Functional Characterization Of Macrophage Migration Inhibitory Factor Derived From Malaria Parasites

Host-derived macrophage migration inhibitory factor(MIF) has been implicated in the pathogenesis of malaria infection,especially in malarial anemia.Recently,two Plasmodium parasite-derived MIFs,P.falciparum MIF and P.berghei MIF,were suggested to modulate host immune-cell activity.However,the precise roles of these parasite-derived MIFs,particularly in combination with the host MIF,during infection remain unknown.In the early study,we identified another MIF from the rodent-specific P. yoelii(PyMIF).This molecule shares only 30%amino acid sequence identity with murine MIF(MmMIF) but the two molecules share a highly conserved three-dimensional structure.Structural analysis showed that although the molecular structure and immune activity of PyMIF was highly similar to that of MmMIF,it had a different substrate binding pattern and much lower tautomerase activity.In this study,we focused on the mechanism of the target cells regulated by PyMIF,including its cell membrane receptor,endocytosis study,the signaling pathway and downstream molecules regulation,whole-cell effects and apoptosis mechanisms,as well as the animal model research.In this study we found that Mouse fibroblasts and macrophages are the target cells of PyMIF.PyMIF could activate MAPK/ERK and PI3K/AKT signal pathway,inhibit AP-1 activation,and has no function on JAK/STAT and NF-κB signal pathway,which is similar to MmMIF.Importantly,although PyMIF and MmMIF work on similar pathway, they acted synergistically to activate the MAPK-ERK1/2 signaling pathway at very low concentration and acted antagonistically at higher concentration.Moreover,they showed different mechanism in inhibiting cell apoptosis and PyMIF significantly regulated more genes transcription than MmMIF did.The study of receptor and endocytosis has not been completed for the difficulty in protein expression and purification and protein labeling.In the mouse model study,Plasmodium parasite-derived MIF can be detected in the peripheral blood of mice infected with different malaria parasites strains,and the Plasmodium parasite-derived MIF level is associated with the parasitemia.PyMIF protein injected into infected mice resulted in prolonging the parasitemia and increasing the TNF-αand IL-6 levels,but with no effect on the appearance time and number of gametophytes.In addition,after drug treatment to the infected mice in day 6th,PyMIF level in peripheral blood of mice increased for one day,then rapidly declined and completely disappeared on the third day,which may provide the basis for the acquisition time of blood samples at endemic area,and also promoted that Plasmodium parasite-derived MIF may be used as an indicators to the degree of malaria disease.Finally,the study also compared three methods of endotoxin removing from MIF, and proved that RP-C8 column-Non-renaturation method improved by our laboratory is a simple and effective method to remove endotoxin from a small amount of protein in laboratory-scale application,and provided important technical support to the function study.This study provides an important clue to the mechanism research of Plasmodium parasite-derived MIF,and the in vivo research data supplies good reference for sample collection and data analysis.So this paper not only contributes to better understanding of the mechanism of Plasmodium escaping from the host immune response,and providing new ideas in these fields,but also lays a solid foundation for further research work.