| As an opportunistic pathogen,human microsporidia infection mainly occurs in immunocompromised populations,such as AIDS patients and organ transplant patients.Encephalitozoon hellem(E.hellem)is one of the main microsporidia infecting humans,which can cause symptoms such as cholangitis,conjunctivitis and hepatitis.Furthermore,most of the reported cases of Microsporidia encephalitides were caused by E.hellem infection.Therefore,the impact of microsporidia on human health cannot be underestimated,which has received great attention from researchers all over the world.And more and more reports confirm that healthy individuals with normal immunity can also be infected with microsporidia although without obvious clinical symptoms that are easily overlooked.It is proved that microsporidia can evade the host immune response to achieve long-term latency.Early studies on the effects of human Microsporidia on host immune responses focused on adaptive immune responses,with the deepening of research,much more attention has been paid to the role of host innate immune response in resisting microsporidia infection.At present,most studies on microsporidia and the host immune system have used severely combined immunodeficiency SCID mice or immune-related gene knockout mice.These experimental animals have the disadvantages of harsh feeding conditions,high cost,and difficulty in simulating the infection of immune normal individuals in the natural environment.More importantly,due to the lack of immune-related genes,it is difficult for relevant studies to truly reflect the comprehensive interaction between microsporidia and the host immune system.Therefore,we are committed to establishing a simple,stable and more effective wild-type mouse model of human microsporidia infection.The establishment of this model can provide a favorable tool for studying the interaction between human microsporidia infection with the host immune response.This will also have important implications for studying the infection mechanism of microsporidia and finding key targets for the treatment of human microsporidiasis.In this study,we firstly administered transient immunosuppression to C57BL/6 wild-type mice by intraperitoneal injection of immunosuppressive agents,compared the advantages and disadvantages of different immunosuppressive agents to simulate the natural infection environment as much as possible.Then,E.hellem was infected by intraperitoneal injection,and the mice were continuously monitored for changes in body weight,physical signs,microsporidia colonization,and damage to various organs after infection.Through the above tests,we established the validity and stability of this mouse model.Next,this mouse infection model was applied to a preliminary study on the influence of E.hellem infection on host innate immunity.Firstly,the expression of maturation-related molecules and the secretion of inflammatory cytokines in dendritic cells after E.hellem infection were analyzed.To further explore the impact of microsporidia infection on the host immune system,we used this mouse model to conduct experiments in mice co-infected with E.hellem and other pathogens,preliminarily established the infection method and infection dose.Through this study,it provides a reliable tool and a solid foundation for the research group to elucidate the interaction between Microsporidia and the host innate immune system.The main findings are as follows:1.Establishment of wild-type murine model of E.hellem infection.By reviewing relevant literature reports,it was found that there were two main types of mouse models of microsporidia infection:severe combined immunodeficiency mice(SCID)or immunosuppressed mice.Trough the comparison and analysis of the acquisition methods of these two model mice,our model mice were determined to be simple and easy-to-operate immunosuppressed.The reported methods for obtaining immunosuppressed mice are mainly drug treatment methods.Therefore,we have comprehensively evaluated and compared the advantages and disadvantages of different immunosuppressive drugs,mainly for the two commonly used drugs,cyclophosphamide and dexamethasone.The characteristics of the drug were analyzed,and the comprehensive evaluation was carried out from the aspects of different administration methods(such as intraperitoneal injection,gavage,etc.),administration dosage and administration frequency,finally screened out the best drug treatment method.The specific method is as follows:firstly,the E.hellem used for infecting mice is raised and purified by the existing mature method in our laboratory,and the fresh spores of the purified E.hellem are subjected to germination and cell infection experiments to ensure the pre-infection.The spores of E.hellem have infectious activity,and by reading the literature of related mouse models,the infectious dose of E.hellem was determined,that is,1×10~7 microsporidia were inoculated into mice.Then,C57BL/6 wild-type mice were immunosuppressed by intraperitoneal injection of 5 mg/kg of dexamethasone sodium phosphate,injected continuously for 6 days.And on the 7th day,the mice in the infected group were injected with 1×10~7 purified E.hellem by intraperitoneal injection,and the infection was repeated once to increase the infection probability.So far,we have initially established an immunosuppressed wild-type mouse model of E.hellem infection,and further related experiments are needed to ensure the stability and practicability of this mouse model.2.Validation of the wild-type murine model of E.hellem infection.After establishing the model,we need to verify the validity and stability of the model.First,we analyzed the body weight of mice on the 8th and 17th days after E.hellem infection,and compared with the LPS positive control group,we found that the body weight of the mice did not change significantly after E.hellem infection.Then on the 17th day after E.hellem infection,the mice were dissected and sampled,spleen DCs of the mice were extracted and E.hellem infection was confirmed through PCR detection performing with E.hellem-specific primers.Then we continued to observe the colonization of E.hellem in mouse colon,liver and spleen tissues through tissue sections.IFA results showed that spores of E.hellem were observed in colon and liver tissues of infected mice;H&E staining results also showed that granulomatous lesions were formed in the colon,liver and spleen of infected mice.In addition,E.hellem infection can cause host renal failure,enteritis and hematological diseases.Therefore,we tested the infection of E.hellem by sampling liver,kidney,peripheral blood,feces and urine of infected mice,and using universal primers that can simultaneously detect 4species of human microsporidia to perform real-time quantitative PCR detection condition.Finally,E.hellem infection could be detected by real-time PCR in peripheral blood and fecal samples of infected mice,but not completely.However,the infection of E.hellem could be completely detected by real-time PCR from the urine samples of infected mice,which indicated that E.hellem could be stably infected in this mouse model.The above test results fully prove that the immunosuppressed wild-type mice we established are relatively stable,and the model mice can be used for subsequent related experiments.3.The application of the wild-type mouse model of E.hellem infection.After validating the established model,we also need to apply the model in practical research.Firstly,we used this model to study the effect of E.hellem infection on the host innate immune response.Dendritic cells(DCs)are the most important antigen-presenting cells(APC)in the innate immune system,capable of phagocytosing pathogens and presenting them to T cells.Therefore,we mainly analyzed the effect of E.hellem infection on host DCs.In normal immune responses,various stimuli from different sources induce the maturation of DCs through various pathways,and mature DCs mediate helper T cell activation through the surface antigen-presenting molecule MHC II and the intercellular adhesion molecule DC-SIGN.First,we extracted mouse spleen dendritic cells for culture and collected the supernatant,and then ELISA was used to detect the secretion of inflammatory cytokines IL-6 and IL-12 by DCs;afterwards,flow cytometry was used to detect the expression of mature markers CD40 and CD86 on the surface of DCs.The results showed that compared with the LPS positive control group,E.hellem infection significantly inhibited the up-regulated expression of CD40 and CD86,the mature markers of DCs;at the same time,it significantly inhibited the up-regulated expression of IL-6 by DCs,and inhibited the up-regulated expression of IL-12 by DCs,but no significant difference.Furthermore,we used this model to explore the effect of E.hellem infection on the host response to other pathogens.We injected E.hellem-infected model mice with LPS by intraperitoneal injection to study the immune changes of E.hellem infection in response to LPS stimulation.Simultaneously,we established a mouse model of co-infection between E.hellem and Staphylococcus aureus(S.aureus)to explore whether the regulation of the innate immune system after E.hellem infection affects the host immune response to other pathogens or not.Body weight analysis showed that compared with the LPS positive control group,the weight of the E.hellem-infected mice decreased significantly,and the later recovery was significantly slower;compared with the S.aureus positive control group,the co-infected mice lost weight and the later recovery was significantly slower.RT-qPCR analysis showed that compared with S.aureus and LPS positive control groups,E.hellem infection significantly inhibited the up-regulated expression of antigen-presenting molecule MHC II and intercellular adhesion molecule DC-SIGN on the surface of DCs.It suggests that after E.hellem infection,the immune function of DCs may be inhibited by staying in the host,so that the immunity of the host is reduced and the ability to respond to other pathogens is weakened. |
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