The Function And Mechanism Of Autophagy Protein 5 Of DCs In Controlling Malaria Blood Stage Infection

Background:Malaria is one of the three-devastating infectious diseases in the world.According to the WHO’s 2022 annual report,the number of malaria cases worldwide is about 247 million,resulting in 619 000 deaths（WHO:World Malaria Report 2022）.Although the number of new malaria cases and deaths worldwide are declining,the situation of malaria prevention and control is still not optimistic with the emergence and spread of artemisinin-resistant Plasmodium and Anopheles mosquitoes.More importantly,artemisinin does not prevent malaria infection,Malaria prevention ultimately depends on an effective malaria vaccine.However there is no safe and effective malaria vaccine licensed,the main reason for this is that the host anti Plasmodium immune protection mechanism is still unclear.Plasmodium-specific CD4⁺T/CD8⁺T cells responses are known to play an important role in protecting the host against the malaria blood stage infection.Studies have confirmed that Plasmodium-specific CD4⁺T cells can not only enhance the phagocytosis of p RBCs by macrophages by secreting IFN-γ;At the same time,it can also assist B cells to secrete Plasmodium-specific antibodies,enhance the neutralization of merozoites and the phagocytosis of p RBCs by macrophages.CD8⁺T cells can target and kill reticulocytes infected with Plasmodium through cytotoxicity,and it can also enhance the phagocytosis of p RBCs by macrophages in a Fas L-dependent manner.Therefore,elucidating the regulatory mechanism of Plasmodium-specific CD4⁺T/CD8⁺T cells activation can provide an important theoretical basis to design effective malaria vaccines.Dendritic cells（DCs）are known to play a key role in activating Plasmodium-specific CD4⁺T/CD8⁺T cells.Studies have shown that classical DCs（c DCs）subsets rather than plasma cell-like DCs（pDCs）are responsible for the activation of Plasmodium-specific CD4⁺T/CD8⁺T cells by providing antigen signals,co-stimulatory molecular signals,and cytokine signals.Autophagy proteins are involved not only in the regulation of classical autophagy,but also in the regulation of non-canonical autophagy,which can regulating DCs antigen presentation and T cell activation processes.However,the role and mechanism of ATGs and their mediated autophagy and LAP in controlling malaria blood stage infection are unclear.Therefore,we analyzed the differential expression of DCs autophagy protein ATGs through RNA-seq before and after host infection,and further explored the effect and mechanism of up-regulated ATG5 in host resistance to malaria blood stage infection,which can provide the necessary theoretical basis for the successful development of malaria vaccines.Contents and methods:1.To detect the change of ATGs in the spleen DCs of infected mice,firstly used transcriptomics and quantitative PCR technology to analyze the differential expression of DCs autophagy proteins ATGs before and after Plasmodium blood stage infection.2.To investigate the role of DCs autophagy protein ATG5 and its mediated autophagy or LAP in host anti-Plasmodium blood stage infection,constructed ATG5 condition-deficient mice(ATG5^f/fCD11c-Cre^+/+),autophagy condition-deficient mice(ULK1^f/fCD11c-Cre^+/+)and LAP condition-deficient mice(Rubcn^f/fCD11c-Cre^+/+)in DCs,and observed their Plasmodium infection.3.In order to explore the mechanism of inhibiting the development of Plasmodium blood stage in the host by the deletion of autophagy protein ATG5 from DCs,the activity of Plasmodium-specific CD4⁺T cells,CD8⁺T cells and antibodies in ATG5^f/fCD11c-Cre^+/+mice were detected,and in vivo depletion experiments confirmed that the enhanced CD4⁺T/CD8⁺T cells responses were contributed to the well-controlled Plasmodium in the ATG5^f/fCD11c-Cre^+/+mice.4.In order to explore the specific molecular mechanism of ATG5^f/fCD11c-Cre^+/+infected mice to enhance the activation of Plasmodium-specific CD4⁺T/CD8⁺T cells,possible potential effector molecules were identified by flow cytometry and single-cell sequencing.5.In order to explore the role and mechanism of DCs autophagy protein ATG5 in host resistance to Plasmodium chronic infection,the chronic infection model of Plasmodium chabaudi（P.chabaudi）was used to carry out blood stage infection in ATG5^f/fCD11c-Cre^+/+mice and observe the changes of parasitemia,and the molecular mechanism of host control in chronic infection was preliminarily discussed.Results:1.RNA-seq of Spleen DCs in Plasmodium yoelii 17XNL-infected mice showed that the levels of ATG5 instead of other autophagy proteins increased significantly after infection,D3and D6.Subsequently,quantitative PCR was used to further confirm that Plasmodium yoelii17XNL infection can selectively induce host DCs to express ATG5.2.Plasmodium yoelii 17XNL infected ATG5 condition-deficient mice(ATG5^f/fCD11c-Cre^+/+),autophagy condition-deficient mice(ULK1^f/fCD11c-Cre^+/+)and LAP condition-deficient mice(Rubcn^f/fCD11c-Cre^+/+)models found that DCs deletion ATG5can significantly inhibit the growth of Plasmodium in the host,while DCs deletion autophagy or LAP does not affect parasitemia in host.3.Flow cytometry and ELISA detection experiments showed that ATG5^f/fCD11c-Cre^+/+infected mice had significantly higher proliferation and IFN-γsecretion numbers of Plasmodium-specific CD4⁺/CD8⁺T cells than those of the control group.The number of Spleen germinal center B cells（GCBs）and the plasma titers of Plasmodium-specific antibodies increased with the progression of infection,but there were no significant differences compared with the control group.4.In vivo depletion experiment showed that after antibody specifically removed CD4⁺T or CD8⁺T cells in ATG5^f/fCD11c-Cre^+/+infected mice,parasitemia significantly elevated to control level.5.Flow cytometry detected the expression of MHC molecules and co-stimulatory molecules on the surface of spleen c DCs of ATG5^f/fCD11c-Cre^+/+infected mice,and it was found that the expression levels of MHC molecules and co-stimulatory molecules on the spleen c DCs surface of ATG5^f/fCD11c-Cre^+/+infected mice increased significantly with the infection process,but there was no statistical difference with the control group.6.Single-cell sequencing of CD11c⁺cell and flow cytometry showed that compared with the control group,the phagocytosis function of spleen c DCs in ATG5^f/fCD11c-Cre^+/+infected mice was significantly enhanced,and the pattern recognition receptor DC-SIGN（CD209a）on the c DCs surface increased significantly with infection.7.The P.chabaudi chronic infection models showed no significant change in parasitemia of ATG5^f/fCD11c-Cre^+/+infected mice compared with the control group.8.ELISA detection and flow cytometry showed that the serum colony-stimulating factor（CSF）1 of P.chabaudi infected mice reached the highest level in the chronic infection stage,while there was no significant difference in the CSF1 receptor（CSF1R）expressed on the surface of innate immune cells.After depletion of CSF1 or CSF1R in vivo,it was found that the parasitemia in infected mice increased rapidly in chronic infection stage,and flow cytometry further found the frequency of spleen red pulp macrophages（RPMs）and non-classical monocytes（NCMs）cells decreased significantly.Conclusion:1.DCs autophagy protein ATG5 regulates the growth of Plasmodium yoelii in an autophagy-independent manner during blood stage.2.DCs autophagy protein ATG5 affects host Plasmodium-specific CD4⁺T/CD8⁺T cell activation.3.DCs autophagy protein ATG5 may regulate the expression of DC-SIGN.4.DCs autophagy protein ATG5 is not involved in the controlling of P.chabaudi chronic infection.