However, production of TNF-, an inflammasome-independent cytokine, was not affected, suggesting that knocking down of inhibited the release of IL-1inflammasome activation. Open in a separate window Figure 1 Screening of HCK as a regulator for the NLRP3 inflammasome. 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. (IL-1and IL-18 requires two distinct signals: the signal driven by pattern recognition receptors, which induces the expression of pro-IL-1and pro-IL-18 mRNAs (signal 1) and activation of the inflammasome (signal 2), which stimulates the cleavage of caspase 1 (Davis and Ting, 2010; Schroder and Tschopp, 2010; Lamkanfi and Dixit, 2012). Many studies have shown that the NLRP3 inflammasome is involved in diseases such as type 2 diabetes, atherosclerosis, colitis, Parkinsons disease, and multiple sclerosis, as well as psychological disorders (Guarda et?al., 2009; Duewell et?al., 2010; Lamkanfi and PITPNM1 Dixit, 2012; Heneka et?al., 2013; Zhou et?al., 2016). Various protein kinases, including PAK1, PKA, PKC, PKD, PKR, SIS3 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). A protein tyrosine phosphatase, non-receptor type 22 (PTPN22), interacts with and dephosphorylates NLRP3 at tyrosine 861, thereby activating the inflammasome (Spalinger et?al., 2016). Phosphatase PP2A dephosphorylates NLRP3 at serine 295 and negatively regulates its SIS3 activity (Stutz et?al., 2017). Interestingly, phosphorylation of NLRP3 at serine 295 by different upstream kinases differentially regulates NLRP3 inflammasome activation. PKA-induced NLRP3 phosphorylation at serine 295 significantly blocks nigericin-induced inflammasome activation, although PKD-induced serine 295 phosphorylation at the Golgi apparatus is required for inflammasome activation. In addition to NLRP3, phosphorylation of ASC and caspase 1 is also involved in NLRP3 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). In addition, Pyk2, BTK, IKKin the murine macrophage cell line BAC1.2F5 interferes with the lipopolysaccharide (LPS)-induced expression, without altering TNF- release (Scholz et?al., 2000). Furthermore, A419259, a specific pharmacological inhibitor of HCK, ameliorated bone destruction associated with inflammation (Kim et?al., 2020). Therefore, we speculated that HCK might also be involved in the regulation of microglia (resident macrophages of the central nervous system) function and progression of Alzheimers disease-like neuropathology (Lim et?al., 2018). However, the underlying mechanism is not known. Hence, in this study, we aimed to investigate the role of HCK in NLRP3 inflammasome activation. HCK physically interacted with NLRP3, leading to the induction of ASC oligomerization and caspase- 1 activation in a kinase activity-dependent manner for 5?min, and the red blood cells were lysed in ammonium chloride-potassium (ACK) buffer (0.154 M NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) for 3?min. Subsequently, the cells were centrifuged at 350for 5?min and resuspended in complete basic high glucose DMEM supplemented with macrophage colony stimulating factor (MCSF) (20 ng/ml) (Peprotech). The medium was changed with fresh MCSF-supplemented medium after every 3 days. The cells were ready for use in a week and were cultured for 3 weeks. Murine peritoneal macrophages (PMs) were prepared as below. Briefly, mice were sacrificed and then immersed in 75% ethanol for 5?min. Five milliliters Roswell Park Memorial Institute (RPMI)-1640 medium SIS3 (Gibco) was injected into the abdominal cavities after clipping the abdominal skin and exposing the abdomens. Next, the mice were shaken mildly to extract the liquid from the SIS3 abdominal cavities. The collected cells were centrifuged at 350 for 5?min, and the re-suspended cells were cultured and maintained in complete RPMI-1640 medium (supplemented with heat-inactivated 10% FBS and 1% penicillin/streptomycin). Microglia were prepared from the cortex of mice at the P0 stage as described previously (Cheng et?al., 2017; Wu et?al., 2019). The microglia were maintained in basic SIS3 high glucose DMEM supplemented with heat-inactivated 20% FBS and 1% penicillin/streptomycin (Gibco). Regents and Antibodies LPS (L2630) and ATP (A7699) were purchased from Sigma-Aldrich. Nigericin (HY-127019),.