Preliminary Study On Immune Responses Induced By Exosomes From Haemonchus Contortus In Goat

Haemonchus contortus is a kind of gastrointestinal parasite that causes great harm to cattle and sheep.It often causes anaemia and death of lamb and leads to huge economic loss.For a long time,the controlling of this parasitic disease(so called Haemonchosis)was mainly based on drug treatment.However,with the emergence of increasing problems such as resistant strains and veterinary drug residues,immunoprophylaxis is urgently needed to control haemonchosis.Although some progress has been made in the development of vaccine against haemonchosis,no vaccines(DNA or proteins)are available for controlling this disease.The reason may be due to the fact that the mechanisms of infection and immunity elicited by H.contortus in hosts are still not fully understood,specially we have failed to obtain a comprehensive understanding of both protective components(cells and antigens)derived from parasites and immunobiologic properties of these cells/molecules exposed to immune system in the hosts.So this situation might restrict the progress of development of vaccine with high level of protective effect.Therefore,it is needed to undertake researches on screening and identification of antigens with high protective effect.Previous studies have shown that immunization of radiation-attenuated(RA)H.contortus larvae can induce a high level of protective immunity against larval challenge in sheep or goats,whereas no protective effect can be observed in hosts infected with normal H.contortus larvae.Therefore,RA larvae vaccine becomes an ideal model for elucidating mechanisms of high protective immunity in hosts.In a recent investigation,we have observed that normal and RA H.contortus larvae/cells cultured in vitro could show apoptotic and necrotic phenotypes,respectively,suggesting that immunobiologic functions and properties of the cells/molecules derived from RA H.contortus larvae are different from that of cells/molecules from normal larvae.However,cellular and molecular mechanisms responsible for this kind of difference were not very clear as yet.Exosomes from most of cells are unique 30-100nm vesicles containing proteins and RNA that have intercellular communication and immune-related functions.Recent studies have shown that exosomes derived from normal protozoa and nematode parasites can suppress innate immune responses in hosts.Furthermore,excretory-secretory antigens from helminth(trematode and nematode parasites)are also able to suppress immune responses in hosts,whereas immunization with RA helminth larvae can induce high levels of protective immunity in hosts,suggesting that molecular components and immunobiologic functions of exosomes from RA helminth larvae might be different from that of exosomes from normal helminth larvae.Therefore,we speculated that there are differences in molecular composition and expression of exosomes between RA and normal H.contortus larvae,and this situation can lead to different immune responses in hosts.However,this hypothesis remains to be experimentally validated.Therefore,we carried out a preliminary study on identification and immunobiologic functions of exosomes from both RA and normal H.contortus larvae.In this present study,firstly,we collected 18d-H.contortus larvae from sheep and these normal or being irradiated with UV(400?W/cm~2 for 4 min larvae were cultured for 12h,and supernants were collected,ultrafiltered and concentrated.Then the exosomes were isolated using isolation kit,the morphological properties of exosomes from both normal and RA H.contortus larvae were identified by transmission electron microscopy.We found that these two groups of exosomes have morphological properties of typical exosomes.Secondly,aforementioned two groups of exosomes were analyzed by label-free quantitative proteomics method,and it was found that expression levels of some danger-associated molecular patterns(DAMPs)such as HSP70,HSP90 and CRT molecules in exosome of RA group were higher than that of DAMPs in exosome of normal group.Lastly,immunobiologic functions of aforementioned two groups of exosomes were investigated by testing expressions of cytokins in goat/sheep PBMCs stimulated with 5?g and 20?g of exosomes,respectively,and we found that exosome from RA laevae can upregulate expression of pro-inflammatory cytokines IFN?and IL-4in sheep PBMC,and suppress expression of anti-inflammatory cytokine IL-10 and regulatory cytokine IL-17;while exosome from normal laevae can inhibit the expression of pro-inflammatory cytokines IFN?and IL-4,and upregulate the expression of anti-inflammatory IL-10 and regulatory cytokines in sheep PBMCs.We also found that exosome from normal laevae can reduce the secretion of NO,and exosome from RA laevae can increase secretion of NO by PBMCs.In summary,we have found that exosome from RA laevae had an increased content of DAMPs molecules,and could induce inflammatory response in immune cells of host;whereas exosome from normal laevae could suppress inflammatory response.The results suggested that molecular components and immunobiologic functions of exosome from RA larvae are different from that of exosome from normal larvae.It provided the basis for further elucidating mechanisms of high protective immunity induced by the RA H.contortus larvae vaccine.