Pro-inflammatory phenotype of B cells with depleted mitochondrial DNA
FISCHER R. 1, URBANCZYK S. 1, TAUDTE V. 2, BARIS O. 3, EKICI A. 4, WIRTZ S. 5, MIELENZ D. 1
1 Division of Molecular Immunology, Department of Internal Medicine III, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany; 2 Institute of Laboratory Medicine and Pathobiochemistry, Molecular Diagnostics, University of Marburg, Marburg, Germany; 3 UMR CNRS 6015/INSERM U1083, MitoVasc, University of Angers, Angers, France; 4 Institute of Human Genetics, Universitätsklinikum Erlangen, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany; 5 Dept. of Internal Medicine I, Universitätsklinikum Erlangen, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany
Mitochondrial DNA (mtDNA) encodes for essential subunits of the respiratory chain complexes of oxidative phosphorylation (OxPhos). Preliminary work showed that mtDNA is depleted during B cell maturation by CD23Cre-mediated expression of a dominant-negative K320E variant of mitochondrial helicase TWINKLE (DNT). This impairs OxPhos and humoral immunity but exaggerates glycolysis. Depletion of mtDNA by DNT increased fumarate and stabilized hypoxia-inducible factor 1-alpha (HIF1a), which could alter gene expression, likewise, the flux of glucose should be deviated in DNT B cells.
1) To identify genes regulated by OxPhos in B cells by comparing the gene expression profile of LPS-activated CD23Cre B cells with CD23Cre-DNT B cells.
2) To trace the aberrant flux of glucose in CD23Cre-DNT B cells.
RNA-Seq was performed with 48h LPS-activated CD23Cre and CD23Cre-DNT B cells. For glucose flux analysis, cells under similar conditions were labelled with
12C- or 13C-labelled glucose for another 12h and metabolites were analyzed by mass spectrometry.
Over 5000 genes are differentially regulated between CD23Cre and CD23Cre-DNT B cells. We found a strong induction of the integrated stress response (ISR), indicated by up-regulation of ATF-5, TRIB-3 and ASNS (asparaginsynthetase), suggesting that DNT expression affects amino acid metabolism. ASNS metabolizes L-aspartate to asparagine. Consistent with high ASNS expression, L-aspartate was strongly reduced in DNT B cells and less labelled with glucose. Moreover, we found a strong induction of the IL-6 pathway. In line with the RNA data, IL-6 was also significantly increased in the supernatant of DNT cells.
Reduction of OxPhos by DNT expression in B cells alters gene expression, glucose usage and amino acid metabolism. Therefore, OxPhos is required to prevent the ISR and exacerbation of IL-6 expression and production. This argues for an inflammatory phenotype of DNT B cells.
Supported by grants from the DFG (TRR130, MI9939/5-2, to D.M.)