The Disruption of the Endothelial Barrier Contributes to Acute Lung Injury Induced by Coxsackievirus A2 Infection in Mice

Figures

• 

  Figure 1. CVA2 infection led to acute lung injury in mice. (A–C) Body weights, survival rates, and mean clinical scores of mice in the experiments under optimized conditions (dosage (104 TCID50/mouse), inoculation route (intramuscularly), and age (5-day-old BALB/c mice)) were monitored and recorded daily after inoculation with CVA2 until 15 dpi. Control animals were administered with equal culture medium (n = 10 per group). (D) Clinical manifestations of mice after CVA2 infection at 7 dpi. (D(a)): The representative clinical signs (weight loss, reduced mobility, ataxia, and single or double hind limb paralysis) induced by CVA2 infection in mice. The control mice treated with medium were healthy throughout the experiment. (D(b)): Normal lung tissue from control mouse. (D(c)): The lung tissue from CVA2 infected mouse (dark red with obvious bleeding and enlargement). (E) EB extravasation assay was performed to detect the pulmonary vascular permeability expressed as the ratio of EB content (µg) to the wet weight (g) of the lung tissues (n = 6 per group). (F) The relative expression of CVA2 VP1 mRNA was detected by qPCR (n = 4 per group). (G) TUNEL assay detected lung apoptosis at 7 dpi by…

• 

  Figure 2. Histopathological changes of lung tissues after CVA2 infection. (A) Masson’s staining and HE were used to observe the pathological changes of lung tissue in mice at 3 dpi and 7 dpi. The black arrows indicate characteristic lesions. The alveoli collapsed, local pulmonary consolidation and diffused glial protein increase were observed in lungs of CVA2-infected mice. The infected lumens of alveoli were filled with edema fluid and red cells. No histological changes were observed in the lungs of uninfected mice. (B) The integrity of the pulmonary epithelium and morphological alterations of junctions was studied by TEM in both CVA2 infection and control groups. The arrows indicate tight junctions between endothelial cells. All experiments were repeated three times.

• 

  Figure 3. CVA2 infection led to the degradation of tight junction proteins in lung tissues. At 7 dpi, mice (n = 3 per group) were euthanized, and related proteins of isolated lungs were determined. (A) Immunofluorescence staining of ZO-1 (red); occludin (red) and immunohistochemical staining of claudin-5 were conducted. The arrows indicate the pulmonary microvessel. The relative expression levels of ZO-1 (B), occludin (C) and claudin-5 (D) compared to control group were calculated by Image J software. (E) The immunoblot bands of occludin and claudin-5. (F) The quantification of occludin and claudin-5. n = 3 per group, data represent median with range. * p < 0.01; ** p < 0.01; *** p < 0.001; **** p < 0.0001 vs. control. All the experiments were repeated at least three times.

• 

  Figure 4. Effect of CVA2 infection on the mRNA levels of biomarkers of vascular endothelial cell dysfunction. qPCR was conducted to measure the expression levels of the related genes in lung tissues (3 dpi and 7 dpi) normalized to β-actin expression level. n = 3 or 4 per group, data represent median with range. * p < 0.05; ** p < 0.01 vs. control. All the experiments were repeated at least three times.

• 

  Figure 5. Inflammatory cell infiltration and cytokines expression induced by CVA2 infection. (A) CD11b and F4/80 positive cells in lung tissues were stained by IF and quantified by Image J software (n = 3 per group). (B) The expression levels of some cytokines (IL-6, IL-1β, MCP-1, and TNF-α) in tissue lysates of lungs by ELISA. n = 4 per group, data represent the median with range. * p < 0.05; **** p < 0.0001 vs. control. All the experiments were repeated at least three times.

• 

  Figure 6. CVA2 infection activated p38-MAPK signal pathway in lung tissues. At 3 dpi and 7 dpi, control and infected mice were euthanized and isolated lung tissues for WB analysis. The immunoblot bands (A) and quantification (B) of related proteins in MAPK, NF-κB and PI3K/Akt signal pathways. pAKT was normalized by β-actin and others were normalized by p65, ERK1/2, or p38, respectively. n = 3 per group, data represent the median with range. * p < 0.05 vs. control. All the experiments were repeated at least three times.

• 

  Figure 7. A hypothetical schematic of the pathogenesis.