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Dysregulation of hepatic microRNA expression in C57BL/6 mice affected by excretory-secretory products of Fasciola gigantica.

ABSTRACT: The excretory-secretory products released by the liver fluke Fasciola gigantica (FgESPs) play important roles in regulating the host immune response during the infection. Identification of hepatic miRNAs altered by FgESPs may improve our understanding of the pathogenesis of F. gigantica infection. In this study, we investigated the alterations in the hepatic microRNAs (miRNAs) in mice treated with FgESPs using high-throughput small RNA (sRNA) sequencing and bioinformatics analysis. The expression of seven miRNAs was confirmed by quantitative stem-loop reverse transcription quantitative PCR (qRT-PCR). A total of 1,313 miRNAs were identified in the liver of mice, and the differentially expressed (DE) miRNAs varied across the time lapsed post exposure to FgESPs. We identified 67, 154 and 53 dysregulated miRNAs at 1, 4 and 12 weeks post-exposure, respectively. 5 miRNAs (miR-126a-3p, miR-150-5p, miR-155-5p, miR-181a-5p and miR-362-3p) were commonly dysregulated at the three time points. We also found that most of the DE miRNAs were induced by FgESPs in the mouse liver after 4 weeks of exposure. These were subjected to Gene Ontology (GO) enrichment analysis, which showed that the predicted targets of the hepatic DE miRNAs of mice 4 weeks of FgESPs injection were enriched in GO terms, including cell membrane, ion binding, cellular communication, organelle and DNA damage. KEGG analysis indicated that the predicted targets of the most downregulated miRNAs were involved in 15 neural activity-related pathways, 6 digestion-related pathways, 20 immune response-related pathways and 17 cancer-related pathways. These data provide new insights into how FgESPs can dysregulate hepatic miRNAs, which play important roles in modulating several aspects of F. gigantica pathogenesis.

SUBMITTER: Shi W

PROVIDER: S-EPMC7775122 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Dysregulation of hepatic microRNA expression in C57BL/6 mice affected by excretory-secretory products of Fasciola gigantica.

Shi Wei W He Jun-Jun JJ Mei Xue-Fang XF Lu Ke-Jing KJ Zeng Zi-Xuan ZX Zhang Yao-Yao YY Sheng Zhao-An ZA Elsheikha Hany M HM Huang Wei-Yi WY Zhu Xing-Quan XQ

PLoS neglected tropical diseases 20201217 12

The excretory-secretory products released by the liver fluke Fasciola gigantica (FgESPs) play important roles in regulating the host immune response during the infection. Identification of hepatic miRNAs altered by FgESPs may improve our understanding of the pathogenesis of F. gigantica infection. In this study, we investigated the alterations in the hepatic microRNAs (miRNAs) in mice treated with FgESPs using high-throughput small RNA (sRNA) sequencing and bioinformatics analysis. The expressio ...[more]

PMID: 33332355

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Project description:BackgroundFasciola gigantica infection threatens the health of both humans and animals in the world. The excretory/secretory products (ESPs) of this fluke has been reported to impair the activation and maturation of immune cells. We have previously shown the influence of F. gigantica ESPs (FgESPs) on the maturation of buffalo dendritic cells (DCs). However, the underlying mechanisms remain unclear. The objective of this study was to investigate the potency of FgESPs in shifting the differentiation and immune functions of buffalo DCs.MethodsBuffalo DCs were incubated with FgESPs directly or further co-cultured with lymphocytes in vitro. qRT-PCR was employed to determine the gene expression profile of DCs or the mixed cells, and an ELISA was used to measure cytokine levels in the supernatants. Hoechst and Giemsa staining assays, transmission electron microscopy, caspase-3/7 activity test and histone methylation test were performed to determine DC phenotyping, apoptosis and methylation. To investigate the mechanism involved with DNA methylation, a Co-IP assay and immunofluorescent staining assay were performed to observe if there was any direct interaction between FgESPs and DNMT1/TET1 in buffalo DCs, while RNAi technology was employed to knockdown DNMT1 and TET1 in order to evaluate any different influence of FgESPs on DCs when these genes were absent.ResultsqRT-PCR and ELISA data together demonstrated the upregulation of DC2 and Th2/Treg markers in DCs alone and DCs with a mixed lymphocyte reaction (MLR), suggesting a bias of DC2 that potentially directed Th2 differentiation in vitro. DC apoptosis was also found and evidenced morphologically and biochemically, which might be a source of tolerogenic DCs that led to Treg differentiation. In addition, FgESPs induced methylation level changes of histones H3K4 and H3K9, which correlate with DNA methylation. Co-IP and immunofluorescent subcellular localization assays showed no direct interaction between the FgESPs and DNMT1/TET1 in buffalo DCs. The productions of IL-6 and IL-12 were found separately altered by the knockdown of DNMT1 and TET1 in DCs after FgESPs treatment.ConclusionsFgESPs may induce the DC2 phenotype or the apoptosis of buffalo DCs to induce the downstream Th2/Treg response of T cells, possibly through a DNMT1- or TET1-dependent manner(s).

Project description:BackgroundExcretory/secretory products (ESPs) released by parasites influence the development and functions of host dendritic cells (DCs). However, little is known about changes of DNA (hydroxy)methylation on DC development during Fasciola gigantica infection. The present study aimed to investigate whether F. gigantica ESPs (FgESPs) affects the development and functions of buffalo DCs through altering the DNA (hydroxy)methylation of DCs.MethodsBuffalo DCs were prepared from peripheral blood mononuclear cells (PBMCs) and characterized using scanning and transmission electron microscopy (SEM/TEM) and quantitative reverse transcriptional PCR (qRT-RCR). DCs were treated with 200 μg/ml of FgESPs in vitro, following DNA extraction. The DNA methylome and hydroxymethylome were profiled based on (hydroxy)methylated DNA immunoprecipitation sequencing [(h)MeDIP-Seq] and bioinformatics analyses. qRT-RCR was also performed to assess the gene transcription levels of interest.ResultsFgESPs markedly suppressed DC maturation evidenced by morphological changes and downregulated gene expression of CD1a and MHC II. Totals of 5432 and 360 genes with significant changes in the 5-methylcytosine (5-mC) and the 5-hydroxymethylcytosine (5-hmC) levels, respectively, were identified in buffalo DCs in response to FgESPs challenge. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that these differentially expressed genes were highly enriched in pathways associated with immune response. Some cancer-related pathways were also indicated. There were 111 genes demonstrating changes in both 5-mC and 5-hmC levels, 12 of which were interconnected and enriched in 12 pathways. The transcription of hypermethylated genes TLR2, TLR4 and IL-12B were downregulated or in a decreasing trend, while the mRNA level of high-hydroxymethylated TNF gene was upregulated in buffalo DCs post-exposure to FgESPs in vitro.ConclusionsTo our knowledge, the present study provides for the first time a unique genome-wide profile of DNA (hydroxy)methylation for DCs that interact with FgESPs, and suggests a possible mechanism of FgESPs in suppressing DC maturation and functions that are involved in TLR signaling.

Project description:Widespread Fasciola gigantica infection in buffaloes has caused great economic losses in buffalo farming. Studies on F. gigantica excretory and secretory products (FgESP) have highlighted their importance in F. gigantica parasitism and their potential in vaccine development. Identifying FgESP components involved in F. gigantica-buffalo interactions during different periods is important for developing effective strategies against fasciolosis. Buffaloes were assigned to non-infection (n = 3, as control group) and infection (n = 3) groups. The infection group was orally administrated 250 metacercariae. Sera were collected at 3, 10, and 16 weeks post-infection (wpi) for the non-infection group and at 0 (pre-infection), 1, 3, 6, 8, 10, 13, and 16 wpi for the infection group. FgESP components interacting with sera from the non-infection and infection groups assay were pulled down by co-IP and identified using LC-MS/MS. Interacting FgESP components in infection group were subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway and gene ontology (GO) functional annotation to infer their potential functions. Proteins of FgESP components identified in the non-infection group at 3, 10, and 16 wpi accounted for 80.5%, 84.3%, and 82.1% of all proteins identified in these 3 time points, respectively, indicating surroundings did not affect buffalo immune response during maintenance. 490 proteins were identified in the infection group, of which 87 were consistently identified at 7 time points. Following GO analysis showed that most of these 87 proteins were in biological processes, while KEGG analysis showed they mainly functioned in metabolism and cellular processing, some of which were thought to functions throughout the infection process. The numbers of specific interactors identified for each week were 1 (n = 12), 3 (n = 5), 6 (n = 8), 8 (n = 15), 10 (n = 23), 13 (n = 22), and 16 (n = 14) wpi, some of which were thought to functions in specific infection precess. This study screened the antigenic targets in FgESP during a dense time course over a long period. These findings may enhance the understanding of molecular F. gigantica-buffalo interactions and help identify new potential vaccine and drug target candidates.

Dataset Information

Dysregulation of hepatic microRNA expression in C57BL/6 mice affected by excretory-secretory products of Fasciola gigantica.

Publications

Dysregulation of hepatic microRNA expression in C57BL/6 mice affected by excretory-secretory products of Fasciola gigantica.

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