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Negative regulation of the NLRP3 inflammasome by A20 protects against arthritis.

Vande Walle L, Van Opdenbosch N, Jacques P, Fossoul A, Verheugen E, Vogel P, Beyaert R, Elewaut D, Kanneganti TD, van Loo G, Lamkanfi M - Nature (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Medical Protein Research, VIB, Ghent B-9000, Belgium [2] Department of Biochemistry, Ghent University, Ghent B-9000, Belgium.

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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Nlrp3 and caspase-1 deletion rescues arthritis in A20myel-KO micea, 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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Figure 4: Nlrp3 and caspase-1 deletion rescues arthritis in A20myel-KO micea, 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).

Mentions: Having established A20 as a negative regulator of Nlrp3 inflammasome activation, we hypothesized that excessive Nlrp3 activation might drive RA pathology in A20-deficient mice upstream of IL1R1. To test this hypothesis, Nlrp3−/− mice were crossed into A20myel-KO mice and the levels of 4 RA-relevant cytokines (IL-1α, IL-1β, IL-6 and TNF) were monitored. Although IL-1α levels were not significantly different in A20-sufficient and A20myel-KO mice (Extended Data figure 4a), the latter mice had significantly higher levels of IL-1β in circulation (Fig. 4a). In addition, the levels of IL-6 and TNF were also significantly higher in A20myel-KO mice compared to A20flox/flox littermates (Extended Data figure 4b, c). Notably, deletion of Nlrp3 in A20myel-KO mice markedly reduced IL-1β secretion to baseline levels of A20flox/flox mice, thereby demonstrating that the Nlrp3 inflammasome contributes critically to excessive IL-1β production in A20myel-KO mice in vivo (Fig. 4a). Intriguingly, A20myel-KONlrp3−/− mice also were protected from excessive IL-6 production, suggesting that high IL-6 levels are consequent to excessive inflammasome-mediated IL-1β production (Extended Data figure 4b). In contrast, TNF production was not significantly affected in A20myel-KONlrp3−/− and A20myel-KOCasp1−/− mice (Extended Data figure 4c). In agreement, TNF-R1 signaling was previously shown to be dispensable for RA pathology in A20myel-KO mice, whereas IL-6 neutralization provided protection2.


Negative regulation of the NLRP3 inflammasome by A20 protects against arthritis.

Vande Walle L, Van Opdenbosch N, Jacques P, Fossoul A, Verheugen E, Vogel P, Beyaert R, Elewaut D, Kanneganti TD, van Loo G, Lamkanfi M - Nature (2014)

Nlrp3 and caspase-1 deletion rescues arthritis in A20myel-KO micea, 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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Figure 4: Nlrp3 and caspase-1 deletion rescues arthritis in A20myel-KO micea, 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).
Mentions: Having established A20 as a negative regulator of Nlrp3 inflammasome activation, we hypothesized that excessive Nlrp3 activation might drive RA pathology in A20-deficient mice upstream of IL1R1. To test this hypothesis, Nlrp3−/− mice were crossed into A20myel-KO mice and the levels of 4 RA-relevant cytokines (IL-1α, IL-1β, IL-6 and TNF) were monitored. Although IL-1α levels were not significantly different in A20-sufficient and A20myel-KO mice (Extended Data figure 4a), the latter mice had significantly higher levels of IL-1β in circulation (Fig. 4a). In addition, the levels of IL-6 and TNF were also significantly higher in A20myel-KO mice compared to A20flox/flox littermates (Extended Data figure 4b, c). Notably, deletion of Nlrp3 in A20myel-KO mice markedly reduced IL-1β secretion to baseline levels of A20flox/flox mice, thereby demonstrating that the Nlrp3 inflammasome contributes critically to excessive IL-1β production in A20myel-KO mice in vivo (Fig. 4a). Intriguingly, A20myel-KONlrp3−/− mice also were protected from excessive IL-6 production, suggesting that high IL-6 levels are consequent to excessive inflammasome-mediated IL-1β production (Extended Data figure 4b). In contrast, TNF production was not significantly affected in A20myel-KONlrp3−/− and A20myel-KOCasp1−/− mice (Extended Data figure 4c). In agreement, TNF-R1 signaling was previously shown to be dispensable for RA pathology in A20myel-KO mice, whereas IL-6 neutralization provided protection2.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Medical Protein Research, VIB, Ghent B-9000, Belgium [2] Department of Biochemistry, Ghent University, Ghent B-9000, Belgium.

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.

Show MeSH
Related in: MedlinePlus