Elsevier

Fish & Shellfish Immunology

Volume 101, June 2020, Pages 216-224
Fish & Shellfish Immunology

Full length article
ROS induced by spring viraemia of carp virus activate the inflammatory response via the MAPK/AP-1 and PI3K signaling pathways

https://doi.org/10.1016/j.fsi.2020.03.056Get rights and content

Highlights

  • ROS involve in the SVCV-induced inflammatory response in EPC cells.

  • MAPK/AP-1 signaling pathways participate in the inflammatory response induced by SVCV infection.

  • The PI3K signaling pathway involves in the expression of chemokine IL-8 in the SVCV-induced inflammatory response.

  • Inflammatory signaling pathways of MAPK/AP-1 and PI3K are related to SVCV replication.

Abstract

Spring viraemia of carp virus (SVCV) can cause a high mortality in common carp (Cyprinus carpio), and its main pathological processes include the inflammatory response. However, the detailed mechanism is still unclear. Reactive oxygen species (ROS) have been shown to play critical roles in the immune response, including inflammation, in different models. Our previous studies have demonstrated that SVCV infection results in the accumulation of ROS, including H2O2, in epithelioma papulosum cyprini (EPC) cells. In this study, we aimed to explore the relationship between H2O2 accumulation and inflammation during SVCV infection. After EPC cells were infected with SVCV, the expression levels of the inflammatory factors tumor necrosis factor (TNF)-α, cyclooxygenase (COX)-2, and interleukin (IL)-8 were up-regulated, while the expression of the anti-inflammatory factor interleukin (IL)-10 was down-regulated, compared with that in mock-infected EPC cells. The antioxidant N-acetyl-l-cysteine (NAC) could dampen the increased TNF-ɑ and COX-2 expression induced by SVCV and H2O2, suggesting a relationship between ROS accumulation and inflammation during SVCV infection. Dual luciferase reporter assays demonstrated that SVCV could not activate the NF-κB pathway. In addition, inhibition of NF-κB by pyrrolidine dithiocarbamate (PDTC) treatment had no effect on the expression of inflammatory factors. Furthermore, inhibition of the ERK, JNK, and p38MAPK signaling pathways by U0126, SP600125, and SB203580, respectively, reduced the expression of TNF-ɑ, COX-2, and IL-8, indicating that these three signaling pathways were all involved in the inflammatory response after SVCV infection. In addition, the PI3K signaling pathway was involved in the expression of the chemokine IL-8 in the SVCV-induced inflammatory response. We also showed that inhibition of the MAPK or PI3K signaling pathway facilitated the expression of SVCV-G as well as increased the SVCV viral titer. Altogether these results reveal the mechanism of the SVCV-mediated inflammatory response. Thus, targeting these signaling pathways may provide novel treatment strategies for SVCV-mediated diseases.

Introduction

Spring viraemia of carp virus (SVCV), designated as carp sprivivirus by the International Committee on Taxonomy of Viruses in 2018, is a single-stranded negative-strand RNA virus that belonging to the genus Sprivivirus in the family Rhabdoviridae. The genome of SVCV is approximately 11 kb in length and encodes five proteins, including nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and viral RNA-dependent RNA polymerase (L). SVCV is the etiological agent of spring viraemia of carp (SVC), which causes a high mortality in cyprinids, mainly common carp (Cyprinus carpio) [1]. Currently, no drugs or vaccines can control SVCV infection. Therefore, a better understanding the host response during SVCV infection is needed to develop strategies against this viral infection.

Viral infection with viruses such as hepatitis C virus (HCV), human papillomavirus, red-spotted grouper nervous necrosis virus, and grass carp reovirus can produce large amounts of reactive oxygen species (ROS) and induce oxidative stress [[2], [3], [4], [5]]. ROS can elicit extreme biological damage to the host if redox homeostasis is not properly maintained. For example, ROS have been shown to cause DNA damage and apoptosis in a Trichoplusia Tn5B1-4 cell model [6]. However, ROS also have been demonstrated to play important roles in the immune response of different models including mouse, zebrafish, and Caenorhabditis elegans [[7], [8], [9]]. For instance, hydrogen peroxide (H2O2) acts as a long-range signaling molecule that is important for attracting leukocytes to wound sites in zebrafish [7], and mitochondrial ROS play an important role in anti-bacterial immunity and wound healing in C. elegans [8].

Inflammation is part of the complex biological response of body tissues to harmful stimuli, such as pathogens, damaged cells, and irritants. The function of inflammation is to eliminate the initial cause of cell injury, clear out necrotic cells and tissues damaged from the original insult or the inflammatory process, and initiate tissue repair [10]. ROS generated during viral infection are signaling mediators that are important for activation of the phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinase (MAPK), and nuclear factor kappa B (NF-κB) pathways, resulting in the expression of the inflammatory factors tumor necrosis factor (TNF)-α and interleukin (IL)-8 [7,11,12]. As an important component of ROS, H2O2 can induce an inflammatory response through the MAPK and NF-κB pathways thus increasing cyclooxygenase (COX)-2 and inducible nitric oxide synthase expression [13,14]. In mammals, the NLRP3 inflammasome pathway is also involved in ROS-induced inflammation [15].

Oxidative stress is one of the pathogenic mechanisms of SVCV [[16], [17], [18]]. Our previous studies have shown that SVCV infection induces oxidative stress, causes H2O2 accumulation, and, simultaneously, down-regulates the expression of heme oxygenase-1 (HO-1) [16,17]. In addition, we have confirmed that SVCV impairs the function of mitochondrial complex III, resulting in the accumulation of H2O2 [18]. Furthermore, fish infected by SVCV show lethal hemorrhagic swim-bladder inflammation and peritonitis [1], but the detailed mechanism is not well understood. Here, we aimed to explore the relationship between H2O2 accumulation and inflammation upon SVCV infection. Moreover, the effect of ROS induced by SVCV infection on the MAPK/AP-1 and PI3K pathways. These findings will provide new insights into the mechanism of inflammation upon SVCV infection and may contribute to the control of SVCV.

Section snippets

Materials and methods

Cell cultures and reagents. Epithelioma papulosum cyprini (EPC; ATCC: CRL-2872) cells are SVCV-sensitive and were cultured in Eagle's minimum essential medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher) at 28 °C in a humidified 5% CO2 atmosphere. SVCV (ATCC: VR-1390) was a kind gift from Professor Yuanan Lu (University of Hawaii at Manoa). N-acetyl-l-cysteine (NAC) was purchased from the Beyotime Institute of Biotechnology (China). SP600125, U0126,

SVCV infection induces the inflammatory response in EPC cells

To determine whether the inflammatory response was occurred following SVCV infection, the expression levels of the pro-inflammatory factors including TNF-α, (COX)-2, and IL-8 as well as the anti-inflammatory factor IL-10 were measured in EPC cells. The RT-qPCR results showed that the expression levels of TNF-ɑ and COX-2 in SVCV-infected cells were significantly increased by 6.54–38.6-fold and 2.34–16.02-fold, respectively, compared with the mock-infected group (Fig. 1). Similar observations

Discussion

SVCV causes lethal hemorrhagic swim-bladder inflammation and peritonitis as well as a high mortality in cyprinids, suggesting the importance of inflammation in its pathogenicity [1]. This study demonstrated that SVCV could activate the expression of molecules associated with inflammation and that ROS induced by SVCV infection could mediate the inflammatory response via the MAPK/AP-1 and PI3K pathways (Fig. 7).

Previous studies have shown that SVCV induces oxidative stress in EPC cells and

CRediT authorship contribution statement

Jie Sun: Investigation, Writing - original draft. Jingwen Wang: Methodology. Lijuan Li: Writing - review & editing. Zhixin Wu: Methodology. Xiaoxuan Chen: Methodology. Junfa Yuan: Conceptualization, Writing - review & editing.

Declaration of competing interest

The authors declare no financial or commercial conflicts of interest.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (31872598) and the Fundamental Research Funds for the Central Universities (2662017PY041).

References (46)

  • C.M. Yang et al.

    Japanese encephalitis virus induces matrix metalloproteinase-9 expression via a ROS/c-Src/PDGFR/PI3K/Akt/MAPKs-dependent AP-1 pathway in rat brain astrocytes

    J. Neuroinflammation

    (2012)
  • H.J. Jeong et al.

    Down-regulation of MAPK/NF-kappaB signaling underlies anti-inflammatory response induced by transduced PEP-1-Prx2 proteins in LPS-induced Raw 264.7 and TPA-induced mouse ear edema model

    Int. Immunopharm.

    (2014)
  • S.H. Korn et al.

    Cytokine-induced activation of nuclear factor-kappa B is inhibited by hydrogen peroxide through oxidative inactivation of IkappaB kinase

    J. Biol. Chem.

    (2001)
  • A.R. Clark et al.

    Post-transcriptional regulation of gene expression by mitogen-activated protein kinase p38

    FEBS (Fed. Eur. Biochem. Soc.) Lett.

    (2003)
  • D.J. Papachristou et al.

    Activation of the JNK–AP-1 signal transduction pathway is associated with pathogenesis and progression of human osteosarcomas

    Bone

    (2003)
  • P.T. Hawkins et al.

    PI3K signalling in inflammation

    Biochim. Biophys. Acta

    (2015)
  • X.Y. Gong et al.

    SVCV infection triggers fish IFN response through RLR signaling pathway

    Fish Shellfish Immunol.

    (2019)
  • S.A. Read et al.

    Virus induced inflammation and cancer development

    Canc. Lett.

    (2014)
  • M. Guo et al.

    Q. Qin, c-Jun N-terminal kinases 3 (JNK3) from orange-spotted grouper, Epinephelus coioides, inhibiting the replication of Singapore grouper iridovirus (SGIV) and SGIV-induced apoptosis

    Dev. Comp. Immunol.

    (2016)
  • J.S. Peiris et al.

    Innate immune responses to influenza A H5N1: friend or foe?

    Trends Immunol.

    (2009)
  • W. Ahne et al.

    Spring viremia of carp (SVC)

    Dis. Aquat. Org.

    (2002)
  • C.W. Chang et al.

    Betanodavirus induces oxidative stress-mediated cell death that prevented by anti-oxidants and zfcatalase in fish cells

    PloS One

    (2011)
  • D. Lai et al.

    Localization of HPV-18 E2 at mitochondrial membranes induces ROS release and modulates host cell metabolism

    PloS One

    (2013)
  • Cited by (19)

    • Characterization of a novel activating protein-1 (AP-1) gene and the association of its single nucleotide polymorphisms with vibrio resistance in Tegillarca granosa

      2022, Fish and Shellfish Immunology
      Citation Excerpt :

      AP-1 plays an important role in the occurrence of skin and liver tumors and can regulate apoptosis by regulating genes such as Bax and Bcl [15,16]. Although studies on AP-1 in aquatic species have been scarce, recent investigations have implicated this transcription factor in antibacterial, antiviral, as well as antifungal immunity of fish such as the orange-spotted grouper (Epinephelus coioides) [17], carp (Cyprinus carpio) [18,19], grass carp (Ctenopharyngodon idella) [20], and redlip mullet (Liza haematocheila) [21] and marine invertebrates such as the sea cucumber (Apostichopus japonicus) [22], Pacific white shrimp (Litopenaeus vannamei) [23,24], hard clam (Mercenaria) [25], abalone (Haliotis disus) [26], and Hong Kong oyster (Crassostrea hongkongensis) [27], AP-1 caused the production of lysozyme and defensins in the giant river prawn (Macrobrachium rosenbergii) [28]. In Pacific oyster (Crassostrea gigas), AP-1 regulated the expression of members of the IL17 family of inflammatory cytokines [29].

    • Heterosis versus breakdown in fish hybrids revealed by one-parental species-associated viral infection

      2022, Aquaculture
      Citation Excerpt :

      However, the knowledge on mechanisms involved in hybrid vigour or hybrid breakdown in relation to parasite disease in interspecific fish hybrids are almost unknown. Concerning SVCV, its effect on host immunity is widely studied and mostly the mechanisms related to the innate immunity have been documented as associated with host susceptibility (e.g. Adamek et al., 2012; Gong et al., 2019; Li et al., 2019; Wang et al., 2019; Sun et al., 2020; Gao et al., 2021). Multiple immune pathways of hosts were shown to be involved in SVCV infection.

    • Investigation on the mechanisms of biochanin A alleviate PM10-induced acute pulmonary cell injury

      2021, Ecotoxicology and Environmental Safety
      Citation Excerpt :

      One of the main pathological features of PM10 induced pulmonary inflammation is excessive polymorphonuclear neutrophils (PMNs) infiltration of lung tissue. IL-8 is a key factor in local inflammation caused by PMNs penetrating the pulmonary interstitium and alveolar space (Mantecca et al., 2010; Sun et al., 2020). TNF-α is produced in the initial stages of the inflammatory response and induces the release of other inflammatory factors, and PM10 exposure causes its levels to spike (Cui et al., 2021; Elsayed et al., 2020).

    View all citing articles on Scopus
    View full text