STAT3 governs the HIF-1α response in IL-15 primed human NK cells.

Natural killer (NK) cells mediate innate host defense against microbial infection and cancer. Hypoxia and low glucose are characteristic for these tissue lesions but do not affect early interferon (IFN) γ and CC chemokine release by interleukin 15 (IL-15) primed human NK cells in vitro. Hypoxia inducible factor 1α (HIF-1α) mediates cellular adaption to hypoxia. Its production is supported by mechanistic target of rapamycin complex 1 (mTORC1) and signal transducer and activator of transcription 3 (STAT3). We used chemical inhibition to probe the importance of mTORC1 and STAT3 for the hypoxia response and of STAT3 for the cytokine response in isolated and IL-15 primed human NK cells. Cellular responses were assayed by magnetic bead array, RT-PCR, western blotting, flow cytometry, and metabolic flux analysis. STAT3 but not mTORC1 activation was essential for HIF-1α accumulation, glycolysis, and oxygen consumption. In both primed normoxic and hypoxic NK cells, STAT3 inhibition reduced the secretion of CCL3, CCL4 and CCL5, and it interfered with IL-12/IL-18 stimulated IFNγ production, but it did not affect cytotoxic granule degranulation up on target cell contact. We conclude that IL-15 priming promotes the HIF-1α dependent hypoxia response and the early cytokine response in NK cells predominantly through STAT3 signaling.

Innate Cytokine Induced Early Release of IFNγ and CC Chemokines from Hypoxic Human NK Cells Is Independent of Glucose.

Natural killer (NK) cells are among the first innate immune cells to arrive at sites of tissue inflammation and regulate the immune response to infection and tumors by the release of cytokines including interferon (IFN)γ. In vitro exposure to the innate cytokines interleukin 15 (IL-15) and IL-12/IL-18 enhances NK cell IFNγ production which, beyond 16 h of culture, was shown to depend on metabolic switching to glycolysis. NK effector responses are, however, rapid by comparison. Therefore, we sought to evaluate the importance of glycolysis for shorter-term IFNγ production, considering glucose deprivation and hypoxia as adverse tissue inflammation associated conditions. Treatments with IL-15 for 6 and 16 h were equally effective in priming early IFNγ production in human NK cells in response to secondary IL-12/IL-18 stimulation. Short-term priming was not associated with glycolytic switching but induced the release of IFNγ and, additionally, CCL3, CCL4 and CCL5 from both normoxic and hypoxic NK cells in an equally efficient and, unexpectedly, glucose independent manner. We conclude that release of IFNγ and CC chemokines in the early innate immune response is a metabolically autonomous NK effector program.

Carbonyl reductase 1 is a predominant doxorubicin reductase in the human liver.

A first step in the enzymatic disposition of the antineoplastic drug doxorubicin (DOX) is the reduction to doxorubicinol (DOX-OL). Because DOX-OL is less antineoplastic but more cardiotoxic than the parent compound, the individual rate of this reaction may affect the antitumor effect and the risk of DOX-induced heart failure. Using purified enzymes and human tissues we determined enzymes generating DOX-OL and interindividual differences in their activities. Human tissues express at least two DOX-reducing enzymes. High-clearance organs (kidney, liver, and the gastrointestinal tract) express an enzyme with an apparent Km of approximately 140 microM. Of six enzymes found to reduce DOX, Km values in this range are exhibited by carbonyl reductase 1 (CBR1) and aldo-keto reductase (AKR) 1C3. CBR1 is expressed in these three organs at higher levels than AKR1C3, whereas AKR1C3 has higher catalytic efficiency. However, inhibition constants for DOX reduction with 4-amino-1-tert-butyl-3-(2-hydroxyphenyl)pyrazolo[3,4-d]pyrimidine (an inhibitor that can discriminate between CBR1 and AKR1C3) were identical for CBR1 and human liver cytosol, but not for AKR1C3. These results suggest that CBR1 is a predominant hepatic DOX reductase. In cytosols from 80 human livers, the expression level of CBR1 and the activity of DOX reduction varied >70- and 22-fold, respectively, but showed no association with CBR1 gene variants found in these samples. Instead, the interindividual differences in CBR1 expression and activity may be mediated by environmental factors acting via recently identified xenobiotic response elements in the CBR1 promoter. The variability in the CBR1 expression may affect outcomes of therapies with DOX, as well as with other CBR1 substrates.