The MTOC is “speck”-tacular Inflammasome complexes are formed in response to pathogen-associated molecules. They initiate both the maturation of inflammatory cytokines and pyroptosis, a type of programmed cell death. One notable feature for inflammasome activation is the formation of a single supramolecular punctum (or “speck”) in each affected cell. However, the location and mechanism of speck formation is poorly understood. Magupalli et al. report that for NLRP3- and pyrin-mediated inflammasomes, their assembly and downstream functions occur at the microtubule-organizing center (MTOC). This process requires the dynein adaptor HDAC6, which is also a central player in aggresome formation and autophagosomal degradation at the MTOC. This work links several important cellular processes and provides clues for how inflammasomes are efficiently regulated. Science, this issue p. eaas8995 NLRP3 and pyrin inflammasome activation provides an unexpected parallel between formation of physiological and pathological aggregates. INTRODUCTION Canonical inflammasomes are multicomponent protein complexes that play key roles in immune surveillance of infections and danger by activating caspase-1, which cleaves interleukin 1β (IL-1β) and the pore-forming protein gasdermin D, leading to cytokine maturation and pyroptosis. The nucleotide-binding domain, leucine-rich repeat, and pyrin domain–containing protein 3 (NLRP3) can be activated by the bacterial toxin nigericin, extracellular ATP, and various particulates such as monosodium urate (MSU) crystals, alum, silica, and amyloids, whereas the pyrin inflammasome can be stimulated by the Rho-glucosylation activity of Clostridium difficile toxin B. One important hallmark for inflammasome activation is the formation of a single supramolecular punctum (also known as a speck) per cell. However, the location and trafficking of such puncta remain unknown. RATIONALE To decode the site and the molecular machinery in inflammasome assembly and activation in macrophages, we aimed to visualize inflammasome assembly by cellular imaging complemented by pharmacological inhibition and targeted deletion in cells and in mice. RESULTS We found that NLRP3 and pyrin inflammasomes are assembled at the centrosome, also known as the microtubule-organizing center (MTOC), of each cell, which serves as the major site for caspase-1 activation and IL-1β conversion. Pharmacological inhibitors of microtubule polymerization, dynein ATPase and the dynein adaptor histone deacetylase 6 (HDAC6), and targeted deletion or knockdown of Hdac6 compromised the assembly and activation of these inflammasomes. Reconstitution of Hdac6−/− macrophages showed that the ubiquitin-binding ability of HDAC6, but not its deacetylase activity, is required for NLRP3 and pyrin inflammasome activation. In mice, Hdac6 deficiency reduced lipopolysaccharide- and MSU-induced inflammation to a similar extent as direct NLRP3 inhibition, which demonstrates the requirement of HDAC6 and the microtubule retrograde transport machinery for NLRP3 activation. By contrast, AIM2 and NLRC4 inflammasome puncta do not localize at the MTOC, and these inflammasomes do not require microtubule retrograde transport for their activation. Thus, our studies revealed a specific mechanism of inflammasome activation for NLRP3 and pyrin. For the NLRP3 inflammasome, MTOC localization may facilitate association with the centrosome-localized kinase NEK7 to enhance inflammasome assembly. We found that in Hdac6−/− macrophages, NLRP3 is trapped as small speckles at the trans-Golgi network (TGN), a previously recognized common site for NLRP3 association upon induction by multiple NLRP3 stimulators. These data suggested that HDAC6-mediated microtubule transport delivers NLRP3 from TGN to the MTOC. The NLRP3 inflammasome formed at the MTOC colocalizes with LC3b, an autophagy marker, and an autophagy inhibitor enhances NLRP3-induced IL-1β secretion. CONCLUSION Our study reveals an unexpected parallel between HDAC6-dependent assembly of NLRP3 and pyrin inflammasomes and the formation of aggresomes at the MTOC for autophagic degradation of ubiquitinated pathological aggregates. The dual activating and inhibiting roles played by the MTOC localization of NLRP3 and pyrin inflammasomes may be critical for achieving balanced inflammasome regulation. HDAC6- and microtubule-dependent assembly and activation of NLRP3 and pyrin inflammasomes. Multiple stimuli for NLRP3 (MSU, silica, nigericin, and ATP) and pyrin (Rho GTPase modification) are shown on the top part of the cell. NLRP3 and pyrin inflammasome components are represented in pink and light blue, respectively. NLRP3 inflammasome assembly may involve two steps: initial oligomerization at the TGN and further assembly into a single punctum with the centrosomal kinase NEK7, the adaptor ASC, and caspase-1 at the MTOC. The latter is achieved after being retrograde transported on the microtubule by the HDAC6-dynein machinery. Pyrin inflammasome activation also requires HDAC6, dynein, and microtubules. Assembled inflammasomes at the MTOC are subject to degradation by autophagy in a manner similar to aggresomes that degrade pathological aggregates. [Figure was created with BioRender (https://BioRender.com).] Inflammasomes are supramolecular complexes that play key roles in immune surveillance. This is accomplished by the activation of inflammatory caspases, which leads to the proteolytic maturation of interleukin 1β (IL-1β) and pyroptosis. Here, we show that nucleotide-binding domain, leucine-rich repeat, and pyrin domain–containing protein 3 (NLRP3)- and pyrin-mediated inflammasome assembly, caspase activation, and IL-1β conversion occur at the microtubule-organizing center (MTOC). Furthermore, the dynein adapter histone deacetylase 6 (HDAC6) is indispensable for the microtubule transport and assembly of these inflammasomes both in vitro and in mice. Because HDAC6 can transport ubiquitinated pathological aggregates to the MTOC for aggresome formation and autophagosomal degradation, its role in NLRP3 and pyrin inflammasome activation also provides an inherent mechanism for the down-regulation of these inflammasomes by autophagy. This work suggests an unexpected parallel between the formation of physiological and pathological aggregates.

中文翻译：

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HDAC6 介导 NLRP3 和 pyrin 炎症小体激活的聚集体样机制

MTOC 是“斑点”-tacular Inflammasome 复合物是响应病原体相关分子而形成的。它们启动炎症细胞因子的成熟和细胞焦亡，这是一种程序性细胞死亡。炎症小体激活的一个显着特征是在每个受影响的细胞中形成单个超分子点（或“斑点”）。然而，人们对斑点形成的位置和机制知之甚少。马古帕利等人。报告说，对于 NLRP3 和 pyrin 介导的炎症小体，它们的组装和下游功能发生在微管组织中心 (MTOC)。这个过程需要动力蛋白适配器 HDAC6，它也是 MTOC 聚集体形成和自噬体降解的核心参与者。这项工作将几个重要的细胞过程联系起来，并为炎症小体如何被有效调节提供了线索。科学，这个问题 p。eaas8995 NLRP3 和 pyrin 炎症小体激活在生理和病理聚集体的形成之间提供了意想不到的平行。引言 典型炎症小体是多组分蛋白质复合物，通过激活 caspase-1，在感染和危险的免疫监视中发挥关键作用，caspase-1 裂解白细胞介素 1β (IL-1β) 和成孔蛋白 gasdermin D，导致细胞因子成熟和细胞焦亡。核苷酸结合结构域、富含亮氨酸的重复序列和含吡喃结构域的蛋白 3 (NLRP3) 可以被细菌毒素尼日利亚菌素、细胞外 ATP 和各种颗粒物激活，如尿酸钠 (MSU) 晶体、明矾、二氧化硅和淀粉样蛋白，而 pyrin 炎症小体可以被艰难梭菌毒素 B 的 Rho-葡萄糖基化活性刺激。炎症小体激活的一个重要标志是每个细胞形成单个超分子点（也称为斑点）。然而，这种斑点的位置和贩运仍然未知。基本原理为了解码巨噬细胞炎症小体组装和激活的位点和分子机制，我们的目标是通过细胞成像来可视化炎症小体组装，辅以药理抑制和细胞和小鼠的靶向缺失。结果 我们发现 NLRP3 和 pyrin 炎症小体组装在每个细胞的中心体，也称为微管组织中心 (MTOC)，它是 caspase-1 激活和 IL-1β 转化的主要位点。微管聚合、动力蛋白 ATP 酶和动力蛋白接头组蛋白脱乙酰酶 6 (HDAC6) 的药理学抑制剂以及 Hdac6 的靶向缺失或敲低会破坏这些炎症小体的组装和激活。Hdac6-/- 巨噬细胞的重建表明，NLRP3 和 pyrin 炎症小体激活需要 HDAC6 的泛素结合能力，而不是其脱乙酰酶活性。在小鼠中，Hdac6 缺乏将脂多糖和 MSU 诱导的炎症减少到与直接 NLRP3 抑制相似的程度，这表明 HDAC6 和微管逆行转运机制需要 NLRP3 激活。相比之下，AIM2 和 NLRC4 炎症小体点不定位在 MTOC，并且这些炎症小体不需要微管逆行运输来激活。因此，我们的研究揭示了 NLRP3 和 pyrin 炎症小体激活的特定机制。对于 NLRP3 炎症小体，MTOC 定位可能促进与中心体定位激酶 NEK7 的结合，以增强炎症小体的组装。我们发现，在 Hdac6−/− 巨噬细胞中，NLRP3 被捕获为反式高尔基网络 (TGN) 处的小斑点，TGN 是先前公认的在被多个 NLRP3 刺激物诱导后 NLRP3 关联的常见位点。这些数据表明 HDAC6 介导的微管运输将 NLRP3 从 TGN 传递到 MTOC。在 MTOC 处形成的 NLRP3 炎性体与 LC3b（一种自噬标记物）共定位，自噬抑制剂可增强 NLRP3 诱导的 IL-1β 分泌。结论我们的研究揭示了 NLRP3 和 pyrin 炎症小体的 HDAC6 依赖性组装与泛素化病理聚集体自噬降解在 MTOC 处形成的聚集体之间的意外平行。NLRP3 和 pyrin 炎症小体的 MTOC 定位所发挥的双重激活和抑制作用可能对于实现平衡的炎症小体调节至关重要。HDAC6 和微管依赖性组装和 NLRP3 和 pyrin 炎症小体的激活。NLRP3（MSU、二氧化硅、尼日利亚菌素和 ATP）和 pyrin（Rho GTPase 修饰）的多重刺激显示在细胞的顶部。NLRP3 和 pyrin 炎性体成分分别以粉红色和浅蓝色表示。NLRP3炎性体组装可能涉及两个步骤：在 TGN 处进行初始寡聚化，并在 MTOC 处与中心体激酶 NEK7、接头 ASC 和 caspase-1 进一步组装成单个泪点。后者是在 HDAC6-动力蛋白机制在微管上逆行运输后实现的。Pyrin 炎症小体激活还需要 HDAC6、动力蛋白和微管。在 MTOC 组装的炎症小体以类似于降解病理聚集体的聚集体的方式被自噬降解。[图是使用 BioRender (https://BioRender.com) 创建的。] 炎症小体是在免疫监视中起关键作用的超分子复合物。这是通过激活炎性半胱天冬酶来实现的，这会导致白细胞介素 1β (IL-1β) 的蛋白水解成熟和细胞焦亡。在这里，我们展示了核苷酸结合域，富含亮氨酸的重复序列，和 pyrin 结构域蛋白 3 (NLRP3) 和 pyrin 介导的炎症小体组装、半胱天冬酶激活和 IL-1β 转化发生在微管组织中心 (MTOC)。此外，动力蛋白接头组蛋白脱乙酰酶 6 (HDAC6) 对于这些炎性体在体外和小鼠中的微管运输和组装是必不可少的。由于 HDAC6 可以将泛素化病理聚集体运输到 MTOC 以形成聚集体和自噬体降解，因此其在 NLRP3 和 pyrin 炎症小体激活中的作用也为自噬下调这些炎症小体提供了内在机制。这项工作表明生理和病理聚集体的形成之间存在意想不到的相似之处。和 IL-1β 转化发生在微管组织中心 (MTOC)。此外，动力蛋白接头组蛋白脱乙酰酶 6 (HDAC6) 对于这些炎性体在体外和小鼠中的微管运输和组装是必不可少的。由于 HDAC6 可以将泛素化病理聚集体运输到 MTOC 以形成聚集体和自噬体降解，因此其在 NLRP3 和 pyrin 炎症小体激活中的作用也为自噬下调这些炎症小体提供了内在机制。这项工作表明生理和病理聚集体的形成之间存在意想不到的相似之处。和 IL-1β 转化发生在微管组织中心 (MTOC)。此外，动力蛋白接头组蛋白脱乙酰酶 6 (HDAC6) 对于这些炎性体在体外和小鼠中的微管运输和组装是必不可少的。由于 HDAC6 可以将泛素化病理聚集体运输到 MTOC 以形成聚集体和自噬体降解，因此其在 NLRP3 和 pyrin 炎症小体激活中的作用也为自噬下调这些炎症小体提供了内在机制。这项工作表明生理和病理聚集体的形成之间存在意想不到的相似之处。动力蛋白接头组蛋白脱乙酰酶 6 (HDAC6) 对于这些炎性体在体外和小鼠中的微管运输和组装是必不可少的。由于 HDAC6 可以将泛素化病理聚集体转运至 MTOC 以形成聚集体和自噬体降解，因此其在 NLRP3 和 pyrin 炎症小体激活中的作用也为自噬下调这些炎症小体提供了内在机制。这项工作表明生理和病理聚集体的形成之间存在意想不到的相似之处。动力蛋白接头组蛋白脱乙酰酶 6 (HDAC6) 对于这些炎性体在体外和小鼠中的微管运输和组装是必不可少的。由于 HDAC6 可以将泛素化病理聚集体运输到 MTOC 以形成聚集体和自噬体降解，因此其在 NLRP3 和 pyrin 炎症小体激活中的作用也为自噬下调这些炎症小体提供了内在机制。这项工作表明生理和病理聚集体的形成之间存在意想不到的相似之处。