Ligand-Independent TREM-1 Blockade Ameliorates Pulmonary Inflammation and Fibrosis

Abstract Rationale: Triggering receptor expressed on myeloid cells-1 (TREM-1) amplifies inflammation and is upregulated in patients with acute respiratory distress syndrome (ARDS) and pulmonary fibrosis (PF). Multiplicity and promiscuity of TREM-1 ligands significantly increase the risk of failure of traditional, ligand-dependent approaches to TREM-1 inhibition (e.g., anti-TREM-1 blocking antibodies and decoy peptides) in clinical efficacy testing. Despite promising preclinical data and safety in humans, the first clinical inhibitor of TREM-1, a decoy peptide LR12 (nangibotide), failed in phase IIb sepsis trial. To improve preclinical to clinical translatability, we designed a first-in-class, ligand-independent TREM-1 inhibitory peptide sequence GF9 that can be used as a free peptide GF9 or as a part of a 31-mer peptide GA31 that upon interaction with lipid(s), self-assembles into macrophage-targeted lipopeptide nanocomplexes (GA31-LPC). While GF9 inhibits TREM-1 on all TREM-1-expressing myeloid cells (pan-TREM-1 blockade), GA31-LPC inhibits TREM-1 predominantly on macrophages (targeted TREM-1 blockade). In this study, we tested GF9 and GA31-LPC for their ability to prevent and/or treat pulmonary inflammation and fibrosis in rats with a lipopolysaccharide (LPS)-induced pulmonary neutrophilia and mice with bleomycin-induced PF. Methods: Acute lung injury in rats was induced via oropharyngeal LPS aspiration. TREM-1 inhibitors were administered intraperitoneally (i.p.) 1 h before or 1 h after LPS challenge. Upon euthanization 6 h post-LPS, cytokines and cell composition were analyzed in bronchoalveolar lavage fluid (BALF). Terminal plasma cytokines were also tested. Pulmonary inflammation and fibrosis in mice were induced by intratracheal administration of bleomycin (at Day 0). TREM-1 inhibitors were administered i.p. daily for 14 days starting either Day 1 (prevention) or Day 15 (treatment). Cell composition, hydroxyproline and total protein content in BALF were analyzed at Days 7 and 22 in the prevention model and at Day 28 in the treatment model. Results: Targeted TREM-1 blockade with GA31-LPC substantially reduced LPS-induced neutrophil accumulation in the rat lungs when administered after but not before LPS, while GF9 did not exhibit any significant preventative or therapeutic effect. Notably, GF9 and GA31-LPC differently affected plasma and BALF cytokine expression levels. In mice with bleomycin-induced PF, TREM-1 blockade reduced the rate of progression and reversed fibrosis in the prevention and treatment models, respectively, with the extent dependent on the inhibitor used. Conclusions: Ligand-independent TREM-1 blockade for the first time has been shown to ameliorate pulmonary inflammation and fibrosis in animal models, warranting further development of a novel, first-in-class TREM-1 therapy for human ARDS and PF.