C6 peptide blockade of Hv1 channels inhibits neutrophil migration into the lungs to suppress Pseudomonas aeruginosa-induced acute lung injury

Acute Lung Injury (ALI) and its most severe form, Acute Respiratory Distress Syndrome (ARDS), are critical pulmonary conditions characterized by life-threatening acute hypoxic respiratory failure, affecting over three million individuals globally each year. ALI involves alveolar inflammation and disruption of the alveolar-capillary barrier, primarily driven by neutrophil infiltration and the release of inflammatory mediators. In our previous study using a lipopolysaccharide (LPS)-induced mouse model of ALI, we demonstrated that C6, a peptide inhibitor of voltage-gated proton channels (Hv1), ameliorates lung injury, identifying Hv1 as a potential therapeutic target. However, (i) whether the anti-inflammatory effects of C6 are translatable to a clinically relevant live bacterial infection model, and (ii) the molecular mechanisms underlying these anti-inflammatory effects, remain unknown, and are a crucial next step towards targeted rational drug development. To induce ALI, we used an intratracheal Pseudomonas aeruginosa infection model, a gram-negative bacterium relevant in ventilated and immunocompromised patients. A separate group of infected mice also received intravenous treatment with C6 (4 mg/kg). Lung injury severity was evaluated using histopathological analysis. Bronchoalveolar lavage (BAL) fluid was collected to quantify neutrophil infiltration and proinflammatory cytokines concentrations. In addition, reactive oxygen species (ROS) production and intracellular calcium levels in BAL neutrophils were measured. RNA sequencing of BAL neutrophils was conducted to assess C6-induced transcriptional changes. Key findings were validated in vitro using human neutrophils. C6 mitigates P. aeruginosa-induced ALI in mice by reducing neutrophil infiltration into the alveolar space by ~ 86%, improving lung injury scores, decreasing BAL fluid proinflammatory cytokine levels, and suppressing neutrophil ROS production and intracellular calcium levels. RNA sequencing of BAL neutrophils revealed 51 downregulated genes, including key regulators of neutrophil migration, cytokine release, and ROS production; only three genes were upregulated and they also have roles in neutrophil immune defense. In human neutrophils, C6 similarly inhibited chemotaxis and reduced ROS and cytokine release, and calcium influx. Targeting Hv1 with C6 effectively protects against P. aeruginosa-induced ALI by limiting neutrophil recruitment and activation. These findings establish C6 as a promising therapeutic candidate against infectious ALI and provide important mechanistic insights into its immunomodulatory effects on neutrophils.