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American Journal of Respiratory and Critical Care Medicine logoLink to American Journal of Respiratory and Critical Care Medicine
. 2021 Mar 30;204(11):1259–1273. doi: 10.1164/rccm.202010-3754OC

Pulmonary Innate Lymphoid Cell Responses during Rhinovirus-induced Asthma Exacerbations In Vivo: A Clinical Trial

Jaideep Dhariwal 1,2,3,✉, Aoife Cameron 1,2, Ernie Wong 1,2, Malte Paulsen 4, Maria-Belen Trujillo-Torralbo 1,2, Ajerico del Rosario 1,2, Eteri Bakhsoliani 1,2, Tatiana Kebadze 1,2, Mark Almond 1,2, Hugo Farne 1,2, Leila Gogsadze 1,2, Julia Aniscenko 1,2, Batika M J Rana 2,5, Trevor T Hansel 1,2, David J Jackson 2,4,5, Onn Min Kon 1,2, Michael R Edwards 1,2, Roberto Solari 1,2, David J Cousins 2,5,6, Ross P Walton 1,2,*, Sebastian L Johnston, on behalf of the MRC-GSK Strategic Alliance Consortium1,2,*; on behalf of the MRC-GSK Strategic Alliance Consortium
PMCID: PMC8786078  PMID: 34469272

Abstract

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 and at Day 3, and Day 8 after inoculation. Pulmonary ILC1s and ILC2s were quantified in bronchoalveolar lavage using flow cytometry. The ratio of bronchoalveolar lavage 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 in healthy subjects. At Day 8, ILC2s significantly increased from baseline in both groups, which was significantly higher in patients with 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 patients with asthma correlated with clinical exacerbation severity and type 2 cytokines in nasal mucosal lining fluid.

Conclusions

An ILC2-predominant inflammatory profile in patients with 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.

Keywords: innate lymphoid cells, ILC1, ILC2, asthma, rhinovirus

At a Glance Commentary

Scientific Knowledge on the Subject

Enrichment of type 2 innate lymphoid cells (ILC2s) has been previously shown in patients with asthma, including in the nasal mucosa in response to allergen challenge. The potential role of ILC2s in rhinovirus-induced asthma exacerbations is not well-characterized, including the kinetics and relative contributions of different ILC subsets in response to rhinovirus infection.

What This Study Adds to the Field

This study demonstrates differential ILC1 and ILC2 responses in the lower airways of patients with asthma and healthy subjects following experimental rhinovirus challenge. An ILC2-predominant response was associated with greater severity of rhinovirus infection in asthma, as determined by greater reductions in lung function and increased upper and lower respiratory symptoms, as well as increased type 2 cytokine and chemokine expression.

Rhinovirus (RV) infections are the major trigger of asthma exacerbations. Clinical models of experimental RV challenge permit controlled study of RV-induced asthma exacerbations with accurate monitoring of clinical symptoms, lung function, and airway inflammation. Previous studies have evaluated patients with mild and moderate asthma (1), with varying asthma control (2). To reflect the significantly greater morbidity associated with more severe asthma and poor asthma control (3), we performed experimental RV challenge in patients with moderate asthma that was not well-controlled. Whereas previous studies performed bronchoscopy at a single time point following RV challenge (Day 4) (1, 2), we report bronchoscopy results at Days 3 and 8 after inoculation, allowing the evaluation of lower airway responses over time.

RV induction of the type 2 cytokines IL-4, IL-5, and IL-13 has been demonstrated in clinical and animal models of asthma exacerbation (2, 4). The magnitude of type 2 responses following RV infection correlated with exacerbation severity (2). Clinical trials and real-world experience of biologics targeting type 2 cytokines have demonstrated substantial reductions in asthma exacerbation rates in severe asthma (5–17). This evidence suggests enhanced type 2 cytokine responses following respiratory virus infection may be implicated in the pathogenesis of asthma exacerbations.

Identification of type 2 innate lymphoid cells (ILC2s) as a potent, early source of type 2 cytokines has led to the suggestion that they may play a key role in the generation of the type 2 pathology seen in asthma and asthma exacerbations. The majority of the evidence regarding the role of ILC2s as effector cells of the innate immune system derives from animal models (18–23). In clinical studies, bronchoalveolar lavage (BAL) ILC2s have been identified in patients with asthma (24, 25). Our group previously reported the identification of ILC2s in the nasal mucosa, which were increased in response to allergen provocation in patients with asthma (26).

Virus infection typically involves activation of type 1, IFN-γ–producing CD4+ T cells. A corresponding ILC1 subset capable of secreting IFN-γ was initially identified in the gut mucosa (27, 28) and later reported in the lung (29, 30), with potential roles in antiviral responses (31, 32). The secretion of IFN-γ is known to suppress type 2 immune responses, yet this does not align mechanistically with the observations regarding type 2 immunopathology in virus-mediated asthma exacerbations. It is well established that counterregulation of type 1/type 2 responses exists for cells of the adaptive immune response (33–35), with additional evidence that similar regulation occurs for ILC1/ILC2 subsets (36–38). However, there are no data on the frequencies of pulmonary ILC1s or ILC2s during clinical respiratory virus infections or virus-induced asthma exacerbations.

In the present study, we used a clinical RV challenge model of virus-induced asthma exacerbation to investigate pulmonary ILC and clinical responses at baseline and following RV infection in patients with asthma and compared their responses to those of healthy control subjects.

Some of the results of this study have been previously reported in the form of abstracts (39, 40).

Methods

Study Approval

This study was approved by the London Bridge Research Ethics Committee (12LO/1278). Written informed consent was obtained from all subjects prior to their participation. Patients were recruited into the study between February 2013 and May 2015.

Study Participants

Nonsmoking patients with atopic asthma and nonsmoking, nonatopic healthy subjects were eligible for the study. Subjects were aged 18–55 years with absent serum-neutralizing antibodies to RV-16 at screening. Patients with asthma were required to have a doctor diagnosis of asthma treated with inhaled corticosteroids at a daily dose of 100 μg fluticasone or equivalent, airway hyperresponsiveness or a bronchodilator response of ⩾12%, a worsening of asthma symptoms with infection since last change in asthma therapy, and an Asthma Control Questionnaire–6 (ACQ-6) score of >0.75. Patients with asthma and pollen sensitization were inoculated with virus outside of the hayfever season. Full inclusion and exclusion criteria are available in the online supplement.

Study Design

Subjects underwent baseline sampling including bronchoscopy 2 weeks before inoculation with RV-16 on Day 0. Patients were inoculated with 100 TCID50 RV-16 administered nasally via an atomizer as previously described (2). Bronchoscopies were performed on Days 3 and 8 after inoculation. Subjects completed daily diary cards of upper and lower respiratory symptoms from baseline sampling until 6 weeks after inoculation as previously described (1, 2). Subjects attended clinic visits on Days 1–8, 11, 15, and 42 after inoculation for assessment and sampling (see Figure E1 in the online supplement).

Clinical Sampling and Assessments

Spirometry was performed daily using a PiKo-1 spirometer (nSpire Health). RV was detected using PCR of nasal lavage and BAL samples as described (1, 2). Nasosorption and bronchosorption were performed as described (2), and levels of IL-33, type 1 (IFN-γ, IL-12, CXCL10/IP10 and CXCL11/ITAC) and type 2 (IL-4, IL-5, IL-9, IL-13, CCL17/TARC, CCL22/MDC) soluble mediators were measured (Meso Scale Discovery). Further details on study design are available in the online supplement.

Flow Cytometric Analysis

BAL cells were processed immediately after bronchoscopy. Cells were filtered, washed, and incubated in human serum before undergoing surface staining followed by an initial primary sort using a Becton Dickinson Aria IIIU Cell Sorter to identify four cell populations of interest (HLA-DR− lineage− cells, HLA-DR+ lineage− cells, CD19+ cells, and CD3+ cells) (Table E2 and Figure E2). Following the primary sort, HLA-DR− lineage− cells were filtered, washed, and incubated in human serum (Sigma-Aldrich) before surface staining for ILC2s (Lin−, CD123−, FCεRIα−, CD45+ , CD127+ , CRTH2+) and ILC1s (Lin−, CD123−, FCεRIα−, CD45+, CD127+, CRTH2−, c-Kit−, NKp44−). ILC populations were then sorted and analyzed using a Becton Dickinson Aria IIIU Cell Sorter (Figure 1). Positive/negative staining was determined using fluorescence minus one controls. The number of ILCs/mL BAL were calculated for each patient by multiplying their percentage of total live cells by the total number of cells stained, taking into account the total BAL volume. Further details are available in the online supplement.

Figure 1.

Figure 1.

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Gating strategy for identification of type 1 innate lymphoid cells (ILC1) and ILC2 populations. Shown is a representative flow cytometric plot identifying the gating strategy used to identify ILC1 and ILC2 populations. ILC populations were isolated and collected using fluorescence-activated cell sorting. ILC2s were identified as lineage (CD2/CD3/CD14/CD16/CD19/CD56/CD235a) negative, CD123−ve, FcεRI−ve, CD127+ve, CD45+ve, CRTH2+ve cells. ILC1s were identified as lineage (CD2/CD3/CD14/CD16/CD19/CD56/CD235a) negative, CD123−ve, FcεRI−ve, CD127+ve, CD45+ve, CRTH2−ve, c-kit−ve, NKp44–ve cells. FSC = forward scatter; SSC = side scatter.

Statistical Analysis

Statistical analysis was performed using GraphPad Prism 9 (GraphPad Software). Data are presented as mean ± (standard error of mean [SEM]) if normally distributed or median (interquartile range) if nonparametric. Differences within groups were determined using Wilcoxon’s signed rank test. Differences between groups were determined using Mann-Whitney tests. For analysis of three or more unmatched groups, a Kruskal-Wallace nonparametric ANOVA was first performed. Two-way ANOVA with Bonferroni’s multiple posttest comparison was used to test differences for parametric data. Correlations were investigated using Spearman’s rank correlation test for nonparametric data. Differences were considered significant at P < 0.05. All reported P values are two-sided.

Results

Fifteen patients with asthma and 14 healthy subjects were inoculated with RV-16. Overall, 23/29 (79%) subjects (11/15 patients with asthma and 12/14 healthy subjects) had confirmed RV-16 infection, evidenced by presence of RV-16–specific serum neutralizing antibodies 6 weeks after inoculation and/or RV-16 detection in nasal lavage or BAL at any time point after inoculation. Subjects without confirmed RV-16 infection were excluded from further analysis. Among patients with asthma with positive RV-16 infection, eight were poorly controlled, and three were partially controlled (41). Baseline characteristics of successfully infected subjects are shown in Table 1.

Table 1.

Baseline Characteristics of Study Volunteers

Characteristic Healthy Subjects (n = 12) Subjects with Asthma (n = 11) P Value
Age, yr 27 ± 3 31 ± 3 —
Sex, %     —
 Male 67 64  
 Female 33 36  
Baseline FEV1      
Percentage predicted 99 ± 3 90 ± 6 0.03
Baseline histamine PC20, mg/mL >8 1.63 ± 0.69 —
Baseline ACQ score — 1.59 ± 0.14 —
 ACQ ⩾ 0.75 but < 1.5, n — 3 —
 ACQ ⩾ 1.5, n — 8 —
Dose ICS (BDP or equivalent), μg — 873 ± 213 —
Total serum IgE, IU/ml      
 Median 28 201 <0.001
 Interquartile Range 10–58 152–352  
Skin Prick Test responses      
Number of positive responses, %      
 Grass — 8 (73) N/A
 HDM — 11 (100)
 Mugwort — 0 (0)
 Cladosporium — 2 (18)
 Alternaria — 2 (18)
 Aspergillus — 0 (0)
 Birch — 6 (55)
 Three Tree — 7 (64)
 Cat — 5 (45)
 Dog — 2 (18)
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Definition of abbreviations: ACQ = Asthma Control Questionnaire; BDP = beclomethasone dipropionate; HDM = house dust mite; ICS = inhaled corticosteroid; IU = international units; N/A = not applicable; PC20 = provocative concentration of histamine to provoke a 20% reduction in FEV1.

Data presented as mean ± SEM, unless otherwise stated.

Patients with Asthma That Is Not Well-Controlled Experience Greater Respiratory Morbidity and Nasal Viral Load after RV-16 Infection

After RV-16 inoculation, patients with asthma experienced greater, more prolonged upper and lower respiratory symptoms and greater reductions in peak expiratory flow (PEF) and forced expiratory volume in 1 second (FEV1) than healthy subjects (Figures 2A–2D). Patients with asthma had an increase in airway hyperresponsiveness on Day 6 after RV infection compared with baseline (Figure E3). Nasal viral load was significantly higher (nearly 40-fold) at Day 4 in patients with asthma than in healthy subjects: median 5.81 × 106 copies/ml (2.79 × 105–1.80 × 107) versus 1.55 × 105 copies/ml (1.38 × 104–8.35 × 105); P = 0.032 (Figure 2E). Higher baseline ACQ6 scores were associated with higher peak nasal viral load in patients with asthma (Figure 2F). Bronchial viral load was below the limit of detection in the majority of patients (Figure E4).

Figure 2.

Figure 2.

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Increased virus-induced morbidity and greater virus load in patients with asthma following viral challenge. (A–C) Rhinovirus infection resulted in greater upper (A) and lower (B) respiratory symptoms in patients with asthma accompanied by greater and more prolonged reductions in their morning peak expiratory flow (PEF), expressed as the percentage change from baseline (C). (D) The maximal fall in FEV1 during the 2-week period after inoculation was greater in asthma. (E) Nasal lavage virus load was significantly greater in asthma at Day 4 after inoculation. (F) Peak nasal lavage virus load in asthma correlated with baseline preinfection Asthma Control Questionnaire (ACQ). Between group statistical analyses were performed using Mann-Whitney U tests. Data displayed as (A–D) means and (E) box and whisker plots. Boxes display medians and interquartile range, with whiskers representing maximum and minimum values. P values: * < 0.05 and ** < 0.01. Correlations were performed using the nonparametric Spearman rank correlation.

Type 1 Cytokines and Chemokines Are Increased in Airways of Patients with Asthma and Healthy Subjects during RV-16 Infection

Both groups had significantly increased nasal IFN-γ during RV infection (patients with asthma, P = 0.002; healthy subjects, P = 0.001) (Figure 3A). Bronchial IFN-γ was increased in patients with asthma from baseline to Day 8 (P = 0.012), while there was a trend for increased IFN-γ in healthy subjects from baseline to Day 3 (P = 0.08) (Figure 3B). No correlations were observed between BAL IFN-γ and ILC numbers, virus load, or symptom scores at any time point.

Figure 3.

Figure 3.

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Increased type I cytokines/chemokines in upper and lower airways of both patients with asthma and healthy subjects after virus inoculation. (A–H) Rhinovirus infection resulted in induction of upper and lower airways type I cytokines and chemokines in both patients with asthma and healthy subjects. Nasal cytokine values are presented as the baseline level, the maximal level during the 2-week infection period, and the Day 42 level for each individual. Bronchial cytokine levels are presented at the baseline and postinfection Day 3 and Day 8 time points. P values: * < 0.05, ** < 0.01, and *** < 0.001; values from 0.05 to 0.1 are given numerically, values >0.1 are not displayed. IP-10 = interferon-γ–inducible protein 10 kD; ITAC = interferon-inducible T-cell alpha chemoattractant.

Both groups had significantly increased nasal CXCL10/IP-10, CXCL11/ITAC, and IL-12 during infection (all P < 0.01) (Figures 3C, 3E, and 3G). Bronchial CXCL10/IP-10 increased in healthy subjects from baseline to Day 8 (P = 0.027). There were similar trends in patients with asthma for increased CXCL10/IP10 (P = 0.055) and CXCL11/ITAC (P = 0.055) from baseline to Day 8 (Figures 3D, 3F, and 3H). A time course for mediators evaluated is included in the online supplement (Figure E5).

Nasal IL-33 Is Induced by RV-16 Infection in Asthma

Patients with asthma had a significant increase in nasal IL-33 during RV infection (P = 0.006) (Figure 4A). IL-33 levels increased during infection in healthy subjects, but the increase was not statistically significant (P = 0.301) (Figure 4A). Peak nasal IL-33 correlated to peak nasal viral load in asthma (P = 0.002, r = 0.867). There was no baseline difference between groups in bronchial IL-33, and levels did not change significantly at Days 3 or 8 in either group (Figure 4B).

Figure 4.

Figure 4.

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Patients with asthma have greater airway type 2 cytokines/chemokines at baseline and during viral infection. (A–J) Rhinovirus infection in patients with asthma resulted in the induction of IL-33, type 2 cytokines, and chemokines in vivo. Nasal cytokine values are presented as the baseline level, the maximal level during the 2-week infection period, and the Day 42 level for each individual. Bronchial cytokine levels are presented at the baseline and postinfection Day 3 and Day 8 time points. P values: * < 0.05, ** < 0.01, and *** < 0.001; values from 0.05 to 0.1 are given numerically, values >0.1 are not displayed. MDC = macrophage-derived chemokine; ns = not significant; TARC = thymus and activation-regulated chemokine.

Patients with Asthma Have Greater Type 2 Cytokines and Chemokines in Nasal Mucosal Lining Fluid at Baseline and during RV-16 Infection

Patients with asthma had increased nasal IL-5 at baseline compared with healthy subjects (P = 0.001) (Figure 4C). There were no differences in baseline nasal CCL17/TARC, CCL22/MDC, IL-4, IL-9, or IL-13 between patients with asthma and healthy subjects (Figures 4E, 4G, 4I, and 4J). Patients with asthma had significantly increased nasal IL-4 (P = 0.008), IL-5 (P = 0.001), IL-9 (P = 0.016), IL-13 (P = 0.004) and CCL22/MDC (P = 0.031) during infection (Figures 4C, 4G, 4I, 4J, and E6). Healthy subjects had significantly increased nasal CCL17/TARC (P < 0.05), and there was a trend for increased nasal CCL17/TARC in patients with asthma (P = 0.06) (Figure 4E).

Patients with asthma had significantly higher nasal IL-5 compared with healthy subjects during infection (P < 0.0001), which was maintained through Day 42 (P = 0.045) (Figure 4C). A similar trend was observed for increased nasal IL-13 in patients with asthma compared with healthy subjects during infection (P = 0.08) (Figure 4J). Patients with asthma had significantly higher bronchial IL-5 at baseline, Day 3, and Day 8 (all P < 0.05) (Figure 4D) and CCL17/TARC at Day 8 (P = 0.005) (Figure 4F) compared with healthy subjects. Bronchial IL-4, IL-9, and IL-13 levels were mostly undetectable, and data are not shown for these mediators. A time course of nasal mediators is included in the online supplement (Figure E7).

Pulmonary ILC2s Are Increased in Patients with Asthma at Baseline and Further Increased during RV-16 Infection

Representative ILC2 flow plots from healthy subjects and patients with asthma are displayed in Figure 5. At baseline, patients with asthma had significantly more BAL ILC2s compared with healthy subjects: 225.9 cells/ml (20.9–586.3) versus 32.7 cells/ml (9.3–68.2), respectively (P = 0.037) (Figure 6A). BAL ILC2s increased in both groups during infection. In healthy subjects, BAL ILC2s increased to 324.8 cells/ml (22–725.6) at Day 3 (P = 0.002) and 439.8 cells/ml (207.2–565.1) at Day 8 (P = 0.003). In patients with asthma, BAL ILC2s increased to 421.5 cells/ml (38.4–1695) at Day 3 (P = 0.11) and 1,913 cells/ml (380.5–3270) at Day 8 (P = 0.032). ILC2s at Day 8 were significantly higher in patients with asthma than in healthy subjects (P = 0.044) (Figure 6A).

Figure 5.

Figure 5.

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Changes in type 2 innate lymphoid cell populations following rhinovirus-16 infection. Shown are representative flow cytometric plots identifying the type 2 innate lymphoid cell population at baseline, Day 3, and Day 8 after rhinovirus infection in a healthy subject and in a patient with asthma.

Figure 6.

Figure 6.

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Patients with asthma have greater numbers of lower airway type 2 innate lymphoid cells (ILC2s) at baseline and during rhinovirus infection. (A and B) Numbers of lower airway ILC2s were significantly greater in asthma at baseline, whereas numbers of ILC1s at baseline were not significantly different between groups. Rhinovirus infection resulted in increased numbers of both bronchial ILC1 and ILC2 populations with both ILC1s and ILC2s increased early on Day 3 during rhinovirus infection in healthy subjects, but later on Day 8 in patients with asthma. (C) There was a greater ratio of ILC2s to ILC1s in patients with asthma both at baseline and following rhinovirus infection. ILC2 : ILC1 ratio generated for each subject at each bronchial sampling time point. P values: * < 0.05, ** < 0.01, and *** < 0.001; values from 0.05 to 0.1 are given numerically, values >0.1 are not displayed.

There were no significant differences in BAL ILC1s between groups at baseline: 295.3 cells/ml (50.8–620.2) in healthy subjects versus 152.7 cells/ml (46.9–1798) in patients with asthma (P = 0.487). In healthy subjects, BAL ILC1s increased to 2,416 cells/ml (1675–3949) at Day 3 (P = 0.001) and 2,014 cells/ml (620.7–3336) at Day 8 (P = 0.002). In patients with asthma, ILC1s increased to 668.6 cells/ml (322.9–2316) at Day 3 (P = 0.375) and 1,599 cells/ml (1036–4109) at Day 8 (P = 0.042) (Figure 6B). There was a trend for enrichment of ILC1s at Day 3 in healthy subjects compared with patients with asthma (P = 0.059) (Figure 6B). Type 3 ILCs (ILC3s) were also identified as part of our gating strategy. ILC3 numbers increased after infection in healthy subjects but not in patients with asthma. These results are included in the online supplement (Figure E8).

ILC2 : ILC1 Ratios Are Greater in Patients with Asthma at Baseline and during RV-16 Infection

The ratio of BAL ILC2 : ILC1 numbers at baseline and Days 3 and 8 time points was assessed as a marker of their relative contributions to the inflammatory response. Patients with asthma had significantly higher ILC2 : ILC1 ratios than healthy subjects at baseline: 0.25 (0.22–1.26) versus 0.13 (0.05–0.29), respectively (P = 0.024); and Day 8: 0.44 (0.36–1.5) versus 0.17 (0.14–0.33), respectively (P = 0.005); similar trends were observed at Day 3: 0.39 (0.13–2.03) versus 0.16 (0.08–0.24]), respectively (P = 0.059) (Figure 5C). The ratio of ILC2 : ILC1 did not change significantly in either group after infection. In all healthy subjects, ILC1s outnumbered ILC2s at every time point assessed. Although most patients with asthma had more ILC1s compared with ILC2s, three subjects had more ILC2s than ILC1s as evidenced by an ILC2 : ILC1 ratio of >1 (Figure 6C).

Pulmonary ILC Ratios Are Associated with Exacerbation Severity, Viral Load, and Type 2 Cytokine Levels

Higher ILC2 : ILC1 ratios at Day 3 significantly correlated with peak nasal viral loads at Day 4 (P = 0.006, r = 0.622) (Figure 7A). Higher ILC2 : ILC1 ratios at baseline, Day 3, and Day 8 significantly correlated with increased total lower respiratory symptom score during infection: P = 0.031, r = 0.472; P < 0.001, r = 0.730; P = 0.024, r = 0.490, respectively (Figures 7B–7D). Higher ILC2:ILC1 ratios at Day 3 significantly correlated with increased changes in PEF on Day 10 (P = 0.042, r = −0.459); Day 11 (P = 0.019, r = −0.520); Day 12 (P = 0.011, r = −0.553); Day 13 (P = 0.020, r = −0.517); and Day 14 (P = 0.026, r = −0.496). Higher baseline ILC2 : ILC1 ratios significantly correlated with increased change in FEV1 at Day 4 after inoculation (P = 0.026, r = −0.485).

Figure 7.

Figure 7.

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Lower airway innate lymphoid cell (ILC) ratios correlate with clinical markers of infection severity and induction of type 2 soluble mediators. ILC2 : ILC1 ratios at baseline, Day 3, and Day 8 time points correlated with (A) virus load and (B–D) total lower respiratory symptom scores (LRSSs). Total lower respiratory symptom scores were calculated by summation of daily scores over the 14 days after virus inoculation. ILC2 : ILC1 ratios at baseline, Day 3, and Day 8 time points correlated with the type 2 cytokine IL-5 in vivo. ILC2 : ILC1 ratios were generated for each subject at each bronchial sampling time point. Correlations were performed using the nonparametric Spearman rank correlation.

There was a significant relationship between the pulmonary ILC2 : ILC1 ratio at baseline and IL-5 levels in nasal mucosal lining fluid at baseline (P = 0.006, r = 0.557) (Figure 7E). There were multiple positive correlations between pulmonary ILC2 : ILC1 ratios at baseline, Day 3, and to a lesser extent Day 8, with the levels of type 2 cytokines, chemokines, and IL-33 in nasal mucosal lining fluid during infection (Table 2). The strongest relationship was observed with IL-5. There was a strong relationship between the pulmonary ILC2 : ILC1 ratio at baseline and nasal IL-5 levels on multiple days during infection (Table 2 and Figure 7F). Similar relationships were observed between pulmonary ILC2 : ILC1 ratios on Days 3 and 8 and nasal IL-5 levels (Table 2 and Figures 7G and 7H). There were also significant relationships between pulmonary ILC2 : ILC1 ratio at baseline, Day 3, and Day 8 and bronchial IL-5, CCL17/TARC, and CCL22/MDC concentrations (Table E3).

Table 2.

Correlations between ILC2 : ILC1 Ratios, IL-33, and Type 2 Soluble Mediator Levels in Nasal Mucosal Lining Fluid during Rhinovirus Infection

Baseline ILC2:ILC1 Ratio
IL-33  
 Day 0 r = 0.489, P = 0.018
IL-5  
 Day 0 r = 0.557, P = 0.006
 Day 2 r = 0.415, P = 0.049
 Day 3 r = 0.356, P = 0.096
 Day 4 r = 0.430, P = 0.041
 Day 5 r = 0.545, P = 0.007
 Day 6 r = 0.538, P = 0.008
 Day 11 r = 0.561, P = 0.008
 Day 15 r = 0.474, P = 0.026
IL-13  
 Day 5 r = 0.433, P = 0.039
IL-4  
 Day 6 r = 0.543, P = 0.008
IL-9  
 Day 4
 r = 0.420, P = 0.046
Day 3 ILC2:ILC1 Ratio
IL-5  
 Day 3 r = 0.446, P = 0.038
 Day 4 r = 0.589, P = 0.004
IL-13  
 Day 4 r = 0.532, P = 0.011
 Day 8 r = 0.500, P = 0.018
IL-4  
 Day 6 r = 0.544, P = 0.009
 Day 8 r = 0.461, P = 0.031
IL-9  
 Day 3 r = 0.634, P = 0.002
 Day 4 r = 0.379, P = 0.082
MDC  
 Day 1 r = 0.470, P = 0.027
TARC  
 Day 1 r = 0.478, P = 0.025
 Day 2
 r = −0.499, P = 0.018
Day 8 ILC2:ILC1 Ratio
IL-5  
 Day 11 r = 0.635, P = 0.002
IL-13  
 Day 11 r = 0.544, P = 0.089
MDC  
 Day 5 r = 0.420, P = 0.046
 Day 6 r = 0.463 P = 0.026
TARC  
 Day 6 r = 0.478, P = 0.021
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Definition of abbreviations: ILC = innate lymphoid cell; MDC = macrophage-derived chemokine; TARC = thymus and activation-regulated chemokine.

Boldface text indicates significant P values.

Discussion

RV infections are a major cause of asthma exacerbations, and experimental viral challenge studies have contributed to our understanding of the mechanisms underlying the morbidity of RV-associated asthma exacerbations. Here, we characterized clinical and immunological outcomes of experimental RV infection in patients with moderate asthma, the most severe asthma cohort studied under these conditions to date, and demonstrated a correlation between poorer asthma control at baseline and severity of RV infection. We also demonstrated upregulation of pulmonary ILC responses in both patients with asthma and healthy subjects after RV infection, with predominance for ILC2 responses in asthma that was associated with more severe viral infection and clinical outcomes, and type 2 cytokine expression.

After RV infection, peak viral load occurred on Day 4 after inoculation in both patients with asthma and healthy control subjects, which was significantly higher (by ∼40-fold) in patients with asthma than in healthy control subjects. Similarly, patients with asthma experienced significantly more severe upper and lower respiratory symptoms and greater reductions in lung function compared with healthy subjects. It was demonstrated that poor asthma control at baseline, as defined by a higher ACQ score, significantly correlated with peak viral load on Day 4, indicating patients with more severe asthma disease have an increased risk of severe RV infection and therefore greater severity of asthma exacerbations. These findings are in keeping with observations in naturally occurring RV infection and experimental RV challenge studies, confirming that patients with asthma are more susceptible to greater RV infection–associated morbidity than otherwise healthy individuals. There is conflicting evidence regarding whether patients with asthma are more susceptible to RV infection in terms of having greater virus loads, as some previous studies have not detected increased virus loads (42, 43). However, our group has demonstrated increased viral load in patients with asthma both in the current study and in our previous RV challenge study in mild/moderate asthma, in which the greater virus loads were also accompanied by significantly more severe cold and asthma symptoms (2). Bronchial viral load was below the limit of detection in the majority of patients studied. However, virus was detected more often in BAL from patients with asthma than in BAL from healthy subjects, and the greatest BAL viral load was identified in a patient with asthma at Day 3. It is likely that quantification of BAL virus load was impacted by the significant dilution and variable return associated with BAL sampling in these patients.

It is possible that the increase in upper respiratory symptom scores (URSS) seen in patients with asthma at baseline is related to coexisting allergic rhinitis. However, it is difficult to ascertain if the increases seen in URSS during the course of RV infection relate to allergic rhinitis, asthma or a combination of both. Patients with asthma and pollen sensitization were challenged with virus outside of tree and grass pollen seasons to reduce the impact of allergic rhinitis on symptoms during the study period. The higher viral load identified in patients with asthma compared with healthy subjects may suggest greater virus-induced cytopathic effects as an explanation for the increase in symptoms seen during the course of viral infection. Further work is needed to interrogate the relative contribution of atopic and asthma status to upper respiratory symptoms following respiratory viral challenge.

Enrichment of ILC2s in BAL at baseline was observed in patients with asthma but not in healthy subjects, which was significantly increased after RV infection. We also observed increased type 2 cytokines IL-5 and IL-13 levels after RV infection in patients with asthma. ILC2s have been demonstrated to be a potent early source of IL-5 and IL-13, compared with T cells, in response to stimulation by the epithelial-derived cytokines IL-25 and IL-33 (21, 22). It has been previously demonstrated that RV infection increases IL-25 and IL-33 expression in the airways of patients with asthma (2, 4). We have similarly demonstrated RV induction of IL-33 in patients with asthma, which correlated with peak viral load at Day 4. The enrichment of ILC2s at baseline in asthma reported here, in our previous study and by others, suggests that their presence during RV infection could result in their rapid expansion in response to virus-induced IL-25 and IL-33 and subsequent production of cytokines associated with asthma immunopathology. The further increase in ILC2s at Day 8 in patients with asthma suggests that there are likely additional pathways activated by virus, which peaked at Day 4 in asthma. Levels of the type 2 chemokines CCL17/TARC and CCL22/MDC at Day 5 and Day 6, respectively, correlated with ILC2:ILC1 ratios at Day 8, which suggests they may also contribute to ILC2 recruitment in asthma. In healthy subjects, ILC2s were increased after RV infection at Day 3. However, increases in IL-33, IL-5, and IL-13 did not reach statistically significant levels, and the absence of significant lower respiratory symptoms would suggest ILC2s are not causing significant lower airway immunopathology in these subjects. Further studies sampling at different time points are needed to determine the kinetics and effector functionality of ILC2 responses after RV infection.

We observed delayed accumulation of BAL ILC1s in patients with asthma compared with healthy subjects, with significant increases observed at Day 8 compared with Day 3, respectively. These ILC1 kinetics were associated with similar trends in BAL IFN-γ, which peaked at Day 8 in patients with asthma and Day 3 in healthy subjects. Previous studies have demonstrated impaired innate antiviral responses in patients with asthma, including impaired innate IFN production by lung cells, as a potential mechanism of more severe and prolonged viral infections. The antiviral role of ILC1s has been previously demonstrated in preclinical models (31, 32); whether the delayed ILC1 response to RV infection in patients with asthma is associated with the earlier and higher peak viral load and prolonged clinical symptoms compared with healthy subjects warrants further investigation. Increased bronchial IFN-γ in patients with asthma has previously been reported following virus challenge, with the magnitude of the IFN-γ response in both nasal and bronchial samples being relatively greater than that of the type 2 cytokines, although induction of type 2 cytokines and chemokines was observed in subjects with asthma alone (44). It is not known whether the increased IFN-γ observed in BAL contributes to exacerbation pathogenesis and/or is a consequence of the immune response to the increased viral load seen in subjects with asthma. The clinical manifestations of an exacerbation are likely accounted for by the balance between type 2 and type 1 responses with no single cell or mediator exerting an effect in isolation.

Given the differences in kinetics of ILC2 and ILC1 responses between patients with asthma and healthy subjects, ILC2 : ILC1 ratios were generated to better understand their relative contributions to the response to RV infection (45). We found that a higher ILC2 : ILC1 ratio was associated with greater exacerbation severity as determined by greater reductions in lung function and increased upper and lower respiratory symptoms. There were also strong correlations between pulmonary ILC2 : ILC1 ratios and pulmonary levels of IL-5 at multiple time points after RV infection. Together, this suggests that enrichment of ILC2s and type 2 inflammation may contribute to greater RV-induced asthma exacerbation severity. We recognize that other cells of the adaptive immune response, including T cells, may be responsible for production of the type 2 mediators presented in the manuscript. The objective of this analysis was to focus on the relationship between RV infection and ILC responses in patients with asthma. The correlations between ILCs, type 2 cytokines, and clinical symptoms described within this manuscript further support ILC involvement in the response to RV infection.

Clinical exacerbations, ILC2 accumulation, and type 2 inflammation occurred in patients with asthma despite the permitted use of inhaled corticosteroids during the study, suggesting the limitations of standard-of-care asthma management strategies on clinical outcomes and key immunological pathways in asthma. BAL ILC2s have been shown to demonstrate steroid resistance (25), and increased frequencies of ILC2s have been identified from blood and sputum samples in patients with severe eosinophilic asthma requiring treatment with maintenance oral corticosteroids, despite suppression of blood eosinophils (46). These findings suggest that ILC2s may represent a persistent cellular source of IL-5 and IL-13 driving airways eosinophilia and airway hyperresponsiveness in patients with severe asthma. Biologic therapies targeting these specific cytokine pathways have been shown to be effective in specific subpopulations of patients with asthma (10, 13, 47–49). Targeting ILC2s directly or upstream of their activation could have the potential to provide a broader therapeutic effect by modulating downstream effector functions of multiple ILC2-derived cytokines and therefore represents an attractive therapeutic target. A phase 3 clinical trial demonstrated an antithymic stromal lymphopoietin antibody significantly reduced annual exacerbation rates in patients with severe asthma compared with standard of care (50).

Two bronchoscopies were performed during the acute infectious period following RV inoculation, and this may have impacted clinical outcomes as observed by increased lower respiratory symptom scores in both groups on days in which bronchoscopies were performed (Days 3 and 8). Although other studies corrected symptom scores for the potential effect of a single bronchoscopy (1, 2), we reported uncorrected symptom scores, as these data demonstrated important differences between groups, with consistently higher symptom scores in patients with asthma than healthy subjects following inoculation, regardless of bronchoscopy days. In this study, although we observed significant induction of both type 1 and type 2 mediators in the nose, the responses seen in the lower airways were more variable. Despite performing two bronchoscopies after inoculation in this study to increase lower airway sampling compared with previous RV challenge studies, nasal sampling has the advantage that it can be performed daily (2). A previous experimental challenge study by our group, identified significant increases from baseline in bronchial IL-5, IFN-γ, IP-10, and ITAC on Day 4 after infection (2), which would support the idea that peak bronchial levels occur after Day 3 and before Day 8. It is therefore possible that we have not sampled the lower airway at the correct time point after RV infection to identify increases in bronchial mediators. In addition to these differences in timing of sampling the lower airway, Jackson and colleagues recruited a larger number of patients with asthma, of whom just under half were ICS naive (2). It is also possible that the differences in the study populations and treatment could explain the responses observed in bronchial mediators in the current study. However, in addition to reductions in lung function and PEF, we have identified a significant increase in airways hyperresponsiveness at Day 6 after RV infection compared with baseline in subjects with asthma alone. It is likely that these changes relate to increases in bronchial levels of type 2 mediators such as IL-5 and IL-13 during RV infection. In addition, nasal mediators have been shown to correlate with inflammatory cells in the lower airway in asthma (51), and we have previously demonstrated increased nasal mediators after RV inoculation correlate with lower airway symptoms (2). These findings support nasal mediator levels as being reflective of lower airway inflammation.

In summary, this study has identified ILC1s and ILC2s in the lower airways of patients with asthma and healthy subjects and demonstrated differential enrichment of these innate cell populations after RV infection between asthma and otherwise healthy individuals. ILC2 responses were found to correlate with clinical markers of exacerbation severity in asthma, supporting a potential clinical role for this novel innate cell population in the pathology of virus-induced asthma exacerbations. These findings highlight the need for further investigation of the mechanistic role of ILC2s during asthma exacerbations to support further identification of novel therapeutic targets to address this unmet need.

Acknowledgments

Acknowledgment

The authors thank the St. Mary’s Flow Cytometry Core Facility, National Heart and Lung Institute, and Imperial College London for support and access to equipment. Editorial support was provided by Yee-Man Ching, Ph.D.

Footnotes

Supported by a Medical Research Council (MRC) and GlaxoSmithKline Strategic Alliance Program grant G1100238, the National Institute of Health Research (NIHR) Biomedical Research Centre funding scheme, and MRC and Asthma UK Centre grant G1000758. S.L.J. is the Asthma UK Clinical Chair (grant CH11SJ) and is an NIHR Senior Investigator.

Author Contributions: J.D. performed the clinical procedures of the study. M.-B.T.-T. and A.d.R. assisted with screening volunteers and sampling. E.W., M.A., H.F., and O.M.K. assisted with research bronchoscopies. S.L.J., O.M.K., T.T.H., and D.J.J. supervised clinical aspects of the study. J.D., T.K., J.A., L.G., and A.C. performed clinical sample processing. M.P. performed fluorescence-activated cell sorting of innate lymphoid cells. J.D. conducted flow cytometric analysis. R.P.W., D.J.C., and B.M.J.R. advised on flow cytometric analysis. J.D. and E.B. conducted Meso-Scale Discovery analysis. T.T.H., R.P.W., and M.R.E. supervised laboratory processing and analysis. S.L.J. and R.S. conceived and designed the study.

This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org.

Originally Published in Press as DOI: 10.1164/rccm.202010-3754OC on September 1, 2021

Author disclosures are available with the text of this article at www.atsjournals.org.

Contributor Information

on behalf of the MRC-GSK Strategic Alliance Consortium:

Sebastian L. Johnston, Roberto Solari, Michael R. Edwards, Paul Lavender, Ross P. Walton, Hannah Gould, David Cousins, Antoon J. van Oosterhout, Jaideep Dhariwal, Aoife Cameron, Nathan W. Bartlett, Patrick Mallia, David J. Jackson, Maria-Belen Trujillo-Torralbo, Jerico del Rosario, Janet L. Smith, Matthew J. Edwards, Karen Affleck, Nil Turan Jurdzinski, Veronique Birault, Peter McErlean, Yu-Chang Wu, Nadine Upton, and Ismael Ranz Jimenez

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