Mechanism Of Ranitidine As An Adjuvant To Regulate Macrophages Polarization And Activate Anti-tumor Immunity

Objective: The immune escape mechanism of tumor cells leads to the immune tolerance of the body to tumor.Although there are many immunotherapy regiments used alone or in combination with conventional clinical methods,they are not effective in finding related antigens and actively activating the body’s own immunity.Adjuvants can enhance the immunogenicity of antigens,increase the duration of immune response,and promote the production of immune memory,thus improving the quality of immune response and increasing the protective efficacy of vaccines.They have become an indispensable component in modern vaccine research and development.Finding effective and safe adjuvants for tumor vaccines plays a key role in breaking the immune tolerance of tumors and promoting the immune clearance of tumors.In the screening process of adjuvants,we found that H2 receptor blockers,which inhibit gastric acid secretion,have a general immune enhancement effect.Therefore,Ranitidine(RAN),with good water solubility,became the first choice of adjuvant drugs in this study.Through this study,we will clarify that RAN as an adjuvant can enhance the killing function of T cells to tumors by promoting macrophage antigen phagocytosis and M1 polarization,providing a solid experimental basis for the research and development of tumor vaccines based on RAN as an adjuvant,and also provide a new idea for the research of H2 receptor blockers as adjuvants.Methods:1.Flow cytometry was used to detect the effect of adding RAN to the model antigen ovalbumin(OVA)on the expression levels of the M1 surface marker CD86,Major Histocompatibility complex I(MHC I)and Major Histocompatibility complex II(MHC II)in RAW264.7 cells,and to detect the secretion level of M1 related cytokine: TNF-α,IL-12p40,IL-12p70,IL1-β,CXCL1.Flow cytometry and immunofluorescence techniques were used to observe the effect of RAN on the endocytosis of OVA antigen in RAW264.7 cells,as well as the co localization of the precursor protein Rab5 with OVA antigen.2.Western Blot was used to detect the activation of PI3K-AKT2 pathway when RAN promoted RAW264.7 cells to phagocytosis antigen(OVA)and polarized to M1.Western blot,flow cytometry,and immunofluorescence techniques were used todetect the effect of RAN+OVA co stimulation on M1 type cytokine secretion and endocytosis of OVA antigen in RAW264.7 cells after inhibiting the PI3K-AKT2 signaling pathway.The phosphorylation levels of NF-κB and GSK3β / Dynamin1 signal related proteins were detected by Western blot after inhibiting of PI3K-AKT2 signaling pathway.3.In vivo,RAN+OVA antigen was used as adjuvant vaccine in the experimental group.Flow cytometry was used to analyze the expression levels of CD80,CD86,MHC Ⅰ and MHC Ⅱ in immunized mice macrophage.The expression levels of p-STAT1/STAT1 and p-STAT4/STAT4 in spleen cells of mice after three days of immunization were detected by Western blot assay.4.The levels of related cytokines secreted by Th1 cells,Treg cells and CD8+T cells in spleen cells of mice co-immunized with RAN as adjuvant and OVA antigen were detected by flow cytometry.5.Seven days after the second immunization with RAN+OVA antigen,the mice were subcutaneously injected with melanoma cells(chicken OVA gene modified)B16(B16-OVA)to establish melanoma bearing mice model.Recorded the tumor growth size and survival rate,and evaluate the anti-tumor effect of RAN as an adjuvant.6.Seven days after the second immunization with RAN+OVA antigen,mouse splenocytes were co-cultured with B16-OVA tumor cells in vitro,and the killing rate in vitro of splenocytes was detected by flow cytometry,and the tumor growth size and survival rate were recorded to evaluate whether RAN as an adjuvant inhibited tumor growth by enhancing CTL function in unimmunized tumor-bearing mice.Results:1.In vitro cell experiments showed that the average fluorescence intensity of CD86,MHC Ⅰ and MHC Ⅱ markers were significantly up-regulated after RAN plus OVA co-stimulated macrophages,and the levels of CXCL1,IL1-β,TNF-α,IL12p40 and IL12p70 cytokines in the supernatant of macrophages were all up-regulated.It significantly promoted the M1 polarization of RAW264.7 cells.Flow cytometry and immunofluorescence showed that the co-localization of OVA antigen with Rab5 was significantly enhanced under the stimulation of RAN,that is,the endocytosis of macrophages was enhanced.2.Western blot analysis showed that RAN+OVA significantly promoted the phosphorylation levels of PI3 K and AKT2 in macrophages.Western blot and flow cytometry analysis showed that inhibition of the PI3 K pathway blocked the activation of macrophage PI3K/AKT signaling pathway induced by RAN+OVA,inhibited thesecretion of M1 type related inflammatory factors and antigen endocytosis,confirming that RAN+OVA promotes macrophage M1 polarization and antigen phagocytosis through the PI3K/AKT signaling pathway.Western blot detection showed that RAN+OVA enhanced phosphorylation level of NF-κB p65 subunit and promoted GSK3β Ser9 phosphorylation and downregulated the phosphorylation of Dynmamin1.After inhibiting the PI3K-AKT signaling pathway,there was no statistical difference between the drug treatment groups.It was suggested that after activating PI3K/AKT signaling pathway,RAN+OVA could induce macrophage polarization to M1 and enhance macrophage endocytosis by regulating NF-κB and GSK3β / Dynamin1 pathway respectively.3.After co-immunization of mice with RAN as an adjuvant and OVA antigen for 3days,the average fluorescence intensity of M1 type macrophage surface markers CD80,CD86,MHC I,and MHC II in spleen cells was significantly upregulated,indicating that RAN as an adjuvant could promote the polarization of macrophages towards M1 in mice.Western blot analysis showed that RAN,as an adjuvant,could significantly upregulate the phosphorylation levels of STAT1 and STAT4 in mouse spleen cells.4.Flow cytometry further confirmed that RAN could enhance the expression levels of Th1 cytokines IFN-γ and TNF-α,and inhibit the secretion of anti-inflammatory cytokines IL-10 and TGF-β in vivo.Meanwhile,the expression levels of CD8+T cells IFN-γ,Granzyme B and TNF-α cytokines were significantly up-regulated.5.The tumor growth curve and survival curve indicated that the RAN+OVA antigen immunized mice exhibit slowly tumor growth and highly survival rate.6.The results of killing experiment in vitro showed that the function of CTL could be enhanced by co-culture of immunized spleen cells and tumor cells in vitroThrough the B16-OVA tumor-bearing experiment on the immunized mice,the tumor growth curve and survival curve were recorded.It was found that the tumor growth of the mice immunized with RAN+OVA antigen was slow and the survival rate was high.6.The results of killing experiment in vitro showed that the function of CTL could be enhanced by co-culture of immunized spleen cells and tumor cells in vitro.The immunized CD8+T cells were infused back into the unimmunized tumor-bearing mice,and the tumor growth was observed and the survival curve was recorded.Conclusion: This study demonstrated that RAN as an adjuvant of tumor vaccine had the effect of preventing tumor growth in B16-OVA tumor-bearing mice.Itsmechanism may be related to RAN activating PI3K-AKT2 signaling pathway,promoting M1 polarization and antigen endocytosis of macrophages,and thus activating the body’s CTL function to play a role in tumor killing and effectively improve the differentiation of Th1 cells and the expression of CD8+T cells killer cytokines.This new discovery may lay a theoretical foundation for the application of RAN as a vaccine adjuvant in anti-tumor immunotherapy.