miR-369-3p Modulates Intestinal Inflammatory Response via BRCC3/NLRP3 Inflammasome Axis

Abstract

Inflammasomes are multiprotein complexes expressed by immune cells in response to distinct stimuli that trigger inflammatory responses and the release of pro-inflammatory cytokines. Evidence suggests a different role of inflammasome NLRP3 in IBD. NLRP3 inflammasome activation can be controlled by post-translational modifications such as ubiquitination through BRCC3. The aim of this study was to investigate the effect of miR-369-3p on the expression and activation of NLRP3 inflammasomes via BRCC3 regulation. After bioinformatics prediction of Brcc3 as a gene target of miR-369-3p, in vitro, we validated its modulation in bone marrow-derived macrophages (BMDM). The increase in miR-369-3p significantly reduced BRCC3 gene and protein expression. This modulation, in turn, reduced the expression of NLRP3 and blocked the recruitment of ASC adaptor protein by NLRP3. As a result, miR-369-3p reduced the activity of Caspase-1 by the inflammasome, decreasing the cleavage of pro-IL-1β and pro-IL-18. These results support a novel mechanism that seems to act on post-translational modification of NLRP3 inflammasome activation by BRCC3. This may be an interesting new target in the personalized treatment of inflammatory disorders, including IBD.

Keywords: BRCC3; NLRP3; inflammasome; miR-369-3p; microRNA.

Figure 1

miR-369-3p targets Brrc3. (A) Sequence alignments of miR-369-3p and binding sites in 3′ UTR of Brcc3 mRNA. (B) The mRNA expression levels of Brcc3 evaluated by qRT-PCR in BMDM cells transfected with 30 nM and 50 nM of miR-369-3p mimic both in the basal condition and after inflammasome activation. (C) Western blot analysis of BRCC3 protein expression after miR-369-3p mimic transfection in unstimulated BMDM cells as well as following inflammasome activation of the BMDM cell line. To normalize data, GAPDH was used as housekeeping protein. Data are representative of four independent experiments. Raw data of the independent experiments of Western blot were reported in Supplementary File S1. The histograms correspond to mean ± SEM. (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 5

miR-369-3p reduced the activation and release of caspase-1. (A) Western blot analysis of pro-CASP1 protein expression after miR-369-3p mimic transfection following LPS and Nigericin stimulations in the BMDM cell line. To normalize data, GAPDH was used as housekeeping protein. Raw data of the independent experiments of Western blot were reported in Supplementary File S1. (B) Caspase-1 activity after LPS and Nigericin stimulations in the mimic transfected BMDM cell line. (C) In vitro cell viability assay of BMDM cells transiently transfected with miR-369-3p mimic and stimulated with LPS and Nigericin. (* p < 0.05; ** p < 0.01; *** p < 0.0001).

Figure 2

miR-369-3p regulates Nlrp3 mRNA and protein expression. (A) The mRNA expression levels of Nlrp3 evaluated by qRT-PCR in BMDM cells transfected with 30 nM and 50 nM of miR-369-3p mimic both in the basal condition and after inflammasome-activating stimulations. (B) Western blot analysis of NLRP3 protein expression after miR-369-3p mimic transfection in unstimulated BMDM cells as well as following inflammasome activation in the BMDM cell line. To normalize data, GAPDH was used as housekeeping protein. Data are representative of four independent experiments. Raw data of the independent experiments of Western blot were reported in Supplementary File S1. The histograms correspond to mean ± SEM. (* p < 0.05; ** p < 0.01).

Figure 3

Immunofluorescence staining of ASC in BMDM cell cultures after miR-369-3p mimic transfection. Representative images of BMDM transfected with miR-369-3p mimic and stimulated with LPS 1 μg/mL for 4 h then Nigericin 20 μM for 30 min. Mock condition and transfected conditions were acquired by fluorescence microscopy. Red spots represent the expression of ASC, while DAPI (blue) correspond to cells’ nuclei. White arrows point to ASC expression. Original magnification, ×20. Scale bar presents 50 μm.

Figure 4

miR-369-3p modulated the deubiquitination of NLRP3 by BRCC3. (A) Endogenous NLRP3 immunoprecipitates were analyzed for ubiquitination. miR-369-3p modulated the deubiquitination of NLRP3 by BRCC3 after miR-369-3p transient transfection and LPS and Nigericin stimulation. (B) Expression of NLRP3 and BRCC3 in BMDM lysates after miR-369-3p transient transfection and LPS and Nigericin stimulation (input samples). (C) Histograms of NLRP3 and BRCC3 expression from lysates of BMDM treated with miR-369-3p mimic and stimulated with LPS and Nigericin. To normalize data, GAPDH was used as housekeeping protein. Raw data of the independent experiments of Western blot were reported in Supplementary File S1. The histograms correspond to mean ± SEM. Data are representative of four independent experiments. (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 6

miR-369-3p modulated the release of pro-inflammatory cytokines, NLRP3 inflammasome-related. miR-369-3p induction in BMDM after mimic transfection led to a significant decrease in IL-1β (A), IL-18 (B) production in response to LPS and LPS and Nigericin stimulation. Data are representative of four independent experiments. (* p < 0.05; ** p < 0.01).

Figure 7

BRCC3 and NLRP3 and ASC expression in UC patients. (A) BRCC3, NLRP3, and ASC protein expression in formalin-fixed, paraffin-embedded tissues obtained from healthy controls and ulcerative colitis (UC) patients. Original magnification, ×4. (B) Inflammatory score representing the expression levels of BRCC3, NLRP3, and ASC proteins in the immune infiltrate (* p < 0.01; *** p < 0.001).