Short Term Hypoxia Synergizes with Interleukin 15 Priming in Driving Glycolytic Gene Transcription and Supports Human Natural Killer Cell Activities

Abstract

Natural killer (NK) cells induce apoptosis in infected and transformed cells and are important producers of immunoregulatory cytokines. Therefore, they operate under low oxygen conditions (hypoxia) in inflammatory and tumor environments. In vitro studies of NK cells are, however, commonly performed in ambient air (normoxia). We used global gene expression profiling to evaluate changes in transcriptional pathways in primary human NK cells following short term culture under hypoxia compared with normoxia and in response to interleukin 15 (IL-15) priming using a 2 ؋ 2 factorial design. The largest contrasts observed were priming dependences for associations between hypoxia and the hypoxia-inducible factor (Hif) 1 signaling and glycolysis pathways. RT-PCR confirmed positive synergistic hypoxia/IL-15 interactions for genes of key regulatory and metabolic enzymes. IL-15 primes NK cells for effector functions, which were recently demonstrated to depend on glycolytic switching. We did not, however, observe important increases in glycolytic flux through hypoxia and priming alone. Chemical Hif-1␣ inhibition suggested equal importance of this transcription factor for glycolysis and energy production under normoxia and hypoxia. Hypoxia promoted secretion of CC chemokines Ccl3/4/5 and macrophage migration inhibitory factor. Unexpectedly, hypoxia also stimulated migration of NK cells through the extracellular matrix and shifted amounts of susceptible leukemia target cells toward late apoptosis in a cell killing assay. We conclude that short term hypoxia supports these activities by positively interacting with NK cell priming at the level of glycolytic gene transcription. Hypoxic conditioning of NK cells may thus benefit their use in cell-based immunotherapy of cancer.

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

“…In this regard, it is notable that IFNγ production in mouse NK cells treated for 6 h with IL-12/IL-18 was shown previously to remain unaffected by glucose deprivation or chemical ATP-synthase inhibition by oligomycin [49]. Accordingly, human NK cells primed with IL-15 for 6 h show only a very moderate increase in glycolysis [7]. Nevertheless, such short-term IL-15 exposure and hypoxia transcriptionally upregulate hypoxia inducible factor (HIF-1α) and glycolysis pathways in a synergistic fashion [7].…”

“…Approval for this study was obtained by the Ethics Committee Medical Faculty Mannheim (approval number 2016-521N-MA). For all experiments, NK cells were isolated, their purity was evaluated and cells were cultured as described [7]. In brief, immunomagnetic cell separation (NK-Cell Isolation Kit, Miltenyi Biotec, Bergisch Gladbach, Germany) yielded >99% CD45 + leukocytes that stained ≥93% for NK cells (CD56 + CD3 − ) and ≤1% each for T cells (CD3 + ), B cells (CD19 + ), granulocytes (CD15 + ) and monocytes (CD14 + ) by flow cytometry.…”

“…Accordingly, human NK cells primed with IL-15 for 6 h show only a very moderate increase in glycolysis [7]. Nevertheless, such short-term IL-15 exposure and hypoxia transcriptionally upregulate hypoxia inducible factor (HIF-1α) and glycolysis pathways in a synergistic fashion [7]. In agreement with this, treatment of human NK cells with IL-15 for 4 h under chemical hypoxia augments protein levels of HIF-1α [50], the major mediator of cellular adaption to hypoxia [51].…”

“…They develop predominantly from the common lymphoid progenitor [4]. NK cell development, survival and homeostasis depend on interleukin 15 (IL-15) [5,6] which also promotes NK cell migration [7,8] and enhances their effector functions in response to secondary stimulation, referred to as priming [9][10][11][12].…”

Molecular definition of group 1 innate lymphoid cells in the mouse uterus

“…In addition, Tgfbi , encoding transforming growth factor-beta induced protein (TGFBIp, BIGH3), was also upregulated in trNK cells. UpSet pointed to Slc16a1 , encoding the cell exporter of lactate MCT1, to be uniquely upregulated in trNK cells, emphasizing the potential importance of anaerobic glycolysis for fueling uterine CD49a + cells (Velásquez et al 2016). Biglycan, encoded by Bgn , is a CD44 ligand that may be involved in the recruitment of circulating CD16 -ve NK cells into human endometrium (Kitaya & Yasuo 2008) and upregulated in trNK cells ( Figure S3F ).…”