Inhibition of Interleukin-12 p40 Transcription and NF-κB Activation by Nitric Oxide in Murine Macrophages and Dendritic Cells

Abstract

Nitric oxide (NO), an important effector molecule of the innate immune system, can also regulate adaptive immunity. In this study, the molecular effects of NO on the toll-like receptor signaling pathway were determined using interleukin-12 (IL-12) as an immunologically relevant target gene. The principal conclusion of these experiments is that NO inhibits IL-1 receptor-associated kinase (IRAK) activity and attenuates the molecular interaction between tumor necrosis factor receptor-associated factor-6 and IRAK. As a consequence, the NO donor S-nitroso-N-acetylpenicillamine (SNAP) inhibits lipopolysaccharide (LPS)-induced IL-12 p40 mRNA expression, protein production, and promoter activity in murine macrophages, dendritic cells, and the murine macrophage cell line RAW 264.7. Splenocytes from inducible nitric-oxide synthase-deficient mice demonstrate markedly increased IL-12 p40 protein and mRNA expression compared with wild type splenocytes. The inhibitory action of NO on IL-12 p40 is independent of the cytokine IL-10. The effects of NO can be directly attributed to inhibition of NF-κB activation through IRAK-dependent pathways. Accordingly, SNAP strongly reduces LPS-induced NF-κB DNA binding to the p40 promoter and inhibits LPS-induced IκB phosphorylation. Similarly, NO attenuates IL-1β-induced NF-κB activation. These experiments provide another example of how an innate immune molecule may have a profound effect on adaptive immunity.