Mitochondrial transcription factor A, an endogenous danger signal, promotes TNFα release via RAGE- and TLR9-responsive plasmacytoid dendritic cells.
OBJECTIVE
Mitochondrial transcription factor A (TFAM) is normally bound to and remains associated with mitochondrial DNA (mtDNA) when released from damaged cells. We hypothesized that TFAM, bound to mtDNA (or equivalent CpG-enriched DNA), amplifies TNFα release from TLR9-expressing plasmacytoid dendritic cells (pDCs) by engaging RAGE.


METHODS
Murine Flt3 ligand-expanded splenocytes obtained from C57BL/6 mice were treated with recombinant human TFAM, alone or in combination with CpG-enriched DNA with subsequent TNFα release measured by ELISA. The role of RAGE was determined by pre-treatment with soluble RAGE or heparin or by employing matching RAGE (-/-) splenocytes. TLR9 signaling was evaluated using a specific TLR9-blocking oligonucleotide and by inhibiting endosomal processing, PI3K and NF-κB. Additional studies examined whether heparin sulfate moieties or endothelin converting enzyme-1 (ECE-1)-dependent recycling of endosomal receptors were required for TFAM and CpG DNA recognition.


RESULTS
TFAM augmented splenocyte TNFα release in response to CpGA DNA, which was strongly dependent upon pDCs and regulated by RAGE and TLR9 receptors. Putative TLR9 signaling pathways, including endosomal acidification and signaling through PI3K and NF-κB, were essential for splenocyte TNFα release in response to TFAM+CpGA DNA. Interestingly, TNFα release depended upon endothelin converting enzyme (ECE)-1, which cleaves and presumably activates TLR9 within endosomes. Recognition of the TFAM-CpGA DNA complex was dependent upon heparin sulfate moieties, and recombinant TFAM Box 1 and Box 2 proteins were equivalent in terms of augmenting TNFα release.


CONCLUSIONS
TFAM promoted TNFα release in a splenocyte culture model representing complex cell-cell interactions in vivo with pDCs playing a critical role. To our knowledge, this study is the first to incriminate ECE-1-dependent endosomal cleavage of TLR9 as a critical step in the signaling pathway leading to TNFα release. These findings, and others reported herein, significantly advance our understanding of sterile immune responses triggered by mitochondrial danger signals.
Dorothy M. Davis Heart and Lung Research Institute, Division of Pulmonary, Allergy, Critical Care, and Sleep Medicine, Wexner Medical Center, the Ohio State University, Columbus, Ohio, United States of America.
Mark W
MW
Julian
Guohong
G
Shao
Zachary C
ZC
Vangundy
Tracey L
TL
Papenfuss
Elliott D
ED
Crouser
D000818:Animals; D002460:Cell Line; D018899:CpG Islands; D004272:DNA, Mitochondrial / Q000276:immunology / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003713:Dendritic Cells / Q000276:immunology / Q000378:metabolism*; D024243:HMGB1 Protein / Q000378:metabolism; D024261:HMGB2 Protein / Q000378:metabolism; D006801:Humans; D008297:Male; D051379:Mice; D024101:Mitochondrial Proteins / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008954:Models, Biological; D016328:NF-kappa B / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011485:Protein Binding; D015398:Signal Transduction; D013154:Spleen / Q000166:cytology / Q000378:metabolism; D051217:Toll-Like Receptor 9 / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D004272:DNA, Mitochondrial; D004268:DNA-Binding Proteins; D024243:HMGB1 Protein; D024261:HMGB2 Protein; D024101:Mitochondrial Proteins; D016328:NF-kappa B; D051217:Toll-Like Receptor 9; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; C422562:mitochondrial transcription factor A; D019869:Phosphatidylinositol 3-Kinases; C498977:Mok protein, mouse; D020928:Mitogen-Activated Protein Kinases