Inflammasome-independent role of NLRP12 in suppressing colonic inflammation regulated by Blimp-1

Abstract

NLRP12 is a member of the Nod-like receptor (NLR). Previous studies have reported enhanced colitis-associated inflammatory responses in NLRP12-deficient mice. In this study, we sought to investigate the role of NLRP12 in DSS-stimulated proinflammatory response in dendritic cells and mice colitis, and the molecular mechanisms involved in the development of the inflammation. Our results showed that down-regulation of NLRP12 is required for DSS-induced release of proinflammatory cytokines IL-1β and TNF-α; that PR domain zinc finger protein 1 (also known as Blimp-1) induces NLRP12 down-regulation during DSS-induced proinflammatory response and colitis; and that TLR4 is implicated in the up-regulation of Blimp-1 that led to the down-regulation of NLRP12 expression in DSS-induced colitis. Taken together, the results suggest that the TLR4-Blimp-1 axis promotes DSS induced experimental colitis through the down-regulation of NLRP12.

Keywords: Blimp-1; NLRP12; TLR4; colitis; inflammation.

Figure 1. DSS induces partially caspase-1 dependent release of IL-1β in murine dendritic cells

(A) DC2.4 cell lines were stimulated with different concentrations of DSS for 12 h or 24 h. IL-1β concentration in the supernatant was determined by ELISA. (B) DC2.4 cell lines were treated with 5% DSS at the indicated time points, and IL-1β was determined in the supernatant by ELISA. Detection of IL-1β (C) and TNF-α (D) release in BMDCs and BMDMs upon 5% DSS treatment at different time points. (E) Western blot analysis of pro-caspase-1 p45 and bioactive form of caspase-1 p20 in BMDCs after DSS stimulation. (F) Effect of the caspase-1 specific inhibitor Z-YVAD-FMK on IL-1β release in response to DSS treatment in mice BMDCs. Data represent the mean ± SD of triplicate samples from one of three independent experiments. *p < 0.05, significantly different from control under the same experimental conditions.

Figure 2. NLRP12 inhibits the release of pro-inflammatory cytokines upon DSS stimulation

(A) Measurement by quantitative PCR or western blot (B) of the expression of NLRP12 in BMDCs at the indicated time points after exposure to DSS. ELISA analysis of IL-1β and TNF-α (C) released in cell culture supernatants of NLRP12-knock-down DC2.4 cells upon DSS stimulation. (D) Western blot analysis of pro-caspase-1 p45 and bioactive form of caspase-1 p20 in NLRP12-knock-down DC2.4 cells upon DSS treatment. (E) Western blot analysis shows the effect of NLRP12 knock-down on the LPS+ATP-induced caspase-1 activation. (F) ELISA analysis of the effect of NLRP12 knock-down on LPS+ATP-induced IL-1β release in DC2.4 cells. (G) ELISA analysis of IL-1β and TNF-α released in the cell culture supernatants of ASC knock-down DC2.4 cells treated with DSS. (H) p-IκBα/IκBα ratio was evaluated in wild-type dendritic cells and NLRP12 knock-down dendritic cells after DSS stimulation. Data were performed in triplicate and expressed as the mean ± SD, and are representative of three separate experiments. *p < 0.05 and **p < 0.01, significantly different from control under the same experimental conditions, n.s., no significant.

Figure 3. Blimp-1 binds to the NLRP12 promoter and down-regulates its expression during DSS stimulation

Measurement by quantitative PCR (A) or western blot analysis (B) of the expression of Blimp-1 in BMDCs after exposure to DSS. (C) ELISA analysis of IL-1β and TNF-α released in cell culture supernatants of Blimp-1-knock-down D2.4 cells treated with DSS. (D) EMSA analysis of the Blimp-1 binding site in the NLRP12 promoter (1: free DNA; 2: cells treated with PBS; 3: cells treated with DSS; 4: cells treated with TNF-α for detecting NF-κB as positive control), the top arrow suggest the complex of protein with labeled probe, and the bottom arrow indicate probe alone. Data are mean ± SD of triplicate samples. *p < 0.05, significantly different from control under the same experimental conditions.

Figure 4. TLR4 participates in Blimp-1 mediated NLRP12 down-regulation in DSS-induced mice colitis

(A) ELISA analysis of IL-1β and TNF-α secretion by BMDCs from wild-type and TLR4^−/− mice in response to DSS treatment. (B) Western blot analysis of Blimp-1 and NLRP12 expression in BMDCs from wild-type and TLR4^−/− mice upon DSS stimulation. (C–E) Body weight (C) and Diseases Activity Index (E) were scored daily, and colonic length (D) was measured at the seventh day of DSS administration. (F) Representative images of H&E staining of colon section from (1) PBS control, (2) wild-type treated with DSS, (3) TLR4^−/− mice, and (4) TLR4^−/− at the seventh day of DSS treatment. (G) Blimp-1 and NLRP12 levels were evaluated in colons harvested from wild-type and TLR4−/− mice at day 7 after colitis initiation. (H) Distal colons collected at the seventh day of DSS administration were used to determine the level of pro-inflammatory cytokines IL-1β and TNF-α. Data were as the mean ± SD, and are representative of three separate experiments. *p < 0.05 and **p < 0.01, significantly different from control under the same experimental conditions, n = 6.

Figure 5. Schematic diagram illustrating the hypothetical molecular signaling triggered by DSS stimulation

DSS leads to NLRP12 decrease through up-regulation of Blimp-1 in dendritic cells. Down-regulation of NLRP12 increases NF-κB activation, which results in an increase in Pro- IL-1β level. Meanwhile, DSS-induced NLRP3 inflammasome activation leads to caspase-1-mediated maturation of IL-1β.