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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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A20 inhibits Nlrp3 inflammasome priminga, 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).
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Figure 3: A20 inhibits Nlrp3 inflammasome priminga, 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).

Mentions: Activation of the Nlrp3 inflammasome in wild-type macrophages is tightly regulated at different levels. A priming signal (referred to as step 1 and usually provided by TLRs) upregulates Nlrp3 expression levels along with the inflammasome substrate proIL-1β via the pro-inflammatory transcription factor NF-κB4. A20 negatively regulates LPS-induced NF-κB activation (refs.5-8 and Extended Data figure 2a), which also was reflected in increased secretion of the NF-κB-dependent cytokines IL-6 and TNF in A20myel-KO macrophages (Extended Data figure 2b, c). We further showed A20-deficiency to markedly enhance LPS-induced mRNA expression levels of Nlrp3 (Fig. 3a) and proIL-1β (Fig. 3b). In contrast, LPS-induced transcript levels of caspase-1 and the inflammasome adaptor ASC were respectively mildly upregulated and normal in A20myel-KO macrophages (Extended Data figure 3a, b). Analysis of protein expression levels confirmed Nlrp3 and proIL-1β to be significantly higher in LPS-primed A20myel-KO macrophages compared to wild-type cells, whereas caspase-1 and ASC were not differentially modulated in the two genotypes (Fig. 3c).


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)

A20 inhibits Nlrp3 inflammasome priminga, 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).
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Related In: Results  -  Collection

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Figure 3: A20 inhibits Nlrp3 inflammasome priminga, 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).
Mentions: Activation of the Nlrp3 inflammasome in wild-type macrophages is tightly regulated at different levels. A priming signal (referred to as step 1 and usually provided by TLRs) upregulates Nlrp3 expression levels along with the inflammasome substrate proIL-1β via the pro-inflammatory transcription factor NF-κB4. A20 negatively regulates LPS-induced NF-κB activation (refs.5-8 and Extended Data figure 2a), which also was reflected in increased secretion of the NF-κB-dependent cytokines IL-6 and TNF in A20myel-KO macrophages (Extended Data figure 2b, c). We further showed A20-deficiency to markedly enhance LPS-induced mRNA expression levels of Nlrp3 (Fig. 3a) and proIL-1β (Fig. 3b). In contrast, LPS-induced transcript levels of caspase-1 and the inflammasome adaptor ASC were respectively mildly upregulated and normal in A20myel-KO macrophages (Extended Data figure 3a, b). Analysis of protein expression levels confirmed Nlrp3 and proIL-1β to be significantly higher in LPS-primed A20myel-KO macrophages compared to wild-type cells, whereas caspase-1 and ASC were not differentially modulated in the two genotypes (Fig. 3c).

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