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

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

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

Consort Flow Chart.

<p>Consort Flow Chart.</p

Levels of interleukins (IL) in nasosorption eluates presented as area under the curve (AUC) for times 0.5-10h in relation to topical nasal challenge (n = 14 or 15).

<p>AUC is derived from from 0.5 since the baseline sample at -0.5h is pre-nasal lavage and is highly variable due to difference in individuals in terms of nasal microbes and inflammatory mediators. A) IL-1β as medians with quartiles; B) IL-6 as medians with quartiles; C) IL-1β AUC data for individual subjects; D) IL- AUC data for individual subjects. Note the idiosyncratic large responses of subject 2009 after nasal challenge with 10μg LPS, and subject 3101 after nasal challenge with 30μg LPS, these responses being too large to be presented on the y axis.</p

Nasal lipopolysaccharide challenge study design and procedures.

<p>14 subjects completed the entire protocol (<i>per protocol</i>), while 1 subject only received nasal challenges at placebo and LPS. TNSS–total nasal symptom score. AE–adverse events</p

Idiosyncratic large nasal responses to lipopolysaccharide (LPS) on 2 occasions.

<p>Raw data for levels of chemokines and cytokines (pg/ml) in nasal mucosal lining fluid (MLF). Data is presented for individual subjects: A. Subject 2009 after nasal challenge with 10μg LPS. B. Subject 3101 after nasal challenge with 30μg LPS. Note that baseline levels of cytokines and chemokines at -0.5h are markedly raised, and come down to only a small extent after nasal lavage at 0.5h. It is suggested that these individuals may have had a non-symptomatic alteration in their nasal microbial flora prior to LPS challenge on these opccasions: possibly the individuals had a subclinical viral infection or bacterial colonisation.</p

ICAM-1 mRNA abundance within nasal curettage samples after nasal LPS challenge, Data is shown as means with 95% confidence intervals.

<p>Relative abundance of expression with Reverse Transcriptase quantitative Polymerase Chain Reaction (RT-qPCR) using normalized data and (glyceraldehyde-3-phosphate dehydrogenase) (GAPDH) as a housekeeper gene (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135363#sec008" target="_blank">Methods</a>).</p

Median levels of chemokines and cytokines (pg/ml) in nasal mucosal lining fluid (MLF) are shown after nasal spray challenge with 4 different doses of LPS and a placebo (see key below).

<p>Lower limits of detection (LLOD) for cytokines and chemokines were as follows: IL-1β (5.0pg/ml), IL-6 (5.0pg/ml), CXCL8/IL-8 (10.0pg/ml), CCL3/MIP-1α (5.0pg/ml), CCL2/MCP-1 (25.0pg/ml), IL-10 (5.0pg/ml). Time -0.5h refers to nasosorption performed 30 min prior to topical nasal challenge, and this varies considerably between individuals due to variations in individual’s microbial flora and mucosal immune responses. Nasal lavage was performed after nasosorption at -0.5h, but prior to nasal challenge, in order to partially wash the nose free of baseline inflammatory microbes and mediators, but causes detectable levels of inflammatory mediators at 0.5h to decrease markedly from -0.5h. There is a tendency for nasosorption levels of mediators to gradually increase from 0.5h to 10h in the placebo arm, but levels of inflammatory cytokines and chemokines after LPS are generally higher.</p

Nasal lavage leukocyte counts (medians expressed as cells/ml) following nasal LPS challenge.

<p>Cells were counted in a modified Neubauer chamber, and a leukocyte differential performed on a stained cytospin observed under a light microscope.</p