Changes Of Intestinal Microflora And Cellular Immune And Its Mechanism In Malaria Mice In Early Erythrocytic Stage

Background:In tropical developing countries or regions,the infection itself and the enteritis and diarrhoea caused by malaria pose a serious threat to human health.The mechanism by which malaria causes intestinal complications is not fully understood.Host-protozoan and host-microbe interactions are known to alter the host’s immune defense against pathogen infection.At present,the influence of Plasmodium on gut microbiota of vertebrate hosts is rarely reported.The research results of Japanese scholars showed that P.berghei ANKA caused gut microbiota changes in C57BL/6 mice and Balb/c mice,which were believed to be gut microbiota disorders or disorders which were related to intestinal lesions of mice.Studies have shown that gut microbiota changes,immune responses and Plasmodium infection of the same mice from different businesses were still different,let alone the relevant changes among different mice.In addition,in animal experiments,gut microbiota can reduce the burden of the blood-stage malaria parasite and regulate the severity of malaria.Therefore,we believe that gut microbiota changes may not necessarily be due to microflora disorders or disorders.CD8~+T cells have antimalarial effect on Plasmodium in the exo-erythrocytic stage,but their effect on Plasmodium in the erythrocytic stage is controversial.Therefore,the effect of CD8~+T cells on plasmodium in the erythrocytic stage is worth studying.Interleukin 17-CD8~+T cells(Tc17 cells or Tc17)are a subset of CD8~+T cells.It is known that Tc17 has anti-tumor and anti-pathogen effects,but there are no reports on the interaction between Tc17 and malaria parasites in early erythrocytic stage.Preliminary results showed that Plasmodium can increase the percentage of Tc17 cells in the peripheral blood and spleen of mice.CD8~+regulatory T cells(CD8~+Treg or T8reg)have the functions of inhibiting il-17 secretion and Tc17 differentiation,etc.Currently,there are no reports on the interaction between T8reg and Plasmodium in the erythrocytic stage.Objuctive:1.To investigate the changes of gut microbiota of the host of Plasmodium in the erythrocytic stage and the relationship between the changes of gut microbiota and the course of malaria.2.To investigate the changes of CD8+T cells,Tc17 cells and T8reg in peripheral blood and spleen of mice in the early early erythrocytic stage,and to study the interaction and mechanism of between Tc17 and Plasmodium in the early erythrocytic stage.Methods:1.Effects of Plasmodium in the erythrocytic stage on gut microbiota of mice:establishment of animal model,deinsectization treatment,collection of fecal samples in different time periods,DNA extraction,polymerase chain reaction(PCR)amplification and product purification,product quantification and homogeneity,establishment of PE library,Illumunx sequencing 16rRNA,bioinformatics analysis.2.Effects of Plasmodium in early erythrocytic stage on Tc17 in peripheral blood and spleen of mice in normal environment:animal models were constructed,specimens were sampled and then detected by flow cytometry.3.Effects of infusion of Tc17 on malaria mice and Plasmodium in erythrocytic stage under normal conditions:isolation of CD8~+T cells,construction of animal models,induction of Tc17 production,infusion of Tc17 into mice,construction of animal models,thin film smears which was used to check the infection of Plasmodium,and the behavioral status and time of death of the malaria mice were observed.4.Effects of Plasmodium in erythrocytic stage on Tc17 in vitro:(1)Constructed animal models,and then isolated Plasmodium-infected red blood cells(iRBC),iRBC-containing whole blood cells,and normal mouse spleen CD8~+T cells,and then co-cultured iRBC and CD8~+T cells for 3 days and co-cultured whole blood cells containing iRBC and CD8~+T cells for 3 days,and then specimens were detected by flow cytometry;(2)Animal model was constructed,iRBC and malaria mouse spleen CD8~+T cells(iCD8~+T cells)were isolated,and then iRBC and iCD8~+T cells were co-cultured for 3 days,and then specimens were detected by flow cytometry;(3)Animal models were constructed,iRBC and iCD8~+T cells were isolated,and iRBC and iCD8~+T cells were co-cultured for 1 day,and then specimens were detected by flow cytometry.(4)Animal models were constructed,iRBC and iCD8~+T cells were isolated,and iRBC and iCD8+T cells were co-cultured and added transforming growth factor beta(TGF-β)and Interleukin6(IL-6)for 1 day,and then specimens were detected by flow cytometry.5.Effects of Plasmodium in erythrocytic stage on CD8~+CD25~+Foxp3~+T cells(T8reg)in peripheral blood and spleen of mice in normal environment:constructed animal models,sampled and measured by flow cytometry.Results:1.The gut microbiota of mice was changed after the mice were infected with Plasmodium.After the elimination of Plasmodium,the gut microbiota of mice was changed and the composition of microflora could not be restored to the pre-infection state.The dominant bacterium in the pre-infectious gut microbiota of mice gradually become non-dominant bacterium after recovery from infection,while the non-dominant bacterium in the pre-infectious gut microbiota gradually become dominant bacterium after recovery from infection.This suggests that the change of gut microbiota was not necessarily a disorder or disorder of gut microbiota,but rather a reconstruction or remodeling of gut microbiota homeostasis.2.OTU265-bacterium is a lactobacillus,not a pathogen.OTU234-bacterium and OTU265-bacterium are the dominant bacteria after cure and after health respectively,and their change trend is opposite,so it cannot be excluded that they may be competitive.Unfortunately,it is impossible to isolate and culture OTU234-bacterium,so follow-up studies cannot be carried out.3.OTU147-bacterium increased during malaria and returned to healthy levels after malaria was cured.It is worth studying whether the changes of this bacterium belong to disorder or self-steady state reconstruction.However,because the bacterium could not be isolated and cultured,follow-up studies could not be carried out.3.In the normal environment,Plasmodium in erythrocytic stage promoted the increase of the percentage of Tc17 in peripheral blood and spleen of mice,and the percentage of Tc17 in peripheral blood increased gradually,while the percentage of Tc17 in spleen increased first gradually increased and then returned to normal levels.4.Infusion of Tc17 in normal environment delayed the emergence of Plasmodium in the peripheral blood of mice by 1 day(P<0.05).The delay time of death in every mouse was delayed(P<0.05).5.Effects of Plasmodium in erythrocytic stage on Tc17 in vitro:(1)iRBC and CD8~+T cells were co-cultured for 3 days to promote the production of Tc17(P<0.05).With the increase of iRBC,the percentage of Tc17 increased first and then decreased(P<0.05).After whole blood cells containing iRBC and CD8~+T cells were co-cultured for 3 days,and in the case of a small number of whole blood cells,the percentage of Tc17 was promoted(P<0.05).(2)iRBC and iCD8~+T cells were co-cultured for 3 days to promote the percentage of Tc17 was decreased(P<0.05).(3)iRBC and iCD8~+T cells were co-cultured for 1 day to promote the percentage of Tc17 was increased(P<0.05).(4)TGF-and il-6 promoted plasmodium to increase the proportion of Tc17(P<0.05).6.Effects of Plasmodium in erythrocytic stage on CD8~+CD25~+Foxp3~+T cells(T8reg)in peripheral blood and spleen of mice in normal environment:(1)The proportion of T8reg increased in the first two days(P<0.05).With the increase of the number of Plasmodium parasites,the increase trend showed a downward trend,and returned to the normal level on the third day.(2)In the spleen,the changes of T8reg were consistent with those of Tc17,while in the peripheral blood,the changes were contrary.Conclusion:1.Plasmodium causes changes in the composition and proportion of gut microbiota.2.This change is more likely to be a reconstruction or remodeling of gut microbiota homeostasis than a disorder or disorder of gut microbiota.3.Both OTU234-bacterium and OTU147-bacterium are worthy of study when isolation and culture techniques allow.4.Plasmodium in erythrocytic stage promotes the increase of Tc17 in mice;5.Plasmodium in erythrocytic stage promotes the increase of Tc17 level,which is affected by the malaria parasite itself,the number of malaria parasites,the state of CD8~+T cells,TGF-βand IL-6,and T8reg.These may be one of immune evasion mechanism of Plasmodium.6.Tc17 has antimalarial effect,but CD8~+T cells do not.