Hypermetabolic macrophages in rheumatoid arthritis and coronary artery disease due to glycogen synthase kinase 3b inactivation

Abstract

Objectives: Accelerated atherosclerotic disease typically complicates rheumatoid arthritis (RA), leading to premature cardiovascular death. Inflammatory macrophages are key effector cells in both rheumatoid synovitis and the plaques of coronary artery disease (CAD). Whether both diseases share macrophage-dependent pathogenic mechanisms is unknown.

Methods: Patients with RA or CAD (at least one myocardial infarction) and healthy age-matched controls were recruited into the study. Peripheral blood CD14+ monocytes were differentiated into macrophages. Metabolic profiles were assessed by Seahorse Analyzer, intracellular ATP concentrations were quantified and mitochondrial protein localisation was determined by confocal image analysis.

Results: In macrophages from patients with RA or CAD, mitochondria consumed more oxygen, generated more ATP and built tight interorganelle connections with the endoplasmic reticulum, forming mitochondria-associated membranes (MAM). Calcium transfer through MAM sites sustained mitochondrial hyperactivity and was dependent on inactivation of glycogen synthase kinase 3b (GSK3b), a serine/threonine kinase functioning as a metabolic switch. In patient-derived macrophages, inactivated pGSK3b-Ser9 co-precipitated with the mitochondrial fraction. Immunostaining of atherosclerotic plaques and synovial lesions confirmed that most macrophages had inactivated GSK3b. MAM formation and GSK3b inactivation sustained production of the collagenase cathepsin K, a macrophage effector function closely correlated with clinical disease activity in RA and CAD.

Conclusions: Re-organisation of the macrophage metabolism in patients with RA and CAD drives unopposed oxygen consumption and ultimately, excessive production of tissue-destructive enzymes. The underlying molecular defect relates to the deactivation of GSK3b, which controls mitochondrial fuel influx and as such represents a potential therapeutic target for anti-inflammatory therapy.

Keywords: atherosclerosis; cardiovascular disease; inflammation; rheumatoid arthritis.

Figure 1.. Increased mitochondrial activity and ATP production in macrophages from patients with RA and CAD

(A) Summarized curves of OCR tracings from Seahorse experiments for all study cohorts (HC; n=7. Patients with RA or CAD; n=10 each). Baseline Respiration (B), respiration coupled to ATP production (C), respiratory spare capacity (D), and maximal respiration (E) were calculated based on OCR. (F) Seahorse analyzer-derived ECAR values and (G) ECAR to OCR ratios. (H, I) Representative dot plots and MFIs from TMRM staining indicative for mitochondrial membrane potential from 6 samples in each group. (J, K) MitoSOX Red staining indicative for mitochondrial ROS. Representative contour plots from RA and CAD macrophages compared to a control sample (green) and summary results from 6 samples in each group. (L) Intracellular ATP concentrations per 20,000 activated macrophages, 6 samples each group. Unpaired t-test was applied. *, P<0.05; **, P<0.01; ***, P<0.001. All bar graphs show mean ± SEM.

Figure 2.. Enhanced mitochondria-associated membrane (MAM) formation and intensified ER-mitochondria calcium transfer in RA and CAD macrophages

(A) Confocal microscopy of activated macrophages stained with anti-GRP75 (green) and anti-IP3R (red). White boxes represent enlarged areas in the right panel. (B) Quantification of GRP75/IP3R co-localization. Summarized data from six healthy, five RA, and five CAD samples. (C) Representative histogram of mitochondrial calcium uptake after calcium was released from the ER with 50 μM ATP; (D) bar graphs summarizes results from 10 experiments (peak value F1 divided by baseline level F0). (E-G) Mitochondrial membrane potential (n=7 each group), mitochondrial ROS (n=7 each group), and intracellular ATP (n=6 each group) measured after inhibiting mitochondrial calcium influx with Ru360 (10μM). Unpaired t-test (B-D) and paired t-test (E-H). Scale bar 10 μm. *, P<0.05; **, P<0.01; ***, P<0.001. All bar graphs show mean ± SEM.

Figure 3.. GSK3b is deactivated in patient-derived macrophages

(A) Representative histograms of phosphoflow for pGSK3b-S9 from activated macrophages. Bar graph (B) summarizes results from 6 experiments with unstimulated and from 8 experiments with activated macrophages. (C) Confocal microscopy of GSK3b-Ser9 (red) in activated macrophages. Nuclei were localized by DAPI (blue). (D) Quantification of phosphoflow for pGSK3b-Ser9 in fresh monocytes from 5 experiments. (E) Isolation of mitochondrial fractions and immunoblotting of proteins with antibodies against pGSK3b-S9 and mitochondrial transcription factor A (mTFA) as control protein. Assay was performed 3 times with four healthy, four RA, and four CAD samples. (F) Representative curves and (G) quantification of mitochondrial calcium uptake from 6 experiments after treatment of macrophages from healthy individuals with inhibitor SB216763 (10 μM). (H) Intracellular accumulation of β-catenin in activated macrophages (n=6 in each group). (I) Phosphoflow of Akt-S473. Bar graph summarizes MFIs from 6 samples in each group. Unpaired t-test was applied. *, P<0.05; **, P<0.01; ***, P<0.001. All bar graphs as well as box and whisker diagrams show mean ± SEM. Scale bar 20 μm.

Figure 4.. GSK3b is deactivated in macrophages infiltrating into RA synovial tissue and into the atherosclerotic plaque

Tissue sections were immunostained with anti-CD68 (red), anti-GSK3b-Ser9 (green), and DAPI (blue) and analyzed by fluorescence microscopy. (A) RA synovitis from a 36-year-old female patient with nodular disease taken from the left wrist. (B) Atherosclerotic fibro-calcified atheroma from a 76-year-old male patient symptomatic carotid stenosis. Control stainings show tissue sections of (C) liver, (D) colon, and (E) lung. In liver tissue, cholangiocytes stained positive for GSK3-Ser9. Scale bar 50 μm.

Figure 5.. GSK3b regulates mitochondrial activity

OCRs (A-E) and ECARs (F) of macrophages from healthy donors (n=7) were measured as in figure 1 after they were pre-treated with vehicle or the inhibitor SB216763 (10 μM, 24 hrs). (G) Representative dot plot and (H) summarizing bar graph of the quantification of mitochondrial membrane potential (n=6). (I) Representative histogram of mitochondrial ROS and (J) summarized results from six experiments. (H) Intracellular ATP concentrations in 20,000 activated macrophages (n=8). All values are mean ± SEM. Paired t-test was applied. *, P<0.05; **, P<0.01; ***, P<0.001.

Figure 6.. Mitochondrial activity and MAM function drive macrophage cathepsin K production

(A) Gene expression measured by RT-PCR in LPS/IFN-γ-stimulated macrophages (n=6 in each group). Results are fold change compared to controls. (B) Cathepsin K enzyme activity measured in activated macrophages (n=8 each group). Healthy macrophages were treated with SB216763 (10 μM) as indicated, other cells were treated with vehicle. (C) Correlation of vitro cathepsin K activity with disease activity (CADI score) of 14 RA patients. (D) Association of the extent of cardiovascular disease (number of affected coronary arteries) of 15 CAD patients with cathepsin K activity. (E-G) Gene expression of activated macrophages from six healthy donors, SB216763 (10 μM) or vehicle was added as indicated. (H-I) Cathepsin K activity of RA and CAD macrophages treated with Ru360 (10 μM); results from seven samples each group. Unpaired t-test (A-B) and paired t-test (B, E-I) were applied. In figures (C-D) Pearson correlation coefficient was applied. *, P<0.05; **, P<0.01; ***, P<0.001. All bar graphs show mean ± SEM.