The role for neutrophil extracellular traps in cystic fibrosis autoimmunity
Sladjana Skopelja, B. JoNell Hamilton, Jonathan D. Jones, Mei-Ling Yang, Mark Mamula, Alix Ashare, Alex H. Gifford, William F.C. Rigby
Sladjana Skopelja, B. JoNell Hamilton, Jonathan D. Jones, Mei-Ling Yang, Mark Mamula, Alix Ashare, Alex H. Gifford, William F.C. Rigby
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Research Article Immunology

The role for neutrophil extracellular traps in cystic fibrosis autoimmunity

Abstract

While respiratory failure in cystic fibrosis (CF) frequently associates with chronic infection by Pseudomonas aeruginosa, no single factor predicts the extent of lung damage in CF. To elucidate other causes, we studied the autoantibody profile in CF and rheumatoid arthritis (RA) patients, given the similar association of airway inflammation and autoimmunity in RA. Even though we observed that bactericidal permeability-increasing protein (BPI), carbamylated proteins, and citrullinated proteins all localized to the neutrophil extracellular traps (NETs), which are implicated in the development of autoimmunity, our study demonstrates striking autoantibody specificity in CF. Particularly, CF patients developed anti-BPI autoantibodies but hardly any anti-citrullinated protein autoantibodies (ACPA). In contrast, ACPA-positive RA patients exhibited no reactivity with BPI. Interestingly, anti-carbamylated protein autoantibodies (ACarPA) were found in both cohorts but did not cross-react with BPI. Contrary to ACPA and ACarPA, anti-BPI autoantibodies recognized the BPI C-terminus in the absence of posttranslational modifications. In fact, we discovered that P. aeruginosa–mediated NET formation results in BPI cleavage by P. aeruginosa elastase, which suggests a novel mechanism in the development of autoimmunity to BPI. In accordance with this model, autoantibodies associated with presence of P. aeruginosa on sputum culture. Finally, our results provide a role for autoimmunity in CF disease severity, as autoantibody levels associate with diminished lung function.

Authors

Sladjana Skopelja, B. JoNell Hamilton, Jonathan D. Jones, Mei-Ling Yang, Mark Mamula, Alix Ashare, Alex H. Gifford, William F.C. Rigby

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Figure 4

BPI is cleaved in a P.

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BPI is cleaved in a P. aeruginosastrain-dependent manner. (A) Healthy n...
aeruginosastrain-dependent manner. (A) Healthy neutrophils were treated with NET-inducing agents, PMA (20 nM), P. aeruginosa PA14 (100 MOI), or glucose oxidase (GO, 2 U/ml), or left untreated (NT) for 1 hour. Total BPI protein (55 kDa) and smaller protein fragments were detected in 20 μg of insoluble and soluble protein extracts via immunoblot with a mouse anti-human BPI antibody directed at aa 227–254 epitope (i.e., BPI C-terminus hinge region). (B) Total BPI and protein fragments were detected by immunoblot in soluble protein extracts (10 μg) from neutrophils treated with PMA or increasing MOIs of P. aeruginosa strains PAO1 (0.1, 1, 10, and 100 MOI) and PA14 (1, 10, and 100 MOI) with the antibody used in A. (C) The extent of BPI cleavage detected in soluble protein extracts (10 μg) from neutrophils following incubation with P. aeruginosa strains PAO1 and PA14, wild type strains (WT), or elastase deficient strains (ΔlasB or ΔlasR) (10 MOI) for 1 hour. (D) Total BPI and protein fragments were detected by immunoblot in BAL samples (20 μg protein/sample) with the antibody used in A. The immunoblots in A–C are representative images of n = 3 experiments. NET, neutrophil extracellular trap; BPI, bactericidal permeability-increasing protein; BAL, bronchoalveolar lavage.