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Phosphorylation of the adaptor ASC acts as a molecular switch that controls the formation of speck-like aggregates and inflammasome activity

Nature Immunology volume 14, pages 1247–1255 (2013)Cite this article

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Abstract

The inflammasome adaptor ASC contributes to innate immunity through the activation of caspase-1. Here we found that signaling pathways dependent on the kinases Syk and Jnk were required for the activation of caspase-1 via the ASC-dependent inflammasomes NLRP3 and AIM2. Inhibition of Syk or Jnk abolished the formation of ASC specks without affecting the interaction of ASC with NLRP3. ASC was phosphorylated during inflammasome activation in a Syk- and Jnk-dependent manner, which suggested that Syk and Jnk are upstream of ASC phosphorylation. Moreover, phosphorylation of Tyr144 in mouse ASC was critical for speck formation and caspase-1 activation. Our results suggest that phosphorylation of ASC controls inflammasome activity through the formation of ASC specks.

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Figure 1: Syk and Jnk are required for IL-18 secretion mediated by NLRP3 and AIM2 but not that mediated by NLRC4.
Figure 2: Involvement of Syk and Jnk in NLRP3- and AIM2-mediated activation of caspase-1.
Figure 3: Requirement for signaling via Syk and Jnk for ASC aggregation.
Figure 4: Phosphorylation of ASC in macrophages after activation of the inflammasome.
Figure 5: Identification of amino acid residues in ASC critical for its biological activities.
Figure 6: Critical roles of the possible phosphorylation sites of ASC in macrophages.
Figure 7: Identification of phosphorylation sites in ASC.
Figure 8: Involvement of Syk and Jnk in inflammatory responses to MSU in vivo.

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Acknowledgements

We thank J. Tschopp and the Institute for Arthritis Research for permission to use Nlrp3−/− mice; S.Taniguchi (Shinshu University) for Pycard−/− mice; K. Kuida (Millennium Pharmaceuticals) for Casp1−/− mice; T. Saito (RIKEN RCAI) for Card9−/− mice; K. Kawasaki (Doshisha Women's College) for S. Typhimurium 14028; H. Tsutsui (Hyogo Medical University) for Nlrp3−/−, Pycard−/− and Casp1−/− mice; H. Hara (Saga University) for Card9−/− mice; M. Matsuura (Kyoto University) for S. Typhimurium 14028 and for U937 cells; H. Tanizaki (Kyoto University) for RAW264.7 cells; and K. Sada and Y. Tohyama for advice on Syk experiments. Supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Ministry of Health, Labour and Welfare of Japan, the Japan Society for the Promotion of Science and Medical Research Council UK (U117527252 for E.S. and V.T.).

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  1. Hideki Hara and Kohsuke Tsuchiya: H.H. and K.T. contributed equally to this work.

Authors and Affiliations

  1. Department of Microbiology, Kyoto University Graduate School of Medicine, Kyoto, Japan

    Hideki Hara, Kohsuke Tsuchiya, Ikuo Kawamura, Rendong Fang, Eduardo Hernandez-Cuellar, Yanna Shen & Masao Mitsuyama

  2. School of Laboratory Medicine, Tianjin Medical University, Tianjin, China

    Yanna Shen

  3. Department of Immunology and Intractable Immunology Research Center, Tokyo Medical University, Tokyo, Japan

    Junichiro Mizuguchi

  4. MRC National Institute for Medical Research, London, UK

    Edina Schweighoffer & Victor Tybulewicz

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Contributions

H.H. initiated the study and designed and did the experiments with macrophages and peritonitis; K.T. designed and did the experiments with HEK293 cells; K.T., H.H. and M.M. wrote the manuscript; I.K. provided advice; R.F., E.H.-C. and Y.S. contributed to the experiments; J.M. provided the Mapk8−/− and Mapk9−/− mice; E.S. and V.T. provided the Syk+/+, Syk+/− and Syk−/− fetal liver cells; and M.M. supervised the project.

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Correspondence to Masao Mitsuyama.

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Integrated supplementary information

Supplementary Figure 1 Inhibition of Syk or Jnk do not affect the expression of inflammasome molecules.

(a,d,f) Immunoblot analysis of inflammasome molecules or (b,c,e) Enzyme-linked immunosorbent assayof IL-18 in peritoneal macrophages (a,d–f), bone marrow–derived macrophages(b), or U937 cells (c) primed with LPS for 4 h (a), followed by stimulation with nigericin for 90 min (b–d), or alum for 6 h (e,f). The indicated kinase inhibitors were added to the cultures 1 h before stimulation. CL, cell lysates; SN, supernatants. Data are shown as the means ± s.d. of triplicate samples of one experiment representative of three independent experiments. Data were analyzed by one-way ANOVA with Bonferroni multiple comparison test (b,c,e). * P < 0.01 and ** P < 0.001.

Supplementary Figure 2 Knockdown of Syk or Mapk8-Mapk9 in primary macrophages.

(a,b,e,f) Immunoblot analysis of caspase-1 or (c,d) enzyme-linked immunosorbent assayof IL-18 in peritoneal macrophages unprimed (a,b), primed with LPS for 4 h, followed by stimulation with nigericin for 90 min (c,e), or unprimed macrophages stimulated with poly(dA:dT) for 3 h (d,f). Macrophages were transfected with siRNAs for 48 h. Control, negative control siRNA; CL, cell lysates; SN, supernatants; ND, not detected. Data are shown as the means ± s.d. of triplicate samples of one experiment representative of three independent experiments. Data were analyzed by one-way ANOVA with Bonferroni multiple comparison test (c,d). * P < 0.001.

Supplementary Figure 3 Syk is not required for NLRP3 inflammasome activation in dendritic cells.

(a,b) Enzyme-linked immunosorbent assayof IL-18 or (c,d) immunoblot analysis of inflammasome molecules in bone marrow–derived dendritic cells(a,c,d) or bone marrow–derived macrophages(b,e) primed with LPS for 4 h, followed by stimulation with nigericin for 90 min. Bone marrow–derived macrophageswere prepared using L-cell conditioned medium. CL, cell lysates; SN, supernatants. Data are shown as the means ± s.d. of triplicate samples of one experiment representative of two independent experiments. Data were analyzed by two-tailed unpaired t test with Welch's correction (a,b). * P < 0.01.

Supplementary Figure 4 Implication that Syk contributes to inflammasome activity through an unknown mechanism.

(a–g) Immunoblot analysis of kinases, (h–j) enzyme-linked immunosorbent assayof IL-18, or (k) FACS analysis of mitochondrial ROS in peritoneal macrophages primed with LPS for 4 h, followed by stimulation with nigericin for the indicated times (a,c,e,g,h,j,k), or unprimed macrophages stimulated with poly(dA:dT) for 3 h (b,d,f,i,j). The kinase inhibitors and BHA (25 mM) were added to the cultures 1 h before stimulation (a–e,j,k). The cells were incubated with nigericin for 20 min in the presence of MitoSOX (5 mM) and analyzed on a flow cytometer (k). Data are shown as the means ± s.d. of triplicate samples of one experiment. Data shown in e,f,h–k are representative of three independent experiments and those in a–d,g are representative of two independent experiments. Data were analyzed by two-tailed unpaired t test with Welch's correction (h–j). * P < 0.001.

Supplementary Figure 5 Requirement of Syk and Jnk for ASC speck formation induced by poly(dA:dT).

(a–d) ASC staining or (e,f) immunoblot analysis of ASC in peritoneal macrophages primed with LPS for 4 h, followed by stimulation with nigericin for 90 min (a,b), or unprimed macrophages stimulated with poly(dA:dT) for the indicated times (c–f). The kinase inhibitors were added to the cultures 1 h before stimulation (c–f). ASC is shown in green, nuclei in blue (a–c). The number of ASC speck-positive cell was counted and normalized to that of dimethyl sulfoxide(d). Triton-soluble (S) and Triton-insoluble (I) fractions (right margin; e) or Triton-insoluble fractions treated with DSS (I + DSS; f).Data are shown as the means ± s.d. of triplicate samples of one experiment. Data shown in c–f are representative of three independent experiments and those in a,b are representative of two independent experiments. Data were analyzed by Kruskal-Wallis test with Dunn's multiple comparison test (d). Scale bar, 10 mm. * P < 0.05.

Supplementary Figure 6 Prediction of phosphorylation sites in mouse ASC.

Possible phosphorylation sites in the amino acid sequence of mouse ASC were predicted by using the online program NetPhos 2.0. The threshold is 0.5.

Supplementary Figure 7 Reconstitution of inflammasome system in HEK293 cells.

(a, b) Immunoblot analysis of inflammasome molecules in reconstituted HEK293 cells transfected as described in Fig. 5a,b.

Supplementary Figure 8 Involvement of ASC and Jnk in inflammatory responses to MSU and Alum in vivo.

(a–f) Infiltration of inflammatory cells in the peritoneal cavity induced by intraperitoneal injection of MSU (a–c) or alum (d–f) at 6 h after injection. Two hours before and 30 min later administration of the irritants, the mice were intraperitoneally treated with Jnk inhibitor. (g–l) Infiltration of inflammatory cells in the peritoneal cavity induced by intraperitoneal injection of KC or PBS at 1.5 h after injection. Absolute numbers of PECs (a,d,g,j), Gr-1+ F4/80– neutrophils (b,e,h,k), and F4/80+ monocytes and macrophages (c,f,i,l) in the peritoneum were then determined. Data are shown as dots, and the bars indicate the means ± s.d. (n = 7 for a–f; n = 5 for g–l; n = 3 for PBS control in g–l). Data were analyzed by one-way ANOVA with Bonferroni (a–d,f) or Tukey-Kramer (g–l) multiple comparison test, or Kruskal-Wallis test with Dunn's multiple comparison test (e). NS, no significant difference. * P < 0.05 , ** P < 0.01 and *** P< 0.001.

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Hara, H., Tsuchiya, K., Kawamura, I. et al. Phosphorylation of the adaptor ASC acts as a molecular switch that controls the formation of speck-like aggregates and inflammasome activity. Nat Immunol 14, 1247–1255 (2013). https://doi.org/10.1038/ni.2749

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