Cell culture oxygen levels affect B cell migration through HIF-1a signaling

Abstract

Abstract B lymphocytes in vitro encounter oxygen levels far in excess of what they experience in vivo. Room air incubator O2 is not 21%, the accepted level for outdoor O2[1]. High CO2 and humidity drive incubator O2 down (16–19%), with variability from door openings. However, in vivo O2 is far lower even (tissues 0–7%, blood 5–10%). Hypoxia-induced factor 1 alpha (HIF-1a) is a tightly regulated transcription factor responsive to O2 levels [2]. Downstream of HIF-1a are genes tied to the unfolded protein response, metabolism, proliferation, and migration. A therapeutic target in multiple myeloma [3], HIF-1a protein is stabilized at low O2 levels and rapidly degraded at higher O2 levels. We previously showed in primary human B cells and myeloma cell lines, that HIF-1a protein levels stabilize at physioxic 1% and 4% oxygen, but not at 19% oxygen. B cell migration in response to the CXCR4 ligand, CXCL12, was decreased at low O2 levels. Here we seek to add direct evidence to HIF-1a participation in B cell migration changes by using a lentiviral vector to produce stable transfectants expressing a HIF-1a shRNA to genetically ablate HIF-1a. We used C-chamber sub-chambers and Pro-Ox 110 gas controllers to stably control the gas levels inside a standard incubator. At 1% O2, HIF-1a shRNA expression resulted in undetectable HIF-1a protein levels, and a correlating increase in CXCR4-mediated B cell migration as compared to a lentiviral control vector or non-transfected cells. This adds genetic evidence that directly links incubator O2 levels, HIF-1a activity and B cell migration in vitro.