Abstract Rationale Propionate (C3), a metabolite produced only by the gut microbiota, is a regulator of inflammatory signaling in various pulmonary diseases. Previously, we found that propionate suppressed interleukin-6 (IL-6) protein induction in murine and human alveolar macrophages (AM) exposed to lipopolysaccharide (LPS), with restoration of OX-PHOS metabolism and upregulation of peroxisome proliferator-activated receptor gamma (PPARγ), a regulator of cellular metabolism and a hallmark of anti-inflammatory macrophages. In this study, we investigated the signaling pathways underlying the metabolic reprogramming and polarization of LPS challenged AMs from a pro-inflammatory to an anti-inflammatory phenotype in response to propionate in AMs. Methods The murine AM cell line, MH-S, was stimulated for 24 hours with 50ng/mL LPS and/or various concentrations of C3 (0.03, 0.3 or 3mM). Cytokine (IL-6 and IL-10) levels in the supernatants at the endpoint were evaluated by using ELISA. Cells were interrogated by qPCRs to evaluate the mRNA expression of inflammatory and anti-inflammatory phenotype markers and potential signaling pathway candidates, including peroxisome proliferator-activated receptor gamma (Pparγ), retinoid X receptor alpha (RXRα), C-X3-C motif chemokine receptor 1 (Cx3cr1) and interleukin-10 (IL-10).To assess the metabolic dependency of inflammatory responses, MH-S cells were stimulated with 3mM C3 for 4 hours before being co-treated overnight with 50ng/mL LPS ± 3mM C3 ± 0.1μM 2-Deoxy-D-Glucose, a glycolysis inhibitor, or ± 0.05μM Oligomycin, an OX-PHOS inhibitor. The supernatants at the endpoint were used to evaluate IL-6 protein levels using ELISA. Results mRNA expression levels of Pparγ, Rxrα and Cx3cr1 increased in a C3 dose-dependent manner, particularly with high dose C3, both with and without LPS stimulation while induction of IL-10 by C3 occurred only in the context of LPS injury (Fig.1A). IL-6 protein levels were significantly decreased in a dose-responsive fashion in the presence of C3 in the context of concurrent LPS challenge. In contrast, IL-10 protein levels were significantly elevated in the presence of 3mM C3 (Fig.1B).Using inhibitors of glycolysis and OX-PHOS, we observed that IL-6 protein levels increased when glycolysis was unopposed in the LPS+Oligomycin group. C3 was able to suppress LPS-induced inflammatory responses, but this suppression was weaker when OX-PHOS was blocked and glycolysis was unopposed (Fig.1C). Conclusions Our findings suggest that propionate activates PPARγ signaling pathways and likely reprograms resting AMs. In the context of LPS injury, propionate suppresses inflammatory IL-6 responses while promoting anti-inflammatory IL-10 cytokine production. These effects appear to require active oxidative phosphorylation metabolic programming in the AMs. This abstract is funded by: None
Doan et al. (Fri,) studied this question.