Cluster analysis of nasal cytokines during rhinovirus infection identifies different immunophenotypes in both children and adults with allergic asthma.

BACKGROUND: Infection with rhinovirus (RV) is a major risk factor for disease exacerbations in patients with allergic asthma. This study analysed a broad set of cytokines in the noses of children and adults with asthma during RV infection in order to identify immunophenotypes that may link to virus-induced episodes. METHODS: Nasal wash specimens were analysed in children (n = 279 [healthy, n = 125; stable asthma, n = 64; wheeze, n = 90], ages 2-12) who presented to a hospital emergency department, and in adults (n = 44 [healthy, n = 13; asthma, n = 31], ages 18-38) who were experimentally infected with RV, including a subset who received anti-IgE. Cytokines were measured by multiplex bead assay and data analysed by univariate and multivariate methods to test relationships to viral load, allergic status, airway inflammation, and clinical outcomes. RESULTS: Analysis of a core set of 7 cytokines (IL-6, CXCL8/IL-8, IL-15, EGF, G-CSF, CXCL10/IP-10 and CCL22/MDC) revealed higher levels in children with acute wheeze versus those with stable asthma or controls. Multivariate analysis identified two clusters that were enriched for acutely wheezing children; one displaying high viral load ("RV-high") with robust secretion of CXCL10, and the other displaying high IgE with elevated EGF, CXCL8 and both eosinophil- and neutrophil-derived mediators. Broader assessment of 39 cytokines confirmed that children with acute wheeze were not deficient in type 1 anti-viral responses. Analysis of 18 nasal cytokines in adults with asthma who received RV challenge identified two clusters; one that was "RV-high" and linked to robust induction of anti-viral cytokines and anti-IgE; and the other associated with more severe symptoms and a higher inflammatory state featuring eosinophil and neutrophil factors. CONCLUSIONS: The results confirm the presence of different immunophenotypes linked to parameters of airway disease in both children and adults with asthma who are infected with RV. Such discrepancies may reflect the ability to regulate anti-viral responses.

Human TH1 and TH2 cells targeting rhinovirus and allergen coordinately promote allergic asthma.

BACKGROUND: Allergic asthmatic subjects are uniquely susceptible to acute wheezing episodes provoked by rhinovirus. However, the underlying immune mechanisms and interaction between rhinovirus and allergy remain enigmatic, and current paradigms are controversial. OBJECTIVE: We sought to perform a comprehensive analysis of type 1 and type 2 innate and adaptive responses in allergic asthmatic subjects infected with rhinovirus. METHODS: Circulating virus-specific TH1 cells and allergen-specific TH2 cells were precisely monitored before and after rhinovirus challenge in allergic asthmatic subjects (total IgE, 133-4692 IU/mL; n = 28) and healthy nonallergic controls (n = 12) using peptide/MHCII tetramers. T cells were sampled for up to 11 weeks to capture steady-state and postinfection phases. T-cell responses were analyzed in parallel with 18 cytokines in the nose, upper and lower airway symptoms, and lung function. The influence of in vivo IgE blockade was also examined. RESULTS: In uninfected asthmatic subjects, higher numbers of circulating virus-specific PD-1+ TH1 cells, but not allergen-specific TH2 cells, were linked to worse lung function. Rhinovirus infection induced an amplified antiviral TH1 response in asthmatic subjects versus controls, with synchronized allergen-specific TH2 expansion, and production of type 1 and 2 cytokines in the nose. In contrast, TH2 responses were absent in infected asthmatic subjects who had normal lung function, and in those receiving anti-IgE. Across all subjects, early induction of a minimal set of nasal cytokines that discriminated high responders (G-CSF, IFN-γ, TNF-α) correlated with both egress of circulating virus-specific TH1 cells and worse symptoms. CONCLUSIONS: Rhinovirus induces robust TH1 responses in allergic asthmatic subjects that may promote disease, even after the infection resolves.

Understanding the asthmatic response to an experimental rhinovirus infection: Exploring the effects of blocking IgE.

BACKGROUND: Rhinovirus frequently causes asthma exacerbations among children and young adults who are allergic. The interaction between allergen and rhinovirus-induced symptoms and inflammation over time is unclear. OBJECTIVE: Our aim was to compare the response to an experimental inoculation with rhinovirus-16 in allergic asthmatics with the response in healthy controls and to evaluate the effects of administrating omalizumab before and during the infection. METHODS: Two clinical trials were run in parallel. In one of these trials, the response to an experimental inoculation with rhinovirus-16 among asthmatics with high levels of total IgE was compared to the response in healthy controls. The other trial compared the effects of administering omalizumab versus placebo to asthmatics in a randomized, double-blind placebo-controlled investigation. The primary outcome for both trials compared lower respiratory tract symptoms (LRTSs) between study groups over the first 4 days of infection. RESULTS: Frequent comparisons of symptoms, lung function, and blood eosinophil counts revealed differences that were more pronounced among allergic asthmatics than among controls by days 2 and 3 after virus inoculation. Additionally, an augmentation of upper respiratory tract symptom scores and LRTS scores occurred among the atopic asthmatics versus the controls during the resolution of symptoms (P < .01 for upper respiratory symptom tract scores and P < .001 for LRTS scores). The beneficial effects of administering omalizumab on reducing LRTSs and improving lung function were strongest over the first 4 days. CONCLUSIONS: LRTSs and blood eosinophil counts were augmented and lung function was reduced among allergic asthmatics early after rhinovirus inoculation but increased late in the infection during symptom resolution. The effect of administering omalizumab on the response to rhinovirus was most pronounced during the early/innate phase of the infection.

Comparison of viral load in individuals with and without asthma during infections with rhinovirus.

RATIONALE: Most virus-induced attacks of asthma are caused by rhinoviruses (RVs). OBJECTIVES: To determine whether people with asthma are susceptible to an increased viral load during RV infection. METHODS: Seventy-four children (4-18 yr old) were enrolled; 28 with wheezing, 32 with acute rhinitis, and 14 without respiratory tract symptoms. Nasal washes were evaluated using quantitative polymerase chain reaction for RV to judge viral load along with gene sequencing to identify strains of RV. Soluble intercellular adhesion molecule-1, IFN-λ1, and eosinophil cationic protein in nasal washes, along with blood eosinophil counts and total and allergen-specific IgE in sera, were also evaluated. Similar assessments were done in 24 young adults (16 with asthma, 8 without) who participated in an experimental challenge with RV (serotype 16). MEASUREMENTS AND MAIN RESULTS: Fifty-seven percent of wheezing children and 56% with acute rhinitis had nasal washes testing positive for RV. The geometric mean of viral loads by quantitative polymerase chain reaction in washes from wheezing children was 2.8-fold lower, but did not differ significantly from children with rhinitis (7,718 and 21,612 copies of viral RNA per microliter nasal wash, respectively; P = 0.48). The odds for wheezing were increased if children who tested positive for RV were sensitized to one or more allergens (odds ratio, 3.9; P = 0.02). Similarly, neither peak nor cumulative viral loads differed significantly in washes from adults with asthma compared with those without asthma during the experimental RV challenge. CONCLUSIONS: During acute symptoms, children infected with RV enrolled for wheezing or acute rhinitis had similar viral loads in their nasal washes, as did adults with and without asthma infected with RV-16 experimentally.

High titers of IgE antibody to dust mite allergen and risk for wheezing among asthmatic children infected with rhinovirus.

BACKGROUND: The relevance of allergic sensitization, as judged by titers of serum IgE antibodies, to the risk of an asthma exacerbation caused by rhinovirus is unclear. OBJECTIVE: We sought to examine the prevalence of rhinovirus infections in relation to the atopic status of children treated for wheezing in Costa Rica, a country with an increased asthma burden. METHODS: The children enrolled (n= 287) were 7 through 12 years old. They included 96 with acute wheezing, 65 with stable asthma, and 126 nonasthmatic control subjects. PCR methods, including gene sequencing to identify rhinovirus strains, were used to identify viral pathogens in nasal washes. Results were examined in relation to wheezing, IgE, allergen-specific IgE antibody, and fraction of exhaled nitric oxide levels. RESULTS: Sixty-four percent of wheezing children compared with 13% of children with stable asthma and 13% of nonasthmatic control subjects had positive test results for rhinovirus (P< .001 for both comparisons). Among wheezing subjects, 75% of the rhinoviruses detected were group C strains. High titers of IgE antibodies to dust mite allergen (especially Dermatophagoides species) were common and correlated significantly with total IgE and fraction of exhaled nitric oxide levels. The greatest risk for wheezing was observed among children with titers of IgE antibodies to dust mite of 17.5 IU/mL or greater who tested positive for rhinovirus (odds ratio for wheezing, 31.5; 95% CI, 8.3-108; P< .001). CONCLUSIONS: High titers of IgE antibody to dust mite allergen were common and significantly increased the risk for acute wheezing provoked by rhinovirus among asthmatic children.

Rhinovirus infection results in stronger and more persistent genomic dysregulation: Evidence for altered innate immune response in asthmatics at baseline, early in infection, and during convalescence.

BACKGROUND: Rhinovirus (HRV) is associated with the large majority of virus-induced asthma exacerbations in children and young adults, but the mechanisms remain poorly defined. METHODS: Asthmatics and non-asthmatic controls were inoculated with HRV-A16, and nasal epithelial samples were obtained 7 days before, 36 hours after, and 7 days after viral inoculation. RNA was extracted and subjected to RNA-seq analysis. RESULTS: At baseline, 57 genes were differentially expressed between asthmatics and controls, and the asthmatics had decreased expression of viral replication inhibitors and increased expression of genes involved in inflammation. At 36 hours (before the emergence of peak symptoms), 1329 genes were significantly altered from baseline in the asthmatics compared to 62 genes in the controls. At this time point, asthmatics lacked an increase in IL-10 signaling observed in the controls. At 7 days following HRV inoculation, 222 genes were significantly dysregulated in the asthmatics, whereas only 4 genes were dysregulated among controls. At this time point, the controls but not asthmatics demonstrated upregulation of SPINK5. CONCLUSIONS: As judged by the magnitude and persistence of dysregulated genes, asthmatics have a substantially different host response to HRV-A16 infection compared with non-asthmatic controls. Gene expression differences illuminate biologically plausible mechanisms that contribute to a better understanding of the pathogenesis of HRV-induced asthma exacerbations.

Clostridium difficile toxin A alters in vitro-adherent neutrophil morphology and function.

The effects of purified toxin A in vitro on the shape and function of polymorphonuclear leukocytes (PMNL) were examined. Toxin A induced changes in adherent PMNL shape from a compact spherical or pyramidal shape to a thin and rope-like shape. This change in shape was accompanied by rearrangement of the F-actin cytoskeleton into aggregates. Toxin A-treated PMNL exhibited increased adherence and expressed less L-selectin and more Mac-1, compared with untreated PMNL. In contrast to these proinflammatory actions, toxin A impaired both directed and non-directed PMNL migration in response to N-formylmethionylleucylphenylalanine. In addition, toxin A decreased the oxidative activity of adherent PMNL stimulated by recombinant human tumor necrosis factor-alpha. These effects could be explained by toxin A-induced glucosylation of the signaling small-size guanine 5'-triphosphate-binding proteins of the Rho family in human PMNL.

Experimental rhinovirus challenges in adults with mild asthma: response to infection in relation to IgE.

BACKGROUND: Although most children and young adults with asthma are atopic, exacerbations of asthma are frequently associated with viral respiratory tract infections, especially those caused by rhinovirus (HRV). OBJECTIVE: Young atopic adults with mild asthma were evaluated before and during an experimental HRV infection to test the hypothesis that airway inflammation before virus inoculation may be a risk factor for an adverse response to HRV. METHODS: Experimental HRV infections were evaluated in 16 allergic volunteers with mild asthma and 9 nonatopic control patients (age, 18 to 30 years). Before virus inoculation, each participant was screened with tests for lung function, prick skin tests for sensitization to common aeroallergens, measurements of total serum IgE, and serum neutralizing antibody to rhinovirus-16 (the serotype used for inoculation). The response to infection was monitored for 21 days by using symptom diary cards, tests for lung function, and markers of airway inflammation in nasal washes, blood, and expired air. RESULTS: During the infection, asthmatic patients had cumulative upper and lower respiratory tract symptom scores that were significantly greater over the course of 21 days than scores from the control patients. At baseline, the asthmatic patients also had increased sensitivity to methacholine and significantly lower values for FEV(1) (percent predicted) than the control patients (geometric mean and intraquartile values: 87% [79% to 91%] for the asthmatic patients and 101% [90% to 104%] for the control patients, P <.03). Among the patients with mild asthma, 6 had levels of total serum IgE that were substantially elevated (range, 371 to 820 IU/mL) compared with 10 who had lower levels (range, 29 to 124 IU/mL). Those with high levels of IgE had significantly greater lower respiratory tract symptom scores during the initial 4 days of the infection than the low IgE group. They also had higher total blood eosinophil counts at baseline, increased eosinophil cationic protein in their nasal washes (>200 ng/mL), and augmented levels of expired nitric oxide at baseline and during peak cold symptoms. In contrast, levels of soluble intracellular adhesion molecule-1 in nasal wash supernatants from the asthmatic patients with high IgE were diminished, both at baseline and during the infection. CONCLUSIONS: The reduced lung function and increased markers of inflammation observed before virus inoculation in the asthmatic patients who had high levels of total serum IgE may be risk factors for an adverse response to infections with HRV.

Viral infections in relation to age, atopy, and season of admission among children hospitalized for wheezing.

BACKGROUND: Viral respiratory tract infections and atopy are associated with attacks of wheezing during childhood. However, information about the relationship between viral infections and atopy among children whose attacks of wheezing lead to hospitalization is unclear. OBJECTIVE: To evaluate the prevalence of viral respiratory tract pathogens among infants and children hospitalized for wheezing and to analyze the results in relation to the patient's age, atopic characteristics, and season of admission. METHODS: This was a case-control study of children (age 2 months to 18 years) admitted for wheezing to the University of Virginia Medical Center over a period of 12 months. Children without wheezing were enrolled as controls. Nasal secretions were evaluated for viral pathogens by using cultures, PCR tests, and antigen detection. Total IgE and specific IgE antibody to common aeroallergens was measured in serum. RESULTS: Seventy percent of children hospitalized for wheezing before age 3 years (n=79) were admitted between December and March, whereas 46% of children age 3 to 18 years (n=54) were hospitalized between September and November. Among children younger than 3 years, viral pathogens were detected in 84% (66/79) of wheezing children and 55% (42/77) of controls (P <.001). Respiratory syncytial virus was the dominant pathogen during the winter months, but rhinovirus was more common during other months. Total serum IgE levels were generally low, and values from wheezing and control subjects overlapped considerably. Among children 3 years and older, 61% (33/54) of subjects admitted for wheezing tested positive for virus (predominantly rhinovirus), compared with 21% (12/56) of controls (P <.001). The total serum IgE values among wheezing children (geometric mean, 386 IU/mL; 95% CI, 259-573) were substantially elevated compared with those of controls (geometric mean, 38 IU/mL; 95% CI, 26-56; P <.001). A significantly higher percentage of wheezing children compared with controls was sensitized to at least 1 of the inhaled allergens tested: 84% (36/43) compared with 33% (15/45; P <.001). The atopic characteristics of wheezing children who tested positive or negative for virus were similar. CONCLUSIONS: Viral infections were the dominant risk factor for wheezing among children hospitalized before 3 years of age. By comparison, a large majority of the wheezing children age 3 to 18 years had striking atopic characteristics that may be critical as a risk factor for hospitalization and an adverse response to viral infections, especially infections caused by rhinovirus.