Abstract
The inflammasome NLRP6 plays a crucial role in regulating inflammation and host defense against microorganisms in the intestine. However, the molecular mechanisms by which NLRP6 function is inhibited to prevent excessive inflammation remain unclear. Here, we demonstrate that the deubiquitinase Cyld prevents excessive interleukin 18 (IL-18) production in the colonic mucosa by deubiquitinating NLRP6. We show that deubiquitination inhibited the NLRP6–ASC inflammasome complex and regulated the maturation of IL-18. Cyld deficiency in mice resulted in elevated levels of active IL-18 and severe colonic inflammation following Citrobacter rodentium infection. Further, in patients with ulcerative colitis, the concentration of active IL-18 was inversely correlated with CYLD expression. Thus, we have identified a novel regulatory mechanism that inhibits the NLRP6–IL-18 pathway in intestinal inflammation.
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Acknowledgements
We thank E. Burstein for helpful discussions and W. Lancaster for critical reading of the manuscript. This work was supported by the National Institutes of Health (grant R01-DK115668-01), the Cancer Prevention Research Institute of Texas (grants RP160577 and RP190527) and the Baylor Charles A. Sammons Cancer Center and BSWRI-TGEN collaborative grants to K.V.
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S.M., R.K., T.L.E., F.I. and D.L.K. performed the experiments, analyzed the data and helped to prepare the manuscript. V.B. performed MS analysis. G.M., A.L.T. and R.A.F. helped to prepare the manuscript. K.V. conceived the project, designed the experiments and wrote the manuscript.
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Extended data
Extended Data Fig. 1 CYLD mRNA expression in human colonic mucosa.
CYLD mRNA expression from the colonic mucosa of UC patients (n=10 human UC patient samples) and control healthy samples (n=5 healthy control) was measured by real-time PCR and compared (**P=0.0012). Statistics are shown as mean ± SD, with P values determined by Student’s t test (two tail). Data are from one experiment representative of three Experiment with the same samples.
Extended Data Fig. 2 Increased colon length shortening in Cyld−/− mice after C. rodentium infection.
Colons of wild-type vs. Cyld−/− mice infected (inf) with C. rodentium for 10 days. Data are from one experiment representative of three independent experiments with similar results.
Extended Data Fig. 3 Cyld–/– mice exhibit severe TNBS-induced colitis.
(a–c) (a) Body weight change (nsP=0.9666, ***P<0.0001), (b) diarrhea score (***P<0.0001), and (c) colon length (nsP=0.7766, ***P<0.0001) of wild-type vs. Cyld−/− mice (n = 10 mice per group) given intrarectal administration of TNBS. (d) H&E-stained colon sections from TNBS treated wild-type and Cyld−/− mice (scale bars, 50 µm) and (e) Histology scores of the H&E stained sections (n=10 mice per group, nsP=0.7895, ***P<0.0001). (f) Colon tissues from untreated and TNBS treated wild-type and Cyld–/– mice (n=10 mice per group) were cultured and supernatant IL-18 concentrations measured by ELISA and normalized by colon weight (nsP=0.0573, ***P<0.0001). (g) mRNA from colonic mucosa of age- and sex-matched TNBS treated wild-type and Cyld–/– mice (n=10 mice per group) were isolated, and the expression of Il18 was quantified by real-time PCR (nsP=0.7252). (h) Colonic mucosal extracts from TNBS treated wild-type and Cyld–/– mice was subjected to SDS-PAGE. The amounts of pro-IL-18 and mature IL-18 were assessed by immunoblotting with anti-IL-18 antibody. (i) Densitometric analysis of mature IL-18 expression from wild-type and Cyld–/– mice (n=5 mice per group) colon tissue after immunoblotting (***P=0.0009). a,b,c,e,f,g,i Statistics are mean ± SD and P values were determined by Student’s t test (two tail). Data are from one experiment representative of three independent experiments with similar results. Uncropped blots (h) are shown in the Source Data.
Extended Data Fig. 4 Epithelial specific Cyld–/– mice exhibit severe C. rodentium induced colitis.
(a–c) (a) Body weight change (nsP=0.6251, **P=0.0029), (b) diarrhea score (***P=0.0005), and (c) colon length (nsP=0.6433, **P=0.0046) of Cyldfl/fl vs. IEC-Cyld (∆9) (Epithelial specific cyld knockout mice) mice (n = 5 mice per group) infected (inf) with C. rodentium. (d, e) H&E-stained colonic sections from C. rodentium–infected Cyldfl/fl and IEC-Cyld (∆9) mice (scale bars, 50 µm) and histology scores (n = 5 mice per group, nsP=0.99, **P=0.0028). (f) CFU from the organ culture (n = 5 mice per group, *P=0.0241, **P=0.0029, **P=0.0056). (g) Colon tissues from uninfected and infected Cyldfl/fl and IEC-Cyld (∆9) mice were cultured, IL-18 was measured from the supernatant by ELISA and normalized by colon weight (n=5 mice per group, nsP=0.2755, **P=0.0013). (h) Colonic mucosal lysates from infected Cyldfl/fl and IEC-Cyld (∆9) was subjected to SDS-PAGE. The amounts of pro-IL-18 and mature IL-18 were assessed by immunoblotting with anti–IL-18 antibody. a,b,c,e,f,g Statistics are mean ± SD and P values were determined by Student’s t test (Two tail). Data are from one experiment representative of three independent experiments with similar results. Uncropped blots (h) are shown in the Source Data.
Extended Data Fig. 5 Recombinant IL-18 treatment rescues Cyld−/−Il18−/− mice but not Cyld−/− mice from severe colitis.
(a) Body weight change (***P<0.0001) and (b) diarrhea score (***P<0.0001) of wild-type, Cyld−/−, Il18−/−, and Cyld−/−Il18−/− mice (n=8 mice per group) infected (inf) with C. rodentium. (c), Body weight change (***P<0.0001), (d) diarrhea score (***P<0.0001), and (e) colon length (***P<0.0001, **P=0.0039, ***P<0.0001, ***P<0.0001) of wild-type, Cyld−/−, Il18−/− and Cyld−/−Il18−/− mice (n=8 mice per group) infected (inf) with C. rodentium and along with rIL-18. (f) H&E-stained colonic sections from C. rodentium–infected and rIL-18 -treated wild-type, Cyld−/−, Il18−/− and Cyld−/−Il18−/− mice (scale bars, 50 µm) and g, histology scores of the sections (n=8 mice per group, ***P=0.0002, ***P=0.0001). Statistics are mean ± SD, with P values determined by Student’s t test (two tail). Data are from one experiment representative of three independent experiments with similar results.
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Mukherjee, S., Kumar, R., Tsakem Lenou, E. et al. Deubiquitination of NLRP6 inflammasome by Cyld critically regulates intestinal inflammation. Nat Immunol 21, 626–635 (2020). https://doi.org/10.1038/s41590-020-0681-x
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DOI: https://doi.org/10.1038/s41590-020-0681-x
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