Background Early-life human rhinovirus (RV) infection has been linked to asthma development in high risk infants and children. Nevertheless, the role of RV infection in the initiation of asthma remains unclear. Objective We hypothesized that, in contrast to infection of mature BALB/c mice, neonatal infection with RV promotes an IL-25-driven type 2 response which causes persistent mucous metaplasia and airway hyperresponsiveness. Methods Six day-old and eight week-old BALB/c mice were inoculated with sham HeLa cell lysate or RV. Airway responses from 1 to 28 days after infection were assessed by qPCR, ELISA, histology, immunofluorescence microscopy, flow cytometry and methacholine responsiveness. Selected mice were treated with a neutralizing antibody to IL-25. Results Compared to mature mice, RV infection in neonatal mice increased lung IL-13 and IL-25 production whereas IFN-γ, IL-12p40 and TNF-α expression were suppressed. In addition, the population of IL-13-secreting type 2 innate lymphoid cells (ILC2s) was expanded with RV infection in neonatal but not in mature mice. ILC2 cells were the major cell type secreting IL-13 in neonates. Finally, anti-IL-25 neutralizing antibody attenuated ILC2 expansion, mucous hypersecretion and airways responsiveness. Conclusions These findings suggest that early-life viral infection could contribute to asthma development by provoking age-dependent, IL-25-driven type 2 immune responses.

1,1-Bis(3'-indolyl)-1-(p-trifluoromethylphenyl)methane (DIM-C-pPhCF3) and troglitazone activate peroxisome proliferator-activated receptor gamma (PPARgamma) in Panc-28 pancreatic cancer cells and also inhibit cell proliferation. DIM-C-pPhCF3 was more active than troglitazone and was used as a model to investigate the mechanism of PPARgamma-dependent inhibition of Panc-28 cell growth. DIM-C-pPhCF3 significantly inhibited G0/G1-->S phase progression, as determined by FACS analysis, and this was associated with decreased retinoblastoma protein phosphorylation and increased p21 protein and mRNA expression, but no change in p27 or cyclin D1. PPARgamma antagonists blocked DIM-C-pPhCF3-induced growth inhibition and induction of p21 protein, and similar inhibitory effects were observed in Panc-28 cells transfected with a construct (pWWP) containing a -2325 to +8 p21 promoter insert. Deletion analysis of the p21 promoter indicated that PPARgamma-dependent activation of p21 promoter constructs by DIM-C-pPhCF3 required GC-rich sites 3 and 4 in the proximal region (-124 to -60) of the p21 promoter. The results of RNA interference and protein expression/DNA binding assays suggest that DIM-C-pPhCF3 induced p21 expression through a novel mechanism that involves PPARgamma interactions with both Sp1 and Sp4 proteins bound to the proximal GC-rich region of the p21 promoter.

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.

Background Periostin, a secreted extracellular matrix protein, has been localized to deposits of subepithelial fibrosis in asthma, and periostin levels have been linked to elevation of IL-13. Objective We hypothesized that periostin is required for airway inflammatory responses to a physiologic aeroallergen, house dust mite (HDM). Methods We studied F4-F6 B6;129-Postntm1Jmol/J wild-type (+/+) and null (−/−) mice as well as C57BL/6 mice treated with either IgM or OC-20 periostin neutralizing antibody. Mice were exposed to five doses of HDM intranasally over a 16 day period. Results HDM increased airways responsiveness in Postn wild-type but not null mice. In addition, HDM-treated C57BL/6 mice injected with OC-20 had a lower airways responsiveness than HDM-treated mice injected with IgM. Compared to Postn wild-type mice, Postn null mice showed decreases in HDM-induced inflammation and mucous metaplasia, as well as reduced IL-4, IL-25, CD68, Gob5 and periostin mRNA expression. OC-20 antibody gave similar results. HDM exposure increased periostin expression in the airway epithelium, subepithelium, smooth muscle and inflammatory cells. OC-20 blocked the HDM-induced IgE response, and T cells incubated with dendritic cells (DCs) from Postn null mice or treated with OC-20 showed deficient DNA synthesis and IL-13 responses compared to T cells incubated with wild-type DCs. Finally, adoptive transfer of bone marrow-derived DCs from periostin wild-type mice was sufficient to promote allergic responses in F6 periostin null littermates. Conclusions In mice, periostin is required for maximal airways hyperresponsiveness and inflammation following HDM sensitization and challenge. Periostin is required for maximal HDM-induced T cell responses.

Recent studies link early rhinovirus (RV) infections to later asthma development. We hypothesized that neonatal RV infection leads to an IL-13-driven asthma-like phenotype in mice. BALB/c mice were inoculated with RV1B or sham on day 7 of life. Viral RNA persisted in the neonatal lung up to 7 days after infection. Within this time frame, IFNs-α, -β and -γ peaked 1 day after infection, whereas IFN-λ levels persisted. Next, we examined mice on day 35 of life, 28 days after initial infection. Compared to sham-treated controls, virus-inoculated mice demonstrated airways hyperresponsiveness. Lungs from RV-infected mice showed increases in several immune cell populations, as well as the percentages of CD4-positive T cells expressing IFN-γ and of NKp46/CD335+, TCR-β+ cells expressing IL-13. Periodic acid-Schiff and immunohistochemical staining revealed mucous cell metaplasia and muc5AC expression in RV1B- but not sham-inoculated lungs. Mucous metaplasia was accompanied by induction of gob-5, MUC5AC, MUC5B and IL-13 mRNA. By comparison, adult mice infected with RV1B showed no change in IL-13 expression, mucus production or airways responsiveness 28 days after infection. Intraperitoneal administration of anti-IL13 neutralizing antibody attenuated RV-induced mucous metaplasia and methacholine responses, and IL-4R null mice failed to show RV-induced mucous metaplasia. Finally, neonatal RV increased the inflammatory response to subsequent allergic sensitization and challenge. We conclude that neonatal RV1B infection leads to persistent airways inflammation, mucous metaplasia and hyperresponsiveness which are mediated, at least in part, by IL-13.

Human rhinovirus (HRV) infections lead to exacerbations of lower airways disease in asthmatic patients but not in healthy individuals. However, underlying mechanisms remain to be completely elucidated. We hypothesized that the Th2-driven allergic environment enhances HRV-induced CC chemokine production, leading to asthma exacerbations. Ovalbumin (OVA)-sensitized and -challenged mice inoculated with HRV showed significant increases in the expression of lung CC chemokine ligand (CCL)-2/monocyte chemotactic protein (MCP)-1, CCL4/macrophage inflammatory protein (MIP)-1β, CCL7/MCP-3, CCL19/MIP-3β, and CCL20/MIP3α compared with mice treated with OVA alone. Inhibition of CCL2 with neutralizing antibody significantly attenuated HRV-induced airways inflammation and hyperresponsiveness in OVA-treated mice. Immunohistochemical stains showed colocalization of CCL2 with HRV in epithelial cells and CD68-positive macrophages, and flow cytometry showed increased CCL2(+), CD11b(+) cells in the lungs of OVA-treated, HRV-infected mice. Compared with lung macrophages from naïve mice, macrophages from OVA-exposed mice expressed significantly more CCL2 in response to HRV infection ex vivo. Pretreatment of mouse lung macrophages and BEAS-2B human bronchial epithelial cells with interleukin (IL)-4 and IL-13 increased HRV-induced CCL2 expression, and mouse lung macrophages from IL-4 receptor knockout mice showed reduced CCL2 expression in response to HRV, suggesting that exposure to these Th2 cytokines plays a role in the altered HRV response. Finally, bronchoalveolar macrophages from children with asthma elaborated more CCL2 upon ex vivo exposure to HRV than cells from nonasthmatic patients. We conclude that CCL2 production by epithelial cells and macrophages contributes to HRV-induced airway hyperresponsiveness and inflammation in a mouse model of allergic airways disease and may play a role in HRV-induced asthma exacerbations.

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.