The Role Of TLRs Agonists In Plasmodium Yoelii Liver Stage Development

Malaria, the deadliest of the human infectious disease with high incidence and fatality rate, is transmitted by Anopheles mosquito vectors. Sporozoites enter the blood circulation when an infected mosquito bites a host. And then, sporozoites invade liver cells within a few minutes. After a brief period of liver stage development, merozoites enter the bloodstream again, invade the red blood cells (RBC) and reproduce within them, which result in malaria paroxysm. Therefore, the liver stage is important in the lifecycle of Plasmodium. Blocking the proliferation of parasite during the liver stage is critical to prevent malaria transmission. What more, pre-erythrocytic hypnozoite is the major reason for Plasmodium vivax relapse in clinical case. So, the liver stage is the ideal period to develop the intervention measures to block malaria transmission. Inhibition of Plasmodium liver stage development will prevent radically Plasmodium infection. However, there are no effective vaccine and prevention drugs for liver stage malaria and it is an urgent need for new tools and strategies to control malaria transmission.Following the bite of Anopheles mosquito, Plasmodium sporozoites are inoculated into the host. It is necessary for sporozoites to penetrate the liver cells and develop in liver cells. Mouse Plasmodium sporozoites mature in liver cells after 43h post-infection. Following a gradual breakdown of infected liver cells, merozoites are released. Similarly, the cycle of human Plasmodium is about 1 week ~ more than 10 days. Merozoites enter into the blood circulation and develop into blood stage Plasmodium which will not go back to the liver. During blood stages, Plasmodium infected red blood cells repeatedly, which can lead host to produce adaptive immune. However, liver stage is the early and short-term period during the infection course in the host. During this stage, the host can not induce adaptive immunity in time and relies mainly on innate immunity against sporozoite invasion into liver cells. Following sporozoites infection, some innate cells, such as DC, NK, NKT,γδT, were activated to control intrahepatic Plasmodium development through the immune mechannism of cytotoxicity effect or cytokine produciton. What more, the cytokines might work in modulating the adaptive immune response mediated mainly by CD4+ and CD8+ T cells. Therefore, innate immune plays an important role in response to pre-erythrocytic Plasmodium infection and investigation of the host innate immune mechanisms against liver stage malaria is the premise to develop new anti-malaria measures.Toll-like receptors (TLRs) are important pattern-recognition receptors (PRR) on innate cells. The signaling which is mediated by TLRs is crucial part of innate immunity system. TLRs can sense a wide range of microorganisms, such as triacylated lipopeptides (recognized by TLR1/TLR2 heterodimer), diacylated lipopeptides (recognized by TLR2/TLR6 heterodimer), double stranded RNA (dsRNA; recognized by TLR3), lipopolysaccharide (LPS; recognized by TLR4), flagellin from bacterial flagella (recognized by TLR5), single stranded RNA (ssRNA; recognized by TLR7/8), and bacterial DNA containing the unmethylated CpG motif (recognized by TLR9). TLRs are expressed on many innate cells, such as phagocyte, DC, B cell and so on. During the early infection, these innate cells are activated by TLRs through recognition of the microorganisms and suppress pathogen invasion through activation of NF-κB and AP-1 to promote cytokine secretion (IL-12, TNF-, IL-1, IL-6). Nowadays, many studies showed that activation of TLRs signaling pathway in advance could inhibit effectively parasite, bacterial and viral infection. However, it is unclear whether activation of TLRs signaling pathway can inhibit effectively liver stage malaria.Basing on the model of Plasmodium yoelii-BABL/c mouse, we have investigated the role of TLRs agonists in Plasmodium liver stage development though the examination of liver parasite load and parasitemia. The contents and results include the following four aspects:1. To construct the recombinant plasmid of Plasmodium yoelii BY265 18S rRNA. Through analyzing the gene of Plasmodium 18S rRNA, the specific primers of P.y. 18S rRNA was designed according to their conservative strain and the recombinant plasmid was constructed basing on these primers. The sequencing results showed that the fragment length was 839bp and the blast results revealed that the similarity was 98% between the plasmid and P.y. 17XNL 18S rRNA.2. To establish the Real-time PCR for detecting pre-erythrocytic Plasmodium. Firstly, the specific primer and TaqMan probe were designed according to the sequencing results of recombinant plasmid for P.y. BY265 18S rRNA. And then, the liver stage Plasmodium quantitative test platform was established basing on method of Real-time PCR for P.y. 18S rRNA and mouse GAPDH. To evaluate the effect of Real-time PCR, the liver parasite load was quantitatively analyzed after 50, 100, 500 and 1000 sporozoites were inoculated into mice respectively. The data showed that the method could discriminate different liver parasite load and detected liver parasite load in mice challenged with as low as 50 infectious sporozoites.3. To investigate the role of TLRs agonists in Plasmodium liver stage development. To investigate the effect on Plasmodium development with individual TLRs agonist stimulation during pre-erythrocytic stage, we examined the liver Plasmodium burden at 42 hours post-infection and the early parasitemia levels in mice pre-treated by TLRs agonist at 24 hours prior to malaria infection. The results showed that the TLRs agonists had different effect on Plasmodium development:1) With 100 sporozoites infection, the agonist of TLR2, TLR3, TLR4 and TLR9 decreased significantly the liver Plasmodium load. Among all tested TLRs agonists, TLR9 agonist was the strongest inhibitor of Plasmodium liver stage. TLR9 agonist could eliminate about 90% malaria parasite in the liver and no parasitemia was observed within 14 days post-infection in the mice treated by TLR9 agonist. Adding the inoculation dosage of infectious sporozoites, although the liver parasite load and parasitemia were increased, the liver parasite load of CpG group was lower than the control group. So, activation of TLR9 signaling played an important role in the Plasmodium liver stage development.2) On the other hand, TLR5 and TLR7 agonist had no effect on liver stage Plasmodium development. However, the parasitemia of TLR5 group were higher than the control after 8 days post-infection. This indicated that activation of TLR5 signaling pathway might be beneficial to the development of blood stage malaria.4. To explore the immune mechanism of TLR9 and TLR5 agonist on Plasmodium development.1) TLR9 agonist and Kupffer cell: Kupffer cells are obligatory for Plasmodium sporozoite infection of the liver. After gadolinium chloride (GdCl3) blocked specifically the phagocytosis function of kupffer cells (KC), the capacity of TLR9 agonist inhibiting parasite development were decreased significantly. But, administration of GdCl3 failed to completely neutralize CpG-induced immunity against malaria liver stage.2) TLR9 agonist and liver cytokines: Cytokines are key anti-malaria effectors. Further studies found that TLR9 agonist up-regulated hepatic pro-inflammatory cytokines IL-12, IFN-γand TNF-α, as well as down-regulated anti-inflammatory cytokines IL-10 and TGF-β. Therefore, these data indicated that TLR9 agonist inhibited Plasmodium development through induction of cytokine.3) TLR5 agonist and erythrocytic Plasmodium: Here, we do some preliminary study on TLR5 signaling to promote the proliferation of Plasmodium liver stage. The recombinant eukaryotic expression plasmid TLR5 plasmid was transfected into HEK293 cell. And then, these transfected cells were stimulated by the Plasmodium-infected red blood cell (iRBC) and its lysates. Finally, the transfected cells were analyzed by Dual-Luciferase Reporter Assay System. The results showed that the relative ratio of stimulation group were higher than the control. And furtherly, the ratio of full iRBC were higher than the iRBC lysates. The results indicated that iRBC could activate TLR5 signaling and the iRBC member might have TLR5 ligand. The ligand might activate Treg cell to decrease the immune response to pathogen.In the study, we had investigated the role of TLRs agonist on Plasmodium liver stage development and their mechanisms. The results showed that TLRs agonists had different effect on Plasmodium development. And, these results will help us understand the anti-malaria immune mechanism, the relationship between recognition of innate immune and immune escape of Plasmodium, as well as provide the theoretical basis for the prevention and treatment of malaria.