Nghia Pham (1), Flora Finet (1), Twinu Wilson Chirayath (1), Florence Castelli (2), François Fenaille (2), François Brial (1), Augustin Latourte (1), Frédéric Lioté (1), Pascal Richette (1), Thomas Bardin (1), Hang Korng Ea (1)
Affiliation(s):
1. Inserm URM 1132, Hôpital Lariboisière, Paris
2. Laboratoire d’études du métabolisme des médicaments UMS 28, CEA Saclay - Porte Nord, Saclay, Gif-Sur-Yvette
Introduction: Inflammation induced by monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD) crystals depends on interleukin (IL)-1β activated by the NLRP3 inflammasome. The inflammatory response can be modulated by diet. Does intermittent fasting (IF) reduce inflammation induced by MSU and CPPD crystals? Can a metabolite mimic the effect of IF?
Materials and Methods: Crystal-induced inflammation was assessed in the air pouch model in male mice fed either ad libitum or subjected to a two-day IF. Inflammation was measured by cellular infiltration, production of IL-1β and CXCL1 cytokines, and histological analysis.
Metabolomic analyses were performed by high-performance liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) on air pouch membrane and serum samples. Changes in metabolites associated with diet interventions were studied by the Metaboanalyst platform and we established the metabolic profiles for each experimental condition.
The anti-inflammatory effects of spermidine (SPD) were tested in vitro and in vivo. Inflammatory cytokine production (IL-1β, TNFα) was quantified by ELISA; NF-kB activity was assessed using the THP-1 Lucia cell line transfected with a luciferase-expressing plasmid under the NF-kB promoter. In vivo, the anti-inflammatory effect of SPD supplementation was tested.
Results: IF decreased inflammation induced by crystals: a decrease in IL-1β production (MSU 0.0 vs 30.0 pg/ml; CPPD 0.0 vs 47.5 pg/ml, p<0.0001), CXCL-1 (MSU 67.5 vs 186.8 pg/ml, CPPD 156.1 vs 549.5 pg/ml, p<0.005), and cellular infiltration in the AP lavage fluid (APLF) and semi-quantitative histological score of AP membranes. Numerous metabolic pathways were altered by IF in AP membranes and serum such as galactose, starch, sucrose, proline and arginine metabolism. Interestingly, SPD levels were elevated in serum after IF and decreased in AP membranes after crystal stimulation, suggesting its involvement in an anti-inflammatory mechanism.
In vitro, SPD (0-25-50-100µM) dose-dependently decreased IL-1β and TNF-α production induced by MSU and CPPD crystals (IL-1β: MSU 1618 - 941.0 – 614.8 – 392.5 pg/ml, p<0.001; CPPD 1160 - 680.1 – 432.8 – 292.5 pg/ml, p<0.01; TNF-α: MSU 132.1 – 68.6 – 45.1 – 25.4 pg/ml, p<0.01; CPPD 173.9 – 87.27 – 66.47 – 29.67 pg/ml, p<0.01) and NF-kB activity.
In vivo, SPD supplementation in water (6 mM) for 21 days followed by an injection into the SPD pouch (100µg/g mouse) on the day of stimulation significantly reduced inflammation induced by MSU and CPPD crystals: reduced cellular infiltration (MSU 0.5 x 10^6 vs 1.9 x 10^6, p<0.0001; CPPD 0.8 x 10^6 vs 2.7 x 10^6, p<0.001), production of inflammatory cytokines in APLF, and histological inflammation.
Conclusion: IF attenuates microcrystalline inflammation in the murine model by modifying numerous metabolic pathways. SPD appears to mimic the effect of IF and reduces inflammation induced by crystals. Studies are underway to elucidate the mechanisms of inflammation modulation by IF and SPD.