Evaluating the immunomodulatory potential of gut-derived microbial metabolites for therapeutic use in chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is a heterogeneous lung condition affecting 1 in 7 Australians ≥ 40 years (1) and is the third leading cause of death worldwide (2) . COPD exacerbations are the leading cause of preventable hospital admission in Australia (3) , with viral and bacterial infections being the primary cause. The gut and lungs share a mucosal immune system known as the gut-lung axis (4) . The gut contains the bodies largest community of microorganisms, known as the gut microbiota, and disruption to the gut microbiota is implicated in chronic diseases such as Inflammatory Bowel Disease, obesity and type 2 diabetes (5) . Dietary intake modulates the gut microbiota via several pathways. Importantly, gut microbiota produce metabolites via digestion, and these microbial metabolites can modulate the immune system, and exert both beneficial and detrimental effects systemically. Therefore, we aimed to assess the potential of gut-derived microbial metabolites to modify immune cell responses to stimuli known to induce COPD exacerbations. Gut microbial metabolites, including seven tryptophan metabolites, and one secondary bile acid were selected by literature review. In control adults (n = 8), peripheral blood mononuclear cells (PBMC) were isolated via Lymphoprep™ density gradient centrifugation and seeded 2 × 10 6 cells/mL. Metabolite dose-curves (1–100 μM) were generated to determine optimal concentration. Post-3-hour metabolite incubation, PBMCs were stimulated with either lipopolysaccharide (LPS) (24 hr) or influenza-A (H1N1) (48 hr). PBMC production of anti-viral and anti-inflammatory cytokines IFN-γ, IL-6, TNF-α and IL-1β was assessed by DuoSet® ELISA. Cell viability post-metabolite incubation was confirmed via MTT assay. Cytokine dose-curves following metabolite treatment and stimulation were produced (n = 4/metabolite). Metabolite concentrations were selected based on reduction of LPS-induced TNF-α and IL-1β secretion, reduced influenza-A-induced IL-6 secretion, and increased influenza-A-induced IFN-γ secretion. Optimal metabolite concentrations included: secondary bile acid lithocholic acid (1 μM), and tryptophan metabolites nicotinamide (5 μM), indole-3-acetic acid (1 μM), kynurenic acid (5 μM), 3-hydroxyanthranilic acid (10 μM). Tryptophan metabolites cinnabarinic acid was dose-dependently pro-inflammatory, and tryptamine and 2-picolinic acid had no effect. MTT assays (n = 3/metabolite) found metabolites were not cytotoxic at the concentrations tested. These data show that gut-derived microbial metabolites modify immune cell responses to infection. These findings will inform further research which will examine the effects of microbial metabolites in immune cells from adults with COPD and assess the role of the gut microbiota in infectious COPD exacerbations.