The Role And Mechanism Of FGL2 On The Development Of Malaria Blood Stage Parasites

Malaria is still one of the most dangerous infectious diseases in the world to human life and health.The blood stage is where the clinical symptoms and death that are characteristic of malaria appear.Therefore,it is always of great importance and interest in uncovering the proliferation and development mechanism of blood stage malaria parasites.An increasing body of evidence suggests that many immune effector molecules,including immune cells and cytokines and antibodies,play an important role in the resistance to malaria blood stage infection.However,during the long run road of the evolution with host,malaria parasites have also developed a series of precise and perfect immune escape and immunosuppression strategies to evade host immune attack.On the one hand,evasion of host immune system attacks can be done by parasites through hide and molecular mimicry and genetic variation and sequestration.On the other hand,parasites are also good at using the host negative regulatory molecules to suppress host immune response.For example,the expression of PD-1on T cells,B cells and NKT cells and CTLA-4 on the surface of activated T cells have been reported to play an important role in malaria blood stage infection.Their activation will lead to inactivation or exhaustion of immune effector cells and promote the proliferation and development of malaria parasites.Fibrinogen-like protein 2,also known as fibroroleukin,was first found in mice infected with murine hepatitis virus?MHV?,including membrane protein form mFGL 2 and secreted form sFGL2.mFGL2 is mainly expressed in endothelial cells,epithelial cells and macrophages.It can directly activate thrombin to initiate coagulation.sFGL2 is mainly secreted by activated regulatory T cells?Treg?.It plays an immunoregulatory role by binding to the cell surface receptor Fc gamma RIIB,including inhibiting DC maturation followed by impaired T lymphocyte proliferation and cytokine secretion,and promoting B cell apoptosis.The immunosuppressive effect of sFGL2 has been widely confirmed in tumor and some inflammatory diseases in recent years.However,there is no report on the role of sFGL2 in malaria infection.In this study,we first try to detect the expression of sFGL2 in serum of Plamodium falciparum and Plamodium vivax malaria patients and mice infected with Plasmodium chabaudi to observed whether the expression of sFGL2 was up-regulated during malaria blood stage infection.Then,we intend to conduct blood stage infection in FGL2 deficient mice to observe whether the development of blood stage parasites has been affected or not after FGL2 deletion,and to clarify the role of FGL2 in malaria blood stage infection.Finally,the molecular mechanism of FGL2 deletion affecting the proliferation of blood stage parasites will be intensively investigated.1.sFGL2 was significantly up-regulated in malaria blood stage infection1.1.The expression of sFGL2 was significantly up-regulated in blood stage of malaria infection.By detecting the expression of sFGL2 in serum of Plamodium falciparum and Plamodium vivax malaria patients and mice infected with Plasmodium chabaudi,we found that the expression of sFGL2 in the serum of P.falciparum and P.vivax patients was significantly higher than that of the normal people in the malaria epidemic area.This result was reproduced in P.chabaudi infected mice.Moreover,the expression level of sFGL2 was strongly correlated with parasitemia in P.chabaudi infected mice.1.2.Treg cells are the main source of sFGL2 which expression level was up-regulated in malaria blood stage infection.Firstly,FACS was used to detect the frequency of splenic CD4⁺CD25⁺Foxp3⁺Treg cells on the 2,4,6 and 8 days post infection.We found that Treg cells frequency increased along with the proliferation of blood stage parasites.Then,CD4^-CD25^-T cells,CD4⁺CD25^-T cells and CD4⁺CD25⁺T cells were separated by flow cytometry,and the expression of FGL2 mRNA was detected by RT-PCR in different groups of cells.The result showed that compared with CD4^-CD25^-T cells and CD4⁺CD25^-T cells,CD4⁺CD25⁺T cells had a much higher expression of FGL2 mRNA which finally confirmed that CD4⁺CD25⁺Treg cells were the main source of sFGL2 in malaria blood stage infection.2.The development of malaria blood stage parasites was significantly inhibited after FGL2 deletion2.1.The proliferation of parasites in FGL2^-/-infected mice was significantly inhibited.Subsequently,FGL2 knockout mice were infected with Plasmodium chabaudi AS,Plasmodium berghei ANKA and Plasmodium yoelii BY265 of blood stage parasites.The results showed that the development of blood stage parasites in FGL2-deficient mice was attenuated when compared to WT mice regardless of different strains of Plasmodium infection.The parasitemia level was comparable between WT and FGL2^-/-mice when FGL2^-/-mice supplemented with recombinant FGL2 protein.These results strongly suggested that FGL2 plays an important role in malaria blood stage infection,and may contribute to immune evasion of blood stage parasites and promote its development.2.2.The coagulation function of FGL2^-/-mice was not significantly affected.FGL2knockout would lead to the deletion of sFGL2 and mFGL2 at the same time,while the deletion of mFGL 2 might lead to abnormal coagulation function.Therefore,we detected the bleeding time and clotting time of WT and FGL2^-/-mice,and the results showed that the coagulation function of FGL2-/-mice was not significantly affected.3.FGL2 deletion did not enhance the adaptive immunity against malaria blood stage infection3.1 The deletion of FGL2 did not affect the proliferation and function of malaria-specific CD8⁺T cells.The proliferation and function of malaria-specific CD8⁺T cells in spleen of FGL2 knockout mice were analyzed by flow cytometry on the 7,14 and 21 days after infection.The results showed that there was no significant difference between FGL2knockout mice and WT-infected mice.Similarly,FGL2^-/-infected mice had a comparable production of granzyme,perforin and IFN-gamma with WT infected mice.3.2 The deletion of FGL2 did not affect the proliferation and function of malaria-specific CD4⁺T cells.Flow cytometry was used to analyze the activation of malaria-specific CD4⁺T cells on the 7,14 and 21 day after infection.The results showed that there was no significant difference in activation,proliferation and IFN-gamma secretion between FGL2 knockout mice and WT-infected mice on the 7,14 and 21 day after infection.3.3 The deletion of FGL2 did not affect the frequency of GC B cells.In view of the important role of GC B cells in secretion of malaria-specific antibodies against blood stage infection,we detected the frequency and number of splenic GC B cells?B220⁺CD95⁺GL7⁺?in WT and FGL2^-/-infected mice,and found no significant difference between the two groups.3.4 The deletion of FGL2 did not affect the production of malaria-specific antibodies.Since malaria-specific antibodies played a key role in eliminating the blood stage parasites,we detected the titers of malaria-specific antibody IgG and its subclasses IgG1 and IgG2a in the sera of WT and FGL2^-/-infected mice.The results also showed that there was no significant difference between the two groups of infected mice.4.FGL2 deletion significantly enhanced innate immunity against malaria blood stage infection4.1 The significant increase of IFN-gamma expression in FGL2-deficient mice was the main reason of impaired development of blood stage parasites.Since the proliferation of parasites in FGL2-knockout mice was significantly inhibited on the 4 days after infection,we detected the expression of IFN-gamma in serum of WT and FGL2^-/-infected mice on the 2,4and 6 days after infection.The results showed that the expression level of IFN-gamma in FGL2-deficient mice was significantly higher than that in WT mice on the 6 days after infection.To further confirm the role of IFN-gamma in inhibiting the proliferation of blood stage parasites in FGL2 knockout mice,we neutralized IFN-gamma with antagonistic antibody.Compared with FGL2^-/-and isotype IgG groups,the level of parasitemia in FGL2knockout mice restored to WT-infected mice after neutralization of IFN-gamma,indicating that FGL2 knockout mice inhibited the development of blood stage parasites by regulating secretion of IFN-gamma.4.2 NK and NKT cells are the main sources of up-regulated IFN-gamma expression in FGL2^-/-mice serum.We examined the ability and number of spleen NK and NKT cells to secrete IFN-gamma on day 2,4 and 6 after infection.The results showed that the numbers of NK and NKT cells secreting IFN-gamma in spleen of FGL2^-/-mice were significantly higher than those of WT mice on 6 days after infection.In order to further confirm that enhanced NK/NKT secretion of IFN-gamma may help FGL2^-/-mice inhibit the proliferation of blood stage parasites,WT and FGL2^-/-mice were specifically depleted of NK cells and NKT cells with anti-NK1.1 antibody before infection.The results showed that after depletion of NK/NKT cells in FGL2^-/-mice,the level of parasitemia was significantly increased when compared to those FGL2^-/-mice without anti-NK1.1 antibody injection.Therefore,the significant loss of parasites burden in FGL2-/-infected mice was due to the increased secretion of IFN-gamma by NK/NKT cells.4.3 The absence of FGL2 increased the expression of MCP-1 in infected mice,which was the main reason for the increased number of NK/NKT cell.We compared the expression of MCP-1 in serum of WT and FGL2^-/-mice at 2,4 and 6 days after infection,and found that the expression of MCP-1 in FGL2^-/-mice was significantly higher than that in WT mice at 6days post infection.In order to further verify the important role of MCP-1,we used MCP-1antagonistic antibody.The results showed that the absence of MCP-1 completely blocked the recruitment of splenic NK and NKT cells in FGL2^-/-mice.Moreover,after MCP-1 depletion,FGL2^-/-mice had a comparable parasitemia with WT mice.These results suggest that the recruitment of NK/NKT cell mediated by MCP-1 may play an important role in inhibiting the development of blood stage parasites in FGL2^-/-mice.4.4 Macrophages are the main source of up-regulated MCP-1 expression in FGL2^-/-mice serum.Next,we investigated the source of up-regulated MCP-1 cells in FGL2^-/-mice serum.It has been confirmed that MCP-1 is mainly produced by macrophages,so we compared the ability of WT and FGL2^-/-mouse macrophages to produce MCP-1.It was found that the ability of macrophages in FGL2^-/-mice to produce MCP-1 was significantly enhanced when compared to WT mice.Further studies showed that the total number of macrophages secreting MCP-1 in the spleen of FGL2^-/-mice was much higher than that of WT mice.This strongly suggests that the generated MCP-1 has a positive feedback effect on the recruitment of more macrophages to the spleen,thereby promoting the production of MCP-1.5.The molecular mechanism of enhanced innate immunity against malaria blood stage infection in FGL2^-/-mice5.1 sFGL2 can directly inhibit the ability of macrophages to produce MCP-1.The results from FGL2-deficient mice showed that the presence of FGL2 inhibited the ability of macrophages to secrete MCP-1,but there was no direct experimental evidence showed that sFGL2 could directly inhibit the production of MCP-1 by macrophages.Therefore,we added the recombinant mouse FGL2 protein?rFGL2?to RAW264.7 cell line and mouse bone marrow-derived macrophages?BMDM?which were stimulated by pRBC.The expression of MCP-1 was detected by ELISA.The results showed that both RAW264.7 cell line and BMDM had a impaired MCP-1 production after pRBC stimulation with the presence of rFGL2.These results indicated that sFGL2 can directly act on macrophages to inhibit the production of MCP-1.5.2 The ability of sFGL2 to inhibit the production of MCP-1 by macrophages through Fc-gamma RIIB receptor.Fc-gamma RIIB,which is known to be expressed on the surface of many immune cells,is a receptor for sFGL2,and Fc-gamma RIIB receptor also exist on the surface of macrophage.Therefore,we added Fc-gamma RIIB antagonist antibodies in RAW264.7 cells with the presence of pRBC and rFGL2.The results showed that the inhibitory effect of sFGL2 was completely abolished,and the expression of MCP-1 in the well co-incubated with anti-Fc-gamma RIIB antibodies and pRBC and rFGL2 returned to the level of well which had pRBC stimulation alone.This result was confirmed in BMDM.These results demonstrated that up-regulated expression of sFGL2 in blood stage infection plays an inhibitory role through Fc-gamma RIIB receptor.5.3 The inhibitory effect of sFGL2 disappeared in BMDM of mice with macrophage conditioned deletion of Fc-gamma RIIB receptor,and the development of blood stage parasites in mice with macrophage conditioned deletion of Fc-gamma RIIB receptor was significantly inhibited.Subsequently,we constructed mice with macrophage conditioned deletion of Fc-gamma RIIB receptor to verify the inhibitory effect of sFGL2-Fc-gamma RIIB signal.We isolated BMDM from mice with macrophage conditionally deleted Fc-gamma RIIB receptor and stimulated with pRBC and rFGL2.ELISA detection showed that the expression of MCP-1 was not different in the well with or without the presence of rFGL2.This result confirmed that sFGL2 inhibit macrophage to secrete MCP-1 by binding Fc-gamma RIIB receptor.In addition,we also conducted blood stage infection in macrophage Fc-gamma RIIB conditional knockout mice.The results showed that the level of parasitemia in macrophage Fc-gamma RIIB conditional knockout mice was significantly lower than that of WT-control mice during the course of infection.Collectively,our data provide solid evidences that sFGL2 exerted inhibitory effect through Fc-gamma RIIB receptor.5.4 Molecular mechanism of sFGL2-Fc-gamma RIIB signal in inhibiting MCP-1production by macrophages.Firstly,we investigated the molecular mechanism of MCP-1production by macrophages.It is known that intracellular NF-kappa B and MAPK signal pathways are related to MCP-1 production,and the downstream of MAPK signal pathway contains three branches,including ERK,p38 and JNK.By adding a variety of signal molecular inhibitors,including NF-kappa B inhibitor BAY 11-7082,ERK inhibitor SCH772984,p38 inhibitor SB203580 and JNK inhibitor SP600125,to pRBC-stimulated RAW264.7 cells,we found that the addition of these signal inhibitors,except p38,partially blocked the production of MCP-1.This result indicated that NF-kappa B and MAPK signal pathways are both involved in the production of MCP-1 by macrophage induced by pRBC.Next,the activation of signal molecular TAK1 which shared by both signal pathway,and I?B?which belong to the downstream of NF-kappa B signal pathway,and MKK and JNK which belong to the downstream of MAPK signal pathway will be investigated.The activation of these signal molecules under pRBC stimulation with or without the presence of rFGL2 will be detected by Western blot to determine the site and pathway which influenced by sFGL2-Fc gamma RIIB signaling.The results will also be validated in the BMDM of Fc?RIIB^flox/flox Lyz-Cre mice.We first observed the activation of MKK4/7-JNK pathway.The results showed that sFGL2-Fc gamma RIIB signal had no significant effect on the activation of MKK4/7,but could significantly inhibit the activation of JNK,suggesting that sFGL2-Fc gamma RIIB signal might act on the JNK pathway which belongs to the MAPK pathway.However,the identification of whether sFGL2-Fc gamma RIIB signal affects another NF-kappa B pathway or not is still in progress and has not yet been completed.