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. 2014 Aug 7;512(7512):69-73.
doi: 10.1038/nature13322. Epub 2014 Jun 29.

Negative regulation of the NLRP3 inflammasome by A20 protects against arthritis

Lieselotte Vande Walle  1   2 , Nina Van Opdenbosch  1   2 , Peggy Jacques  3 , Amelie Fossoul  1   2 , Eveline Verheugen  3 , Peter Vogel  4 , Rudi Beyaert  5   6 , Dirk Elewaut  3 , Thirumala-Devi Kanneganti  4 , Geert van Loo #  5   6 , Mohamed Lamkanfi #  1   2
Affiliations

Negative regulation of the NLRP3 inflammasome by A20 protects against arthritis

Lieselotte Vande Walle et al. Nature. .

Abstract

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 diseases, 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 (A20(myel-KO) mice) triggers a spontaneous erosive polyarthritis that resembles rheumatoid arthritis in patients. Rheumatoid arthritis in A20(myel-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 A20(myel-KO) mice. These results reveal A20 as a novel negative regulator of Nlrp3 inflammasome activation, and describe A20(myel-KO) mice as the first experimental model to study the role of inflammasomes in the pathology of rheumatoid arthritis.

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Figures

Figure 1
Figure 1. Il1r1-deficiency rescues the arthritis phenotype of A20myel-KO mice
a, b, A20 and β-actin mRNA (a) and protein (b) levels of LPS-stimulated BMDMs. c, Hind paws of 20 weeks old mice. d, e, A20fl/flIL1R1+/+ (n=19), A20myel-KOIL1R1+/+ (n=8), A20myel-KOIL1R1+/− (n=15) and A20myel-KOIL1R1−/− (n=9) mice aged 21-30 weeks were clinically scored for arthritis incidence (d) and severity (e). f, Ankle joints sections stained with haematoxylin and eosin; magnification: 40× (top) and 100x (bottom). g, Histological scores of ankle sections of A20myel-KOIL1R1+/− (n=10) and A20myel-KOIL1R1−/− (n=8). P-values in e and g were determined by Student’s t-test.
Figure 2
Figure 2. Hyperactivation of the Nlrp3, but not the Nlrc4 and AIM2 inflammasomes, in A20-deficient macrophages
a-h, BMDMs were stimulated as described in Methods. Lysates were immunoblotted for caspase-1 (a, c, d) and supernatants analyzed for IL-1β (b, e, f) and LDH (g, h). i-n, BMDMs were treated as described in Methods. Lysates were immunoblotted for caspase-1 (i, k) and supernatants analyzed for LDH (j, l) and IL-1β (m, n). Black arrow, procaspase-1; white arrow, p20. Data represent mean ± s.d. of 1 out of 3 biological replicates, with 3 technical replicates each (*P < 0.05; ***P < 0.001; Student’s t-test).
Figure 3
Figure 3. A20 inhibits Nlrp3 inflammasome priming
a, b, Nlrp3 (a) and proIL-1β (b) mRNA levels of LPS-treated BMDMs. c, Expression of the indicated proteins in BMDMs 6 h after LPS treatment. d, e, Rapid Nlrp3 inflammasome activation as described in Methods. Expression of the indicated proteins (d), and secreted cytokines (e) were determined. f-i, A20myel-KO BMDMs were treated as indicated. Nlrp3 mRNA levels (f), caspase-1 expression (g), secreted IL-1β (h) and LDH activity (i) were determined. Black arrow, procaspase-1; white arrow, p20. Data represent mean ± s.d. of 1 out of 3 biological replicates, with 3 technical replicates each (*P < 0.05; ***P < 0.001; Student’s t-test).
Figure 4
Figure 4. Nlrp3 and caspase-1 deletion rescues arthritis in A20myel-KO mice
a, IL1β serum levels of A20fl/flNlrp3+/+ (n=10), A20myel-KONlrp3+/+ (n=10) and A20myel-KONlrp3−/− (n=10) mice aged 20-35 weeks. b, Hind paws of 30 weeks old mice. c, d, A20myel-KONlrp3+/+ (n = 20) and A20myel-KONlrp3−/−(n=17) mice were scored for arthritis incidence (d) and severity (e). e, Representative ankle joints sections; magnification: 40× (top) and 100x (bottom). f, Histological scores of ankle sections of A20myel-KONlrp3+/+ (n=5) and A20myel-KONlrp3−/− (n=5) mice. g, micro-CT images of hind paws of A20myel-KONlrp3+/+ and A20myel-KONlrp3−/− mice. Solid arrow, bone erosion; empty arrow, intact cartilage and bone. h, IL-1β serum levels of A20fl/flCasp1/11+/+ (n=11), A20myel-KOCasp1/11+/+ (n=26) and A20myel-KOCasp1/11−/− (n=16) mice aged 20-35 weeks. i, Hind paws of 25 weeks old mice. j, k, 15-30 weeks old A20myel-KOCasp1/11+/+ (n=12), A20myel-KOCasp1/11+/−(n=15) and A20myel-KOCasp1/11−/− (n=24) mice were clinically scored for arthritis incidence (j) and severity (k). l, Representative ankle joints sections; magnification: 40× (top) and 100x (bottom). P-values were determined by Student’s t-test (a, d, f, k) and Mann-Whitney test (h).
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