Antibody Treatment against Angiopoietin-Like 4 Reduces Pulmonary Edema and Injury in Secondary Pneumococcal Pneumonia

FIG 1. Secondary pneumococcal infection significantly increases pulmonary edema and tissue damage in a serotype-specific manner. (A) BALB/c mice were infected with a sublethal dose of the PR8 virus, followed by S. pneumoniae infection. Representative H&E images of influenza-infected lung tissue show bronchiolar epithelial damage and alveolar infiltration at 7 dpi. Influenza infection caused cell infiltration into alveolar spaces (black arrows) and bronchiolar epithelial damage (red arrow), compared to the clean alveolar spaces and intact bronchiolar epithelium (blue arrow) in control lung sections from healthy mouse lungs. Scale bar = 50 μm. (B) Representative images of infected lungs showing pulmonary edema and tissue damage. BALB/c mice were infected with a sublethal dose of the PR8 virus, followed by either serotype 3 of S. pneumoniae (Flu+S3), serotype 19F of S. pneumoniae (Flu+19F), or sterile PBS (Flu) at 7 dpi. Mice infected directly with S3 or 19F of S. pneumoniae or sterile PBS (control) were included for comparison. n = 3 independent experiments with 5 mice per time point for each experimental group. Infection-induced edema was evident as early as 9 dpi. Scale bar = 5 mm. (C)…

FIG 2. ANGPTL4 mRNA and protein are significantly upregulated in secondary pneumococcal pneumonia-infected mouse lung tissue. (A) Relative ANGPTL4 mRNA expression in lung tissues infected with the influenza virus and/or S. pneumoniae. rRNA L27 (RPL27) served as the reference or housekeeping gene. Data are represented as the mean ± standard deviation (SD) from at least three independent experiments. (B) Relative cANGPTL4 protein expression in lung tissues infected with the influenza virus and/or S. pneumoniae. Coomassie staining of immunoblots served as loading and transfer controls. Loading controls for the immunoblot analyses were from the same samples. Data are represented as the mean ± SD from at least three independent experiments. (C) Representative immunofluorescent staining for cANGPTL4 in lungs from control and infected mice. Scale bar = 1 mm.

FIG 3. Deficiency in the cANGPTL4 protein reduces pulmonary edema and protects lung tissue integrity in infected mice. (A) Representative image of lung harvested at 11 dpi from ANGPTL4 knockout (−/−), heterozygous (+/−), and wild-type (+/+) mice infected with influenza virus and different pneumococcal serotypes (S3 and 19F). Scale bar = 5 mm. n = 3 independent experiments with 5 mice per time point for each experimental group. (B) Representative H&E images of the indicated primary and secondary pneumococcal infections of lungs from ANGPTL4 knockout (−/−), heterozygous (+/−), and wild-type (+/+) mice. In Flu+19F-infected mice, the ANGPTL4 knockout group showed significant reduction of immune cell infiltration and edema compared with their wild-type counterparts (black versus white arrows); In Flu+S3-infected mice, the wild-type group showed vanished alveolar structures with severe edema, while the ANGPTL4 knockout group preserved the alveolar structures with reduced edema (black versus white arrows). Scale bar = 50 μm. (C) Infected wild-type mice treated with the anti-cANGPTL4 antibody compared with untreated mice. In the Flu+19F-infected mice, anti-cANGPTL4 treatment induced improved…

FIG 4. Cotreatment with antibiotics and specific antibodies ameliorates lung tissue damage and prolongs the survival of infected mice. (A) A Kaplan-Meier survival plot shows the percentage of survival of mice given the various indicated treatments. Five mice were used for each experimental group. (B) Representative H&E images of lungs from mice administered the various indicated treatments. Combined treatment with antibiotics and anti-cANGPTL4 antibody or with anti-cANGPTL4 antibody and antipneumolysin antibody further improved the lung tissue integrity from single treatment with either antibody or antibiotics. Arrows indicate edema, infiltration of immune cells, and tissue damage patterns. Scale bar = 50 μm. (C) Comparative Gene Ontology results analyzed from RNA sequencing data of lung tissues from infected mice (Flu+S3) administered moxifloxacin (a), anti-cANGPTL4 antibody (b), and a combined treatment with antibiotics and antibodies (c). (D) Representative bright-field images of A549 human alveolar epithelial cells cultured with mock IgG treatment (control), pneumolysin, or pneumolysin with an antipneumolysin antibody at the indicated times posttreatment. Cells were tracked for 48 h…

FIG 5. cANGPTL4 immunoneutralization activates immune defenses against bacterial infection. (A) Using mouse lung tissue harvested from mice with a secondary infection of serotype 3 on day 9 post-influenza infection, representative FACS plots are shown for the major groups of immune cells sorted according to their surface markers. The immune cell groups include macrophages (F4/80+ and CD45+), neutrophils (Ly6G+), natural killer cells (NK1.1+), cytotoxic T cells (Tc; CD3+ and CD8+), T helper cells (Th; CD3+ and CD4+), and B cells (CD19+). The graph shows the different relative expression levels of ANGPTL4 as determined by qPCR, with 18S rRNA serving as a reference housekeeping gene. n = 3 independent experiments. (B) Representative transmission electron microscopy images of immune cells from lungs of secondary bacterial pneumonia-infected mice administered the control IgG treatment (Flu+S3) and the anti-cANGPTL4 antibody treatment (Flu+S3+anti-cANGPTL4). White arrows indicate semidigested bacteria. The inset shows a higher-magnification view of a semidigested bacterium inside a lymphocyte. Scale bar = 500 nm. (C) Heat map profile indicating the expression of specific upstream gene…

FIG 6. Elevated cANGPTL4 protein in the bronchoalveolar lavage fluid of patients with pneumonia. Shown are graphs depicting the relative expression of the C-terminal isoform of ANGPTL4 protein (cANGPTL4) in (A) BALF samples (control, n = 16; ARDS, n = 7) and (B) serum samples (control, n = 8; pneumonia, n = 17) from control healthy volunteers and patients with lung disease. n.s., no statistical significance. Representative images of Western blots are shown for BALF and serum samples.