Vitamin D Receptor Inhibits NLRP3 Activation by Impeding Its BRCC3-Mediated Deubiquitination

Zebing Rao, Xin Chen, Junxian Wu, Mengjun Xiao, Jing Zhang, Binghao Wang, Lei Fang, Hongjie Zhang, Xiaoming Wang, Shuo Yang, Yunzi Chen, Zebing Rao, Xin Chen, Junxian Wu, Mengjun Xiao, Jing Zhang, Binghao Wang, Lei Fang, Hongjie Zhang, Xiaoming Wang, Shuo Yang, Yunzi Chen

Abstract

The NLRP3 inflammasome is a multiprotein oligomer responsible for activation of the inflammatory response by promoting the maturation and secretion of the pro-inflammatory cytokines IL-1β and IL-18. Dysregulation of this inflammasome has been linked to several autoimmune diseases, indicating that NLRP3 is tightly regulated to prevent aberrant activation. The regulation of NLRP3 activation remains unclear. Here, we report the identification of vitamin D receptor (VDR) as a negative regulator of NLRP3 oligomerization and activation. VDR can physically bind NLRP3 and block the association of NLRP3 with BRCC3. When BRCC3-mediated deubiquitination of NLRP3 is inhibited by VDR, NLRP3 activation is subsequently inhibited. In the absence of VDR, caspase-1 activation and IL-1β release are increased in response to LPS-induced inflammation or alum-induced peritoneal inflammation, indicating that VDR is a negative regulator of NLRP3 inflammasome activation in vivo. In addition, vitamin D negatively regulates the NLRP3 inflammasome via VDR signaling to effectively inhibit IL-1β secretion. These studies demonstrate that VDR signaling constrains NLRP3 inflammasome activation and might be a potential treatment target for NLRP3 inflammasome-related diseases.

Keywords: BRCC3; NLRP3 inflammasome; VDR; cytokines; deubiquitinating.

Copyright © 2019 Rao, Chen, Wu, Xiao, Zhang, Wang, Fang, Zhang, Wang, Yang and Chen.

Figures

Figure 1
Figure 1
VDR interacts with NLRP3. (A) Mass spectrometry analysis of NLRP3 and VDR peptides after immunoprecipitation with Flag to pull down NLRP3-associated proteins in Flag-NLRP3-overexpressing HEK293T cells. (B) HA-VDR was co-expressed with Flag-tagged NLRP3 in HEK293T cells; proteins were immunoprecipitated and analyzed by immunoblotting. Whole-cell lysates are shown as the input. (C) LPS-primed BMDMs were unstimulated or stimulated with nigericin for 30 min. Cell lysates were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies. (D) Immunofluorescent staining for VDR and NLRP3 in LPS-primed BMDMs treated with or without nigericin. Scale bar, 10 μm. (E) Purified GST-VDR was incubated with purified His-NLRP3 for 2 h. His-NLRP3-Flag bound to GST-VDR was pulled down by glutathione beads and subjected to immunoblot analysis. (F) Wild-type or mutant NLRP3 (PYD, NACHT, or LRR) and HA-VDR were expressed in HEK293T cells, immunoprecipitated, and analyzed by immunoblotting. (G) Wild-type or mutant VDR (DBD or LBD) and Flag-NLRP3 were expressed in HEK293T cells, immunoprecipitated, and analyzed by immunoblotting.
Figure 2
Figure 2
VDR inhibits NLRP3 inflammasome activation. (A) Immunoblot analysis of IL-1β and cleaved caspase-1 (p20) in culture supernatants (SN) of LPS-primed BMDMs (wild-type and Vdr−/−) treated for 4 h and then stimulated with ATP, nigericin, or alum. Immunoblot analysis of NLRP3, ASC, pro-IL-1β and pro-caspase-1 in cell lysates (Lysate). (B–D) IL-1β (B), IL-18 (C), and TNF-α (D) ELISAs using supernatants from LPS-primed BMDMs (wild-type and Vdr−/−) treated for 4 h and then stimulated with ATP, nigericin, or alum. (E) Immunoblot analysis of IL-1β and cleaved caspase-1 (p20) in culture supernatants (SN) of LPS-primed PMs (wild-type and Vdr−/−) treated for 4 h and then stimulated with nigericin or alum. Immunoblot analysis of pro-IL-1β and pro-caspase-1 in cell lysates (Lysate). (F,G) Immunoblot analysis of IL-1β and cleaved caspase-1 (p20) in culture supernatants (SN) of LPS-primed BMDMs (F). Supernatants were analyzed by IL-1β ELISA (G). (H) Pam3CSK4-primed BMDMs (wild-type and Vdr−/−) were stimulated by LPS transfection. Supernatants (SN) and cell extracts (Lysate) were analyzed by immunoblotting. Data are presented as the mean ± SEM; *p < 0.05, **p < 0.01, and ***p < 0.001. Data in (B–D,G) are representative of three independent experiments.
Figure 3
Figure 3
Vitamin D receptor blocks NLRP3 oligomerization and ASC speck formation. (A,B) Representative immunofluorescence images and quantification of endogenous ASC specks (arrows). The data show representative results from three combined independent experiments. Scale bar, 10 μm. (C) ASC oligomerization induced by the indicated stimuli at 0, 5, 10, and 15 min in WT and Vdr−/− macrophages primed with LPS. Data are presented as the mean ± SEM; *p < 0.05. Data in panel B is representative of three independent experiments.
Figure 4
Figure 4
Vitamin D receptor interferes with the BRCC3–NLRP3 interaction. (A) Both WT and Vdr−/− BMDMs were treated with LPS for 4 h. NLRP3 ubiquitination was analyzed. (B) Immunoblot analysis of BRCC3 protein in mock or LPS-primed WT and Vdr−/− BMDMs lysates immunoprecipitated with the anti-NLRP3 antibody. (C,D) HEK293T cells were transfected with the indicated vectors. Samples were immunoprecipitated with the anti-Flag antibody and analyzed by immunoblotting. (E) LPS-primed BMDMs (wild-type and Vdr−/−) transfected with the indicated non-targeting or BRCC3-specific siRNA were unstimulated or stimulated with nigericin for 30 min. Supernatants (SN) and cell extracts (Lysate) were analyzed by immunoblotting. IL-1β ELISA (F). Data are presented as the mean ± SEM; **p < 0.01. Data in (F) is representative of three independent experiments.
Figure 5
Figure 5
Vitamin D receptor inhibits NLRP3 deubiquitination. (A) HEK293T cells were transfected with the indicated vectors. NLRP3 ubiquitination was analyzed. (B) Immunoblot analysis of NLRP3 after treatment of LPS-primed BMDMs (wild-type and Vdr−/−) with CHX (1 μM) for the indicated time. (C) HEK293T cells expressing HA-BRCC3 and Myc-NLRP3 PYD, NACHT, or LRR were co-transfected with VDR as indicated. Myc-NLRP3 immunoprecipitants were analyzed for ubiquitination. (D) HEK293T cells expressing Flag-NLRP3, HA-BRCC3, and HA-Ubiquitin K63 or K48 were collected, and the cell lysates were immunoprecipitated with the anti-Flag antibody to detect ubiquitination.
Figure 6
Figure 6
Vitamin D receptor deficiency promotes LPS-induced systemic inflammation and Alum-induced peritoneal inflammation via suppression of the NLRP3 inflammasome. (A) Survival of VDR+/+NLRP3+/+, VDR−/−NLRP3+/+, VDR+/+NLRP3−/−, and VDR−/−NLRP3−/− mice (n = 10 mice/group) intraperitoneally injected with LPS (8 mg/kg body weight) over a period of 72 h. (B–D) Serum IL-1β, IL-18, and TNFα ELISAs at 6 h after intraperitoneal injection of LPS (8 mg/kg body weight) into VDR+/+NLRP3+/+, VDR−/−NLRP3+/+, VDR+/+NLRP3−/−, and VDR−/−NLRP3−/− mice. (E,F) Representative FACS plots of peritoneal CD11b+ Ly6G+ cells (neutrophils) from VDR+/+NLRP3+/+, VDR−/−NLRP3+/+, VDR+/+NLRP3−/−, and VDR−/−NLRP3−/− mice at 12 h after an intraperitoneal injection with alum (n = 5 mice/group). *p < 0.05, **p < 0.01, ***p < 0.001, NS p > 0.05. Values are the mean ± SEM of five mice per group. Both male and female mice were randomly assigned. Data in (B–D,F) are representative of three independent experiments.
Figure 7
Figure 7
Vitamin D enhances VDR-mediated inhibition of NLRP3 inflammasome activation. (A) Immunoblot analysis of IL-1β and cleaved caspase-1 (p20) in culture supernatants (SN) of LPS-primed BMDMs treated for 3 h with various doses (upper lanes) of 1,25 VD and then stimulated with nigericin. Immunoblot analysis of NLRP3, ASC, pro-IL-1β and pro-caspase-1 in cell lysates (Lysate). (B–D) IL-1β (B), IL-18 (C), and TNF-α (D) in supernatants. (E) LPS-primed BMDMs (wild type and Vdr−/−) were treated with 1,25 vitamin D (1,25 VD, 20 μM) and then stimulated with nigericin. Supernatants (SN) and cell extracts (Lysate) were analyzed by immunoblotting. (F) LPS-primed THP-1 cells were treated with different doses of 1,25 vitamin D (1,25VD) as indicated and then stimulated with nigericin. Supernatants (SN) and cell extracts (Lysate) were analyzed by immunoblotting. (G) The induction of ASC oligomerization by the indicated stimuli in WT and Vdr−/− LPS-primed macrophages. Nigericin stimulation lasted 15 min. (H) LPS-primed BMDMs were treated with or without 20 μM 1,25VD for 3 h. NLRP3 ubiquitination was analyzed. Data are presented as the mean ± SEM; *p < 0.05, **p < 0.01, and ***p < 0.001. Data in (B–D) are representative of three independent experiments.

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