Vitamin D3 alleviates inflammation in ulcerative colitis by activating the VDR-NLRP6 signaling pathway

Abstract

Inflammation is a key factor in the development of ulcerative colitis (UC). 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃, VD₃), as the major active ingredient of vitamin D and an anti-inflammatory activator, is closely related to the initiation and development of UC, but its regulatory mechanism remains unclear. In this study, we carried out histological and physiological analyses in UC patients and UC mice. RNA sequencing (RNA-seq), assays for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), chromatin immunoprecipitation (ChIP) assays and protein and mRNA expression were performed to analyze and identify the potential molecular mechanism in UC mice and lipopolysaccharide (LPS)-induced mouse intestinal epithelial cells (MIECs). Moreover, we established nucleotide-binding oligomerization domain (NOD)-like receptor protein nlrp6 ^-/- mice and siRNA-NLRP6 MIECs to further characterize the role of NLRP6 in anti-inflammation of VD₃. Our study revealed that VD₃ abolished NOD-like receptor protein 6 (NLRP6) inflammasome activation, suppressing NLRP6, apoptosis-associated speck-like protein (ASC) and Caspase-1 levels via the vitamin D receptor (VDR). ChIP and ATAC-seq showed that VDR transcriptionally repressed NLRP6 by binding to vitamin D response elements (VDREs) in the promoter of NLRP6, impairing UC development. Importantly, VD₃ had both preventive and therapeutic effects on the UC mouse model via inhibition of NLRP6 inflammasome activation. Our results demonstrated that VD₃ substantially represses inflammation and the development of UC in vivo. These findings reveal a new mechanism by which VD₃ affects inflammation in UC by regulating the expression of NLRP6 and show the potential clinical use of VD₃ in autoimmune syndromes or other NLRP6 inflammasome-driven inflammatory diseases.

Keywords: ATAC-seq; NLRP6 inflammasome; VD3; VDR; ulcerative colitis.

Figure 1

VDR is negatively correlated with inflammation in UC patients. (A) The pathological features of colitis are depicted by HE staining and TEM observation. (B, C) The contents of IL-1 and IL-18 were analysed through ELISA in UC tissues (B) and LPS-treated cells (C). (D) Schematic diagram illustrating the key factors (CYP27A1, CYP27B1, VDR and CYP24A1) involved in the anabolism and catabolism of 1α,25(OH)₂D₃ and the regulation of target genes. (E) qPCR was used to analyse the mRNA expression of CYP24A1, CYP27A1, CYP27B1 and VDR in colon tissue from UC patients and LPS-treated MIECs. (F, G) Representative Immunohistochemical images (F) and quantified data (G) for VDR in colons from normal and representative UC patients. (H) The expression of VDR was measured by immunofluorescence assay in colon sections from normal and representative UC patients. The data are shown as the means ± SD; n ≥ 3, **P < 0.01, ***P <0.001.

Figure 2

1,25(OH)₂D₃ alleviates DSS-induced UC in mice. (A) Macroscopic appearances and colon lengths of the mice were measured. (B) Mice were given 3% DSS in drinking water for 7 d to induce acute colitis. Body weight loss. (C) The DAI of these mice during the experimental period is depicted. (D) The intestinal permeability rate of FITC-dextran was measured. (E) Representative HE-stained and TEM colon sections. The data are shown as the means ± SD; n ≥ 3, *P < 0.05, **P < 0.01.

Figure 3

Gene expression profile of DSS-induced UC in mice. (A) Volcano plot of differentially expressed genes. X axis: log 2 FC; Y axis: -log10 (FDR). Red represents upregulated genes, and blue represents downregulated genes. (B) GO term analysis of BP, MF, and CC for the genes. The minus logarithm of the P value (x-axis) indicates the significance of the gene set belonging to predefined categories under the coexpression network gene background. The y-axis represents each GO category. (C) A heatmap depicting the gene expression profiles of vitamin B6 metabolism and the pertussis signalling pathway in the colon of the DSS mouse model and healthy controls. X axis: sample name; Y axis: gene name. (D) Enriched upregulated and downregulated genes, as determined by KEGG pathway analysis.

Figure 4

1,25 (OH)₂D₃ inhibits expression of the NLRP6, ASC and Caspase-1 inflammasomes in LPS-treated primary intestinal epithelial cells and DSS-induced UC mice. (A) Western blot analyses were performed to evaluate the expression of the VDR, NLRP6, ASC and Caspase-1 proteins in LPS-primed MIECs treated with VD₃ for 3 h. (C) Western blot analyses were performed to evaluate the expression of the VDR, NLRP6, ASC and Caspase-1 proteins in UC mice treated with VD₃. (B) and (D) The quantification of protein expressions in A and C. (E) Immunohistochemical analysis of VDR, NLRP6, ASC and Caspase-1 expression in colon sections from UC mice treated with VD₃. (F) Quantitative analysis of the average optical density by immunohistochemistry. Scale bar represents 500 μm. The data are shown as the means ± SD; n ≥ 3, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

VDR downregulates NLRP6 expression by inhibiting the transcriptional activity of the NLRP6 promoter. (A) ATAC-seq enrichment from 2500 bp upstream of the TSSs throughout the whole ranges of the NLRP6, ASC and Caspase-1 genes in normal and UC tissues. (B) Presence of the AGTTCA and AGGTCA motifs in the promoters of NLRP6, ASC and Caspase-1 (left) and qChIP-PCR results (right) showing the binding of VDR to the promoter fragments containing the AGTTCA and AGGTCA motifs in the promoters of NLRP6, Caspase-1 and the AGTTCA motifs in the promoters of ASC, respectively. (C) The percentage relative to the input for the enriched fragments of the VDRE ChIP. The data are shown as the mean ± SD, n ≥ 3, **P < 0.01.

Figure 6

1,25(OH)₂D₃ promotes NLRP6 expression in vivo and in vitro. (A) Western blot analyses were performed to evaluate the expression of the VDR, NLRP6, ASC and Caspase-1 proteins in Nlrp6 ^-/- mice and UC mice subsequently treated with VD₃. (B) Western blot analyses were performed to evaluate the expression of the VDR, NLRP6, ASC and Caspase-1 proteins in NLRP6 siRNA MIECs that were then treated with LPS and incubated with VD₃. (C–J) Quantitative analysis of the protein levels. The data are shown as the mean ± SD, n ≥ 3, *P < 0.05, **P < 0.01.

Figure 7

VDR expression is negatively correlated with NLRP6 expression and has the potential to be used for clinical prognosis prediction. (A) Protein–protein association network of the NLRP6-(Caspase-1)/IL-1β signalling pathway in STRING. VDR, NLRP6, ASC and Caspase-1 were selected as input. (B) Correlation of gene expression was analysed in GSE128682 ChiP data. The correlation coefficient ranges from −1 (red colour) to +1 (blue colour). The red region represents absolute negative correlations, afnd the blue region represents absolute positive correlations. Hclust, hierarchical clustering order. A value of 0.05 was chosen as the significance level. (C) Immunohistochemical analysis of ASC, NLRP6 and Caspase-1 expression in normal and UC tissue samples from representative patients. (D) Schematic illustration of VDR-NLRP6 signalling in MIECs. There are three VDREs in the promoter region of NLRP6. The transcription factor VDR transcriptionally represses NLRP6 by binding to the VDRE, reducing NLRP6 expression. The data are shown as the mean ± SD, n ≥ 3, *P < 0.05; **P < 0.01, ***P < 0.001.