Based on our previous research, blocking SYK in monocyte-derived macrophages reduces the production of CXCL1 and M2 polarization of macrophages, thus alleviating chronic inflammation and fibrosis in the liver. Moreover, Trem2 in monocyte-derived macrophages promotes IR inflammatory regression by regulating Cox2/PGE2/Rac1 pathway mediated efferocytosis,and blocking myeloid Trem2 alleviates early liver injury induced by IR[23]. Trem2 activates SYK phosphorylation via Dap12 to induce the inflammatory cascade in macrophages is known to be the classical paradigm.Upon receiving an extracellular stimulus,Trem2 associates with the adapter DAP12,which recruits and activates SYK through its cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM)[24, 25].Therefore, we were curious about the role of SYK in hepatic IR.We observed a significant upregulation of P-SYK and SYK expression in the livers of mice undergoing IR(Fig. 1A-D).Furthermore, the most severe liver damage was observed 6 hours after liver reperfusion, coinciding with a significant increase in P-SYK expression(Fig. 1D,Figugre S1A-C).In this study, we evaluated the effects of three SYK inhibitors on hepatic IRI: GS-9973, R406, and Piceatannol, which have now been shown to have therapeutic promise in hematologic tumors and autoimmune diseases[13, 26, 27].Administration of SYK inhibitors in mice prior to IR 6h significantly inhibited SYK phosphorylation and alleviated liver tissue damage to different degrees(Fig. 1E-H).Among them, mice injected intraperitoneally with GS-9973 and R406 showed less liver tissue damage and a decrease in serum liver enzymes levels than mice treated with Piceatannol, and GS-9973 exhibited the most effective inhibitory effect(Fig. 1I,J).At the same time, the mRNA levels of proinflammatory factors TNFα and IL6 were down-regulated(Fig. 1K,L), and the transcription of IL10 was increased in liver tissue(Fig. 1M).
NETs play a crucial role in regulating the inflammatory response in hepatic IRI.Studies have indicated that SYK phosphorylation at Tyr525/526 is important for the production and function of NETs[17].We collected neutrophils in liver tissue from mice 6h after liver surgery and found increased formation of NETs compared to normal liver tissue(Figure S2A,B).We found that GS-9973 was the most effective in reducing the formation of IR-related NETs in mice compared to the other two SYK inhibitors(Fig. 2A-D).Subsequently,we wondered whether GS-9973 targets and acts on P-SYK localized in neutrophils.Our research indicates that P-SYK is predominantly upregulated in neutrophils in response to liver IR in mice, and this upregulation is significantly inhibited by GS-9973 (Fig. 2E).Expression of P-SYK is also increased in macrophages (Fig. 2E), while no significant expression was observed in hepatocytes(Figure S2C).Furthermore, GS-9973 simultaneously reduces the expression of CXCL1/CXCL2(Figure S1D,E).We therefore wondered whether pharmacological inhibition of SYK suppresses NETs production by affecting neutrophil recruitment.Thus,we intraperitoneally injected SYK inhibitors into mice prior to liver IR, and showed that inhibition of SYK significantly reduced the number of neutrophils in the liver (Fig. 2F,G, H,I,Figure S1F), and the concentration of in vivo dosing affected its recruitment efficacy (Fig. 2K).The level of P-SYK was positively correlated with the level of Ly6G (Figure S1G). We next wondered whether GS-9973 regulates the pathologic process of NETs formation.We pretreated mouse bone marrow neutrophils with three SYK inhibitors in vitro for 2h, followed by stimulation with PMA and LPS. We found a reduction in NETs production compared to the control group, with GS-9973 showing a higher inhibitory effect on NETs (Fig. 2J, L).GS-9973 clearly demonstrated inhibition of SYK activation in vitro (Figure S2D, E). Consistent results were also obtained in human peripheral blood neutrophils (Figure S2F). Therefore, we demonstrate that GS-9973 not only inhibits neutrophil recruitment but also directly acts on neutrophils to regulate NETs formation.
The formation of NETs regulated by SYK depends on PKM2/P-STAT3
STAT3 is a key participant in NETosis induced by liver inflammation, as experiments have shown that STAT3 overexpression or PMA treatment both promote the formation of NETs in the colon cancer-derived neutrophils[28].PKM2 mainly exists in the cytoplasm in the form of a tetramer, which plays a pyruvate kinase activity.When receiving stimulus signals, PKM2 is allosteric transferred to the nucleus in the form of a dimer, which plays a protein kinase activity and regulates the transcription and expression of downstream genes, including proinflammatory genes. PKM2 entering the nucleus can activate STAT3 and promote the inflammatory process[29, 30].We found that nuclear translocation and expression of P-STAT3 in the liver after IR were significantly increased and markedly inhibited by GS-9973(Fig. 3A-C).We then inquired whether the inhibitory effect of GS-9973 on P-STAT3 depends on the inhibition of PKM2 nuclear translocation.It is known that SYK-dependent phosphorylation of PKM2 at Tyr105 and its nuclear translocation regulate CRC-associated MDSCs[31].The expression of PKM2 was significantly up-regulated in neutrophils in response to IRI, and its nuclear translocation was increased. GS-9973 treatment reduced the nuclear expression of PKM2 and inhibited the nuclear translocation of P-STAT3.In addition, confocal immunofluorescence showed that PKM2 and P-STAT3 were co-localized in the nucleus of neutrophils in response to liver inflammation,while GS-9973 inhibited the interaction between PKM2 and P-STAT3 in the nucleus(Fig. 3D-E).To further confirm the interaction between SYK and PKM2, we performed co-immunoprecipitation of neutrophils from patients undergoing hepatic portal occlusion and demonstrated that the stimulation of IRI promoted the interaction of P-SYK with PKM2 (Fig. 3F).TEPP-46, as a known allosteric activator of PKM2, can lead to its tetramerization, block its nuclear translocation,and reduce STAT3 phosphorylation by inhibiting nuclear PKM2 accumulation[32, 33].We observed that neutrophils treated with TEPP-46 detected lower levels of P-STAT3 and NETs production in response to PMA stimulation relative to controls (Fig. 3G,H).Stattic is a potent STAT3 inhibitor known to suppress STAT3 phosphorylation and activation.Treatment with Stattic resulted in a decrease in the production of P-STAT3 and NETs in neutrophils stimulated with PMA(Fig. 3I,J).In vivo experiments also showed that preoperative administration of TEPP-46 and Stattic had a protective effect on liver IRI in mice (Figure S3D,E). Additionally, the combined use of TEPP-46 or Stattic with GS-9973 did not further significantly reduce NETs formation (Figure S3A-C).Therefore, we conclude that GS-9973 regulates NETs production through the PKM2/P-STAT3 pathway, thereby affecting hepatic IRI.
Targeting SYK of monocyte-derived macrophages reduces neutrophil recruitment and IL1β-mediated NETosis.The expression of P-SYK was also upregulated in macrophages in IR livers and was inhibited by GS-9973(Fig.
2E), prompting our interest in understanding the role and mechanism of macrophages in this process.GS-9973 has previously been shown to reduce the recruitment of neutrophils in liver IRI,We wondered about whether GS-9973 influences neutrophil recruitment by regulating SYK in macrophages.We injected siSYK mixed with mannose-conjugated polymer (JetPEI-polyplus) in mice via tail vein before IR to knockdown SYK in liver monocyte-derived macrophages(MoMFs),This method was elaborated in our previous study[
16,
34,
35].We found that targeting of MoMF SYK also reduced the transcription of CXCL1 and CXCL2 and neutrophil recruitment in the liver after IR (Fig.
4A-C).Moreover,we found that the levels of proinflammatory cytokines TNFα, IL6, and IL1β
, especially IL1β, were significantly down-regulated in the liver (Fig.
4D-F), and the level of IL1β in the serum of mice was also significantly down-regulated (Fig.
4G).IL1β is mainly activated by the NLRP3 inflammasome, and studies have shown that macrophage-derived NLRP3 inflammasome activation and IL1β release can induce NETosis[
36].SYK has been shown to activate the NLRP3 inflammasome in many studies.Here,we hypothesized that macrophage SYK may regulate NLRP3 inflammasome activation, affecting the production of IL1β,and thereby regulating NETosis.We found that inhibition of SYK in macrophages significantly suppressed the expression of NLRP3,cleaved Caspase-1,cleaved IL1β in mouse liver (Fig.
4H).Knockdown of SYK in raw264.7 cells resulted in decreased secretion of IL1β in response to LPS in vitro (Figure S3F). Pre-treatment of mice with MCC950 (a NLRP3 inflammasome inhibitor) (20 mg/kg, ip) or Anakinra (an interleukin-1 receptor (IL-1R) antagonist) (20 mg/kg, ip) before IR,alleviated liver injury after IR (Figure S3G, H), reduced the production of NLRP3 inflammasomes (Figure S3I),and diminished NETs formation in the liver (Figure S3J-L).In vitro, BMDMs transfected with siSYK were treated with LPS + ATP, followed by incubation in fresh medium without LPS or ATP for 1 hour to allow the release of pro-inflammatory factors.Then, neutrophils were transferred to the macrophage culture medium.We observed that knocking down SYK in macrophages reduced the NETs formation induced by macrophages. Addition of IL1β (100 ng/ml) (Sigma, I5271-5UG) reversed the inhibitory effect of GS-9973 on NETs formation (Fig.
4I).These findings suggest that GS-9973 can diminish NETs formation by inhibiting SYK-mediated activation of the NLRP3 inflammasome and IL1β in macrophages.
GS-9973 alleviates IR-driven liver tumor recurrence.
We have previously shown that targeting SGK1 alleviates recurrence of liver metastasis after hepatic IR[6].To investigate the effect of pharmacological inhibition of SYK on liver tumor recurrence after IRI, we established standardized mouse models of colorectal liver metastasis(CRLM) and HCC recurrence.Mice were pre-treated with GS-9973 via intraperitoneal injection, followed by splenic injection of MC38 or Hepa1-6 cells 4 hours later.After 15min of circulation, the spleen was ligated and removed, and 70% hepatic ischemia-reperfusion was performed simultaneously.GS-9973 was intraperitoneally injected once 3 days after surgery, and the liver was harvested 2 weeks later.We observed that GS-9973 significantly reduced liver tumor metastasis and recurrence(Fig. 5A), and detected a decrease in NETs formation in liver tissues (Fig. 5C-F), a reduction in the proportion of neutrophils in tumor tissues (Fig. 5B).Furthermore, the phosphorylation levels of SYK and STAT3 in neutrophils were notably downregulated (Fig. 5H, I).Mouse bone marrow neutrophils were pretreated with GS-9973 or DMSO and co-cultured with MC38 or Hepa1-6.The results showed that GS-9973 significantly reduced the formation of NETs in the co-culture system (Fig. 5G).It is known that Tumor-associated neutrophils (TAN) in HCC induce CD4 + T cells to express TGFβ, which promotes Treg cell differentiation and T cell exhaustion, thereby suppressing anti-tumor immune responses[37].We also observed a decrease in the number of Treg cells in tumor tissues after inhibiting SYK(Fig. 5J).Meanwhile,the mRNA and protein levels of TGFβ were significantly down-regulated in liver tumor tissues of GS9973-treated mice (Fig. 5K,L).Therefore, we speculate that inhibiting SYK mitigates liver tumor metastasis by reducing the recruitment of neutrophils and the formation of NETs.Additionally, by downregulating TGFβ, it affects the recruitment of Treg cells in tumor tissue, further reducing tumor metastasis.
Expression of peripheral P-SYK associated with the prognosis of patients after liver surgery.
To explore whether the abundance of SYK can serve as a predictor for the prognosis of patients undergoing partial hepatectomy,we collected peripheral blood samples from patients on the first day after hepatectomy, and measured the mean fluorescence intensity (MFI) of P-SYK in peripheral blood neutrophils by flow cytometry.We found that P-SYK expression in neutrophils was significantly increased postoperatively compared to preoperative levels (Fig. 6A-B).Moreover, the MFI of P-SYK in peripheral blood was positively correlated with the postoperative serum ALT and AST levels(Fig. 6C-D).In summary, our study suggests that SYK in neutrophils and macrophages is a promising target for alleviating liver IR injury and preventing liver tumor recurrence.GS-9973, as a specific small molecule inhibitor of SYK, may provide additional benefits for patients with liver cancer (Fig. 6E).