Proteomic analyses reveal that Orf virus induces the activation and maturation of mouse bone marrow-derived dendritic cells
Introduction
Orf virus (ORFV), a member of the genus Parapoxvirus, is an ovoid-shaped epitheliotrophic linear double-stranded DNA (dsDNA) virus with a genome of approximately 138 kbp encoding 132 genes (Siegemund et al., 2009; Spyrou and Valiakos, 2015). It causes ecthyma contagiosum (contagious pustular dermatitis) by infecting damaged or scarified skin in sheep and goats worldwide and occasionally infects humans, cats, dogs, and some wild animals (Frandsen et al., 2011; Haig and McInnes, 2002; Kuhl et al., 2003). ORFV infection is self-limiting and the primary lesions usually resolve spontaneously within 3–4 weeks; However, it can be highly lethal in lambs or kids when there is a mixed or secondary infection with other pathogens, resulting in serious economic losses (Haig and McInnes, 2002; Abu and Housawi, 2009). The mechanisms to establish repeated and persistent infections in vivo are almost completely unknown (Zhao et al., 2017). In addition, ORFV has strong immunomodulatory activities and has evolved a strategy for immune evasion via the development of an array of virulence factors (Bergqvist et al., 2017; Weber et al., 2013). Several studies have shown that treatment with inactivated ORFV leads to the activation of protective innate immune mechanisms (Siegemund et al., 2009). ORFV has been shown to stimulate the immune system, leading to release of inflammatory cytokines (Büttner et al., 1995). To date, the host immune response to ORFV has been extensively studied, yet many aspects of the complex host-virus interactions remain unclear.
Priming of the adaptive immune response requires prior activation of the innate immune response. Dendritic cells (DCs) play a vital role in the interaction between the innate and the adaptive immune response (Von Buttlar et al., 2014). DCs are professional antigen-presenting cells and their main function is to take up and process exogenous antigens (Ags) in the peripheral tissues to present them to T cells after migration to the draining lymph nodes (Florez-Grau et al., 2018; Foulon and Foucras, 2008). Immature DCs reside in nonlymphoid tissues where they can capture and process Ags. Thereafter, DCs migrate to the T cell-containing areas of lymphoid organs where they lose their Ag-processing activity and mature to become potent immunostimulatory cells (Cella et al., 1997). Fully mature DCs have the ability to upregulate several immune-related surface molecules, such as co-stimulatory receptors (CD40, CD86), adhesion molecules, and integrin receptors to prime naïve T cells, but have less capacity to internalize Ags than immature DCs (Cella et al., 1997; Chiesa et al., 2011). Mature DCs express increased levels of CD11c and DCs are frequently identified by staining for CD11c (Gil-Pulido and Zernecke, 2017; Son et al., 2002). The function of DCs is the outposts of immune surveillance as they can trigger primary immune reactions against infectious pathogens, including viruses.
Recently, studies have focused on the influence of ORFV on mouse bone marrow-derived dendritic cells (BMDCs). Several reports have demonstrated that ORFV potently promotes the expression of pro-inflammatory cytokines and co-stimulatory molecules of bone marrow-derived DCs. For example, Siegemund et al. (Siegemund et al., 2009) observed that inactivated PPVO can induce release of tumor necrosis factor alpha (TNF-α) and interleukin-12/23p40 (IL-12/23p40) and upregulation of major histocompatibility complex class II (MHC-II), MHC-I, and CD86 by bone marrow-derived conventional DCs (BM-cDCs). von Buttlar et al.11 demonstrated that PPVO induces expression of pro-inflammatory cytokines such as IFN-α and IFN-γ, increases co-stimulatory marker CD86 by bone marrow-derived plasmacytoid DCs (BM-pDCs). However, the effect of ORFV on BMDCs maturation and subsequent innate immune response is poorly understood. To address this issue, the current study used immature BMDCs to assess the early events that occur after DCs encounter ORFV and to investigate the ability of ORFV-stimulated DCs to initiate primary T cell responses. We observed that ORFV can activate maturation and function of DCs. Furthermore, we carried out an analysis of proteome changes in DCs following ORFV stimulation via iTRAQ LC-MS/MS. It is reported that a large dataset of proteome profiles of DCs was detected after stimulation with ORFV; These findings provide enhanced understanding of the mechanisms towards ORFV-mediated activation of DCs maturation. and function and represents proteomic analysis of ORFV-induced activation and maturation of DCs.
Section snippets
Reagents
RPMI Medium 1640 was purchased from Gibco (Grand Island, NY, USA). 10× RBC Lysis Buffer was purchased from BioGems (Westlake Village, CA, USA). LPS was purchased from Sigma-Aldrich (St. Louis, MO, USA). Tris-base, SDS, and the 2-D Quantification Kit were purchased from GE Healthcare (Piscataway, NJ, USA). The iTRAQ Reagents Kit was acquired from Applied Biosystems (Foster City, CA, USA). Formic acid (FA) was purchased from TEDIA (Fairfield, OH, USA).
Indirect immunofluorescence assay
ImDCs on day six in culture were harvested
Confirmation of ORFV enter DCs by IFA
ORFV did not induce a typical cytopathic effect (CPE) in DCs. Whether the virus can enter DC cells was confirmed by the detection of ORFV antigen using IFA. The results clearly showed green fluorescence in DCs inoculated by ORFV at 12, 24, 36, and 48 h. While the mock-infected cells showed no fluorescence (Fig. 1). Furthermore, the fluorescence intensity of the ORFV-inoculated DCs reached their peak at 24 h with no further increase over time. Therefore, ORFV- inoculated DCs at 24 h were chosen
Discussion
The activation of DCs by orthopoxviruses such as VACV, MVA, and ECTV has been well investigated (Samuelsson et al., 2008; Waibler et al., 2007). ORFV is a member of the genus Parapoxvirus which displays only limited homology with orthopoxviruses in terms of virion morphology, protein composition, and genome organization (Lutz et al., 1999; Rehfeld et al., 2018). Therefore, it is of great importance to characterize ORFV interactions with BMDCs to elucidate the mechanisms of DC activation by the
Acknowledgements
The authors acknowledge the use of the facilities of the Instrument Center, a core facility of the Lanzhou Veterinary Research Institute. And the authors would like to thank Dr. Xingquan Zhu for helpful discussions.
This study was supported by the National Key Research and Development Programme (2017YFD0500903, 2018YFD0502000) and Inner Mongolia Science and Technology Major Project “Bami Meat Sheep Industrialization Technology Research and Integration Application.”
Declaration of Competing Interest
I declare on behalf of all authors that the work described was original research that has not been published previously, and not under consideration for published elsewhere, in whole or in part. All the authors listed have approved the manuscript that is enclosed.
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