Toll-Like Receptor 9-Mediated Protection of Enterovirus 71 Infection in Mice Is Due to the Release of Danger-Associated Molecular Patterns

Abstract

Enterovirus 71 (EV71), a positive-stranded RNA virus, is the major cause of hand, foot, and mouth disease (HFMD) with severe neurological symptoms. Antiviral type I interferon (alpha/beta interferon [IFN-␣/␤]) responses initiated from innate receptor signaling are inhibited by EV71-encoded proteases. It is less well understood whether EV71-induced apoptosis provides a signal to activate type I interferon responses as a host defensive mechanism. In this report, we found that EV71 alone cannot activate Toll-like receptor 9 (TLR9) signaling, but supernatant from EV71-infected cells is capable of activating TLR9. We hypothesized that TLR9-activating signaling from plasmacytoid dendritic cells (pDCs) may contribute to host defense mechanisms. To test our hypothesis, Flt3 ligand-cultured DCs (Flt3L-DCs) from both wild-type (WT) and TLR9 knockout (TLR9KO) mice were infected with EV71. More viral particles were produced in TLR9KO mice than by WT mice. In contrast, alpha interferon (IFN-␣), monocyte chemotactic protein 1 (MCP-1), tumor necrosis factor-alpha (TNF-␣), IFN-␥, interleukin 6 (IL-6), and IL-10 levels were increased in Flt3L-DCs from WT mice infected with EV71 compared with TLR9KO mice. Seven-day-old TLR9KO mice infected with a non-mouse-adapted EV71 strain developed neurological lesion-related symptoms, including hind-limb paralysis, slowness, ataxia, and lethargy, but WT mice did not present with these symptoms. Lung, brain, small intestine, forelimb, and hind-limb tissues collected from TLR9KO mice exhibited significantly higher viral loads than equivalent tissues collected from WT mice. Histopathologic damage was observed in brain, small intestine, forelimb, and hind-limb tissues collected from TLR9KO mice infected with EV71. Our findings demonstrate that TLR9 is an important host defense molecule during EV71 infection. IMPORTANCEThe host innate immune system is equipped with pattern recognition receptors (PRRs), which are useful for defending the host against invading pathogens. During enterovirus 71 (EV71) infection, the innate immune system is activated by pathogen-associated molecular patterns (PAMPs), which include viral RNA or DNA, and these PAMPs are recognized by PRRs. Toll-like receptor 3 (TLR3) and TLR7/8 recognize viral nucleic acids, and TLR9 senses unmethylated CpG DNA or pathogen-derived DNA. These PRRs stimulate the production of type I interferons (IFNs) to counteract viral infection, and they are the major source of antiviral alpha interferon (IFN-␣) production in pDCs, which can produce 200-to 1,000-fold more IFN-␣ than any other immune cell type. In addition to PAMPs, danger-associated molecular patterns (DAMPs) are known to be potent activators of innate immune signaling, including TLR9. We found that EV71 induces cellular apoptosis, resulting in tissue damage; the endogenous DNA from dead cells may activate the innate immune system through TLR9. Therefore, our study provides new insights into EV71-induced apoptosis, which stimulates TLR9 in EV71-associated infections.

Innate Immunity Evasion by Enteroviruses: Insights into Virus-Host Interaction

“…TLR9 is also important for immune responses against EV-A71 infection as TLR9 knockout mice display more serious symptoms than wild type mice after EV-A71 infection [ 55 ]. Interestingly, EV-A71 infection cannot directly activate TLR9-mediated innate immune responses, which are stimulated by EV-A71-induced danger-associated molecular patterns (DAMPs) release.…”

Innate Immunity and Immune Evasion by Enterovirus 71

“…Recently it has been reported that TLR9 mediated protection in EV71 infected mice is due to the release of danger-associated molecular patterns (DAMPs). The endogenous DNA of apoptotic cells in the supernatant of EV71 infected cells is capable of activating TLR9 signaling and release of IFN-α, IFN-γ, MCP-1, TNF-α, IL-6, and IL-10 [ 111 ]. Therefore, TLR agonist may play an important role in the host defense against EV71 infection.…”

Molecular Pathogenicity of Enteroviruses Causing Neurological Disease

“…Induction of an antiviral immune state in non-infected cells is mediated by TLR9 detection of released DAMPS from virally infected cells. This was observed for EV-A71 where an immune response was evoked in uninfected neighboring cells (Hsiao et al, 2014) as a mechanism to prevent viral spread and tissue damage. To circumvent these immune responses, EVs can influence the cell to induce autophagy which degrades the membrane bound TLR7 sensor .…”