Figure 4. BHB suppresses NLRP3-mediated inflammatory disease in vivo and the inflammasome in in human monocytes.

(a) Analysis of IL-1β and IL-18 secretion in culture supernatants of human monocytes stimulated with vehicle or LPS (1μg/mL) for 4h in presence of increasing concentrations of BHB (n =6/treatment). (b) BHB-complexed nanolipogels (nLGs) block the NLRP3 inflammasome activation and caspase-1 cleavage (n=3, repeated twice). (c) Frequency of CD45⁺ and Gr1⁺ immune cells in the peritoneum of mice treated with MSU (3 mg) and BHB-nLGs (125 mg/kg/bw), as assessed by FACS (N =6/group). (d) IL-1β secretion from peritoneal cells cultured overnight and (e) serum IL-1β levels from mice challenged with MSU and treated with BHB-nLGs (n =6/group) (f) Western blot analysis of caspase-1 and IL-1β activation in the BM cells stimulated in presence of LPS and BHB-nLGs from mice harbouring the MWS mutation NLRP3^A350V and (g) FCAS mutation (n = 6, repeated twice) (h)Representative immunoblot analysis of disuccinimidyl suberate (DSS) cross-linked ASC in the Nonidet P-40-insoluble pellet of BMDM from FCAS mice (n = 6) that were primed with LPS (4 h) and treated with increasing concentrations of BHB-nLGs. (i) Neutrophil numbers in peritoneum of FCAS mice fed a chow or ketone diester diet (1,3-butanediol) for one week. (n =6/group).Data are expressed as mean ± S.E.M (*P < 0.05) and statistical differences between means and the effects of treatments were determined by one-way ANOVA using Tukey's test (a,d, e) and t-test (k).