The Study Of Syk And JNK On Regulating The Inflammasomes Activation Upon Listeria Monocytogenes Infection In Murine Macrophages

ObjectiveThe aim of this study is to clarify the roles of syk kinase and other approved common signaling kinases including JNK, p38MAPK and PI3K in inflammasomes activation in mouse peritoneal macrophages during Listeria monocytogenes (LM) infection.MethodsMurine peritoneal macrophages were randomly divided into BAY treatment group, SP treatment group, SB treatment group, WO treatment group, no treatment group and negative control group (NI), triplicate well/group. BAY treatment group, SP treatment group, SB treatment group, WO treatment group peritoneal marophages were pretreated with syk, JNK, P38MAPK, or PI3K kinase inhibitors (BAY117082/SP600125/SB203580/wotamine) for1h, each group macrophages were infected with WT-LM for24h besides NI group, the level of IL-18in the supernatant was detected by ELISA kit, the activation condition of ASC in the infected-macrophages cytoplasm was observed under fluorescence microscope. Furthermore, phosphorylation level of syk and JNK protein kinase at different time point during WT-LM infection were determined by western blot, and we compared p-syk and p-JNK during LM mutants (Δhly and Δhly::hly) infection. BAY treatment group and SP treatment group macrophages were stimulated by ATP for3h, the supernatant was collected for detecting the level of IL-18and ASC-speck in the cytoplasm were observed under immunofluorescence microscope. SP treatment group macrophages were stimulated by Poly (dA:dT) or flagellin, then the protein level of IL-18in the supernatant was tested by ELISA kit respectively.ResultsIL-18protein level produced by peritoneal macrophages in BAY treatment group was significantly reduced compared with that in macrophages without pretreatment (P<0.05). Interestingly, we found that the level of IL-18in SP treatment group was decreased significantly (P<0.05) and the reduced extent was similar with BAY treatment group. In contrast, there was no significant difference in IL-18production between SB treatment group or WO treatment and no treatment group (P>0.05) Meanwhile, the percentage of ASC-speck positive cells in BAY treatment group and SP treatment group were all obviously almost diminished compared with that in no treatment group (P<0.01). Furthermore, we found that the phosphorylated JNK was significantly increased at10min, peak at40min, and persisted for at least120min after WT-LM infection, and p-syk was detected at5min, which confirmed the critical roles of JNK and syk in the inflammasomes activation upon LM infection, western blot results found that p-JNK in the Δhly mutant strains infection was still significant expression at40min, but in80min and120min sharply reduced, while p-syk expression trend changed, the peak of expression after Δhly mutant strains infection was delayed, the highest in the30min, the results showed that LLO, the main virulence factors of LM, may be the important control targets of the syk and JNK signaling pathway in regulating the process of inflammasomes after LM infection. To further explore whether syk and JNK played the roles in the LM infection specifically, we examined IL-18level and the percentage of ASC-speck after ATP stimulation, and we found that IL-18level and the percentage of ASC-speck were significantly reduced in SP treatment group compared with no treatment group (P<0.05), whereas no diference was observed between BAY treatment group and no treatment group. In addition, we applied Poly (dA:dT) and flagellin to stimulate SP treatment group macrophages, respectively. The results showed that the protein level of IL-18was reduced after Poly (dA:dT) stimulation (P<0.05), but not flagellin.ConclusionsIn a word, Syk kinase signaling and JNK kinase signaling are required for the inflammasomes activation upon Listeria monocytogenes infection. And the regulation processes are closely associated with LLO. Moreover, the results deduce that syk kinase signaling specifically regulates the inflammasome activation upon listeria infection, whereas JNK kinase signaling may be play a universal role in regulating NLRP3-or AIM2-dependent inflammasome.