscle cell size, contractile protein expression, and contractile function, as well on the roles of glycogen synthase kinase (GSK)-3[beta] and p70 ribosomal S6 kinase (p70S6K), two proteins involved in translational control, in this process. Unlike epidermal growth factor, BMP-4, TGF-[beta]1, 5-HT, and ET-1 each increased smooth muscle cell size, contractile protein expression, fractional cell shortening, and GSK-3[beta] phosphorylation. GSK-3[beta] inhibition by lithium or SB-216763 increased cell size, protein synthesis, and contractile protein expression. Expression of a non-phosphorylatable GSK-3[beta] mutant blocked BMP-4-, TGF-[beta]1-, 5-HT-, and ET-1-induced cell size enlargement, suggesting that GSK-3[beta] phosphorylation is required and sufficient for cellular hypertrophy. However, BMP-4, TGF-[beta]1, 5-HT, and ET-1 stimulation was accompanied by an increase in serum response factor transcriptional activation but not eIF2 phosphorylation, suggesting that GSK-3[beta]-mediated hypertrophy occurs via transcriptional, not translational, control. Finally, BMP-4, TGF-[beta]1, 5-HT, and ET-1 treatment induced phosphorylation of p70S6K and ribosomal protein S6, and siRNAs against p70S6K and S6 blocked the hypertrophic response. We conclude that mediators implicated in the pathogenesis of PAH induce pulmonary arterial smooth muscle hypertrophy. Identification of the signaling pathways regulating vascular smooth muscle hypertrophy may define new therapeutic targets for PAH. bone morphogenetic proteins; transforming growth factor-[beta]1; serotonin; endothelin-1 doi: 10.1152/ajplung.00108.2009.

We have isolated mesenchymal stem cells (MSCs) from tracheal aspirates of premature infants with respiratory distress. We examined the capacity of MSCs to differentiate into myofibroblasts, cells that participate in lung development, injury, and repair. Gene expression was measured by array, qPCR, immunoblot, and immunocytochemistry. Unstimulated MSCs expressed mRNAs encoding contractile (e.g., ACTA2, TAGLN), extra-cellular matrix (COL1A1 and ELN), and actin-binding (DBN1, PXN) proteins, consistent with a myofibroblast phenotype, although there was little translation into immunoreactive protein. Incubation in serum-free medium increased contractile protein (ACTA2, MYH11) gene expression. MSC-conditioned medium showed substantial levels of TGF-[beta]1, and treatment of serum-deprived cells with a type I activin receptor-like kinase inhibitor, SB-431542, attenuated the expression of genes encoding contractile and extracellular matrix proteins. Treatment of MSCs with TGF-[beta]1 further induced the expression of mRNAs encoding contractile (ACTA2, MYH11, TAGLN, DES) and extracellular matrix proteins (FN1, ELN, COL1A1, COL1A2), and increased the protein expression of [alpha]-smooth muscle actin, myosin heavy chain, and SM22. In contrast, human bone marrow-derived MSCs failed to undergo TGF-[beta]1-induced myofibroblastic differentiation. Finally, primary cells from tracheal aspirates behaved in an identical manner as later passage cells. We conclude that human neonatal lung MSCs demonstrate an mRNA expression pattern characteristic of myofibroblast progenitor cells. Autocrine production of TGF-[beta]1 further drives myofibroblastic differentiation, suggesting that, in the absence of other signals, fibrosis represents the 'default program' for neonatal lung MSC gene expression. These data are consistent with the notion that MSCs play a key role in neonatal lung injury and repair. alveolarization; bronchopulmonary dysplasia; myofibroblast; neonate doi: 10.1152/ajplung.00347.2009.

Increased airway smooth muscle (ASM) mass, a characteristic finding in asthma, may be caused by hyperplasia or hypertrophy. Cell growth requires increased translation of contractile apparatus mRNA, which is controlled, in part, by glycogen synthase kinase (GSK)-3[beta], a constitutively active kinase that inhibits eukaryotic initiation factor-2 activity and binding of methionyl tRNA to the ribosome. Phosphorylation of GSK-3[beta] inactivates it, enhancing translation. We sought to quantify the contributions of hyperplasia and hypertrophy to increased ASM mass in ovalbumin (OVA)-sensitized and -challenged BALB/c mice and the role of GSK-3[beta] in this process. Immunofluorescent probes, confocal microscopy, and stereological methods were used to analyze the number and volume of cells expressing [alpha]-smooth muscle actin and phospho-[Ser.sup.9] GSK-3[beta] (pGSK). OVA treatment caused a 3-fold increase in ASM fractional unit volume or volume density (Vv) (PBS, 0.006 [+ or -] 0.0003; OVA, 0.014 [+ or -] 0.001), a 1.5-fold increase in ASM number per unit volume (Nv), and a 59% increase in volume per cell (Vv/Nv) (PBS, 824 [+ or -] 76 [micro] [m.sup.3]; OVA, 1,310 [+ or -] 183 [micro][m.sup.3]). In OVA-treated mice, there was a 12-fold increase in the Vv of pGSK (+) ASM, a 5-fold increase in the Nv of pGSK (+) ASM, and a 1.6-fold increase in Vv/Nv. Lung homogenates from OVA-treated mice showed increased GSK-3[beta] phosphorylation and lower GSK-3[beta] activity. Both hyperplasia and hypertrophy are responsible for increased ASM mass in OVA-treated mice. Phosphorylation and inactivation of GSK-3[beta] are associated with ASM hypertrophy, suggesting that this kinase may play a role in asthmatic airway remodeling. ovalbumin; remodeling; stereology

Rhinovirus (RV) infections trigger exacerbations of airways disease, but underlying mechanisms remain unknown. We hypothesized that RV and cytokines present in inflamed airways combine to induce augmented airway epithelial cell chemokine expression, promoting further inflammation. To test this hypothesis in a cellular system, we examined the combined effects of RV39 and TNF-[alpha], a cytokine increased in asthma and chronic obstructive pulmonary disease, on airway epithelial cell pro-inflammatory gene expression. Costimulation of 16HBE14o--human bronchial epithelial cells and primary mucociliary-differentiated tracheal epithelial cells with RV and TNF-[alpha] induced synergistic increases in IL-8 and epithelial neutrophil attractant-78 production. Similar synergism was observed for IL-8 promoter activity, demonstrating that the effect is transcriptionally mediated. Whereas increases in ICAM-1 expression and viral load were noted 16-24 h after costimulation, cooperative effects between RV39 and TNF-[alpha] were evident 4 h after stimulation and maintained despite incubation with blocking antibody to ICAM-1 given 2 h postinfection or UV irradiation of virus, implying that effects were not solely due to changes in ICAM-1 expression. Furthermore, RV39 infection induced phosphorylation of ERK and transactivation of the IL-8 promoter AP-1 site, which functions as a basal level enhancer, leading to enhanced TNF-[alpha] responses. We conclude that RV infection and TNF-[alpha] stimulation induce cooperative increases in epithelial cell chemokine expression, providing a cellular mechanism for RV-induced exacerbations of airways disease. asthma; ERK; chronic obstructive pulmonary disease; intercellular adhesion molecule-1 ; interleukin-8

Airway smooth muscle hypertrophy appears to be present in severe asthma. However, the effect of corticosteroids on airway smooth muscle cell size or contractile protein expression has not been studied. We examined the effects of dexamethasone, fluticasone, and salmeterol on contractile protein expression in transforming growth factor (TGF)-[beta]-treated primary bronchial smooth muscle cells. Dexamethasone and fluticasone, but not salmeterol, each reduced expression of [alpha]-smooth muscle actin and the short isoform of myosin light chain kinase. Steady-state [alpha]-actin mRNA level and stability were unchanged, consistent with posttranscriptional control. Fluticasone significantly decreased [alpha]-actin protein synthesis following treatment with the transcriptional inhibitor actinomycin D, indicative of an inhibitory effect on mRNA translation. Fluticasone also significantly increased [alpha]-actin protein turnover. Finally, fluticasone reduced TGF-[beta]-induced incorporation of [alpha]-actin into filamentous actin, cell length, and cell shortening in response to ACh and KCl. We conclude that glucocorticoids reduce human airway smooth muscle a-smooth muscle actin expression and incorporation into contractile filaments, as well as contractile function, in part by attenuation of mRNA translation and enhancement of protein degradation. corticosteroids; fluticasone; hypertrophy; salmeterol; transforming growth factor-[beta]]

Abstract Background Recent studies have indicated the presence of multipotent mesenchymal stromal cells (MSCs) in human lung diseases. Excess airway smooth muscle, myofibroblasts and activated fibroblasts have each been noted in asthma, suggesting that mesenchymal progenitor cells play a role in asthma pathogenesis. We therefore sought to determine whether MSCs are present in the lungs of ovalbumin (OVA)-sensitized and challenged mice, a model of allergic airways disease. Methods Balb/c mice were sensitized and challenged with PBS or OVA over a 25 day period. Flow cytometry as well as colony forming and differentiation potential were used to analyze the emergence of MSCs along with gene expression studies using immunochemical analyses, quantitative polymerase chain reaction (qPCR), and gene expression beadchips. Results A CD45-negative subset of cells expressed Stro-1, Sca-1, CD73 and CD105. Selection for these markers and negative selection against CD45 yielded a population of cells capable of adipogenic, osteogenic and chondrogenic differentiation. Lungs from OVA-treated mice demonstrated a greater average colony forming unit-fibroblast (CFU-F) than control mice. Sorted cells differed from unsorted lung adherent cells, exhibiting a pattern of gene expression nearly identical to bone marrow-derived sorted cells. Finally, cells isolated from the bronchoalveolar lavage of a human asthma patient showed identical patterns of cell surface markers and differentiation potential. Conclusions In summary, allergen sensitization and challenge is accompanied by an increase of MSCs resident in the lungs that may regulate inflammatory and fibrotic responses.