Fig. 4: Lactate activates SAA via the GPR81-NF-κB pathway.
a FC and Padj of the levels of differential metabolites of MRS-broth cultured with Tg-L.m. (Ribhigh-L.m.) compared with MRS-broth alone without bacteria detected by untargeted metabolomics. Color legends indicate up-regulate (red) or down-regulate (green) metabolites in MRS-broth cultured with Tg L.m. (MRS + Ribhigh-L.m.); noDiff, no difference between groups. b Lactate level in the 24-h anaerobic MRS culture medium of Riblow-L.m and Ribhigh-L.m. ***P < 0.001 by Student’s t-test. c Schematic design of in vitro and in vivo assays on host–bacteria interaction using Ribhigh-L.m and Riblow-L.m. d Lactate changes in the 48 h L.m. MRS-HT29 condition medium (+Ribhigh-L.m.) vs the MRS-HT29 condition medium without L.m. culture (Con). ***P < 0.001 by Student’s t-test. e The in vivo intestine lactate levels of the recipient GF mice that received FMT of single Riblow-L.m. and Ribhigh-L.m. species. * P < 0.05, by Student’s t-test. f Effect of 10 mM lactate on the protein level of GPR81, phosphorylated NF-κB p65 (p-NF-κB p65), and SAA in HT29 cells. Cells were treated with lactate for 2 h followed by cell lysis and western blotted with the indicated antibodies. s, short exposure; l, long exposure. g Effect of transient knockdown of GPR81 (small interference RNA, or siGPR81) on the protein level of GPR81, p-NF-κB p65, and SAA in HT29 cells that stimulated by 2 h treatment of 10 mM lactate. All siRNAs were added to HT29 cells with RNAimax 6 h before stimulating by lactate. After 2 h stimulation, HT29 cells were subjected to western blotting with the indicated antibodies. h–j Ileum IHC staining results of GPR81 (h), p-NF-κB p65 (i), and SAA (j) of the recipient WT mice transplanted with two-week oral gavage of 1 × 109 CFU/mL single-bacteria suspension of Ribhigh-L.m. Representative images were shown on the left, with statistical results shown on the right. ***P < 0.001, by Student’s t-test. n = 9–10 per group. k Plasma SAA levels from the recipient germ-free (GF) mice transplanted with two-week oral gavage of 1 × 109 CFU/mL single-bacteria suspension of Riblow-and Ribhigh-L.m., detected using ELISA. PBS was given the same volume as bacteria suspension as control. *P < 0.05, by Mann–Whitley test, n = 8 per group. l Blood Th1 cell frequency of the recipient GF mice transplanted with two-week oral gavage of 1 × 109 CFU/mL single-bacteria suspension of Riblow-and Ribhigh-L.m. PBS was given the same volume as bacteria suspension as control. *P < 0.05, by Mann–Whitley test, n = 8 per group. All recipient mice in FMT assays are male mice. Data are represented as mean ± SEM.
