Abstract Rationale Chronic obstructive pulmonary disease (COPD) is the third leading causeof death worldwide, therefore there is a large unmet medical need for therapeutic options particularly in patients who frequently develop microbial exacerbations. IL-36 cytokines have been discovered to promote pro-inflammatory pathways in COPD. Pro- IL-36 cytokines are known to be cleaved and activated by neutrophil-derived or pathogen-derived proteases in cell-free conditions. However, the relevance of these findings to acute bacterial exacerbations of COPD with neutrophilic inflammation has remained to be addressed. We sought to determine whether 1) IL-36 isoforms are cleaved and activated by neutrophil and bacterial-derived proteases and 2) IL-36 receptor activity is induced in human sputum from COPD patients. Methods A HEK-Blue reporter cell line expressing the IL-36 receptor (IL-36R) was utilized in these studies. Briefly, a protease source, i.e. neutrophil extracellular traps (NET) derived from blood neutrophils and a cytokine source, i.e. recombinant human IL-36Ɣ, were incubated together at 37 °C in the presence or absence of protease inhibitors before being added to HEK-Blue cell culture. For bacterial-derived proteases we harvested culture filtrates (CF) from non-typeable haemophilus influenzae (NTHi), a common pathogen associated with COPD exacerbations, to be incubated with recombinant human IL-36Ɣ. Production of secreted embryonic alkaline phosphatase(SEAP) was quantified as a measure of IL-36R activity. Western blotting determined the cleavage pattern of full-length IL-36Ɣ under various proteolytic conditions. Additionally, IL-36 receptor activity was assessed upon treating HEK-Blue reporter cells with sputum collected from COPD patients in the presence or absence of protease inhibitors. Results The HEK-Blue reporter cell line was validated utilizing pro- and truncated IL-36 isoforms. There was ∼30-fold increase in signaling activity with truncated IL-36ɑ/β/Ɣ than the pro-IL-36 isoforms alone. Utilizing a neutralizing antibody for IL-36 receptor, we demonstrated that SEAP production was IL-36-dependent in this assay. CF from NTHi and NET induced IL-36ɑ/β/Ɣ-medaited IL-36 signaling which were blunted by treatment with a pan-protease inhibitor. Our Western blot analysis revealed IL-36Ɣ cleavage by neutrophil elastase (NE) or NET that was blocked by protease inhibitors. In human COPD sputum samples, IL-36 signaling was detectable and it was suppressed by protease inhibitors. Conclusion IL-36 maturation is regulated by both neutrophil and pathogen-derived proteases suggestive of a novel pro-inflammatory mechanism in COPD. This abstract is funded by: Genentech Inc.
Movassagh et al. (Fri,) studied this question.