| Objective To establish a kind of rhesus monkey animal model that can be used to study commensal bacteria.Then,to study the relationship between the changes of the immune system induced by the depletion of commensal bacteria and the changes of Coxsackievirus A16 infection.Methods To establish a primate model with depleted commensal bacteria,rhesus monkeys were fed with a combination of four kinds of broad-spectrum antibiotics,such as aminoglycosides,penicillins,glycopeptides and nitroimidazoles.The real-time PCR and 16S amplicon sequencing techniques were used to detect the diversity and abundance of commensal bacteria in intestine and upper respiratory tract.Genome sequencing and metabolomics were used to analyze the microbial metabolites and the composition of intestinal virome.After CA16 infection,we observed the clinical symptoms,including fever,hand,foot and mouth herpes in rhesus monkeys.RT-qPCR was used to detect viremia and viral load in tissues.H&E staining was used to observe the pathological changes of tissues and organs.The immune system changes and CA16 antigen in rhesus monkeys were detected by flow cytometry,liquid bio-chip technique,expression profiling chip and immunofluorescence method.Results This study was divided into two parts according to the objectives.In the first part of the study about the establishment of the animal model,the results of real-time PCR showed the relative abundance of total 16S rRNA gene decreased by more than 80%on the 9th day after antibiotic treatment(ABX)and the abundance of the bacteria remained on the low level.According to the sequencing results,the OTU level of intestinal commensal bacteria decreased by more than 90%on the 9th day after ABX and the dominant bacteria was transformed into Escherichia-Shigella 14 days after ABX.The abundance of the intestinal bacteria was on the low level after ABX and had no harmful effects on the health of the host.The sequencing results of respiratory samples showed the composition and structure of the commensal bactera in upper respiratory tract of rhesus monkeys were changed at the genus level after ABX,but they were still abundant and diverse.In addition,the results of the virome and the metabolome showed that the richness of phage and DNA virus was significantly decreased after ABX,and there was a positive correlation between the abundance of virome and the diversity of commensal bacteria.After antibiotic treatment,tryptophan,a kind of bacterial metabolite,decreased significantly,while the levels of ornithine,mannitol,threonol and glycosaminoglycan increased significantly.The results of peripheral immune cells showed that the number of T lymphocytes increased significantly and the proportion of B lymphocytes decreased in ABX rhesus monkeys.The transcription profiling of PBMCs in ABX rhesus monkeys showed that the expression levels of IL13 gene,VC AM1 gene and IL1 gene were increased by more than 5 times,and the expression levels of other innate immune-related genes were also significantly changed.In the second part of the study about CA16 infection,the rhesus monkeys with lack of gut commensal bacteria could be infected by CA16.There were clinical symptoms,such as elevated body temperature,herpes around mouth and hand 3～4 days after CA16 infection.Antibiotic-treated rhesus monkeys showed viremia on the 3rd day after infection,whereas normal rhesus monkeys showed viremia on the 5th day after infection.The viremia of antibiotic-treated rhesus monkeys lasted longer and the peak value of viral load was higher compared to conventional monkeys.The results of viral loads in major tissues of antibiotic-treated rhesus monkeys showed the peak value occurs later and higher and the clearance time was longer.Pathological changes were also more serious in antibiotic-treated rhesus monkeys.After CA16 infection,ABX-treated rhesus monkeys did not produce effective neutralizing antibody,and the level of B cells secreting IgG was lower than that of normal rhesus monkeys.Immunofluorescence results confirmed that the depletion of commensal bacteria increased the number of CD3+ T lymphocytes and infected virus particles in the small intestine.The results of immune system of antibiotic-treated rhesus monkeys showed that the proportion of CD3+,CD4+and CD8+T lymphocytes was higher than the normal level before infection,and the levels of CD8+and CD20+lymphocytes were lower than conventional monkeys in the early stage of CA16 infection,while the proportion of Thl lymphocytes decreased significantly.The proportion of Th2 lymphocytes and the levels of various cytokines increased significantly in the middle and late stages of infection.The Thl7/Treg ratio as well as interleukin(IL)-17A levels were changed in CA16 infected monkeys.Compared with normal rhesus monkeys,the proportion of CD3+CD4+IL17A+,CD3+CD4+GATA3+cells increased in small intestine of ABX-treated rhesus monkeys after CA16 infection,while the proportion of CD3+CD4+IFNy+cells decreased,and the proportion of CD4+CD25+lymphocytes had no significant difference between this two groups.Conclusion The results of this study indicated that short-term feeding of high-dose combined antibiotics can effectively deplete the gut commensal bacteria in rhesus monkeys and establish a rhesus animal model for intestinal bacteria research without affecting the health of the host.At the same time,the depletion of commensal bacteria affected the level of bacterial metabolites,the composition of virome and the balance of the immune system in rhesus monkeys.After CA16 virus infection,the antibiotic-treated rhesus monkeys could be infected and have clinical symptoms as same as conventional rhesus monkeys.However,the antibiotic-treated rhesus monkeys had more severe viremia and viral load after CA16 infection and had a longer clearance time.These results may be due to the changes of virus-associated bacterial metabolic products,the high proportion of CD4+T lymphocytes,the decrease of CD20+lymphocytes,and the inability of CD8+T lymphocytes to respond during the early stages of viral infection.In particular,the enhancement of Th2 and Th17 immune response and the up-regulation of various cytokine levels may affect the host inflammatory reaction,the production of antibodies and pathological injury.,thus lead to the persistent infection of CA16.In conclusion,this study confirms that the absence of intestinal commensal bacteria in rhesus monkeys may affect the infection process and pathological changes of CA16 by regulating the balances of Th2/Thl and Th17/Treg immune response of the host.However,the exact mechanism of intestinal bacteria affecting the health of the host has not yet been elucidated,and the mechanisms of interaction between the microbiome-host-CA16 infection still needs to be further studied. |
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