Characterisation of airway inflammation and proteomes associated with cystic fibrosis-related diabetes

Abstract

Background: Cystic fibrosis (CF)-related diabetes (CFRD) is the most common extrapulmonary complication in CF. CFRD is associated with low lung function, but the underlying mechanisms are poorly understood. The aim of the present study was to compare airway inflammation and the airway proteome in people with CF (pwCF) with and without CFRD.

Methods: Sputum samples from pwCF were analysed for neutrophil elastase (NE) activity with a chromogenic assay, inflammatory cytokines using Meso Scale and bacterial load via quantitative PCR of the 16S rRNA gene. The sputum proteome was characterised by liquid chromatography-mass spectrometry.

Results: 33 pwCF were included in the study, of which 55% had CFRD. The CFRD group had significantly lower lung function and higher sputum levels of NE, interleukin (IL)-8 and IL-1β, whereas IL-6 levels were lower compared to pwCF without CFRD. Proteome analysis identified 27 sputum proteins linked to CFRD, mainly involved in neutrophil degranulation. Given that lung function could be a possible confounding factor, we matched pwCF with and without CFRD based on lung function. In these lung function-matched cohorts, IL-8 and IL-6 levels did not differ significantly, but IL-1β showed a trend towards higher levels in the CFRD group. 10 CFRD-associated proteins were significantly more abundant in the CFRD group, including prothymosin α, which plays a role in diabetes and insulin release.

Conclusion: CFRD is associated with lower lung function, increased sputum levels of NE, IL-8 and IL-1β, and specific protein profiles.

FIGURE 1

Neutrophil elastase (NE) activity and expression of inflammatory cytokines in cystic fibrosis (CF) sputum. Sputum samples from people with CF with (n=18) or without CF-related diabetes (CFRD) (n=15) were analysed for NE activity using a) a chromogenic assay, and the inflammatory cytokines b) interleukin (IL)-8, c) IL-1β, d) IL-6, e) IL-17 and f) tumour necrosis factor-α (TNF-α) using Meso Scale U-PLEX assays, and g) bacterial DNA using qPCR of the 16SDNA gene. Red dots represent patients matched on lung function. Bars represent median values. NS: not significant. *p<0.05; **p<0.01.

FIGURE 2

Sputum proteins significantly associated with cystic fibrosis (CF)-related diabetes (CFRD). a) Volcano plot showing proteins significantly associated with CFRD and non-CFRD. Statistical significance was determined using a two-tailed t-test with an FDR of 0.05 to correct for multiple comparisons. Coloured dots (blue and red) represent proteins significantly associated with CFRD or non-CFRD. b–c) Gene enrichment analyses of the 27 proteins significantly associated with CFRD (b) and 83 proteins associated with non-CFRD (c) using Metascape.

FIGURE 3

Abundancies of sputum proteins associated with cystic fibrosis (CF)-related diabetes (CFRD) or non-CFRD in patients matched on lung function. Protein abundancies in the sputum proteome from people with CF with and without CFRD in the lung function-matched cohort. The graphs show Log2-normalised intensities in sputum of a) prothymosin α, b) α₁-antitrypsin, c) ezrin and d) TGFBI. *p<0.05.