Prolonged in vitro anti-bacterial, anti-inflammatory, and surfactant-promoting effects of volatile anesthetics

Abstract

Background: Volatile anesthetics are gaining recognition for their benefits in long-term sedation of mechanically ventilated patients with bacterial pneumonia and acute respiratory distress syndrome. In addition to their sedative role, they also exhibit anti-bacterial and anti-inflammatory properties, though the mechanisms behind these effects remain only partially understood. In vitro studies examining the prolonged impact of volatile anesthetics on bacterial growth, inflammatory cytokine response, and surfactant proteins - key to maintaining lung homeostasis - are still lacking.

Methods: Using an anaerobic chamber setup, we evaluated the effects of the most commonly used volatile anesthetics, Sevoflurane and Desflurane, at clinically relevant concentrations on the growth of Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus. Bacterial growth was monitored over 24 h, assessing OD₆₀₀, CFU/ml, and growth rate during the log phase. In the same setup, but with aerobic conditions, we investigated the immunomodulatory properties of both anesthetics on human A549 cells, either with or without bacterial lipopolysaccharide (LPS, 1 µg/ml) stimulation. Over 48 h, we analyzed pro-inflammatory chemokine release using ELISA and assessed surfactant protein expression with Western blot analysis.

Results: Sevoflurane and Desflurane significantly reduced Pseudomonas aeruginosa growth as expressed consistently in OD₆₀₀ and CFU/ml starting after 12 h. Both volatile anesthetics also significantly reduced Staphylococcus aureus OD₆₀₀ starting after 21 h. Sevoflurane (p < 0.01) and Desflurane (p < 0.001) counteracted LPS-induced interleukin-8 release by A549 cells after 48 h and significantly ( p < 0.01 and p < 0.05) enhanced the expression of the propeptide of surfactant protein C after 24 h.

Conclusions: Prolonged anti-bacterial and anti-inflammatory effects of Sevoflurane and Desflurane include both the reduction of Pseudomonas aeruginosa and Staphylococcus aureus growth as well as the inhibition of LPS-induced chemokine release by A549 epithelial cells paralleled by an increase of surfactant protein expression. These effects highlight the potential of volatile anesthetics beyond sedation in supporting lung function in ventilated patients with respiratory failure.

Keywords: Pseudomonas aeruginosa; A549 cells; ARDS; Cytokines; Desflurane; IL-8; Pneumonia; Sevoflurane; Surfactant protein; Volatile anesthetics.

Fi. 1

Model of experimental set up. (A) Control gases (Ctrl) varied based on the type of culture: for bacterial cultures, an anaerobic environment was maintained (95% N₂ + 5% CO₂), while A549 cell cultures were kept in an aerobic environment (95% room air + 5% CO₂). The gas mixture also served as carrier gas for the application of two commonly used volatile anesthetics (VA). The concentration of Desflurane (Des, blue) and Sevoflurane (Sev, yellow) was measured by an external anesthesia gas detector which determined the concentration at the outgoing channel of gas conduction (exhaustion air, red). Gas concentrations were adjusted to MAC₅₀, (2.1 to 2.2% for Sev and 6.0% for Des) within the anaerobic chamber. (B, l) Bacterial culture flasks with eftPseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E. coli), and Staphylococcus aureus (S. aureus) as well as (B, right) 6-well plates with A549 cell cultures were put into the anaerobic container after gas equilibrium was achieved. In the A549 cell culture 3 wells were additionally exposed to lipopolysaccharide (LPS) of E. coli (1 µg/ml). (C, left) Analysis of optical density at 600 nm wavelength (OD₆₀₀) and colony forming units per milliliter bacterial suspension (CFU/ml) were performed after 9, 12, 15, 18, 21, and 24 h, respectively. (B, right) Analysis of Enzyme-linked Immunosorbent Assay (ELISA) and Western Blot analysis of pro-SP-C protein expression were performed 8, 16, 24, and 48 h after VA exposure

Fig. 2

Bacterial growth after exposure to volatile anesthetics Measures of (A, C, E) adjusted optical density at 600 nm wavelength (OD₆₀₀) and (B, D, F) colony forming units (CFU) per milliliter (ml) bacterial suspension are depicted for three bacterial strains associated with hospital-acquired pneumonia: two Gram-negative strains, Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli), and one Gram-positive strain, Staphylococcus aureus (S. aureus). Data collection was performed 9, 12, 15, 18, 21, and 24 h after exposure to volatile anesthetics (Sevoflurane: yellow, Desflurane: blue) or Control gas (grey). Concentrations are depicted as CFU/ml. Ctrl: Control gas, Sev: Sevoflurane, Des: Desflurane. All results of are presented as mean +/- standard error of the mean (SEM) for n = 5–7. Statistical significances are depicted as p < 0.05 *, p < 0.01 **, p < 0.001 ***. Asterisks account for $ = Sev, # = Des

Fig. 3

Prolonged effects of volatile anesthetics on IL-8 release by A549 cells under basal and LPS-induced conditions (A) Baseline interleukin (IL)-8 release in the presence of control gas (Ctrl; consisting of 95% room-air and 5% CO2), 6.0% Desflurane (Des), or 2.1-2.2% Sevoflurane (Sev). (B) IL-8 release in the presence of 1 µg/ml lipopolysaccharide (LPS) and Ctrl, Des, or Sev. (C) To assess, if volatile anesthetics (VA) exposure has an anti-inflammatory effect on LPS-exposed A549 cells, the difference in IL-8 concentration (pg/ml) between untreated and LPS-treated conditions was analyzed (for further details on the calculation method see results section). Measures were obtained throughout prolonged exposure (8, 16, 24, and 48 h) to VA. All results of are presented as mean +/- standard error of the mean (SEM) for n = 6 experiments. Statistical significances are depicted as p <0.05*, p <0.01**, p <0.001***. Asterisks account for + = Ctrl, $ = Sev, # = Des.

Fig. 4

Prolonged effects of volatile anesthetics on pro-SP-C expression by A549 cells under basal and LPS-induced conditions A549 cells (1.5 × 10⁵ cells/well) were treated with control gas (Ctrl; consisting of 95% room-air and 5% CO₂), 6.0% Desflurane (Des), or 2.1–2.2% Sevoflurane (Sev) in in the absence (B, left) or presence of 1 µg/ml lipopolysaccharide (LPS) (B, right) for the times indicated. Equal amounts of cell lysates were subjected to SDS-PAGE and immunoblotted for propeptide of surfactant protein C (pro-SP-C) and β-actin. (A) Full blots are provided in Supplement 4. For comparison, the results presented in arbitrary units (a.u.) were put into relation to those of Ctrl at each time point. Measures were obtained throughout prolonged exposure (8, 16, 24, and 48 h) to volatile anesthetics (VA). All results of are presented as mean +/- standard error of the mean (SEM) for n = 4–6. Statistical significances are depicted as p < 0.05*, p < 0.01**, p < 0.001***. Asterisks account for + = Ctrl, $ = Sev, # = Des

Fig. 5

Synopsis of volatile anesthetics effect from literature and own findings. Based on the results of our experiments, the following mechanisms are proposed: Volatile anesthetics (VA), Sevoflurane (Sev, yellow) and Desflurane (Des, blue), exhibit both anti-inflammatory and anti-bacterial effects in our in vitro model of ARDS. Results from this in vitro study included in the overview figure are marked with an *. All other elements are derived from the following literature [6, 33, 42, 50]. (Upper panel) At the alveolar level, VA exert various effects: (I) They stimulate alveolar epithelial (AEC) Type II cells [1], leading to increased surfactant protein C (SP-C) production *. Since SP-C binds bacterial components like lipopolysaccharide (LPS) *, this enhances [2] the clearance of bacterial components. (II) Additionally, VAs inhibit [3] LPS-induced, toll-like receptor (TLR)−4-mediated production of pro-inflammatory cytokines * and [4] interleukin (IL)−8-driven recruitment of neutrophils. Together, these actions reduce both local and systemic inflammation in acute respiratory distress syndrome (ARDS). (Lower panel) VAs also directly affect bacterial growth *, biofilm formation, and autoaggregation, particularly in P. aeruginosa. AM: Alveolar macrophages