Abstract
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).
Highlights
Fasciola gigantica infection threatens the health of both humans and animals in the world
F. gigantica excre‐ tory/secretory products (ESPs) (FgESPs) possibly induces a DC2 phenotype that activates Th2 and Treg cell differentiation in vitro In order to determine the direction of dendritic cell (DC) differentiation induced by FgESPs, DCs were incubated with FgESPs in vitro for 48 h, while phosphate-buffered solution (PBS) served as controls
The results showed that the mRNA levels of surface molecular markers and cytokine of DC1 (CD86, interferon gamma (IFN-γ)) and DCreg (C1QA, STAB1) were significantly lower than those in PBS-treated DCs (t(2) = 9.888, P = 0.0101 for CD86; t(2) = 5.464, P = 0.0319 for IFN-γ; t(2) = 8.350, P = 0.0140
Summary
Fasciola gigantica infection threatens the health of both humans and animals in the world. The objective of this study was to investigate the potency of FgESPs in shifting the differentiation and immune functions of buffalo DCs. Fascioliasis caused by Fasciola hepatica in temperate regions and Fasciola gigantica in tropical regions has been considered an important but neglected zoonosis with an increasing number of people and livestock animals infected around the world [1]. Helminths and helminth-derived molecules condition DCs to generate a non-classical maturation during infection, and prime a protective Th2 or Treg response which cause less damage to either the parasite or the host [14, 15] These multicellular flukes, unlike protozoans and microorganisms, release excretory-secretory products (ESPs) to influence the host immune system as a useful strategy for survival. Fasciola gigantica and its products are known to elicit a modest Th2 response to attenuate the harmful Th1-type and
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