The Roles And Mechanisms Of Exosomes And Circular RNAs In The Pathogenesis Of Cryptococcosis

Chapter 1 Differential expression profiles of lnc RNAs and m RNAs in PBMCs of cryptococcal meningitis patientsAims: Lnc RNAs play a vital role in the pathological and physiological process.This chapter aimed to explore the involvement of lnc RNAs in cryptococcal meningitis.Methods: Microarray was performed in cryptococcal meningitis patients,and then,GO and KEGG pathways were analyzed.Co-expression relationship between lnc RNAs and m RNAs was explored.The expressions of the lnc RNAs and m RNAs,and their changes after treatment were detected by PCR.Results: A total of 325 mRNAs?201 upregulated and 124 downregulated?and 497 lnc RNAs?263 upregulated and 234 downregulated?were identified.The top three enriched GO terms for the m RNAs were arachidonic acid binding,activin receptor binding,and replication fork protection complex.The top three pathways in KEGG were asthma,one carbon pool by folate,and allograft rejection.A total of 305 coexpression relationships were found between 108 lnc RNAs and 87 m RNAs.Lnc RNADPY19L1p1 was significantly increased in patients and decreased after treatment.ROC analysis revealed DPY19L1p1 was a potential diagnostic marker?AUCROC = 0.9389?.Furthermore,the target genes of DPY19L1p1 in cis or trans regulation were mainly involved in immune?related pathways like the interleukin signaling pathway.Conclusions: This chapter analyzed the differential lnc RNA profile in cryptococcal meningitis patients and revealed DPY19L1p1 could be used for treatment evaluation and disease diagnosis.Chapter 2 Contents and functional analysis of extracellular vesicles derived from Cryptococcus neoformans-infected macrophagesAims: Exosomes play a vital role in the cell to cell communication.This present chapter aimed to explore the role of M1 phenotype macrophage-derived exosomes on the immunological function of na?ve macrophage during Cryptocococcus neoformans infection.Methods: Isolated and purified the exosomes from BMDMs under three different conditions: live Cryptocococcus neoformans infection,heat-killed Cryptocococcus neoformans infection and non-infection.First,DLS and TEM were used to identify the size and shape features of these exosomes;Micro BCA kit and MPLEx technique were used to identify the proteomics,lipids and metabolics in these exosomes;Then,co-cultured these exosomes with na?ve BMDM to test their impacts on the phagocytosis,killing function,non-lytic exocytosis and transcriptome changes.Finally,infection mice models were constructed to test whether these exosomes could impact the progress of cryptococcosis.Results: The distributions of these three different originating exosomes were very similar,around 50 nm via both DLS and TEM.No obvious difference were found among these three exosomes in both protein and lipid concentrations.The results of proteomics showed that 46 protein components were differentially expressed in exosomes from three sources,among which ECM receptor interaction signaling pathway was the most enriched.The results of lipids showed that the levels of phosphatidylcholine?PC?,phosphatidylethanolamine?PE?and sphingomyelin?SM?in exosomes from three different sources were significantly different.There was no significant difference in metabolomic results among three groups of exosomes from different sources.The incorporation of exosomes by BMDM happened as early as 30 min upon co-incubation and tended to increase over time.Three kinds of exosomes from different sources can all significantly enhanced the phagocytosis,killing and non-lytic exocytosis of inactivated macrophages and the production of cytokines.Among them,the exosomes secreted by M1 macrophages infected with live Cryptococcus neoformans had the most significant effect.Three kinds of exosomes from different sources?live C.neoformans,hk C.neoformans,without C.neoformans?could significantly change the transciptome of BMDM with 40 differentially expressed genes,54 differentially expressed genes,18 differentially expressed genes,respectively.In vivo animal models,it was found that these exosomes could significantly reduce the local fungal burden both in the brains and lungs of mice infected with Cryptococcus neoformans.Conclusions: After cryptococcal infection,M1-polarized macrophages can induce the naive macrophages to M1 phenotype via effective anti-cryptococcal function by secreting biological exosomes.Chapter 3 The role of circ₀001806 as ce RNA in mi RNA-126/ADM axis in cryptococcosisAims: Explored the role of circ RNAs in Cryptocococcus infection and its regulation mechanism.Methods: circ RNA microarray was used to screen and identify differentially expressed circ RNAs in PBMC of cryptococcal meningitis and healthy controls,and the differential expressed circ RNAs were verified by real-time fluorescent quantitative PCR in a larger sample size.In vivo cryptococcosis mouse model was constructed and the role of circ RNA was studied.Then,primary T cells,B cells and monocytes in peripheral blood were sorted by magnetic beads to clarify the subcellular expression and distribution of circ RNA,combined with high-throughput sequencing technology,the functional target genes of circ RNA were systematically analyzed.Finally,RNA pull-down,si RNA transfection,luciferase report and other methods were used to explore the mechanism of circ RNA.Results:?1?400 circRNAs were differentially expressed in cryptococcal meningitis patients,with P < 0.05 and fold change ? 2 as the cut-off value.Among them,212 circ RNAs were significantly up-regulated,and 188 circ RNAs were significantly down-regulated.Compared with healthy control group,the expression of circ₀001806 in PBMC of cryptococcal meningitis patients was significantly decreased.?2?In vivo model of cryptococcosis in mice,compared with the control group,the fungal burdens of lungs and brains in mice injected with circ₀001806 inhibitory interfering RNA increased significantly;?3?circ₀001806 was mainly expressed in the cytoplasm of T cells.?4?Decreased circ₀001806 could inhibit T cell apoptosis,promote cell cycle G1 S arrest,impair ADM expression,and regulate genes involved in like Mannose type O-glycan ynthesis,fatty acid biosyntheses and nitrogen in T cells after Cryptococcus neoformans infection;?5?During Cryptococcus neoformans infection,the binding of circ₀001806 to micro RNA-126 in T cells significantly increased and co-localized in the cytoplasm;?6?circ₀001806 regulated the expression of ADM and cell cycle and apoptosis in T cells after Cryptococcus neoformans infection partially through the sponge adsorption of micro RNA-126.Conclusions: The low expression of circ₀001806 in cryptococcal meningitis patients may decreased the adsorbtion with micro RNA-126,thus enhancing its inhibition role on ADM gene,promoting cell apoptosis and ultimately leading to less anti-cryptococcal ability during infection.