Negative regulation of the NLRP3 inflammasome by A20 protects against arthritis


Rheumatoid arthritis is a chronic autoinflammatory disease that affects 1–2% of the world’s population and is characterized by widespread joint inflammation. Interleukin-1 is an important mediator of cartilage destruction in rheumatic diseases1, but our understanding of the upstream mechanisms leading to production of interleukin-1β in rheumatoid arthritis is limited by the absence of suitable mouse models of the disease in which inflammasomes contribute to pathology. Myeloid-cell-specific deletion of the rheumatoid arthritis susceptibility gene A20/Tnfaip3 in mice (A20myel-KO mice) triggers a spontaneous erosive polyarthritis that resembles rheumatoid arthritis in patients2. Rheumatoid arthritis in A20myel-KO mice is not rescued by deletion of tumour necrosis factor receptor 1 (ref. 2). Here we show, however, that it crucially relies on the Nlrp3 inflammasome and interleukin-1 receptor signalling. Macrophages lacking A20 have increased basal and lipopolysaccharide-induced expression levels of the inflammasome adaptor Nlrp3 and proIL-1β. As a result, A20-deficiency in macrophages significantly enhances Nlrp3 inflammasome-mediated caspase-1 activation, pyroptosis and interleukin-1β secretion by soluble and crystalline Nlrp3 stimuli. In contrast, activation of the Nlrc4 and AIM2 inflammasomes is not altered. Importantly, increased Nlrp3 inflammasome activation contributes to the pathology of rheumatoid arthritis in vivo, because deletion of Nlrp3, caspase-1 and the interleukin-1 receptor markedly protects against rheumatoid-arthritis-associated inflammation and cartilage destruction in A20myel-KO mice. These results reveal A20 as a novel negative regulator of Nlrp3 inflammasome activation, and describe A20myel-KO mice as the first experimental model to study the role of inflammasomes in the pathology of rheumatoid arthritis.

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Figure 1: Il1r1 deficiency rescues the arthritis phenotype of A20myel-KO mice.
Figure 2: Hyperactivation of the Nlrp3 but not the Nlrc4 and AIM2 inflammasomes, in A20-deficient macrophages.
Figure 3: A20 inhibits Nlrp3 inflammasome priming.
Figure 4: Nlrp3 and caspase-1 deletion rescues arthritis in A20myel-KO mice.


  1. 1

    Burger, D., Dayer, J. M., Palmer, G. & Gabay, C. Is IL-1 a good therapeutic target in the treatment of arthritis? Best Pract. Res. Clin. Rheumatol. 20, 879–896 (2006)

    CAS  Article  Google Scholar 

  2. 2

    Matmati, M. et al. A20 (TNFAIP3) deficiency in myeloid cells triggers erosive polyarthritis resembling rheumatoid arthritis. Nature Genet. 43, 908–912 (2011)

    CAS  Article  Google Scholar 

  3. 3

    Lamkanfi, M. & Dixit, V. M. Inflammasomes and their roles in health and disease. Annu. Rev. Cell Dev. Biol. 28, 137–161 (2012)

    CAS  Article  Google Scholar 

  4. 4

    Bauernfeind, F. G. et al. Cutting edge: NF-κB activating pattern recognition and cytokine receptors license NLRP3 inflammasome activation by regulating NLRP3 expression. J. Immunol. 183, 787–791 (2009)

    CAS  Article  Google Scholar 

  5. 5

    Wertz, I. E. et al. De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-κB signalling. Nature 430, 694–699 (2004)

    ADS  CAS  Article  Google Scholar 

  6. 6

    Vereecke, L., Beyaert, R. & van Loo, G. The ubiquitin-editing enzyme A20 (TNFAIP3) is a central regulator of immunopathology. Trends Immunol. 30, 383–391 (2009)

    CAS  Article  Google Scholar 

  7. 7

    Lee, E. G. et al. Failure to regulate TNF-induced NF-κB and cell death responses in A20-deficient mice. Science 289, 2350–2354 (2000)

    ADS  CAS  Article  Google Scholar 

  8. 8

    Boone, D. L. et al. The ubiquitin-modifying enzyme A20 is required for termination of Toll-like receptor responses. Nature Immunol. 5, 1052–1060 (2004)

    CAS  Article  Google Scholar 

  9. 9

    Fernandes-Alnemri, T. et al. Cutting edge: TLR signaling licenses IRAK1 for rapid activation of the NLRP3 inflammasome. J. Immunol. 191, 3995–3999 (2013)

    CAS  Article  Google Scholar 

  10. 10

    Juliana, C. et al. Non-transcriptional priming and deubiquitination regulate NLRP3 inflammasome activation. J. Biol. Chem. 287, 36617–36622 (2012)

    CAS  Article  Google Scholar 

  11. 11

    Lin, K. M. et al. IRAK-1 bypasses priming and directly links TLRs to rapid NLRP3 inflammasome activation. Proc. Natl Acad. Sci. USA 111, 775–780 (2014)

    ADS  CAS  Article  Google Scholar 

  12. 12

    Schroder, K. et al. Acute lipopolysaccharide priming boosts inflammasome activation independently of inflammasome sensor induction. Immunobiology 217, 1325–1329 (2012)

    CAS  Article  Google Scholar 

  13. 13

    Ippagunta, S. K. et al. Inflammasome-independent role of apoptosis-associated speck-like protein containing a CARD (ASC) in T cell priming is critical for collagen-induced arthritis. J. Biol. Chem. 285, 12454–12462 (2010)

    CAS  Article  Google Scholar 

  14. 14

    Kolly, L. et al. Inflammatory role of ASC in antigen-induced arthritis is independent of caspase-1, NALP-3, and IPAF. J. Immunol. 183, 4003–4012 (2009)

    CAS  Article  Google Scholar 

  15. 15

    Consortium, W. T. C. C. Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls. Nature 447, 661–678 (2007)

    Article  Google Scholar 

  16. 16

    Dieguez-Gonzalez, R. et al. Analysis of TNFAIP3, a feedback inhibitor of nuclear factor-κB and the neighbor intergenic 6q23 region in rheumatoid arthritis susceptibility. Arthritis Res. Ther. 11, R42 (2009)

    Article  Google Scholar 

  17. 17

    Plenge, R. M. et al. Two independent alleles at 6q23 associated with risk of rheumatoid arthritis. Nature Genet. 39, 1477–1482 (2007)

    CAS  Article  Google Scholar 

  18. 18

    Thomson, W. et al. Rheumatoid arthritis association at 6q23. Nature Genet. 39, 1431–1433 (2007)

    CAS  Article  Google Scholar 

  19. 19

    Ben Hamad, M. et al. Association study of CARD8 (p.C10X) and NLRP3 (p.Q705K) variants with rheumatoid arthritis in French and Tunisian populations. Int. J. Immunogenet. 39, 131–136 (2012)

    CAS  Article  Google Scholar 

  20. 20

    Kastbom, A. et al. Genetic variation in proteins of the cryopyrin inflammasome influences susceptibility and severity of rheumatoid arthritis (the Swedish TIRA project). Rheumatology (Oxford) 47, 415–417 (2008)

    CAS  Article  Google Scholar 

  21. 21

    Mathews, R. J. et al. Evidence of NLRP3-inflammasome activation in rheumatoid arthritis (RA); genetic variants within the NLRP3-inflammasome complex in relation to susceptibility to RA and response to anti-TNF treatment. Ann. Rheum. Dis. 73, 1202–1210 (2014)

    CAS  Article  Google Scholar 

  22. 22

    Mariathasan, S. et al. Cryopyrin activates the inflammasome in response to toxins and ATP. Nature 440, 228–232 (2006)

    ADS  CAS  Article  Google Scholar 

  23. 23

    Schott, W. H. et al. Caspase-1 is not required for type 1 diabetes in the NOD mouse. Diabetes 53, 99–104 (2004)

    CAS  Article  Google Scholar 

  24. 24

    Glaccum, M. B. et al. Phenotypic and functional characterization of mice that lack the type I receptor for IL-1. J. Immunol. 159, 3364–3371 (1997)

    CAS  PubMed  Google Scholar 

  25. 25

    Burke, J. R. et al. BMS-345541 is a highly selective inhibitor of I kappa B kinase that binds at an allosteric site of the enzyme and blocks NF-kappa B-dependent transcription in mice. J. Biol. Chem. 278, 1450–1456 (2003)

    CAS  Article  Google Scholar 

  26. 26

    Podolin, P. L. et al. Attenuation of murine collagen-induced arthritis by a novel, potent, selective small molecule inhibitor of IκB kinase 2, TPCA-1 (2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide), occurs via reduction of proinflammatory cytokines and antigen-induced T cell proliferation. J. Pharmacol. Exp. Ther. 312, 373–381 (2005)

    CAS  Article  Google Scholar 

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We thank R. Flavell and V. Dixit for supplying mutant mice. L.V.W. is a postdoctoral fellow with the Fund for Scientific Research-Flanders (FWO). This work was supported by the Ghent University Concerted Research Actions (grant BOF14/GOA/013) and grants from the European Research Council (Grant 281600) and the FWO (grants G030212N, and to M.L., and by FWO research grants (Odysseus-G091908, G061910N and G016812N) and a Queen Elisabeth Medical Foundation grant to G.V.L., and by the Ghent University Group-ID MRP to G.V.L., R.B. and D.E. T.-D.K. is supported by grants from the National Institutes of Health (AR056296, CA163507 and AI101935) and the American Lebanese Syrian Associated Charities.

Author information




L.V.W., G.V.L. and M.L. designed the study; L.V.W., N.V., A.F., P.J., E.V. and P.V. performed experiments; L.V.W., N.V., P.J., P.V., G.V.L., R.B., D.E., T.-D.K. and M.L. analysed data and wrote the manuscript; T.-D.K. provided essential reagents and scientific insight; M.L. oversaw the project.

Corresponding author

Correspondence to Mohamed Lamkanfi.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 A20 deficiency does not cause spontaneous Nlrp3 inflammasome activation.

ac, Wild-type and A20myel-KO BMDMs were stimulated with 5 μg ml–1 LPS for 3 h, treated with 5 mM ATP or 20 μM nigericin for 60 min, or stimulated with 5 μg ml–1 LPS for 3 h and then treated with 5 mM ATP or 20 μM nigericin for 60 min. Cell extracts were immunoblotted for caspase-1 (a) and culture supernatants were analysed for secretion of IL-1β (b) and IL-18 (c). Black arrows on western blots denote procaspase-1 (p45); white arrows denote the processed p20 subunit (p20). ELISA data are shown as mean ± s.d. of one out of three biological replicates, with three technical replicates each (***P < 0.001; Student’s t-test).

Extended Data Figure 2 A20 negatively regulates NF-κB activation.

a, Wild-type and A20myel-KO BMDMs were incubated with 5 μg ml–1 LPS for the indicated durations before cell extracts were prepared and immunoblotted with the indicated antibodies. b, c, Wild-type and A20myel-KO BMDMs were stimulated with 5 μg ml–1 LPS for 3 h, treated with 5 mM ATP or 20 μM nigericin for 60 min, or stimulated with 5 μg ml–1 LPS for 3 h and then treated with 5 mM ATP or 20 μM nigericin for 60 min. Culture supernatants were analysed for IL-6 secretion (b) and TNF secretion (c). ELISA data are shown as mean ± s.d. of one out of three biological replicates, with three technical replicates each (***P < 0.001; Student’s t-test).

Extended Data Figure 3 Quantification of ASC, caspase-1 and proIL-18 mRNA expression in wild-type and A20 deficient macrophages.

ac, Wild-type and A20myel-KO BMDMs were stimulated with 5 μg ml–1 LPS for 3 h before mRNA levels of caspase-1 (a), ASC (b) and proIL-18 (c) were analysed by qRT–PCR. Data are shown as mean ± s.d. of one out of three biological replicates, with three technical replicates each (*P < 0.05; ***P < 0.001; Student’s t-test).

Extended Data Figure 4 Comparison of serum titres of inflammatory cytokines between A20myel-KO mice and A20myel-KONlrp3−/− or A20myel-KOCasp1/11−/− mice.

ac, Levels of IL-1α (a), IL-6 (b) and TNF (c) in serum of A20fl/fl (n = 10), A20myel-KO (n = 10), A20myel-KOCasp1/11−/− (n = 12) and A20myel-KONlrp3−/− (n = 9) mice between 20 and 35 weeks of age. P values were determined by Mann–Whitney U test (b) and Student’s t-test (c).

Extended Data Figure 5 The ubiquitin-editing enzyme A20 negatively regulates Nlrp3 inflammasome activation.

The NLR member Nlrp3, the inflammatory cytokine proIL-1β and the ubiquitin-editing enzyme A20 are expressed at low levels in resting macrophages. Binding of the TLR4 ligand LPS to its receptor triggers phosphorylation and rapid degradation of IκBα, allowing translocation of NF-κB to the nucleus, and NF-κB-mediated upregulation of proIL-1β, proIL-18, Nlrp3 and A20. A20 prevents excessive Nlrp3 inflammasome activation by dampening basal and LPS-induced NF-κB-mediated upregulation of Nlrp3. As such, A20 reduces the pool of Nlrp3 that is available for inflammasome assembly. In addition, it limits the levels of the inflammasome substrates proIL-1β and proIL-18.

Extended Data Table 1 Arthritic histopathology of A20myel-KOIl1r1+/− and A20myel-KOIl1r1−/− mice
Extended Data Table 2 Arthritic histopathology of A20myel-KONlrp3+/+ and A20myel-KONlrp3−/− mice

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Vande Walle, L., Van Opdenbosch, N., Jacques, P. et al. Negative regulation of the NLRP3 inflammasome by A20 protects against arthritis. Nature 512, 69–73 (2014).

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