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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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, 188.8.131.52.N.00 and 184.108.40.206.N.00) 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.
The authors declare no competing financial interests.
Extended data figures and tables
a–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. 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).
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.
a–c, 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.
a–c, 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.
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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). https://doi.org/10.1038/nature13322
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