Characterization of ecotin homologs from Campylobacter rectus and Campylobacter showae.

Ecotin, first described in Escherichia coli, is a potent inhibitor of a broad range of serine proteases including those typically released by the innate immune system such as neutrophil elastase (NE). Here we describe the identification of ecotin orthologs in various Campylobacter species, including Campylobacter rectus and Campylobacter showae residing in the oral cavity and implicated in the development and progression of periodontal disease in humans. To investigate the function of these ecotins in vitro, the orthologs from C. rectus and C. showae were recombinantly expressed and purified from E. coli. Using CmeA degradation/protection assays, fluorescence resonance energy transfer and NE activity assays, we found that ecotins from C. rectus and C. showae inhibit NE, factor Xa and trypsin, but not the Campylobacter jejuni serine protease HtrA or its ortholog in E. coli, DegP. To further evaluate ecotin function in vivo, an E. coli ecotin-deficient mutant was complemented with the C. rectus and C. showae homologs. Using a neutrophil killing assay, we demonstrate that the low survival rate of the E. coli ecotin-deficient mutant can be rescued upon expression of ecotins from C. rectus and C. showae. In addition, the C. rectus and C. showae ecotins partially compensate for loss of N-glycosylation and increased protease susceptibility in the related pathogen, Campylobacter jejuni, thus implicating a similar role for these proteins in the native host to cope with the protease-rich environment of the oral cavity.

Systemic levels of anti-PAD4 autoantibodies correlate with airway obstruction in cystic fibrosis.

Cystic fibrosis (CF) airway disease is characterized by the long-term presence of neutrophil granulocytes. Formation of neutrophil extracellular traps (NETs) and/or autoantibodies directed against extracellular components of NETs are possible contributors to neutrophil-mediated lung damage in CF. The goal of this study was to measure their levels in CF adults compared to healthy controls and subjects with rheumatologic diseases known to develop NET-related autoantibodies and pathologies, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Sera were analyzed from the following number of subjects: 37 CF, 23 healthy controls (HC), 20 RA, and 21 SLE. CF had elevated serum myeloperoxidase (MPO) concentrations (347.5±56.1 ng/ml, mean+/-S.E.M., p = .0132) compared to HC (144.5±14.6 ng/ml) but not of neutrophil elastase (NE) complexed with alpha-1-antitrypsin, cell-free DNA or NE-DNA complexes. The peptidylarginine deiminase 4 (PAD4) enzyme is required for NET formation and associated DNA release in neutrophils. Serum levels of anti-PAD4 antibodies (Ab) were elevated in CF (p = .0147) compared to HC and showed an inverse correlation with a measure of lung function, FEV1% predicted (r = -0.5020, p = .015), as did MPO levels (r = -0.4801, p = .0026). Anti-PAD4 Ab levels in CF sera associated with lung infection by P. aeruginosa, but not that by S. aureus, age, sex, CF-related diabetes or the presence of musculoskeletal pain. Serum levels of anti-citrullinated protein Abs (ACPAs) and anti-nucleosome Abs were not elevated in CF compared to HC (p = .7498, p = .0678). In summary, adult CF subjects develop an autoimmune response against NET components that correlates with worsening lung disease.