Abstract
Background Hypoxia is a potent molecular signal for cellular metabolism, mitochondrial function, and migration. Conditions of low oxygen tension trigger regulatory cascades mediated via the highly conserved HIF-1α post-translational modification system. In the adaptive immune response, B cells (Bc) are activated and differentiate under hypoxic conditions within lymph node germinal centers, and subsequently migrate to other compartments. During migration, they traverse through changing oxygen levels, ranging from 1-5% in the lymph node to 5-13% in the peripheral blood. Interestingly, the calcineurin inhibitor cyclosporine A is known to stimulate prolyl hydroxylase activity, resulting in HIF-1α destabilization and may alter Bc responses directly. Over 60% of patients taking calcineurin immunosuppressant medications have hypo-gammaglobulinemia and poor vaccine responses, putting them at high risk of infection with significantly increased morbidity and mortality.
Results We demonstrate that O2 tension is a previously unrecognized Bc regulatory switch, altering CXCR4 chemokine receptor signaling in activated Bc through HIF-1α expression and controlling critical aspects of Bc migration. Our data demonstrate that calcineurin inhibition hinders this O2 regulatory switch in primary human Bc.
Conclusion This previously unrecognized effect of calcineurin inhibition directly on human Bc has significant and direct clinical implications.
ABBREVIATIONS
- AF
- ammonium formate
- AGC
- automatic gain control
- BCA
- bicinchoninic acid
- CD
- cluster of differentiation
- CNI
- Calcineurin inhibitors
- CyA
- cyclosporine A
- DMOG
- dimethyloxallyl glycine
- DTT
- dithiothreitol
- ECL
- enhanced chemiluminescence
- EDTA
- ethylenediaminetetraacetic acid
- FAK
- Focal adhesion kinase
- FBS
- fetal bovine serum
- FDR
- false discovery rate
- GC
- Germinal center
- HCD
- higherenergy collisional dissociation
- HIF
- Hypoxia inducible factor
- HPLC
- high performance liquid chromatography
- IL-2
- Interleukin 2
- HRP
- horseradish peroxidase
- LN
- Lymph node
- IMDM
- Iscove’s modified Dulbecco’s medium
- MAPK
- Mitogenactivated protein kinase
- MKP-1
- MAP kinase phosphatase 1
- NFAT
- Nuclear factor of activated T-cells
- O/N
- Overnight
- PBMC
- peripheral blood mononuclear cell
- PBS
- phosphate-buffered saline
- PHD
- prolyl-4-hydroxylase
- PVDF
- polyvinylidene difluoride
- RBCs
- Red blood cells
- RGS1
- Regulator Of G Protein Signaling 1
- RLU
- relative luminescent units
- RPMI
- Roswell Park Memorial Institute
- RT
- room temperature
- SCS
- single cell suspension
- shRNA
- Small hairpin ribonucleic acid
- TBS
- tris-buffered saline
- TBS-T
- TBS with tween-20
- TEAB
- triethylammonium bicarbonate
- TFA
- trifluoroacetic acid
- TMT
- tandem mass tag