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10: MITOCHONDRIAL TRANSCRIPTION FACTOR A (TFAM), A DANGER SIGNAL RELEASED FROM DAMAGED CELLS, PROMOTES AN INNATE IMMUNE IN THROUGH RAGE AND TLR9 SIGNALING PATHWAYS

Julian, Mark; Shao, Guohong; Vangundy, Zack; Papenfuss, Tracey; Crouser, Elliott

doi: 10.1097/01.ccm.0000424266.18598.ea
Oral Abstract Session: ABSTRACT Only

Introduction: In the context of acute cell damage, TFAM, normally tightly bound to mitochondrial DNA, is shown to promote Type I interferon release from dendritic cells through RAGE and TLR9 (Julian et al. J Immunol 2012; 189:433-43.), which has implications for the adaptive immune response. However, it is unclear if TFAM also promotes innate immune responses.

Hypothesis: We hypothesize that TFAM promotes innate immune responses by enhancing the recognition of DNA through RAGE and TLR9-dependent mechanisms.

Methods: Murine splenocyte cultures were used to determine if highly purified recombinant human TFAM promotes TNF&#945 release (measured by ELISA) alone or in combination with immunogenic (CpG) DNA. The role of RAGE was determined by pretreatment with soluble RAGE (sRAGE), use of RAGE (-/-) splenocytes, or heparin. The role of TLR9 was tested with a specific TLR9-blocking oligonucleotide, and by inhibiting putative TLR9 signaling mechanisms including inhibition of endosomal acidification (chloroquine), PI3K (LY294002) and NFkB (Bay). PI3K and NFkB phosphorylation status were verified by western blot. Additional studies were performed to determine if heparin sulfate moieties required for RAGE signaling (heparin lyase) or endothelin converting enzyme-1 (ECE-1)-dependent recycling of endosomal receptors were engaged in TFAM and DNA recognition.

Results: Our data confirms that TFAM augments TNF&#945 release from splenocyte cultures in response to DNA through RAGE and TLR9 signaling pathways, including endosomal acidification, PI3K and NFkB signaling. Moreover, inhibition of ECE-1 or cleavage of heparin sulfate moieties completely blocked the immune response. Furthermore, depletion of dendritic cells from the splenocyte preparation was shown to inhibit TNF&#945 release in response to TFAM and DNA. Recombinant TFAM, alone, did not promote TNF&#945 release under these conditions.

Conclusions: TFAM is shown to augment the innate immune response to DNA through mechanisms involving RAGE and TLR9 signaling pathways, and is dependent, at least in part, upon dendritic cells. This study is the first, to our knowledge, to incriminate ECE-1-dependent endosomal recycling of TLR9, which amplifies antigen-mediated signaling (Roosterman et al. PNAS 2007; 104:11838-43.), in the context of immune responses. These findings have important implications for understanding sterile immune responses triggered by mitochondrial danger signals. Supported, in part, by NIH grants: R21-AI083912 and RO3-AI62740 (EDC), and by DHLRI Davis Endowment Fund.

The Ohio State University Medical Center

The Ohio State University

Ohio State University Medical Center

© 2012 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins