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.

Figure 1. BPI and carbamylated proteins are localized on neutrophil extracellular traps.

(A) PMA treatment (600 nM, 2 hours) of healthy neutrophils induced formation of neutrophil extracellular traps (NETs), characterized by extrusion of DNA (DAPI, blue) and neutrophil elastase (AI488, green). (B and C) Cy3 and Al488 alone were used to determine the level of nonspecific staining. Immunocytochemistry staining demonstrated that (D) citrullinated proteins (Cy3, red), (E) carbamylated proteins (Cy3, red), and (F) BPI-Al488 localize to the DNA strands of the NETs. (G–I) Neutrophil elastase (Al488) and BPI-Cy3 colocalize in the NETs. (J–L) Neutrophil elastase (Al488) and BPI-Cy3 colocalize in untreated PMNs. Scale bar: 10 μm. NE, neutrophil elastase; BPI, bactericidal permeability-increasing protein; Cit, citrullinated proteins; Carb, carbamylated proteins.

Figure 2. Autoantibody profile and specificity in CF patients.

(A) Anti-nBPI IgG levels are higher in cystic fibrosis (CF) patients compared with those in healthy sera (HS) and rheumatoid arthritis (RA) patients by 1-way ANOVA, with Bonferroni post-hoc (HS [n = 23], mean = 0.15; CF [n = 38], mean = 0.41; RA [n = 50], mean = 0.10); dotted line represents positive cutoff determined as mean + 2 SD of HS cohort. (B) ACPA IgG levels are higher in RA patients compared with CF patients by t test (CF [n = 38], geometric mean [CI = 95%] = 6.47 U/ml; RA [n = 50], geometric mean [CI = 95%] = 430.5 U/ml); samples with ACPA levels >20 U/ml were considered to be ACPA positive. (C) IgM rheumatoid factor (RF) levels are higher in RA patients compared with CF patients by t test (CF [n = 38], geometric mean [CI = 95%] = 10.9 U/ml; RA [n = 50], geometric mean [CI = 95%] = 122 U/ml); samples from patients with IgM RF concentrations >25 U/ml were considered to be IgM RF positive. (D) ACarPA IgG are present at comparable levels in CF and RA patients by t test (CF [n = 38], geometric mean [CI = 95%] = 17.2 U/ml; RA [n = 50], geometric mean [CI = 95%] = 15.4 U/ml); samples with ACarPA concentrations >18 U/ml were considered to be ACarPA positive (measured by Inova Diagnostics). (A–D) Error bars represent mean ± SEM; ****P < 0.0001. (E) Reactivity of CF sera to denatured fibrinogen (Fib), carbamylated fibrinogen (carbam. Fib.), and nBPI (1 μg) was evaluated by immunoblot. Representative CF sera bind to nBPI and carbamylated fibrinogen with specificity. ACPA, anti-citrullinated protein autoantibodies; ACarPA, anti-carbamylated protein autoantibodies; nBPI, neutrophil-purified BPI.

Figure 3. Anti-BPI autoantibodies in CF target the C-terminus of BPI.

(A) Crystal structure of BPI protein (RCSB Protein Data Bank, accession number P17213.4); diagram shows N-terminus (NT-BPI, aa 10–240) and C-terminus (CTBPI, aa 260–430). (B) Reactivity of cystic fibrosis (CF) sera to the recombinant CTBPI correlates with reactivity to the intact nBPI, as determined by Pearson correlation analysis (r = 0.690, P < 0.0001, n = 38). (C) Recombinant CTBPI and nBPI (1μg) were detected by immunoblot using monoclonal anti-BPI antibody specific for the epitope mapping to aa 227–254. (D) Representative immunoblots confirm reactivity of CF sera to the total BPI (55 kDa, left lane) and to recombinant CTBPI protein (~30 kDa, right lane). nBPI = neutrophil-purified bactericidal permeability-increasing protein.

Figure 4. BPI is cleaved in a P.

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.

Figure 5. Anti-BPI IgG correlates with FEV1 and anti-Cif immune response to P.

aeruginosain a US adult CF cohort. (A) Anti-CTBPI IgG levels determined by ELISA negatively correlate with pulmonary function in cystic fibrosis (CF) patients (FEV1 percent predicted), as determined by Pearson correlation analysis (n = 38, r = –0.664, P < 0.0001). P. aeruginosa infection status of patients is denoted: P. aeruginosa⁺ (white circle) and P. aeruginosa^– (black circle). (B) Anti-BPI IgG titers were measured in representative CF bronchoalveolar lavage (BAL) samples that previously demonstrated serum anti-BPI reactivity by ELISA (ANOVA, n = 3), ***P < 0.001. (C) Higher anti-CTBPI IgG levels associate with positive P. aeruginosa sputum culture (PA⁺) in CF patients (n = 38), (D) particularly with presence of the mucoid P. aeruginosa strains (mPA) (n = 38). (E) Anti-BPI IgG titers are higher in CF patients that do not carry the F508del homozygous CFTR gene mutation (n = 38). (F) Anti-Cif (10 μg/ml) IgG levels are higher in CF patients with anti-BPI autoantibodies (Anti-BPI IgG⁺) compared with patients without reactivity to BPI (Anti-BPI IgG^–) (n = 28). (C–F) Significance between two groups was determined by Student’s t test with Welch’s correction; *P < 0.05, **P < 0.01, ***P < 0.001; error bars represent mean ± SEM. FEV 1%, forced expiratory volume in 1 second percent predicted; Cif, CFTR inhibitory factor; BPI, bactericidal permeability-increasing protein.