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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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Hyperactivation of the Nlrp3, but not the Nlrc4 and AIM2 inflammasomes, in A20-deficient macrophagesa-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).
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Figure 2: Hyperactivation of the Nlrp3, but not the Nlrc4 and AIM2 inflammasomes, in A20-deficient macrophagesa-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).

Mentions: Macrophages are a prime source of proIL-1β, and generally depend on caspase-1 for maturation and secretion of the biologically active cytokine. Caspase-1 is produced as a cytosolic zymogen, the activation of which is controlled by different inflammasomes3. To study the role of A20 in inflammasome signaling, we assessed caspase-1 processing in bone marrow-derived macrophages (BMDMs) of wild-type and A20myel-KO mice. Notably, caspase-1 activation was substantially increased in LPS-primed A20myel-KO macrophages that were treated with soluble (ATP and nigericin) or crystalline (silica) stimuli of the Nlrp3 inflammasome compared to wild-type BMDMs (Fig. 2a). Concurrently, the levels of secreted IL-1β in the culture medium of ATP- and nigericin-treated A20myel-KO macrophages were about twice those of wild-type cells, and silica triggered nearly four times higher levels of secreted IL-1β (Fig. 2b). Enhanced caspase-1 autoprocessing (Fig. 2c, d) and IL-1β secretion (Fig. 2e, f) by the Nlrp3 inflammasome was evident within 10 minutes after ATP or nigericin addition, and continued to increase in a time-dependent fashion. Similarly, the induction of caspase-1-dependent pyroptosis was enhanced in A20myel-KO macrophages (Fig. 2g, h). Despite their hypersensitivity for Nlrp3 inflammasome activation, A20myel-KO macrophages failed to process caspase-1, and secrete IL-1β and IL-18 when treated with LPS, ATP or nigericin alone (Extended Data figure 1). The increased responsiveness of A20myel-KO macrophages towards inflammasome activation was restricted to the Nlrp3 inflammasome because caspase-1 processing and pyroptotic cell death by the Nlrc4 inflammasome were similarly induced in wild-type and A20myel-KO macrophages that had been infected with Salmonella Typhimurium (S. Typhimurium) (Fig. 2i, j). Similarly, stimulation of the AIM2 inflammasome with cytosolic dsDNA did not result in differential levels of caspase-1 processing and pyroptosis induction in wild-type and A20myel-KO macrophages (Fig. 2k, l). It is worth noting, however, that despite normal caspase-1 activation and pyroptosis levels in response to S. Typhimurium infection and dsDNA transfection, secretion of IL-1β in response to these treatments was consistently higher in A20myel-KO macrophages compared to wild-type controls (Fig. 2m, n), which could be explained by increased induction of proIL-1β mRNA in A20myel-KO macrophages (data not shown). Together, these results demonstrate that A20 negatively regulates activation of caspase-1 by the Nlrp3 inflammasome, but not by the Nlrc4 and AIM2 inflammasomes.


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)

Hyperactivation of the Nlrp3, but not the Nlrc4 and AIM2 inflammasomes, in A20-deficient macrophagesa-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).
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Related In: Results  -  Collection

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Figure 2: Hyperactivation of the Nlrp3, but not the Nlrc4 and AIM2 inflammasomes, in A20-deficient macrophagesa-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).
Mentions: Macrophages are a prime source of proIL-1β, and generally depend on caspase-1 for maturation and secretion of the biologically active cytokine. Caspase-1 is produced as a cytosolic zymogen, the activation of which is controlled by different inflammasomes3. To study the role of A20 in inflammasome signaling, we assessed caspase-1 processing in bone marrow-derived macrophages (BMDMs) of wild-type and A20myel-KO mice. Notably, caspase-1 activation was substantially increased in LPS-primed A20myel-KO macrophages that were treated with soluble (ATP and nigericin) or crystalline (silica) stimuli of the Nlrp3 inflammasome compared to wild-type BMDMs (Fig. 2a). Concurrently, the levels of secreted IL-1β in the culture medium of ATP- and nigericin-treated A20myel-KO macrophages were about twice those of wild-type cells, and silica triggered nearly four times higher levels of secreted IL-1β (Fig. 2b). Enhanced caspase-1 autoprocessing (Fig. 2c, d) and IL-1β secretion (Fig. 2e, f) by the Nlrp3 inflammasome was evident within 10 minutes after ATP or nigericin addition, and continued to increase in a time-dependent fashion. Similarly, the induction of caspase-1-dependent pyroptosis was enhanced in A20myel-KO macrophages (Fig. 2g, h). Despite their hypersensitivity for Nlrp3 inflammasome activation, A20myel-KO macrophages failed to process caspase-1, and secrete IL-1β and IL-18 when treated with LPS, ATP or nigericin alone (Extended Data figure 1). The increased responsiveness of A20myel-KO macrophages towards inflammasome activation was restricted to the Nlrp3 inflammasome because caspase-1 processing and pyroptotic cell death by the Nlrc4 inflammasome were similarly induced in wild-type and A20myel-KO macrophages that had been infected with Salmonella Typhimurium (S. Typhimurium) (Fig. 2i, j). Similarly, stimulation of the AIM2 inflammasome with cytosolic dsDNA did not result in differential levels of caspase-1 processing and pyroptosis induction in wild-type and A20myel-KO macrophages (Fig. 2k, l). It is worth noting, however, that despite normal caspase-1 activation and pyroptosis levels in response to S. Typhimurium infection and dsDNA transfection, secretion of IL-1β in response to these treatments was consistently higher in A20myel-KO macrophages compared to wild-type controls (Fig. 2m, n), which could be explained by increased induction of proIL-1β mRNA in A20myel-KO macrophages (data not shown). Together, these results demonstrate that A20 negatively regulates activation of caspase-1 by the Nlrp3 inflammasome, but not by the Nlrc4 and AIM2 inflammasomes.

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