Enzymes and inhibitors in Lung Disease

Enzymes and inhibitors in lung disease. Source: D. Jenne

The well-known neutrophil serine proteases (NSPs), elastase (NE) and proteinase 3 (PR3), and the fourth member, NSP4, recently discovered by us, cleave a large number of potential substrates, but the biological and pathological relevance of these cleavages is still not known. Substrate libraries prepared from complex natural protein mixtures and proteomic approaches will permit us to map and discriminate the substrate preferences of these functionally related proteases. Progranulin is one of the targets of NE and PR3 under in vivo conditions, while other inflammatory mediators may also be proteolytically trimmed locally to isoforms with enhanced biological activity.

Using experimental models of lung inflammation, we are searching for NSP-specific proteolytic events in lung extracts and lavage fluids. In genetically modified mice, that lack one or a combination of these protease genes, we will identify bioprocessing defects and functional alterations in response to cigarette smoke, lipopolysaccharide or exogenous elastase exposure.

As a consequence and complication of chronic inflammatory diseases, autoantibodies to affected organs and activated neutrophils can develop. Such autoantibodies eventually result in generalized inflammation and organ failure. For example, autoantibodies to proteinase 3 (PR3-ANCA) and myeloperoxidase (MPO-ANCA), hallmarks of Wegener’s granulomatosis (also called granulomatosis with polyangiitis, GPA) and microscopic polyangiitis, respectively, trigger neutrophil activation and tissue damage during disease exacerbations.

Posttranslational modifications of self-proteins by oxidation and limited proteolysis may initiate autoimmune responses which later also spread to the native self-antigen. Moreover, neutrophil extracellular traps formed by dying neutrophils are suggested to enhance autoimmune responses to neutrophil granule proteins in small vessel vasculitis and in other chronic auto-inflammatory conditions.

Projects currently pursued include the recombinant production of hPR3 variants to distinguish and evaluate the pathogenic potential of individual ANCA responses, development of a mouse or macaque model for PR3-ANCA testing in Wegener’s granulomatosis.

Similarly, we are developing a mouse model for the transfer of human MPO-ANCA. The neutrophil-derived MPO antigen is a homodimer with two light and two heavy chains after several processing steps of a single chain precursor. These processing steps are being explored and their relevance for autoantibody formation, recognition and neutrophil activation will be assessed.

Our current view on NSPs is that they collectively accelerate innate immune responses and adaptive protection against microbial pathogens. All four neutrophil serine proteases, however, are not essential to their host under normal living conditions, but contribute significantly to inflammation-associated tissue damage, e. g. in the lung and bronchi, when neutrophils and macrophages accumulate. Hence these proteases are attractive targets for therapeutic interventions with tailor-made inhibitors which will be further evaluated in preclinical models. To this end, inhibitor variants with tailor-made profiles for the specific inhibition of NE, PR3 and NSP4 are being developped, characterized and optimized.