Signalling via CD131 regulates pulmonary inflammation, alveolar cell death and emphysema in COPD

Abstract

Background: COPD is a heterogenous disease where chronic inflammation is implicated in airway remodelling and emphysema. The CD131 receptor is indispensable for signalling by the β common (βc) family of cytokines encompassing granulocyte-macrophage colony-stimulating factor, interleukin (IL)-5 and IL-3, which instigate both type-2 and non-type-2 inflammatory responses. This study aims to determine whether antagonising CD131 signalling can prevent pulmonary inflammation, alveolar cell death and emphysema development.

Methods: We performed in-house and in silico transcriptomic analysis to investigate the gene expression of CD131 (CSF2RB) and pathway enrichment for βc cytokine signalling in blood, sputum and lung biopsies of COPD patients. To model emphysema, transgenic mice expressing human CD131 were exposed to elastase or cigarette smoke (CS) and a fully human monoclonal antibody (CSL311) was employed to inhibit CD131 signalling.

Results: CD131 gene expression was significantly increased in COPD, along with an enrichment of gene set for βc cytokine signalling. In transgenic mice subjected to emphysema models, CD131 antagonism effectively prevented lung injury, alveolar cell death and emphysema development. Mechanistically, RNA sequencing identified pathway enrichment for myeloid cell activation, type-2 immune response and macrophage alternative activation in elastase-induced emphysematous mice, mirroring human COPD. Blocking CD131 signalling almost completely reversed the global gene expression alterations associated with emphysema development.

Conclusions: CD131 signalling orchestrates pulmonary inflammation in COPD, resulting in immunopathology that underpins emphysema and lung function decline. Antagonising CD131 therefore represents a unique strategy to simultaneously target multiple pathogenic myeloid cell populations.

FIGURE 1

CD131 gene expression is increased in the lungs and blood of COPD. a) In silico analysis of the CD131 gene − CSF2RB expression was performed on human sputum samples (GSE148004) and lung biopsies (GSE47460) from healthy subjects and COPD patients. GSE57148 was explored to query CSF2RB expression in healthy smokers versus COPD patients. b) Single-sample gene set variation analysis (GSVA) was performed on lung biopsy data (GSE47460) from the Lung Genomics Research Consortium to assess the activity of “IL-3, IL-5, and GM-CSF signalling” (βc cytokine signalling), “GM-CSF pathway”, “IL-5 pathway” and “IL-3 pathway” in COPD compared with healthy controls, where an enrichment score (ES) was computed for individual samples. c) Whole blood was collected from patients with COPD and from healthy donors (control). Flow cytometry was performed on white blood cells to quantify immune cell subsets (gating strategy provided in supplementary figure S1). d) RNA-seq was performed on blood samples and analysed for the enrichment of “IL-3, IL-5 and GM-CSF signalling” (βc cytokine signalling) using GSVA, where an ES was computed for individual samples. The ES was then further analysed across COPD subsets based on disease severity using the Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria or COPD subsets classified as eosinophilic (sputum eosinophil % ≥3% or blood eosinophil counts ≥300 cells per μL and non-eosinophilic. n=30 for control; n=29 for COPD. IL: interleukin; GM-CSF: granulocyte−macrophage colony-stimulating factor. *: p<0.05, **: p<0.01, ***: p<0.001 by Mann–Whitney U-test or Kruskal–Wallis test followed by Dunn's tests.

FIGURE 2

CD131 antagonism prevents myeloid cell expansion and the associated lung injury in porcine pancreas elastase (PPE)-induced emphysema model. a) To evaluate CD131 signalling in driving emphysema, hβcTg mice were intranasally instilled with 0.75 U PPE on day 0 and intravenously injected with 50 mg·kg⁻¹ CD131 blocking antibody CSL311 or isotype control (ISO) on day 1, 3, 5, 12 and 19 respectively. 4 days after instillation, mice were culled to measure the inflammatory response. 21 days after instillation, mice were culled to assess lung function and histopathology. b) On day 4, lung tissues were digested for flow cytometry analysis, with representative plots shown and total number of neutrophils, monocyte-derived macrophages (mo-Macs), eosinophils and resident alveolar macrophages (rAMs) quantified. c) Lung injury markers, lactate dehydrogenase (LDH) and serum albumin were measured in the bronchoalveolar lavage fluid (BALF). d) Lung expression of epithelial markers Ager, Sftpc and Foxj1 were measure by quantitative PCR with reverse transcriptase (RT–qPCR). n=6; data are mean±sem; *: p<0.05, **: p<0.01, ***: p<0.001 by one-way ANOVA with Dunnett's tests or two-way ANOVA. VEH: vehicle.

FIGURE 3

CD131 antagonism prevents transcriptomic alterations in porcine pancreas elastase (PPE)-induced emphysema model. a) Bulk RNA-seq was performed on frozen lungs from hβcTg mice on day 4 post-PPE instillation. The number of differentially expressed genes (DEGs) obtained from two-group comparisons are depicted in the Venn diagram: PPE-ISO (isotype control) versus control (SAL) in red; PPE-CSL311 versus PPE-ISO in blue; PPE-CSL311 versus SAL in green (left panel). Expression level of the top 1000 most upregulated and top 1000 most downregulated DEGs from PPE-ISO versus SAL comparison are demonstrated in individual mice as heatmap across all three groups (right panel). b) All DEGs are also shown in volcano plots as orange dots for upregulation and blue dots for downregulation. Grey dots are non-DEGs. c) Gene set enrichment analysis (GSEA) was performed on pre-ranked gene lists based on differential expression statistics derived from comparing COPD and healthy samples (GSE47460) and from PPE-ISO versus SAL, using Hallmark, Ontology and Curated gene sets from the Molecular Signatures Database. Pathways significantly enriched in both COPD and PPE model are shown in dot plots, where dot size indicates the gene ratio within a pathway, and colour reflects the adjusted p-values. d) A normalised enrichment score (NES) was calculated for enriched pathways in the comparisons PPE-ISO versus SAL and PPE-CSL311 versus PPE-ISO, with positive NES values indicating pathway activation, negative values indicating deactivation and colour denoting the statistical significance of the enrichment.

FIGURE 4

CD131 antagonism prevents porcine pancreas elastase (PPE)-induced lung function decline, hyperinflation and alveolar space enlargement. a) On day 21 post-PPE instillation, respiratory mechanics were measured in anaesthetised mice using Flexivent FX1. Pre-designed scripts were used to obtain inspiratory capacity (IC). Pressure–volume loop was performed to obtain a PV-loop curve and quasi-static compliance (Cst). Forced oscillation was performed to measure tissue elastance (H). Following lung function tests, the mice were killed and inflated with neutral buffered formalin. b) Lungs were excised and water displacement by the lung was measured using the Archimedes principle. c) Lungs were then processed, sectioned and stained with haematoxylin and eosin (H&E) to measure the airspace enlargement using the mean linear intercept method. d) A TUNEL assay was performed to assess lung cell death using 4,6-diamidino-2-phenylindole (DAPI) as a counterstain. TUNEL-positive cells were detected in green. Red arrows denote TUNEL-positive cells in airways; yellow arrows denote TUNNEL-positive cells in lung parenchyma; green arrows denote TUNEL-positive cells in lung vasculature. The number of TUNEL-positive cells per cm² tissue area was calculated. n=6; data are mean±sem; *: p<0.05, **: p<0.01, ***: p<0.001 by one-way ANOVA with Dunnett's tests or two-way ANOVA. ISO: isotype control; VEH: vehicle.

FIGURE 5

CD131 antagonism prevents cigarette smoke (CS)-induced lung function decline, hyperinflation and alveolar space enlargement. a) To evaluate CD131 signalling in driving emphysema, hβcTg mice were exposed to CS 5 days per week for 8 weeks. Mice were intravenously injected with 50 mg·kg⁻¹ CD131 blocking antibody CSL311 or isotype control (ISO) weekly. b) At the end of the CS exposure, respiratory mechanics were measured in anesthetised mice using Flexivent FX1. Pressure–volume loop was performed to obtain PV-loop curve and quasi-static compliance (Cst). Forced oscillation was performed to measure tissue elastance (H). Following lung function tests, mice were culled and inflated with neutral buffered formalin. c) Lungs were excised and water displacement by the lung was measured using the Archimedes principle. d) Lungs were then processed, sectioned and stained with haematoxylin and eosin (H&E) to measure the airspace enlargement using the mean linear intercept method. n=6; data are mean±sem; *: p<0.05, **: p<0.01, ***: p<0.001 by one-way ANOVA with Dunnett's tests or two-way ANOVA.