Fig 1.
The study design and timeline for culture treatment.
Fig 2.
Effects of S1 protein and exposure duration on co-cultures of HPAEpics and AMs.
AM, alveolar macrophage; HPAEpic, human pulmonary alveolar epithelial cell; IL-6, interleukin-6; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; MDA, malonaldehyde; NC, negative control; TNF-ɑ, tumor necrosis factor-ɑ. Symbols and error bars on the connecting lines in the line chart represent means and standard deviations. Co-cultured cells were exposed to complete medium (NC), or three concentrations (0.1, 1, and 10 μg/mL) of S1 protein (S_0.1, S_1, and S_10), or 10 μg/mL of LPS (positive control); and were harvested at 1, 2, and 3 days post-exposure. Two-way analysis of variance was used, P values were adjusted with Bonferroni correction for multiple comparisons, n = 6 per condition. *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significant. aMain exposure effect; bmain exposure duration effect; abinteraction. (A) LDH release, aP < 0.001, bP < 0.001, abP < 0.001. (B) IL-6 production, aP < 0.001, bP < 0.001, abP < 0.01. (C) TNF-ɑ production, aP < 0.001, bP < 0.001, abP < 0.001. (D) MDA production, aP = 0.10, bP < 0.001, abP < 0.05.
Fig 3.
Pathology and morphology of cell injury in co-cultures or monocultures 3 days after exposure to S1 protein or LPS.
AM, alveolar macrophage; HPAEpic, human pulmonary alveolar epithelial cell; LPS, lipopolysaccharide; NC, negative control. Three exposure groups, NC, S_10, LPS, were exposed to complete medium, or S1 protein (10 μg/mL), or LPS (10 μg/mL). Images of cultured cells under optical microscope (400 x) were based on thinprep liquid-based cytology test, stained with H&E. The pathology and morphology changes of cell injury included that the cytoplasm was swollen, dissolved, and reduced (blue arrow), and the nucleus became smaller, degenerated, vacuolated, and irregular, with a wrinkled nuclear membrane (red arrow). (A) Monocultured AMs. (B) Monocultured HPAEpics. (C) Co-cultured HPAEpics and AMs.
Fig 4.
Effects of cell culture patterns on injury and inflammation induced by the S1 protein.
AM, alveolar macrophage; HPAEpic, human pulmonary alveolar epithelial cell; IL-6, interleukin-6; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; MDA, malonaldehyde; NC, negative control; TNF-ɑ, tumor necrosis factor-ɑ. Symbols and error bars on the connecting lines in the line chart represent means and standard deviations. Three exposure groups, NC, LPS, and S_10, were exposed to complete medium, LPS (10 μg/mL), or S1 protein (10 μg/mL). Cells were harvested at 3 days post-exposure. Two-way analysis of variance was used, P values were adjusted with Bonferroni correction for multiple comparisons, n = 6 per condition. *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significant. aMain exposure effect; bmain culture pattern effect; abinteraction. (A) LDH release, aP < 0.001, bP < 0.001, abP < 0.001. (B) IL-6 production, aP < 0.001, bP < 0.001, abP < 0.05. (C) TNF-ɑ production, aP < 0.001, bP < 0.001, abP < 0.01. (D) MDA production, aP = 0.13, bP < 0.001, abP < 0.01.