Chest Infections |

DNA CpG Methylation Contributes to Missing Nitric Oxide Inflammatory Response in Human Alveolar Macrophages FREE TO VIEW

Karol Kremens*, MD; Linda Powers, MS; Robert Philibert, MD; Martha Monick, PhD
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University of Iowa, Coralville, IA

Chest. 2012;142(4_MeetingAbstracts):190A. doi:10.1378/chest.1359634
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SESSION TYPE: Cytokines/Cellular Interactions Posters

PRESENTED ON: Wednesday, October 24, 2012 at 01:30 PM - 02:30 PM

PURPOSE: Alveolar macrophages are primary phagocytic cells of the innate immune system. They are responsible for removal of infectious, toxic and allergic particles from the respiratory tract. Murine macrophages respond to inflammatory stimuli by producing large amounts of nitric oxide via nitric oxide synthase 2 (NOS2). Whether this nitric oxide response is replicated in human macrophages remains controversial.

METHODS: Human alveolar macrophages and murine macrophages Raw264.7 were stimulated with IFN-G and LPS in a time-response and dose-response manner. Presence of NOS2 and NO were measured by mRNA, western and nitric oxide analysis. Downstream modulators of LPS and interferon signaling were measured by protein and RNA analysis (Stat1, IRF-1, ICAM-1). Genome wide methylation status of the alveolar DNA samples was determined using the Illumina Infinium 475K HumanMethylation array.

RESULTS: Stimulation of human alveolar macrophages with a strong inflammatory signal (IFN-γ and LPS) produced no NOS2 mRNA or protein. Consistent with a lack of NOS2, there was no nitric oxide production. In contrast, murine Raw246.7 macrophage stimulation (IFN-G and LPS) led to significant production of NOS2 mRNA and protein as well as nitric oxide. The lack of NOS2 in human alveolar macrophages was not due to a loss of IFN-G / LPS signaling (Stat1, IRF-1) or downstream gene expression (ICAM-1). One possible explanation for the lack of NOS2 and nitric oxide in the human macrophages is epigenetic gene silencing. When we looked at methylation in the promoter/enhancer region and first exon of NOS2, all eight CpG probes from the 475K array were highly methylated (beta values of 0.83 =/- 0.024). This compares, for example, to methylation values of 0.065+/-0.007 in the same region of the expressed ICAM1 gene.

CONCLUSIONS: We observed a lack of production of nitric oxide in human alveolar macrophages in response to IFN- G and LPS. Our data points towards silencing of the NOS2 gene in human alveolar macrophages due to heavy CpG DNA methylation at the 5’end of the human NOS2 gene.

CLINICAL IMPLICATIONS: This data suggests unrecognized and significant differences in how mice and humans handle respiratory pathogens.

DISCLOSURE: The following authors have nothing to disclose: Karol Kremens, Linda Powers, Robert Philibert, Martha Monick

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University of Iowa, Coralville, IA




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