Date: Monday, November 9, 2015
Session Type: ACR Concurrent Abstract Session
Session Time: 2:30PM-4:00PM
Background/Purpose: Neutrophil extracellular trap generation (NETosis) is a reactive oxygen species (ROS)-dependent cell death pathway implicated in autoimmune disorders such as systemic lupus erythematosus (SLE). However, since deficiencies in NADPH oxidase-mediated ROS production is associated with increased, rather than reduced, autoimmunity, this association has been challenged. Since mitochondria are major generators of ROS, we asked the following questions: i) is mitochondrial ROS required for NET formation? ii) what is the relative contribution and oxidative status of mitochondrial DNA in NETs? and iii) what are the immune stimulatory effects of mitochondrial products following RNP immune complex (IC) activation of neutrophils?
Methods: Mitochondrial ROS was quantified by the fluorescent dye MitoSOX. Diphenyleneiodonium (DPI), thenoyltrifluoroacetone (TTFA), rotenone and mitoTEMPO were used as ROS inhibitors. NET formation was quantified by fluorimetry and the extent of DNA oxidation determined using anti-8-oxo-2′-deoxyguanosine (8-OHdG) antibodies by ELISA or immunofluorescence (IF) microscopy. Immunoprecipitation and qPCR (16/18S ratio) were used to assess the origin and properties of NET DNA. To evaluate the inflammatory potential of mitochondrial DNA (mitDNA), oxidized or non-oxidized NET DNA was transfected into THP1 cells or injected into wild type or STING deficient mice.
Results: Following exposure of neutrophils to RNP IC, mitochondria became hypopolarized and translocated to the cell surface. The IC also stimulated mitochondria to produce ROS at levels similar to PMA stimulation. Mitochondrial ROS proved to be necessary for maximal NETosis since the selective inhibitors of mitochondrial ROS, TTFA and MitoTEMPO reduced NETosis by ~50% (p<0.0001, n=8). Furthermore, mitochondrial ROS stimulated by IC caused oxidation of DNA and IF revealed that DNA oxidation occurred predominantly in mitochondrial, rather than chromosomal, DNA. Using a dual immunoprecipitation and qPCR technique, we observed that the oxidized NET DNA displayed a high 16S/18S ratio, demonstrating that it was markedly enriched in mitDNA as compared to non-oxidized NET DNA (p<0.01, n=9). The oxidation of mitDNA was almost completely reversed in the presence of TTFA (p<0.01). When the inflammatory properties of oxidized mitDNA and non-oxidized DNA were compared in vitro, oxidized DNA was much more potent in inducing IL-6 and type I IFNs (p<0.001). Significantly, when oxidized mitDNA was injected into wild type and STING deficient mice, we observed that the oxidized mitDNA stimulated type I IFNs through a pathway that required the DNA sensor, STING (p<0.001).
Conclusion: Mitochondria play an important, previously unappreciated role in immune-mediated NETosis. Not only do they contribute to ROS that promotes NETosis but, also, oxidized mitDNA generated during NETosis has potent inflammatory properties in vitro and in vivo – including stimulation of type I IFN responses mediated through the STING pathway. We suggest that mitochondrial ROS and release of oxidized mitDNA may be instrumental in initiating or perpetuating autoimmunity and type I IFN signature seen in SLE patients.
To cite this abstract in AMA style:Lood C, Blanco LP, Purmalek M, Smith CK, Carmona-Rivera C, Ledbetter J, Kaplan MJ, Elkon KB. Mitochondrial ROS Is a Novel Regulator of Sting-Mediated Type I IFN Production By Governing Extrusion of Oxidized Mitochondrial DNA upon Neutrophil Extracellular Trap Formation. [abstract]. Arthritis Rheumatol. 2015; 67 (suppl 10). https://acrabstracts.org/abstract/mitochondrial-ros-is-a-novel-regulator-of-sting-mediated-type-i-ifn-production-by-governing-extrusion-of-oxidized-mitochondrial-dna-upon-neutrophil-extracellular-trap-formation/. Accessed April 16, 2021.
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