| Haemonchus contortus is an important gastrointestinal nematode parasite and is mainly infected by sheep and goats.It causes anemia,hypoproteinemia and sometimes death in the infected animals.Financial losses due to parasitism are annually estimated to be in the order of hundreds of millions of dollars.The control of livestock parasitism relies mostly on chemical therapy.However,the growing emergence of resistant strains of this parasite has resulted in an urgent need to search for alternative control strategies.Development of molecular vaccines with high effectiveness and safety has became research highlight.However,up to now,no molecular vaccine with high efficacy of protective immunity is available to be applicated in the field.Previous studies had indicated that the high levels of protective immunity could be induced in the livestock(such as cattle,sheep and goats)vaccinated with irradiation-attenuated(RA)Trichostrongylidae and Haemonchus contortus larvae,whereas very lower levels of protective response against reinfection just could be elicited by animals infected with normal nematode larvae,suggesting that RA-larvae-derived cells are of immunogenic properties,whereas normal-larvae-derived cells have tolerogenic features.In addition,in recent years,many studies have demonstrated that anti-tumor protective immunity can be induced by certain agents(e.g.anthracyclines,?-irradiation and hypericin-based photodynamic therapy)in tumor prophylactic vaccination model,and underlying mechanism is that the cancer cells treated with above agents might undergo immunogenic cell death(ICD),and trigger specific signal transduction and effector molecules,which interact with innate immune cells and finally lead to an adaptive immune response against tumor cells.Therefore,we speculate that RA-larvae-derived cells might undergo ICD and elicit a high level of protective immunity in hosts,whereas normal-larvae-derived cells may exhibit mainly apopototic cell death and induce immune tolerance.However,that whether this hypothesis is true or not remains to be experimentally validated.So,in order to provide a basis for furtherelucidating mechanisms of ICD in RA larvae,in this present study,we investigated developmental and morphological features of both RA and normal H.contortus larvae which were in vitro incubated in continuous culture at different times;then we analyzed transcriptomic expression profiles of aforementioned two groups of larvae.Firstly,we collected and cultured H.contortus third-stage larvae(L3),then H.contortus L3 were irradiated by using UV light.The morphological features of both RA and normal H.contortus larvae cultured for 0 day(2 hours),2 days,5 days,10 days,13 days and 16 days were investigated by optical microscope.The results demonstrated that usual morphological features could be observed at above different developmental stages in normal group cultured in vitro.There was no difference in morphology and vitality between RA and normal larvae groups cultured for 2 hours.When cultured for 2 days,two groups of larvae began to undertake exsheathment and the rate of exsheathed L3 of RA group was higher than that of normal group;no morphological differences could be observed in esophaguses and surface of larvae.When cultured for 5 days,some of larvae were able to develop to L4 stage in both RA and normal groups,but the rate of L4 larvae in RA group was lower than that in normal group,and a significant loss of vitality and damage of intestine cell could be also observed in RA larvae.When cultured for 10 days,80 percent of larvae could develop to L4 stage in normal group,whereas in RA group the rate of developmental larvae was only 20 percent,and RA larvae exhibited morphological deform and decrease in vitality.When cultured for 13 days,some of RA larvae lost their vitality and exhibited serious deform including abnormally darkened intestinal tract,rugged tegument and body shrinkage.When cultured for 16 days,the bodies of RA larvae had been disintegrated,only vacuoles were observed in their usual anatomical position,and larvae bad absolutely lost their vitality;whereas most of normal larvae were of their vitality.These results suggest that RA larvae cultured in vitro might show phenotype of necroptotic death,whereas normal larvae show morphological feature of apopototic death.Secondly,in order to further verify whether RA H.contortus larvae could show necroptotic phenotype,we collected and cultured larvae described as mentioned above.Then,we observed ultrastructural changes of both RA and normal larvae by scanning electron microscope(SEM)and transmission electron microscope(TEM).By means of SEM,we found that the times that tegumental surface structures of larvae presented atrophy in RA group are earlier than that in normal group in the early periodof culturing in vitro,and in the later period of culturing,tegument of RA larvae exhibited rugged and distorted structures,and shrinkage and distortion of RA larvae were more serious than that of normal group.Using TEM we also found that no significant differences in morphological structures could be observed between two groups of larvae in the early period of culturing.When cultured for 10 days and 16 days,normal larvae presented apoptotic morphological changes while RA larvae sharply showed typical necroptotic morphological features.These observations further confirm that there are different forms of death between RA and normal larvae.Finally,we analyzed transcriptomic expression profiles of aforementioned two groups of larvae by using RNA-seq,and found that some genes and pathways associated with early apoptosis and cell growth and development are enriched in the early period of culturing,whereas some molecules and pathways related to cellular necrosis could be enriched in the later period.We also further identified three genes(i.e.HSP70,HMG and CRT)associated with ICD,the expression levels of these three genes from RA larvae were higher than that from normal larvae in the early period f culturing.These findings provide a basis for further exploring molecular mechanisms of ICD in RA larvae.Taken,in this study,we found that RA H.contortus larvae could show necroptotic phenotype,we also preliminarily explored molecular mechanisms of ICD in RA larvae,and identified three genes associated with ICD.These data could provide a solid basis for elucidating cellular and molecular mechanisms of ICD in RA H.contortus larvae... |
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