GTS-21, an α7nAChR agonist, increases pulmonary bacterial clearance in mice by restoring hyperoxia-compromised macrophage function

Abstract

Background Mechanical ventilation, in combination with supraphysiological concentrations of oxygen (i.e., hyperoxia), is routinely used to treat patients with respiratory distress, such as COVID-19. However, prolonged exposure to hyperoxia compromises the clearance of invading pathogens by impairing macrophage phagocytosis. Previously, we have shown that the exposure of mice to hyperoxia induces the release of the nuclear protein high mobility group box-1 (HMGB1) into the pulmonary airways. Furthermore, extracellular HMGB1 impairs macrophage phagocytosis and increases the mortality of mice infected with Pseudomonas aeruginosa (PA). The aim of this study was to determine whether GTS-21 (3-(2,4-dimethoxybenzylidene) anabaseine), an α7 nicotinic acetylcholine receptor (α7nAChR) agonist, could (1) inhibit hyperoxia-induced HMGB1 release into the airways; (2) enhance macrophage phagocytosis and (3) increase bacterial clearance from the lungs in a mouse model of ventilator-associated pneumonia. Method GTS-21 (0.04, 0.4, and 4 mg/kg) or saline were administered by intraperitoneal injection to mice that were exposed to hyperoxia (≥ 99% O2) and subsequently challenged with PA. Results The systemic administration of 4 mg/kg i.p. of GTS-21 significantly increased bacterial clearance, decreased acute lung injury and decreased accumulation of airway HMGB1 compared to the saline control. To determine the mechanism of action of GTS-21, RAW 264.7 cells, a macrophage-like cell line, were incubated with different concentrations of GTS-21 in the presence of 95% O2. The phagocytic activity of macrophages was significantly increased by GTS-21 in a dose-dependent manner. In addition, GTS-21 significantly inhibited the cytoplasmic translocation and release of HMGB1 from RAW 264.7 cells and attenuated hyperoxia-induced NF-κB activation in macrophages and mouse lungs exposed to hyperoxia and infected with PA. Conclusions Our results indicate that GTS-21 is efficacious in improving bacterial clearance and reducing acute lung injury via enhancing macrophage function by inhibiting the release of nuclear HMGB1. Therefore, the α7nAChR represents a possible pharmacological target to improve the clinical outcome of patients on ventilators by augmenting host defense against bacterial infections.

The Positive Allosteric Modulation of alpha7-Nicotinic Cholinergic Receptors by GAT107 Increases Bacterial Lung Clearance in Hyperoxic Mice by Decreasing Oxidative Stress in Macrophages

“…Compared to our previous findings, where the administration of 4 mg/kg i.p. of GTS-21 three times a day under the same experimental paradigm utilized in this study [ 37 ], also attenuated hyperoxia-compromised bacterial clearance in mice. In contrast, the twice-daily administration of 3.3 mg/kg i.p.…”

“…Prolonged exposure to hyperoxia can compromise the clearance of pathogenic bacteria in the lungs. [ 37 , 42 , 44 ]. To determine if GAT107 can restore the hyperoxia-compromised clearance of bacteria in the lungs, mice were continuously exposed for 48 h to 95% O 2 (i.e., hyperoxia), administered intraperitoneal (i.p.)…”

“…Our results demonstrated that 3.3 μM of GAT107 significantly attenuated the hyperoxia-induced impairment of macrophage phagocytic function in cultured macrophages and primary BMDMs ( Figure 2 ). Previously, we and others have reported that prolonged exposure to hyperoxia decreases macrophage phagocytic function [ 22 , 37 , 42 , 64 ]. The partial-α7nAChR agonist, GTS-21 (5–50 µM), decreases impaired phagocytic function produced by hyperoxia, which was due, in part, to a decrease in HMGB1 release from macrophages [ 37 ].…”

“…The phagocytosis assay was performed, as previously described, with minor modifications [ 22 , 37 ]. Briefly, RAW 264.7 cells or BMDMs were seeded in 24-well plates and allowed to adhere for 6 h, followed by exposure to 95% O 2 in the absence or presence of GAT107 (3.3 µM) for 24 h. The concentration of 3.3 µM of GAT107 was selected based on preliminary data obtained from phagocytosis assay studies.…”

“…Previously, our lab has shown that GTS-21, a partial agonist of the α7nAChR, significantly increased bacterial clearance and decreased lung injury in hyperoxia-exposed mice with PA pneumonia [ 37 ]. Importantly, GTS-21 activates the cholinergic anti-inflammatory pathway, which has been shown to attenuate the phagocytic function of macrophages compromised by the prolonged exposure to hyperoxia [ 37 ]. However, other clinically relevant endpoints, such as mortality rates, were not significantly affected by GTS-21 administration (unpublished results).…”