Nasal interferon responses to community rhinovirus infections are similar in controls and children with asthma

Abstract

Background:

Rhinovirus (RV) is the main cause of asthma exacerbations in children. Some studies reported that persons with asthma have attenuated interferon (IFN) responses to experimental RV infection compared with healthy individuals. However, responses to community-acquired RV infections in controls and children with asthma have not been compared.

Objective:

To evaluate nasal cytokine responses after natural RV infections in people with asthma and healthy children.

Methods:

We compared nasal cytokine expression among controls and children with asthma during healthy, virus-negative surveillance weeks and self-reported RV-positive sick weeks. A total of 14 controls and 21 patients with asthma were studied. Asthma disease severity was based on symptoms and medication use. Viral genome was detected by multiplex polymerase chain reaction. Nasal cytokine protein levels were determined by multiplex assays.

Results:

Two out of 47 surveillance weeks tested positive for RV, illustrating an asymptomatic infection rate of 5%. A total of 38 of 47 sick weeks (81%) tested positive for the respiratory virus. Of these, 33 (87%) were positive for RV. During well weeks, nasal chemokine (C-X-C motif) ligand 8, interleukin 12 (IL-12), and IL-1β levels were significantly higher in children with asthma than controls. Compared with healthy virus-negative surveillance weeks, chemokine (C-X-C motif) ligand 8, IL-13, and interferon beta increased during colds only in patients with asthma. In both controls and children with asthma, the nasal levels of interferon gamma, interferon lambda-1, IL-1β, IL-8, and IL-10 significantly increased during RV-positive sick weeks. During RV infection, IL-8, IL-1β, and tumor necrosis factor-a levels were strongly correlated.

Conclusion:

In both controls and patients with asthma, natural RV infection results in robust type II and III IFN responses.

Introduction

Respiratory viral infections are responsible for more than 80% of asthma exacerbations in children, with human rhinovirus (RV) being the most common.^1–4 The exact mechanisms of RV-induced viral exacerbation are not fully understood. RV causes a common cold in healthy individuals but may initiate severe bronchoconstriction in patients with asthma. Several lines of evidence suggest that this difference arises from the variable interferon (IFN) expression and an improper shift toward a type 2 inflammatory response. During colds induced by experimental RV infection, patients with asthma with weak peripheral blood monocyte, sputum cell, and CD4 T cell interferon gamma (IFN-γ) responses exhibit more severe symptoms, delayed viral clearance, and reduced airway function,^5–7 consistent with the notion that low IFN-γ responses promote RV-induced exacerbations. In addition, some^8–11 but not all^12–14 ex vivo studies have suggested that epithelial and bronchoalveolar lavage (BAL) cell IFN responses to viral infection are impaired in people with asthma compared with healthy individuals. More importantly, differences in IFN profiles have not been studied in people with asthma and healthy controls after natural RV infection. We, therefore, investigated the cytokine profiles of nasal lining fluid from people with asthma and controls without asthma during naturally acquired RV infections and healthy intervals.

Methods

This observational study evaluated rhinovirus infections among children with physician-diagnosed asthma and children without asthma. Eligible patients were between ages 6 and 17 years and lived within 30 miles of Ann Arbor, Michigan. Patient recruitment and sampling occurred over a 2-year period. This study was approved by the institutional review board (HUM00141779). Written informed consent was obtained from a parent or guardian of all study participants. Children’s written assent was obtained for patients more than 9 years of age.

Specimens were obtained at the patient’s residence. Home visits were scheduled on 3 days during a week when the child was healthy (surveillance weeks) and on 3 days during weeks of self-reported illness (sick weeks). No patient experienced more than 3 illnesses in a single year. A 1-month interval was required between sequential illnesses. The first sick sample was usually taken with 48 hours of the initiation of symptoms. In year 1, nasal aspirates were obtained by squirting sterile saline spray 2 to 5 times (approximately 0.5 mL each) into the patient’s nares.3 Patients were instructed to occlude the opposite nostril and blow their nose into a zippered plastic bag. Aspirates were collected into a zippered plastic bag and 3 mL of M4RT viral transport medium (Remel, Lenexa, Kansas) and 60 μL of protease inhibitor (Roche Diagnostics, Indianapolis, Indiana) at a 50-to-1 dilution were added. In year 2, in addition to nasal aspirates, nasal fluid was obtained using absorbent (40 mm × 4 mm) strips of Leukosorb article (Pall, Port Washington, New York) inserted into the patient’s nose.^15,16 Researchers held the strip in the patient’s nose for 30 seconds while pressing on the outside of the nostril to absorb nasal fluid. Filter paper strips were stored in a dry, sterile 1.5 mL microcentrifuge tube. All samples were placed on −70°C ice packs until arrival at the laboratory, at which point they were transferred to a −70°C freezer.

RNA from aspirates was tested for the virus using the Novaplex II RV16 detection kit (Seegene, Walnut Creek, California). A week was considered RV-positive if at least 1 out of the 3 samples collected during that week tested positive. The 3 aspirate/filter paper samples from each surveillance week or sick week were pooled and cytokines were measured using Meso Scale Diagnostics multiplex biomarker assay kits (Rockville, Maryland). Using this system, interferon alfa-21 (IFN-α2a), interferon beta (IFN-β), interferon lambda 1 (IFN-λ1), IFN-γ1, interleukin (IL)-8, tumor necrosis factor (TNF)-α, IL-1β, IL-2, and IL-12p70 were present in many well samples and nearly all sick samples. However, IL-10, IL-13, IL-5, and TSLP were often undetectable, and IL-4, IL-25, and IL-33 were not detectable in any sample. We, therefore, employed Research and Development Luminex multiplex assays (Minneapolis, Minnesota) to measure IL-4, IL-25, and IL-33. Also, a direct comparison of year 2 nasal aspirate and filter paper samples using the Research and Development system confirmed that filter paper samples were more sensitive (eFig 1). Therefore, for year 2 samples, filter paper readings were used for analysis.

To determine the effects of RV illness on biomarker protein levels, paired samples were compared using Wilcoxon’s matched-pairs signed-rank test. To determine the effects of asthma on biomarker protein levels, sample results were compared using a Mann-Whitney U test (Wilcoxon rank-sum test). When a patient had more than 1 illness week per year, his/her sick week cytokine values were averaged before statistical analysis. Undetectable nasal aspirate cytokine levels were recorded as the value of the minimum detection limit. When there were a large number of missing values, results were categorized into detectable/nondetectable and analyzed as a binary variable using the χ² test. Correlations between different cytokine levels were assessed using Spearman’s correlation coefficient.

Secondary data collection from the medical chart was conducted for assessment of asthma severity and allergen sensitization. Asthma severity was determined according to the Asthma Expert Panel Report 3 and physician assessment. Allergen sensitization was determined by prick skin testing.

Results

A total of 21 children with physician-diagnosed asthma and 14 children without asthma (controls) (total N = 35) were included in the study. Because we allowed siblings to participate, the 35 patients came from 22 unique families. Ten patients participated in both years of the study. Participant demographics and health measures are provided in Table 1 and Table 2. Among participants with asthma, 15 (71%) took inhaled corticosteroids and 9 (43%) had moderate-to-severe persistent asthma. Over the 2-year period, 34 of 35 children provided samples from 1 or 2 surveillance weeks, for a total of 37 surveillance weeks. Twenty-nine of 35 children reported having 1 to 5 colds each for a total of 47 sick weeks.

Table 1.

Participant Baseline Demographics (n = 35)

Demographic	Patients with asthma	Control
N	21	14
Age in y, mean (SD)	10 (2.5)	12 (2.6)
Sex, male n (%)	11 (52)	5 (36)
Race or ethnicity, n (%)
White	14 (67)	10 (71)
African American	4 (19)	0 (0)
Multiracial	2 (10)	4 (29)
Other	1 (5)	0 (0)
Hispanic or Latino	2 (10)	2 (14)
Tobacco smoke exposure, n (%)	7 (33)	2 (14)
Self-reported allergies, n (%)
Hay fever or nasal allergies	17 (81)	2 (14)
Eczema	8 (38)	1 (7)

Table 2.

Initial Surveillance Period Health Measures in Patients With Asthma (n = 21)

Asthma severity, n (%)
Moderate to severe persistent	9 (43)
Mild persistent	7 (33)
Mild intermittent	5 (24)
Asthma medication use in previous 12 months (n, %)
Inhaled corticosteroid	15(71)
Long-acting β agonist	4 (19)
Short-acting β agonist	21(100)
Leukotriene receptor antagonist	8 (38)
Baseline airway function (n = 11)a
FVC, % predicted (SD)	105 (27)
FEV1, % predicted (SD)	94 (26)
FEV1/FVC, actual (SD)	78 (21)
FEF 25%−75%, % predicted (SD)	73 (23)
Allergy tests, n (%), (n = 8)b
Positive	7 (87.5)
Negative	1 (12.5)

Abbreviations: FEF 25%−75%, mean forced expiratory flow between 25% and 75% of the FVC; FEV1, forced expiratory volume in the first second; FVC, forced vital capacity; IgE, immunoglobulin E.

^aIncludes 8 children with moderate or severe persistent asthma and 3 with mild asthma.

^bIncludes 5 skin tests and 3 serum IgE tests.

Healthy surveillance visits were scheduled during periods of low respiratory viral prevalence, mostly July and August (Fig 1, left panel). A week was considered RV-positive if at least 1 out of the 3 samples collected during that week tested positive. Two of 47 surveillance weeks tested positive for RV with an asymptomatic infection rate of 5% (Table 3). Virus-positive samples from respiratory illnesses were mostly collected in September, October, and November (Fig 1, right panel). Thirty-eight of 47 sick weeks (81%) tested positive for the respiratory virus. Of these, 33 (87%) were positive for RV, again illustrating that RV is the most common cause of respiratory viral infections in children with asthma.1–4 Five illness weeks tested positive for viruses other than RV and were excluded from the analysis (2 human coronavirus NL63 and 3 respiratory syncytial virus B). Two RV-positive samples also tested positive for other viruses, 1 with human coronavirus NL63, and 1 with both parainfluenza 1 and respiratory syncytial virus B. Of the 33 RV-positive sick weeks, 12 had 1 virus-positive sample, 15 weeks had 2 positive samples, and 6 had all 3 virus-positive samples. There was no difference in the infection rate between controls and patients with asthma. After viral infection, 5 patients with asthma required additional bronchodilator treatments, and 7 patients with asthma missed school during viral infections.

Figure 1.

Histograms illustrating the monthly distribution of samples collected for well surveillance weeks (left panel) and self-reported sick weeks (right panel). RV-positive samples are shaded in gray. HCoV, human coronavirus; RSV, respiratory syncytial virus; RV, rhinovirus.

Table 3.

Viral Detection Data

Surveillance weeks	Virus-negative	Virus-positive	RV-positive
Control	16	0(0)	0
Asthma	18	2(10)	2(10)
Total	34	2 (5.6)	2 (5.6)
Illness weeks	Virus-negative	Virus-positive	RV-positive
Control	3	14 (82)a	12 (70)b
Asthma	6	24 (73)c	21 (70)d
Total	9	38 (77)	33 (70)

NOTE: Data are expressed as n or n (%).

Abbreviations: RSV, respiratory syncytial virus; RV, rhinovirus.

^aTwo RV-negative illness weeks tested positive for RSV-B and were excluded from the analysis.

^bOne patient had coinfection with parainfluenza type 1.

^cThree illness weeks tested positive for a virus other than RV, 2 with human coronavirus NL63 and 1 with RSV-B; these samples were excluded from the analysis.

^dOne patient had coinfection with human coronavirus NL63.

For cytokine analysis, 3 aspirate/filter paper samples from each surveillance week or sick week were pooled. Furthermore, when a patient had more than 1 illness week per year, his/her sick week cytokine values were averaged before analysis. In this way, we were able to obtained paired well and sick values for subsequent analysis. During well weeks, IL-8, IL-1β, and IL-12 levels were significantly higher in patients with asthma than controls (Fig 2). Next, we analyzed the effect of RV on cytokine expression. Chemokine (C-X-C motif) ligand 8 (CXCL8) and IL-13 significantly increased during colds only in patients with asthma. There also was a trend toward increased IFN-β only in patients with asthma (P = .05). Compared with controls, nasal IL-12 levels in children with asthma were higher during RV infection. In contrast, nasal IL-4 significantly decreased during colds only in controls. Finally, IFN-γ, IFN-λ1, TNFα, IL-1β, and IL-10 increased in both control and asthma groups.

Figure 2.

Nasal fluid cytokine levels. Nasal fluid cytokine levels in controls and in children with asthma during well surveillance periods and RV-positive sick periods. RNA was tested for the virus using the Novaplex II RV16 Detection kit (Seegene, Walnut Creek, California). Cytokines were measured using Meso Scale Diagnostics multiplex biomarker assay kits and Meso Sector S600 reader (Rockville, Maryland) or Research and Development Luminex multiplex assays. Medians and interquartile range are illustrated. The asterisk indicates P <.05, Wilcoxon’s matched-pairs signed-rank test (for paired comparisons between well and RV samples), Mann-Whitney U test (for comparisons between controls and patients with asthma) or χ² Wilcoxon rank-sum test (for IL-13). One-way nonparametric analysis of variance (Kruskal-Wallis test) followed by the 2-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli to correct for multiple comparisons. IFN, interferon; IL, interleukin; TNF, tumor necrosis factor; TSLP, thymic stromal lymphopoietin.

We then assessed cytokine expression in response to RV infection on the basis of the severity of asthma. All patients with moderate or severe persistent asthma had a history of admission to the hospital or emergency department for asthma treatment. These children had a wide range of IFN-λ1 and IFN-γ levels in their nasal lining fluid, with the median value being higher than that of patients with mild intermittent or mild persistent asthma (Fig 3). These data suggest that the lack of a reduced antiviral IFN response in patients with asthma was not because of mild disease. Asthma severity was not associated with differences in any other cytokines (data not given).

Figure 3.

Relationship between asthma severity and nasal IFN levels. Nasal IFN-λ1 and IFN-γ levels during RV-positive sick periods in children with asthma according to asthma severity. Asthma severity was determined according to Asthma Expert Panel Report 3 and physician assessment. Sensitization to specific allergens was determined either by prick skin testing or serum fluorescence enzyme immunoassay. During the course of asthma management, immunoglobulin E levels were measured for 3 patients with moderate-to-severe persistent asthma. Abbreviation: IFN-γ, interferon gamma; IFN-λ1, interferon lambda 1; RV, Rhinovirus.

After RV infection, there was a positive correlation between CXCL8, IL-1β, and TNF-α levels (Fig 4), consistent with the involvement of these cytokines in the antiviral response. Correlations are held in both controls and patients with asthma. There was no correlation between IL-8 and IFN levels.

Figure 4.

Relationships between nasal fluid cytokine levels. Nasal fluid cytokine levels in controls and children with asthma during RV-positive sick periods. IL, interleukin; TNF, tumor necrosis factor.

Discussion

Viral infections are the most common cause of asthma exacerbations in children, and RV is the most frequently identified viral pathogen.^1–4 We evaluated the nasal cytokine responses of 14 controls and 21 children with asthma during healthy surveillance weeks and natural colds. Viral genome was detected by multiplex polymerase chain reaction and nasal cytokine levels were determined by electrochemiluminescence. First, we found that baseline CXCL8, IL-1β, and IL-12 levels were significantly higher in patients with asthma than controls. Second, CXCL8 and IL-13 significantly increased during colds in patients with asthma but not in controls. There also was a trend toward increased IFN-β in patients with asthma. Third, nasal IL-12 levels in children were higher during RV infection in patients with asthma compared with controls. Finally, IFN-γ, IFN-λ1, IL-1β, CXCL8, and IL-10 significantly increased during RV-positive sick weeks in both controls and patients with asthma. Increased sputum and serum levels of the neutrophil chemo-attractant CXCL8 have been associated with atopic, nonatopic, and difficult-to-control asthma in children.^17,18 Increased BAL fluid concentrations of the T_H1 cytokine IL-12 have been found in children with severe asthma.¹⁹ IL-1β is increased in sputum and BAL fluid of patients with neutrophilic²⁰ and T_H2/T_H17-predominant asthma,²¹ consistent with inflammasome activation.²² Thus, in this real-life cohort of children with asthma, most of whom had a history of allergy, we did not find strong evidence for a distinct type 1 or type 2 asthma endotype. Consistent with this, of the type 2 cytokines we assessed, only IL-13 was increased in patients with asthma.

It has been suggested that patients with asthma have a deficient immune response to RV infection, leading to viral-induced exacerbation colds induced by experimental RV infection, patients with asthma with weak IFN-γ responses exhibit more severe cold symptoms and airway dysfunction,^5–7 consistent with the notion that low IFN-γ responses promote RV-induced exacerbations. Unlike IFN-γ, which has a broad range of functions, type I IFNs (including α and β) and type III IFNs (including λ) confer specific antiviral protection to target cells. The IFN-λ1 receptor is primarily limited to epithelial cells.²³ Bronchial epithelial and BAL cells from patients with asthma have exhibited reduced IFN-β and IFN-λ responses to RV infection ex vivo.^8–11 However, alternative studies in human bronchial epithelial cells from patients with and without asthma failed to exhibit a difference in viral-induced IFN protein levels.^12–14 Furthermore, a recent study of nasal and BAL lavage samples after experimental infection with RV exhibit robust IFN-λ responses in patients with asthma, greater than healthy individuals.²⁴ A second group confirmed similar IFN-α and IFN-γ responses in adult patients with and without asthma after intranasal administration of RV.²⁵ As far as we are aware, IFN responses to natural colds have never been compared in healthy children and children with asthma. We found that, after natural RV infection, IFN-λ1 and IFN-γ increased in both control and asthma groups. In addition, nasal IFN-β tended to increase during RV infection in patients with asthma. Together, these data do not illustrate deficient type I (IFN-α and IFN-β), type II (IFN-γ) or type III IFN responses (IFN-λ1) to natural colds in children with asthma. Robust increases in nasal IFN-λ1 were previously found in children with asthma having upper respiratory tract infections.²⁶

Our data are consistent with previous studies evaluating viral loads in patients with asthma and controls. Although the previous assumption was that deficient IFN responses allow higher viral loads in patients with asthma leading to asthma exacerbation, there was no difference in viral load between young adults with asthma and healthy individuals after experimental RV infection.²⁷ In addition, a study of natural colds found a similar burden of RV in controls without atopy vs the asthma group.²⁸

One important limitation of our study was the lack of immunoglobulin E (IgE) data in our cohort. Although 17 of 21 patients with asthma had a positive history of allergic rhinitis, only 8 had allergy skin testing and only 3 had measurements of serum IgE (not illustrated). In cultured epithelial cells infected with RV ex vivo, IFN-λ mRNA expression negatively correlates with total serum IgE.¹¹ IgE decreases plasmacytoid dendritic cell type 1 cytokine and IFN responses to RV infection²⁹ and treatment with omalizumab (anti-IgE) enhances plasmacytoid dendritic cell responses.³⁰ Thus, it could be argued that our patients with asthma did not have sufficient airway inflammation¹⁴ or serum IgE to suppress IFN production. On the other hand, as noted above, 2 previous studies reporting abundant nasal IFN levels after experimental RV infection included patients with high levels of IgE.^24,25 Finally, a recent line of evidence suggests that chronic airway inflammation after RV infection arises not from an impaired IFN response, but from amplified T_H1, and T_H2 responses even after the resolution of the RV infection.²⁵

A second important limitation is the lack of BAL data to corroborate our findings in the nasal airway. Although previous work has found qualitatively similar gene expression profiles,²⁴ nasal cytokine data may not reflect lower airway protein concentrations.

In summary, our data illustrate that type II and III IFN responses are not deficient in children with asthma after natural colds. The IFN response did not seem to depend on asthma severity or atopy status. There was also evidence for increased CXCL8, IL-12, IL-13, and IL-1β in patients with asthma. Although many studies have compared ex vivo and in vivo responses after experimental RV infection, as far as we are aware, this is the first study to compare RV responses after natural colds.