Cellular localization of NLRP3 inflammasome

Abstract

Infl ammasome is a large protein complex activated upon cellular stress or microbial infection, which triggers maturation of pro-inflammatory cytokines interleukin-1β and interleukin-18 through caspase-1 activation. Nod-like receptor family protein 3 (NLRP3) is the most characterized infl ammasome activated by various stimuli. However, the mechanism of its activation is unclear and its exact cellular localization is still unknown. We examined the potential co-localization of NLRP3 infl ammasome with mitochondria and seven other organelles under adenosine triphosphate, nigericin or monosodium urate stimulation in mouse peritoneal macrophages using confocal microscopy approach. Our results revealed that the activated endogenous apoptosis-associated speck-like protein containing a CARD (ASC) pyroptosome forms in the cytoplasm and co-localizes with NLRP3 and caspase-1, but not with any of the organelles screened. This study indicates that the ASC pyroptosome universally localizes within the cytoplasm rather than with any specifi c organelles.

Keywords

NLRP3 / inflammasome / ASC pyroptosome / confocal microscopy / cytoplasmic

References

[1] Agostini, L., Martinon, F., Burns, K., McDermott, M.F., Hawkins, P.N., and Tschopp, J. (2004). NALP3 forms an IL-1beta-processing inflammasome with increased activity in Muckle-Wells autoinflammatory disorder. Immunity 20, 319-325 .10.1016/S1074-7613(04)00046-9
[2] Allen, I.C., Scull, M.A., Moore, C.B., Holl, E.K., McElvania-TeKippe, E., Taxman, D.J., Guthrie, E.H., Pickles, R.J., and Ting, J.P. (2009). The NLRP3 inflammasome mediates in vivo innate immunity to influenza A virus through recognition of viral RNA. Immunity 30, 556-565 .10.1016/j.immuni.2009.02.005
[3] Arlehamn, C.S., Petrilli, V., Gross, O., Tschopp, J., and Evans, T.J. (2010). The role of potassium in inflammasome activation by bacte-ria. J Biol Chem 285, 10508-10518 .10.1074/jbc.M109.067298
[4] Bryan, N.B., Dorfleutner, A., Kramer, S.J., Yun, C., Rojanasakul, Y., and Stehlik, C. (2010). Differential splicing of the apoptosis-asso-ciated speck like protein containing a caspase recruitment domain (ASC) regulates inflammasomes. J Inflamm (Lond) 7, 23.10.1186/1476-9255-7-23
[5] Bryan, N.B., Dorfleutner, A., Rojanasakul, Y., and Stehlik, C. (2009). Activation of masomes requires intracellular redistribution of the apoptotic speck-like protein containing a caspase recruitment domain. J Immunol 182, 3173 -3182 .10.4049/jimmunol.0802367
[6] David, E., Kaufman, J.L., Flowers, C.R., Schafer-Hales, K., Torre, C., Chen, J., Marcus, A.I., Sun, S.Y., Boise, L.H., and Lonial, S. (2010). Tipifarnib sensitizes cells to proteasome inhibition by blocking degradation of bortezomib-induced aggresomes. Blood 116, 5285-5288 .10.1182/blood-2010-03-272393
[7] Dinarello, C.A. (2009). Immunological and matory functions of the interleukin-1 family. Annu Rev Immunol 27, 519-550 .10.1146/annurev.immunol.021908.132612
[8] Dostert, C., Petrilli, V., Van Bruggen, R., Steele, C., Mossman, B.T., and Tschopp, J. (2008). Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica. Science 320, 674-677 .10.1126/science.1156995
[9] Duewell, P., Kono, H., Rayner, K.J., Sirois, C.M., Vladimer, G., Bauernfeind, F.G., Abela, G.S., Franchi, L., Nunez, G., Schnurr, M., . (2010). NLRP3 masomes are required for atherogenesis and activated by cholesterol crystals. Nature 464, 1357-1361 .10.1038/nature08938
[10] Eisenbarth, S.C., Colegio, O.R., O’Connor, W., Sutterwala, F.S., and Flavell, R.A. (2008). Crucial role for the Nalp3 masome in the immunostimulatory properties of aluminium adjuvants. Nature 453, 1122-1126 .10.1038/nature06939
[11] Eng, E.W., Bettio, A., Ibrahim, J., and Harrison, R.E. (2007). MTOC reorientation occurs during FcgammaR-mediated phagocytosis in macrophages. Mol Biol Cell 18, 2389-2399 .10.1091/mbc.E06-12-1128
[12] Fernandes-Alnemri, T., Wu, J., Yu, J.W., Datta, P., Miller, B., Jankows-ki, W., Rosenberg, S., Zhang, J., and Alnemri, E.S. (2007). The py-roptosome: a supramolecular assembly of ASC dimers mediating inflammatory cell death via caspase-1 activation. Cell Death Differ 14, 1590-1604 .10.1038/sj.cdd.4402194
[13] Gross, O., Poeck, H., Bscheider, M., Dostert, C., Hannesschlager, N., Endres, S., Hartmann, G., Tardivel, A., Schweighoffer, E., Tybul-ewicz, V., . (2009). Syk kinase signalling couples to the Nlrp3 masome for anti-fungal host defence. Nature 459, 433-436 .10.1038/nature07965
[14] Hornung, V., and Latz, E. (2010). Critical functions of priming and lyso-somal damage for NLRP3 activation. Eur J Immunol 40, 620-623 .10.1002/eji.200940185
[15] Hu, Y., Mao, K., Zeng, Y., Chen, S., Tao, Z., Yang, C., Sun, S., Wu, X., Meng, G., and Sun, B. (2010). Tripartite-motif protein 30 negatively regulates NLRP3 masome activation by modulating reactive oxygen species production. J Immunol 185, 7699-7705 .10.4049/jimmunol.1001099
[16] Ichinohe, T., Lee, H.K., Ogura, Y., Flavell, R., and Iwasaki, A. (2009). masome recognition of influenza virus is essential for adap-tive immune responses. J Exp Med 206, 79-87 .10.1084/jem.20081667
[17] Latz, E., Schoenemeyer, A., Visintin, A., Fitzgerald, K.A., Monks, B.G., Knetter, C.F., Lien, E., Nilsen, N.J., Espevik, T., and Golenbock, D.T. (2004). TLR9 signals after translocating from the ER to CpG DNAin the lysosome. Nat Immunol 5, 190-198 .10.1038/ni1028
[18] Mariathasan, S., Newton, K., Monack, D.M., Vucic, D., French, D.M., Lee, W.P., Roose-Girma, M., Erickson, S., and Dixit, V.M. (2004). Differential activation of the masome by caspase-1 adaptors ASC and Ipaf. Nature 430, 213-218 .10.1038/nature02664
[19] Martinon, F., Burns, K., and Tschopp, J. (2002). The masome: a molecular platform triggering activation of matory caspases and processing of proIL-beta. Mol Cell 10, 417-426 .10.1016/S1097-2765(02)00599-3
[20] Martinon, F., Petrilli, V., Mayor, A., Tardivel, A., and Tschopp, J. (2006). Gout-associated uric acid crystals activate the NALP3 masome. Nature 440, 237-241 .10.1038/nature04516
[21] Nakahira, K., Haspel, J.A., Rathinam, V.A.K., Lee, S.-J., Dolinay, T., Lam, H.C., Englert, J.A., Rabinovitch, M., Cernadas, M., Kim, H.P., . (2010). Autophagy proteins regulate innate immune respons-es by inhibiting the release of mitochondrial DNA mediated by the NALP3 masome. Nat Immunol 12, 222-230 .10.1038/ni.1980
[22] Pelegrin, P., and Surprenant, A. (2009). Dynamics of macrophage po-larization reveal new mechanism to inhibit IL-1beta release through pyrophosphates. EMBO J 28, 2114-2127 .10.1038/emboj.2009.163
[23] Poeck, H., Bscheider, M., Gross, O., Finger, K., Roth, S., Rebsamen, M., Hannesschlager, N., Schlee, M., Rothenfusser, S., Barchet, W., . (2010). Recognition of RNA virus by RIG-I results in activation of CARD9 and masome signaling for interleukin 1 beta pro-duction. Nat Immunol 11, 63-69 .10.1038/ni.1824
[24] Schroder, K., Zhou, R., and Tschopp, J. (2010). The NLRP3 ma-some: a sensor for metabolic dangerflScience327, 296-300 .
[25] Thomas, P.G., Dash, P., Aldridge, J.R., Ellebedy, A.H., Reynolds, C., Funk, A.J., Martin, W.J., Lamkanfi, M., Webby, R.J., Boyd, K.L., . (2009). The intracellular sensor NLRP3 mediates key innate and healing responses to influenza A virus via the regulation of cas-pase-1. Immunity 30, 566-575 .10.1016/j.immuni.2009.02.006
[26] Webb, J.L., Harvey, M.W., Holden, D.W., and Evans, T.J. (2001). Mac-rophage nitric oxide synthase associates with cortical actin but is not recruited to phagosomes. Infect Immun 69, 6391-6400 .10.1128/IAI.69.10.6391-6400.2001
[27] Wen, H., Gris, D., Lei, Y., Jha, S., Zhang, L., Huang, M.T., Brickey, W.J., and Ting, J.P. (2011). Fatty acid-induced NLRP3-ASC ma-some activation interferes with insulin signaling. Nat Immunol 12, 408-415 .10.1038/ni.2022
[28] Wolff, N.A., Lee, W.K., and Thevenod, F. (2011). Role of Arf1 in endo-somal trafflcking of protein-metal complexes and cadmium-metal-lothionein-1 toxicity in kidney proximal tubule cells. Toxicol Lett 203, 210-218 .10.1016/j.toxlet.2011.03.014
[29] Yuan, R.T., Young, S., Liang, J., Schmid, M.C., Mielgo, A., and Stupack, D.G. (2012). Caspase-8 isoform 6 promotes death effector filament formation independent of microtubules. Apoptosis 17, 229-235 .10.1007/s10495-011-0677-y
[30] Zaki, M.H., Boyd, K.L., Vogel, P., Kastan, M.B., Lamkanfl, M., and Kan-neganti, T.D. (2010). The NLRP3 masome protects against loss of epithelial integrity and mortality during experimental colitis. Immunity 32, 379-391 .10.1016/j.immuni.2010.03.003
[31] Zhou, R., Yazdi, A.S., Menu, P., and Tschopp, J. (2011). A role for mitochondria in NLRP3 inflammasome activation. Nature 469, 221-225 10.1038/nature09663