Elsevier

Cellular Immunology

Volume 285, Issues 1–2, September–October 2013, Pages 100-110
Cellular Immunology

Japanese encephalitis virus infection modulates the expression of suppressors of cytokine signaling (SOCS) in macrophages: Implications for the hosts’ innate immune response

https://doi.org/10.1016/j.cellimm.2013.09.005Get rights and content

Highlights

  • JEV up regulates SOCS level in infected macrophage.

  • Not all SOCS molecules are up regulated, i.e. selective up regulation occurs.

  • JEV up regulates SOCS1, SOCS3 to modulate JAK–STAT antiviral axis.

  • SOCS1 and SOCS3 knockdown can reduce viral load within infected macrophages.

Abstract

Viruses have evolved various mechanisms to subvert the host’s immune system and one of them is preventing the infected cells from sending out chemotactic signals to activate the adaptive immune response. Japanese encephalitis virus (JEV) is a neuropathologic flavivirus that is responsible for significant number of child mortalities in various parts of South-East Asia. In this study we show that JEV modulates suppressors of cytokine signaling (SOCS)1 and 3 expression in macrophages to bring about changes in the JAK–STAT signaling cascade, so as to inhibit proinflammatory cyto/chemokine release. Using real time PCR, immunoblotting and immunofluorescent staining, we show that the expression of type 1 interferons and intracellular expression of viral genes are also affected over time. Also, following the initial activation of SOCS1 and 3, there is production of interferon-inducible anti-viral proteins in the cells which may be responsible for inhibiting viral replication. However, even at later time points, viral genes were still detected from the macrophages, albeit at lesser quantities, than earlier time points, indicative of intracellular persistence of the virus in a latent form. On knocking down SOCS1 and SOCS3 we found a significant decrease in viral gene expression at an early time point, indicating the dysregulation of the signaling cascade leading to increased production of interferon-inducible anti-viral proteins. Taken together, our study provides an insight into the role of JEV infection in modulating the JAK–STAT pathway with the help of SOCS leading to the generation of an antiviral innate immune response.

Introduction

Arthropod-borne flaviviruses are an emerging and re-emerging threat to mankind and causes endemic and epidemic diseases globally, with significant morbidity and mortality [1]. It has been well documented over the years that these viruses are capable of infecting a wide variety of host cells. As the innate immune facet of the host is the first line of defense against these invasions, the viruses must subvert this process so as to be successful in infecting the host. Macrophages are professional phagocytes which are critical to the host’s innate immunity. Apart from the central depots in secondary lymphoid organs, macrophages reside virtually in all tissues. It has been reported in several studies that macrophages can be effectively infected by arthropod-borne flaviviruses [2], [3], [4], [5]. Upon viral sensing or chemotactic signals released by other infected cells, macrophages produce cyto/chemokines and other inflammatory mediators which serve as the activators of adaptive immune system, leading to the ultimate clearance of the virus from the body [6].

Cytokine binding to cell surface receptors results in the initiation of an intracellular signaling cascade that leads to alteration in the transcriptional profile of that cell. These cascades are inextricably linked to the JAK protein tyrosine kinases and their substrates, the signal transducers and activators of transcription (STAT). The activated (phosphorylated) STAT proteins translocate into the nucleus and activate transcription of a range of cytokine responsive genes [7]. However, like most other signaling cascades in any mammalian system, this process is under the influence of intricate regulatory mechanisms controlled by a family of 8 proteins, collectively known as the suppressors of cytokine signaling (SOCS) [8], [9], [10], [11]. Thus, it is not at all surprising that this regulatory mechanism would be a target of viruses in evading the host’s immune system. It has been reported that mice deficient in SOCS1 are prone to severe inflammatory disease, but on the other hand they are resistant to viral infections [12]. Till date, a number of different viruses have been reported to induce SOCS proteins so as to prevent cytokine signalling and inhibit the initiation of an immune response [13]. Among these, HCV is the only reported flavivirus to modulate the expression of SOCS proteins, even though it is not arthropod-borne.

Japanese encephalitis (JE) virus (JEV) is a mosquito-borne flavivirus that is one of the major causes of encephalitic disease in South-East Asia affecting an estimated 67,900 people annually [14]. Persistence of JEV has been demonstrated in cultured mammalian cells [15], [16], as well as in a mouse model [17], [18], [19]. In JE survivors there have been reports of viral persistence long after the alleviation of symptoms [20], [21]. These observations indicate that JEV may have evolved different mechanisms for immune evasion in the host. However, till date, no theory regarding this is either definitive or conclusive. Efforts to decipher the evasion mechanisms of JEV had mostly been concentrated on their effects in macrophages and dendritic cells, two of the most efficient antigen presenting cells in the body, with relation to their viability and ability to present viral antigens to T lymphocytes [22], [23]. However, there is no report available regarding the modulatory effect of JEV on the expression of SOCS proteins leading to alterations in the anti-viral profile of macrophages. A transcriptome analysis of JEV-infected hamster kidney cell line to test the efficacy of an anti-microbial peptide for therapeutic purposes had shown that SOCS6 was upregulated post-infection [24]. In this current investigation we aimed to study the effect of JEV infection in macrophages over a wide time point and the subsequent changes in the expression of some SOCS proteins. From the obtained data, we wish to correlate the altered expression of these proteins with intracellular survival of the virus that might throw light onto the mechanism of its persistence or clearance from macrophages.

Section snippets

Ethics statement

Animals were handled in strict accordance with good animal practice as defined by the committee for the purpose of control and supervision of experimental animals (CPCSEA), Ministry of Environment and Forestry, Government of India as well as institutional animal and ethics committee (IAEC) of National Brain Research Centre. All animal studies were approved by the IAEC of National Brain Research Centre and experimental protocol approval number is NBRC/IAEC/2011/66.

Viruses and cell

The GP78 strain of the virus

Modulation of proinflammatory cyto/chemokine release from macrophages correlates with SOCS1 and SOCS3 expression in the cells post-infection with the virus

F4/80 + ve macrophages were selected post adherence to tissue culture plates (∼92–95% purity, Supplementary Fig. S1). Cytokine bead array using culture supernatants from non-infected, JEV-infected and boiled JEV-treated macrophages were performed to evaluate the time kinetic secretion of some proinflammatory cyto/chemokines. At 6 h post-infection or treatment, it was observed that there was a significant increase in the levels of all the studied cyto/chemokine as compared to the levels in

Discussion

Viruses have evolved various mechanisms to evade detection and destruction of the infected cells by sentinels of the host’s immune system. In a recent study we had demonstrated that at early time points (6, 12 and 24 h) there is no significant alterations in the expression of MHC-1 or the costimulatory molecules CD80 and CD86 on the surface of JEV-infected macrophages compared to non-infected controls [28]. Hence, it may be assumed that during early infection there is no viral antigen

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

This work was in part supported by the National Bioscience Award for Career Development from Department of Biotechnology to AB and by the core grants of the National Brain Research Centre to AB. KD was a recipient of research associateship in biotechnology and life sciences from the Department of Biotechnology, Government of India. AN is the recipient of senior research fellowship from Council for Scientific and Industrial Research, Government of India. The authors would like to thank Kanhaiya

References (43)

  • I. Kurane

    Immune responses to Japanese encephalitis virus

    Curr. Top. Microbiol. Immunol.

    (2002)
  • T.R. Prestwood et al.

    Trafficking and replication patterns reveal splenic macrophages as major targets of dengue virus in mice

    J. Virol.

    (2012)
  • M. Rios et al.

    Monocytes–macrophages are a potential target in human infection with West Nile virus through blood transfusion

    Transfusion

    (2006)
  • P. Matzinger

    The danger model: a renewed sense of self

    Science

    (2002)
  • R. Starr et al.

    A family of cytokine-inducible inhibitors of signalling

    Nature

    (1997)
  • T.A. Endo et al.

    A new protein containing an SH2 domain that inhibits JAK kinases

    Nature

    (1997)
  • T. Naka et al.

    Structure and function of a new STAT-induced STAT inhibitor

    Nature

    (1997)
  • A. Yoshimura et al.

    A novel cytokine-inducible gene CIS encodes an SH2-containing protein that binds to tyrosine-phosphorylated interleukin 3 and erythropoietin receptors

    EMBO J.

    (1995)
  • J.E. Fenner et al.

    Suppressor of cytokine signaling 1 regulates the immune response to infection by a unique inhibition of type I interferon activity

    Nat. Immunol.

    (2006)
  • L.N. Akhtar et al.

    Viral exploitation of host SOCS protein functions

    J. Virol.

    (2011)
  • G.L. Campbell et al.

    Estimated global incidence of Japanese encephalitis: a systematic review

    Bull. World Health Organ.

    (2011)
  • Cited by (34)

    • E3 ligase ASB8 promotes porcine reproductive and respiratory syndrome virus proliferation by stabilizing the viral Nsp1α protein and degrading host IKKβ kinase

      2019, Virology
      Citation Excerpt :

      SOCS family also can act as a negative regulator of cytokine signal (Krebs and Hilton, 2001). SOCS1 would be up-regulated to promote virus production in influenza virus, Japanese encephalitis virus and duck tembusu virus infection (Kundu et al., 2013; Sun et al., 2017; Wei et al., 2016). SOCS3 also involves in signaling suppression and production of IFN in herpes simplex virus infection process (Yokota et al., 2004).

    • Morphine-potentiated cognitive deficits correlate to suppressed hippocampal iNOS RNA expression and an absent type 1 interferon response in LP-BM5 murine AIDS

      2018, Journal of Neuroimmunology
      Citation Excerpt :

      IL-1β was amplified as follows: 95 °C for 10 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 20 s (Zhao et al., 2009). IFN-α and IFN-β were amplified as follows: 95 °C for 3 min, 40 cycles of 95 °C for 30 s, 65 °C for 30 s, and 72 °C for 30 s (Kundu et al., 2013). Gene expression was normalized to GAPDH using the ΔΔCt method.

    • ATM supports gammaherpesvirus replication by attenuating type I interferon pathway

      2017, Virology
      Citation Excerpt :

      Specifically, the focus was placed on three negative IFN regulators that have been shown to be relevant in the context of herpesvirus and other viral infections. SOCS1 is an IFN-inducible Jak kinase inhibitor that suppresses both type I and type II IFN signaling in the context of multiple virus infections (Kundu et al., 2013; Shao et al., 2013; Charoenthongtrakul et al., 2011; Frey et al., 2009). PIAS1 is a SUMO ligase that facilitates viral replication by inhibiting activated Stat1 and IRF-3; it may have additional proviral roles during EBV and HCMV replication (Chang et al., 2004; Liu et al., 2004; Lee et al., 2003; Li et al., 2013).

    • Interaction of the innate immune system with positive-strand RNA virus replication organelles

      2017, Cytokine and Growth Factor Reviews
      Citation Excerpt :

      In addition, several methods are employed by +RNA viruses to interfere with JAK/STAT signaling. For instance, JEV NS5 and WNV NS4 B inhibit phosphorylation and activation of JAK1, and suppressors of JAK1 are induced by viruses such as WNV, JEV, and chikungunya virus [95–99]. However, STAT1 and STAT2 are most heavily targeted.

    View all citing articles on Scopus
    1

    These authors contributed equally to this work.

    2

    Current address: Centre de Recherche de l’Institut Universitaire en Santé Mentale de Québec, Québec, QC G1J 2G3, Canada.

    View full text