Abstract
Activation of the NLRP3 inflammasome results in caspase 1 cleavage, which subsequently leads to IL-1β and IL-18 secretion, as well as pyroptosis, and aberrant activation of the inflammasome is involved in several diseases such as type 2 diabetes, atherosclerosis, multiple sclerosis, Parkinson’s disease, and Alzheimer’s disease. NLRP3 activity is regulated by various kinases. Genetic and pharmacological inhibition of the hematopoietic cell kinase (HCK), a member of the Src family of non-receptor tyrosine kinases (NRTKs) primarily expressed in myeloid cells, has previously been shown to ameliorate inflammation, indicating that it may be involved in the regulation of microglia function. However, the underlying mechanism is not known. Hence, in this study, we aimed to investigate the role of HCK in NLRP3 inflammasome activation. We demonstrated that HCK silencing inhibited NLRP3 inflammasome activation. Furthermore, the HCK-specific inhibitor, A419259, attenuated the release of IL-1β and caspase 1(P20) from the macrophages and microglia and reduced the formation of the apoptosis-associated speck-like protein with a CARD domain (ASC) oligomer. We also observed that HCK binds to full length NLRP3 and its NBD(NACHT) and LRR domains, but not to the PYD domain. In vivo, the HCK inhibitor attenuated the LPS-induced inflammatory response in the liver of LPS-challenged mice. Collectively, these results suggested that HCK plays a critical role in NLRP3 inflammasome activation. Our results will enhance current understanding regarding the effectiveness of HCK inhibitors for treating acute inflammatory diseases.
Highlights
Studies have shown that certain non-receptor tyrosine kinases (NRTKs) are involved in the activation of the Nod-like receptor family protein 3 (NLRP3) inflammasome
The results showed that the knockdown of Hck and Janus protein tyrosine kinase 1 (Jak1) and Jak3 significantly reduced the levels of IL-1b in the supernatant (Figures 1B, C)
These results demonstrated that hematopoietic cell kinase (HCK) was involved in NLRP3 inflammasome activation and subsequent IL1b production, indicating that NLRP3 inflammasome activation depended on HCK kinase activity
Summary
The Nod-like receptor family protein 3 (NLRP3) inflammasome is a cytosolic protein complex composed of NLRP3, the adaptor protein apoptosis-associated speck-like protein with a CARD domain (ASC), and caspase 1, which are rapidly assembled in response to both infection with pathogens and endogenous “danger signals” such as monosodium urate, alum, silica, reactive oxygen species, amyloid b, and cholesterol (Duewell et al, 2010; Spreafico et al, 2010; Tschopp and Schroder, 2010; Peeters et al, 2013; Cho et al, 2014; Shaw et al, 2014; Kong et al, 2017). Various protein kinases, including PAK1, PKA, PKC, PKD, PKR, BTK, PyK2, IRAK, Syk, and JNK1 have been reported to be required for inflammasome activation (Basak et al, 2005; Lu et al, 2012; Ito et al, 2015; Chung et al, 2016; Spalinger et al, 2016; Swanson and Ting, 2016; Stutz et al, 2017; Magnotti et al, 2019). Phosphorylation of NLRP3 at serine 295 by different upstream kinases differentially regulates NLRP3 inflammasome activation. Studies have shown that Syk is involved in the tyrosine phosphorylation of murine ASC, which promotes ASC speck formation and NLRP3/AIM2 activation (Chung et al, 2016). Phosphorylation of NLRP3 by specific kinases diversely regulates inflammasome activation in the presence of different stimuli, which controls the temporal and spatial activation of the NRLP3 inflammasome
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