痛風是一種常見的的關節炎。其原因是由於單鈉尿酸鹽晶體沉積在關節中而導致紅腫發炎。在痛風發炎路徑中，巨噬細胞中的NLRP3蛋白負責下游免疫反應訊號的傳導。而秋水仙鹼作為急性痛風發作治療的的一線藥物，其對巨噬細胞的抑制作用還不是很清楚。Eps8不僅參與在LPS誘導的TLR4信號傳導中，而且還能以不依賴NLRP3發炎體的方式活化caspase-1。其參與在NLRP3發炎體的機制尚未釐清。在本研究中，我們使用THP-1細胞株和免疫沉澱以及西方墨點法來解決這個問題。我們發現秋水仙鹼在不改變NLRP3和IL-1β的表達情況下增強了LPS所誘導的IL-1β分泌，而對MSU所誘導的IL-1β分泌沒有影響。儘管秋水仙鹼會降低Src和FAK激酶的活性，但這種作用與其對IL-1β分泌的活性無關。此外，在LPS處理的THP-1細胞中，秋水仙鹼失去了抑制MSU誘導的IL-1β分泌的能力，而它能夠抑制LPS引發的小鼠腹腔巨噬細胞中IL-1β的分泌。最後，我們發現Eps8在NLRP3發炎體活化過程中與NLRP3、ASC和caspase-1相互作用，該結果支持Eps8參與caspase-1的活化但不參與NLRP3發炎體的形成。

Gout has been reported to be a common arthritis due to the deposition of monosodium urate crystal in joints. In the gout inflammation, NLRP3 protein in macrophages is responsible for the downstream signal transduction. Colchicine served as the first line therapy for acute gout flare, its inhibition on macrophages is still unknown. Eps8 not only shows the significance in TLR4 signaling induced by LPS, but also engages in the caspase-1 activation in a NLRP3 inflammasome independent manner. Whether it participates in the action of NLRP3 inflammasome is not known. In this study we used the THP-1 cell lines and immunoprecipitation as well as western blot to address this issue. We found that instead of inhibiting, colchicine enhances the secretion of IL-1β in LPS-, but not MSU crystal-treated THP1 cells without altering the expression of NLRP3 and IL-1β. Although colchicine decreases the activity of Src and FAK kinases, this effect is not correlated with its activity on IL-1β secretion. Furthermore, in LPS-primed THP-1 cells, colchicine loses the ability to inhibit MSU crystal-induced IL-1β secretion while it is capable to inhibit IL-1β secretion in LPS-primed murine peritoneal macrophages. Finally, we found that Eps8 interacts with NLRP3, ASC and caspase-1 during the activation of NLRP3 inflammasome and Eps8 knockdown inhibits caspase-1 activation as well as IL-1β secretion without affecting the complex formation of NLRP3-ASC-Caspase-1. In summary, our studies support the role of Eps8 in activating caspase-1 instead of the assembly of NLRP3 inflammasome in THP-1 macrophages.

Akira, S. (2006). TLR signaling. Curr Top Microbiol Immunol, 311, 1-16. https://doi.org/10.1007/3-540-32636-7_1

Allen, J. N., Herzyk, D. J., & Wewers, M. D. (1991). Colchicine has opposite effects on interleukin-1 beta and tumor necrosis factor-alpha production. Am J Physiol, 261(4 Pt 1), L315-321. https://doi.org/10.1152/ajplung.1991.261.4.L315

Basak, C., Pathak, S. K., Bhattacharyya, A., Mandal, D., Pathak, S., & Kundu, M. (2005). NF-kappaB- and C/EBPbeta-driven interleukin-1beta gene expression and PAK1-mediated caspase-1 activation play essential roles in interleukin-1beta release from Helicobacter pylori lipopolysaccharide-stimulated macrophages. J Biol Chem, 280(6), 4279-4288. https://doi.org/10.1074/jbc.M412820200

Boucher, D., Monteleone, M., Coll, R. C., Chen, K. W., Ross, C. M., Teo, J. L., Gomez, G. A., Holley, C. L., Bierschenk, D., Stacey, K. J., Yap, A. S., Bezbradica, J. S., & Schroder, K. (2018). Caspase-1 self-cleavage is an intrinsic mechanism to terminate inflammasome activity. J Exp Med, 215(3), 827-840. https://doi.org/10.1084/jem.20172222

Chen, Y. J., Hsieh, M. Y., Chang, M. Y., Chen, H. C., Jan, M. S., Maa, M. C., & Leu, T. H. (2012). Eps8 protein facilitates phagocytosis by increasing TLR4-MyD88 protein interaction in lipopolysaccharide-stimulated macrophages. J Biol Chem, 287(22), 18806-18819. https://doi.org/10.1074/jbc.M112.340935

Chuang, J.-P., Kao, C.-Y., Lee, J.-C., Ling, P., Maa, M.-C., & Leu, T.-H. (2020). EPS8 regulates an NLRP3 inflammasome-independent caspase-1 activation pathway in monosodium urate crystal-treated RAW264.7 macrophages. Biochemical and Biophysical Research Communications, 530(3), 487-493. https://doi.org/https://doi.org/10.1016/j.bbrc.2020.05.084

Devant, P., & Kagan, J. C. (2023). Molecular mechanisms of gasdermin D pore-forming activity. Nat Immunol. https://doi.org/10.1038/s41590-023-01526-w

Ellwanger, K., Becker, E., Kienes, I., Sowa, A., Postma, Y., Cardona Gloria, Y., Weber, A. N. R., & Kufer, T. A. (2018). The NLR family pyrin domain-containing 11 protein contributes to the regulation of inflammatory signaling. J Biol Chem, 293(8), 2701-2710. https://doi.org/10.1074/jbc.RA117.000152

Fry, A. M., O'Regan, L., Sabir, S. R., & Bayliss, R. (2012). Cell cycle regulation by the NEK family of protein kinases. Journal of Cell Science, 125(19), 4423-4433. https://doi.org/10.1242/jcs.111195

He, Y., Zeng, M. Y., Yang, D., Motro, B., & Núñez, G. (2016). NEK7 is an essential mediator of NLRP3 activation downstream of potassium efflux. Nature, 530(7590), 354-357. https://doi.org/10.1038/nature16959

Leung, Y. Y., Yao Hui, L. L., & Kraus, V. B. (2015). Colchicine--Update on mechanisms of action and therapeutic uses. Semin Arthritis Rheum, 45(3), 341-350. https://doi.org/10.1016/j.semarthrit.2015.06.013

Mayes-Hopfinger, L., Enache, A., Xie, J., Huang, C. L., Köchl, R., Tybulewicz, V. L. J., Fernandes-Alnemri, T., & Alnemri, E. S. (2021). Chloride sensing by WNK1 regulates NLRP3 inflammasome activation and pyroptosis. Nat Commun, 12(1), 4546. https://doi.org/10.1038/s41467-021-24784-4

Ming-Chei, M., & Tzeng-Horng, L. (2013). EPS8, an Adaptor Protein Acts as an Oncoprotein in Human Cancer. In T. Kathryn (Ed.), Carcinogenesis (pp. Ch. 5). IntechOpen. https://doi.org/10.5772/54906

Niu, T., De Rosny, C., Chautard, S., Rey, A., Patoli, D., Groslambert, M., Cosson, C., Lagrange, B., Zhang, Z., Visvikis, O., Hacot, S., Hologne, M., Walker, O., Wong, J., Wang, P., Ricci, R., Henry, T., Boyer, L., Petrilli, V., & Py, B. F. (2021). NLRP3 phosphorylation in its LRR domain critically regulates inflammasome assembly. Nature Communications, 12(1), 5862. https://doi.org/10.1038/s41467-021-26142-w

Proell, M., Gerlic, M., Mace, P. D., Reed, J. C., & Riedl, S. J. (2013). The CARD plays a critical role in ASC foci formation and inflammasome signalling. Biochem J, 449(3), 613-621. https://doi.org/10.1042/bj20121198

Ragab, G., Elshahaly, M., & Bardin, T. (2017). Gout: An old disease in new perspective - A review. J Adv Res, 8(5), 495-511. https://doi.org/10.1016/j.jare.2017.04.008

Scita, G., Nordstrom, J., Carbone, R., Tenca, P., Giardina, G., Gutkind, S., Bjarnegård, M., Betsholtz, C., & Di Fiore, P. P. (1999). EPS8 and E3B1 transduce signals from Ras to Rac. Nature, 401(6750), 290-293. https://doi.org/10.1038/45822

Scita, G., Tenca, P., Areces, L. B., Tocchetti, A., Frittoli, E., Giardina, G., Ponzanelli, I., Sini, P., Innocenti, M., & Di Fiore, P. P. (2001). An effector region in Eps8 is responsible for the activation of the Rac-specific GEF activity of Sos-1 and for the proper localization of the Rac-based actin-polymerizing machine. J Cell Biol, 154(5), 1031-1044. https://doi.org/10.1083/jcb.200103146

Seok, J. K., Kang, H. C., Cho, Y. Y., Lee, H. S., & Lee, J. Y. (2020). Regulation of the NLRP3 Inflammasome by Post-Translational Modifications and Small Molecules. Front Immunol, 11, 618231. https://doi.org/10.3389/fimmu.2020.618231

Singh, J. A., & Gaffo, A. (2020). Gout epidemiology and comorbidities. Semin Arthritis Rheum, 50(3s), S11-s16. https://doi.org/10.1016/j.semarthrit.2020.04.008

Song, H., Zhao, C., Yu, Z., Li, Q., Yan, R., Qin, Y., Jia, M., & Zhao, W. (2020). UAF1 deubiquitinase complexes facilitate NLRP3 inflammasome activation by promoting NLRP3 expression. Nature Communications, 11(1), 6042. https://doi.org/10.1038/s41467-020-19939-8

Song, H., Zhao, C., Yu, Z., Li, Q., Yan, R., Qin, Y., Jia, M., & Zhao, W. (2020). UAF1 deubiquitinase complexes facilitate NLRP3 inflammasome activation by promoting NLRP3 expression. Nat Commun, 11(1), 6042. https://doi.org/10.1038/s41467-020-19939-8

Swanson, K. V., Deng, M., & Ting, J. P. (2019). The NLRP3 inflammasome: molecular activation and regulation to therapeutics. Nat Rev Immunol, 19(8), 477-489. https://doi.org/10.1038/s41577-019-0165-0

Wang, D., Zhang, Y., Xu, X., Wu, J., Peng, Y., Li, J., Luo, R., Huang, L., Liu, L., Yu, S., Zhang, N., Lu, B., & Zhao, K. (2021). YAP promotes the activation of NLRP3 inflammasome via blocking K27-linked polyubiquitination of NLRP3. Nature Communications, 12(1), 2674. https://doi.org/10.1038/s41467-021-22987-3