Sodium-potassium-chloride cotransporter 1 as a novel regulator of DAMPs-mediated alveolar epithelial-macrophage crosstalk in LPS-induced lung inflammation

Sepsis is a life-threatening condition characterized by uncontrolled systemic inflammation, in which macrophages play a pivotal role by releasing proinflammatory cytokines that exacerbate organ dysfunction and mortality. When injured, alveolar epithelial cells (AECs) release damage-associated molecular patterns (DAMPs), such as extracellular adenosine triphosphate (ATP), high-mobility group box 1 (HMGB1), and Cyclic GMP-AMP Synthase (cGAS), that further amplify inflammatory cascades and enhance macrophage activation. Sodium-potassium-chloride cotransporter 1 (NKCC1) has been implicated in the pathogenesis of acute lung injury, but its role in AEC-macrophage crosstalk remains poorly defined. To address this, we employed A549 lung epithelial cells and RAW264.7 murine macrophages stimulated with lipopolysaccharide (LPS) in the presence or absence of the NKCC1 inhibitor bumetanide. Five experimental groups were examined: control, bumetanide alone, LPS-A549 conditioned medium (CM), bumetanide pretreatment, and bumetanide post-treatment. LPS stimulation of A549 cells markedly increased extracellular ATP, HMGB1, and cGAS, while LPS-A549 CM triggered RAW264.7 cells to upregulate receptor for advanced glycation end products, P2RX7, Toll-like Receptor 2 and 4, phosphorylation of extracellular signal-regulated kinase and c-Jun N-terminal kinase, and production of interleukin-8 and tumor necrosis factor-α. Enhanced phagocytosis was also observed. Importantly, both pretreatment of A549 cells and post-treatment of RAW264.7 macrophages with bumetanide significantly attenuated these proinflammatory responses (p < 0.05). These findings demonstrate that LPS-induced DAMPs release from epithelial cells drives macrophage activation through inflammatory signaling, while NKCC1 inhibition effectively suppresses these pathways. This study suggests that NKCC1 may act as a regulator of DAMPs-mediated AEC-macrophage interactions.

Alveolar epithelial cells; Macrophages; Sepsis; Sodium–potassium-chloride cotransporter.