Cold-inducible RNA-binding protein through TLR4 signaling induces mitochondrial DNA fragmentation and regulates macrophage cell death after trauma.

Trauma is a major cause of systemic inflammatory response syndrome and multiple organ dysfunction syndrome. Macrophages (Mφ) direct trauma-induced inflammation, and Mφ death critically influences the progression of the inflammatory response. In the current study, we explored an important role of trauma in inducing mitochondrial DNA (mtDNA) damage in Mφ and the subsequent regulation of Mφ death. Using an animal pseudo-fracture trauma model, we demonstrated that tissue damage induced NADPH oxidase activation and increased the release of reactive oxygen species via cold-inducible RNA-binding protein (CIRP)-TLR4-MyD88 signaling. This in turn, activates endonuclease G, which serves as an executor for the fragmentation of mtDNA in Mφ. We further showed that fragmented mtDNA triggered both p62-related autophagy and necroptosis in Mφ. However, autophagy activation also suppressed Mφ necroptosis and pro-inflammatory responses. This study demonstrates a previously unidentified intracellular regulation of Mφ homeostasis in response to trauma.

Trauma is a major cause of systemic inflammatory response syndrome and multiple organ dysfunction syndrome. Macrophages (Mϕ) direct trauma-induced inflammation, and Mϕ death critically influences the progression of the inflammatory response. In the current study, we explored an important role of trauma in inducing mitochondrial DNA (mtDNA) damage in Mϕ and the subsequent regulation of Mϕ death. Using an animal pseudo-fracture trauma model, we demonstrated that tissue damage induced NADPH oxidase activation and increased the release of reactive oxygen species via cold-inducible RNA-binding protein (CIRP)-TLR4-MyD88 signaling. This in turn, activates endonuclease G, which serves as an executor for the fragmentation of mtDNA in Mϕ. We further showed that fragmented mtDNA triggered both p62-related autophagy and necroptosis in Mϕ. However, autophagy activation also suppressed Mϕ necroptosis and pro-inflammatory responses. This study demonstrates a previously unidentified intracellular regulation of Mϕ homeostasis in response to trauma. Traumatic injury represents a significant health care burden worldwide. 1 Initial mortality is usually secondary to major neurologic injury or massive hemorrhage. 2,3 For those victims of severe traumatic injury who survive beyond the initial 24 h, major morbidities and mortality are often secondary to immune dysregulation, which includes increased inflammatory and innate immune signaling, as well as suppression of adaptive immune signaling. The net result leads to organ dysfunction and increased susceptibility to infection with the development of sepsis. [4][5][6][7] Unfortunately, we do not have effective medical intervention for these trauma patients, partly because of an incomplete understanding of the cellular determinants of immune dysregulation following trauma.
Macrophages (Mϕ), as a major innate immune cell population, direct trauma-induced inflammation, and Mϕ death critically influences the progression of inflammatory responses. Mϕ necroptosis has been reported as a response to infection, as well as sterile inflammatory stimuli such as LPS. [8][9][10][11] In turn, necroptotic Mϕ enhance inflammation through release of pro-inflammatory cell contents. 12 Cold-inducible RNA-binding protein (CIRP) is a member of the cold shock protein family. 13 Mouse and human CIRP contain 172 amino-acid residues (95% identical) that form a consensus sequence of N-terminal RAN-binding domain and C-terminal glycine-rich domain of nuclear proteins, which serve as RNA chaperones promoting RNA translation. 14,15 CIRP has been defined as an inflammatory mediator and a damage-associated molecular pattern (DAMP), which highly expresses during trauma and shock, 16,17 and induces a variety of cellular responses including the release of proinflammatory cytokines and endothelial dysfunction. 18 In addition to supplying cellular energy, mitochondria are also involved with other functions including signal transduction, cell differentiation and death, and the maintenance of the cell cycle and cell growth control processes. 19 In mammals, mitochondrial DNA (mtDNA) is circular with 15-17 000 base pairs encoding 37 genes, including 13 genes for respiratory complexes I, III, IV, and V. 20,21 Extracellular mtDNA is a known DAMP mediating inflammatory responses through a TLR9 signaling pathway during sepsis. 22,23 However, the role of intracellular-damaged mtDNA in the regulation of cellular homeostasis of innate immune cells has yet to be addressed.
Endonuclease G is an apoptotic DNase, located in the mitochondrial intermembrane space. 24 The precursor of endonuclease G is an inactive 33 kDa protein, which must be activated by proteolytic cleavage to a 28 kDa form. 25 Degradation of DNA by endonuclease G is independent of caspases. 26 During animal development endonuclease G also functions to degrade mtDNA. 27 In this study, we explored an important role for trauma in inducing mtDNA damage in Mϕ, but not in neutrophils, and subsequently regulating Mϕ death. Using an animal pseudofracture (PF) trauma model, 28 we demonstrate that tissue damage acts through CIRP-TLR4-MyD88 signaling to induce NADPH oxidase activation, which increases release of reactive oxygen species (ROS) and activates endonuclease G. Endonuclease G serves as an executor to fragment mtDNA in the Mϕ, which we further demonstrate triggers both p62-related autophagy and necroptosis in Mϕ, although autophagy activation suppressed Mϕ necroptosis and pro-inflammatory responses. Our study therefore demonstrates a previously unidentified intracellular regulation pathway of Mϕ homeostasis in response to trauma.

PF induces macrophage cytoplasmic DNA fragmentation.
To determine Mϕ DNA damage in response to trauma, WT mice were subjected to PF, 28 a trauma model mimicking long bone fracture, or to hemorrhagic shock with resuscitation (HS/ R), or to both PF and HS/R double hit. We used the TUNEL assay to detect DNA fragmentation as a marker of DNA damage. In the PF model, both alveolar macrophages (AMϕ) and peritoneal macrophages (PMϕ) exhibited TUNEL-positive dots in cytoplasm ( Figure 1a). However, TUNEL staining colocalized with Hoechst stain (nuclear DNA) in HS/R model ( Figure 1a). Furthermore, in mice subjected to the PF and HS/ R double hit TUNEL staining localized to both the nucleus and cytoplasm (Figure 1a). These results suggest that HS/R and PF induce DNA fragmentation in Mϕ differently. To determine whether mediators in the bone crush mixture (BCM) used in the PF model contribute to the effect of PF on Mϕ cytoplasmic DNA fragmentation, BCM (6 ml/kg B.W.) was directly injected into the mouse peritoneal cavity. PMϕ and AMϕ were harvested at 24 h and DNA damage measured by TUNEL assay as above. As shown in Figure 1b, PMϕ cytoplasmic DNA fragmentation significantly increased. Interestingly, BCM i.p. injection also ϕ-induced cytoplasmic DNA fragmentation in AMϕ. This result suggests that BCM can induce cytoplasmic DNA fragmentation in both proximal and distal Mϖ.
The in vivo study was also recapitulated in vitro. Bone marrow-derived macrophage (BMDM) were treated with BCM (40 μl/ml) for 24 h, and as shown in Figure 1c, the percentage of Mϕ with TUNEL-positive cytoplasm increased significantly by 6 h after BCM treatment, and peaked at 24 h. However, LPS-induced DNA fragmentation was localized to the nucleus, which was different from BCM-induced cytoplasmic DNA fragmentation ( Figure 1c). We also directly treated different Mϖ with BCM in vitro. As shown in Figure 1d, BCM-induced cytoplasmic DNA fragmentation in J774.2 mouse monocyte cell line, and AMϖ, and PMϕ. We further separated the BCM into two parts, bone and bone marrow, and found that only the bone marrow was able to induce Mϕ cytoplasmic DNA fragmentation ( Figure 1e). Collectively, these results suggest that PF induces Mϕ cytoplasmic DNA fragmentation and it is bone marrow components of the BCM that are responsible for these effects.
Damaged tissue induces macrophage mtDNA fragmentation. In order to determine the source of fragmented DNA and exclude exogenous DNA, BCM was pretreated with nuclease to remove DNA/RNA. As shown in Figure  2a, nuclease successfully cleared DNA from BCM. However, nuclease-pretreated BCM was still able to induce BMDM cytoplasmic TUNEL dots similarly to non-pretreated groups (Figure 2b). Mϕ endocytosis of exogenous DNA is commonly localized to endosome, 29 so we therefore detected colocalization of fragmented DNA with endosomes that were visualized by staining early endosome antigen 1 (EEA1), which localizes early endosomes and is required for fusion of early and late endosomes. As Figure 2c shows, the fragmented DNA does not localize to endosomes, suggesting an endogenous source of fragmented DNA. Next, we determined whether endogenous fragmented DNA derived from mitochondria. Confocal microscopy showed that BCMinduced fragmented DNA partly colocalized with mitochondria, as stained by Mitotracker, and partly localized to areas surrounding mitochondria suggesting mitochondrial origin ( Figure 2d). Both BCM and nuclease-pretreated BCM also increased levels of mtDNA by 6 h (Figure 2e), but this increase was reversed by 24 h (Figure 2f). To calculate the mtDNA damage rate, as shown in Figure 2g, BCM induces about 5% of total mtDNA damage at 6 h and about 15% mtDNA damage at 24 h.
BCM is essentially a damaged tissue containing multiple DAMPs including the major DAMPs CIRP and HMGB1 as detected with immunoblots ( Figure 2h). To address which of these two DAMPs is the major component inducing macrophage mtDNA fragmentation, we stimulated BMDM with recombinant CIRP or HMGB1. Recombinant CIRP induced TUNEL-positive BMDM cytoplasm similarly to BCM, but recombinant HMGB1 did not have the same effects ( Figure 2i). CIRP (10 μg/ml), rather than recombinant HMGB1, significantly induced increased levels of mtDNA by 6 h. (Figure 2j). Furthermore, while CIRP-induced mtDNA damage increased, CIRP neutralizing antibody reduced the BCMinduced mtDNA damage as compared to the group treated with nonspecific IgG isotype antibody ( Figure 2k). The recombinant HMGB1, again, did not induce the mtDNA damage ( Figure 2k).
To further define the role of the mtDNA responses to BCM, we generated mtDNA-depleted Mϕ. Previous studies showed that treatment of J774.2 Mϕ with ethidium bromide (EtBr) for 7-14 days effectively depletes mtDNA. [30][31][32] We confirmed mtDNA levels in the J774.2 cells decreased to~10% after 100 ng/ml of EtBr treatment for 7-14 days using three different  (Figure 2n). These results indicate that damaged tissue induces Mϕ mtDNA fragmentation, and CIRP, but not HMGB1, serves as a DAMP molecule inducing these changes.
BCM-induced mtDNA fragmentation is mediated through TLR4-MyD88 signaling. CIRP has been reported to signal via TLR4-MyD88 signaling pathways to promote inflammation. 17,33 To address whether PF, through TLR4-MyD88 signaling, induces mtDNA fragmentation, we used TLR4 − / − , MyD88 − / − , TLR2 − / − , and TLR9 − / − mice. As shown in Figure 3a ROS mediates mtDNA fragmentation. Previous studies showed that oxidative stress induces mitochondrial dysfunction and mtDNA damages. 34,35 The NADPH oxidases from NOX family act as a key ROS producers in various cells. 36,37 To address whether NADPH oxidase-derived ROS mediate BCM-induced mtDNA damage, we subjected WT or gp91 phox − / − BMDM to challenge with BCM for 0-24 h. Gp91 phox is one of the five subunits of NADPH oxidase, and gp91 phox − / − cells do not have NADPH oxidase activity. 38 As shown in Figure  Endonuclease G fragments mtDNA in Mϕ after BCM treatment. Previous studies reported that endonuclease G, which locates at mitochondrial inner membrane, fragments chromosomal DNA in caspase-independent apoptosis. 26 Furthermore, the endonuclease G also degrades mtDNA during animal development. 27,39 To determine the role of endonuclease G in mtDNA fragmentation in Mϕ, we measured changes in endonuclease G protein and mRNA levels after BCM stimulation. As shown in Figure 5a, BCM increased protein expression of mature endonuclease G (28 kDa) by 12 h after BCM stimulation. Interestingly, endonuclease G mRNA level was not significantly changed ( Figure 5b). CIRP recombinant protein increased mature endonuclease G expression, while CIRP neutralizing antibody prevented the BCM-and CIRP-induced increase ( Figure 5c). We also determined the cellular location of endonuclease G in WT BMDM after BCM stimulation ( Figure 5d) and show that it colocalizes with mitochondria both before and after BCM stimulation.
To determine whether the endonuclease G expression is dependent on NADPH oxidase, we compared protein levels of mature endonuclease G in WT and gp91 phox − / − BMDM in response to BCM or CIRP. BMDM from gp91 phox − / − mice challenged with BCM ( Figure 5e) or CIRP (Figure 5f) exhibited markedly lower level of mature endonuclease G expression compared to WT BMDM. Furthermore, we investigated the role of endonuclease G in mtDNA fragmentation using endonuclease G knockdown approach. Transfection of small interfering RNA against endonuclease G (siEndoG) successfully knocked down endonuclease G in BMDM as shown by very low levels of endonuclease G mRNA (Figure 5g). Transfection with siEndoG significantly decreased BCMinduced mtDNA damage ( Figure 5h) and TUNEL-positive cytoplasmic staining (Figure 5i) as compared with BMDM transfected with control non-coding siRNA (siNC). These data indicate that NADPH oxidase induces increased expression of mature endonuclease G, which mediates BCM-induced mtDNA damage, therefore suggesting that endonuclease G is the executor of mtDNA fragmentation.
Fragmented mtDNA induces Mϕ autophagy. Autophagy refers to a cell self-regulation process, allowing orderly degradation and recycling of abnormal cell components. 40 Autophagy is known as a major regulator of removal of damaged mitochondria through mitophagy. 41,42 Next, we addressed whether BCM-induced mtDNA fragmentation activates autophagy. First, a time-course study showed that BCM treatment of BMDM increased LC3-II expression at 24 h (Figure 6a). LC3-II is the activated form of LC3 that is incorporated in autophagosomes. 43 Second, CIRP neutralizing antibody suppressed LC3-II formation in response to BCM and CIRP (Figure 6b). Third, there was very little formation of LC3-II in gp91 phox − / − BMDM after BCM or CIRP treatment compared with WT BMDM (Figure 6c) (Figure 6d). To confirm our findings, we also visualized colocalization of LC3 with TUNEL staining in the BMDM treated with BCM or CIRP. Figure 6e shows that both BCM and CIRP can induce LC3 colocalization with TUNEL-positive fragmented DNA. As expected from our results above, when mtDNA was depleted in J774.2 cells, there was no TUNEL staining in the cytoplasm, and so no localization of TUNEL with LC3 ( Figure 6f). These observations suggest CIRP-TLR4 regulates mtDNA fragmentation Z Li et al fragmented mtDNA induces and localizes to the autophagosome. In addition, as Figure 6g shows, BCM also induced mitophagy in BMDM, which can be identified as colocalization of mitochondria (MitoTracker) and lysosomes (LysoTracker). However, this induction of mitophagy was prevented in mtDNA-depleted J774.2 cells (Figure 6g).
The degradation of p62 is often used as an indicator of autophagy. 44 BCM induced increases in expression of p62 mRNA at 3 h (Figure 6h) and in p62 protein expression at 6 h (Figure 6i), which is then followed by decreases in p62 protein expression at 12 and 24 h suggesting activation of autophagy pathways and clearance of p62 (Figure 6i). However, in mtDNA-depleted J774.2 cells BCM failed to induce p62 upregulation (Figure 6j). Again, colocalization of mitochondria, LC3, and p62 following BCM or CIRP stimulation for 24 h was visualized by confocal microscopy as shown in Figure 6k. Taken together, these data suggest that BCM-and CIRP-induced mtDNA fragmentation leads to mitophagy.

Autophagy
prevents fragmented mtDNA-induced macrophage necroptosis. Autophagy is one of the critical mechanisms for the clearance of damaged mtDNA, so it is important for cellular homeostasis. 45 We next determined the role of autophagy in regulation of cell fate in response to mtDNA fragmentation. In LC3 − / − BMDM, in which autophagosomes cannot be induced by mtDNA fragmentation, BCM-induced cell death significantly increased compared with WT BMDM (Figure 7a). To further define the type of the cell death occurring, we measured RIPK1 phosphorylation, as well as colocalization of RIPK1 and RIPK3, which are features of necroptosis. 46 We found that BCM treatment induced higher levels of RIPK1 phosphorylation in LC3 − / − BMDM (Figure 7b), and increased colocalization of RIPK1 and RIPK3 compared with WT BMDM (Figure 7c). Addition of the necroptosis inhibitor necrostatin-1 (Nec-1) significantly blocked BCM-or CIRP-induced cell death in LC3 − / − BMDM ( Figure 7d). These data reveal an important role for autophagy in preventing necroptosis following mtDNA damage.

Necroptotic Mϕ induce inflammatory responses in naive
Mϕ. BCM induced necroptosis in LC3 − / − BMDM and necroptosis is well known as a form of inflammatory cell death. 47,48 To determine the significance of mtDNA fragmentation-induced autophagy in maintaining cellular homeostasis, we investigated the effect of necroptotic Mϕ on neighboring healthy Mϕ. WT or LC3 − / − BMDM were pretreated with BCM for 24 h, and then were cocultured with naive macrophages for 6 or 18 h. As shown in Figure 8, the BCM-pretreated LC3 − / − BMDM, which lack autophagy and exhibit necroptosis, significantly increased inflammatory responses in naive macrophages, including increased IL-1β and NOS2 mRNA expression at 6 h, and increased IL-1β, IL-6 and TNF-α mRNA at 18 h. These results suggest that mtDNA fragmentation-induced autophagy plays a protective role in limiting local inflammation through the regulation of cell death.

Discussion
The mechanisms behind trauma/tissue damage regulation of innate immune responses are not yet fully defined. 16 Our current study explored a mechanism underlying traumainduced regulation of Mϕ death, in which CIRP released from damaged tissue acts through TLR4-MyD88 signaling to induce Mϕ mtDNA fragmentation, via a pathway in which NADPH oxidase-derived ROS served as a major mediator for the induction of endonuclease G, which, in turn, directly mediates mtDNA fragmentation. Fragmented mtDNA then triggered Mϕ autophagy and necroptosis through separate pathways, although autophagy also suppressed Mϕ necroptosis, to attenuate propagation of local inflammation (Figure 9).
Previous studies have shown that during injury extracellular mtDNA serves as a DAMP that induces inflammatory response through TLR9 signaling. 23,49,50 However, the mechanism of intracellular mtDNA damage had not been fully identified. In our current study, we revealed an important role for intracellular fragmented mtDNA in regulating Mϕ death and inflammation.
We defined fragmented DNA in Mϕ cytoplasm after BCM treatment as damaged mtDNA based on the facts that: (1) predeleting exogenous DNA in BCM using nuclease did not prevent the formation of fragmented DNA in Mϕ cytoplasm (Figures 2a and b); (2) fragmented DNA was not endosomal, suggesting it had not been endocytosed (   (Figures 2l-n). We also demonstrated a dynamic alteration in mtDNA quantity after BCM treatment with increased mtDNA in the early phase (6 h), followed by decreased levels in the late phase (24 h).
Overall we detected about 15% mtDNA damage, an indication of mtDNA fragmentation. The observations that CIRP induced mtDNA fragmentation and neutralizing antibody against CIRP prevented the mtDNA fragmentation in response to BCM indicate that CIRP is one of the major components in damaged tissue to induce mtDNA fragmentation.
Our previous studies showed that TLR4-MyD88 signaling mediates NADPH oxidase activation in neutrophils and lung vascular endothelial cells in response to hemorrhagic shock and LPS. 37,51,52 In the current study, we showed that the PF/tissue damage activation of TLR4-MyD88 signaling results in increases in ROS production through the activation of NADPH oxidase in Mϕ. It is noticeable that LPS, CIRP, and HMGB1, all being TLR4 agonists, induce very different responses in terms of DNA damage. For example, LPS causes DNA fragmentation in the nucleus, and CIRP in the mitochondria; HMGB1 did induce DNA fragmentation in the nucleus, though perhaps not as much as LPS. This raises a fundamental biological question on how limited receptor classes can induce such a broad range of different cellular responses, which has generated several hypotheses. A plausible hypothesis is that following receptor activation, different signaling pathways selectively adopt different adaptor proteins to induce different downstream outcomes. It can be assumed that CIRP, HMGB1, and LPS bind to different domain structures of TLR4, which then induce different TLR4 conformational changes and activate different signaling pathways. Indeed, this is an open question that remains to be further elucidated.
The identity of the executor protein downstream of ROS that cleaved mtDNA was not clear. 35 Previous studies showed that endonuclease G can be activated under oxidative stress [53][54][55] and then functions as a chromosomal DNA fragmentation enzyme. 26,56 Studies have also shown that endonuclease G plays a critical role in paternal mtDNA degradation during animal development. 27,39 In this study, we demonstrated that the endonuclease G is an important executor for induction of mtDNA fragmentation, and ROS is required for its activation. Genetic deletion of the NADPH oxidase subunit gp91 phox significantly diminished BCM-and CIRP-induced endonuclease G activation, and knockdown of endonuclease G prevented BCM-induced mtDNA damage and appearance of mtDNA fragmentation in Mϕ cytoplasm.
The current study further determined the protective effect of autophagy in PF/tissue damage. We identified that fragmented mtDNA induces autophagy, which serves as a mechanism to clear the damaged mtDNA. In LC3 − / − BMDM, which cannot initiate the autophagosome formation, the damaged mtDNA promotes the Mϕ necroptosis, which propagates and enhances inflammatory responses by activating neighboring innate immune cells. These observations indicate an important role for autophagy in cellular homeostasis during trauma/tissue damage through the limitation of local inflammation.
In summary, this study demonstrates a novel mechanism underlying trauma-induced mtDNA damage and subsequent self-regulation of cell death and inflammation aiming to maintain cellular homeostasis. Targeting the signaling revealed in this study might serve as a potential approach of therapeutic intervention for the treatment of post-trauma inflammation. In Situ Cell Death Detection Kit, TMR red (TUNEL) was from Roche (12156792910, Indianapolis, IN, USA). Annexin V detection kit was from BD Biosciences. MitoTracker Green FM was from Thermo Fisher Scientific; Dynabeads Protein G Immunoprecipitation Kit (10007D) for immunoprecipitation was from Thermo Fisher Scientific. Transfect reagents, Lipofectamine LTX Reagent with PLUS Reagent (15338100) was purchased from Thermo Fisher Scientific; siNC and siEndoG was purchased from Integrated DNA Technologies (Coralville, IA, USA); Necroptosis inhibitor Necrostatin-1 (BML-AP309-0020) was purchased from Enzo Life Sciences (Farmingdale, NY, USA). Recombinant HMGB1 (1690-HMB-050) was from R&D Systems. Rabbit IgG isotype control (ab171870) was purchased from Abcam. Recombinant mouse CIRP protein and anti-CIRP antibody were from Dr Ping Wang, the Feinstein Institute for Medical Research. 17 The recombinant mouse CIRP protein was detected a residual~10 pg of LPS per μg of CIRP by the Limulus amebocyte lysate assay. 17 And the anti-CIRP Ab was raised in New Zealand white rabbits by standard procedures at Covance (Princeton, NJ, USA). 17 Mouse PF model. The mouse PF model was carried out as previously described. 28,57 Briefly, femurs and tibias were collected from a killed donor mice and then crushed using a sterile mortar and pestle. An amount of 0.5 g of the bone crush material was homogenized in 2 ml sterile phosphate-buffered solution (PBS) to prepare the BCM. The recipient mouse was anesthetized with ketamine (50 mg/ kg B.W.) combined with xylazine (5 mg/kg B.W.) and the thighs were squeezed with a hemostat for 30 s to induce a soft tissue lesion, followed by injection of 0.15 ml of the BCM in the tissue lesion area. Sham animals underwent the same anesthesia procedure and injection of 0.15 ml of normal saline at the thighs.
BMDM isolation and culture. Bone marrow was flushed with prechilled Dulbecco's Modified Eagle Medium (DMEM) from femurs and tibias, which were harvested from WT or gene knockout mice following the previously described method. 58 Briefly, cell pellets were collected by centrifugation at 4°C, and erythrocytes were lysed with RBC lysis buffer (eBioscience, San Diego, CA, USA). The resultant cells were then washed two times with PBS and suspended in BMDM culture medium (DMEM containing 10% FBS complemented with 50 μg/ml penicillin/streptomycin and 10 ng/ml recombinant macrophage-colony stimulating factor (M-CSF; Sigma-Aldrich, St Louis, MO, USA)) at a concentration of 10 6 cells per ml and seeded into 6-cm ultra-low attachment surface plates (Corning Costar, Corning, NY, USA). The BMDM culture medium was changed on day 3 and day 5. BMDM were fully differentiated and ready for use at day 7.
Cell staining. BMDM were seeded in a glass-bottomed Petri dish (P35G-0-10-C, MatTek Corporation, Ashland, MA, USA) and fixed with 4% paraformaldehyde for 15 min at room temperature. After washing with PBS, cells were permeabilized with 0.01% Triton X-100 in PBS for 15 min at room temperature, followed by blocking with 5% bovine serum albumin in PBS for 1 h at room temperature. The cells were stained with TUNEL following manufacturer's instructions or were incubated with a primary antibody at 4°C overnight, followed by incubation with fluorescenceconjugated secondary antibody for 1 h at room temperature. Cell nucleus was stained with Hoechst 33258 (Sigma-Aldrich). The BMDM were then measured by confocal microscopy (Olympus, Fluoview-FV1000, Olympus America Co., Center Valley, PA, USA). Cells were counted in three random fields in each independent experiment. We then quantified the colocalization of the target proteins by Pearson's coefficient using ImageJ version 1.50i.
Western blot. BMDM lysates were separated by 8 and 15% SDS-PAGE, and then transferred onto PVDF membranes. After blocking for 1 h at room temperature with blocking buffer (LI-COR Biosciences, Lincoln, NE, USA), blots were incubated with primary antibody at 4°C overnight, followed by incubation with appropriate secondary antibodies (LI-COR Biosciences) for 1 h. Protein bands were detected using the Odyssey System from LI-COR Biosciences and the intensity of each band was quantified using ImageJ version 1.50i. The intensity of target protein band was normalized with reference protein band and calculated for the fold changing.
Amplification was performed with cycling conditions of 95°C for 15 s then 60°C for 30 s for 40 cycles. After the amplification protocol was completed, PCR product was subjected to melt-curve analysis using Bio-Rad iQ5 software (Bio-Rad). Fold change was calculated using the ΔΔ threshold cycle method and the value for the GAPDH gene, which was normalized to untreated groups. mtDNA damage was calculated by the ratio of long and short segments amplified from mtDNA PCR production. The primers used were: long segments primers-mt long forward, 5′-GCCAGCCTGACC CATAGCCATAATAT-3′, and reverse, 5′-GAGAGATTTTATGGGTGTAATGCGG-3′; short segments primers-mt short forward, 5′-CCCAGCTACTACCATCATTCAA GT-3′, and reverse, 5′-GATGGTTTGGGAGATTGGTTGATGT-3′; Reference segments primers: β-Globin forward, 5′-TTGAGACTGTGATTGGCAATGCCT-3′, and reverse, 5′-CCTTTAATGCCCATCCCGGACT-3′. 59 PCR was done using the TaKaRa Ex Taq DNA polymerase (Clontech Laboratories, Inc., Mountain View, CA, USA) in Applied Biosystems PCR machine (Thermo Fisher Scientific). Amplification was performed with cycling conditions of 94°C for 30 s then 55°C for 60 s and 72°C for 10 min for 30 cycles. PCR product was quantified by using of NanoDrop spectrophotometer (NanoDrop, Wilmington, DE, USA). The mtDNA long and short PCR production normalized to β-Globin, respectively, and calculated the mtDNA damage rate (%) using the following formula: mtDNA damage rate % ð Þ ¼ mtDNA short segment fold induction ð ½ ÀmtDNA long fragment fold inductionÞ=mtDNA short segment fold induction 100% RNA extraction and quantitative real-time PCR. The cells form co-culture bottom wells BMDM were harvested and total RNA was isolated by using TRIzol RNA Isolation Reagents (Thermo Fisher Scientific) by following the instructions. Real-time RT-PCR was done using iTaq Universal SYBR Green Supermix (1725121, Bio-Rad) in a Bio-Rad iQ5 real-time PCR machine (Bio-Rad).