Reduced neutrophil elastase inhibitor elafin and elevated transforming growth factor-β1 are linked to inflammatory response in sputum of cystic fibrosis patients with Pseudomonas aeruginosa

Jan C Thomassen, Tobias Trojan, Maxine Walz, Christina Vohlen, Gregor Fink, Ernst Rietschel, Miguel A Alejandre Alcazar, Silke van Koningsbruggen-Rietschel, Jan C Thomassen, Tobias Trojan, Maxine Walz, Christina Vohlen, Gregor Fink, Ernst Rietschel, Miguel A Alejandre Alcazar, Silke van Koningsbruggen-Rietschel

Abstract

Research question: Pulmonary disease progression in patients with cystic fibrosis (CF) is characterised by inflammation and fibrosis and aggravated by Pseudomonas aeruginosa (Pa). We investigated the impact of Pa specifically on: 1) protease/antiprotease balance; 2) inflammation; and 3) the link of both parameters to clinical parameters of CF patients.

Methods: Transforming growth factor-β1 (TGF-β1), interleukin (IL)-1β, IL-8, neutrophil elastase (NE) and elastase inhibitor elafin were measured (ELISA assays), and gene expression of the NF-κB pathway was assessed (reverse transcriptase PCR) in the sputum of 60 CF patients with a minimum age of 5 years. Spirometry was assessed according to American Thoracic Society guidelines.

Results: Our results demonstrated the following: 1) NE was markedly increased in Pa-positive sputum, whereas elafin was significantly decreased; 2) increased IL-1β/IL-8 levels were associated with both Pa infection and reduced forced expiratory volume in 1 s, and sputum TGF-β1 was elevated in Pa-infected CF patients and linked to an impaired lung function; and 3) gene expression of NF-κB signalling components was increased in sputum of Pa-infected patients, and these findings were positively correlated with IL-8.

Conclusion: Our study links Pa infection to an imbalance of NE and NE inhibitor elafin and increased inflammatory mediators. Moreover, our data demonstrate an association between high TGF-β1 sputum levels and a progress in chronic lung inflammation and pulmonary fibrosis in CF. Controlling the excessive airway inflammation by inhibition of NE and TGF-β1 might be promising therapeutic strategies in future CF therapy and a possible complement to cystic fibrosis transmembrane conductance regulator (CFTR) modulators.

Conflict of interest statement

Conflict of interest: J.C. Thomassen has nothing to disclose. Conflict of interest: T. Trojan has nothing to disclose. Conflict of interest: M. Walz has nothing to disclose. Conflict of interest: C. Vohlen has nothing to disclose. Conflict of interest: G. Fink has nothing to disclose. Conflict of interest: E. Rietschel has nothing to disclose. Conflict of interest: M.A. Alejandre Alcazar has nothing to disclose. Conflict of interest: S. van Koningsbruggen-Rietschel has nothing to disclose.

Copyright ©The authors 2021.

Figures

FIGURE 1
FIGURE 1
Assessment of a) neutrophil elastase (NE) concentrations (ng·mL−1) and b) elafin concentrations (pg·mL−1) in sputum of cystic fibrosis (CF) patients, related to Pseudomonas infection: P. aeruginosa-negative CF patients (n=32) and P. aeruginosa-positive CF patients (n=16). Data presented as median and interquartile range; Mann–Whitney U-test performed. **: p<0.01; ***: p<0.001.
FIGURE 2
FIGURE 2
a) Interleukin (IL)-8 and b) IL-1β concentrations (pg·mL−1) in sputum of cystic fibrosis (CF) patients, related to Pseudomonas infection: P. aeruginosa-negative CF patients (n=34) and P. aeruginosa-positive CF patients (n=18). Data presented as median and interquartile range; Mann–Whitney U-test performed. ****: p<0.0001.
FIGURE 3
FIGURE 3
a) Interleukin (IL)-1β and b) IL-8 concentrations (pg·mL−1) related to forced expiratory volume in 1 s (FEV1) values: elevated IL-1β and IL-8 levels were detected in cystic fibrosis (CF) patients with FEV1 <80% (n=27) compared to CF patients with FEV1 values ≥80% (n=25). Data presented as median and interquartile range; Mann–Whitney U-test performed. **: p<0.01; ****: p<0.0001.
FIGURE 4
FIGURE 4
Pearson correlation between interleukin (IL)-1β and IL-8 concentrations (pg·mL−1) in sputum of cystic fibrosis patients (n=52). A positive correlation between IL-1β and IL-8 levels was detected (r=0.763; ****: p<0.0001).
FIGURE 5
FIGURE 5
Assessment of sputum transforming growth factor-β1 (TGF-β1) concentrations (pg·mL−1) in cystic fibrosis patients by specific TGF-β1 ELISA. Correlation to a) Pseudomonas aeruginosa colonisation (P. aeruginosa-negative: n=33; P. aeruginosa-positive: n=17) and b) forced expiratory volume in 1 s (FEV1) % predicted (FEV1 ≥80%: n=23; FEV1 <80%: n=27). Data presented as median and interquartile range; Mann–Whitney U-test performed. *: p<0.05; ****: p<0.0001.
FIGURE 6
FIGURE 6
Spearman correlation between concentrations of sputum transforming growth factor-β1 (TGF-β1) (pg·mL−1) and proinflammatory cytokines interleukin (IL)-1β and IL-8 in cystic fibrosis patients (n=50). Positive correlations between a) IL-1β and TGF-β1 levels (r=0.707; ****: p<0.0001) and b) IL-8 and TGF-β1 levels (r=0.670; ****: p<0.0001).
FIGURE 7
FIGURE 7
Gene expression of NF-κB signalling cascade mediators in cystic fibrosis sputum cells of 43 patients: p50, p65, IKKα and interleukin (IL)-6 levels were determined by quantitative reverse transcriptase PCR. Significantly elevated mRNA expression of mediators of the NF-κB signalling cascade: a) IKKα, b) IL-6, c) p50 and d) p65. Data presented as median and interquartile range; Mann–Whitney U-test performed. P. aeruginosa: Pseudomonas aeruginosa. *: p<0.05; **: p<0.01.
FIGURE 8
FIGURE 8
Spearman correlations between level of interleukin (IL)-8 and quantitative mRNA expression of p50 and p65. Significant positive correlations between IL-8 levels and quantity of mRNA expression of the NF-κB signalling proteins a) p50 (r=0.402; **: p

FIGURE 9

Simplified presentation of the vicious…

FIGURE 9

Simplified presentation of the vicious circle of chronic inflammation in cystic fibrosis (CF)…

FIGURE 9
Simplified presentation of the vicious circle of chronic inflammation in cystic fibrosis (CF) as a result of high neutrophilic activity, disruption of the homeostatic protease/antiprotease balance and microbial infection with P. aeruginosa in the CF lung causing chronic inflammatory lung disease and pulmonary fibrosis. NE: neutrophil elastase; TGF-β1: transforming growth factor-β1; IL: interleukin.
All figures (9)
FIGURE 9
FIGURE 9
Simplified presentation of the vicious circle of chronic inflammation in cystic fibrosis (CF) as a result of high neutrophilic activity, disruption of the homeostatic protease/antiprotease balance and microbial infection with P. aeruginosa in the CF lung causing chronic inflammatory lung disease and pulmonary fibrosis. NE: neutrophil elastase; TGF-β1: transforming growth factor-β1; IL: interleukin.

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