Oxidative modification enhances the immunostimulatory effects of extracellular mitochondrial DNA on plasmacytoid dendritic cells

Abstract

Oxidative modification enhances the immunostimulatory effects of extracellular mitochondrial DNA on plasmacytoid dendritic cells, Free Radical Biology and Medicine, http://dx.doi.org/10.1016/j. freeradbiomed. 2014.09.028 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.www.elsevier.com/locate/freeradbiomed Abbreviations: 7-AAD, 7-aminoactinomycin-D; 8-oxoG, 8-oxo-7,8-dihydroguanine; 8-oxodG, 8-oxo-7,8-dihydro-2'- well as increased TNF-Į and IL-8 production from the cells. These effects were more apparent when pDCs were exposed to oxidatively modified mtDNA. Neither native nor oxidized mtDNA molecules were able to induce interferon (IFN)-Į secretion from pDCs unless they formed a complex with human cathelicidin LL-37, an antimicrobial peptide.Interestingly, simultaneous administration of a Toll-like receptor (TLR)9 antagonist abrogated the effects of both native and oxidized mtDNAs on human pDCs. In a murine model, oxidized mtDNA also proved a more potent activator of pDCs compared to the native form, except for induction of IFN-Į production. Collectively, we demonstrate here for the first time that elevated levels of 8-oxoG bases in the extracellular mtDNA induced by oxidative stress increase the immunostimulatory capacity of mtDNA on pDCs. 3 HighlightsWe compared the immunostimulatory capacity of native and oxidized mtDNA on pDCs.8-Oxoguanin-containing mtDNA has a greater capacity to activate primary human pDCs.In vivo, oxidized mtDNA also proved a more potent activator of pDC than native mtDNA.

Mitochondrial DNA has a pro-inflammatory role in AMD

“…Practically all the DNA sensors described to date are intracellular 36 , including Toll-like receptor 9 (TLR9), a transmembrane receptor of unmethylated CpG motifs that has been confirmed to be predominantly intracellular rather than on the cell membrane 67,68 , including in ARPE-19 69 . Our findings are also in concordance with previous evidence that extracellular mtDNA has no immunostimulatory effect unless used with a molecule that aids its uptake into the cell 20,70 . In other studies, extracellular mtDNA was mildly immunostimulatory in certain cell types, but only at very high concentrations (>10µg/mL) 13,20 .…”

“…Our findings are also in concordance with previous evidence that extracellular mtDNA has no immunostimulatory effect unless used with a molecule that aids its uptake into the cell 20,70 . In other studies, extracellular mtDNA was mildly immunostimulatory in certain cell types, but only at very high concentrations (>10µg/mL) 13,20 . In ARPE-19 cells, extracellular CpG DNA has been shown to induce IL-8 secretion only at the dose of 84µg/mL, almost 2 orders of magnitude higher than the concentration we used, and the authors attributed the effect to CpG DNA that had been internalized by phagocytosis 69 .…”

“…In particular, mtDNA has been found to induce the pro-inflammatory cytokines IL-6 and IL-8 in vitro and in vivo 12,13,17–20 . Both of these cytokines have been associated with AMD progression 21–25 and the development of choroidal neovascularization (CNV) 26–28 .…”

Mitochondrial DNA Promotes NLRP3 Inflammasome Activation and Contributes to Endothelial Dysfunction and Inflammation in Type 1 Diabetes

“…TLR-9 antagonist reduces phosphorylation of MAPKs, such as p38 and p44/42, and IL-8 levels in human neutrophils stimulated with mDNA extracted from various tissues (Zhang et al, 2010). TLR-9 inhibition also decreases IL-8 and TNF-α in mDNA-stimulated cells from humans and mice (Pazmandi et al, 2014). NLRP3 is another receptor associated with mDNA effects.…”

“…mDNA oxidation has been associated with structural modifications that increase immunogenesis. Oxidized mDNA potentiates increased pro-inflammatory factors (IL-8 and TNF-α) in plasmocytoid dendritic cells (Pazmandi et al, 2014). One of the most common modifications related to DNA oxidation is represented by increased levels of oxidatively modified guanine bases (8-OHdG) (Kuchino et al, 1987).…”