Abstract Rationale Neutrophil elastase (NE) is a major inflammatory mediator in the lungs of patients with CF (pwCF) and patients with COPD. NE inhibits macrophage phagocytosis, induces the release of macrophage extracellular traps (METs), and in the presence of LPS, increases cytokine expression. There are currently no anti-protease therapies available for patients with CF or COPD. We hypothesized that a novel polysulfated hyaluronan, GM-1111, previously shown to potentiate DNase activity and inhibit NE proteolytic activity in CF sputum, would be effective both in vitro and in vivo in blocking NE-induced lung inflammation and restore macrophage dysfunction. Methods Human blood monocytes were isolated from healthy subjects and from pwCF. Cells were cultured in media with serum and GM-CSF to differentiate cells into blood monocyte derived macrophages (hBMDM). Human BMDM were then changed to serum-free media and exposed to NE (50 or 100 nM), LPS (10ng/ml) or control vehicle and to GM-1111 (1.5 μM or 18.2 μM) or control vehicle for varying times for different assays. For phagocytosis assays, after treatment for 18h, uptake of FITC-labeled E. coli K-12 bioparticles was assessed by confocal microscopy and quantitation by FITC: DAPI intensities. For MET release, cells were treated for 2 h, conditioned media was removed, and cells were treated with micrococcal nuclease to release extracellular traps. Quantification of extracellular DNA (ecDNA) in the micrococcal nuclease conditioned media was performed by Quanti-iT PicoGreen dsDNA Assay (Thermo Fisher). To evaluate the impact of GM-1111 on cytokine release, hBMDM were treated with control vehicle, LPS, or NE+LPS in the presence or absence of GM-1111 for 24 h. Cytokine expression (TNFα and IL-6) was measured by ELISA (BioLegend) in the conditioned media. C57BL/6J male mice (8-10 weeks, ∼25 g) (n = 4 per group) were exposed to control vehicle, ii) GM-1111 (630 μM) alone, iii)NE (42 μM), or iv)GM+NE by oropharyngeal aspiration on days 1, 4, and 7. Mice were euthanized on Day 8 and BAL was obtained. BAL cytokines (MCP1 and CXCL1/KC) were measured by ELISA (R cell count and differentials were determined. Results GM-1111 treatment alone had no adverse effects on mice or in-vitro cell viability. GM-1111 restored NE-induced phagocytic dysfunction, prevented MET release in vitro, and reduced the release of cytokines both in vitro and in vivo. Conclusions GM-1111 is a promising anti-protease/ anti-inflammatory therapeutic candidate for CF and COPD lung disease. This abstract is funded by: Supported by the Children’s Hospital Research Institute, VCU, GlycoMira Therapeutics Inc., and NIH R01HL146811-05.
Kummarapurugu et al. (Fri,) studied this question.