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
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.
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.
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.
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.
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.