Research On The Differential Exosomal MiRNAs Of DC2.4 Dendritic Cells Induced By Toxoplasma Gondii Infection

Background:Toxoplasma gondii is an intracellular protozoan that infects humans and other warm-blooded animals.After infection,it can spread rapidly through the blood to all tissues and organs.T.gondii infection stimulates immune cells to rapidly produce IL-12 and IFNγ.Exosomes are a subset of extracellular vesicles released by cells with particle sizes between 30 and 150 nm,which play an important role in cell-to-cell communication.In exosomes,specific macromolecules of proteins,lipids,mRNAs or miRNAs have been detected,some of which are capable of transferring biologically active molecules to recipient cells.When the exosomes are taken up by the target cells,their enriched components will affect the target cells and regulate the target cells.Dendritic cells(DCs)are a type of the immune cells,which are the most powerful professional antigen presenting cells.They can efficiently absorb,process and present antigens.Immature DCs have strong migration ability.Mature DCs can activate the initial T cells effectively and play an indispensable role in the immune system.They are the central link in initiating,regulating and maintaining the immune response.MicroRNAs(miRNAs)are small RNAs of about 20-24 nucleotides in length that are involved in the regulation of gene expression and a number of important life processes,including early development,cell proliferation,apoptosis,and cell death.Methods:Toxoplasma gondii RH tachyzoites were collected in vitro and infected with DC2.4 cells(MOI=3)for 28 hours.Uninfected dendritic cell exosomes(DC-EXO),and Toxoplasma gondii infected dendritic cell exosomes(Tg-DC-EXO)were isolated separately,and the RNA were respectively extracted,and then subjected to high-throughput sequencing.The stable differential expressed miRNA between Tg-DC-EXO and DC-EXO were screened and identified by Q-RT-PCR.The target genes of these stable differential expressed miRNAs were predicted with online softwares of TargetScan and miRanda.The predicted target genes were further analyzed with GO(Gene ontology)enrichment and KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway analysis.Results:DEGseq was used to detect the differential miRNAs based on sequencing,and corrected by multiple hypothesis tests.Those miRNAs with a difference of two times or more and a Q-value of less than or equal to 0.001,were considered as significantly different.A total of 3,434 differentially expressed miRNAs were obtained,and 19 miRNAs with significant differences and stability were screened for further analysis.The target genes of these 19 miRNAs were predicted to be involved in a variety of biological processes and pathways,associated with immune system processes and cell growth,as well as intercellular communication.Conclusion:Infection of DC2.4 cells by Toxoplasma gondii causes changes in the miRNA profile enriched in the exosomes.Those significantly altered miRNAs are associated with the progression of the immune system,particularly the T cell signaling pathway,which makes these differentially expressed miRNAs are critical in studying the immune system’s response to T.gondii infection,and suggests that these miRNAs have potential to be used as biomarkers for detection or prognosis.