LSC Abstract – A functional variant of secretory leukocyte protease inhibitor (SLPI) with improved anti-inflammatory activity against pulmonary infection

Abstract

Secretory leukocyte protease inhibitor (SLPI) is an important respiratory tract protease inhibitor also possessing antimicrobial and anti-inflammatory properties. SLPI has shown promising therapeutic effects in the treatment of cystic fibrosis (CF) lung disease, but its benefits are compromised by its rapid cleavage and inactivation by excessively high levels of neutrophil elastase (NE) in the Pseudomonas -infected CF lung. The aims of this study were to (a) examine the correlation of SLPI levels in paediatric CF patients with NE activity levels and observed lung function, and (b) develop SLPI variants featuring resistance to NE cleavage. We show in a paediatric CF patient cohort that decreased SLPI levels correlate significantly with increased NE activity and decreased FEV1. Two potentially NE-resistant SLPI molecules (SLPI-A16G and SLPI-S15G-A16G) were generated by site-directed mutagenesis, then functionally evaluated using in vitro and in vivo experiments. Both variants showed improved resistance to degradation in the presence of excess NE as well as Pseudomonas -positive CF patient sputum, compared to wild-type (WT) SLPI. Both proteins retained potent NE inhibitory activity, bacterial LPS binding and NF-κB binding ability. Finally we show enhanced anti-inflammatory activity (reduced neutrophil recruitment to the lung) of the SLPI-A16G protein compared to WT SLPI in a model of pulmonary Pseudomonas infection. We propose that the SLPI variants presented in this study may have superior therapeutic potential for novel anti-inflammatory treatment in the CF lung.