Therapeutic effects of high molecular weight hyaluronic acid in severe Pseudomonas aeruginosa pneumonia in ex vivo perfused human lungs

Abstract

We previously reported that extracellular vesicles (EVs) released during Escherichia coli (E. coli) bacterial pneumonia were inflammatory, and administration of high molecular weight hyaluronic acid (HMW HA) suppressed several indices of acute lung injury (ALI) from E. coli pneumonia by binding to these inflammatory EVs. The current study was undertaken to study the therapeutic effects of HMW HA in ex vivo perfused human lungs injured with Pseudomonas aeruginosa (PA)103 bacterial pneumonia. For lungs with baseline alveolar fluid clearance (AFC) <10%/h, HMW HA 1 or 2 mg was injected intravenously after 1 h (n = 4-9), and EVs released during PA pneumonia were collected from the perfusate over 6 h. For lungs with baseline AFC > 10%/h, HMW HA 2 mg was injected intravenously after 1 h (n = 6). In vitro experiments were conducted to evaluate the effects of HA on inflammation and bacterial phagocytosis. For lungs with AFC < 10%/h, administration of HMW HA intravenously significantly restored AFC and numerically decreased protein permeability and alveolar inflammation from PA103 pneumonia but had no effect on bacterial counts at 6 h. However, HMW HA improved bacterial phagocytosis by human monocytes and neutrophils and suppressed the inflammatory properties of EVs released during pneumonia on monocytes. For lungs with AFC > 10%/h, administration of HMW HA intravenously improved AFC from PA103 pneumonia but had no significant effects on protein permeability, inflammation, or bacterial counts. In the presence of impaired alveolar epithelial transport capacity, administration of HMW HA improved the resolution of pulmonary edema from Pseudomonas PA103 bacterial pneumonia.

Keywords: Pseudomonas aeruginosa pneumonia; acute lung injury; alveolar fluid clearance; extracellular vesicles; hyaluronic acid.

Figure 1.

Therapeutic effects of HMW HA on lung injury induced by PA103 instillation in human lungs with baseline AFC < 10%/h. Administration of HMW HA as therapy restored AFC rate and reduced inflammation in the injured alveolus. A: representative histopathological images of lung tissue slices from control, PA103 IT, and PA103 IT + HMW HA IV groups with H&E staining. Instillation of HMW HA IV significantly restored and increased AFC rate in a dose-dependent manner and numerically decreased total lung weight gain and protein permeability in human lungs injured by PA103 bacterial pneumonia. Data were presented as median with IQR, n = 3–9 per treatment groups. Comparisons between groups were made using Dunn’s test following Kruskal–Wallis analysis. B: total white blood cells (WBC), absolute neutrophil counts (ANC), and TNFα levels in BALF in human lungs injured by PA103 bacterial pneumonia with or without HMW HA therapy. There were no apparent effect of HMW HA on WBC and ANC in the BALF. However, HMW HA treatment numerically decreased TNFα levels in the BALF at 6 h. Data were presented as median with IQR, n = 4–9 per treatment groups. Comparisons between groups were made using Dunn’s test following Kruskal–Wallis analysis. C: messenger RNA levels for TNFα, IL-1β, and IL-6 in plasma EVs were measured using qPCR. Administration of HMW HA numerically decreased TNFα and IL-6 mRNA levels in plasma EVs at T1–3 h and T4–6 h vs. the corresponding control group, respectively. Data were presented as median with IQR, n = 3 per group. AFC, alveolar fluid clearance; BALF, bronchoalveolar lavage fluid; EV, extracellular vesicle; H&E, hematoxylin-eosin; HMW HA, high molecular weight hyaluronic acid; IQR, interquartile range; IV, intravenous; PA, Pseudomonas aeruginosa.

Figure 2.

Effect of HMW HA treatment on bacterial CFU counts and hemodynamic and respiratory changes in human lungs injured with PA103 bacteria with baseline AFC < 10%/h. A: HMW HA administration had no significant effects on total bacterial CFU levels in both the BALF or perfusate at 6 h. BALF of the uninjured control lung lobe had a low-baseline bacterial CFU counts, which may be the cause of the initial injury to the alveolar epithelium and endothelium before the experiments. Data were presented as median with IQR, n = 4–9 per treatment groups. Comparison between groups were made by using Dunn’s test following Kruskal–Wallis analysis. B: although treatment with HMW HA numerically increased lung compliance up to 220% at T6 h, there were no significant effects in pulmonary artery pressure, lung compliance, or perfusate Po₂ levels over 6 h or between individual groups. Data were presented as median with IQR, n = 4–9 per treatment groups. AFC, alveolar fluid clearance; BALF, bronchoalveolar lavage fluid; CFU, colony forming units; HMW HA, high molecular weight hyaluronic acid; IQR, interquartile range; PA, Pseudomonas aeruginosa.

Figure 3.

Characterization of plasma EVs released during PA103 bacterial pneumonia in ex vivo perfused human lungs. A: plasma EVs appeared as small membrane bound vesicles by electron microscopy (bar = 200 nm). By flow cytometry, 32.7% of EVs expressed CD9, a marker of exosomes at T6 h, which was higher than T0 h, and 10.9% of EVs expressed CD44, a marker of microvesicles at T6 h. By flow cytometry, EVs isolated from the perfusate following 6 h of injury were predominantly from platelets and endothelial cells. Data were presented as median with IQR, n = 3. B: by NanoSight analyses, >50% of the EVs were approximately 50–250 nm in size at all timepoints. EVs, extracellular vesicles; IQR, interquartile range; PA, Pseudomonas aeruginosa.

Figure 4.

Therapeutic effects of HMW HA on inflammation and PA103 bacterial phagocytosis by human monocytes and neutrophils in vitro. A: plasma EVs from perfused human lungs injured with PA103 bacterial pneumonia with or without HMW HA treatment from T0 to T6 h were added to human blood monocytes separately in vitro, and TNFα levels in the supernatant were measured following coincubation. Compared with monocytes injured with plasma EVs from perfused human lungs injured with bacterial pneumonia, secretion of TNFα by the monocytes was suppressed by treatment of plasma EVs from perfused human lungs injured with bacterial pneumonia and treated with HMW HA. In separate experiments, treatment of exogenous HMW HA at 100 μg/mL significantly decreased TNFα secretion in monocytes injured with plasma EVs (T6 h) from perfused human lungs injured with bacterial pneumonia. Data were presented as median with IQR, n = 4–6 per treatment groups, Comparison between groups were made with Mann–Whitney test. B: effects of HMW HA on phagocytosis of PA103 bacteria by human monocytes and neutrophils. HMW HA administration significantly increased bacteria phagocytosis by monocytes. Data were presented as median with IQR, n = 6–15 per treatment groups. Comparison between groups were made with Mann–Whitney. HMW HA administration also significantly increased bacteria phagocytosis and decreased inflammation by neutrophils. Data were presented as median with IQR, n = 12–14 per group. Comparison between groups were made with Mann–Whitney test. CFU, colony forming units; EVs, extracellular vesicles; HMW HA, high molecular weight hyaluronic acid; IQR, interquartile range; PA, Pseudomonas aeruginosa.

Figure 5.

Therapeutic effects of HMW HA on lung injury induced by PA103 bacterial instillation in human lungs with baseline AFC > 10%/h. A: instillation of HMW HA 2 mg significantly increased AFC rate in human lungs injured by PA103 bacterial pneumonia. Although there were no significant differences in lung weight gain, HMW HA treatment numerically improved protein permeability. Data were presented as medium with IQR, n = 4–6 per treatment groups. Comparison between groups were made using Dunn’s test following Kruskal–Wallis analysis. B: there were no significant differences in absolute neutrophil counts (ANC) and TNFα levels in BALF in human lungs injured by PA103 bacteria pneumonia treated with HMW HA at 6 h. Data were presented as median with IQR, n = 6 per treatment groups. C: HMW HA treatment had no significant effects in PA103 CFU counts in both the BALF and perfusate at 6 h in human lungs injured by PA103 bacteria. Data were presented as median with IQR, n = 6 per treatment groups. Comparison between groups were made using Dunn’s test following Kruskal–Wallis analysis. D: there were no significant effects in pulmonary artery pressure, lung compliance, or perfusate Po₂ levels over 6 h or between individual groups. Data were presented as median with IQR, n = 6 per treatment groups. Comparison vs. corresponding group by Mann–Whitney test. AFC, alveolar fluid clearance; BALF, bronchoalveolar lavage fluid; CFU, colony forming units; HMW HA, high molecular weight hyaluronic acid; IQR, interquartile range; PA, Pseudomonas aeruginosa.