Study On The Role Of C5a/C5aR Signal During Infection Of Cryptosporidium Parvum

Cryptosporidiosis,a zoonotic parasitosis caused by Cryptosporidium infection,seriously threatens the health and life of human and many livestock and poultry.At present,Cryptosporidium has been found in humans and more than 240 animals(including mammals,birds,reptiles and fishes)within more than 100 countries,causing seriously economic and social effects.Cryptosporidium could cause persistent watery and fatal diarrhea in immunodeficiency livestock,poultry and humans,young hosts and transplanted individuals.Infection with Cryptosporidium in healthy animals or adult animals could become a potential transmission source,although it usually manifests to be absent or mild infection,with symptoms as transient or self-limited diarrhea.In view of the wide distribution and severe harm of Cryptosporidium to hosts,lots of works on pathogenic biology,pathogenic characteristics,vaccines and drugs have been done by scholars in China and abroad.However,no effectively preventive measures and drugs have been developed to control cryptosporidiosis.Previous studies showed that the hosts'defense against Cryptosporidium infection mainly depended on cellular immunity,especially CD4~+T cells.Further,previous works also found that the complement cleavage fragment C5a could regulate the differentiation and effect of CD4~+T cells by binding to its receptor C5a R to participate in the anti-infection immune response.However,the role of C5a/C5a R pathway in Cryptosporidium infection is unclear.Here,the present study investigated the regulatory role of C5a/C5a R signal on the main effector cytokines and transcription factors of host CD4~+T cell during Cryptosporidium infection using zoonotic C.parvum infecting suckling BALB/c mice.The results were obtained as following:Firstly,a C.parvum infecting suckling BALB/c mouse model was constructed.The suckling BALB/c mouse aged 5 days was orally infected with 1×10~7 C.parvum oocysts after excystation.Ileum tissues were collected at 6 h,12 h,24 h,36 h,48 h,3 d,4 d,5 d,6 d,7 d,8d,9 d,10 d,15 d,20 d,25 d and 30 d post infection(pi)of C.parvum.Real-time PCR targeting the HSP70 gene of Cryptosporidium was utilized to determine infection.The HE staining was used for histopathological observation,and the acid-fast staining was used to detect oocysts in the contents of the large intestines.The expression of HSP70 gene could be detected from 6hpi to 25 dpi,with the peak at 7 dpi.At 5 dpi and 7 dpi,Cryptosporidium were found in the ileum villi of the suckling mice.The main pathological lesions presented with abscission and atrophy of ileum villi,lost of structural integrity in mucosa of ileum end,significant widen of intercellular space,and infiltration of polymorphonuclear neutrophils and inflammatory cells.A large number of rose red or dark red C.parvum oocysts were observed in the contents of the large intestines at 7 dpi.Secondly,C.parvum infection induced significantly up-regulated m RNA levels of C5a and C5a R in the ileum tissues of suckling BALB/c mice.The ileum tissues were collected from suckling mice infected with C.parvum,and the m RNA levels of C5a and C5a R in these tissues were determined by using Real-time PCR.Compared with the control group,the expression of C5a was significantly up-regulated in the experimental group at 3 dpi,4 dpi,20dpi and 30 dpi,while the expression of C5a R was significantly increased at 12 hpi,36 hpi,4dpi,5 dpi,7 dpi,15 dpi and 30 dpi,with a peak at 5 dpi.Thirdly,C.parvum infection induced Th1 type response in the ileum tissues of suckling BALB/c mice.The relative expression levels of instructive signals and their master transcription factors Th1,Th2,Th17 and Treg cell subsets in the ileum tissues of infected suckling mice were determined and analyzed by using Real-time PCR.The Th1 cell instructive signal IFN-?was significantly up-regulated at 2 dpi,4 dpi,5 dpi,6 dpi,7 dpi,9 dpi,10 dpi,20 dpi,25 dpi and 30 dpi,with a peak at 6 dpi,and its master transcription factors T-bet was significantly up-regulated at 3 dpi,7 dpi,10 dpi,20 dpi and 30 dpi.The m RNA levels of Th2cell instructive signal IL-4 and its master transcription factor GATA-3 showed dynamic changes,but the expression was significantly down-regulated at most of the time points.The expression of Th17 cell instructive signal IL-17 significantly down-regulated at 5 dpi,6 dpi and 8 dpi,but was significantly up-regulated only at 3 dpi.The m RNA levels of its master transcription factor ROR?t was significantly down-regulated at most time points.The Treg cell instructive signal TGF-?was also significantly down-regulated at most time points,with only up-regulated at 20 dpi,and the expression level of its master transcription factor Foxp3was significantly down-regulated at 3 dpi and 4 dpi.Fourthly,C5a/C5a R signal participated in the interaction between C.parvum and ileum tissue of suckling mice by regulating the immune effect of CD4~+T cells.In order to clarify regulatory effect of C5a/C5a R pathway on CD4~+T cells of C.parvum infecting suckling BALB/c mice,in this study,the C5a R inhibitor(PMX 205)was orally given to the suckling mouse at the dose of 3 mg/kg at 6 h before C.parvum infection according to the weight of the suckling mouse.When and after infection with C.parvum oocysts,suckling mice were orally given with PMX 205 at 3-day interval according to the body weight.Real-time PCR detection showed that,compared with the infection group,the instructive signal IFN-?of Th1 cells in the ileum tissues of infected suckling mice in the inhibitor group was significantly down-regulated at most times(2 dpi,4 dpi,6 dpi,7 dpi,9 dpi and 10 dpi),while the expression of its transcription factor T-bet was significantly down-regulated at 7 dpi,9 dpi,10 dpi and 30dpi.The Th2 cell instructive signal IL-4 in the ileum tissues of infected suckling mice was significantly down-regulated at most times,while its transcription factor GATA-3 was significantly down-regulated at 5 dpi and 7 dpi.The Th17 cell instructive signal IL-17 was significantly up-regulated at most times(2 dpi,3 dpi,4 dpi,5 dpi,8 dpi,and 9 dpi),while its transcription factor ROR?t was significantly down-regulated at most times,but was significantly up-regulated at 25 dpi.The Treg cell instructive signal TGF-?was significantly down-regulated at 4 dpi,6 dpi,7 dpi and 25 dpi,while the transcription factor Foxp3 was significantly down-regulated at 7 dpi and 10 dpi,but was significantly up-regulated at 3 dpi.The ileum tissues of mice in the infection group and the inhibitor group were collected at 6dpi for histopathological observation.It was found that C5a/C5a R signal significantly improved the changes of villi diameter and mucosal thickness in ileum tissue of infected suckling mice,but the ratio of villus height to crypt depth was not significantly different.The C5a/C5a R signal could significantly affect the proliferation of C.parvum in ileum tissue cells based on analysis of Cryptosporidium HSP70 m RNA level.In conclusion,C.parvum infection activated C5a/C5a R signal in ileum tissues of suckling BALB/c mice,and this signal was involved in the interaction between C.parvum and ileum tissues of suckling mice by affecting the expression of instructive signals and master transcription factors of Th1,Th2,Th17 and Treg cell subsets of CD4~+T cells in ileum tissues of suckling mice.The results of the present study provided theoretical basis for further exploring hosts'immune mechanism against Cryptosporidium infection,and screening new ideas for effective prevention and control of cryptosporidiosis.