Downregulation of IL-8 and IL-10 by LRRC8A Inhibition through the NOX2-Nrf2-CEBPB Transcriptional Axis in...

M₂-polarized, tumor-associated macrophages (TAMs) produce pro-tumorigenic and angiogenic mediators, such as interleukin-8 (IL-8) and IL-10. Leucine-rich repeat-containing protein 8 members (LRRC8s) form volume-regulated anion channels and play an important role in macrophage functions by regulating cytokine and chemokine production. We herein examined the role of LRRC8A in IL-8 and IL-10 expression in THP-1-differentiated M₂-like macrophages (M₂-MACs), which are a useful tool for investigating TAMs. In M₂-MACs, the pharmacological inhibition of LRRC8A led to hyperpolarizing responses after a transient depolarization phase, followed by a slight elevation in the intracellular concentration of Ca²⁺. Both the small interfering RNA-mediated and pharmacological inhibition of LRRC8A repressed the transcriptional expression of IL-8 and IL-10, resulting in a significant reduction in their secretion. The inhibition of LRRC8A decreased the nuclear translocation of phosphorylated nuclear factor-erythroid 2-related factor 2 (Nrf2), while the activation of Nrf2 reversed the LRRC8A inhibition-induced transcriptional repression of IL-8 and IL-10 in M₂-MACs. We identified the CCAAT/enhancer-binding protein isoform B, CEBPB, as a downstream target of Nrf2 signaling in M₂-MACs. Moreover, among several upstream candidates, the inhibition of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) suppressed the Nrf2-CEBPB transcriptional axis in M₂-MACs. Collectively, the present results indicate that the inhibition of LRRC8A repressed IL-8 and IL-10 transcription in M₂-MACs through the NOX2-Nrf2-CEBPB axis and suggest that LRRC8A inhibitors suppress the IL-10-mediated evasion of tumor immune surveillance and IL-8-mediated metastasis and neovascularization in TAMs.