Background:Regulatory B cells (Bregs) are defective in many auto-immune diseases, i.e. rheumatoid arthritis (RA). The short-chain fatty acid (SCFA) acetate, derived mostly from gut microbial fermentation of dietary fiber, promotes anti-inflammatory regulatory T cells and protects mice from type 1 diabetes and colitis. We hypothesized that acetate could be a good candidate to promote Bregs in auto-immune diseases.Objectives:To assess the effect of acetate on Breg number and function,in vitroandin vivoin mice and humans.Methods:Bregs were defined as IL-10 producing regulatory B cells (B10 cells). Their number was assessed after overnight exposure to acetate (Ac 10 mM) and 4 hours of CpG, ionomycin and PMA in mice and after 24 hours of acetate +/- CpG and 4 hours of ionomycin and PMA in humans. Acetate was given to mice either intraperitoneally (twice at a 12-hour interval) or in drinking water for 3 weeks. Acetate-treated B cells were transferred to mice with collagen-antibody -induced arthritis to assess their function. To decipher the mechanisms behind the effect of acetate, we used inhibitors of GPR43 (CATPB), ATP synthase (oligomycin), glycolysis (2-DG), ACSS2 and ACLY and assessed protein lysine acetylation by flow cytometry on human B cells. Acetate and B10 cells were also assessed before and after a 7-day high-fibre diet in 12 healthy volunteers.Results:In mice, acetate promoted B10 cell differentiation bothin vitro(medians [IQR] 3.1 [0.4-3.7] and 9.9 [5.9-17.6]% of B for CpG and CpG+Ac respectively, p=0.002) andin vivowhen intraperitoneal injected(22 [14-29] and 31 [25-37]% of B for PBS and acetate respectively,p=0.03) or added to drinking water (17 [6-25] and 39 [26-40]% of B for water or acetate respectively, p=0.02). Adoptive transfer of acetate-treated B cells protected mice from arthritis compared to non-exposed B cells (ANOVA p=0.008). Acetate also promoted B10 cells from human blood cells (2.5 [1.6-2.7] and 3.4 [2.6-4.5] for unstimulated [Un] and Ac respectively, p=0.0001). Conversely to CpG, acetate specifically promoted IL-10, with no impact or a decrease of proinflammatory cytokines (IL-6: 17 [5-29]; 12 [3-21] and 40 [20-47]% B cells for Un, Ac and CpG respectively, p<0.01 for all comparisons and TNF-a: 48 [29-61]; 41 [28-67] and 69 [64-78]% B cells for Un, Ac and CpG respectively, p<0.01 for CpG vs Un or Ac, NS for acetate vs Un). Inhibition of GPR43 and ACLY did not impact acetate response, while inhibition of glycolysis significantly decreased its effect. Blockade of ACSS2, converting acetate into acetyl-CoA, decreased acetate-induced B10 cells. Acetate was associated with an increase of protein lysine acetylation which was not observed in presence of CpG alone, suggesting a different mechanism of action (2.0 [1.3-3.4]; 3.3 [2.4-5.4] and 1.4 [0.5-1.7]% B cells for Un, Ac and CpG respectively, p=0.002 for Un vs Ac, NS with CpG). Conversion of acetate into acetyl-CoA could thus be used for the acetylation of cytoplasmic protein, a post-translational modification that regulates key cellular processes, including energy metabolism. In addition, B10 cells had significantly more lysine-acetylated proteins than IL-10negB cells or TNF+B cells (5.3[3.9-7.3]; 3.2 [2.4-5.4] and 3.9 [2.7-6.2] % of B for B10, IL-10negB cells or TNF+B cells respectively, p<0.01 for all comparisons). Finally, dietary fiber supplementation in healthy individuals was associated with increased acetate and B10 cells in the blood, which were significantly correlated (R2=0.20, p=0.02).Conclusion:Our results suggest that acetate induces functional Bregs, through its conversion into acetyl-CoA, used for cell metabolism and protein acetylation. Delivery of acetate or acetate producing diets or bacteria might be a promising approach to restore Bregs in non-communicable diseases such as RA in which they are defective.Disclosure of Interests:Claire DAIEN Grant/research support from: from Pfizer, Abbvie, Roche-Chugaï, Novartis, Abivax, Sandoz, Consultant of: Abbvie, Abivax, BMS, MSD, Roche-Chugaï, Lilly, Novartis, Speakers bureau: Abbvie, Abivax, BMS, MSD, Roche-Chugaï, Lilly, Novartis, Jian Tan: None declared, Rachel Audo: None declared, Julie Mielle: None declared, Laurence Macia: None declared
Assays to Study Enzymatic and Non‐Enzymatic Protein Lysine Acetylation
In Vitro