The Role And Mechanism Of FGL2 In Malaria Infection

Malaria remains one of the global public health problems that seriously threaten human life and health.Malaria mainly occurs in the world's tropical and subtropical regions,which is an important cause of morbidity and mortality in sub Saharan Africa.In a previous report of WHO,200 million cases and more than 400 thousand people died of malaria in 2019?WHO,2019?.The life cycle of malaria includes mosquito stage,liver stage and blood stage.During the two stages of development of Plasmodium in human,some pattern recognition receptors of host immune system can sense the metabolic components of Plasmodium and make corresponding immune response.During the development of liver stage,the signal pathway MDA5-MAVS-IRF3/IRF7 in hepatocytes can recognize Plasmodium RNA and trigger the immune response of type I interferon,thereby killing liver stage Plasmodium by recruiting and inducing NK/NKT cells to secrete IFN-?.In blood stage,both the innate immunity and adaptive immunity can be activated effectively to cope with the proliferation of parasites.In the early stage of infection,dendritic cells?DCs?and macrophages can quickly recognize the invading Plasmodium through TLR2/4 or TLR9receptors,release pro-inflammatory factors,and enhance the killing effect of DCs and macrophages on the intracellular parasites,thereby controlling the proliferation of Plasmodium in the early stage.In the late stage of infection,the activated DCs can prime the adaptive immune response and enhance the clearance of the parasites.For instance,the Plasmodium specific antibody can prevent the schizonts from invading the red blood cells,and enhance the phagocytic capacity of phagocytes through opsonization.CD4⁺T cells kill blood stage parasites by secreting inflammatory factors such as IFN-?and TNF-?,and can effectively assist B cells to produce humoral immune response.CD8⁺T cells produce perforin and granzyme B and other effector molecules,which play crucial roles in the elimination of Plasmodium parasites and control of chronic infection.However,both liver stage and blood stage parasites can still survive and continue to proliferate to complete the life cycle under the host's great degree of immune attack.The main reason is that a series of immune escape or inhibition strategies have been developed in the long-term evolution of Plasmodium.In liver stage,the sporozoites have motility ability to break through the mechanical barrier of the skin and enter the liver by the help of SPECT-1,SPECT-2 and thrombospondin related anonymous protein?TRAP?;have cell traversal?CT?ability to escape phagocytosis through Kupffer cell?KC?,and can regulate the cytokines expression profile of KC,resulting in down-regulation of Th1 and up-regulation of Th2cytokines to ensure safe passage.In addition,circumsporozoite protein?CSP?can interact with low-density lipoprotein receptor related protein?LRP-1?and proteoglycan on the surface of KC,increasing the level of cAMP/EPAC and preventing the formation of ROS.Sporozoites can also induce the apoptosis of KC,or inhibit the antigen-presenting ability of KC by down regulating the expression level of MHC-I molecules and costimulatory molecules on the surface of KC;After cell traversal,the sporozoites can recognize heparin sulfate proteoglycans?HSPGs?,which is the receptor on the surface of hepatocytes,and activate the invasion signal.The protein p52 and p36 released by the microneme can interact with EphA2 receptor of hepatocytes,which is the important to the formation of parasitophorous vacuole?PV?;After the invasion of hepatocytes,the sporozoites are hidden in PV surrounded by the hepatocyte-derived paracitophorus vacuole membrane?PVM?,and the putative dense granule proteins UIS3 and UIS4 are released and transported to PVM.UIS3can help the sporozoites escape from autophagy of hepatocytes.In blood stage,the immune escape mechanisms of Plasmodium are more diverse than in liver stage.On the one hand,blood stage parasites can hide itself from host immune recognition by many means.For example,intracellular parasitism is the most primitive but effective way to hide,which can isolate Plasmodium from host immune cells.Secondly,the blood stage parasites can express the molecule of PfEMP-1 on the surface of infected red blood cells.By binding with the host vascular endothelial cell surface related receptor such as ICAM-1 or CD36,it adheres to the important organs of the host to escape the immune surveillance of the spleen;in addition,the infected red blood cells can"gather"normal red blood cells and platelets to form Rosetting and Clumping respectively to avoid recognition and killing of immune cells.Finally,the blood stage parasites can evade the immune attack by blocking the binding of specific antibodies of Plasmodium through competitive antigen domain,and weakening the immunogenicity of Plasmodium protein through molecular simulation.In different stages of blood infection,Plasmodium expresses variable antigen proteins through var gene,which helps them camouflage in the host immune system.On the other hand,blood stage parasites are very good at manipulating the host immune functional elements.For example,regulatory cells?Treg?with strong immunoregulatory effect,studies from human malaria show that the number of Treg cells in patients will increase continuously due to infection,and the lower Treg cell frequency is related to less parasite load and more favorable disease outcome.Longitudinal studies show that the Treg cell frequency of clinical malaria patients is higher than that of preinfection or recovery patients,and the higher Treg cell frequency before infection is related to the increased risk of severe malaria.In addition,PD-1,which is widely expressed on the surface of DC cells,B cells and T cells,is another negative regulatory molecule with immunosuppressive effect.It is also often used by blood stage parasites to inhibit the antimalaria immune response.Studies from human malaria have shown that the high expression of PD-1 in CD4⁺T cells will lead to their exhaustion.In rodent malaria,it was found that the function of CD4⁺T cells was significantly enhanced after PD-1 was blocked and the blood stage infection could be eliminated rapidly.In addition,PD-1 is also involved in the exhaustion of B cells by blood stage parasites,because it is found that blocking PD-1 can significantly increase the number of CD4⁺Tfh and germinal center B cells?GC B?and produce higher titers of Plasmodium specific antibodies.Recently discovered fibrinogen like protein 2?FGL2?is another negative regulatory molecule with strong immunosuppressive ability.More and more evidences show that FGL2is involved in the pathogenesis of various diseases,such as pregnancy failure,tumor growth,virus infection,allograft rejection and autoimmune diseases.FGL2 has two different structures:membrane-bound FGL2?mFGL2?and soluble FGL2?sFGL2?.mFGL2 is mainly expressed on the surface of macrophages and endothelial cells and is a direct prothrombin with serine protease activity.mFGL2 can cut prothrombin into thrombin through a non-classical way,so as to play a role in promoting coagulation in immune-related coagulation.In contrast,sFGL2 is mainly expressed by CD4⁺CD25⁺regulatory T cells.Different from mFGL2 in function,sFGL2 has immunoregulatory ability.So,is it possible for Plasmodium to suppress or evade immune attack through FGL2?Prior to this study,our group have investigated the role of FGL2 in the blood stage infection.The results showed that the expression of sFGL2 was significantly induced after blood stage infection,and sFGL2 could promote the proliferation of parasites.Moreover,we found that sFGL2 had different mechanisms in malaria infection compared to other diseases,which showed that sFGL2 did not inhibit the adaptive immunity of host against the blood stage infection?the function of antibody,CD4⁺T cells and CD8⁺T cells did not increase after FGL2 deletion?.On the contrary,sFGL2 can inhibit macrophages to produce MCP-1 throughFc?RIIB receptor expressed on the cell surface,significant reducing the accumulation of NKcells and NKT cells in the spleen,resulting in the decrease of IFN-?expression,thereby dampening the innate immunity against blood stage infection,and further promote the proliferation of Plasmodium.Although we have clarified the main line of sFGL2 inhibiting the innate immunity in blood stage infection,there are still many parts that need to be further improved and supplemented.For example,whether the role of sFGL2 in promoting the proliferation of Plasmodium in blood stage is preserved in the infection of other species of Plasmodium,the correlation between the expression of sFGL2 and the parasitemia,the molecular mechanism of MCP-1 production,and the cellular molecular mechanism of sFGL2inhibiting macrophages to produce MCP-1,etc.Therefore,in this paper,we solved the above questions through the first part research.In addition,in view of the important role of FGL2 in malaria blood stage,we further investigated whether FGL2 played a role in the liver stage of infection and preliminarily explored the mechanism of action,and this is the second part of the paper.The first part The role and mechanism of FGL2 in malaria blood stage infection1.The expression of sFGL2 was up-regulated after malaria blood stage infection,and sFGL2 significantly promoted the proliferation of blood stage parasites1.1.The expression of sFGL2 was up-regulated after malaria blood stage infection,and the expression level of sFGL2 was positively correlated with the parasites loadIn mice infected with Plasmodium yoelii yoelii and Plasmodium berghei,sFGL2expression in serum was significantly increased,and the expression level of sFGL2 is consistent with the fluctuation of the parasitemia,which is consistent with the result from Plasmodium chabaudi infection.The results of correlation analysis showed that the expression of sFGL2 was positively correlated with the load of parasites.1.2.sFGL2 is a new way for blood stage parasites to evade host immunityIn the FGL2 knockout mice infected with Plasmodium yoelii yoelii and Plasmodium berghei,rFGL2 supplementation can restore the proliferation ability of parasites to the level of WT mice,which is consistent with the result from Plasmodium chabaudi infection.This result further proved the role of sFGL2 in promoting the development of blood stage.In addition,the results of immunofluorescence showed that there was no significant difference in the expression of fibrin in the spleen of the two kinds of mice before and during the infection.1.3.Regulatory cells?Treg?are one of the main sources of sFGL2In our previous research,results showed that CD4⁺Foxp3⁺CD25⁺Treg cells are one of the main sources of sFGL2.In order to further consolidate this conclusion,we then carried out the adoptive transfer experiment of Treg cells.After the Treg cells from WT mice and FGL2 knockout mice were transferred into the FGL2^-/-infected mice respectively,we found that parasitemia of FGL2^-/-infected mice receiving the Treg cells from WT mice recovered to the level of WT infected mice,while FGL2^-/-infected mice receiving the Treg cells from FGL2 knockout mice had no significant change in parasitemia.2.sFGL2 significantly inhibits innate immunity against malaria blood stage infection2.1.After macrophage depletion,the protective immunity of FGL2^-/-mice against blood stage infection disappearedPrevious studies have confirmed that macrophages play a key role in FGL2^-/-infected mice,which is one of the initiating factors for the enhancement of innate immunity against infection.However,we have not carried out reverse experiments to verify its effect.In this section,by injecting clodronate liposomes to specific deplete macrophages in FGL2^-/-mice,we found that when loss of macrophages,the proliferation ability of parasites in FGL2^-/-mice was significantly enhanced,and parasitemia returned to the level of WT infected mice,and FGL2^-/-mice died 10 days after infection.In addition,the levels of MCP-1 and IFN-?in serum of FGL2^-/-infected mice without macrophages decreased dramatically,even significantly lower than those of WT infected mice.These results once again proved that macrophages play a key role in the innate immunity of FGL2^-/-mice against blood stage infection.2.2.The metabolites of blood stage parasites can be recognized by TLR2 instead of TLR9,which induces macrophages to secrete MCP-1Next,the molecular mechanism of MCP-1 production by macrophages was studied.The BMDM?bone marrow derived macrophases,BMDM?of WT mice and TLR2 knockout mice were isolated and stimulated by PRBC,the lysate of infected Plasmodium erythrocytes,and the expression of MCP-1 was detected.We found that when TLR2 was absent,the ability of BMDM to secrete MCP-1 stimulated by PRBC was significantly lower than that of WT BMDM,while the addition of ODN 2088,the TLR9 antagonist,had no effect on the MCP-1production of WT BMDM.2.3.Malaria blood stage parasites using sFGL2 to inhibit MCP-1 secretion bymacrophages through Fc?RIIB receptorIn the early research,we constructed macrophage Fc?RIIB receptor conditionally deficient mice(Fc?RIIB^fl/fl Lyz2-cre).In vitro experiments showed that sFGL2 inhibited MCP-1 secretion of macrophages through Fc?RIIB receptor.In order to consolidate this conclusion,we have carried out infection in Fc?RIIB^fl/fl Lyz2-cre mice with Plasmodium chabaudi.Using Fc?RIIB^fl/fl mice as the control,we collected the serum of two kinds of mice on the 6 day after infection,and detected the expression of MCP-1 and IFN-?.We found that compared with Fc?RIIB^fl/fl mice,the content of MCP-1 and IFN-?in the serum of Fc?RIIB^fl/fll/fl Lyz2-cre mice increased significantly.2.4.sFGL2-Fc?RIIB signal inhibits macrophages to secrete MCP-1 by inhibiting thephosphorylation of JNK downstream of TLR2 signal pathwayIt is known that the metabolites of blood stage parasites can be recognized by TLR2 and then activated macrophages to secrete MCP-1.NF-?B signaling pathway and MAPK signaling pathway downstream of TLR2 are involved in the production of MCP-1.By detecting the activation of TAK1,p65,MKK4,MKK7,JNK and ERK,we found that only the phosphorylation of JNK in MAPK signaling pathway was significantly inhibited,which indicated that the intracellular site of sFGL2-Fc?RIIB was on JNK.In order to further verifythis conclusion,we conducted the same experiment in Fc?RIIB^fl/fl Lyz2-cre mice.Results inaccordance with the RAW264.7 cell line experiment,the activation of JNK was significantly inhibited in the presence of rFGL2 in BMDM of control mice.However,in the BMDM of Fc?RIIB^fl/fl Lyz2-cre mice,the phosphorylation of JNK is not affected by rFGL2,whichfurther confirms the previous conclusion that sFGL2 plays an inhibitory role through Fc?RIIBreceptor.2.5.sFGL2 may play an important role in human malaria through the same molecular mechanismIn view of the significant increase in the expression of sFGL2 in malaria patients,we next explored the mechanism of sFGL2 in human malaria.By conducting ELISA and Western blotting experiment in human macrophage THP-1,we found that sFGL2 still plays aninhibitory role through Fc?RIIB receptor.Similarly,sFGL2-Fc?RIIB also significantly inhibited the phosphorylation of JNK through Fc?RIIB receptor.These results suggest thatsFGL2 may play an important role in Plasmodium falciparum infection through the same molecular mechanism.The second part The role and mechanism of FGL2 in liver stage of malaria infection1.Malaria liver stage infection led to significant expression of FGL2 in host1.1.The expression level of FGL2 mRNA in the liver of WT mice was significantly increased after malaria liver stage infectionThe expression of FGL2 mRNA in the liver of experimental mice was observed at 42hours after natural infection of Anopheles mosquito bite and inoculation of purified sporozoites.The results showed that the expression of FGL2 mRNA in the livers of the experimental mice was up-regulated after parasites infection,and increased with the dose of sporozoite inoculation.1.2.The expression of mFGL2,rather than sFGL2,may be induced by liver stage infectionBy setting different doses and multiple time points,the serum of mice infected with sporozoites was collected and the content of sFGL2 was detected.The results showed that no matter which dose of sporozoites infection,the concentration of sFGL2 did not change in the whole liver stage,but the expression level of sFGL2 increased significantly after the Plasmodium parasites entered the blood stage,indicating that the liver stage infection would not lead to the increase of the expression of soluble FGL2,and the increased expression of FGL2 mRNA caused by the liver stage infection might come from mFGL2,not sFGL2.2.FGL2 promotes the development of liver stage parasites2.1.The proliferation of liver stage parasites was significantly inhibited after FGL2deletionThen we infected FGL2 knockout mice with sporozoites of Plasmodium yoelii yoelii BY265 strain,and detected the parasites load in the liver of infected mice by quantitative PCR.The results showed that after FGL2 deletion,the proliferation of parasites in the liver was significantly inhibited compared with the control mice,indicating that FGL2 promoted the development of liver stage parasites.2.2.In FGL2 knockout mice infected with sporozoites,blood stage parasites appeared later than that in WT mice and the initial parasitemia was lowerIn order to further determine the role of FGL2,we also observed the emergence time of blood stage parasites and the initial parasitemia of FGL2 knockout mice infected by sporozoites.The results showed that when using low dose of sporozoites infection,the onset of blood stage in FGL2 knockout mice was significantly later than that of WT mice,and some FGL2 knockout mice did not appear blood stage infection.In addition,the initial parasitemia of FGL2 knockout mice was significantly lower than that of WT mice.3.FGL2 promotes the development of liver stage parasites by inhibiting IFN-?secretion3.1.After FGL2 deletion,the expression level of pro-inflammatory cytokines mRNA in the liver of infected mice increased significantlyAfter FGL2 deficiency,the transcription levels of IFN-?and TNF-?in liver of infected mice were significantly higher than those of control mice.There was no significant difference in IL-12 between the two groups,as well as the expression level of anti-inflammatory factor IL-10 mRNA.These results suggest that the decreased parasites load in liver of FGL2knockout mice may be the result of the increased expression of pro-inflammatory factors.3.2.After FGL2 deletion,the level of pro-inflammatory cytokines in the serum of infected mice increased significantlyThe concentrations of six cytokines including pro-inflammatory and anti-inflammatory factors were observed through flow cytometry analysis.The results showed that the concentrations of IFN-?,TNF-?,IL-12 and MCP-1 increased significantly in FGL2^-/-infected mice,consistenting with RT-PCR results.The content of anti-inflammatory factor IL-10 in FGL2^-/-infected mice was also significantly higher than that in control mice.These results further prove that the increased pro-inflammatory factors may play a critical role in FGL2^-/-infected mice against liver stage infection.3.3.IFN-?is the main effector of FGL2 knockout mice against the proliferation of liverstage parasitesDue to pivotal role in limiting liver stage infection,IFN-?then was selected for reverse validation experiment.By injecting anti IFN-?mAb into FGL2 knockout mice,we found that when IFN-?was absent from FGL2 knockout mice,the parasites load returned to WT infected mice.In addition,after depletion of IFN-?,the onset of blood stage of FGL2^-/-infected mice was earlier than that of WT infected mice,and the initial parasitemia of blood stage was significantly higher than that of WT infected mice.These results strongly indicate that IFN-?is the main effector of FGL2 knockout mice against liver stage infection.