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Fig. 4 | Microbiome

Fig. 4

From: Gut microbiota–derived metabolite 3-idoleacetic acid together with LPS induces IL-35+ B cell generation

Fig. 4

Together, IAA and LPS promote the binding of PXR to NFκB p65 and RXR. a Flow cytometry of p35+CD19+ cells in the spleen after exposure to IAA, LPS, LPS + IAA, LPS + IAA + AhR inhibitor (+AHRi), LPS + IAA + PXR inhibitor (+PXRi), or LPS + IAA + NFκB inhibitor (+NFκBi) for 24 h. IsoAb, isotype control; NC, unstimulated control. b Flow cytometry of p35+Ebi3+CD19+ cells in the spleen after exposure to IAA, LPS, LPS + IAA, LPS + IAA + AHR inhibitor (+AHRi), LPS + IAA + PXR inhibitor (+PXRi), or LPS + IAA + NFκB inhibitor (+NFκBi) for 24 h. IsoAb, isotype control; NC, unstimulated control. c ELISA of IL-35 in the supernatants of spleen cells after exposure to different concentrations of IAA in the presence of LPS or different concentrations of LPS in the presence of IAA. d Flow cytometry of CD19+p35+Ebi3+ cells in the adipose tissues of Gram bacteria–deleted WT and huREG4IECtg mice (HuR4) treated with or without LPS. e LPS levels in the blood of Gram bacteria-deleted WT and huREG4IECtg mice (HuR4) treated with or without LPS. f IAA levels in the blood of Gram bacteria-deleted WT and huREG4IECtg mice (HuR4) treated with or without LPS. g LPS concentrations in the sera of WT (WT1), Reg4 KO (R4KO), and huREG4IECtg (HuR4) mice and their littermate controls (WT2) fed (HFD) or not fed (NCD) a HFD for 3 months. h ChIP sequencing analyses of the B lymphocyte WEHI 231 B cells after exposure to LPS, IAA or LPS + IAA for 6 h. i ChIP-PCR of the PXR binding site on the promoter region of p35 in WEHI 231 B cells after exposure to LPS, IAA or LPS + IAA for 6 h. j Immunoblotting of p65, RXRa, and PXR in the cytosolic and nuclear extracts of immunoprecipitants with anti-PXR or anti-p65. Iso, isotypic antibody. k Immunostaining of PXR, p65, and RXRa in WEHI231 B cells after exposure to IAA + LPS. Ctr., no stimulation. The data in a, b, and c were from three independent experiments; the data in dg were from one representative experiment. Student’s t test in a, b, d, e, f, g, and i; mean ± SD; analysis of variance in c. *p < 0.05, **p < 0.01, and ***p < 0.001; NS, no significance

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