Regulation of NLRP3 Inflammasome by Phosphorylation.

Inflammasomes are large multi-protein complexes that mediate the immune responses against pathogen infection and tissue damage ( 1 , 2 ). Canonical inflammasome is mainly composed of the initiator protein such as pattern recognition receptors (PRRs), the adaptor protein called ASC (Apoptosis-associated speck-like protein containing a CARD), and the effector protein, pro-caspase-1 ( 2 – 4 ). In response to the disturbance of tissue homeostasis, PRRs, such as NLRs and ALRs, undergo conformational change and nucleate the oligomerization of monomeric PRR proteins ( 4 ). Once the core PRR oligomer is formed, ASC can then be recruited and interact with the PRR oligomer through their pyrin domains (PYD) ( 5 ). As a platform, the clustered ASC can recruit pro-caspase-1 to the PRR-ASC complex via their caspase-recruitment domain (CARD) ( 6 ). This recruitment converts dormant pro-caspase-1 into active caspase-1 by the proximity-mediated cleavage ( 7 ). Active caspase-1 then promotes the proteolytic cleavage of inflammatory cytokines including pro-interleukin-1β (pro-IL-1β) and pro-IL-18, and also the Gasdermin D (GSDMD) ( 8 , 9 ). The pore-forming activity of GSDMD can then trigger pyroptosis, facilitating the secretion of a serious of cytokines including cleaved IL-1β, IL-18, and IL-1α ( 10 , 11 ). So far, five different types of inflammasomes have been identified, distinguished by their core PRRs, which are NLRP1, NLRP3, NLRC4, Pyrin, and Absent in Melanoma 2 (AIM2) ( 3 , 12 – 18 ). Although sharing highly resembled downstream pathways, each inflammasome responds to different kind of stimuli ( 19 ). Particularly, NLRP3 inflammasome senses a wide array of stimuli such as ATP, toxins, RNA viruses, and crystals ( 20 – 24 ). Moreover, aberrant activation of NLRP3 caused by mutations has been implicated in the pathogenesis of Cryopyrin-associated periodic syndrome (CAPS) and other inflammatory diseases ( 25 – 27 ). Therefore, the molecular mechanism of NLRP3 inflammasome activation has been extensively investigated. To data, a two-step model of NLRP3 activation has been well documented, which is triggered by two sequential signals ( 3 , 28 – 30 ). Unlike other PRRs, the basal level of NLRP3 in macrophage is quite low ( 30 ). Before activation, NLRP3 needs to be “primed” by Toll-like receptor (TLR) agonists such as lipopolysaccharide (LPS). Activation of TLR signaling not only transcriptionally upregulates NLRP3 expression, but also post-transcriptionally activates NLRP3 by phosphorylation and deubiquitination. This step has been referred to as “priming.” The second step, defined as “activation,” can be induced by several potent stimuli such as pore-forming toxins, leading to the oligomerization of NLRP3 and the subsequent assembly of inflammasome. Therefore the activation of NLRP3 inflammasome is tightly regulated by different mechanisms mediated by interacting proteins and/or modifications ( 31 – 34 ). Of note, growing evidence highlights that phosphorylation, one of the most abundant PTM, serves as one of the predominant signals controlling inflammasome activation ( 35 ). Several inflammasome components, such as the core PRRs like NLRP3, NLRC4, Pyrin, the adaptor ASC, and the effector caspase-1, are all reported to be regulated by phosphorylation ( 32 , 35 – 44 ). Here, we summarize the progress in understanding the role that phosphorylation plays in the regulation of NLRP3 inflammasome activation, including how phosphorylation can affect the activity of NLRP3 and other inflammasome components in both priming and activation steps (Figure 1 ). Regulation of NLRP3 inflammasome by phosphorylation. The phosphorylation modifications of NLRP3 (Nod-like receptor protein 3), ASC and pro-caspase-1 are indicated. The phosphorylations regulated by signal 1 are highlighted in red, while signal 2-regulated phosphorylations are in black. Briefly, during priming process, NLRP3 can be dephosphorylated at S5 by PP2A, and phosphorylated at S198 by JNK1. ASC is phosphorylated at S16 and S193 by IKKα, and phosphorylated at S58 by IKKi. H. pylori -derived LPS can activate PAK1, which in turn phosphorylates pro-caspase-1 at S376. Upon signal 2 stimulation, PTPN22 dephosphorylates NLRP3 at Y861, while ASC undergoes phosphorylation at Y146. Both of these events are required for inflammasome assembly. NLRP3 S295 phosphorylation triggered by PKD promotes the release of NLRP3 from mitochondria-associated membranes, and also negatively regulates NLRP3 activation in a PKA-dependent manner. All residue numbers refer to the human proteins. ASC, apoptosis-associated speck-like protein containing a CARD; BTK, Bruton‘s tyrosine kinase; EP4, E-prostanoid 4; IKK, IκB kinase; JNK1, c-Jun N-terminal kinase 1; P, phosphate; PAK1, p21-activated kinase 1; PGE 2 , prostaglandin E 2 ; PKA, protein kinase A; PKD, protein kinase D; PP2A, protein phosphatase2A; PTPN22, protein tyrosine phosphatase, non-receptor type 22; Pyk2, proline-rich tyrosine kinase 2; Syk, Spleen tyrosine kinase; TLR, Toll-like receptor.

NS drafted the manuscript. TL supervised and edited the manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

In the past decade, extensive efforts have been put into the exploring of the regulation mechanisms of NLRP3 inflammasome. Although many interacting proteins and post-transcriptional modifications have been suggested to be essential for either the priming of NLRP3 or its activation, the detailed mechanisms of NLRP3 regulation are still needed to be elucidated. Of note, many studies that focus on the PTM regulation of NLRP3 highly rely on the in vitro system, therefore the contradictories may be caused due to the different methodologies utilized in either the generation of stable cell lines, or other function assays. Future studies performed in vivo would provide a more convincing clue for understanding the regulation of NLRP3 inflammasome. Given the fact that the aberrant NLRP3 inflammasome activation is involved in the pathology of various inflammatory disorders, including CAPS, gout, type 2 diabetes, obesity, as well as neurological diseases, what is the unified mechanism that modulates the NLRP3 activation in response to a wide spectrum of stimuli need to be further investigated. Finally, clinical investigation of compounds such as MCC950 and the ketone metabolite β-hydroxybutyrate (BHB), which specifically targets NLRP3, may lead to the development of therapies against NLRP3-related diseases. Investigation into the mechanism of NLRP3 inflammasome activation will aid the development of NLRP3-targeted compounds for treatment of NLRP3-driven diseases.

In addition to NLRP3, ASC phosphorylation may also occur during the activation step. Hara et al. suggested that Syk/JNK1/JNK2-mediated ASC phosphorylation plays a role in the pyroptosome formation of NLRP3 and AIM2, but not NLRC4, inflammasome ( 37 ). Inhibition of Syk or JNK abolished the formation of ASC specks, while does not affect the interaction of ASC with NLRP3. By mutagenesis assay, Hara et al. identified the Tyr144 of ASC as a possible phosphorylation site, which is critical for speck formation. Of note, the role of Syk and JNK proposed in this study is contradictory to previous findings. Gross et al. reported that Syk is not required for ATP or nigericin-induced NLRP3 inflammasome activation, but required for anti-fungal host defenses ( 77 ). Moreover, JNK2 showed an inhibitory effect on NLRP3 inflammasome activation ( 78 ). JNK1, as a serine/threonine-specific protein kinase, is not likely the kinase that mediates the Tyr144 phosphorylation. In addition, the role of ASC in the activation of NLRC4 inflammasome is still under debate, as the N terminal CARD domain of NLRC4 is sufficient for the recruitment of NLRC4. According to the in vitro reconstitution assay, NLRC4 can directly activate caspase-1 independent of ASC expression ( 79 ). However, ASC oligomerization has indeed been observed in primary BMDMs triggered with NLRC4 stimuli. Furthermore, knockout of ASC significantly disrupts the NLRC4 inflammasome activation in BMDMs ( 80 ). In contrast to these observations, Hara et al. showed that NLRC4 inflammasome activation is not regulated by Syk/JNK1/JNK2-mediated ASC phosphorylation, indicating the binding pattern of ASC and NLRC4 may be different from that of ASC and other PYD-containing PRRs. Indeed, Chung et al. reported that Pyk2, downstream kinase of Syk, directly phosphorylates ASC at Tyr146 ( 44 ). Upon nigericin treatment, Pyk2 is activated by phosphorylation at Tyr402. The phosphorylated Pyk2 co-localizes with ASC, facilitating the formation of ASC oligomerization by Tyr146 phosphorylation. Moreover, Pyk2 inhibitor PF-431396 downregulates NLRP3- and AIM2-induced IL-1β secretion and pyroptosis ( 44 ). In addition, the clinical-trial-tested Pyk2/FAK dual inhibitor PF-562271 mitigates monosodium urate-mediated peritonitis in vivo . Syk signaling can be activated by integrins ( 81 ). Consistently, Integrin α5β1 has been reported to activate inflammasome by direct interaction with a bacterial surface protein ( 82 ), suggesting that the Syk signaling plays a critical role in the regulation of inflammasome activation. Whether other tyrosine phosphorylations such NLRP3 Tyr861 may be regulated by Syk remains to be further clarified. Besides Pyk2, Bruton's tyrosine kinase (BTK) can also be triggered by Syk ( 83 ). Ito et al. performed a screening of pharmacological signal inhibitors, and found that BTK is an essential regulator of the NLRP3 inflammasome ( 84 ). BTK inhibition substantially reduced ASC redistribution into the Triton X-insoluble fraction in macrophages in which NLRP3 inflammasome is activated. Moreover, BTK-induced redistribution of ASC can be severely reduced in the presence of the BTK inhibitor. In addition, a recent study indicates that the approved BTK inhibitor ibrutinib blocks the secretion of IL-1β in cells derived from patients with Muckle-Wells syndrome ( 85 ). Together with the fact that BTK is a tyrosine kinase, BTK kinase activity is also likely to be required for ASC phosphorylation. Further studies using a specific ASC phosphorylation antibody would promote our understanding of the role of this PTM regulation in ASC oligomerization.