RV-specific antibody responses in Tbet deficient and wild type mice.

<p>Wild type and Tbet-/- mice were infected intranasally with RV1B or sham infected with PBS. Blood was collected 14 days after infection. (A&B) RV1B-binding IgG2c (A) and IgG1 (B) in sera was measured by ELISA. (C) Neutralisation of RV1B infection of Ohio HeLa cells by pooled sera assessed by crystal violet cell viability staining. ATCC ctl: control reference guinea pig anti-sera. Top dashed line in C, uninfected cells control. Bottom dashed line in C, RV infected cells control. Data represent results from 5–6 pooled sera per treatment group in a single experiment, representative of 3 independent experiments.</p

Asthma-like airways inflammation is CD4+ T cell dependent.

<p>Tbet-/- mice were infected intranasally with RV1B or sham infected with PBS. In addition, mice were systemically depleted of CD4 expressing cells (anti-CD4), or treated with isotype control antibody (isotype) 3hrs prior to infection. Tissues were harvested at 7 days post-challenge. (A) Lung flow cytometry staining for CD3+CD4+ T cells. (B-D) Levels of cytokines IL-4 (B), IL-13 (C) and IL-17a (D) in lung tissue measured by Taqman qPCR. (E) Total BAL cell counts and (F) eosinophil counts in BAL measured by cytospin assay. (G) MUC5AC levels in BAL measured by ELISA. (H) Representative PAS staining for mucus in lung tissue sections. Scale bars 50μm. n = 12–15 mice/group (mice for which CD4+ cell depletion was not successful were excluded from all analyses (n = 5 of 30)). ***p<0.001, **p<0.01, *p<0.05, n.s. not significant.</p

Helper T cell responses in Tbet deficient and wild type mice.

<p>Wild type and Tbet-/- mice were infected intranasally with RV1B or sham infected with PBS. (A-D) Intranuclear flow cytometry staining for transcription factors Tbet (A), GATA-3 (B), RORγt (C) and FOXP3 (D) in CD3+CD4+ lung T cells, 2 and 7 days post-infection. (E-H) Intracellular flow cytometry staining for cytokines IFN-γ (E), IL-13 (F), IL-17A (G) and IL-10 (H) in lung CD3+CD4+ cells stimulated with PMA and ionomycin, on day 7 post-infection. (I-L) RNA was extracted from lung tissue harvested on day 7 post-infection and expression of IFN-γ (I), IL-13 (J), IL-17a (K) and IL-10 (L) mRNA was quantified by Taqman qPCR. n = 8–9 mice/group.***p<0.001, **p<0.01, *p<0.05, n.s. not significant.</p

Pulmonary Innate Lymphoid Cell Responses during Rhinovirus-induced Asthma Exacerbations In Vivo

Rationale Type 2 innate lymphoid cells (ILC2s) are significant sources of type 2 cytokines, which are implicated in the pathogenesis of asthma and asthma exacerbations. The role of ILC2s in virus-induced asthma exacerbations is not well-characterized. Objectives To characterize pulmonary ILC responses following experimental rhinovirus challenge in patients with moderate asthma and healthy subjects. Methods Patients with moderate asthma and healthy subjects were inoculated with rhinovirus-16, and underwent bronchoscopy at baseline, day 3 and day 8 post-inoculation. Pulmonary ILC1s and ILC2s were quantified in bronchoalveolar lavage (BAL) using flow cytometry. The ratio of BAL ILC2:ILC1 was assessed to determine their relative contributions to the clinical and immune response to rhinovirus challenge. Measurements and Main Results At baseline, ILC2s were significantly higher in patients with asthma than healthy subjects. At day 8, ILC2s significantly increased from baseline in both groups, which was significantly higher in asthma than in healthy subjects (all comparisons P<0.05). In healthy subjects, ILC1s increased from baseline at day 3 (P=0.001), while in patients with asthma, ILC1s increased from baseline at day 8 (P=0.042). Patients with asthma had significantly higher ILC2:ILC1 ratios at baseline (P=0.024) and day 8 (P=0.005). Increased ILC2:ILC1 ratio in asthma correlated with clinical exacerbation severity and type 2 cytokines in nasal mucosal lining fluid. Conclusions An ILC2-predominant inflammatory profile in asthma was associated with increased severity and duration of rhinovirus infection compared with healthy subjects, supporting the potential role of ILC2s in the pathogenesis of virus-induced asthma exacerbations

IL-33-dependent type 2 inflammation during rhinovirus-induced asthma exacerbations in vivo

Rationale: Rhinoviruses are the major cause of asthma exacerbations; however, its underlying mechanisms are poorly understood. We hypothesized that the epithelial cell–derived cytokine IL-33 plays a central role in exacerbation pathogenesis through augmentation of type 2 inflammation. Objectives: To assess whether rhinovirus induces a type 2 inflammatory response in asthma in vivo and to define a role for IL-33 in this pathway. Methods: We used a human experimental model of rhinovirus infection and novel airway sampling techniques to measure IL-4, IL-5, IL-13, and IL-33 levels in the asthmatic and healthy airways during a rhinovirus infection. Additionally, we cultured human T cells and type 2 innate lymphoid cells (ILC2s) with the supernatants of rhinovirus- infected bronchial epithelial cells (BECs) to assess type 2 cytokine production in the presence or absence of IL-33 receptor blockade. Measurements and Main Results: IL-4, IL-5, IL-13, and IL-33 are all induced by rhinovirus in the asthmatic airway in vivo and relate to exacerbation severity. Further, induction of IL-33 correlates with viral load and IL-5 and IL-13 levels. Rhinovirus infection of human primary BECs induced IL-33, and culture of human T cells and ILC2s with supernatants of rhinovirus-infected BECs strongly induced type 2 cytokines. This induction was entirely dependent on IL-33. Conclusions: IL-33 and type 2 cytokines are induced during a rhinovirus-induced asthma exacerbation in vivo. Virus-induced IL-33 and IL-33 – responsive T cells and ILC2s are key mechanistic links between viral infection and exacerbation of asthma. IL-33 inhibition is a novel therapeutic approach for asthma exacerbations