High serum granulocyte-colony stimulating factor characterises neutrophilic COPD exacerbations associated with dysbiosis

Abstract

Introduction: COPD exacerbations are heterogeneous and can be triggered by bacterial, viral, or noninfectious insults. Exacerbations are also heterogeneous in neutrophilic or eosinophilic inflammatory responses. A noninvasive peripheral biomarker of COPD exacerbations characterised by bacterial/neutrophilic inflammation is lacking. Granulocyte-colony stimulating factor (G-CSF) is a key cytokine elevated during bacterial infection and mediates survival, proliferation, differentiation and function of neutrophils.

Objective: We hypothesised that high peripheral G-CSF would be indicative of COPD exacerbations with a neutrophilic and bacterial phenotype associated with microbial dysbiosis.

Methods: Serum G-CSF was measured during hospitalised exacerbation (day 0 or D0) and after 30 days of recovery (Day30 or D30) in 37 subjects. In a second cohort, serum and sputum cytokines were measured in 59 COPD patients during stable disease, at exacerbation, and at 2-weeks and 6-weeks following exacerbation.

Results: Serum G-CSF was increased during exacerbation in a subset of patients. These exacerbations were enriched for bacterial but not viral or type-2 biologies. The median serum G-CSF level was 1.6-fold higher in bacterial exacerbation compared to nonbacterial exacerbation (22 pg·mL-1 versus 13 pg·mL-1, p=0.0007). Serum G-CSF classified bacterial exacerbations with an area under the curve (AUC) for the receiver operating characteristic (ROC) curve equal to 0.76. Exacerbations with a two-fold or greater increase in serum G-CSF were characterised by neutrophilic inflammation, with increased sputum and blood neutrophils, and high sputum interleukin (IL)-1β, IL-6 and serum amyloid A1 (SAA1) levels. These exacerbations were preceded by dysbiosis, with decreased microbiome diversity and enrichment of respiratory pathogens such as Haemophilus and Moraxella. Furthermore, serum G-CSF at exacerbation classified neutrophilic-dysbiotic exacerbations (AUC for the ROC curve equal to 0.75).

Conclusions: High serum G-CSF enriches for COPD exacerbations characterised by neutrophilic inflammation with underlying bacterial dysbiosis.

Conflict of interest statement

Conflict of interest: A. Chakrabarti reports personal fees from and stock in Genentech, Inc., during the conduct of the study and outside the submitted work. Conflict of interest: J.S. Mar reports personal fees from and equity in Genentech, Inc., outside the submitted work. Conflict of interest: D.F. Choy is an employee of Genentech, Inc. Conflict of interest: Y. Cao reports personal fees from and stock in Genentech, Inc., during the conduct of the study and outside the submitted work. Conflict of interest: N. Rathore reports personal fees from and stock in Genentech, Inc., during the conduct of the study and outside the submitted work. Conflict of interest: X. Yang reports personal fees from Genentech, Inc., during the conduct of the study and outside the submitted work. Conflict of interest: G.W. Tew reports personal fees from Genentech, Inc., during the conduct of the study and outside the submitted work. Conflict of interest: O. Li is a salaried employee of Genentech, Inc. and received nonfinancial support from Genentech, Inc., during the conduct of the study. Conflict of interest: P.G. Woodruff reports consulting fees from Regeneron, Sanofi, Glenmark Pharma, Theravance and NGM Pharma, and visiting professor honoraria from Amgen and Genentech, outside the submitted work. Conflict of interest: C.E. Brightling is an employee of the University of Leicester. Conflict of interest: M. Grimbaldeston is an employee of Genentech, Inc., and reports personal fees and nonfinancial support during the conduct of the study. Conflict of interest: S.A. Christenson reports consulting fees from AstraZeneca, GlaxoSmithKline, Amgen and Glenmark; personal fees for invited lectures from Sunovion and Genentech; and personal fees for writing for UpToDate, all outside the submitted work. Conflict of interest: M. Bafadhel reports grants from AstraZeneca; honoraria for consulting and advisory boards, as well as travel to conferences, from AstraZeneca, Chiesi and GSK. She is a scientific advisor to and minor shareholder in AlbusHealth, all outside the submitted work. Conflict of interest: C.M. Rosenberger reports personal fees from and stock in Genentech, Inc., during the conduct of the study and outside the submitted work.

Copyright ©The authors 2021.

Figures

FIGURE 1

Serum granulocyte-colony stimulating factor (G-CSF) is significantly elevated during COPD exacerbation. a) Serum G-CSF was measured at exacerbation (Day0 (D0), n=54) and recovery (Day30 (D30) post-exacerbation, n=52) from a hospitalised acute exacerbation of COPD (AECOPD) cohort (the LEUKO cohort). Paired exacerbation and recovery samples were available for 37 patients and analysis was performed on pooled treatment arms as there was no obvious treatment advantage of zileuton compared with placebo. p-Values were determined by the Wilcoxon matched-pairs signed-rank test. b) Among the 37 paired samples, 21 subjects had a two-fold or greater decrease in serum G-CSF at recovery (median values±95% confidence intervals (CIs) are shown and p-values were determined by the Mann–Whitney U-test. c) A heatmap of Spearman's correlations between the fold-change of the biomarkers at exacerbation compared to a preceding stable visit. Each square represents the correlation between the feature heading the column with the feature heading the row. The number shown in a given square indicates the corresponding Spearman correlation coefficient between the two features. CRP: C-reactive protein; SAA1: serum amyloid A1; TNF-α: tumour necrosis factor-α; IL: interleukin.

FIGURE 2

Elevated levels of serum granulocyte-colony stimulating factor (G-CSF) during exacerbation are non-eosinophilic and predominantly associated with infection. a) Serum G-CSF was measured for 74 exacerbations with preceding stable visits and at 2-week and 6-week follow-up visits for 54 subjects (the MRC cohort). Serum G-CSF measurements were categorised between b) non-eosinophilic and c) eosinophilic exacerbations. Serum G-CSF measurements were compared between exacerbations strictly associated with d) bacterial infection (n=19), e) viral infection (n=7), or f) conditions described as “infectious” (bacterial, viral or bacterial/viral co-infection (n=36)). Statistical significance is shown by p-values generated by the Wilcoxon matched-pairs signed-rank test.

FIGURE 3

Exacerbations categorised by granulocyte-colony stimulating factor (G-CSF) induction are characterised by higher biomarkers of neutrophilic inflammation. G-CSF high and low groups are categorised based on a two-fold or higher increase in G-CSF on exacerbation compared to a preceding stable visit. a) Blood neutrophil fold-change is the change in blood neutrophils on exacerbation compared to a preceding stable visit. b) Absolute blood neutrophil count at exacerbation. c) Serum interleukin (IL)-6 at exacerbation. d) Serum amyloid A1 (SAA1) at exacerbation. e) Sputum neutrophil percentage at exacerbation. f) Sputum IL-6 at exacerbation. The p-values shown are determined by Mann–Whitney U-test. g) Heatmap of Spearman's correlations between biomarkers at exacerbation. h) Heatmap of Spearman's correlations between biomarker fold-changes at exacerbation compared to the preceding stable visit. Each square represents the correlation between the feature heading the column and the feature heading the row. The number shown in a given square indicates the corresponding Spearman correlation coefficient between the two features. IFN: interferon; IP-10: IFN-γ induced protein-10; TNF-α; tumour necrosis factor-α; CRP: C-reactive protein.

FIGURE 4

High granulocyte-colony stimulating factor (G-CSF) levels at exacerbation are associated with higher frequency of bacterial infection. Biomarker levels were compared between bacterial versus non-bacterial exacerbations. a) G-CSF fold change between exacerbation and a preceding stable visit. b) Serum G-CSF at exacerbation. c) Spearman's correlation plot of sputum interleukin (IL)-1β at exacerbation versus serum G-CSF at exacerbation. d) Sputum interleukin IL-1β at exacerbation. e) Spearman's correlation plot of serum C-reactive protein (CRP) at exacerbation versus serum G-CSF at exacerbation. f) Serum CRP at exacerbation. g) Receiver operating characteristic (ROC) curve illustrating biomarkers that positively predict bacterial exacerbation. For non-correlation plots, median values±95% confidence intervals (CIs) are shown. p-Values are determined by the Mann–Whitney U-test. AUC: area under the curve.

FIGURE 5

An altered sputum microbiome precedes exacerbations characterised by bacteria or a two-fold or greater increase in serum granulocyte-colony stimulating factor (G-CSF). a) Shannon's diversity index for bacterial exacerbation. b) Non-metric multi-dimensional scaling (NMDS) ordination plot of Bray–Curtis distances for bacterial exacerbation. Dashed ellipses represent the 95% confidence interval (CI) for the centroid of each stratification group. c) Haemophilus abundance for sputum microbiota during stable disease prior to bacterial exacerbation. d) Moraxella abundance for sputum microbiota during stable disease prior to bacterial exacerbation. e) Shannon's diversity index for a two-fold or greater increase in serum G-CSF. f) NMDS ordination plot of Bray–Curtis distances for a two-fold or greater increase in serum G-CSF. Dashed ellipses represent the 95% CI for the centroid of each stratification group. g) Haemophilus abundance for sputum microbiota during stable disease prior to a two-fold or greater increase in serum G-CSF. h) Moraxella abundance for sputum microbiota during stable disease prior to a two-fold or greater increase in serum G-CSF. Cohort sizes are as follows: for bacterial exacerbation (a–d), No (n=17), Yes (n=22). For exacerbations characterised by a two-fold or greater increase in G-CSF (e–h), Low (n=33), High (n=13). For panels a) and e), group mean±standard error (se) values are shown and p-values are determined by the Wilcoxon rank-sum test. For panels b) and (f), permutational ANOVA was calculated. For panels c), d), g) and h), the y-axes have been fixed to the limits of log10(RA) for these samples, group mean log10(RA)±se values are depicted and statistical significance is determined by DESeq2 and adjusted for false discoveries. RA: relative abundance.

FIGURE 6

Serum granulocyte-colony stimulating factor (G-CSF) levels at exacerbation stratify neutrophil-dysbiotic exacerbations. a) Serum G-CSF, b) blood neutrophil and c) serum C-reactive protein (CRP) levels at exacerbation were compared for neutrophil and neutrophil-high (sputum neutrophils ≥61%) COPD exacerbations. d) Serum G-CSF, e) blood neutrophil and f) serum CRP levels at exacerbation were also compared between neutrophil-dysbiotic COPD exacerbations (relative abundance of Haemophilus and Moraxella >0.41), neutrophil-balanced and neutrophil-low (sputum neutrophils <61%) COPD exacerbations. A receiver operating characteristic (ROC) curve g) was also produced to classify neutrophil-dysbiotic COPD exacerbations. p-Values were determined by the Mann–Whitney U-test. AUC: area under the curve.

FIGURE 7

Interleukin (IL)-1β treatment directly induces genes conducive of neutrophilic inflammation. Normal human bronchial epithelial cells were differentiated at the air–liquid interface and treated with IL-1β (10 ng·mL−1) for 24 h. Cytokine levels were then measured from cell supernatants by Luminex-based enzyme-linked immunosorbent assay (ELISA) (a–c). RNA was also isolated and gene expression was examined by RNA sequencing (d–h). Statistical significance was determined by the unpaired t-test with Welch's correction. CSF: colony stimulating factor; CXCL: chemokine (C-X-C motif) ligand. rpkm: reads per kb transcript per million mapped reads.