| Backgrounds:Hepatocellular carcinoma(HCC)is a kind of primary liver cancer with high mortality.It is one of the most common malignant tumors in the world,especially in Asia,Africa and southern Europe.Especially in China,the incidence rate of liver cancer ranks highest in the world.At present,transplantation is the most effective method for the treatment of liver cancer,but the tumor recurrence rate and metastasis rate are high after transplantation.Therefore,it is urgent to find a new treatment.In recent years,tumor immunotherapy has made great breakthrough.The blocking therapy of immune checkpoint(PD-L1/PD-1,CTLA4/CD28)and adoptive immunotherapy of CRR-T cells have been applied in clinic with great success.However,these immunotherapies are not perfect.Blocking the immune checkpoint only works in about 30%of cancer patients.As for CAR-T,tumor cells of patients must express specific tumor antigen.Therefore,it is necessary to study the anti-tumor effect of other immune cells.Activated macrophages have anti-tumor effect.It will be a new strategy to develop their application in tumor immunotherapy.As an important member of the innate immune system,macrophages have the function of clearing foreign bodies,necrotic cells and organelles in the body,and can be differentiated into anti-tumor cells by appropriate stimulation.One of the mechanisms of anti-tumor of macrophages is the phagocytosis of tumor cells,which depends on the balance of anti phagocytic signal and pro phagocytic signal.Calreticulin(CRT)is highly expressed in tumor cell membrane,and activated macrophages express the receptor of CAR-T,LRP1.The specific binding of LRP1 with CRT can promote phagocytosis of macrophages to tumor cells;Tumor cells tend to overexpress CD47,which can interact with SIRPα,a receptor expressed by macrophages.In addition,it can also inhibit the phagocytosis of macrophages.According to this mechanism,we adopt the strategy of targeting CD47 or SIRPa to block the immune checkpoint CD47/SIRPα.This kind of treatment of cancer has entered the stage of clinical trials.We wonder whether the phagocytic function of macrophages will also be enhanced under the stimulation of cGAS-STING agonist,in addition to the increased expression level of type I interferon.If cGAS-STING agonist can indeed enhance the phagocytic function of macrophages,how can it be enhanced,and whether this enhancement is dependent of IFN-β?So we explored the effect and mechanism of cGAS-STING agonist on macrophage phagocytosis.After the experiments,we found that cGAS-STING agonist can effectively enhance the phagocytosis of macrophages,and this enhancement does not depend on IFN-β,but is related to the signal regulatory protein SIRPa on the surface of macrophages.SIRPa can bind to CD47 highly expressed on the surface of cancer cells.CD47 is the don’t eat me signal of cancer cells,and the combination of CD47 and SIRPa will inhibit the phagocytosis.Therefore,cGAS-STING agonist can enhance the phagocytic function by down regulating the expression of SIRPα.This paper first revealed the relationship between the activation of cGAS-STING pathway and phagocytic function of macrophages,provided a potential adoptive immunotherapy,which may be significant in the clinical treatment of hepatocarcinoma.ObjectiveThe aim of this study is to investigate the effect of activation of cGAS-STING pathway on macrophage phagocytosis and its mechanismMethods and results(Ⅰ)ISD and 2’3’-cGAMP activate cGAS-STING signaling pathway in macrophages1.ISD and 2’3’-cGAMP promot the expression of IFN-β and MX1 genes in THP-1 and BMDM cellsTwo kinds of cGAS agonists,ISD and 2’3’-cGAMP,were used to stimulate THP-1 cells(treated with PMA)and BMDM cells.After 4 hours,the RNA was collected and the cDNA was obtained by reverse transcription.The expression levels of IFN-βand MX1 genes were detected by RT-PCR.The results showed that ISD and 2’3’-cGAMP could significantly increase the expression of IFN-β and MX1 in THP-1 and BMDM cells.2.ISD and 2’3’-cGAMP promot the expression of p-IRF3 in THP-1 cellsTwo kinds of cGAS agonists,ISD and 2’3’-cGAMP,were used to stimulate THP-1 cells(treated with PMA).After 4 hours,the protein was harvested,and the change of p-IRF3 at protein level was detected by Western blot.The results showed that ISD and 2’3’-cGAMP could significantly increase the expression of p-IRF3 of THP-1 cells.(Ⅱ)ISD and 2’3’-cGAMP enhance macrophage phagocytosis of hepatoma cells1.ISD and 2’3’-cGAMP enhance the phagocytosis of HCCLM3 by THP-1The THP-1 cells treated by PMA were stimulated by ISD and 2’3’-cGAMP for 24 hours.The stimulated THP-1 cells and HCCLM3 cells were cultured in serum-free medium for 2 hours in a ratio of 1:2.The proportion of macrophages engulfed by HCC cells to total macrophages was measured by flow cytometry.The results showed that ISD and 2’3’-cGAMP could significantly enhance the phagocytosis of THP-1.2.ISD and 2’3’-cGAMP enhance the phagocytosis of HCCLM3 by BMDMThe differentiated BMDM cells were stimulated by ISD and 2’3’-cGAMP for 24 hours.The macrophages and HCCLM3 were cultured in serum-free medium for 2 hours in a ratio of 1:2.The proportion of phagocytes engulfed by hepatoma cells to total macrophages was detected by flow cytometry and double fluorescence labeling.The results showed that both ISD and 2’3’-cGAMP could significantly enhance the phagocytosis of BMDM.(Ⅲ)The enhancement of phagocytosis is not dependent on IFN-β1.ODN 2216 stimulates IFN-β production in macrophagesThe THP-1 cells and BMDM cells were stimulated by TLR9 agonist ODN 2216 RNA was collected 24 hours later,and cDNA was obtained by reverse transcription and RT-PCR.The results showed that ODN 2216 significantly increased the expression of IFN-β and MX1 in THP-1 and BMDM.2.IFN β has no influnce on the phagocytosis of HCCLM3 by THP-1The THP-1 cells treated by PMA were stimulated by IFN-β for 24 hours.The stimulated THP-1 cells and HCCLM3 cells were cultured in serum-free medium for 2 hours in a ratio of 1:2.The proportion of macrophages engulfed by hepatoma cells in total macrophages was measured by flow cytometry.The results showed that IFN-βstimulation reduced the phagocytic function of THP-1 cells,which indicated that the enhancement of phagocytic capacity of cGAS-STING agonists was not related to the IFN β produced downstream3.IFN β has no influnce on the phagocytosis of HCCLM3 by BMDMIFN β was used to stimulate the differentiated BMDM cells for 24 hours.The macrophages and HCCLM3 were cultured in serum-free medium for 2 hours in 1:2 ratio.The proportion of macrophages engulfed by hepatoma cells in total macrophages was detected by flow cytometry and double fluorescence labeling.The results showed that IFN β stimulation had no effect on the phagocytosis of BMDM,which further indicated that the enhancement of phagocytosis of macrophages after activation of cGAS-STING pathway was not related to IFN β.4.ODN 2216 has no influnce on the phagocytosis of HCCLM3 by THP-1The THP-1 cells treated by PMA were stimulated by ODN 2216 for 24 hours.The THP-1 cells and HCCLM3 cells were cultured in serum-free medium for 2 hours in a ratio of 1:2.The proportion of macrophages engulfed by hepatoma cells in total macrophages was detected by flow cytometry.The results showed that ODN 2216 had no effect on THP-1 phagocytosis,which further showed that the enhancement of THP-1 phagocytosis was not caused by IFN-β,but related to the activation of cGAS-STING pathway itself.5.ODN 2216 has no influnce on the phagocytosis of HCCLM3 by BMDMThe differentiated BMDM was stimulated by ODN 2216 for 24 hours.The macrophages and HCCLM3 were cultured in serum-free medium for 2 hours in 1:2 ratio.The proportion of macrophages engulfed by HCC cells in total macrophages was detected by flow cytometry and double fluorescence labeling.The results showed that ODN 2216 stimulation had no effect on BMDM phagocytosis(Ⅳ)cGAS-STING agonist enhances the phagocytosis of HCCLM3 by macrophages through CD47-SIRPa axis1.cGAS-STING agonist enhances the phagocytosis through inhibiting the CD47/SIRPa axis1)The enhancement of phagocytosis is not related to LRP1 on the surface of macrophagesTHP-1 cells and BMDM cells treated with PMA were stimulated with ISD and 2’3’-cGAMP.After 24 hours,RNA was collected and cDNA was obtained by reverse transcription.The expression of LRP1 gene was detected by RT-PCR.The results showed that the LRP1 levels of THP-1 and BMDM did not change significantly after ISD and 2’3’-cGAMP stimulation.This suggests that the enhancement of phagocytic capacity of macrophages by cGAS agonists is independent of LRP12)The enhancement of phagocytosis is related to SIRP α on the surface of macrophagesThe THP-1 cells and BMDM cells treated with PMA were stimulated with ISD and 2’3’一cGAMP.After 24 hours,the RNA was collected and the cDNA was obtained by reverse transcription.The expression of SIRP a gene was detected by RT-PCR.The results showed that ISD and 2’3’-cGAMP could significantly down regulate SIRP αmRNA expression in THP-1 and BMDM cells.This suggests that cGAS agonists may regulate macrophage phagocytosis through SIRP α-CD47 axis.2.The enhancement of phagocytosis of HCCLM3 by macrophages depends on the CD47/SIRPa axis1)Construction of CD47 knockout cell lineThe CD47 knockout HCCLM3 cell line was constructed by crispr-cas9 technique,and the CD47 knockout efficiency was detected by flow cytometry and Western blot.2)cGAS-STING agonist could not enhance the phagocytosis of CD47 knockout HCCLM3 by THP-1THP-1 cells were stimulated with ISD and 2’3’-cGAMP for 24 hours,and then incubated in serum-free medium with HCCLM3 and cd47ko cells at the ratio of 1:2 for 2 hours.The percentage of macrophages phagocytizing hepatoma cells was detected by flow cytometry.The results showed that the phagocytic rate of THP-1 cells stimulated by ISD and 2’3’-cGAMP in HCCLM3 was significantly higher than that in the control group,but there was no significant difference in the phagocytic rate between the control group and the stimulation group in HCCLM3 with cd47ko,which indicated that the enhancement of phagocytic function of macrophages by cGAS agonist really depended on the combination of CD47 and SIRP α.3)cGAS-STING agonist could not enhance the phagocytosis of CD47 knockout HCCLM3 by BMDMBMDM cells were stimulated with ISD and 2’3’-cGAMP for 24 hours.The stimulated macrophages were cultured in serum-free medium with HCCLM3 and HCCLM3 of cd47ko at the ratio of 1:2 for 2 hours.The ratio of macrophages phagocytizing hepatoma cells to total macrophages was detected by flow cytometry and double fluorescent labeling.The results showed that the enhanced phagocytosis of BMDM only occurred in HCCLM3 of wild type,but not in HCCLM3 of cd47ko.This further shows that the enhancement of phagocytic function of macrophages by cGAS agonists is indeed dependent on the combination of CD47 and SIRP α.Conclusion1.cGAS-STING agonist enhances the phagocytosis of HCCLM3 by THP-1 and BMDM cells;2.cGAS-STING agonist enhances the phagocytosis of HCCLM3 by THP-1 and BMDM through CD47/SIRPa axis;3.cGAS-STING agonist enhances phagocytosis of macrophages,and it is independent of IFN-β.Innovation and significance1.We found that cGAS-STING signaling pathway can enhance the phagocytosis of HCCLM3 by macrophages thruough CD47-SIRPα axis;2.Our research may provide a new method for tumor treatment. |
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