MALT1 inhibitors prevent the development of DSS-induced experimental colitis in mice via inhibiting NF-κB and NLRP3 inflammasome activation

Abstract

Mucosa-associated-lymphoid-tissue lymphoma-translocation gene 1 (MALT1), a paracaspase and essential regulator for nuclear factor kB (NF-κB) activation, plays an important role in innate and adaptive immunity. Suppression of MALT1 protease activity with small molecule inhibitors showed promising efficacies in subtypes of B cell lymphoma and improvement in experimental autoimmune encephalomyelitis model. However, whether MALT1 inhibitors could ameliorate colitis remains unclear. In the present study, we examined the pharmacological effect of two specific MALT1 inhibitors MI-2 and mepazine on the dextran sulfate sodium (DSS)-induced experimental colitis in mice, followed by mechanistic analysis on NF-κB and NLRP3 inflammasome activation. Treatment with MI-2 and mepazine dose-dependently attenuated symptoms of colitis in mice, evidenced by reduction in the elevated disease activity index, the shortening of colon length as well as the histopathologic improvement. Moreover, protein and mRNA levels of DSS-induced proinflammatory cytokines in colon, including TNF, IL-1β, IL-6, IL-18, IL-17A and IFN-γ, were markedly suppressed by MALT1 inhibitors. The underlying mechanisms for the protective effect of MALT1 inhibitors in DSS-induced colitis may be attributed to its inhibition on NF-κB and NLRP3 inflammasome activation in macrophages. The in vitro study showed that MALT1 inhibitors decreased production of IL-1β/IL-18 in phorbol myristate acetate-differentiated THP-1 cells and bone marrow derived macrophage via suppressing the activation of NF-κB and NLRP3 inflammasome. Taken together, our results demonstrated that inhibition of the protease activity of MALT1 might be a viable strategy to treat inflammatory bowel disease and the NLRP3 inflammasome and NF-κB activation are critical components in MALT1 signaling cascades in this disease model.

Keywords: IL-1β; MALT1; NF-κB; NLRP3 inflammasome; colitis.

Figure 1. MALT1 inhibitors treatment ameliorated DSS-induced experimental colitis in mice

(A) Chemical structure of MALT1 inhibitors MI-2 and Mepazine. Mice were given 2.5% DSS in drinking water for 7 days, then mice were provided with water for another 3 days before sacrificed. The mice in each group were treated with MI-2 or Mepazine (i.p., 15 and 30 mg/kg) or sulfasalazine (i.g., 200 mg/kg) for day 0–10. (B) Bodyweight change and disease activity index (DAI) (C) were calculated (n = 6 per group). (D, E) Macroscopic appearances and the length of colons from each group of mice were measured. Data are presented as means ± SEM. *P < 0.05, **P < 0.01 vs. DSS-treated alone group at the same day. ^#P < 0.01 vs. normal group.

Figure 2. MALT1 inhibitors treatment prevented DSS-induced colon damage in mice

(A) Serial sections of colon tissues were stained with H&E. Green arrow indicated mononuclear cell infiltration and black arrow indicated globet cell damage. (B) Histopathological scores of each group were determined. Data are presented as means ± SEM. *P < 0.05, **P < 0.01 vs. DSS-treated alone group at the same day. ^#P < 0.01 vs. normal group.

Figure 3. MALT1 inhibitors suppressed proinflammatory cytokine production in colon tissues from DSS-colitis mice

Protein levels of cytokines including IFN-γ, TNF-α, IL-1β, and IL-6 in colonic homogenate were determined by ELISA. Data are presented as means ± SEM (n = 6). *P < 0.05, **P < 0.01 vs. DSS-treated alone group.

Figure 4. MALT1 inhibitors decreased activations of NF-κB signaling pathways in colon tissues from DSS-colitis mice

(A, B) Serial sections of colon tissues were stained with p-p65 and COX2. (C) Colonic homogenate from each group of mice were subjected to western blot. Data are presented as means ± SEM. *P < 0.05, **P < 0.01 vs. DSS-treated group.

Figure 5. MALT1 inhibitors inhibited NLRP3 inflammasome activation in mice with DSS-induced colitis

(A) Sections of colonic tissue were immunostained with DAPI (blue) and anti-CD11b-FITC (green) and observed by confocal laser-scanning microscope, 100×. (B) Spleen cells were extracted from colitis mice in each group at day 7 and CASP1 activation in CD11b+cells was analyzed by FACS staining. (C) Th1/Th17 differentiation in mice with DSS-induced colitis. Splenocytes was isolated from each group of mice and restimulated with PMA/ionomycin/monensin for 4 h. Th1 and Th17 cells were analyzed by intracellular staining of IFN-γ and IL-17A in the CD4 gate. Numbers in quadrants indicate percent cells in each throughout. Data are presented as means ± SEM. *P < 0.05 vs. DSS-treated group.

Figure 6. MALT1 inhibitors decreased activation of NF-kB signaling pathway in vitro

(A, B) Peritoneal macrophage cells were treated with LPS alone or with MI-2, Mapezine for 1 h. Then proteins were collected and phosphorylation of p65, IκBα, IKKαβ were analyzed by western blot. (C) Peritoneal macrophage cells were treated with LPS alone or with MI-2 and Mapezine (4 μM) for 1 h, the localization of p65 was examined by immunofluorescence stain. (D) Peritoneal macrophage cells were treated with LPS alone or with MI-2 and Mapezine for 12 h, mRNA level of IL-1β and IL-18 were analyzed by RT-PCR. *P < 0.05, **P < 0.01 vs. LPS group.

Figure 7. MI-2 inhibited IL-1β/IL-18 processing in THP-1 cells

(A) LPS-primed THP-1/BMDM/peritoneal macrophage cells were treated with MI-2 or Mepazine (1, 2, 4, 8 μM) or CsA (5 μM) for 1 h, following by 1 h incubation of 5 mM ATP. Released IL-1β/IL-18 in the supernatant were analyzed by ELISA. Data are presented as means ± SEM of three different experiments. (B) LPS-primed THP-1 cells were treated with MI-2 or Mepazine (1, 2, 4, 8 μM) or CsA (5 μM) for 1 h, following by 1 h incubation of 5 mM ATP. Protein levels of pro-CASP1, cleaved CASP1, ASC and NRLP3 were determined by western blot. (C) LPS-primed THP-1 cells were treated with MI-2 or CsA (5 μM) for 1 h, following by 1 h incubation of 5 mM ATP. Protein levels of pro-CASP1, CASP1 p10, ASC and NRLP3 were determined by Western blot. (D, E) LPS-primed THP-1 cells were treated with MI-2 (1, 2, 4, 8 μM) or CsA (5 μM) for 1 h, following by 1 h incubation of 500 μg/ml MSU or 2 h incubation of 10 μM Nigericin. Protein levels of pro-CASP1, CASP1 p10, were determined by western blot. *P < 0.05, **P < 0.01 vs. LPS+ATP/MSU/Nigericin group.

Figure 8. MI-2 suppressed NLRP3 inflammasome complex assembly induced by ATP

(A) THP-1 cells were treated with MI-2 (4 μM) for 1 h, following by 1 h incubation of 5 mM ATP. ASC oligomerization and redistribution was determined by immunoblot of ASC in cross-linked pellets (upper panels) and in cell lysates (lower panels). (B) THP-1 cells were treated with MI-2, following by 15 min ATP treatment. Cell lysates were immunoprecipitated with anti-ASC. (C) LPS-primed adherent bone marrow derived macrophages (BMDM) were treated with MI-2 (4 μM) for 1 h, followed by 5 mM ATP treatment for 15 min. The treated cells were analyzed by immunofluorescence cytochemistry (×100).

Figure 9. Illustration for the mechanism underlying MALT1 inhibitors for improvement of DSS-induced colitis in mice

MALT1 inhibitor MI-2 can inhibit both NF-κB and NLRP3 inflammasome activation in vivo and vitro, thus decrease the protein and mRNA levels proinflammatory cytokines and finally alleviate colitis.