Type2 Innate Lymphoid Cells in Induced Sputum from Children with Severe Asthma

Type2 innate lymphoid cells in induced sputum from children with severe asthma

1,2P. Nagakumar, 1L. Denney, 1,2L. Fleming, 1,2A. Bush, 1CM. Lloyd* and 1,2S. Saglani*

1National Heart & Lung Institute, Imperial College London, UK. 2 Royal Brompton Hospital, London, UK

*Equal contribution

Correspondence:

Dr Sejal Saglani

Reader in Respiratory Paediatrics

368 Sir Alexander Fleming Building

National Heart & Lung Institute

Imperial College London

Exhibition Road

London SW7 2AZ

Tel: 020 7594 3167 Fax: 020 7594 3119

Abbreviations:

STRA: severe therapy resistant asthma, ILC: innate lymphoid cells, BAL: bronchoalveolar lavage, LRTI: lower respiratory tract infections

Keywords:

Severe asthma; innate lymphoid cells; children; sputum; Bronchoalveolar lavage

Capsule Summary (35 words)

Significantly more type2 innate lymphoid cells were identified in bronchoalveolar lavage and induced sputum compared to peripheral blood from children with severe asthma. However, the majority of airway innate lymphoid cells were CD127 negative.

To The Editor:

Innate lymphoid cells (ILC) are characterised by their lymphoid morphology and absence of lymphocyte lineage surface markers (1). Group 2 ILCs (ILC2) expressing CD127 and CRTH2 are induced by the epithelial cytokines IL-33 and IL-25 and are implicated in the pathogenesis of allergic airways disease in murine models. ILC2s have been identified in cord blood (2), tonsils and nasal polyps from patients with chronic rhinoosinusitis(3) and in peripheral blood and bronchoalveolar lavage (BAL) from adults with asthma(4, 5). However, there are no reports of ILC2s in paediatric airways.

We have shown increased submucosal expression of IL-33 in children with severe therapy resistant asthma (STRA) which was related to the presence of airway remodelling (6). Furthermore, direct inhaled administration of IL-33 to neonatal mice resulted in increased pulmonary IL-13 producing ILCs concomitant with airway remodelling (6). Since an important role for IL-33 is apparent in paediatric STRA, we aimed to determine the presence of ILC2s in the airways of children with STRA in order to assess their potential role as therapeutic targets. Children with STRA and a disease control group of children with recurrent lower respiratory tract infections (LRTI), but no atopy or asthma, undergoing clinically indicated investigations (bronchoscopy, BAL, blood tests and induced sputum) were compared. The study was approved by the regional ethics committee; written informed consent was obtained. (See online repository table E1, E2 for clinical and demographic details). Eleven children with STRA (median age 13.2yr [8.2-16.4]) and 16 with LRTI (median age 5.1yr [1.1-16.1]) undergoing clinically indicated flexible bronchoscopy were included. Sputum induction was also performed in 13 children with STRA, (12.4yr [8.9-15.8]) and 6 with LRTI, (12.6yr [8.116.2]). Of these, 7 STRA patients and 3 LRTI patients had BAL, blood and sputum collected on the same day. 5/13 STRA patients in the sputum cohort were on Omalizumab. Sputum and BAL samples were processed and stained as previously described (7) (see online repository) and were analysed by flow cytometry (BD Fortessa) using Flowjo software (v10) (see online repository Figure E1 for gating strategy).

The proportion of CD45+, Lineage negative (CD3-CD19-CD14-CD16-CD20-CD26-) cells were similar in BAL and peripheral blood (PBMCs) in the STRA children (Figure 1A). However, the proportion of ILCs and ILC2s (CD45+Lin-CD127+, CD45+Lin-CRTH2+ and CD45+Lin-CD127+CRTH2+) was significantly higher in BAL from STRA patients compared to LRTI (Figure 1B-C). The proportion of CD3+CD4+ cells was higher in BAL from children with STRA (Figure1D) but CD3+CD8+ cells were higher in BAL from children with LRTI (p=0.01), (data not shown).

Analysis of cells in induced sputum is an attractive non-invasive method for assessment of lower airway inflammation (7). It can be used to assess serial changes after interventions, which is particularly relevant in children since bronchoscopy requires general anaesthesia. Although numerous paediatric studies have reported use of induced sputum cytology in asthma (8), sputum analysis by flow cytometry is scarce and to date, innate lymphoid like cells (CD34+ IL-5+/IL-13+) have only been reported in sputum from adults with mild asthma (9). Sputum induction was performed as previously described (8). The immunophenotyping protocol used for BAL samples was refined to exclude NK cells (Figure E2, E3). ILC2s expressing CD127 and CRTH2 were present in induced sputum from STRA patients (n=13) (Figure 2). In contrast, neither CD3+ T cells nor Lin-CD45+ cells were present in sputum from children with LRTI (n=6) (data not shown). The proportion of ILC2s that expressed CD127+ and/or CRTH2 in STRA sputum was <0.5% of the lymphoid gate (Figure 2B), while total ILCs (Lin-CD45+) in sputum was 40 times higher (Figure 2A), suggesting a significant number of Lin-CD45+CD127- cells are present in STRA. CD3+CD4+ cells were present in higher proportion than CD3+CD8+ cells in sputum from STRA children (p=0.02) (Figure 2C). The proportion of Th2 cells (CD4+CRTH2+) in sputum from STRA children was significantly higher than ILC2s (Figure 2D). 5/13 STRA patients were on anti-IgE antibody treatment, but despite this ILCs were detected. Strikingly, ten-fold more ILC2s were present in sputum than in peripheral blood from children with STRA (Figure 2E), which is perhaps because of preferential trafficking of ILCs to mucosal sites. There were no correlations between the proportions of ILC2s and the patient clinical characteristics or demographic data.

The strength of our study is the inclusion of STRA patients in whom multiple methods to ensure adherence to inhaled steroids, minimise allergen exposure and other comorbidities were employed. BAL, peripheral blood and sputum were obtained from clinically stable patients undergoing elective investigations reflecting real life clinical practice. But despite this, ILCs were identified and significantly more were present in STRA compared to LRTI. The absence of an acute challenge such as allergen exposure or infection may partially explain the low proportion of lineage negative cells expressing ILC2 markers like CD127 and CRTH2. However, the proportion of ILCs in peripheral blood and BAL were similar to that reported in the literature. The significant proportion of lineage negative cells which did not express any of the currently accepted ILC2 surface markers therefore warrants further investigation. Due to a scarcity of reports of ILCs in human samples, we were unable to relate the proportion of Lin- CD127- cells in BAL and sputum from our patients to previous findings. An acknowledged limitation is the absence of healthy controls. However, performance of BAL or sputum induction in healthy children poses ethical challenges; therefore non-atopic disease controls were included, similar to adult studies (5).

In summary, we report for the first time the presence of group2 innate lymphoid cells in BAL, induced sputum and peripheral blood from children with STRA. There were significantly more pulmonary ILC2s in BAL and induced sputum than peripheral blood and a significantly higher proportion of ILC2s were present in STRA compared to patients without asthma. Identification of ILCs in induced sputum provides an exciting opportunity to assess changes during exacerbation and following interventions in the future. ILC2s were not identified in patients with LRTI suggesting they play a specific role in allergic airway diseases. The presence of pulmonary, rather than systemic ILCs, which concurs with elevated IL-33 in children with STRA (6) suggests therapies designed to target these cells locally should be pursued.

Prasad Nagakumar

Laura Denney

Louise Fleming

Andrew Bush

Clare M Lloyd

Sejal Saglani

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