Deubiquitination of NLRP6 inflammasome by Cyld critically regulates intestinal inflammation

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Cyld−/− mice show severe colitis induced by C. rodentium.
Fig. 2: NLRP6 directly interacts with Cyld.
Fig. 3: Cyld controls the deubiquitination of the NLRP6 protein.
Fig. 4: NLRP6 association with ASC is pronounced in the absence of Cyld.
Fig. 5: Increased IL-18 production in Cyld−/− mice following C. rodentium infection.
Fig. 6: Deletion of IL-18 and NLRP6 rescues elevated IL-18 levels in colon tissues of Cyld−/− mice ex vivo.
Fig. 7: Heterozygous deletion of Il18 or Nlrp6 rescues Cyld−/− mice.
Fig. 8: Cyld regulates maturation of IL-18 in colonic epithelial cells and the level of active IL-18 in the colonic mucosa of patients with UC.

Data availability

The data that support the findings of this study are available from the corresponding author upon request. Source data for Figs. 18 and Extended Data Figs. 1 and 35 are provided with the paper.

References

  1. 1.

    Neurath, M. F. Cytokines in inflammatory bowel disease. Nat. Rev. Immunol. 14, 329–342 (2014).

  2. 2.

    Broz, P. & Dixit, V. M. Inflammasomes: mechanism of assembly, regulation and signalling. Nat. Rev. Immunol. 16, 407–420 (2016).

  3. 3.

    Davis, B. K., Wen, H. & Ting, J. P. The inflammasome NLRs in immunity, inflammation, and associated diseases. Annu. Rev. Immunol. 29, 707–735 (2011).

  4. 4.

    Kanneganti, T. D., Lamkanfi, M. & Nunez, G. Intracellular NOD-like receptors in host defense and disease. Immunity 27, 549–559 (2007).

  5. 5.

    Chen, G. Y., Liu, M., Wang, F., Bertin, J. & Nunez, G. A functional role for Nlrp6 in intestinal inflammation and tumorigenesis. J. Immunol. 186, 7187–7194 (2011).

  6. 6.

    Elinav, E. et al. NLRP6 inflammasome regulates colonic microbial ecology and risk for colitis. Cell 145, 745–757 (2011).

  7. 7.

    Levy, M. et al. Microbiota-modulated metabolites shape the intestinal microenvironment by regulating NLRP6 inflammasome signaling. Cell 163, 1428–1443 (2015).

  8. 8.

    Hara, H. et al. The NLRP6 inflammasome recognizes lipoteichoic acid and regulates Gram-positive pathogen infection. Cell 175, 1651–1664.e14 (2018).

  9. 9.

    Venuprasad, K., Zeng, M., Baughan, S. L. & Massoumi, R. Multifaceted role of the ubiquitin ligase Itch in immune regulation. Immunol. Cell Biol. 93, 452–460 (2015).

  10. 10.

    Massoumi, R. Ubiquitin chain cleavage: CYLD at work. Trends Biochem. Sci. 35, 392–399 (2010).

  11. 11.

    Costello, C. M. et al. Dissection of the inflammatory bowel disease transcriptome using genome-wide cDNA microarrays. PLoS Med. 2, e199 (2005).

  12. 12.

    Cleynen, I. et al. Genetic and microbial factors modulating the ubiquitin proteasome system in inflammatory bowel disease. Gut 63, 1265–1274 (2014).

  13. 13.

    Kathania, M. et al. Itch inhibits IL-17-mediated colon inflammation and tumorigenesis by ROR-γt ubiquitination. Nat. Immunol. 17, 997–1004 (2016).

  14. 14.

    Peng, D. J. et al. Noncanonical K27-linked polyubiquitination of TIEG1 regulates Foxp3 expression and tumor growth. J. Immunol. 186, 5638–5647 (2011).

  15. 15.

    Mevissen, T. E. et al. OTU deubiquitinases reveal mechanisms of linkage specificity and enable ubiquitin chain restriction analysis. Cell 154, 169–184 (2013).

  16. 16.

    Hrdinka, M. et al. CYLD limits Lys63- and Met1-linked ubiquitin at receptor complexes to regulate innate immune signaling. Cell Rep. 14, 2846–2858 (2016).

  17. 17.

    Shen, C. et al. Molecular mechanism for NLRP6 inflammasome assembly and activation. Proc. Natl Acad. Sci. USA 116, 2052–2057 (2019).

  18. 18.

    Dixon, L. J., Berk, M., Thapaliya, S., Papouchado, B. G. & Feldstein, A. E. Caspase-1-mediated regulation of fibrogenesis in diet-induced steatohepatitis. Lab. Investig. 92, 713–723 (2012).

  19. 19.

    Singh, A. K. et al. SUMOylation of ROR-γ inhibits IL-17 expression and inflammation via HDAC2. Nat. Commun. 9, 4515 (2018).

  20. 20.

    Ten Hove, T. et al. Blockade of endogenous IL-18 ameliorates TNBS-induced colitis by decreasing local TNF-α production in mice. Gastroenterology 121, 1372–1379 (2001).

  21. 21.

    Kanai, T. et al. Macrophage-derived IL-18-mediated intestinal inflammation in the murine model of Crohn’s disease. Gastroenterology 121, 875–888 (2001).

  22. 22.

    Wlodarska, M. et al. NLRP6 inflammasome orchestrates the colonic host-microbial interface by regulating goblet cell mucus secretion. Cell 156, 1045–1059 (2014).

  23. 23.

    Karatzas, D. N. et al. Inactivation of CYLD in intestinal epithelial cells exacerbates colitis-associated colorectal carcinogenesis - a short report. Cell Oncol. 39, 287–293 (2016).

  24. 24.

    Siegmund, B. Interleukin-18 in intestinal inflammation: friend and foe? Immunity 32, 300–302 (2010).

  25. 25.

    Nowarski, R. et al. Epithelial IL-18 equilibrium controls barrier function in colitis. Cell 163, 1444–1456 (2015).

  26. 26.

    Liu, Z. et al. Role of inflammasomes in host defense against Citrobacter rodentium infection. J. Biol. Chem. 287, 16955–16964 (2012).

  27. 27.

    Lochner, M. & Forster, I. Anti-interleukin-18 therapy in murine models of inflammatory bowel disease. Pathobiology 70, 164–169 (2002).

  28. 28.

    Siegmund, B. et al. Neutralization of interleukin-18 reduces severity in murine colitis and intestinal IFN-γ and TNF-α production. Am. J. Physiol. Regul. Integr. Comp. Physiol. 281, R1264–R1273 (2001).

  29. 29.

    Ahmed, N. et al. The E3 ligase Itch and deubiquitinase Cyld act together to regulate Tak1 and inflammation. Nat. Immunol. 12, 1176–1183 (2011).

  30. 30.

    Leach, S. T. et al. Local and systemic interleukin-18 and interleukin-18-binding protein in children with inflammatory bowel disease. Inflamm. Bowel Dis. 14, 68–74 (2008).

  31. 31.

    Leon, A. J. et al. High levels of proinflammatory cytokines, but not markers of tissue injury, in unaffected intestinal areas from patients with IBD. Mediators Inflamm. 2009, 580450 (2009).

  32. 32.

    Monteleone, G. et al. Bioactive IL-18 expression is up-regulated in Crohn’s disease. J. Immunol. 163, 143–147 (1999).

  33. 33.

    Pizarro, T. T. et al. IL-18, a novel immunoregulatory cytokine, is up-regulated in Crohn’s disease: expression and localization in intestinal mucosal cells. J. Immunol. 162, 6829–6835 (1999).

  34. 34.

    Reuter, B. K. & Pizarro, T. T. Commentary: the role of the IL-18 system and other members of the IL-1R/TLR superfamily in innate mucosal immunity and the pathogenesis of inflammatory bowel disease: friend or foe? Eur. J. Immunol. 34, 2347–2355 (2004).

  35. 35.

    Man, S. M. Inflammasomes in the gastrointestinal tract: infection, cancer and gut microbiota homeostasis. Nat. Rev. Gastroenterol. Hepatol. 15, 721–737 (2018).

  36. 36.

    Deretic, V., Saitoh, T. & Akira, S. Autophagy in infection, inflammation and immunity. Nat. Rev. Immunol. 13, 722–737 (2013).

  37. 37.

    Yan, Y. et al. Dopamine controls systemic inflammation through inhibition of NLRP3 inflammasome. Cell 160, 62–73 (2015).

  38. 38.

    Han, S. et al. Lipopolysaccharide primes the NALP3 inflammasome by inhibiting its ubiquitination and degradation mediated by the SCFFBXL2 E3 ligase. J. Biol. Chem. 290, 18124–18133 (2015).

  39. 39.

    Py, B. F., Kim, M. S., Vakifahmetoglu-Norberg, H. & Yuan, J. Deubiquitination of NLRP3 by BRCC3 critically regulates inflammasome activity. Mol. Cell 49, 331–338 (2013).

  40. 40.

    Hu, Z. et al. Crystal structure of NLRC4 reveals its autoinhibition mechanism. Science 341, 172–175 (2013).

  41. 41.

    Salcedo, R. et al. MyD88-mediated signaling prevents development of adenocarcinomas of the colon: role of interleukin 18. J. Exp. Med. 207, 1625–1636 (2010).

  42. 42.

    Takagi, H. et al. Contrasting action of IL-12 and IL-18 in the development of dextran sodium sulphate colitis in mice. Scand. J. Gastroenterol. 38, 837–844 (2003).

  43. 43.

    Tucker, T. A. et al. Transient transfection of polarized epithelial monolayers with CFTR and reporter genes using efficacious lipids. Am. J. Physiol. Cell Physiol. 284, C791–C804 (2003).

  44. 44.

    Komander, D. et al. The structure of the CYLD USP domain explains its specificity for Lys63-linked polyubiquitin and reveals a B box module. Mol. Cell 29, 451–464 (2008).

  45. 45.

    Iggo, R. & Richard, E. Lentiviral transduction of mammary epithelial cells. Methods Mol. Biol. 1293, 137–160 (2015).

  46. 46.

    Ohta, Y., Hamada, Y. & Katsuoka, K. Expression of IL-18 in psoriasis. Arch. Dermatol. Res. 293, 334–342 (2001).

Download references

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.

Author information

Affiliations

Authors

Contributions

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.

Corresponding author

Correspondence to K. Venuprasad.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information L. A. Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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. Source data

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. 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. 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. Source data

Supplementary information

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Unprocessed immunoblots.

Source Data Fig. 3

Unprocessed immunoblots.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 4

Unprocessed immunoblots.

Source Data Fig. 5

Statistical source data.

Source Data Fig. 5

Unprocessed immunoblots.

Source Data Fig. 6

Statistical source data.

Source Data Fig. 7

Statistical source data.

Source Data Fig. 8

Statistical source data.

Source Data Fig. 8

Unprocessed immunoblots.

Source Data Extended Data Fig. 1

Statistical source data.

Source Data Extended Data Fig. 3

Statistical source data.

Source Data Extended Data Fig. 3

Unprocessed immunoblots.

Source Data Extended Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 4

Unprocessed immunoblots.

Source Data Extended Data Fig. 5

Statistical source data.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation