Dynamin-Related Protein 1 Is Involved in Mitochondrial Damage, Defective Mitophagy, and NLRP3 Inflammasome Activation Induced by MSU Crystals

Excessive generation of reactive oxygen species (ROS) has great impacts on MSU crystal-induced inflammation. Drp1-dependent mitochondrial fission is closely associated with mitochondrial ROS levels. However, whether Drp1 signaling contributes to MSU crystal-induced inflammation remains unclear. Mice bone marrow-derived macrophages (BMDMs) were primed with LPS and then stimulated with MSU suspensions for 12 h. The protein levels associated with mitochondrial dynamics, oxidative stress, and mitophagy were detected by Western blot. BMDMs were loaded with MitoTracker Green probe to detect mitochondrial morphology. To measure mitochondrial reactive oxygen species (ROS) and total ROS levels, cells were loaded, respectively, with MitoSOX and DHE probes. The effects of Mito-TEMPO, an antioxidant that targets the mitochondria or DRP1 inhibitor (Mdivi-1) on MSU crystal-induced peritonitis and arthritis mouse models, were evaluated. Our study revealed that MSU crystal stimulation resulted in elevation of mitochondrial fragmentation of BMDMs. Treatment with Mito-TEMPO or Drp1 knockdown significantly ameliorated the mitochondrial damage induced by MSU crystals. BMDMs exposure to MSU crystals increased the expression of auto/mitophagy marker proteins and promoted the fusion of mitophagosomes with lysosomes, leading to accumulation of mitolysosomes. Drp1 knockdown alleviated defective mitophagy and activation of the NLRP3 inflammasome in MSU crystal-treated BMDMs. This study indicates that there is crosstalk between mitochondrial ROS and Drp1 signaling in MSU crystal-induced inflammation. Drp1 signaling is involved in MSU crystal-induced mitochondrial damage, impaired mitophagy and NLRP3 inflammasome activation.


Introduction
Gout is the most common form of inflammatory arthritis worldwide, and it is caused by the deposition of monosodium urate (MSU) crystals in joints and surrounding tissues [1]. In high-income countries, the prevalence of gout appears to have stabilized [2,3]. However, the incidence of gout is still increasing in mainland China [4]. The main treatment options for gout flare up are colchicine, nonsteroidal anti-inflammatory drugs (NSAIDs), or corticosteroids [5][6][7]. However, these traditional treatments have many side effects, and it is still difficult to treat gout [8]. IL-1 inhibitors are commonly used in patients with intolerable side effects or contraindications to first-line antiinflammatory therapies, but patients must be closely monitored for adverse reactions or intolerance.
MSU crystals stimulate innate immune pathways by damage-associated molecular patterns. In addition, studies have confirmed that excessive ROS production plays a key role in mediating inflammation in gout patients [9,10]. However, the exact molecular mechanism by which ROS regulate the inflammatory response induced by MSU crystals remains unknown. Mitochondria are the main sources of ROS, and damaged mitochondria produce higher ROS levels. Mitochondrial networks are dynamic and respond to metabolic signals by increasing the mass of the network (biogenesis) as well as by dividing existing mitochondria to form a larger network (fission) [11]. Mitochondrial biogenesis increases mitochondrial mass, and mitochondrial fission increases the actual number of mitochondria.
Dynamin-related protein 1 (Drp1) plays an important role during the process of mitochondrial fission [12][13][14][15]. Phosphorylated and activated Drp1 is recruited from the cytoplasm to a division site near the outer mitochondrial membrane, subsequently initiating fission. An increasing number of studies have shown that Drp1 activation leads to an increase in mitochondrial fission, thereby accelerating ROS production, while Drp1 inhibition leads to decreased ROS levels [16]. Drp1-mediated fission is considered to be the convergence point of ROS-dependent pathological processes. ROS may be upstream molecules of Drp1 activation, resulting in mitochondrial fission [13]. Under conditions of excessive ROS levels, enhanced mitochondrial fragmentation can also be observed, and this increased mitochondrial fragmentation is accompanied by increased Drp1 activity [14]. Therefore, the impact of Drp1 on ROS production appears to be bidirectional: on the one hand, Drp1may act as a receiver of ROS signals, and on the other hand, it stimulates ROS production by regulating mitochondrial fission.
Previous studies have suggested that mitochondrial fission occurs first, and then mitophagosomes are formed [22]. Based on these reports, we summarized the relationship among mitochondrial damage, mitochondrial fission, and mitophagy caused by oxidative stress (Supplementary Figure 1). However, the link between mitochondrial fission and mitophagy in MSU crystal-induced inflammation is poorly understood. The underlying molecular mechanism by which MSU crystals affect mitophagy is also unclear. Based on these facts, in the present study, we explored whether there was crosstalk between mitochondrial ROS and Drp1 signaling in response to exposure to MSU crystals. The roles of Drp1 signaling in MSU crystal-induced mitochondrial damage, mitophagy, and NLRP3 inflammasome activation were also assessed. Our data showed that Drp1 knockdown could improve MSU crystal-induced inflammatory response by inhibiting mitochondrial damage, impaired mitophagy, and NLRP3 inflammasome activation.

Materials and Methods
2.1. Mice. C57BL/6 mice aged 8 to 10 weeks were ordered from the Dossy Experimental Animals Company (Chengdu, China). All the animal experiments were performed in accordance with the Guidelines for the Care and Use of Experimental Animals and were approved by the Ethics Committee of North Sichuan Medical College.

Immunofluorescence and Confocal
Microscopy. BMDMs were fixed in 4% paraformaldehyde (PFA), permeabilized with 1× PBS supplemented with 0.03% Triton X-100, and blocked with 1× PBS supplemented with 2% bovine serum albumin (BSA) and 5% normal horse serum. Primary antibodies were added and incubated at 4°C overnight in blocking buffer. Secondary Alexa-conjugated antibodies from Jackson ImmunoResearch Laboratories were added and incubated at room temperature for 1 h. The nuclei were counterstained with DAPI. The samples were imaged by laser confocal microscope (Olympus FV3000).

ELISA Analysis.
The IL-1β levels in mouse peritoneal lavage fluids samples and cell culture supernatants were determined by ELISA kits (R&D Systems) following the manufacturer's instructions.
2.5. Immunoblotting Assay. The primary antibodies used in this study were listed by Supplementary materials. Mitochondria were isolated using mitochondria isolation kit (Beyotime, Shanghai, China, c3601). Whole cell lysates were prepared in RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl,1% NP-40, 1% sodium deoxycholate, 0.1% SDS) supplemented with a protease inhibitor cocktail (Roche). The protein concentrations were determined using a BCA Protein Assay Kit (Pierce, 23225). Equal amounts of protein 2 Oxidative Medicine and Cellular Longevity were separated by SDS-PAGE and transferred to PVDF membranes. Then, the membranes were blocked with 5% BSA in 1× TBST for 1 h at room temperature and incubated with relevant antibodies overnight. The secondary antibodies were added and incubated for 1 h at room temperature, and the protein bands were detected using chemiluminescence reagents (Bio-Rad). The ASC oligomer crosslinking assay was performed as previously described [24]. Cells were lysed with PBS supplemented with 0.5% Triton X-100 and a protease inhibitor cocktail (Roche). Cell lysates were centrifuged at 10,000 rpm for 15 min at 4°C. The supernatants were transferred to new tubes (TritonX-100 soluble fractions). The Triton X-100-insoluble pellets were washed with PBS three times and then suspended in 200 μl of PBS. The pellets were then crosslinked by adding 2 mM disuccinimidyl suberate (DSS) (Thermo Fisher Scientific) at room temperature for 30 min. The crosslinked pellets were further centrifuged at 10,000 rpm for 15 min and directly dissolved in SDS sample buffer for SDS-PAGE.
2.6. Mitochondrial Function Detection. Mitochondrial morphology was observed by laser confocal microscope after MitoTracker Green probe staining. (Beyotime, Shanghai, China, C1048). Mitochondrial membrane potential was measured using a JC-1 probe (Beyotime, Shanghai, China, C2006). Mitochondrial reactive oxygen species (mtROS) production was measured using MitoSOX (Invitrogen) probe. Total intracellular ROS levels were measured using DHE (BestBio, BB-47051) probe. Cells were loaded with the corresponding probe and then washed three times. The cells were resuspended in PBS, and then the fluorescence intensity was measured with flow cytometry (FCM, Novo-Cyte 2060R) according to manufacturer's instructions. For laser confocal microscopy (LCM) assessment, DAPI was used for nuclear counterstaining, and the cells were washed three times with PBS. Images were obtained using laser confocal microscope (Olympus FV3000).
The oxygen consumption rate was measured using Seahorse XFe24 Extracellular Flux Assay Kits (Seahorse Bioscience, Billerica, MA, USA) as previously described [25]. BMDMs were cultured in Seahorse XFe24 cell culture microplates at a density of 2 × 10 5 cells/well in DMEM supplemented with 10% FBS and antibiotics. After stimulation with MSU crystals (100 μg/ml) for 12 h, the culture medium was replaced with unbuffered DMEM supplemented with 2 mMl glutamine, 10 mM glucose, and 2 mM pyruvate. The "Flux Pak" cartridge was hydrated with XF Calibrant Solution by overnight incubation in a non-CO 2 incubator at 37°C. Oxygen consumption rate was measured, and the respiration rate was analyzed with step by step injections of mitochondrial complex inhibitors such as 1.5 μM oligomycin A (56 μl), 2 μM FCCP (62 μl), and 0.5 μM rotenone-antimycinA cocktail (69 μl) following the manufacturer's protocol. These mitochondrial complex inhibitors were provided with the Agilent Seahorse XF Cell Mito Stress Test Kit (Seahorse Bioscience, 103015-100). The data were normalized to total protein concentrations as measured using the Bradford assay. Data analysis was performed with the Seahorse Wave 2.2.0 software package (Seahorse Bioscience).

The mt-Keima Mitophagy Detection Assay.
To develop stable expression cell lines, BMDMs were incubated with mt-Keima-COX8 lentiviruses (GENECHEM, Shanghai, China, LV01230-2a) at a multiplicity of infection (MOI) of 80, and polybrene was added to facilitate infection. At 24 h postinfection, surviving cells were selected with culture medium supplemented with puromycin (6 μg/ml) to generate stable cell lines. BMDMs stably expressing mt-Keima-COX8 were treated with Mito-TEMPO or transfected with Drp1 siRNA, primed with LPS, and then stimulated with MSU crystals. For LCM detection, after the BMDMs were washed with PBS for three times, culture medium was added and LCM was used for imaging and analysis. The mitophagic ratio was calculated by measuring the red mean fluorescence intensity (MFI)/green mean fluorescence intensity ratio (excitation 561 : 488). For FCM detection, the cells were resuspended, and the cell suspensions were incubated on ice and measured with a flow cytometry (NovoCyte 2060R). Events were preselected for viable, single-cell populations, which were detected by excitation at 405 and emission at 610/620 nm. Fluorescent cells (20,000 per sample) were collected and analyzed for dual excitation at 488 (pH 7) and 561 (pH 4) nm with 582/515 and 610/620 nm emission filters, respectively. By analyzing the MFI (561) nm/MFI (488) nm ratio, the mitophagic ratio could be analyzed. Data processing was performed with FlowJo (v10, Tree Star) software.
The C57BL/6 mice were intraperitoneally injected with Mito-TEMPO (20 mg/kg, Sigma-Aldrich, SML0737) or Mdivi-1(25 mg/kg, GLPBIO, GC10200) and then injected the foot pad with MSU suspension (1 mg in 50 μL PBS). Twenty-four hours after injection with the MSU suspension, the swelling of the foot pad was measured with digital vernier caliper, and then the mice were sacrificed. Joint index evaluation was described previously [26]. evaluate the infiltration of inflammatory cells. For the immunofluorescence assay, briefly, foot pad sections were hydrated and boiled at 95°C in citrate buffer for 25 min. The sections were blocked with 2% BSA/PBS supplemented with 5% normal horse serum for 1 h at room temperature. Primary antibodies against MPO, CD11b, and DRP1 were incubated in blocking solution overnight at 4°C. After washing, Alexa secondary antibodies were added and incubated for 1 h at room temperature. The sections were washed with PBS and incubated with DAPI. The sections were imaged using laser confocal microscope.
2.10. Statistical Analysis. The values are expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA (analysis of variance). All the statistical analyses were assessed using the GraphPad Prism software (Version 6.01). Values were considered statistically significant when P < 0:05.

Mito-TEMPO Improves Mitochondrial Dynamic
Damage in MSU Crystal-Treated BMDMs. Mitochondrial ROS can affect mitochondrial homeostasis. As mitochondria are the highly dynamic organelles, we analyzed whether Mito-TEMPO could affect mitochondrial dynamic balance in MSU crystal-treated BMDMs. By TEM, we detected the mitochondrial morphology in BMDMs. As shown in Figure 1(a), mitochondria were smaller and fragmented in BMDMs treated with MSU crystals. Mito-TEMPO treatment significantly alleviated mitochondrial fragmentation induced by MSU crystals. In addition, the mitochondrial morphology stained with MitoTracker Green (a probe that emits fluorescence) was assessed by laser confocal microscopy (LCM). A large number of small and round mitochon-dria was observed in BMDMs stimulated by MSU crystals. We also analyzed the aspect ratio (AR) of mitochondria. The mitochondrial AR analysis showed a decreasing trend in BMDMs stimulated with MSU crystals (Figure 1(b)), suggesting that MSU crystals accelerated mitochondrial fission. Mito-TEMPO successfully prevented mitochondrial morphological abnormalities in MSU crystal-treated BMDMs ( Figure 1(b)). These data showed that Mito-TEMPO effectively relieved mitochondrial morphological damage induced by MSU crystals. Disruption of mitochondrial dynamics affects mitochondrial morphology. Dynamin-related protein 1 (Drp1) is a known regulator of mitochondrial fission. The phosphorylation of Drp1 (p-Drp1) at Ser616 promotes Drp1 recruitment to mitochondria and subsequent mitochondrial fission [27]. Therefore, we investigated the effect of Mito-TEMPO on the mitochondrial distribution of MSU crystal-induced Drp1 protein. LCM imaging analysis revealed that MSU crystal exposure obviously promoted the elevation of Drp1 protein located in mitochondria, indicating the initiation of the fission pathway, and Mito-TEMPO pretreatment significantly reduced the fluorescence signal of Drp1 in mitochondria (Figure 1(c)). More importantly, western blot data showed that the expression of p-DRP1 (s616) in the mitochondrial fraction of BMDMs was decreased after Mito-TEMPO pretreatment (Figure 1(d)). We also explored the role of Mito-TEMPO on the mitochondrial fusion proteins (MFN1, MFN2, and OPA1) and mitochondrial fission protein Fis1 levels. Mito-TEMPO reversed the MSU crystal-induced increase in the Fis1 protein expression and the decrease in MFN1, MFN2, and OPA1 protein levels ( Figure 1(e)). These data indicated that Mito-TEMPO might relieve mitochondrial dynamic damage by improving the imbalance of mitochondrial fission-fusion proteins.   Oxidative Medicine and Cellular Longevity

Drp1 Signaling Is Involved in Mitochondrial Dysfunction.
We first detected the impact of Drp1 knockdown on mitochondrial morphology induced by MSU crystals. Drp1 was inhibited in BMDMs transfected with targeted Drp1 siRNA. (Figure 2(a)). The LCM imaging revealed that the enhanced mitochondrial aspect ratio in MSU crystal-treated BMDMs was also largely reversed by Drp1 knockdown (Figure 2(b)). Since the excessive mitochondrial fission mediated by Drp1 could exacerbate the production of mitochondrial ROS [28], we also observed that Drp1 knockdown significantly decreased mitochondrial ROS and total intracellular ROS generation in MSU crystal-treated BMDMs (Figures 2(c) and 2(d)). We sought to explore whether Drp1 knockdown mediated antioxidant protein expression induced by MSU crystals. SOD1, SOD2, CAT, and GPX1 are all intracellular antioxidant proteins. It is interesting to note that after MSU crystal stimulation, SOD1, CAT, and GPX1 protein levels decreased, while the protein levels of SOD2 increased. SOD2 is an important mitochondrial antioxidant enzyme residing in the mitochondrial matrix. In MSU crystal-induced BMDMs, Drp1 knockdown almost restored the expression of these antioxidant proteins (Figure 2(e)). These data suggested that there was crosstalk between mitochondrial ROS and Drp1 signaling in MSU crystal-induced inflammation. Excessive ROS levels usually cause the Δψm to collapse by disrupting mitochondrial membrane potential homeostasis. The loss of mitochondrial membrane potential induced by MSU crystals was partially recovered in Drp1 knockdown BMDMs (Figure 2(f)). Drp1 is involved in regulating mitochondrial dynamics induced by MSU crystals, and mitochondrial dynamics play an important role in the response of cells to exogenous stress. In this study, mitochondrial stress was assessed by monitoring the real-time oxygen consumption rate (OCR) using the Seahorse XFe24 Extracellular Flux Analyzer. Interestingly, mitochondrial basal respiration, ATP production,     Oxidative Medicine and Cellular Longevity maximal respiration rate, spare capacity, and proton leak were significantly decreased in the BMDMs exposed to MSU crystals (Figures 2(g) and 2(h)). Drp1 knockdown significantly attenuated the mitochondrial stress induced by MSU crystals (Figures 2(g) and 2(h)). Furthermore, the Drp1 overexpression exacerbated the mitochondrial stress induced by MSU crystals (Supplementary Figures 2(a)-2(b)).

MSU Crystals Promote Mitophagy Initiation and
Increase Mitolysosome Formation. MSU crystal stimulation results in mitochondrial damage, which requires clearance through mitophagy. Next, we attempted to detect the effect of MSU crystal exposure on the initiation of mitophagy. The protein expression of mitophagy markers, such as PINK1, PRKN, and OPTN, was evaluated using immunoblotting of protein lysates from BMDMs stimulated with different doses of MSU crystals (25, 50 and 75 μg/ml) for 12 h. Interestingly, MSU crystals almost dose-dependently promoted the expression of PINK1, PRKN, and OPTN ( Figure 3(a)). Next, we performed a time-course experiment to determine the optimal time point at which MSU crystalinduced upregulation of the mitophagy marker protein expression in BMDMs is as follows. BMDMs were stimulated with 75 μg/ml MSU crystals for the indicated time points (0, 4, 8, 12 h), and cell lysates were used to measure the mitophagy marker protein expression by immunoblotting. The PINK1 protein expression almost reached its peak 8 h after exposure to MSU crystals, while PRKN and OPTN protein levels peaked at approximately 12 h (Figure 3(b)).
In the process of mitophagy, depolarized mitochondria cannot import and degrade PINK1, which is activated by the PRKN protein and modified by the OPTN protein, finally resulting in its elimination via the BECN1/ MAP1LC3B-dependent autophagy process. Next, we aimed to monitor the expression of proteins involved in the autophagy process, including autophagosome initiation marker (BECN1), autophagosome formation marker (MAP1LC3B), and autophagy degradation marker (SQSTM1) in BMDMs stimulated with MSU crystals (75 μg/ml) for varying time points. The expression of the autophagy markers BECN1, MAP1LC3B-II, and SQSTM1 was significantly increased in BMDMs exposed to MSU crystals over a period of up to 12 hours (Figure 3(b)).
Next, we measured functional mitophagy using a mitochondrial targeted mt-Keima probe, which is an indicator of mitochondria colocalization with mature autolysosomes. Since the mt-Keima probe is resistant to lysosomal proteases and exhibits reversible color changes at acidic pH levels, the mt-Keima probe can be used to assess autolysosome maturation. The high excitation peak ratio (561: 488) of mt-Keima is shown in pseudo, indicating the presence of mitochondria in mature autolysosomes. Using this assay, we observed that the number of red mt-Keima puncta was significantly increased in BMDMs exposed to MSU crystals, indicating an increased level of mitolysosomes (Figure 3(c)). Bafilomycin A1 is a known inhibitor of the late phase of autophagy and can inhibit the fusion of phagosomes and lysosomes. As shown in Figure 3(d), in non-MSU crystal treatment BMDMs, the levels of LC3B-II and SQSTM1 were significantly higher after the application of bafilomycin A1 (Figure 3(d)). Nevertheless, the levels of LC3B-II and SQSTM1 were not further increased by the application of bafilomycin A1 in the BMDMs treated with MSU crystals, compared with MSU crystal-induced cells (Figure 3(d)). All these findings suggest that MSU crystals might accelerate the fusion of mitophagosmes and lysosomes to form mitolysosomes, but autophagic flux is blocked in MSU crystal-induced BMDMs.

Silence of Drp1 Expression Rescued Defective Mitophagy
Induced by MSU Crystals. In the present study, we further investigate the role of Drp1 in MSU crystal-induced mitophagy response. As expected, the immunoblotting data of mitochondrial extracts showed that the PINK1, MAP1LC3B-II, and SQSTM1 proteins were highly expressed    (Figure 4(a)). Drp1 knockdown also greatly inhibited MSU crystal-induced expression of PRKN, OPTN, and BECN1 (Figure 4(b)). Drp1 was overexpressed in BMDMs transfected with corresponding plasmid. (Figure 4(c)). However, the Drp1 overexpression in BMDMs aggravated the expression of mito/autophagy markers PINK1 and BECN1 induced by MSU crystals (Figure 4(d)).
Next, we attempted to genetically block autophagy to confirm the role of this process in MSU crystal-induced mitophagy. BMDMs were transfected with either BECN1 siRNA or Ctrl siRNA ( Figure 5(a)). DRP1 and mito/ autophagy marker protein levels were measured by immunoblotting. Downregulation of BECN1 almost had no effect of DRP1 protein level induced by MSU crystals (Figure 5(b)). However, the MSU crystal-induced upregulation of mito/autophagy marker protein (including PINK1, PRKN, OPTN, MAP1LC3B-II and SQSTM1) expression was prevented in BMDMs transfected with BECN1 siRNA (Figure 5(c)). The effect of mitophagy initiation signaling on the expression of DRP1 and mito/ autophagy marker proteins was also detected. BMDMs were transfected with either Ctrl siRNA or PINK1 siRNA ( Figure 5(d)). Further western blot data showed that PINK1 knockdown had little effect on the MSU crystalmediated DRP1 protein levels and the autophagy initiation marker BECN1 (Figure 5(e)) but prevented the upregulation of the mitophagy markers (PRKN and OPTN) and the autophagy markers (MAP1LC3B-II and SQSTM1) ( Figure 5(f)). These data imply that Drp1 signaling is upstream of autophagy initiation and PINK1/PRKN-mediated priming of damaged mitochondria.
Subsequently, we further explored the effect of Drp1 knockdown on the mitophagic response through multiple assays. First, we detected the impact of Drp1 knockdown on the expression of the mitochondrial marker proteins Tom20 and Cyto C in BMDMs treated with MSU crystals. They are commonly used as mitochondrial markers to reflect damage mitochondrial clearance and mitophagic rate [29]. Silence of Drp1 alleviated the elevation of Tom20 and Cyto C protein levels induced by MSU crystals (Figure 6(a)). Second, we examined mitophagy by costaining mitochondria (labeled by MitoTracker Red) and LC3 (an autophagy marker) to reflect formation of mitophagosomes. The immunofluorescence staining showed that Drp1 knockdown decreased colocalization of LC3 + puncta with MitoTracker in BMDMs treated with MSU crystals (Figure 6(b)), indicating that silence of Drp1 inhibited the accumulation of damaged mitochondria and mitophagosomes. Third, we used the mitophagy reporter Mito-Keima to quantitatively detect mitophagic activity. After the stable expression of Mito-Keima-COX8 plasmid by BMDMs, interfered with Drp1 siRNA or NC siRNA, and then stimulated with MSU crystals, LCM imaging data and flow cytometry analysis, respectively, indicated that NC siRNA and Drp1 siRNA BMDMs almost exhibited similar mitophagy at baseline level without stimulation of MSU crystals, as reflected by similar percentage of the cells with a high 561/405 nM ratio (Figures 6(c) and 6(d)). As expected, downregulation of Drp1 markedly decreased the percentage of cells with the high 561/488 nm ratio in BMDMs treated with MSU crystals (Figures 6(c) and 6(d)). Fourth, we monitored autophagic rate to analyze LC3-II and P62 protein levels by adding autophagosome (mitophagosome) and lysosome fusion inhibitor (bafilomycin A1). Western blot data indicated that Drp1 knockdown in the presence of MSU crystals effectively prevented the accumulation of LC3-II and P62 in BMDMs treated with bafilomycin A1 (Figure 6(e)). Taken together, these findings suggest that silence of Drp1 effectively relieved impaired mitophagy induced by MSU crystals.

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Oxidative Medicine and Cellular Longevity 3.5. Drp1 Knockdown Relieved NLRP3 Inflammasome Activation Induced by MSU Crystals. Since Drp1 signaling is involved in mitochondrial fitness and mitophagy, next, we investigated whether Drp1 knockdown ameliorated NLRP3 inflammasome activation induced by MSU crystals. Drp1 knockdown inhibited IL-1β secretion (Figure 7(a)) and moderately decreased the expression of NLRP3 which is the important component of NLRP3 inflammasome, but Drp1 knockdown had almost no effect on the protein expression of pro-IL-1β and pro-caspase-1 in LPS-primed BMDMs treated with MSU crystals (Figure 7(b)). Importantly, Drp1 knockdown blunted Ccspase-1 activation and pro-IL-1β processing into mature IL-1β in LPS-primed BMDMs incubated with MSU crystals (Figure 7(b)). ASC oligomerization leading to ASC speck formation mediates caspase-1 activation [30,31]. The effect of Drp1 knockdown on ASC oligomerization and subsequent ASC speck formation was also analyzed. ASC oligomerization was measured by immunoblotting analysis of the DSS-crosslinked insoluble BMDM lysate fraction. In LPS-primed BMDMs treated with MSU crystals, Drp1 knockdown blocked ASC oligomerization (Figure 7(c)), and similar results were obtained by immunofluorescence staining of ASC speck information (Figure 7(d)).
3.6. Silence of PINK1 or BECN1 Fails to Suppress Mitochondrial Damage in MSU Crystal-Treated BMDMs. We attempted to study the role of auto/mitophagy in   (Figure 8(a)) and decrease in the OCR (Figure 8(b)), suggesting that MSU crystal-mediated mitochondrial dysfunction was upstream of the mitophagy process. Blockage of mitophagy can exacerbate inflammation [19][20][21]. MSU crystal exposure induced IL-1β secretion, which was decreased after silence of BECN1 or PINK1 (Figure 8(c)), suggesting that BECN1 or PINK1 might be involved in NLRP3 inflammasome activation induced by MSU crystals.  The level of IL-1β was detected by ELISA in the culture supernatants. The data are presented as mean ± SEM from 3 independent experiments. * P < 0:05 vs. without MSU crystal treatment + Ctrl siRNA; # P < 0:05 vs. MSU crystal treatment + vehicle. 16 Oxidative Medicine and Cellular Longevity  involved in MSU crystal-induced mouse arthritis and peritonitis models. Mouse models of arthritis and peritonitis were established by injecting MSU crystals into the foot pad and the abdominal cavity, respectively, and the mice were pretreated with Mito-TEMPO or Mdivi-1 by intraperitoneal injection. A peritonitis model in C57BL/6 mice was used to assess the role of Mito-TEMPO or Mdivi-1 on inflammatory cell influx and IL-1β production. As shown in Figure 9(a), MSU crystals accelerated the infiltration of leukocytes (CD45 + ), macrophages (CD11b + F4/80 + ), and neutrophils (CD11b + Ly-6G + ) into the peritoneal cavity. The number of leukocytes, macrophages, and neutrophils was greatly diminished after Mito-TEMPO or Mdivi-1 pretreatment (Figure 9(a)). In contrast with vehicle treatment, Mito-TEMPO or Mdivi-1 pretreatment also obviously suppressed the MSU crystal-induced IL-1β secretion in peritoneal lavage fluids (Figure 9(c)). However, we noted that Mdivi-1 treatment had no significant difference in inflammatory cell migration and IL-1β secretion in the peritonitis model compared with Mito-TEMPO treatment.
Injection of a certain dose of MSU suspensions into the mouse foot pad can also lead to inflammatory response. Our data showed significant reduction in MSU crystalinduced foot pad swelling after Mito-TEMPO or Mdivi-1 pretreatment (Figure 9(b)). H&E staining showed that there were a large number of immune cells infiltrated into the tissue sections of the foot pad injected with the MSU suspen-sions, but Mito-TEMPO or Mdivi-1 administration noticeably reduced immune cell infiltration (Figure 9(d)). After immunofluorescence staining of foot pad tissue sections, LCM imaging data showed that Mito-TEMPO or Mdivi-1 pretreatment reduced the distribution of inflammatory cell (MPO and CD11b + -positive cells) (Figure 9(e)). We also noted that almost no DRP1 positive cells were distributed in the foot pad of Ctrl mice, while a number of DRP1-positive cells infiltrated the foot pad in MSU suspension treated mice, Mito-TEMPO or Mdivi-1 pretreatment decreased the number of DRP1-positive cells in foot pad of MSU suspension treated mice (Figure 9(e)). This suggested that in vivo MSU treatment promoted Drp1 expression, and Mito-TEMPO or Mdivi-1 could reduce Drp1 protein levels. Thus, all these data indicated that pretreatment with Mito-TEMPO or Mdivi-1 notably relieved the inflammation induced by MSU crystals in vivo, and that targeting mitochondrial ROS or Drp1 activity may be potential candidates for the treatment of gouty arthritis in the future.

Discussion
Previous work has documented the role of oxidative stress and mitochondrial dysfunction in inflammatory responses induced by MSU crystals. However, the molecular mechanism of mitochondrial ROS involvement in MSU crystalinduced inflammatory response is not fully understood. Although crosstalk between Drp1 signaling and ROS has