Plasminogen deficiency reduces disease severity and immune responses in enterovirus A71-infected mice

Abstract

Enterovirus A71 (EV-A71) is a causative agent of hand, foot, and mouth diseases. EV-A71 infections may result in severe neurological complications in children. Although several receptors or attachment molecules for EV-A71 have been identified, EV-A71 can still infect host cells even after blocking these receptors with antibodies. We have previously identified plasminogen (PLG), a circulating zymogen of plasmin, as a cell membrane-associated EV-A71-interacting glycoprotein. We confirmed that anti-PLG antibodies could reduce the binding of EV-A71 to RD cells as anti-SCARB2 and anti-nucleolin. Knockdown of PLG reduced EV-A71 binding to RD cells, and preincubation of PLG with EV-A71 increased virus binding. Enzyme-linked immunosorbent assay and surface plasmon resonance assays demonstrated the direct binding of PLG to EV-A71. We further evaluated the biological characteristics of EV-A71-infected PLG knockout (heterozygous) and wild-type mice. We found that the clinical scores and mortality of WT mice were higher than those of PLG-knockout mice after EV-A71 infection. The viral loads in the spinal cord of PLG knockout mice were lower than those in WT mice 6 days post-infection. EV-A71-associated cytokines such as IL-1β, IL-6, MCP-1, IL-10, and IFN-γ were investigated. Serum IL-10 and MCP-1 expression were significantly higher in EV-71-infected WT mice than in PLG knockout mice, and MCP-1 may be one of the critical chemokines that induce intense inflammation and chemoattracts leukocytes. Our findings reveal a possible role for PLG in EV-A71 infection/pathogenesis and shed light on developing novel therapeutic approaches and drugs to prevent EV-A71 infection.IMPORTANCEUnderstanding the pathogenesis of enterovirus A71 (EV-A71) for developing novel drugs or therapeutic approaches has always been a significant issue. In this study, we demonstrated the interactions between plasminogen (PLG) and EV-A71, characterized the biological effects of EV-A71-infected PLG knockout mice, and evaluated their immune response. We found that EV-A71 caused more severe tissue damage than PLG knockout mice in skeletal muscle, spinal cord, and brain stem. Higher virus protein was observed in these tissues of WT mice. The reduced clinical scores, mortality, and cytokine expression suggested PLG may be involved in EV-A71 infection-induced cytokine storm. The findings and animal model in the current study provide the new drug target for anti-EV-A71 drug discovery.

Keywords: enterovirus A71; plasminogen.

Fig 1

Anti-plasminogen antibody blocks the binding of EV-A71 to RD cells. (A) RD cells were treated with different concentrations of antibodies, including anti-SACRB2, anti-NCL, anti-PLG, and anti-SELS antibodies, for 1 h, followed by infection with EV-A71 for 3 h at 4°C. The bound virus was detected by flow cytometry with anti-EV-A71 antibody and FITC-conjugated second antibody. Preincubation of antibodies significantly reduced the binding of EV-A71 to RD cells in a dose-dependent manner. (B) PLG-virus preincubation facilitates EV-A71 binding to RD cells. (C) Knockdown of PLG attenuates EV-A71 binding to RD cells. (D) Virus-protein interaction detected by ELISA assay. Purified EV-A71 and BSA were diluted from 1 pg/50 µL to 10 ng/50 µL. The 100 pg/100 µL of PLG was added to each well. EV-A71 directly interacts with PLG. (E) The plates were coated with 1 ng/well of PLG. The purified EV-A71 was diluted in twofold serial dilution. The virus-protein interaction was detected by an SPR assay reader (EnSpire). (F) The virus-protein interaction was detected by the SPR assay reader (BIACore). The data were expressed as the mean ± SD of each group (n ≥ 3). *P < 0.05.

Fig 2

The clinical scores, survival rates, and tissue viral loads of PLG-KO and WT mice in EV-A71 infection. Seven-day-old WT and PLG-KO mice were infected with the 5 × 10⁴ pfu/mouse of EV71 strain. The clinical score (A) and the survival rates (B) of the WT and PLG-KO mice were measured for 2 weeks. PLG-KO mice showed lower clinical scores and mortality rates than WT mice. Clinical scores: 0, healthy; 1, reduced motility; 2, limb weakness; 3, limb paralysis; 4, moribund; and 5, death. Seven-day-old WT and PLG-KO mice were infected with 5 × 10⁴ pfu/mouse of EV-A71 MP4 strain to evaluate the viral loads in the brain stem, spinal cord, and skeletal muscle. The WT (n = 5) and PLG-KO mice (n = 5) were sacrificed 3 days (C) and 6 days (D) post-infection. The virus titers were determined by plaque assay. The mean of virus titers (log10 pfu/g) in WT mice was red, and in PLG-KO mice was blue. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 3

PLG-KO decreases the disease progression of EV-A71-associated neuropathy. Seven-day-old WT and PLG-KO mice were infected with 5 × 10⁴ pfu/mouse EV-A71 intraperitoneally. Mice were sacrificed on days 2 (A) and 6 (B). Paraffin-embedded skeletal muscle, spinal cord, and brain stem sections were examined with H&E stain at 200× magnifications. WT mice show cell neuropathy, such as proliferation, tissue damage, and lymphocytic infiltration in their nervous system at day 6 post-infection (arrow indicated). Negative control (mock): without virus infection.

Fig 4

PLG-KO decreases EV-A71 VP1 protein expression in tissues. Seven-day-old WT and PLG-KO mice were infected with 5 × 10⁴ pfu/mouse EV-A71 intraperitoneally. Mice were sacrificed on day 6. Paraffin-embedded skeletal muscle, spinal cord, and brain stem sections were examined with IHC stain at 200× magnifications. IHC staining detected the most vital expression of viral VP1 protein in skeletal muscle, spinal cord, and brain stem in the WT mice (arrow indicated). Negative control (mock): without virus infection.

Fig 5

Cytokine expression in the serum of WT and PLG-KO mice. Seven-day-old WT (white blocks) and PLG-KO (black blocks) mice were infected with 5 × 10⁴ pfu/mouse EV-A71 intraperitoneally (n = 5). Mice were sacrificed at 2, 4, and 6 days post-infection, and serum/spinal cord samples were collected. The expression of cytokines was evaluated. Cytokine concentrations were presented as pg/mL of the specimen. (A) IL-1β, (B) IL-6, (C) MCP-1, (D) IL-10, (E) IFN-γ, and (F) MCP-1 in the spinal cord. The nonparametric Mann-Whitney U-test determined each group’s statistical significance, the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.