Molecular Evidence Of Macrophage Polarization Associated With The Progress Of Schistosoma Japonicum Infection

Schistosomiasis is the2nd most common parasitic disease in the world, withalmost700million individuals at risk, about200million people infected and over halfof whom having various degrees of morbidity, which leads to a burden that might beas high as20million disability-adjusted life-years and results in more than20thousand deaths annually. In China, though great progress has been made incontrolling schistosomiasis japonica over the past50years, there still are more than300thousand cases of schistosomiasis reported in the seven endemic provinces by theend of2010. Therefore, prevention and control of schistosomiasis is still greatchallenges in China.S. japonicum causes serious damages of host organs, mainly by inducingimmune responses. Researchers are gradually uncovering the complex interplaybetween this helminth and its appropriate hosts including human. It has been reportedthat in the early stages of the infection, host immune responses are Th1-dominant andthen shifted to Th2-type associated with a series of immune suppressions after eggproduction by adult worms. But so far, the mechanism underlying this immunephenomenon has not been fully elucidated. In this respect, one of major concerns isthe regulatory role of macrophage plying in the immune responses induced by theinfection of the parasite, about which very little has so far been known. It has been found that macrophage can differentiate into different subtypes in thedifferent micro-environments. There are two major subtypes of macrophage: M1andM2. M1is classically activated macrophages that can directly phagocytose anddestroy pathogens and malignant cells. It can also activate adaptive immuneresponses by secretion of proinflammatory cytokines and chemokines and acting asprofessional antigen-presenting cell. M2is alternatively activated macropahgesinduced by M-CSF, IL-4,-13,-10, glucocorticoid, TGF-beta, vitamin D3, PGE2, etc.M2has low antigen presentation capacity and can downregulate adapative immuneresponses by secretion of IL-10, CCL17, CCL18and TGF-beta. Obiviously, M1andM2can give rise to positive or negative regulatory signals respectively to the immuneresponses and it was important to determine whether the polarization of macrophagemay be linked to the change of antigen micro-environments before and after eggproduction during the infection of schistosome and then regulate the development ofadaptive immune responses, including Th polarization shift. In this study, we try tofind the molecular evidence of Mφ polarization associated with the change of antigenmicro-environments before and after egg production during the infection ofschistosome by use of experiments in vitro (macrophage cell line RAW264.7) and invivo (experimental infection of mice with S. japonicum).The overall experimental design is：in vitro to stimulate RAW264.7(M0) withclassic M1inducer IFN-γ and M2inducer IL-4and then identify polarization propertyof RAW264.7with surface marks by FACS, cytokine secretion by ELISA and geneexpression by RT-PCR, to establish a Mφ polarization recognition system；Next, invitro to stimulate RAW264.7with S. japonicum antigens derived from differentdevelopmental stages，in vivo to obtain mouse peritoneal macrophages at differentstages of S. japonicum infection, then identify polarization property of these antigenstimulated macrophages and peritoneal macrophages through the arginine metabolicpathway, surface markers and cytokine secretion, to prove that antigens derived fromdifferent developmental stages of S. japonicum can induce macrophages to polarizeinto different subtypes; Then use ELISA to investigate the expression of effectory/inhibitory molecules of mouse peritoneal macrophages taken from different stages of S. japonicum infection; through TLRs blocking experiments, to explore therelationship of TLR4signal pathway and macrophage polarization; Finally, after S.japonicum antigens stimulation, the co-stimulatory molecules CD40, CD86andco-suppression molecular PD-L1expression of M1and M2will be detected.The main results are as follows:1. Under our laboratory conditions, we established a macrophage polarizationrecognition system successfully. M1inducer IFN-γ and M2inducer IL-4canstimulate RAW264.7to M1/M2polarization.2. Antigens derived from different developmental stages of S. japonicuminduced Mφ polarization. CD16/32, IL-12and inos expression increasedsignificantly in RAW264.7following NCA and ACA stimulation compared withcontrol group(p<0.01), but there were no significant difference between NCA andACA stimulated groups. CD206, IL-10, arg1increased significantly (p<0.01) inRAW264.7following SEA stimulation compared with control group. These resultsshowed that ACA and NCA could induce M1polarization, SEA could induce M2polarization, SWAP could induce both M1and M2because of its complicatedingredients (contaminated with SEA).3. Mφ polarization existed during the process of schistosome infection. Thehighest expression of M1surface marker CD16/32appeared at3weeks after infection,gradually reduced from6weeks after infection(p<0.01); M2surface marker CD206expression increased at3weeks after infection, reaching to the highest level at9weeks after infection. Interestingly, during3to9weeks after infection, we observedthat the peritoneal macrophage divided into two groups according to the F4/80fluorescent intensity. Because we could not find the right technology to separate thesetwo groups of macrophage, and identify the polarization properties of each group ofcells, we detected IL-12, IL-10secretion, inos, arg1mRNA expression to identifypolarization properties. The results showed that high level of IL-12was secretedbefore eggs laid, then gradually reduced after egg production, while IL-10secretion significantly increased at6weeks after infection. inos mRNA expression reached tothe highest level at3weeks after infection，arg1mRNA expression significantlyincreased after egg production. These results showed that M1polarization dominatedbefore egg production while M2dominated after egg production.4. Macrophages polarization regulated immune responses through secretingcytokines after S. japonicum infection. Macrophages secreted cytokines of positiveeffect (TNF alpha, IL-12) in dominant before egg production, but molecules ofnegative effect (IL-10, TGF-beta) after egg production.5. TLR4signal pathway may have effect on the macrophages polarization.When TLR2pathway was blocked, there were no significant influence in CD16/32，CD206expression, cytokines IL-10，IL-12secretion and inos，arg1expression. ButWhen TLR4pathway was blocked, NCA stimulated Mφ expressed lower level ofCD16/32and inos compared to the control group (P <0.01), prompting that TLR4signal pathway effect on the M1polarization. The expression of CD206, IL-10, arg1of macrophages after SEA stimulation have no significant changes compared to thecontrol group, the reason needs further exploration.6. The modulation of balance between co-stimulatory molecule CD40, CD86and co-suppression molecule PD-L1might regulate macrophage polarization.Both INF-γ and IL-4could stimulate macrophages to express PD-L1, CD40andCD86; INF-γ stimulated macrophages to express significantly higher level of PD-L1,CD40and CD86than IL-4did(P <0.01); NCA stimulated macrophages expresssignificantly higher level of of PD-L1and CD86than SEA and SWAP stimulatedmacrophages; On the role of costimulatory signals involved in macrophagepolarization and regulation, in the present study, is only stay on the experimentalresults of intuitionistic description and inference and there are no direct evidence toprove the linkage of positive or negative co-stimulatory signals to the macrophagepolarization properties of regulation, which may take place at an unknown level involving the modulation of balance between co-stimulatory molecule andco-suppression molecule.In summary, this study explored the relationship of S. japonicum antigens andformation of macrophage polarization and the impact of antigen environment changeduring schistosome infection on macrophage polarization modulation, for future frommacrophages polarization properties of regulation, looking for development of newimmunosuppressive agents based on the parasite components provide pilotexperimental arguments.