The Prliminary Study On The Innate Immune Response Mechanism Of DC Against Cryptosporidium Parvum Mediated By TLR4

Cryptosporidiosis is one of the important protozoan disease with worldwide distribution,which can infect human being and many animals,and it can cause severe diarrhea to them.Cumulative data indicate that the width and severity of its harmfulness are obvious.Self-limiting infection can be found to those who are immune-complete,but those are immune-deficient individuals couldn’t clear it after infected such as AIDS patients,etc.which lead to chronic infection accompanying with severe diarrhea.However,there still is no effective measures to treat and control it by using drugs and vaccines.Nowadays,the immune mechanism on C.parvum is still not completely clear,but many studies showed that dendritic cells(Dendritic cells,DC)in the innate immunity plays an important role,Toll-like receptors(TLRs)from DCs are a class of important pattern recognition receptors that recognize a variety of pathogen-associated molecular patterns(PAMPs),and also can regulate natural immune and adaptive immune responses.TLRs recognition abilities to different pathogens is different,and TLR4 plays an important role in anti-parasitic infection.Additionally,the data have shown there are a plenty of glycoprotein antigens from C.parvum on its surface,which can provides the basis for TLR4 recognition to C.parvum and induce the innate immune response,but there still is no detail report on the mechanism of TLR4 respond to C.parvum.Based on the above mentioned,this study was to investigate the mechanism of TLR4-mediated DC immune response to C.parvum to elucidate its importance in C.parvum congenital immune.Part 1:Cloning and expression of murine TLR4 gene.The TLR4 gene was amplified by RT-PCR,Then,TLR4 gene was connected with vector p GEX-4T to construct the recombinant plasmid of p GEX-4T-TLR4,and it was expressed by induction of IPTG.Part 2:The test was to carry out TLR4-mediated DC adhesion to C.parvum in vitro.Firstly,C.parvum oocysts were isolated and purified by gradient centrifugation,the oocysts were digested by sodium hypochlorite to acquire sporozoites,and its sporozoites were marked by 5(6)-carboxy diacetic acid fluorescein succinimide Ester(CFSE).At the same time,bone marrow-derived dendritic cells from mouse were isolated and cultured in6 well plates for 7 d,then 1 m L 1×10~6 DCs mixed with the 1 m L 2×10~5 marked sporozoites and 0.5 m L TLR4(final concentration is 1μg/m L)in the group of TLR4antibody seal,1 m L 2×10~5 DCs mixed with the 1 ml 2×10~5 marked sporozoites in the group of TLR4 antibody unseal,the blank control group was added with 1.5 m L RPMI1640 medium,three repeats also were set up in every group in the trial.After DCs were infected by sporozoites for 2 h,the CD11c~+level of DCs was detected by flow cytometry to analyze the maturation degree of dendritic cells in each group.After infected 24 h,the adhesion of Cryptosporidium sporozoites to dendritic cells was examined by fluorescence microscopy.In vivo test,3 groups were set up,namely the group of blank control(BC),the group of TLR4 antibody seal(TAS),the group of TLR4 antibody unseal(TAU).TAS and TAU were infected by C.parvum,the discharges of C.parvum oocysts were examined 2 days later,the examination continued for 6 days.After the infection at 5 d,8 d and 12 d,the proliferation of T lymphocytes from spleen and the expression of TLR4,IL-4,IL-6,IL-12,IL-18 and IFN-γfrom peripheral blood was determined to explore the relation of TLR4 to C.parvum infection,and the data were analyzed by statistical method.The results showed that the murine TLR4 gene was successfully cloned with the size of753bp,its homology highly was 99%comparing with the reported sequences from Gen Bank(NM021297.3).Recombinant expression plasmid p GEX-4T-TLR4 was gained,and it could be expressed by induction of IPTG,the protein size was about 54 k Da;Flow cytometry detection results showed that the expression levels of CD11c~+between TAS and TAU were 67.67±1.80 and 83.37±3.73 respectively,they were higher than that of the blank control,the difference was obvious(P<0.05),and the difference between TAS and TAU also were obvious(P<0.05).The results of CD11c~+expression showed that the peak in TAS was lower than that of in TAU,which indicated that the DC’s maturity degree in TAS was lower than that of TAU;In the trial on C.parvum sporozoite adhesion to DCs,it was found C.parvum sporozoite could directly adhere to DC,however the adhesion ability in TAS was lower than that of TAU.After the mouse were challenged by C.parvum,the isolated T lymphocytes from spleen respectively were labeled with PE anti-mouse CD3,APC anti-mouse CD4,FITC anti-mouse CD8a,the CD3CD4CD8 were determined by flow cytometry.It indicated that CD4~+CD8~-/CD8~+CD4~-expression between TAS and TAU separately was 20.62±0.723/70.99±1.132 and 50.11±1.651/30.28±2.829,they were higher than that of the blank control,the difference in CD4~+CD8~-was obvious compared with the control(P<0.05),and the difference between TAS and TAU also were obvious(P<0.05),it displayed that TLR4 could promote the proliferation of T lymphocytes.In cytokines expression,it showed the higher expression level at the 8th day after infection,the levels of TLR4,IL-4,IL-12,IL-18 and IFN-γin the TAU improved 4.2,4.3,11.7,6.1and 7.7 times compared with TAS’s,but it was no obvious difference in IL-6 between TAU and TAS.Finally,TLR4 distribution and expression in different organs was detected by immunohistochemistry,the results indicated TLR4 expression level in TAU was the highest one,following as TAS,blank control group by immunohistochemistry.In a conclusion,it suggested that TLR4 could mediate DC to recognize and adhere to C.parvum,and Th1 immunoreaction by TLR4 induction to C.parvum was the main path.It could provide theoretical basis to understand the mechanism on innate immunity of C.parvum.