Elsevier

Antiviral Research

Volume 169, September 2019, 104554
Antiviral Research

Altered monocyte response to the dengue virus in those with varying severity of past dengue infection

https://doi.org/10.1016/j.antiviral.2019.104554Get rights and content

Highlights

  • Monocytes of individuals with past severe dengue produce more pro-inflammatory cytokines when infected with dengue virus.

  • Viral loads in monocyte culture supernatants positively correlated with IL-1β cytokines in individuals with past severe dengue.

  • Viral loads in monocyte culture supernatants inversely correlated with proinflammatory cytokines in individuals with past non-severe dengue.

  • RIG-I and NLRP-3 were upregulated in individuals with past severe dengue when infected with non-immune serotypes.

Abstract

Objective

We sought to investigate the differences in monocyte immune responses to the dengue virus (DENV) in those who previously had either severe disease (past SD) or non-severe dengue (past NSD) following a secondary dengue infection.

Method

Monocytes from healthy individuals who had either past SD (n = 6) or past NSD (n = 6) were infected at MOI one with all four DENV serotypes following incubation with autologous serum. 36-hours post infection, levels of inflammatory cytokines and viral loads were measured in the supernatant and expression of genes involved in viral sensing and interferon signaling was determined.

Results

Monocytes of individuals with past SD produced significantly higher viral loads (p = 0.0426 and cytokines (IL-10 p = 0.008, IL-1β p = 0.008 and IL-6 p = 0.0411) when infected with DENV serotypes they were not immune to, compared to those who has past NSD. Monocytes of individuals with past SD also produced significantly higher viral loads (p = 0.022) and cytokines (IL-10 p < 0.0001, IL-1β < 0.0001 and IL-6 p < 0.0001) when infected with DENV serotypes they were previously exposed to, despite the monocytes being infected in the presence of autologous serum. A significant upregulation of NLRP3 (p = 0.005), RIG-I (0.0004) and IFNB-1 (0.01) genes were observed in those who had past SD compared to past NSD when infected with non-immune DENV serotypes.

Conclusion

Monocytes from those with past SD appear to show marked differences in viral loads, viral sensing and production of inflammatory mediators in response to the DENV, when compared to those who experienced past NSD, suggesting that initial innate immune responses may influence the disease outcome.

Introduction

Dengue viral infections represent one of the most rapidly emerging mosquito borne viral infections in the world, with an estimated annual global cost of $8.9 billion (Shepard et al., 2016). It is estimated that 390 million individuals are infected with the dengue virus (DENV) annually of which 96 million manifest as apparent dengue infections (Bhatt et al., 2013). Intense monitoring with meticulous fluid control is currently the only option in the management of dengue infection, as specific treatments for dengue are not yet available. Therefore, it is important to further understand dengue pathogenesis in order to develop drugs for the treatment of acute dengue infection.

Infection with the DENV is associated with a self-limiting illness in the majority of individuals. However, it can cause severe clinical disease manifestations such as dengue haemorrhagic fever (DHF) and organ involvement in up to 10–25% of individuals, depending on serotype and population (Fernando et al., 2016; Lee et al., 2016). Although disease enhancement due to the presence of non-neutralizing antibodies and possibly cross-reactive T cells is thought to lead to severe disease (Guzman et al., 2013), DHF and fatalities have also been reported in primary dengue infection in the absence of DENV specific antibodies or T cells (Ong et al., 2007; Singla et al., 2016). In addition, it has been shown that inapparent dengue infection occurs in an equal proportion of those experiencing a primary or secondary dengue infection (Grange et al., 2014). Since the likelihood of developing an inapparent infection was shown to be the same for those who have an acute primary or secondary dengue infection, the contribution of non-neutralizing cross reactive antibodies, which are present in those with a secondary dengue infection, in the pathogenesis of severe clinical disease should be further investigated (Grange et al., 2014). On the other hand, it could be an altered initial immune response to the DENV by immune cells readily infected by the virus, such as monocytes and dendritic cells, that could lead to either severe disease or asymptomatic infection.

Following the bite of a DENV-infected mosquito, the DENV infects immune cells such as dendritic cells, monocytes and mast cells, which in turn produce massive quantities of inflammatory cytokines and lipid mediators (Malavige and Ogg, 2017). The initial phase of viral replication is known as the febrile phase, which is then followed by a critical phase in some individuals that is characterized by fluid leakage (WHO, 2011). Patients who proceed to this vascular leakage phase (critical phase) are known to have developed DHF, whereas those who proceed to the recovery phase without any clinically apparent vascular leakage are diagnosed as having dengue fever (DF) (WHO, 2011). Inflammatory mediators such as platelet activating factor (PAF), IL-1β, TNFα, VEGF, and chymase produced by innate immune cells are thought to act on the vascular endothelium leading to endothelial dysfunction, which subsequently leads to vascular leakage (Malavige and Ogg, 2017; Tissera et al., 2017). Inflammatory mediators that lead to vascular leakage are highest at the critical phase (defervescence) of illness in patients with DHF (Jeewandara et al., 2015b; Kamaladasa et al., 2016; van de Weg et al., 2014). On the other hand, there is contradicting data showing that mediators and enzymes responsible for the production of these mediators are significantly elevated very early (day 2–4 since onset of symptoms) illness in those who proceed to develop DHF (Fernando et al., 2016; Jeewandara et al., 2017; Zanini et al., 2018). Therefore, in order to fully understand the reasons why some individuals develop DHF, while some develop milder clinical disease, it would be important to understand the events that occur during early infection and the differences in responses of immune cells infected with the DENV.

Studies both in vitro and in patients with acute dengue have shown that monocytes are target cells for DENV (Srikiatkhachorn et al., 2012; Zanini et al., 2018). Separately, an expansion of CD14 + CD16 + monocytes in acute dengue has been shown to be associated with severe clinical disease and to produce high levels of proinflammatory cytokines (Kwissa et al., 2014). In addition, monocytes have also been shown to induce differentiation of B cells into plasmablasts, further modulating disease pathogenesis (Kwissa et al., 2014). In addition, expression of CD163 and IFIT1 by monocytes in early infection (before the critical phase) was associated with severe disease. Therefore, monocytes appear to play a significant role in the development of severe dengue. In this study, to understand the role of monocytes in contributing to severe dengue, we investigated the responses of primary human monocytes of individuals who had developed either past severe dengue or past non-apparent dengue during a secondary dengue infection. Following infection of primary human monocytes in the presence of autologous serum, we examined the quantity of virus, differences in cytokine production and differences in gene expression in these two groups of individuals.

Section snippets

Human subjects

Twelve healthy adult individuals who previously had either asymptomatic dengue/mild dengue (n = 6) or DHF (n = 6) were recruited for this study. Healthy dengue seropositive individuals, who had never been hospitalized due to a febrile infection and therefore likely had an asymptomatic or mild infection) and were considered as having past non-severe dengue (past NSD). Individuals with past DHF were also healthy individuals who had previous DHF diagnosed according to WHO 2011 guidelines (past

Immunity to DENV serotypes in this cohort of healthy donors

Of the 12 healthy individuals recruited, 6 had had an episode of DHF in the past and were considered to have past SD and were found to be immune to two DENV serotypes (Table 1), based on the results of the cultured T cell ELISpot assays. The other 6 healthy individuals were seropositive for the DENV and were also found to be immune to two DENV serotypes (Table 2), despite them never having been hospitalized for a febrile illness and not being aware of an infection (past NSD). Therefore, each

Discussion

In this study we have investigated the differences in the responses to the DENV from monocytes from individuals who had past SD and past NSD. Although all individuals were healthy at the time of recruitment and had the episode of DHF more than 1 year ago, at the time of recruitment to the study, marked differences in the responses to the DENV were seen. Monocytes of individuals with past SD produced significantly higher viral loads and inflammatory cytokines (IL-1β, IL-6 and IL-10) when

Acknowledgements

Funding was provided by the Centre for Dengue Research, University of Sri Jayewardenepura, University of Sri Jayewardenapura, National Science Foundation, Sri Lanka (RPHS/2016/D-06) and by the Medical Research Council (UK). Graham Ogg receives support from the National Institute for Health Research Oxford Biomedical Research Centre (BRC). Katja Fink received funding from A*STAR.

References (51)

  • A. Baum et al.

    Induction of type I interferon by RNA viruses: cellular receptors and their substrates

    Amino Acids

    (2010)
  • S. Bhatt et al.

    The global distribution and burden of dengue

    Nature

    (2013)
  • S.T. Chen et al.

    CLEC5A is critical for dengue-virus-induced lethal disease

    Nature

    (2008)
  • Y.C. Chen et al.

    Bacterial lipopolysaccharide inhibits dengue virus infection of primary human monocytes/macrophages by blockade of virus entry via a CD14-dependent mechanism

    J. Virol.

    (1999)
  • R. de Alwis et al.

    Identification of human neutralizing antibodies that bind to complex epitopes on dengue virions

    Proc. Natl. Acad. Sci. U.S.A.

    (2012)
  • R. de Alwis et al.

    Dengue viruses are enhanced by distinct populations of serotype cross-reactive antibodies in human immune sera

    PLoS Pathog.

    (2014)
  • W. Dejnirattisai et al.

    A new class of highly potent, broadly neutralizing antibodies isolated from viremic patients infected with dengue virus

    Nat. Immunol.

    (2015)
  • M.S. Diamond et al.

    Infection of human cells by dengue virus is modulated by different cell types and viral strains

    J. Virol.

    (2000)
  • M.S. Diamond et al.

    Modulation of Dengue virus infection in human cells by alpha, beta, and gamma interferons

    J. Virol.

    (2000)
  • S. Fernando et al.

    Patterns and causes of liver involvement in acute dengue infection

    BMC Infect. Dis.

    (2016)
  • L. Grange et al.

    Epidemiological risk factors associated with high global frequency of inapparent dengue virus infections

    Front. Immunol.

    (2014)
  • M.G. Guzman et al.

    Secondary infection as a risk factor for dengue hemorrhagic fever/dengue shock syndrome: an historical perspective and role of antibody-dependent enhancement of infection

    Arch. Virol.

    (2013)
  • C. Jeewandara et al.

    Functionality of dengue virus specific memory T cell responses in individuals who were hospitalized or who had mild or subclinical dengue infection

    PLoS Neglected Trop. Dis.

    (2015)
  • C. Jeewandara et al.

    Secretory phospholipase A2 in the pathogenesis of acute dengue infection

    Immun Inflamm Dis

    (2017)
  • C. Jeewandara et al.

    Platelet activating factor contributes to vascular leak in acute dengue infection

    PLoS Neglected Trop. Dis.

    (2015)
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