A Preliminary Study On The Activation Of Ovine Macrophage By Haemonchus Contortus HSP70 And CRT Proteins

Haemonchus contortus is a kind of parasite of the gastrointestinal tract of cattle and ovine,resulting in serious economic losses to the livestock industry.At present,the control of Haemonchus contortus mainly relies on benzimidazole repellents,but studies have shown that drug-resistant strains have emerged.It is urgent to develop new strategies for the effective control of Haemonchus contortus.Vaccines are one of the most studied fields,but the lack of comprehensive understanding of the immunobiological characteristics from the protective components of worms is one of the main reasons for the restriction of vaccine research.Therefore,the study of the interaction between Haemonchus contortus and the host is particularly important for the development of effective vaccines.Previous research in our laboratory found that Haemonchus contortus calreticulin(Hc-CRT)and Haemonchus contortus heat shock protein 70(Hc-HSP70)could cause Th2 type immune response of Peripheral Blood Mononuclear Cells(PBMC)of ovine.We further investigated whether Hc-CRT and Hc-HSP70 proteins can stimulate the maturation and differentiation of host macrophages and cause immune responses mediated by CD4+ T cells.In this experiment,PBMC were obtained from ovine's whole blood,and macrophages from PBMC were cultured in vitro.The macrophage viability was verified by phagocytic chicken erythrocyte experiment,and the positive rate of CD14 was verified by flow cytometry method.After the recombinant proteins Hc-CRT and Hc-HSP70 stimulated ovine macrophages,we analyzed the expression levels of M1 polarization-related factors INOS,IL-6,and M2 polarization-related factors Arg-1 and IL-10 by Real time-PCR.We found that Hc-CRT and Hc-Hsp70 could up-regulate the expression levels of Arg-1 and IL-10,and down-regulate the expression levels of IL-6,which was similar to the results of ELISA.Therefore,Hc-CRT and Hc-HSP70 proteins could effectively activate macrophages,and promote macrophages-mediated protective immune response in the host.Then,Hc-CRT and Hc-HSP70 stimulated the sorted macrophages,and incubated with CD4+ T cells to detect the expression levels of CD4+ T cytokines IFN-? and IL-4.We found that the expression levels of IFN-? and IL-4 were significantly increased.The results showed that Hc-CRT and Hc-HSP70 could promote the functional maturation of macrophages,activate CD4+ T cells,and induce a mixed immune response of Th1 and Th2 in the host.This study provides a new direction for the interaction between Haemonchus contortus and ovine macrophages,and provides a theoretical basis for further elucidating the potential mechanism and specific vaccines of Haemonchus contortus induced immune response.