Functions Of TLR2,TLR4,TLR7,and TLR9 In Cellular Immunity,humoral Immunity,and Immunological Memory Formation During BALB/c Mice Infected With Plasmodium Chabaudi

Objective:An estimated 200 million cases of malaria occur worldwide and cause400,000 deaths globally every year with approximately 70%of fatal malaria cases occurring in children less than five years old.Although there has been a significant advance in the understanding of acquired immunity in malaria,the innate immune response to malaria has not been intensely scrutinized.Blood stage infections lead to production of both pro-inflammatory and anti-inflammatory mediators by the host immune system.Studies performed in humans and in rodent malaria models have demonstrated that CD4⁺T cells mediate the major protective immunity in malaria infection.Innate cytokines such as Tumor Necrosis Factorα（TNF-α）and type I interferons（IFN-I）restrict parasite growth during infection.Chronic malaria infections usually promote immunological memory formation,and activated immunological memory cells rapidly produce high-affinity antibodies to protect against re-infections.Toll-like receptors（TLRs）as one of pattern-recognition receptors（PRRs）involved in detection and rapid response to plasmodium and play a pivotal role in regulating the balance between Th1 and Th2 types of response essential for the survivability of the host.Thirteen TLRs are identified so far and 10 are known to be functional in humans.Among these,TLR4,TLR7 and TLR9 are known to detect malarial antigens and induce anti-malarial immune response.The ligands for these receptors are highly conserved molecules are recognized by TLRs,TLR4 mediates the recognition of the LPS（lipopolysaccharide）,TLR7 recognizes a plasmodium-derived RNA molecule,while TLR9 mediates the recognition of the CpG motif in bacterial DNA.Although TLRs activation promotes both innate inflammatory responses as well as the induction of adaptive immunity,the effects of changes in TLRs activity on regulation of immune responses and development of protective immunity and the establishment and maintenance of immunological memory during malaria infections remain poorly understood.The purpose of this study was to investigate the effects of changes in TLRs activity on immune system activation and the establishment of long-lasting immune memory.Methods:1.TLRs intervention and infection.24 hours before Plasmodium chabaudi parasites infection,each BALB/c mouse in the TLRs agonists-treated group was intraperitoneally injected with TLRs agonists or inhibitors.For TLR4 and TLR7intervention groups,each mouse was intraperitoneally injected with agonist or inhibitor of 10μg for TLR2 group,15μg for TLR4 group and 1mg for TLR7 group,as control group,same volume of PBS was injected;For TLR9 intervention group,each mouse was intraperitoneally injected with TLR9 agonist 50μg,as control group,same volume of control DNA fragment was injected.For each group,five mice were intraperitoneally challenged with 10⁶ parasites-infected erythrocytes（pRBCs）per a malaria infection model and re-infected with 10⁶ pRBCs at day 40 when the mice had recovered from the initial infections.Parasitemia was monitored every other day during the initial infections and re-infections.2.Measurement of IgG subtypes in serum.Serum IgG1 and IgG2a levels in infected mice were measured after initial infections and re-infections.For each group,five mice were euthanized,and sera were collected for IgG1 and IgG2a assays using a commercial enzyme linked immunosorbent assay（ELISA）kits according to the manufacturer’s protocol.The concentration of IgG1 and IgG2a were indirectly expressed by the value of OD₄₅₀.3.Measurement of cytokines.Levels of IFN-γ,TNF-α,TGF-βand IL-10 in splenocytes were measured using ELISA and real-time PCR after initial infection.For each group,five mice were euthanized and the splenocytes culture was performed, after culturing for 48h,the culture supernatants were collected and cytokines were measured using ELISA kits according to the manufacturer’s protocol.The concentration of cytokines was calculated via a standard curve and all cytokines had pg/ml sensitivity.4.Flow cytometry analysis.After initial infections and re-infections,flow cytometry of mouse spleen cells.For each experimental group,three mice were sacrificed for flow cytometric detections.All the antibodies used in the experiments were stained according to the antibody staining instructions.Subsets of spleen cells were defined as:TLR2⁺CD11c⁺MHCII⁺（TLR2 activated DCs）,TLR4⁺CD11c⁺ MHCII⁺（TLR4 activated DCs）,TLR7⁺CD11c⁺MHCII⁺（TLR7 activated DCs）,TLR9⁺CD11c⁺MHCII⁺（TLR9 activated DCs）,CD3⁺CD4⁺IFN-γ⁺（Th1）,CD3⁺CD4⁺IL-4⁺（Th2）,CD4⁺CD25⁺Foxp3⁺（Tregs）,CD4⁺IFN-γ⁺PD-1⁺（Th1 expressing PD1）,CD4⁺IL-4⁺PD-1⁺（Th2 expressing PD-1）,B220⁺IgG1⁺（IgG1class switching B cells）,B220⁺IgG2a⁺（IgG2a class switching B cells）,CD138⁺B220⁺IgG1⁺（IgG1class switching plasma cells）,CD138⁺B220⁺IgG2a⁺（IgG2aclass switching plasma cells）,CD4⁺CD44⁺CD62L⁺（Tcm）,CD4⁺CD44⁺CD62L^-（Tem）and CD4⁺PD-1⁺CXCR5⁺（Tfh）.5.Statistical analyses.Data are presented as mean±standard error of the mean（SEM）.Statistical significance was analyzed using a Student’s t-test or one-way ANOVA（SPSS 17.0）.A value of P<0.05 was considered significant.Results:1.TLR4,TLR7 and TLR9 activation alleviate parasitemia in Pc infected mice.For all infected mice parasitemias began to rise after the initial infection and peaked at days 6 to 9,and then declined.Almost all infected mice recovered from the initial infections by day 30,and no mice died before 40 days.TLR4,TLR7 and TLR9activation effectively reduces infection rate in mice with initial infections. Parasitemias peaked around 10 days after re-infection,and were lower than the initial infection in all groups.Following pre-treatment with TLR7 and TLR9 agonists the parasitemias dropped significantly at during re-infections.2.TLR4,TLR7 and TLR9activation effectively activate DCs during initial infection.The expression of TLRs on DCs was drastically increased in TLR4,TLR7 and TLR9 agonists groups compare with each control group,and MHC II expression on DCs was significantly increased after infection compare with un-infection in all groups.The MHC II on DCs in TLR4,TLR7 and TLR9 agonists-treated groups was over-expression compared with the control groups on the day 5 after infection.3.TLR4,TLR7 and TLR9 activation stimulate Th1 cells activation during initial infection.On day 0,day 5,day10 and day15 after initial infection,the percentage of Th1 cells in spleens was examined.Th1cells were significantly expanded after infection compare with un-infection in all groups.The percentage of Th1 cells was significantly elevated in TLR4,TLR7 and TLR9 agonists-treated groups compare with each control groups.4.TLR4,TLR7 and TLR9 activation promote IFN-γand TNF-αexpression during initial infection.IFN-γand TNF-αexpression levels in splenocytes were detected after initial infection.IFN-γand TNF-αbegan expressed after infection and reach the peak between day 3 and day5 when the parasitemia was most serious.IFN-γand TNF-αproduction was significantly increased in TLR4,TLR7 and TLR9 agonists-treated mice on the day 3or day 5 after infection.5.TLR4,TLR7 and TLR9 activation stimulate Th2 cells activation during initial infection.After initial infection,the percentage of Th2 cells in spleens was examined.Th2 cells were significantly expanded after infection compare with un-infection in all groups.The percentage of Th1 cells was significantly elevated in TLR4,TLR7 and TLR9 agonists-treated groups compare with each control groups.6.TLR4,TLR7 and TLR9 activation block Tregs expansion and reduce TGF-βand IL-10 production.The proportion of Tregs in splenocytes of infected mice increased significantly.Similar results also appeared in the expression levels of anti-inflammatory cytokines IL-10 and TGF-βin the spleens.Compared with each control groups,the percentage of Tregs of TLR4,TLR7 and TLR9agonists-treated groups in spleens was decreased significantly.The expression levels of IL-10 and TGF-βwere significant lower in TLR4,TLR7 and TLR9 agonists-treated mice.7.TLR4,TLR7 and TLR9 activation reduce PD-1 expression in Th1 and Th2 cells.PD-1 expression in Th1 and Th2 cells were analysis after initial infection.The expression of PD-1 was significantly increased in all groups after infection.Compared with each control groups,fewer Th1 and Th2 cells expressing PD-1 in TLR4,TLR7and TLR9 agonists-treated groups.8.TLR7 and TLR9 activation effectively increase the content of IgG1 and IgG2a in the serum during initial infection and re-infection.The concentration of IgG1 and IgG2a in the serum of all infected mice increased after initial infection and re-infection especially IgG1,In TLR7 and TLR9 agonist treated groups,the concentration of IgG1 in serum was significantly higher than that of the control groups.There was also a significant increase in the concentration of IgG2a in serum in TLR9 agonist treated mice.9.TLR7 and TLR9 activation promote memory B cells with IgG1 or IgG2a class switching.Memory B cells were significantly expanded after infections compare with uninfected controls in all groups.Compared with the control group,pre-treatment with TLR7 agonists promoted IgG1 switching,but there was no difference in IgG2a class switching.Memory B cells with IgG1 and IgG2a class switching significantly increased in the TLR9 agonist treated group.10.TLR7 and TLR9 activation promotes secretion of IgG1 and IgG2a by plasma cells.The number of plasma cells with IgG1 or IgG2a class switching in the spleen of all mice increased significantly after initial infections and re-infections.Similar with the results of memory B cells in spleen,pre-treatment with TLR7 and TLR9 agonists promoted the switching of plasma cells to IgG1 or IgG2a.11.TLR7 and TLR9activation effectively promote Tcm and Tem expansion.Memory T cells were significantly expanded after infections compare with un-infected controls in all groups.The proportions of Tcm and Tem were significantly elevated in TLR7 and TLR9 agonist treated groups compared with respective control groups.12.TLR7 and TLR9activation effectively stimulate Tfh cells activation.Compared with uninfected mice,the number of Tfh in spleen increased after infections.Pre-treatment with TLR7 and TLR9 agonists effectively stimulated Tfh activation in spleen,the inhibitor-treated group showed opposite results.Conclution:1.TLR4,TLR7 and TLR9 activation alleviate parasitemia in Pc infected mice.2.TLR4,TLR7 and TLR9 activation effectively activate DCs during initial infection.3.TLR4,TLR7 and TLR9 activation stimulate Th1 and Th2 cells activation during initial infection.4.TLR4,TLR7 and TLR9 activation block Tregs expansion and reduce TGF-βand IL-10 production.5.TLR7 and TLR9 activation effectively increase the content of IgG1 and IgG2a in the serum during initial infection and re-infection.6.TLR7 and TLR9 activation effectively promote Tcm,Tem and Tfh cells expansion.