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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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Il1r1-deficiency rescues the arthritis phenotype of A20myel-KO micea, 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.
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Figure 1: Il1r1-deficiency rescues the arthritis phenotype of A20myel-KO micea, 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.

Mentions: A20 was deleted in myeloid cells by crossing A20flox/flox mice into Lysosyme M (LysM)-Cre recombinase-expressing mice. Unlike wild-type macrophages, A20myel-KO macrophages failed to induce A20 mRNA and protein expression in response to the TLR4 ligand LPS (Fig. 1a, b), demonstrating the effectiveness of LysM-driven deletion of A20 in myeloid cells. Arthritis development in A20myel-KO mice was shown to be TNFR1-independent, whereas deletion of MyD88 markedly protected against RA pathology2. As this signaling adaptor operates downstream of both TLRs and IL-1R, we crossed IL1R1−/− mice into A20myel-KO mice to assess the contribution of IL-1 signaling to arthritis pathogenesis. As expected, wild-type mice (A20flox/floxIL1R1+/+) did not develop arthritis, whereas A20myel-KO mice spontaneously developed an arthritic phenotype (Fig. 1c). The incidence of A20myel-KO mice developing arthritis was 100% (Fig. 1d). In sharp contrast, A20myel-KOIL1R1−/− mice were virtually devoid of clinical signs of arthritis (Fig. 1c, d). In agreement, clinical scoring of disease severity confirmed A20myel-KOIL1R1+/+ mice to develop severe arthritic disease (high clinical score) whereas A20myel-KOIL1R1−/−mice were markedly protected (clinical score 0). A20myel-KO littermates that were heterozygous for IL1R1 expression showed an intermediate arthritic phenotype between those of A20myel-KOIL1R1+/+ and A20myel-KOIL1R1−/− mice (Fig. 1d, e). These clinical assessments were supported by a histological examination of representative ankle joints. Hematoxylin and eosin-stained tissue sections of diseased A20myel-KOIL1R1+/− mice showed significant synovial and periarticular inflammation and high levels of infiltrated mononuclear cells, which was associated with extensive cartilage and bone destruction (Fig. 1f). In marked contrast, ankle joints of A20myel-KOIL1R1−/− littermates were strongly protected from arthritic histopathology and contained significantly reduced numbers of infiltrating inflammatory cells (Fig. 1f). Semi-quantitative scoring of these histological parameters confirmed that the severity of arthritis was substantially lower in A20myel-KOIL1R1−/− mice relative to A20myel-KOIL1R1+/− littermates (Fig. 1g and Extended Data Table 1). These results demonstrate that IL-1 production is detrimental for arthritis pathogenesis in mice with a myeloid cell-restricted deletion in A20.


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)

Il1r1-deficiency rescues the arthritis phenotype of A20myel-KO micea, 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4126806&req=5

Figure 1: Il1r1-deficiency rescues the arthritis phenotype of A20myel-KO micea, 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.
Mentions: A20 was deleted in myeloid cells by crossing A20flox/flox mice into Lysosyme M (LysM)-Cre recombinase-expressing mice. Unlike wild-type macrophages, A20myel-KO macrophages failed to induce A20 mRNA and protein expression in response to the TLR4 ligand LPS (Fig. 1a, b), demonstrating the effectiveness of LysM-driven deletion of A20 in myeloid cells. Arthritis development in A20myel-KO mice was shown to be TNFR1-independent, whereas deletion of MyD88 markedly protected against RA pathology2. As this signaling adaptor operates downstream of both TLRs and IL-1R, we crossed IL1R1−/− mice into A20myel-KO mice to assess the contribution of IL-1 signaling to arthritis pathogenesis. As expected, wild-type mice (A20flox/floxIL1R1+/+) did not develop arthritis, whereas A20myel-KO mice spontaneously developed an arthritic phenotype (Fig. 1c). The incidence of A20myel-KO mice developing arthritis was 100% (Fig. 1d). In sharp contrast, A20myel-KOIL1R1−/− mice were virtually devoid of clinical signs of arthritis (Fig. 1c, d). In agreement, clinical scoring of disease severity confirmed A20myel-KOIL1R1+/+ mice to develop severe arthritic disease (high clinical score) whereas A20myel-KOIL1R1−/−mice were markedly protected (clinical score 0). A20myel-KO littermates that were heterozygous for IL1R1 expression showed an intermediate arthritic phenotype between those of A20myel-KOIL1R1+/+ and A20myel-KOIL1R1−/− mice (Fig. 1d, e). These clinical assessments were supported by a histological examination of representative ankle joints. Hematoxylin and eosin-stained tissue sections of diseased A20myel-KOIL1R1+/− mice showed significant synovial and periarticular inflammation and high levels of infiltrated mononuclear cells, which was associated with extensive cartilage and bone destruction (Fig. 1f). In marked contrast, ankle joints of A20myel-KOIL1R1−/− littermates were strongly protected from arthritic histopathology and contained significantly reduced numbers of infiltrating inflammatory cells (Fig. 1f). Semi-quantitative scoring of these histological parameters confirmed that the severity of arthritis was substantially lower in A20myel-KOIL1R1−/− mice relative to A20myel-KOIL1R1+/− littermates (Fig. 1g and Extended Data Table 1). These results demonstrate that IL-1 production is detrimental for arthritis pathogenesis in mice with a myeloid cell-restricted deletion in A20.

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