| Several characteristics of HIV-1 infection,including massive CD4+T cell depletion in lamina propria,structural and functional damage of the gut-associated immune system,destruction of intestinal epithelial cells,microbial translocation and sustained immune activation,make it considered as a disease of the Gustrointestinal tract(GI).It is of great significance to promote the innate immunity of intestinal system and establish a specific anti-HIV-1 immune response at the surface of the intestinal system.IL-22/IL-22R1 and XCL1/XCR1 are two important pairs of mucosal related molecules.Accumulated studies have shown that IL-22-IL-22R1,an important pair of innate immune molecules in the mucosal system,has a potential therapeutic regulatory effects,for which play an important role in inflammation,anti-bacterial infection and tissue maintenance and repair.XCL1-XCR1 plays an important role in regulating the composition of intestinal T cells and DCs,and participating in intestinal antigen presentation and maintaining intestinal immune homeostasis.Not only that,XCL1 is also considered to be a potential mucosal immune adjuvant.These findings are undoubtedly obtained in mouse models,as an irreplaceable animal model for human diseases,rhesus macaques are still poorly understood particularly.In order to make better use of rhesus macaques to study the role of IL-22 in HIV infection and the application of XCL1 in HIV-1 vaccine development,we have accomplished the following investigations:To understand the molecular characteristics and functions of rhesus macaque IL-22 and its receptor IL-22R1,the nucleotide sequences of IL-22 and IL-22R1 cDNA were cloned using RT-PCR and RACE amplification methods.The full-length of IL-22 cDNA is 1163 bp(69 bp-5' UTR,540 bp-ORF and 554 bp-3' UTR).The genomic mapping revealed that the IL-22 gene contains five exons,locates on chromosome 11,and forms IFN/IL-10 related cytokine gene clusters with genes such as IFN-y and IL-26.The full length of rhesus macaque IL-22R1 cDNA is 2802 bp(32 bp-5' UTR,1725 bp-ORF and 1045 bp-3'UTR).The IL-22R1 gene contains seven exons,locates on chromosome 1,and promoter analysis revealed that IL-22R1 does not have a conserved TATA box,but there are two CpG islands upstream of the promoter sequence.The amino acid of rhesus IL-22 and IL-22R1 shows 95.5%and 96%similarity with that of human IL-22 and IL-22R1,respectively.In addition,other conserved sites such as cysteine,N-glycosylation sites,and ligand-receptor binding sites are all consistent with that of human IL-22 and IL-22R1.Real-time RT-PCR was used to analyze the expression and distribution of IL-22 and IL-22R1 in different tissues of normal rhesus macaques.It was found that both IL-22 and IL-22R1 are highly expressed in various parts of the intestinal tract,which indicated that IL-22-IL-22R1 plays an important role in the intestinal immune system.To further investigate the mucosal epithelial associated bioactivities of rhesus macaque IL-22,and compare its difference with that of human IL-22.In this study,rhesus macaque IL-22 was purified from E.coli system.Functional experiments with intestinal epithelial cells stimulated by IL-22 revealed that both rhesus macaque IL-22 and human IL-22 could activate downstream signaling molecules STAT3 at Tyr705 without affecting Ser727 phosphorylation.In contrast,the peak of STAT3 Tyr705 phosphorylation initiated by rhesus IL-22 appeared earlier than that of human IL-22.In addition,rhesus IL-22 and human IL-22 can promote the expression of a various antimicrobial peptides by epithelial cells,such as BD-2,S100A8,S100A9,Reg?? and Muc1.Not only that,rhesus IL-22 could also promote cell proliferation at low concentration,while inhibited cell proliferation at high concentration.What's more,apoptosis experiments demonstrated that IL-22 had no significant effect on cell apoptosis nor expression of anti-apoptosis associated proteins.To elucidate the pathological changes of IL-22 associated molecules after SIV/SHIV infection.In this study,we detected the expression of IL-22 and IL-22R1 in six parts of the intestinal tract by real-time RT-PCR.It was found that there was a significant down-regulation of IL-22 in the colonic tissue,while its receptor IL-22R1 has obviously increased expression,which is also corresponding to existing literatures confirming that IL-22 producing cells including Th17,Th22,and ILC3s were depleted after SIV/HIV infection.While its receptor IL-22R1 shows the opposite expression tendency,the underlined mechanism and functional effect were worth exploring.To understand the mechanism of up-regulated of IL-22R1,further studies were carried out with the colonic tissues where there was a significant changes in the expression of both IL-22 and IL-22R1.After screening for the expression of multiple inflammatory factors,it was found that IL-1? also showed up-regulated expression in synchronization with IL-22R1.In vitro experiments demonstrated that IL-1? significantly up-regulates the expression of IL-22R1 at the transcriptional level in a concentration dependent manner.The RNA stability experiment showed that the degradation curve of IL-22R1 mRNA in IL-1?stimulated cells was almost the same with that of non-stimulated group.What's more,the effect of IL-1? stimulation on IL-22R1 promoter activity was not observed by luciferase report assay.Considering the mucosal barrier damage after SIV/SHIV infection,epithelial cells infiltrate in a complex microenviroment composed of a variety of inflammatory factors,microbial metabolites,and viral proteins.Therefore,further investigation on the synergistically effect with LPS and Gp140 were carried out with IECs.The expression of IL-22R1 was significantly up-regulated by co-stimulation with LPS and Gp 140.the promoter of IL-22R1 would be significantly activated under the co-stimulation with LPS,Gp140,and IL-1? through the luciferase report assay.To further explore the effect of IL-22 and its receptor IL-22R1 on the expression of downstream signaling molecules and associated antibacterial proteins.We further examined the expression and phosphorylation of the downstream signaling molecule STAT3,and found that STAT3 was significantly up-regulated both at mRNA and protein expression levels.Not only that,STAT3 showed more pronounced phosphorylation at Tyr705 after SIV/SHIV infection.After continuing to screen the expression of downstream antibacterial peptides and mucin molecules,the expression of Mucl was increased as well,and has a positive correlation with that of STAT3.At the same time,in order to explore the biological effects of up-regulated expression of IL-22R1 after SIV/SHIV infection,over-expressed IL-22R1 cell model were constructed with the adenoviral packaging plasmid containing IL-22R1 ORF(open reading frame).After stimulated with equal amounts of IL-22,much stronger STAT3 signal activation was observed in IL-22R1 overexpressed cells.Furthermore,compared with adenovirus control cells,overexpression of IL-22R1 cells showed significant cell proliferation after 2 days of continuous culture.To use the advantages of XCL1 as a mucosal adjuvant and XCL1-fused vaccines targeting XCR1+DCs(CD8+DCs),which could promoting the humoral and cellular immune responses against antigen,and explore its application in the development of HIV-1 mucosal vaccines,we have accomplished following studies.First,the cDNA sequence of XCL1 and its specific receptor XCR1 of rhesus macaques were cloned,analyzed and compared with that of human XCL1 and XCR1.Second,the optimized antigenic sequence(V1V2)of the SIVmac239 strain was fused to the C-terminal of XCL1,and the vaccine plasmid(pl.0-XCL1-linker-V1V2)was constructed by inserting the fragments of XCL1-V1V2 into the p1.0 vaccine vector.At the same time,XCL1,V1V2 and structural mutant plasmid LVC11A were also constructed.Each of the vaccine plasmids was confirmed to be able to express effectively by flow cytometry and Western blot.In order to enhance the mucosal immune response,four vaccine plasmids were packaged with chitosan.The gel retardation and package efficiency analysis confirmed that the vaccine plasmid could be effectively encapsulated in chitosan.Transmission electron microscopy showed that the chitosan-plasmid encapsulated microspheres were uniform in size(200-350nm).The DNase I enzyme digestion experiment confirmed that the entrapped nanoparticles could effectively protect the plasmid DNA from enzyme degradation.Finally,rhesus macaques were immunized with the nanoparticles coated with chitosan by intranasal spray.Very weakly of serum IgG and oral IgA has been detected after the third immunization.In the intestine,total IgA and specific IgA against V1V2 region were detected,and there was a higher level of titer produced in LV-targeting plasmid-immunized group than that of V1V2-immunized group.In addition,intranasal immunization with XCL1 DNA carrying the V1V2 domain also induced a stronger cellular immune response.Although the levels of specific sera IgG and IgA immunized with targeting plasmids were higher than that of other groups,there is no statistical difference between different groups.The total IgA levels and specific anti-V1V2 IgA levels in saliva were lower and there was no regularity in antibody levels between different groups.Interestingly,the total IgA antibodies produced in the intestinal tract and the specific anti-V1V2 IgA levels were significantly up-regulated,and there was a significant difference between the LV immunized group and control groups.In addition,the fusion targeting plasmid LV immunized group also caused a weak CD8+T cellular immune response.In summary,the sequences of rhesus macaque IL-22 and its receptor IL-22R1 were cloned and compared with that of human in terms of sequence characteristics and tissue expression.At the same time,in vitro studies demonstrated that rhesus IL-22 can play a key role in intestinal functions,such as induce the phosphorylation of STAT3,up-regulate the expression of antimicrobial peptides and enhance cell proliferation.Those would benefit for the researches of HIV-1 related intestinal mucosal repair by using rhesus macaque IL-22.This study comprehensively analyzed the effects of SIV/SHIV infection on intestinal IL-22,IL-22R1,inflammatory factors,and their downstream signaling molecules and antimicrobial peptides.It was demonstrated that IL-1? would promote the expression of IL-22R1 through activation of IL-22R1 promoter activity under the synergistic effect of LPS and Gp140 in vitro.Furthermore,over-expressed IL-22R1 would enhance signal activation and cell proliferative capacity to cells,which will provide a basis for the pathogenesis of HIV-1-induced intestinal pathology.In this study,XCL1 was used to carry V1V2 antigen to target its specific receptor XCR1,and combined with nano-encapsulating technology to obtain significant intestinal IgA and specific anti-V1V2 IgA levels in nasal cavity immunization.This study provides guidance for promoting HIV-1 vaccine in enhancing mucosal immune response. |
PDF Full Text Request