The Reduced Oligomerization of MAVS Mediated by ROS Enhances the Cellular Radioresistance

Although the mitochondrial antiviral signaling protein (MAVS), located in the mitochondrial outmembrane, is believed to be a signaling adaptor with antiviral feature firstly, it has been shown that suppression of MAVS enhanced radioresistance. The mechanisms underlying this radioresistance remain unclear. Our current study demonstrated that knockdown of MAVS alleviated the radiation-induced mitochondrial dysfunction (mitochondrial membrane potential disruption and ATP production), downregulated the expressions of proapoptotic proteins, and reduced the generation of ROS in cells after irradiation. Furthermore, inhibition of mitochondrial ROS by the mitochondria-targeted antioxidant MitoQ reduced amounts of oligomerized MAVS after irradiation compared with the control group and also prevented the incidence of MN and increased the survival fraction of normal A549 cells after irradiation. To our knowledge, it is the first report to indicate that MAVS, an innate immune signaling molecule, is involved in radiation response via its oligomerization mediated by radiation-induced ROS, which may be a potential target for the precise radiotherapy or radioprotection.


Introduction
Mitochondrial antiviral signaling protein (MAVS), a signaling adaptor with antiviral feature in the mitochondrial membrane, is critical for host defenses against viral infection. The homeotypic interaction between the domains of a caspase recruitment domain (CARD) of MAVS and the CARD of RIG-I forms protein aggregates on the surface of the mitochondria that can further activate MAVS proteins to form functional clusters to propagate antiviral innate immune response [1]. These high molecular weight MAVS complexes then recruit the IKK and TBK1/IKKi complexes to induce transcriptional expression of type I interferon (IFN) by promoting the nuclear translocation of the NF-κB and IRF3/7 transcription factors, respectively, and thus elicit the innate antiviral response (F. [2,3]). Radiation-mediated NF-κB activation can directly induce expression of several proinflam-matory cytokines including TNF, IL-1α, and IL-1β [4][5][6]. Thereby, radiotherapy can also activate innate and adaptive immune responses against tumors [7][8][9][10]. MAVS is involved in IFN-beta and IFN-stimulated gene expression in the response to ionizing radiation (IR). It was reported that physiologic responses to radio-/chemotherapy converge on an antiviral program in recruitment of the RLR pathway by a sncRNA-(small nuclear RNAs U1 and U2-) dependent activation of RIG-I which commences cytotoxic IFN signaling, and suppression of MAVS conferred radioresistance in normal and cancer cells [11]. However, the underlying mechanisms on MAVS suppression resulting in radioresistance remain poorly understood.
Mitochondria are involved in many important cell processes including cell respiration, reactive oxygen species (ROS) production, and apoptosis induction. Accumulating data indicate that IR damages the mitochondrial structure (mass, morphology) and induces the dysfunctions of mitochondria in cells, such as the disorder of cellular respiration, changes of calcium balance and membrane potential, and elevation of ROS level, which result in the radiosensitivity [12][13][14]. MAVS is predominantly localized and executes its functions at the outer membrane of the mitochondria. It is not clear whether the change of MAVS expression influences the mitochondrial functions responding to IR and results in the radiosensitivity. Hou et al. reported that increased cellular ROS promoted MAVS forming functional prion-like aggregates to activate and propagate antiviral innate immune response [1,15]. Conversely, repression of mitochondrial ROS (mtROS) production by cytochrome C oxidase complex subunit 5 inhibits MAVS aggregation and the downstream NF-κB and IRF3/7 signaling pathway [16]. IR can induce the generation of ROS. Whether and how did the increase of radiosenstivity partially via the activation of MAVS aggregation by radiation induce ROS?
In the present work, we attempted to investigate the mechanisms of radioresistance in cells after MAVS suppression. Our results demonstrated that suppression of MAVS alleviated the radiation-induced mitochondrial dysfunction and reduced the generation of ROS, compared to the normal cells. We also observed that more large aggregates of MAVS formed after IR and these MAVS aggregates lead to a gain of function in activating downstream factors. Radiationinduced MAVS oligomerization was inhibited by the addition of MitoQ, a mitochondria-targeted antioxidant, and decreased the radiosensitivity. Our results suggest a mechanism that MAVS oligomerization induced by radiation plays a role in the induction of radiosenstivity.

Mitochondrial
Membrane Potential (MMP) Measurement. The status of the cellular mitochondrial membrane potential (ΔΨm) was evaluated using JC-1 (BD Biosciences) and was quantified by the microplate reader (Tecan Infinite 200 M) [18]. Briefly, after being transfected with MAVS siRNA oligos for 48 h and followed by 2 Gy X-ray irradiation for 24 h, the cells (~1 × 10 6 cells/mL) were washed once with PBS and harvested into a centrifuge tube. Cells were resuspended in 500 μL JC-1 working solution, incubated at 37°C for 15 min, then washed twice with 1x assay buffer, and resuspended each cell pellet in 500 μL of 1x assay 2 Oxidative Medicine and Cellular Longevity buffer. Microplate reader and immunofluorescence microscopy (Axio Imager Z2) analysis at 10x magnification were performed after staining.
2.8. Cellular ATP Content. The intracellular ATP content was evaluated by measuring the luminescence with an ATP determination kit (Thermo Fisher, USA) according to the manufacturer's protocol. Briefly, the MAVS knockdown cells were irradiated by 0 and 2 Gy X-rays and then incubated for 1, 12, and 24 h. The cells were lysed in RIPA buffer on ice (Beyotime, China), and then, the supernatants were collected into 1.5 mL tubes. The chemiluminescence from each well was measured with an Infinite 200 Microplate Reader (Tecan, Switzerland) set at 25°C. The standard curve for a series of ATP concentrations was confirmed. ATP concentrations of sample were calculated from the standard curve. To eliminate errors caused by differences in sample content, we use a Bradford protein assay kit to normalize protein quantity. The amount of ATP was expressed as mmol/g of protein.
2.9. Apoptosis Assays. At the end of all interventions, A549 (>1 × 10 5 ) cells were collected and stained with Annexin V-FITC/propidium iodide (PI) Kit (BD, USA) according to the manufacturer's protocol. The apoptosis of A549 was measured by a flow cytometer FlowSight (Amnis, USA).

ROS Level Measurement.
The fluorescent probe, 2′7′-dichlorofluorescin (DCFH-DA, Sigma, USA), was employed to quantify the level of ROS as described [19]. The medium of the cells to be detected was removed at the designed time points. Cells were washed once with 1x PBS solution and then stained with 10 μM DCFH-DA for 30 min at 37°C in the dark. The fluorescence images were taken under the fluorescence microscopy (Axio Imager Z2), and the relative fluorescence intensity for each treatment was quantified by means of ImageJ software.
2.11. Semidenaturing Detergent Agarose Gel Electrophoresis (SDD-AGE). SDD-AGE was performed according to a published protocol with minor modifications [20]. Briefly, equal amounts of the whole cell lysate were resuspended in one-third volume of 4x loading buffer consisting of 20% glycerol, 8% SDS, and 0.01% bromophenol blue in 2x TAE. Samples were incubated for 5 min at room temperature and loaded onto a vertical 1.5% agarose gel containing a final concentration of 0.1% SDS. Migration was performed for 35 min with a constant voltage of 100 V at 4°C. Proteins were transferred to PVDF membranes using a liquid transfer system in preparation for western blotting analysis. In order to confirm the consistency of the sample amounts, save half of the sample volume and boil it for western blotting analysis.
2.12. Level of Cytokine Measurement by ELISA Analysis. The cellular media of each treated group were collected at the indicated time points, and the levels of IL6, TNF-α, and IL-1β in the media were measured using ELISA kits (eBioscience) following the manufacturer's instructions. The fold changes of IL-6, TNF-α, and IL-1β levels were analyzed among different treatment group.
2.13. Statistical Analysis. The data were presented as mean ± SD of three independent experiments at least. The statistical significance (P value) was determined using Student's t-test for single comparisons and analysis of variance (ANOVA) for statistical comparison between different groups. If the P value < 0.05 was considered statistically significant between two sample comparison.

MAVS Is Involved in Radiation
Response. The levels of MAVS expression in different cell lines (A549, BEAS-2B, HepG2, and MCF7) were also analyzed. Figure 1(a) shows that the expressions of MAVS in BEAS-2B and MCF7 cells were lower. The expressions of MAVS were upregulated in both A549 (Figure 1(b)) and BEAS-2B (Figure 1(c)) cells at 1 h after X-ray radiation and then decreased. After being transfected with siRNA, the expression of MAVS in two cell lines was silenced and the upregulations of MAVS expressions induced by radiation were suppressed effectively, compared to the normal irradiated cells. Further, colony formation assays revealed that knockdown of MAVS gene increased the survival fraction of A549 (Figure 1(d)) and BEAS-2B (Figure 1(e)) after irradiation, compared to the normal control (NC) irradiation group. Consistently, knockdown of MAVS gene greatly diminished the incidence of MN in A549 and BEAS-2B cells after irradiation, compared to those NC cells after radiation (Figure 1(f)). These results confirm that MAVS responds to radiation and knockdown of MAVS increases the radioresistance.

Knockdown of MAVS Attenuates Radiation-Triggered
MMP Disruption. MMP is a key indicator of mitochondrial function and activity because it reflects the process of elector transport and oxidative phosphorylation and is the driving force for mitochondrial ATP synthesis. Consequently, mitochondrial depolarization exhibited decreased red fluorescence and enhanced green fluorescence, and a collapse in the ΔΨm is indicated by a reduction in the red/green fluorescence intensity ratio [21]. To investigate the effects of MAVS suppression on mitochondrial functions after irradiation, we carried out the measurement of MMP. Figure 2(a) shows that the states of JC-1 monomers (green color cell) and JC-1 aggregates (red color cell) in normal or MAVS knockdown cell lines after irradiation. Figure 2(b) shows the knockdown of MAVS prevented the radiation-induced decrease in the red-to-green fluorescence intensity ratio of JC-1 staining measured by the microplate reader. After irradiation, the fluorescence ratio of JC-1 aggregates dramatically decreased in the irradiated normal A549 cell lines at 24 h time point, while these ratios did not change obviously in the irradiated cells with MAVS knockdown, compared to their control cells. These results suggest that knockdown of MAVS attenuates radiation-triggered MMP disruption.

Knockdown of MAVS Downregulates the Expressions of
Apoptosis-Related Protein. Mitochondrial damage facilitates cytochrome C (cyto C) release from mitochondria into the cytoplasm and activates Bcl-2 family proteins, which leads to activation of the caspase cascade (apoptotic markers) and cellular apoptosis [22]. Our above results showed that knockdown of MAVS led to the alleviation of radiationtriggered MMP disruption. Thus, the expression levels of mitochondrial apoptosis-associated proteins, such as cyto C, caspase-3, Bax, and Bcl-2, were determined by western blot to deduce the role of MAVS suppression in the radiation-induced cell apoptosis. Figure 3   Overexpression of Bcl2 promotes cellular survival, and overexpression of Bax enhances the cellular apoptosis. Thus, the higher the ratio of Bcl2/Bax, the more cellular survival it reflects [23]. In comparison to the normal A549, the ratio of Bcl2/Bax is slightly higher in MAVS knockdown A549 cells at 6 and 12 h after irradiation (Figure 3(b)). These results indicate that knockdown of MAVS suppresses the expressions of apoptosis-associated proteins and protect the cells from the radiation-induced death. Meanwhile, our results from flow cytometry confirmed that knockdown of MAVS inhibited cell apoptosis after X-ray radiation (Figure 3(c)).

Knockdown of MAVS Inhibits the Generation of
Radiation-Induced ROS. Generation of higher level of ROS in cells is related to cell apoptosis [24]. To determine the relationship between the knockdown of MAVS and the level of ROS after irradiation, we measured the ROS level in cells at 15 min, 1, and 6 h after exposed to 2 Gy X-rays. Figure 4 shows that the ROS level increased in normal A549 cells at 15 min after irradiation, while it remained relatively constant in the MAVS knockdown cells after irradiation. Compared to the irradiated MAVS knockdown A549 cells, the ROS level in the normal cells increased by 3.0-fold at 15 min after irradiation. There were no big differences between normal and MAVS knockdown A549 cells at 1 and 6 h after irradiation.  Values are presented as mean ± SD. * P < 0:05, * * P < 0:01, and * * * P < 0:001 vs. the control group; # P < 0:05, ## P < 0:01, and ### P < 0:001 vs. the irradiation group. 7 Oxidative Medicine and Cellular Longevity 3.6. The Mitochondria-Targeted Antioxidant MitoQ Reduces MAVS Oligomerization. Study demonstrated that increased cellular ROS promoted MAVS aggregates to activate the downstream signaling response [1]. To evaluate the influence of radiation or radiation-induced ROS on MAVS oligomerization, we adapted a SDD-AGE system to detect MAVS aggregates after irradiation. MitoQ is a mitochondriatargeted antioxidant, which contains ubiquinol as the active antioxidant component [25]. Cells pretreated with 1 μm MitoQ for 2 h dramatically reduced MAVS oligomerization, compared to the X-ray radiation alone group, which is at least a 75% of decrease in the amount of MAVS oligomers ( Figure 5), while there is no increase of MAVS oligomers in MAVS knockdown A549 cells after irradiation. These results suggest that radiation-induced ROS activate the oligomerization of MAVS and the phenomenon will be reduced if the MAVS is suppressed.

The Pretreatment of MitoQ Reduces the Cellular
Radiosensitivity. Figure 6(a) shows that the incidence of MN was greatly diminished in A549 cells pretreated with MitoQ, compared to the knockdown of MAVS after 1 Gy or 2 Gy X-ray irradiation. Further, survival fraction of A549 cells pretreated with MitoQ and then irradiation was increased, compared to the irradiated cells alone (Figure 6(b)). MitoQ did not affect the biological effect of MAVS knockdown cell lines after irradiation.

Knockdown of MAVS Inhibits the Generation of IL6 and
Expression of IFN. Cytokines, such as IL6, TGFα, and IL1β, are the downstream of MAVS signaling pathway and involve in radiation induced sensitivity. Thus, we detected the relative level of these cytokines in normal and MAVS knockdown cells after irradiation. Irradiation stimulation reduced the level of IL6 at early time (15 min after) then induced the upregulation of level of IL6 at 6 h time point. Knockdown of MAVS suppressed the level of IL6 after irradiation, compared to the irradiated normal cells (Figure 7(a)). The levels of TGFα and IL1β were not obviously different between the normal and MAVS knockdown cells after irradiation (data not shown). Figure 7(b) shows that knockdown of MAVS suppressed the expression of IFN and also inhibited the expression of total IRF3 (IFN regulatory factor-3) and pho-IRF3, suggesting that MAVS knockdown suppresses the inflammation signaling pathway and may contribute to the radioresistance.

Discussion
IR, because of its cell killing capability, has been utilized to therapy cancers, and radiotherapy is one of the three main therapies (surgery, chemotherapy, and radiotherapy) for many types of tumor [26]. Radiotherapy can increase innate and adaptive immune responses against tumors, and the type I IFN signaling pathway is involved in this process [27]. It was reported that MAVS is necessary for IFN-beta induction and interferon-stimulated gene expression in the response to IR and suppression of MAVS conferred radioresistance in normal and cancer cells [11]. However, the mechanisms underlying this process are largely unknown.
In the current study, we first verified the phenomenon of suppression of MAVS conferring radioresistance by means of clone survival and MN assay. Further, because MAVS is a protein which localized the outer membrane of mitochondrial, we attempted to investigate the influences of suppression of MAVS on mitochondrial functions to probe the potential mechanisms. Although we also observed a slight changes of MMP in MAVS-silenced cells, the cellular collapse of the ΔΨm in the irradiated normal cells was dramatically more than that in the irradiated MAVS-silenced cells at 24 h after irradiation. The content of ATP in normal cells after irradiation dramatically decreased, but the decrease was relieved in the MAVS-silenced cells after irradiation. Mitochondrial dysfunction can lead to the release of Cyto C and cell apoptosis. Our results demonstrated that the expressions of apoptosis protein Cyto C and active caspase-3 in the irradiated MAVS knockdown cell line were lower and the ratio of Bcl2/Bax proteins was higher than that of the irradiated normal cells, indicating that apoptosis decreased in MAVS knockdown cells after irradiation. These data suggest that knockdown of MAVS not only inhibit the radiationinduced mitochondrial dysfunction but also block the transmission of cellular apoptosis signaling pathway, which results in the subsequent radioresistance and may also indicate one of mechanisms on radioresistance.
In general case, except for the direct damage to DNA and other cellular components, IR also instantaneously causes the formation of water radiolysis products that contain ROS (chemical process occurred in the extremely early stage of radiation). ROS are also suggested to be released from biological sources in irradiated cells [28]. In our study, we observed that generation of radiation-induced ROS in MAVS knockdown cells decreased by 3.0-fold at 15 min after irradiation. Buskiewicz et al. reported that chemically generated oxidative stress (using glucose oxidase) stimulated the formation of MAVS oligomers, which led to mitochondrial hyperpolarization and decreased ATP production and spare respiratory capacity [29]. MAVS oligomerization directly suppresses the function of mitochondrial complexes I to IV and trigger the deficiency of ATP and the effusion of Cyto C [30]. Based on their findings, we speculate that MAVS involves in radiosensitivity through its oligomerization mediated by ROS. According to their measure method, we first observed that the irradiation increased the oligomerization of MAVS in A549, which was significantly inhibited by MitoQ pretreatment, and there was no increased oligomer of MAVS in the knockdown cell. MitoQ pretreatment also decreased the radiation-induced fractions of MN and increased the survival of normal A549 cells. Additionally, knockdown of MAVS also suppressed the expression of IRF3, type I IFNs, and almost eliminated pho-IRF3. Radiation stimulation induced the upregulation of IL6 level at 6 h after IR. Knockdown of MAVS suppressed the increase of the level of IL6 after IR. These results suggest that oligomerization of MAVS mediated by radiation-induced ROS form prion-like aggregates to induce the mitochondrial dysfunction and the activation NF-κB and IFN-type I. Suppression of MAVS reduces MAVS oligomer, thus decreasing radiation-induced mitochondrial dysfunction and inhibiting the pathway of cell apoptosis and the release of part of proinflammation cytokines, as well as the generation of mtROS again,  The survival analysis of negative vector and MAVS-silenced A549 cells pretreated with MitoQ after exposed to 0, 1, 2, 4, and 6 Gy X-rays. Data are presented as mean ± SD. * P < 0:05, * * P < 0:01, and * * * P < 0:001 vs. the irradiation group. 9 Oxidative Medicine and Cellular Longevity resulting in the radioresistance. These findings imply a model that ROS generation during initial chemical progress after irradiation could be an initial upstream signaling factor of MAVS responding to radiation, and then this kind of ROS-mediating initial MAVS oligomerization induces the increase of mtROS, which in turn induces more oligomers of MAVS and expand the radiationinduced damage (Figure 8). In order to confirm the conclusion, we also constructed the overexpression MAVS plasmid and transfected to A549 or BEAS-2B cell lines but unfortunately could not get stable over expression cell lines. From previous experiment results, we speculated the occurrence of mitochondrial dysfunction and apoptosis in MAVS overexpression, resulting to apoptosis or necrosis in the MAVS overexpression cell lines.

Conclusions
MAVS, an innate immune signaling molecule, is involved in radiation response via its oligomerization mediated by radiation-induced ROS. Knockdown of MAVS alleviates the radiation-induced mitochondrial dysfunction and decreases the expressions of proapoptotic proteins in cells after irradiation, which results in the radioresistance. Our study implies that MAVS may be a potential target for the precise radiotherapy or radioprotection.

Data Availability
The data used to support the findings of this study are available from the corresponding authors upon request.

Disclosure
Dong Pan's present address is Department of Dermatology, Duke University Medical Center, Durham, NC 27710, USA.

Conflicts of Interest
The authors do not have any conflicts of interest.

Authors' Contributions
Yarong Du designed and performed experiments, analyzed the results, and wrote the manuscript. Dong Pan, Rong Jia, Yaxiong Chen, and Cong Jia helped with the experiments, data interpretation, and manuscript preparation. Jufang Wang and Burong Hu involved in the experimental design, data analysis, and manuscript revise. All authors read and approved the final manuscript.  Figure 7: Associated immune factor was detected by ELISA assay (a) and western blotting (b) in negative vector and MAVS-silenced A549 cells at the indicated times after X-ray radiation. Data are presented as mean ± SD. * P < 0:05, * * P < 0:01, and * * * P < 0:001 vs. the irradiation group.  Oxidative Medicine and Cellular Longevity