IFN-γ Primes RAW264 Macrophages and Human Monocytes for Enhanced Oxidant Production in Response to CpG DNA via Metabolic Signaling: Roles of TLR9 and Myeloperoxidase Trafficking

Abstract

Macrophages and monocytes are activated by CpG DNA motifs to produce NO, which is enhanced dramatically by IFN-γ. We hypothesize that synergistic cellular responses to IFN-γ and CpG DNA are due to cross-talk between metabolic signaling pathways of leukocytes. Adherent RAW264.7 macrophages and human monocytes exhibited NAD(P)H autofluorescence oscillation periods of ∼20 s. IFN-γ increased the oscillatory amplitude, which was required for CpG DNA-mediated metabolic changes. These alterations in metabolic dynamics required the appropriate combinations of murine/human TLR9 and murine/human-specific CpG DNA. Other factors that also promoted an increase in metabolic oscillatory amplitude could substitute for IFN-γ. Because recent studies have shown that the metabolic frequency is coupled to the hexose monophosphate shunt, and the amplitude is coupled to the peroxidase cycle, we tested the hypothesis that myeloperoxidase (MPO) participates in IFN-γ priming for oxidant production. MPO inhibitors blocked cell responses to IFN-γ and CpG DNA. In the absence of IFN-γ exposure, the effects of CpG DNA could be duplicated by MPO addition to cell samples. Moreover, monocytes from MPO knockout mice were metabolically unresponsive to IFN-γ and CpG DNA. NAD(P)H frequency doubling responses due to CpG DNA were blocked by an inhibitor of the hexose monophosphate shunt. Because NAD(P)H participates in electron trafficking to NO and superoxide anions, we tested oxidant production. Although CpG DNA alone had no effect, IFN-γ plus CpG enhanced NO and reactive oxygen metabolite release compared with IFN-γ treatment alone. We suggest that amplitude and frequency modulation of cellular metabolic oscillations contribute to intracellular signaling synergy.

IL-17 and IFN-γ Mediate the Elicitation of Contact Hypersensitivity Responses by Different Mechanisms and Both Are Required for Optimal Responses

“…Inducible nitric oxide synthase (iNOS) and myeloperoxidase (MPO), which are primarily produced by macrophages and granulocytes (36, 37), have been reported to play roles in cutaneous inflammations and CHS responses (38–40). IFN-γ is a stimulator for both iNOS and MPO (37, 41) whereas it is unknown whether IL-17 regulates ROS activities in inflammatory tissues. In the hapten challenged skin, iNOS and MPO activities were significantly inhibited in IFN-γR−/− mice compared to wild type mice (Fig.…”

Monocyte chemoattractant protein-1 influences trauma-hemorrhage-induced distal organ damage via regulation of keratinocyte-derived chemokine production

“…Because myeloperoxidase is not exclusively found in neutrophils but also in macrophages (2,40), the neutrophil content in lung and liver was determined by immunohistochemistry staining. Fresh tissue samples were fixed in 4% buffered formalin and embedded in paraffin.…”

Innate immune recognition of, and regulation by, DNA

“…Although the precise mechanism involved in this species-specific TLR9 expression pattern remains unclear, it should be noted that distinct cis-and trans-regulation at the promoter region of the gene encoding TLR9 has been reported [51,52]. Moreover, growing recent evidence indicates that TLR9 expression and/or function can be upregulated in B cells [53] and other cell types, such as macrophages or cDCs stimulated with IFNb [52] or IFNg [54,55], monocytes infected with Yersinia pestis [56], granulocytes stimulated with granulocyte-macrophage colony-stimulating factor [57,58], and even epithelial cells cultured with transforming growth factor (TGF)a [59]. These observations might underscore the potential contribution of these various cell types, other than B cells and pDCs, to TLR9-mediated protective or pathogenic roles in infection and other immunological disorders.…”