Role of mitochondrial DNA damage in cigarette smoke extract-induced innate immunity and its clinical application to atherosclerosis screening

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Role of mitochondrial DNA damage in cigarette smoke
extract-induced innate immunity and its clinical
application to atherosclerosis screening
（タバコ煙抽出物によるミトコンドリア DNA 損傷と動脈硬
化症スクリーニングへの臨床応用）

主指導教員：池上 浩司教授
（医系科学研究科

解剖学及び発生生物学）

副指導教員：田代 聡教授
（原爆放射線医科学研究所
副指導教員：石田
(医系科学研究科

上田

細胞修復制御）
万里准教授

心臓血管生理医学)

桂太郎

（医系科学研究科医歯薬学専攻）

Smoking is an independent risk factor for atherosclerosis, yet the mechanism by which
smoking causes atherosclerosis remains unclear. Several reports suggest that DNA damage
is involved in the development of atherosclerosis. We have previously reported that nuclear
DNA damage was increased in peripheral mononuclear cells of smokers compared to those of
non-smokers. The objective of this study is to investigate the effect of cigarette smoke
extract (CSE) on both nuclear and mitochondrial DNA damage and the subsequent cellular
response.
We prepared CSE by dissolving eight cigarettes smoke in 15 ml of PBS and
administered to human umbilical vein endothelial cells (HUVECs). DNA double-strand
breaks (DSBs), estimated by immunofluorescence staining using a phosphorylated histone
H2AX antibody, was significantly increased at 72 hours by CSE, which is relatively slow
compared to other DSBs-inducing stimuli such as H2O2. Oxidative DNA damage, estimated
by immunofluorescence staining using 8-hydroxy-2’-deoxyguanosine (8-OXO-dG) antibody,
was increased in both nucleus and cytosol by CSE addition. We speculated that the cytosolic
staining might indicate oxidative damage to the mitochondrial DNA. Co-immunostaining
using 8-OXO-dG antibody, Mitotracker, and DAPI, followed by confocal microscopy revealed
that the mitochondrial DNA was oxidatively damaged. Mitochondrial membrane potential
was evaluated by the JC-1 assay, and mitochondrial dysfunction was observed by CSE
addition. It is known that stimuli that cause mitochondrial dysfunction increase
mitochondrial outer membrane permeability called mitochondrial outer membrane
permeabilization (MOMP) mediated by the activation of BAK/BAX, which results in the
release of mitochondrial contents such as cytochrome c and activation of caspases. Activated
caspase-3 cleaves inhibitor of caspase-activated deoxyribonuclease (ICAD), which induces
translocation of caspase-activated deoxyribonuclease (CAD) to the nucleus, resulting in
DNA fragmentation and apoptosis. However, recent reports have shown that this MOMP
does not occur in all mitochondria depending on the stimulus, but only a part of
mitochondria, called minority MOMP, and that the cells survive because of partial DNA
fragmentation (i.e., DSBs). We hypothesized that the increase in DSBs as late as 72 hours
might be due to this mechanism, and examined the downstream signaling of the apoptotic
pathway. We found significant accumulation of active BAX in the mitochondria of cells
treated with CSE, and CSE treatment significantly activated caspase-3. In addition, CSE
treatment resulted in decreased cytosolic expression of ICAD and nuclear translocation of
CAD.
We next examined whether the accumulation of DNA in the cytosol was occurred as a
result of DNA damage using double-strand DNA (dsDNA) antibody, and found that dsDNA
antibody was stained in the cytosol of cells treated with CSE. To know the origin of the

accumulated cytosolic DNA, real-time PCR analysis was performed using specific primers
for nucleus or mitochondria. The results revealed that the accumulated DNA in the cytosol
was derived not only from the nucleus but also from mitochondria. Accumulated cytosolic
DNA is known to induce inflammation through the innate sensing pathways. We examined
whether CSE activated cGAS-STING pathways. The production of cGAMP, a second
messenger in cGAS signaling, was increased by CSE. In addition, TBK1, a protein
downstream of the cGAS-STING pathway, was phosphorylated. Although CSE addition
increased mRNA expression of IL-6, IL-1a, IFN-b, and MCP-1 within 3 days, we thought
that cGAS-STING pathway may be involved in chronic inflammation. Therefore, we
continued to administer CSE for 7 days and found that only the increase in IL-6 was
maintained even at 7 days after the addition of CSE. We next examined whether the
persistent increase in IL-6 mRNA expression was dependent on the activation of the
cGAS-STING pathway. HUVECs were transfected either with siRNAs against cGAS or
negative control siRNA. The increase of IL-6 mRNA expression was suppressed by siRNAs
of cGAS, while IL-1a, IFN-b, and MCP-1 were not suppressed, indicating that CSE-induced
persistent IL-6 expression was mediated by cGAS-STING pathway.
Cytosolic DNA is incorporated into extracellular vesicles and thus, cell-free DNA
(cfDNA) in human peripheral blood has been reported to be altered in association with
several diseases. We confirmed that cfDNA was increased in the culture medium after CSE
administration, and then, measured cfDNA in the blood of smokers to support the
significance of our in-vitro experiments. The cfDNA derived from not only nuclear DNA but
also mitochondrial DNA were found to be increased in the blood of smokers compared to that
of age-matched non-smokers. To further investigate whether cfDNA in blood could be a new
biomarker reflecting the condition of vascular endothelium, we measured cfDNA in the
blood of atherosclerosis patients. Both nuclear-cfDNA and mitochondrial-cfDNA were
increased in patients with atherosclerosis. In conclusion, this study showed that persistent
exposure to CSE induces not only nuclear but also mitochondrial DNA damage, which leads
to cytosolic DNA accumulation, and evokes chronic inflammation via the cGAS-STING
pathway. We also showed that damaged DNA is released not only into the cytosol but also
into the extracellular space. Furthermore, we found that cfDNA was increased in the blood
of smokers. Finally, our study suggests that cfDNA in blood may be a new biomarker
reflecting the condition of vascular endothelium, because cfDNA in blood is also increased in
atherosclerotic patients.