| Cryptosporidium,one of the zoonotic protozoans,mainly infects host gastrointestinal epithelial cells.The infection can damage the intestinal barrier and absorption function in the host,causing cryptosporidiosis with diarrhea as the primary symptom and seriously threatening the health of humans and animals worldwide.However,commercial vaccines and specific drugs have not yet been developed to control cryptosporidiosis.Since the development of cryptosporidiosis is closely related to the host’s immune status,understanding the pathogenic mechanism of Cryptosporidium and the immune defense mechanism of the host would provide a theoretical basis for screening drugs and vaccines candidates.During Cryptosporidium infection,the intestinal mucosal barrier formed by intestinal epithelial cells and tight intercellular junctions is the first line of defense against pathogenic invasion in the intestines.More importantly,intestinal epithelial cells can also exert non-specific protection against infections by recruiting innate immune cells and producing cytokines,initiating and regulating the types and intensities of adaptive immune responses.The adaptive immune response is the key for hosts to clear Cryptosporidium infection and recovery,in which the T cell immune response dominated by CD4~+T cells played a leading role in defense against Cryptosporidium infection.Previous studies found that anaphylatoxin C3a could bind to the specific receptor C3a R on the surface of immune cells/non-immune cells.They played an essential role in a variety of diseases,such as mediating inflammatory responses and tissue damages,regulating tissue regeneration and fibrosis,regulating the proliferation and differentiation of CD4~+T cells.However,C3a/C3a R signaling mechanisms during C.parvum infection have not been elucidated.In this study,the expression levels of C3a and C3a R in the ileum tissues of mice infected with C.parvum were analyzed using a 5-day-old BALB/c mouse model infected with C.parvum.A C3a R antagonist(SB290157)was used to block C3a/C3a R signaling to study the effect and potential regulatory mechanisms of C3a/C3a R signaling on the proliferation of C.parvum in mouse ileum tissues.The following results were obtained:1.A C.parvum infection in the BALB/c mouse model was optimized.The excystation rate of C.parvum could be significantly increased to 85%by increasing the concentration of sodium taurocholate in the excystation fluid and the excystation time.The 5-day-old BALB/c mice were orally infected with C.parvum oocysts,which were excysted using optimized excystation conditions.C.parvum infection in ileum tissues of mice lasted for 29days,with the peak at 6 days post infection(dpi).The infection in large intestinal contents at6 dpi was detected by acid-fast staining,showing red spherical or oval C.parvum oocysts.Molecular biological detection showed that C.parvum infection existed in both ileum and contents within large intestines,and the genotype of the parasite was identified as IId A15G1,consistent with the initially infected oocysts.The histomorphopathological observation in the ileum tissues of mice infected C.parvum showed that Cryptosporidium parasitized the brush border of ileum villi of mice at 6 dpi.In addition,ileum villi were shortened in length,thicker in villi diameter,decreased in the ratio of villi length to crypt depth,and neutrophil and lymphocyte infiltration were seen in the lamina propria.2.The expression levels of C3a and C3a R in the ileum tissues of mice infected C.parvum were determined.The m RNA levels of C3a and C3a R in ileum tissues of mice infected with C.parvum were determined by using q RT-PCR,and the expression of C3a was significantly up-regulated at most of the time points(1 dpi,2 dpi,4 dpi,29 dpi,30 dpi,35dpi,and 36 dpi).However,its receptor C3a R was significantly down-regulated at 1 dpi,2dpi,4 dpi,6 dpi,and 8 dpi,and significantly up-regulated from 10 dpi.Western blotting showed that the protein levels of C3a R were also significantly down-regulated in ileum tissues at 2 dpi and 6 dpi,and immunohistochemical staining showed that C3a R was mainly distributed in ileum epithelial cells.3.The potential mechanisms of C3a/C3a R signaling affecting the proliferation of C.parvum in ileum tissues of mice were clarified.5-day-old BALB/c mouse was intraperitoneally injected with 10 mg/kg C3a R antagonist(SB290157)3 h before infection according to the mouse’s body weights to reduce the expression of C3a R in ileum tissues.Then,the BALB/c mouse was orally infected with 1×10~7 C.parvum oocysts to establish a C.parvum infecting C3a R-inhibited mouse model.After inhibition of C3a R,the m RNA levels of the Cryptosporidium 18S r RNA gene were significantly up-regulated in the ileum tissues of mice infected with C.parvum.The ileum villi length in mice infected C.parvum shortened,villi diameter and mucosal thickness increased,and the ratio of villus length to crypt depth decreased.Further studies found that although m RNA levels of ZO-1,Claudin3,and Occludin in ileum tissues of mice infected with C.parvum were significantly down-regulated,compared with the single infection group,Occludin was significantly down-regulated at most time points(4 dpi,6 dpi,8 dpi,9 dpi,10 dpi,and 29 dpi)in the ileum tissues of mice infected with C.parvum in the C3a R inhibition group,with significant up-regulation only at 1 dpi,35 dpi and 36 dpi.However,both ZO-1 and Claudin3 showed dynamic changes,with no noticeable change trends.The m RNA levels of the cell proliferation marker Ki67 and intestinal stem cell marker Lgr5 in ileum tissues of mice infected with C.parvum were significantly down-regulated.However,compared with the single infection group,the expression levels of Lgr5 in the ileum tissues of mice infected with C.parvum in the C3a R inhibition group were still significantly down-regulated at 6 dpi,8 dpi,29 dpi,30 dpi,and 35 dpi,while Ki67 were significantly up-regulated at 1 dpi,2 dpi,4dpi,6 dpi,29 dpi,and 36 dpi,and significantly down-regulated only at 9 dpi and 35 dpi.The expression levels of the Th1 cell instructive signal IFN-γwere significantly up-regulated,and the Treg cell instructive signal TGF-βwas significantly down-regulated in ileum tissues of mice infected with C.parvum.However,compared with the single infection group,the relative expressions levels of IFN-γand TGF-βin ileum tissues of mice infected with C.parvum in the C3a R inhibition group were significantly increased.These findings suggested that C3a/C3a R signaling would affect the proliferation of C.parvum in mouse ileum tissues by regulating the expressions of Occludin,Lgr5,Ki67,IFN-γ,and TGF-β.In conclusion,this study optimized the model of C.parvum infection in BALB/c mice,and,using this in vivo model,the potential mechanisms of C3a/C3a R signaling affecting the proliferation of C.parvum in ileum tissues of mice were preliminarily elucidated.The results in this study provide a theoretical basis for further exploring the interaction mechanisms between host and Cryptosporidium,and for screening drug targets and vaccine candidates against cryptosporidiosis. |
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