The coenzyme Q10 analog idebenone attenuates murine lupus by improving mitochondrial metabolism and reducing inflammation.

Abstract

Objective:

A role for mitochondrial dysfunction has been proposed in the immune dysregulation and organ damage characteristic of systemic lupus erythematosus (SLE). Idebenone is a coenzyme Q10 synthetic quinone analog and an antioxidant that has been used in humans to treat diverse diseases where mitochondrial function is impaired. This study assessed if idebenone ameliorates murine lupus.

Methods:

Idebenone was administered orally to MRL/lpr mice at two different doses (either 1 or 1.5 g/kg diet) for 8 weeks. At peak disease activity, clinical, immunologic and metabolic parameters were analyzed and compared to untreated mice. Results were confirmed in the lupus-prone NZM2328 mouse model.

Results:

In MRL/lpr mice, idebenone-treated mice showed significant attenuation in mortality (p<0.01) and in several features including glomerular inflammation and fibrosis (p<0.05) and renal function, in association with decreases in renal IL-17A and mature IL-18 expression. Levels of splenic proinflammatory cytokines and inflammasome-related genes were significantly decreased by idebenone, while no obvious toxicity was observed. Idebenone inhibited neutrophil extracellular trap formation in lupus mouse and human neutrophils. Idebenone also improved mitochondrial metabolism (30% increase in basal respiration and ATP production), reduced heart lipid peroxidation in half, and significantly improved endothelium-dependent vasorelaxation. NZM2328 mice exposed to idebenone also displayed improvements in renal and systemic inflammation.

Conclusions:

Idebenone ameliorates murine lupus disease activity and organ damage severity supporting the hypothesis that agents that modulate mitochondrial biology may have a therapeutic role in SLE.

Introduction

An important role for mitochondrial dysfunction in the pathogenesis of systemic lupus erythematous (SLE) has been described by our group and others (reviewed in (1)). Enhanced synthesis of mitochondrial reactive oxygen species (mROS) by lupus neutrophil subsets is associated with enhanced formation of neutrophil extracellular traps (NETs) that are enriched in oxidized mitochondrial DNA (mDNA). This oxidized mDNA promotes type I interferon (IFN) responses through activation of the cGAS-STING pathway (2). Mitochondrial dysfunction has also been reported in lupus-prone mice at early ages, before overt immune dysregulation and tissue damage become apparent (3). Furthermore, in vivo administration of mROS scavengers ameliorates murine lupus (2). These observations indicate that drugs improving mitochondrial homeostasis may have therapeutic benefits in SLE.

Idebenone (2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinonenoben) is a drug previously tested in clinical trials and already approved in some countries for the treatment of certain diseases associated with mitochondrial dysfunction (4–7) including Leber’s hereditary optic neuropathy and Duchenne muscular dystrophy. It is a synthetic quinone analog compound of coenzyme Q₁₀ (CoQ₁₀) with a shorter aliphatic chain and is considered a potent antioxidant that protects cells against enhanced ROS toxicity (8, 9). Idebenone improves electron transfer chain function by bypassing a deficient complex I activity and enhancing the amount of ATP synthesized, thereby improving mitochondrial physiology (10). The potential therapeutic benefit of idebenone has been tested in various murine models of disease (11–13). We hypothesized that idebenone could modulate SLE pathology through regulation of mitochondrial function and ROS synthesis and tested the effect of this drug in murine models of lupus.

Materials and methods

Animal procedures and diet.

The NIAMS Animal Care and Use Committee (ACUCA) approved all animal procedures for these experiments (protocol # A016-05-26). Female lupus-prone MRL/MpJ-Fas^lpr/J mice (MRL/lpr, stock #000485, n=10 per group per experiment) and control female MRL/MpJ (MpJ, stock #000486) were purchased from The Jackson Laboratories (Bar Harbor, ME). The control diet was the standard NIH-31 diet (14), while idebenone (Cayman Chemical, Ann Arbor, MI)-supplemented diet (Envigo, Madison, WI) contained either 1 g (low dose) or 1.5 g (high dose) idebenone /kg NIH-31 diet and a blue dye (FD&C Green #3, CAS# 2353-45-9, Chemical Formula: C37H34N2Na2O10S3) to monitor intake. MRL/lpr mice started idebenone diet at 9 weeks of age. Animals were kept in the diet for approximately 2 months (MRL/lpr) until peak in disease manifestations, followed by euthanasia. All experiments compared high and low dose idenenone to control group, while a more limited set of experiments (diet intake and complex II activity) were performed comparing only low dose of idebenone to control group (supplementary data). In addition, a more limited set of experiments were performed in NZM2328 lupus-prone mice (breeding pair originally obtained by Dr. Shu-Man Fu, University of Virginia), to assess efficacy beyond a single mouse model. These animals (10 per group) were treated from week 10 to week 38 with low dose idebenone containing diet (1.0 g/kg diet) or normal control NIH-31 diet.

Anti-dsDNA, anti-RNP, and anti-SSA quantification.

Serum concentrations of autoantibodies were calculated as before (2) using commercially available ELISA kits (catalog #5110, #5410, and #5710 from Alpha Diagnostic International, San Antonio, TX, respectively). Briefly, serum was diluted (1:125) in NSB buffer and the assay was done following manufacturer’s instructions.

Assessment of kidney function and histology.

Kidney slides were evaluated blindly by a veterinary pathologist for severity, fibrosis, inflammation, and glomerulosclerosis, and the scores were calculated as previously described (15). Renal immune complex deposition was quantified as previously described (2) using an Alexa fluor 594-F(ab’)2-goat anti-mouse IgG (catalog#A11020, Thermofisher, Waltham, MA) and a FITC-anti-murine C3 antibody (catalog#GC3–90F-Z, Immunology Consultants Laboratories, Portland, OR). Frozen kidney sections were also stained with Alexa fluor-488 rat anti-mouse IL17A antibody from BD (San Jose, CA). Nuclei were stained with Hoechst (1:500, Life Technologies). For quantification, three random images were obtained from each stained frozen section. The images were analyzed with Image J software selecting the glomerular compartment to quantify mean pixels for each fluorescence channel used.

To quantify serum creatinine and eliminate the influence of chromogens in mouse serum that interfere with the classic Jaffe method for creatinine detection, a HPLC assay was used as previously described (16). Briefly, 5 μl serum were treated with 0.5 ml acetonitrile, centrifuged at 4°C at 13,000 × g for 20 min, and supernatants were dried by SpeedVac and resuspended in mobile phase (5 mM sodium acetate, pH 5.1). Duplicates were run on a 100 × 4.1 mm PRP-X200 column (Hamilton, Reno, NV) and isocratically eluted at 2 ml/min in an Agilent 1100 system, with UV detection at 234 nm. Absolute quantitation was determined with a standard curve of 2–50 ng creatinine (r² = 0.999).

Determination of endothelium-dependent vasorelaxation.

Endothelium-dependent vasorelaxation of thoracic aortas was accomplished as previously described by our group (15). Clean aorta rings were mounted in a myograph DMV device and allowed to stabilize under 7 mN isometric condition. After exposure to potassium buffer, aortic rings were pre-contracted with phenylephrine and the relaxation was evaluated adding increasing amounts of acetylcholine.

Assessment of pro-inflammatory gene expression.

mRNA was isolated from frozen spleens and quantification of pro-inflammatory and IFN regulated genes was performed as previously described (2). Briefly, tissues were homogenized in RLT lysis buffer and RNA isolated with RNA Easy kit (QIAGEN), following manufacturer’s instructions. cDNA was synthesized using 1 ug of RNA, BIORAD iScript kit, and an ABI thermocycler. qRT PCR was performed using BIORAD reagents and instructions, and a CFX96 BIORAD real time thermocycler. Fold gene expression for each gene was calculated using Actb (beta-actin) as house-keeping gene and Ct from tissue of mice under the control diet for delta delta calculations. The following primers were used: Actb (forward: 5’-CCA ACC GCG AGA AGA TGA-3′, reverse: 5’-CCA GAG GCG TAC AGG GAT AG-3′); Ifna1(forward: 5’- AAG GAC AGG CAG GAC TTT GGA TTC-3′, reverse: 5′- GAT CTC GCA GCA CAG GGA TGG-3′); Ifnb (forward: 5′- AAG AGT TAC ACT GCC TTT GCC ATC-3′, reverse: 5′- CAC TGT CTG CTG GTG GAG TTC ATC-3′); ll6 (forward: 5′- TGG CTA AGG ACC AAG ACC ATC CAA-3′, reverse: 5′- AAC GCA CTA GGT TTG CCG AGT AGA-3′); Mx1 (forward: 5′- GAT CCG ACT TCA CTT CCA GAT GG-3′, reverse: 5′- CAT CTC AGT GGT AGT CAA CCC-3′); Tnf (forward: 5′- CCC TCA CAC TCA GAT CAT CTT CT-3′, reverse: 5′- GCT ACG ACG TGG GCT ACA G-3′); Il1b (forward: 5′- CCC TGC AGC TGG AGA GTG TGG A-3′, reverse: 5′- CTG AGC GAC CTG TCT TGG CCG-3′); Il12b (forward: 5′-AGA AAG GTC CGT TCC TCG TAG-3′, reverse: 5′-AGC CAA CCA AGC AGA AGA CAG-3′); Pycard (forward: 5′-AAC CCA AGC AAG ATG CGG AAG-3′, reverse: 5′-TTA GGG CCT GGA GGA GCA AG-3′); Nlrp3 (forward: 5′-CTT CTC TGA TGA GGC CCA AG-3′, reverse: 5-GCA GCA AAC TGG AAA GGA AG-3′); Il10 (forward: 5′-CCA GTT TTA CCT GGT AGA AGT GAT G-3′, reverse: 5′-TGT CTA GGT CCT GGA GTC CAG CAG ACT-3′); Mpo (forward: 5′-TGC TCT CGA ACA AAG AGG GT-3′, reverse: 5′-CTC CTC ACC AAC CGC TCC-3′), and Il18 (forward: 5′-ACT GTA CAA CCG CAG TAA TAC GC-3′, reverse: 5′-AGT GAA CAT TAC AGA TTT ATC CC-3′). The mitochondrial/nuclear transcription ratio was measured using the following primers: 16S or Mrnr2 (forward: 5′-CTA GAA ACC CCG AAA CCA AA-3′, reverse: 5′-CCA GCT ATC ACC AAG CTC GT-3′) and beta-2-microglobulin B2m (forward: 5′-ATG GGA AGC CGA ACA TAC TG-3′, reverse: 5′-CAG TCT CAG TGG GGG TGA AT-3′).

Quantification of NETs and mROS in bone marrow (BM) neutrophils.

The isolation of BM derived neutrophils, quantification of NET and mROS were performed as previously described by us (2). Briefly, hindlimb bone marrow neutrophils were purified by Percoll gradient. Cells were seeded in a 96 well-plate (200,000 cells /100 ul/well) in triplicates for each dye and allowed to NET in the presence of SYTOX (externalized DNA, 1 uM final concentration), Quant-It Picogreen (total DNA, stock solution diluted 250 times) and MitoSox (mROS final concentration 200 ng/ml) (all from Thermofisher). Fluorescence was measured at different time points for each dye, at earliest time point 485/520 (Picogreen), 1h 510/580 (Mitosox), and 2h 485/520 (SYTOX), using a FLUOstar Omega BMG Labtech (Cary, NC) plate reader. Picogreen measurement was used as the initial number of cells or total DNA.

Phenotyping of splenocytes by flow cytometry.

After euthanasia splenocytes were isolated and, following red blood lysis using ACK buffer (GIBCO, Life technologies), single-cell suspensions were stained with Live/Dead Aqua (Thermofisher) diluted in PBS on ice for 15 minutes and washed with FACS buffer (PBS + 2% FBS). Cells were then incubated with TruStain FcX (Biolegend, San Diego, CA) on ice for 10 minutes before staining with the following antibodies (Biolegend) on ice for 15 minutes: APC CD8a (53–6.7), FITC CD19 (6D5), PerCP/Cy5.5 CD45 (30-F11), PE CD62L (MEL-14), BV421 CD4 (GK1.5), APC/Cy7 CD44 (IM7), PE/Cy7 CD3 (17A2). Cells were washed twice in FACS and immediately collected on LSRFortessa (BD Franklin Lakes, NJ). Data was analyzed using FlowJo software.

Isolation of human neutrophils and characterization of NET formation.

Human peripheral blood neutrophils were isolated from subjects who fulfilled revised ACR criteria for SLE (17) as previously described (2). All subjects gave informed consent to participate in a NIAMS/NIDDK IRB approved protocol. Peripheral blood was obtained by venipuncture and normal density neutrophils were purified from sedimented red blood cells (RBCs) following Ficoll gradient using dextran 10% solution. RBCs were lysed using sodium chloride hypo- and hyper-tonic solutions. Lupus low-density granulocytes (LDGs) were isolated by negative selection from the PBMC layer following Ficoll gradient, as previously described by our group (18). NETs were detected by immunofluorescence as described (2). Neutrophils seeded in coverslips or coverslip chambers were incubated for 90 min at 37°C with or without idebenone (10 μM), fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, followed by 0.5% gelatin for 20 min. Cells were stained with antibodies directed against human neutrophil elastase (1:1,000, ab21595, Abcam, Cambridge, MA), Hoechst 33342 (1:1,000, Life Technologies, Waltham, MA), and secondary antibodies (1:500, Alexa Fluor 488 (A31570), Life Technologies). After mounting (Prolong, Life Technologies), cells were visualized and imaged using a confocal Zeiss LSM780 microscope.

Mitochondrial metabolism analysis by Seahorse.

Bone marrow derived neutrophils or splenocytes were plated on Cell-Tak coated Seahorse culture plates (300.000 cells/well) in Seahorse XF RPMI medium (pH 7.4). XF analysis was performed at 37°C with no CO2 using the XF-96e analyzer (Agilent) as per manufacturer’s instructions. Mitochondrial stress test assay was performed using Seahorse XF RPMI medium (pH 7.4) supplemented with 25 mM glucose, 1 mM sodium pyruvate, and 2 mM L-glutamine. Cells were treated serially with oligomycin (5 μM), FCCP (1 μM), and rotenone (100 nM) and antimycin A (10 uM), and oxygen consumption rates (OCR) were measured over time. The cell numbers at assay completion were normalized to DNA content using CyQuant dye (Thermofischer). Wave, Excel, and Graph Pad Prism software were used to analyze the data. Mann-Whitney U test was performed to calculate significance.

Measurement of cardiac lipid peroxidation.

Lipid peroxidation was measured in half hearts as previously described (19). Briefly, half hearts were homogenized in buffer Tris-HCl (0.1 M, pH 7.4) and incubated with 10% trichloroacetic acid for 15 min on ice. After centrifugation at 2,200 × g for 15 min, supernatants and standard solutions were mixed with equal volume of thiobarbituric acid (0.67% w/v) and incubated for 10 min at 95°C. Standard was prepared by base two serial dilutions with tetramethoxypropane solution starting from 500 uM in water. Absorbance was measured at 532 nm. Protein concentration was measured in the homogenized tissue using the BCA protein assay from Thermofisher following manufacturer’s instructions.

Kidney expression of mature IL18.

Protein detection from renal tissue preserved at −80°C was performed as previously described (20). Briefly, 50 μg of total protein were resolved in a 4–12% NuPAGE Bis-Tris gradient gel (Invitrogen). Proteins were immobilized onto a nitrocellulose membrane (Invitrogen) and blocked with 10% BSA for 30 min at room temperature. Membrane was incubated with rabbit anti-mouse IL-18 clone H-173 (1:500, Santa Cruz) or anti–tubulin (1:1,000, Cell Signaling Technology) overnight at 4°C. After three washes with PBS-Tween, membrane was probed with secondary IRDye800 goat anti-mouse or IRDye800 goat anti-rabbit antibodies (1:10,000, Li-Cor Biosciences, Lincoln, NE) for 1 h at room temperature. Proteins were detected using the Li-Cor Odyssey Infrared Imaging System following manufacturer’s instructions (Li-Cor Biosciences).

Results

Assessment of intake and tolerability of idebenone-supplemented diet.

The mice displayed adequate intake of idebenone containing diet, as measured by incorporation of blue dye into feces and intestinal lumen (supplementary Figures 1A–C). MRL/lpr mice started idebenone diet at 9 weeks of age. Two doses of idebenone were tested. Assuming each 20 g mouse consumes approximately 3–5 g diet per day, the dose received per mouse was in the range of 150 mg (low dose)-375 mg (high dose) /day/kg. Mice tolerated diet well without any obvious signs of toxicity. At euthanasia, the weight for idebenone-treated mice was slightly higher than for mice fed the control diet (supplementary figure 1D).

Idebenone improves survival and renal pathology in murine lupus.

Mice were not kept alive beyond 17 weeks of age, in order to follow institutional regulations of animal care to prevent discomfort from neck lymphadenopathy in this animal model. Nevertheless, we observed that mice in both low and high dose idebenone diet survive throughout week 17, while several early deaths occurred in the control group, as shown by the Kaplan Meier survival curve (Figure 1A). One of the most prominent features of lupus is the development of glomerulonephritis. To explore the effect of idebenone on kidney function, we quantified serum creatinine and found that MRL/lpr mice fed high dose idebenone displayed significantly lower serum creatinine concentration (Figure 1B), similar to levels in the MpJ non-autoimmune control strain, while low dose idebenone did not significantly differ from control diet mice. Histologic analysis of kidneys (representative images in supplementary figure 2) revealed that low dose idebenone significantly reduced severity of glomerulonephritis (Figure 1C) and glomerulosclerosis (Figure 1F). High dose idebenone was associated with significant reductions in the glomerular fibrosis score (Figure 1D). No reductions in the inflammation score were observed with either dose of idebenone compared to untreated mice (Figure 1E). Similarly, renal immune complex deposition was not attenuated by either dose of idebenone compared to animals in control diet. Despite significant improvements in renal function and reductions in glomerular fibrosis, there was enhanced renal immune complex deposition in the high and low dose idebenone groups (Figures 1G–K). Treatment with either dose of idebenone significantly reduced the expression of mature IL18 cytokine compared to the control diet group, suggesting that idebenone may modulate inflammasome activation in lupus kidney (Figure 2A and 2B). In addition, both doses of idebenone significantly reduced kidney IL-17A levels (Figure 2C). These results suggest that idebenone can improve kidney function and ameliorate renal damage characteristic of SLE. Further support for the beneficial effects of idebenone in lupus renal disease were obtained by administering low dose idebenone to NZM2328 mice, which displayed significant reductions in immune complex deposition and renal inflammation (supplementary Figure 3B–3E). Overall, these results indicate that idebenone administration modulates lupus glomerulonephritis in murine systems and that the mechanisms may be complex and differ between mouse strains.

Figure 1: Idebenone improves renal disease in murine lupus.

(A) Kaplan-Meier survival curves indicate protection from death achieved by low and high dose idebenone diet. Log-rank (Mantel-Cox) test. **:P<0.01; n=10 mice/group. (B) Serum creatinine in MRL/lpr or MpJ mice determined at euthanasia (n=40/ treatment and n=5 for MpJ). (C) Severity kidney pathology score at euthanasia (n=20/treatment for MRL/lpr; n=3 for MpJ). (D) Fibrosis kidney pathology scores at euthanasia (n=20/treatment for MRL/lpr; n=3 for MpJ). (E) Inflammation kidney pathology score at euthanasia (n=20/treatment for MRL/lpr; n=3 for MpJ). (F) Glomerulosclerosis kidney pathology score at euthanasia (n=20/treatment for MRL/lpr; n=3 for MpJ). Images G through J are representative of renal IgG (red) and C3 (green) deposition; nuclei are stained with Hoechst (blue). (G) Control diet; (H) low dose idebenone; (I) high dose idebenone; (J) MpJ. (K) Quantification of glomerular immune complex (IgG and C3) deposition. Results represent mean ± SEM. Mann-Whitney U-test was used to compare the control to the idebenone diets. *:P<0.05; **: P <0.01, and ****: P <0.0001.

Figure 2: Idebenone modulates inflammasome-associated molecules and IL17 levels in lupus kidney.

(A) Western blot of kidney proteins displays control diet (n=4) and idebenone diets (n=5/group) expression of pro-IL18, mature IL18, and tubulin. (B) Mature IL18 was quantified by densitometry. (C) IL17A quantification in glomeruli (four tissue slides /condition, n=10 per treatment and n=3 for MpJ). Results represent mean ± SEM. Unpaired student’s t-test was used to compare control diet to the idebenone groups. *: P <0.05; **: P <0.01.

Idebenone does not reduce circulating autoantibodies but improves endothelium-dependent vasorelaxation and reduces cardiac lipid peroxidation.

Neither high nor low doses of idebenone modify serum anti-nRNP, -SSA, or -dsDNA autoantibody levels in MRL/lpr (Figures 3A–3C) or NZM2328 mice (not shown). In contrast, both doses of idebenone induced significant improvements in endothelium-dependent vasorelaxation (Figure 3D), suggesting that this compound can beneficially modulate lupus vasculopathy. Further supporting that idebenone improves cardiovascular physiology, cardiac lipid peroxidation (a phenomenon associated with enhanced oxidative damage and cardiovascular disease) was significantly diminished by both doses of idebenone compared to control diet (Figure 3E). These results indicate that idebenone significantly improves cardiovascular risk parameters in lupus-prone mice.

Figure 3. Effect of idebenone on circulating autoantibodies, endothelium-dependent vasorelaxation and cardiac lipid peroxidation.

Serum anti-nRNP (A), anti-SSA (B), anti-dsDNA (C) were quantified in MRL/lpr and MpJ sera at euthanasia (n=10 / group). (D) Endothelium-dependent vasorelaxation of aortic rings was evaluated in MRL/lpr (n=3 /group) by measuring the relaxation of the phenylephrine (PE) pre-contracted aortas in response to acetylcholine (Ach) using a myograph. Results represent mean ± SEM. Statistical analysis was done with 2way ANOVA multiple comparisons and Tukey’s multiple comparisons test in D to compare control versus idebenone diets. P values as indicated at the end of this legend. (C) Cardiac tissue lipid peroxidation quantification by thiobarbituric acid assay (TBARS) was performed (n=10 mice per treatment). Results represent mean ± SEM. Mann-Whitney U-test was applied to compare control versus idebenone diet groups. *: P <0.05; **: P <0.01; and ***: P <0.001.

Idebenone attenuates inflammatory pathways and immune dysregulation.

Spleen size, a parameter of murine lupus disease activity, was reduced with both doses of idebenone, being significant with the lower dose (Figure 4A). There were no significant differences in total splenic T cell numbers between control diet and idebenone diets (Figure 4B). Similarly, there were no significant changes in numbers of double negative (DN; CD4- CD8-) T cells (Figure 4C). However, when assessing maturation and activation status, low dose idebenone significantly reduced numbers of effector memory T cells (CD44+, CD62L-, Figures 4D–E) and effector memory CD4+ T helper cells (Figure 4F, supplementary Figure 4); in contrast high dose idebenone did not affect spleen cells significantly compared to control diet. There were no differences between control and either idebenone diet in other splenic cell subsets including B cells, plasma cells, naïve B cells, marginal zone B cells, plasmablast, plasmacytoid dendritic cells, monocytes, and neutrophils (supplementary Figure 5). These results suggest that idebenone preferentially reduces the number of effector memory CD4+ T cells in lupus mice and that the effect differs between doses of the compound.

Figure 4: Idebenone decreases spleen size, effector memory T cells, and modulates inflammatory gene expression.

(A) Size of spleen relative to total weight in MRL/lpr; Percentage of splenic (B) T cells (CD45+CD3+), (C) DN T cells (CD45+CD3+CD4-CD8-), (D) memory T cells (CD45+CD3+CD4+CD8-CD44+CD62L-), (E) effector memory T cells (CD45+CD3+CD44+CD62L-), and (F) effector memory CD4+ T cells (CD45+CD3+CD4+CD8-CD44+CD62L-). Results represent mean ± SEM. Mann-Whitney U-test used to compare controls with low and high dose idebenone diet (n=10/group). * P: <0.05, and ****: P <0.0001. Expression of representative type I IFN-regulated genes, proinflammatory cytokines, and inflammasome-related genes in MRL/lpr splenocytes from mice treated with low dose (G) or high dose (H) idebenone diet. Results represent mean ± SEM of 4 mice per group, and the bar graph results show fold changes in idebenone groups adjusted for control diet group (normalized to a value of 1). Mann-Whitney U-test was used, *: P <0.05; **: P <0.01; ***: P <0.001.

Gene expression analysis in splenocytes showed that selected type I IFNs and type I IFN-regulated genes and other proinflammatory genes (such as Tnf and lL6) were not significantly modified by low dose idebenone, while Il18, Il17a and Il1B gene expression were significantly decreased (Figure 4G) compared to control diet. High dose idebenone diet significantly inhibited Il18, Pycard, IL17a, Ifna, Ifnb, and Il6 expression, while it significantly induced Il12b, Tnf, and Mpo (Figure 4H) compared to control diet. Supporting these findings, in NZM2328 mice, low dose idebenone also reduced splenomegaly significantly (supplementary Figure 3A) and decreased splenocyte gene expression of Ifna1, Mx1, Il6, Il1B and Il10 (supplementary Figure 3F). These results indicate that idebenone may modify inflammatory gene expression in lupus-prone mice.

Idebenone modulates mitochondrial metabolism in lupus-prone mice.

We quantified idebenone modulation of mitochondrial metabolism in splenocytes and bone marrow-derived neutrophils using a Seahorse Analyzer. In both splenocytes (Figures 5A–C) and neutrophils (Figures 5E–G), low dose idebenone significantly enhanced mitochondrial basal respiration (Figures 5A and 5E), maximal respiration (Figures 5B and 5F), and ATP production (Figures 5C and 5G) compared to control diet. Trends were similar in the high dose idebenone group but did not reach statistical significance (Figure 5). Low dose idebenone increased the ratio of mitochondrial gene/nuclear gene transcription (16S / B2m) but the effect was the opposite with high dose idebenone (Figure 5D). Overall, these results suggest that mitochondrial immunometabolism is improved by idebenone in lupus prone mice. To further characterize the pathways modulated by idebenone in murine lupus, we measured the specific activity of mitochondrial complex II in splenic extracts and found it to be enhanced in splenocytes from MRL/lpr exposed to low dose idebenone, compared to control diet, and inhibited by both complex II specific inhibitors: malonate and TTFA (supplementary Figure 6).

Figure 5: Mitochondrial immunometabolism is modulated by idebenone.

Mitochondrial metabolism was analyzed in splenocytes (A through D) and bone marrow derived neutrophils (E through G). Shown in (A) and (E) basal respiration; (B) and (F) maximal respiration and (C) and (G) ATP production, (n=10 per group). (D) The ratio of transcription between the mitochondrial gene 16S over the nuclear gene B2m was analyzed by qRT-PCR in splenocytes (n=10 per group). Shown are mean ± SEM. Mann-Whitney U-test was applied to compare control versus idebenone diet groups. *: P <0.05 and **: P <0.01.

Idebenone modulates lupus murine and human NET formation.

The antioxidant and immunometabolism modulation capacity of idebenone could potentially modify the amount of mROS synthesized by myeloid cells. As mROS are implicated in pathogenic NET formation (2), we quantified this process ex vivo in bone marrow-derived neutrophils (BMDN) from idebenone-treated mice at euthanasia (Figure 6A). Spontaneous NET formation by neutrophils from MRL/lpr mice was significantly decreased in neutrophils from the high dose idebenone-treated mice but not by the low dose idebenone group (Figure 6A), while no differences were observed between groups with ionophore-induced NET formation. Similarly, mROS synthesis in MRL/lpr neutrophils was significantly decreased in the high dose idebenone group but not by the low dose idebenone (Figure 6B) compared to control diet. These results suggest that mROS production is enhanced in murine lupus neutrophils and that idebenone can modulate NET formation, at least in part, by reducing mROS generation.

Figure 6: Idebenone modulates spontaneous murine and human NET formation and murine mitochondrial ROS synthesis.

(A) Ex vivo spontaneous and ionophore (A23187)-induced BM MRL/lpr neutrophil NET formation at 2 h post-stimulation quantified by SYTOX plate assay (n=10 /group). (B) Mitochondrial ROS synthesis assessed by ratio of MitoSox/ Picogreen fluorescence in MRL/lpr BM neutrophils (n=10 /group). Results represent mean ± SEM. Mann-Whitney U-test was applied to compare control versus idebenone diet groups. *:P<0.05; **: P <0.01, and ***: P <0.001. Assessment of NET formation in human lupus LDGs and NDGs in the presence or absence of idebenone, as quantified by immunofluorescence. LDGs (C and D) and NDGs (E and F) without idebenone treatment (C and E) or with idebenone (10 uM) (D and F) after 2 h incubation. Image representative of 3 different patients analyzed with similar results. Green fluorescence corresponds to neutrophil elastase and blue fluorescence stains DNA; upper panels are single fluorochromes and the bottom left panel shows the merged fluorescence. Magnification is 40×. Bar shown in each image corresponds to 10 um.

The in vitro effect of idebenone on NET formation was also analyzed in human SLE neutrophils. We previously described that a pathogenic neutrophil subset in SLE, called low density granulocytes (LDGs), is primed to form NETs in a mROS-dependent manner, while NET formation in normal dense granulocytes (NDGs, less prone to NET) is mROS-independent (2). Idebenone significantly inhibited the spontaneous NET formation of LDGs, while it did not affect this process in NDGs (Figures 6C through 6F). These observations suggest that the beneficial effects of idebenone are operational when given to dysfunctional/inflamed cells and that human lupus neutrophils can be modulated by this drug.

Discussion

A key role for mitochondrial dysfunction has been suggested in the pathogenesis of lupus and other autoimmune diseases (2, 21–24). The present work further supports that the use of compounds that modulate mitochondrial function and are also antioxidants may attenuate lupus-associated organ damage and immune dysregulation associated to this disease. Importantly, idebenone was well-tolerated and modified survival in murine lupus. Some aspects of lupus immune dysregulation, organ damage and immunometabolism were similarly modified by both doses of idebenone while other aspects were differentially modulated by only one dose or showed opposite effects. This suggests that idebenone’s putative pleiotropic roles in immune function, inflammation, tissue repair/fibrosis and oxidative damage may be in several instances dose-dependent and this should be taken into consideration for potential future studies, both pre-clinical and clinical.

With regards to regulation of immune responses, idebenone led to the decrease of effector memory CD4+ T cells in MRL/lpr mice. The mechanisms by which idebenone specifically modifies the number of these T cells is unclear and could be direct (through effects on cell metabolism that impact the phenotype/differentiation of these cells) or indirect through reduction of cytokines that impact their development (25–27). Despite the reduction in activated T cells by idebenone, autoantibody levels were not affected by this compound. The reason for these differential effects on specific cell subsets remain unclear and could potentially be related to the timing of administration, to distinct effects on subsets of T cells involved in direct stimulation of B cell function, or to lack of direct impact on autoreactive B cells and/or long lived plasma cells.

Of note, endothelium-dependent vasorelaxation significantly improved with idebenone administration. As endothelial dysfunction, vasculopathy, and premature atherosclerosis are increased in human SLE, these findings suggest that idebenone could have potential beneficial effects in vascular health in this disease (28–30). This is also emphasized by the observation of decreased cardiac lipid peroxidation by idebenone, a phenomenon that may also promote enhanced vascular health (31). Recent work implicates inflammasome activation in the pathogenesis of murine and human SLE and in vasculopathy (32). Idebenone downregulated mRNAs of components of the inflammasome pathway and reduced the expression of mature IL18 in kidney tissue. As one of the putative mechanisms of action of idebenone is through bypassing a dysfunctional mitochondrial complex I, it may dampen enhanced mROS synthesis and subsequent inflammasome activation (33). Indeed, both splenocytes and neutrophils showed improved mitochondrial function in animals treated with low dose idebenone. Importantly, idebenone had an inhibitory effect on NET formation in proinflammatory neutrophil subsets from lupus patients but not in other neutrophil subsets not considered to play a major role in SLE pathogenesis. This could be reassuring when considering testing this or similar compounds in SLE subjects in the future, as the drug may preferentially target aberrant immune subsets. Idebenone has been reported to be safe in treatment of humans suffering from a variety of conditions associated with mitochondrial dysfunction, at doses ranging from 6 to 225 times higher than the doses used in this study. This provides good therapeutic rationale to potentially test this drug in SLE in the future (34).

Another potentially immunomodulatory effect of idebenone in SLE could be secondary to the reduction of IL17 expression in spleen and kidneys. Blockade of the IL-17 pathway has been reported to reduce renal disease and immune dysregulation in murine and human SLE (35–38). The observations that idebenone modulated renal involvement in two mouse models of SLE implicate that targeting mitochondrial dysfunction can have pleiotropic effects on organ damage or high relevance to this disease. Again, the effects on kidney pathology varied between the two doses of idebenone and the mouse model studied, indicating that future studies should focus on further characterizing the effects of this compound on tissue repair and fibrosis/evolution to end-stage renal disease. Low dose idebenone reduced significantly the expression of Il10 in NZM2328 and this finding may be relevant in the context of the putative role of IL-10 in the pathogenesis of SLE (39).

In conclusion, idebenone improves immune dysregulation, organ damage, vasculopathy and mitochondrial function in murine SLE through pleiotropic effects on the immune compartment, immunometabolism and tissue damage. Our study suggests that targeting mitochondrial dysfunction should be further explored as a putative strategy in lupus and potentially other autoimmune conditions.

Key messages.

Mitochondrial dysfunction might play a relevant role in SLE disease. Idebenone is already approved for treatment of some human diseases where mitochondria are dysfunctional. Idebenone was tested in murine lupus and was found to mitigate kidney damage, improve vascular dysfunction, inflammatory cascades and neutrophil dysregulation. Idebenone may have a potential therapeutic role in SLE.