A Preliminary Study On The Cellular And Molecular Basis Of Apoptosis And Necrosis Of Radiation Attenuated Haemonchus Contortus Larvae

Haemonchus contortus is a major gastrointestinal parasite of goats and other ruminants.It causes anaemia,weight loss,and death after animal infection,causing huge economic losses to herdsmen.The current main control method is the use of chemical drugs to drive insects,but with the emergence of drug-resistant strains,the role of chemical drugs is gradually diminishing.Therefore,immunoprophylaxis,especially the development of molecular vaccines,has attracted the attention of researchers.But so far no highly protective molecular vaccine that can be applied to the field has been developed.A higher protective effect can be indeed achieved in sheep/goats immunized with X-rays/UV-attenuated H.contortus third-stage larvae,whereas a high anti-reinfection effect can't be induced in the host infected with normal larvae.This may be due to the fact that the cells or molecules derived from the irradiated H.contortus may have better immunogenicity,while the normal larvae have tolerogenic properties.In addition,in recent years,in the study of anti-tumor immunity,it has also found that animals immunized with tumor cells treated with light irradiation and drugs can also obtain higher levels of anti-tumor effects.The mechanism may be that the treated tumor cells initiate an immunogenic cell death program which triggers specific signaling and effector molecules(such as exposure and release of the stress molecules calreticulin Hc-CRT and HSP70 and ATP molecules),and these molecules interact with innate immune cells,eventually induce an anti-tumor immune response.Therefore,we speculated that irradiation-attenuated H.contortus third-stage larvae may have immunogenic cell death(necrotic apoptosis)and can stimulate the host's protective immune response,whereas normal nematode larvae cells are mainly apoptotic,mainly induce host immune tolerance.To this end,in this study,we compared the changes of apoptosis and necrosis of irradiated and normal larvae at the cellular and molecular levels,and carried out a preliminary study on the immunobiological functions of the stress molecules Hc-CRT and HSP70.The aim is to provide a basis for further elucidation of the mechanism of immunogenic cell death in irradiation-attenuated larvae.First,we carried out a comparative analysis of changes in apoptosis and necrosis of irradiated and normal larvae.Radiation-attenuated and normal L3 larvae were in vitro cultured for 0 d,2 d,5d,10 d and 13 d,respectively,these larvae and supernatants were harvested.After larvae were digested by trypsin,cells were harvested.The changes of necrosis and apoptosis of cells from L3 larvae at different time points mentioned above were detected by using flow cytometry.The results showed that the proportion of cells(early and late)apoptosis in the normal group reached a maximum at 2 days and then rapidly decreased;The proportion of apoptosis(early and late)in cells in the irradiation group reached its maximum at 5 days and then gradually decreased.And compared with the normal group,the early apoptosis rate of cells in the irradiation group was greater than that of the normal group at the above different time points;the proportion of late apoptosis in the irradiated group was significantly higher than that in the normal group at the late stage of in vitro culture(5 days,10 days,and 13days).The supernatant was detected for the expression of ATP(one of the characteristic molecules of cells undergoing necrotizing/immunogenic apoptosis).The results showed that with the extension of culture time,the content of ATP in the normal group gradually decreased,while the ATP content in the irradiated group had large fluctuations.However,compared with the normal group,the ATP content of the irradiation group was higher than that of the normal group at each of the above time points,especially at the 2nd and 10 th days,the difference was very significant.The results suggested that the larvae of the irradiation group showed a clear necrotizing/immunogenic apoptotic characteristic,whereas the larvae of the normal group had a conventional apoptotic characteristic.Secondly,using immunohistochemical techniques,we compared the expression characteristics of Hc-CRT and Hc-HSP70 in irradiated and normal H.contortus larvae.The results showed that Hc-CRT was mainly concentrated in the intestinal tract and muscle except epidermis and other non-cellular sites.The expression difference was mainly concentrated in the middle period.The expression level of CRT in the irradiated group was higher than that in the normal group at 2 days,and it was at 5days.10 and 13 days expression levels were lower than normal group.HSP70 mainly distributed in the subcutaneous tissues and intestinal tracts of the worms.The expression of HSP70 in the two groups of larvae increased first and then decreased as the culture time prolonged.The expression of HSP70 was higher in the irradiation group than that in the normal group on days 0,2,13,and 16 except for 5 days.It is suggested that the expressions and distribution of stress molecules Hc-CRT and Hc-HSP70 from RA H.contortus larvae,have necrotic/immunogenic apoptosis characteristics.Finally,the functions of Hc-CRT and Hc-HSP70 activated goat PBMCs were compared using molecular and cellular immunological techniques.The results showed that Hc-CRT and Hc-Hsp70 could stimulate the proliferation of goat PBMC cells in vitro;promote the expressions of Th1-related cytokines IFN-? and TNF-? in goat PBMC;and inhibit expressions of Th2-type-related cytokines IL-13 and IL-4,as well as regulatory cytokine IL-17.And Hc-CRT and Hc-Hsp70 also promote goat PBMC to produce more NO.It suggests that both Hc-CRT and Hc-HSP70 can promote anti-inflammatory immune responses in goat PBMCs.In conclusion,in this study,we found that cells from irradiated H.contortus larvae exhibit characteristics of immunogenic cell death;and also found that the immunogenicity-related stress molecules Hc-CRT and Hc-HSP70 could promote anti-inflammatory immune responses in goat PBMCs.This laid a foundation for further elucidating the cellular and molecular mechanisms of immunogenic cell death in irradiated H.contortus larvae.