Monocyte Chemotactic Factors in the Airways of Patients With Mild Asthma Before and After an Allergen Challenge

Allergic asthma is characterized by eosinophilic airway inflammation, but a number of individuals, especially subjects with severe asthma, may have primarily neutrophilic inflammation. Murine studies suggest that blood monocytes (Mo) facilitate eosinophil and neutrophil recruitment to the lungs [1, 2] and their depletion decreases airway inflammation and hyperresponsiveness. However, we know little about the role of Mo in human asthma. Induced sputum from patients with asthma has increased numbers of Mo compared to healthy controls [3], while airway Mo correlate with indicators of inflammation [4]. The triggers for Mo recruitment to the airways are poorly understood. Here, we measured Mo chemotactic factors in mild asthmatics before and after an allergen challenge, and developed an ex vivo assay to assess their biological significance. With ethics approval and informed consent, we studied the presence of Mo chemotactic factors in pre- and 24 h post-allergen challenge (24 h) induced sputum from subjects with mild asthma (Table S1). Eosinophils increased 24 h after allergen challenge, while other immune cells remained unchanged (Figure S1). Chemokine CC-motif ligand (CCL)2, CCL3, CCL4, CCL13, CCL17, and CCL22 were detected in induced sputum, but only CCL4 and CCL17 increased at 24 h compared to baseline (Figure 1A,B; Figure S2); CCL17 increased only in dual responders, not in those with isolated early responses (Figure 1C,D). Changes in other chemotactic factors may have been missed due to the timing of sampling. To test the biological significance of these Mo chemotactic factors, we developed a Mo chemotactic assay using peripheral blood mononuclear cells (PBMC) from healthy volunteers stimulated with sputum supernatants from asthmatic subjects; migrating Mo were identified among migrating PBMC by flow cytometry (gating strategy in Figure S3). Increasing dilutions of sputum supernatants mediated chemotaxis but not chemokinesis (Figure S4A,B). Migrated cells were Mo-enriched, indicating sputum supernatants preferentially attract Mo over lymphocytes (Figure S5A,B). We observed an increase in both the numbers of migrating Mo (Figure 2A) and the % of total seeded Mo that migrated (Figure 2B) toward 24 h sputum supernatants than pre-challenge supernatants. The results were consistent between donors, with the majority of the 24 h sputum supernatants inducing higher chemotaxis compared to corresponding pre-challenge supernatants. Sputum supernatants showed lymphocyte chemotactic activity, which was unchanged 24 h post-challenge (Figure 2C,D). We then tested a CCR4 and a CCR2/CCR5 antagonist to block the effects of CCL17 and CCL4, respectively. Both the CCR4 antagonist C021 (0.14 μM; Figure 2E) and the CCR2/CCR5 antagonist TAK (30 nM; Figure 2F) inhibited Mo chemotaxis toward 24 h sputum supernatants, with a slightly greater effect when combined (Figure S6). Our findings demonstrate that induced sputum from patients with mild asthma following allergen challenge induces enhanced Mo chemotaxis, likely driven by increased CCL4 and CCL17 levels, though the involvement of additional factors cannot be ruled out. Preferential increase of CCL17 in dual responders suggests its role in driving late responses. Our results represent airway changes in subjects with mild asthma, and their generalizability may not be directly obvious. However, elevated levels of Mo chemotactic factors, such as CCL13 and CCL4 have been found also in the airways of patients with asthma compared to healthy controls [5, 6] and CCL17 and CCL22 are elevated in BAL after allergen challenge in asthma [7], supporting the notion of Mo accumulation contributing to airway inflammation. Further research is required to understand whether Mo chemotactic factors are associated with asthma severity and/or control. Therapeutic approaches to block Mo recruitment in the airways in asthma by blocking Mo chemotactic molecules and/or their receptors should be tested in humans as have been done in mice [1, 2] and primates [8]. Nami Shrestha Palikhe contributed to study design, performed flow cytometry, measured monocyte chemotactic factors, conducted chemotaxis assays, analyzed the data, and drafted and finalized the manuscript. Yingqi Wu isolated peripheral blood monocytes for the chemotaxis assays. Karen J. Howie, Caitlin Stevens, Jennifer Mitchell, and Lesley Wiltshire recruited subjects, conducted allergen challenges in 12 participants, and performed sputum induction and processing for supernatant collection. Gail M. Gauvreau prepared ethics documentation, recruited patients at McMaster, contributed to study design, supervised clinical data collection, and reviewed the manuscript draft. Harissios Vliagoftis oversaw study design, supervised data collection and all experiments, and authored the final manuscript draft. This research was supported by grant funding to H.V. from CIHR (133475) and the GlaxoSmithKline–CIHR Research Chair in Airway Inflammation (143901). The authors declare no conflicts of interest. The data that support the findings of this study are available from the corresponding author upon reasonable request. Appendix S1: all70192-sup-0001-AppendixS1.docx. 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