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. 2018 Jun 5;8(1):8618.
doi: 10.1038/s41598-018-26775-w.

MCC950, a Specific Small Molecule Inhibitor of NLRP3 Inflammasome Attenuates Colonic Inflammation in Spontaneous Colitis Mice

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Free PMC article

MCC950, a Specific Small Molecule Inhibitor of NLRP3 Inflammasome Attenuates Colonic Inflammation in Spontaneous Colitis Mice

Agampodi Promoda Perera et al. Sci Rep. .
Free PMC article

Abstract

MCC950 a potent, highly specific small molecule inhibitor of canonical and noncanonical activation of NLRP3 inflammasome has been evaluated in a multitude of NLRP3 driven inflammatory diseases. However, the effect of MCC950 on colonic inflammation has not yet been reported. In the present study we investigated the effect of MCC950 in a spontaneous chronic colitis mouse model Winnie, which mimics human ulcerative colitis. Oral administration of 40 mg/kg MCC950 commencing at Winnie week seven for three weeks significantly improved body weight gain, colon length, colon weight to body weight ratio, disease activity index and histopathological scores. MCC950 significantly suppressed release of proinflammatory cytokines IL-1β, IL-18, IL1-α, IFNγ, TNF-α, IL6, IL17, chemokine MIP1a and Nitric Oxide in colonic explants. Moreover, MCC950 resulted in a significant decrease of IL-1β release and activation of caspase-1 in colonic explants and macrophage cells isolated from Winnie. Complete inhibition with MCC950 in Winnie colonic explants shows, for the first time, the contribution of inflammatory effects resulting exclusively from canonical and noncanonical NLRP3 inflammasome activation in colitis. Taken together, our results illustrate the efficacy of MCC950 in the treatment of murine ulcerative colitis and provides avenue for a potential novel therapeutic agent for human inflammatory bowel diseases.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
The effect of MCC950 on NLRP3 inflammasome activation in murine macrophages. Production of IL-1β (a) C57BL/6 and Winnie BMDMs (b) C57BL/6 and Winnie IP Macrophages (c) Winnie MLNs (d) Winnie LPMCs. Unprimed (UN), primed with 10 ng/ml LPS and treated with MCC950 (MCC) (ac) 0.01 µM, (d) 1 µM) and Glyburide (Gly) (200 µM) and stimulated with ATP and Nigericin as measured by ELISA. (e) Production of TNF-α in Winnie BMDM supernatants treated with MCC950 0.01 µM and glyburide 200 µM and stimulated with ATP and Nigericin as measured by ELISA. Data are expressed as the mean ± sem of three independent experiments carried out in duplicates. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA with Tukey’s post-hoc test). (f) Western blots of cell lysates and supernatants from C57BL/6 and Winnie BMDMs primed with 10 ng/ml LPS and treated with MCC950 (0.01 µM) or glyburide (200 µM) and stimulated with ATP. These results are representative of three independent experiments.
Figure 2
Figure 2
MCC950 inhibits NLRP3 inflammasome activation in colonic explants. (a) Production of IL-1β from Winnie proximal and distal colons stimulated with 10 ng/ml LPS and treated with MCC950 (MCC) (0.001–10 µM) and Glyburide (Gly) 200 µM as measured by ELISA. (b)Percentage of IL-1β release of Winnie proximal and distal colons stimulated with LPS no treatment compared to treated with MCC950 (10 µM) as measured by ELISA. (c) Production of TNF-α for proximal and distal colonic explant supernatants treated with MCC950 (1 µM) and glyburide 200 µM as measured by ELISA. Data are expressed as the mean ± SEM of five independent experiments carried out in duplicates. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA with Tukey’s post-hoc test). (d) Western blots of tissue lysates and supernatants from proximal and distal colons stimulated with 10 ng/ml LPS and treated with MCC950 (1 µM) or glyburide (200 µM). These results are representative of three independent experiments.
Figure 3
Figure 3
Effect of MCC950 on Winnie. Winnie at 10 week were weighed on the day of termination. Lengths of the freshly removed colons from each group were measured from ileocecal junction to rectum. The weight of the colons after removing luminal content was recorded. (a) Macroscopic appearances and (b) Colon Length for each group. (c) Ratio of colon weight over body weight. Data are expressed as the mean ± SEM (n = 10 per group) *P < 0.05, **P < 0.01 (two-tailed Student’s t test). (d) Body weight of mice was measured every 3 days and presented as a percentage of their initial weight. Data are represented as means ± SEM (n = 10 per group) repeated-measures analysis of variance (ANOVA).
Figure 4
Figure 4
MCC950 treatment improves colitis in 10 week old Winnie. (a) Disease activity index. (b) Comparison of summed inflammation scores between control and treatment Winnie mice. PC, proximal colon, MC, middle colon, DC, Distal Colon. Data are represented as means ± SEM (n = 10 per group) *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA with Tukey’s post-hoc test). (c–e) Representative Winnie control proximal, middle and distal colon sections stained with hematoxylin and eosin at 100x and 400x. (c) Lamina propria inflammatory cell infiltrates (black arrow). (d) Epithelial surface damage (red arrow), goblet cell loss (black arrow). (e) Crypt abscesses with neutrophils in the lumen and nearly intact epithelium (red arrow) or damaged epithelium and complete crypt loss (blue arrow) and crypt architectural distortion (black arrow). (fh) Representative MCC950 treated Winnie proximal, middle and distal colon sections stained with hematoxylin and eosin at 100x and 400x.
Figure 5
Figure 5
MCC950 suppressed NLRP3 activated proinflammatory cytokine levels in colon explant of Winnie mice. Protein levels of cytokines (a) IL-1β (b) IL18 in proximal and distal colon explant supernatants as determined by Bio-plex. Data presented as means ± SEM (n = 3 per group) *P < 0.05, **P < 0.01 (two-tailed Student’s t test). The mean values of fold change in mRNA expression levels for (c) IL-1β (d) IL18 in MCC950 treated Winnie proximal and distal colon tissue are shown relative to the untreated Winnie proximal and distal control samples respectively. Both control and treated values were normalised to those of the internal control Gapdh, with treated values representing the fold change relative to that of controls, which was converted to 1. Data are expressed as the mean ± SEM (n = 4 per group) *P < 0.05, **P < 0.01 ***P < 0.001 (one sample t test).
Figure 6
Figure 6
MCC950 suppressed proinflammatory cytokine and chemokine production in colon tissues but not in blood serum. Protein levels of cytokines including (a) IL1α (b) MIP1a (c) IL17 (d) IFNγ (e) TNF-α in explant supernatants (f) IL-1β, IL-1α, MIP1a, IL17, IFNγ, and TNF-α in blood serum were determined by Bio-plex. Data are presented as means ± SEM (n = 3) *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed Student’s t test).
Figure 7
Figure 7
J774A.1 cells were primed with 100 ng/ml LPS and inhibited with 1 µM MCC950 or 200 µM Glyburide then stimulated with 5 mM ATP. (a) The phosphorylation state of NEK7 was analysed using Phos-tag SDS-PAGE. These results are representative of three independent experiments. (b) ELISA analysis of IL-1β in the culture supernatant of J774A.1 cells treated as in (a). Data are expressed as the mean ± sem of three independent experiments carried out in duplicates. ***P < 0.001 (one-way ANOVA with Tukey’s post-hoc test).

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References

    1. Ponder A, Long MD. A clinical review of recent findings in the epidemiology of inflammatory bowel disease. Clin Epidemiol. 2013;5:237–247. - PMC - PubMed
    1. Terzić J, Grivennikov S, Karin E, Karin M. Inflammation and Colon Cancer. Gastroenterology. 2010;138:2101–2114.e2105. doi: 10.1053/j.gastro.2010.01.058. - DOI - PubMed
    1. Elinav E, Thaiss CA, Flavell RA. Analysis of microbiota alterations in inflammasome-deficient mice. Methods Mol Biol. 2013;1040:185–194. doi: 10.1007/978-1-62703-523-1_14. - DOI - PubMed
    1. Schroder K, Tschopp J. The inflammasomes. Cell. 2010;140:821–832. doi: 10.1016/j.cell.2010.01.040. - DOI - PubMed
    1. Martinon F, Mayor A, Tschopp J. The Inflammasomes: Guardians of the Body. Annual Review of Immunology. 2009;27:229–265. doi: 10.1146/annurev.immunol.021908.132715. - DOI - PubMed

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