doi: 10.1136/ard-2022-223626.
L-arginine metabolism inhibits arthritis and inflammatory bone loss
Affiliations
- PMID: 37775153
- PMCID: PMC10803985
- DOI: 10.1136/ard-2022-223626
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L-arginine metabolism inhibits arthritis and inflammatory bone loss
Ann Rheum Dis.
.
Abstract
Objectives: To investigate the effect of the L-arginine metabolism on arthritis and inflammation-mediated bone loss.
Methods: L-arginine was applied to three arthritis models (collagen-induced arthritis, serum-induced arthritis and human TNF transgenic mice). Inflammation was assessed clinically and histologically, while bone changes were quantified by μCT and histomorphometry. In vitro, effects of L-arginine on osteoclast differentiation were analysed by RNA-seq and mass spectrometry (MS). Seahorse, Single Cell ENergetIc metabolism by profilIng Translation inHibition and transmission electron microscopy were used for detecting metabolic changes in osteoclasts. Moreover, arginine-associated metabolites were measured in the serum of rheumatoid arthritis (RA) and pre-RA patients.
Results: L-arginine inhibited arthritis and bone loss in all three models and directly blocked TNFα-induced murine and human osteoclastogenesis. RNA-seq and MS analyses indicated that L-arginine switched glycolysis to oxidative phosphorylation in inflammatory osteoclasts leading to increased ATP production, purine metabolism and elevated inosine and hypoxanthine levels. Adenosine deaminase inhibitors blocking inosine and hypoxanthine production abolished the inhibition of L-arginine on osteoclastogenesis in vitro and in vivo. Altered arginine levels were also found in RA and pre-RA patients.
Conclusion: Our study demonstrated that L-arginine ameliorates arthritis and bone erosion through metabolic reprogramming and perturbation of purine metabolism in osteoclasts.
Keywords: Arthritis; Inflammation; Osteoporosis.
© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.
Conflict of interest statement
Competing interests: None declared.
Figures
L-arginine protects from bone loss in three murine arthritis models. (A) Arthritis was induced by K/BxN serum transfer into C57BL/6 J mice and 40 g/L L-arginine was supplemented in drinking water, either simultaneously with the K/BxN serum transfer (SIA/L-Arginine day 0) or 4 days after serum transfer (SIA/L-arginine day 4). Mice were sacrificed at day 10 after serum transfer; (B) Arthritis score and quantification of area under the curve (AUC) of arthritis of SIA, SIA/L-arginine day 0 and SIA/L-arginine day 4 groups (n=15–25/group); (C, D) Representative tibial μCT images (C) and quantification of bone volume/total volume (BV/TV), bone density trabecular numbers (Tb.) (N) trabecular thickness (Tb. Th) and trabecular separation (Tb. Sp) (D) in control, control/L-arginine, SIA, SIA/L-arginine day 0 and SIA/L-arginine day 4 groups (n=5–9/group). Scale bars: 1 mm; (E, F) Representative images of tartrate-resistant acid phosphatase (TRAP) staining (E) and quantification of osteoclast surface/bone surface (Oc.S/BS), osteoclast number/tissue area (N.Oc/T.Ar) and osteoclast number/bone perimeter (N.Oc./B.Pm.) (F) in control, control/L-arginine, SIA, SIA/L-arginine day 0 and SIA/L-arginine day 4 groups (n=5–9/group). Scale bars: 500 µm; (G) Collagen-induced arthritis (CIA) was triggered by immunisation with an emulsion of complete Freund’s adjuvant and type II collagen (CII) into DBA/1 J mice, supplemented with 40 g/L L-arginine, after the onset of arthritis (day 23). Mice were sacrificed at day 39 after first immunisation; (H) Arthritis scores and quantification of AUC of CIA and CIA/L-arginine groups (n=7–9/group); (I, J) Representative tibial μCT images (I) and quantification of BV/TV, Tb. N, Tb. Th and Tb. Sp (J) in controls, control/L-Arginine, CIA and CIA/L-arginine groups (n=4–9/group). Scale bars: 1 mm; (K, L) Representative images of tartrate-resistant acid phosphatase (TRAP) staining (K) and quantification of Oc.S/BS, N.Oc/T.Ar and N.Oc./B.Pm. (L) in controls, control/L-arginine, CIA and CIA/L-arginine groups (n=4–9/group). Scale bars: 500 µm; (M) Human TNF-transgenic (hTNFtg) mice developed spontaneously erosive arthritis aggravated with age. 40 g/L L-arginine was supplemented within drinking water 6 weeks after birth. Mice were sacrificed 12 weeks after birth; (N, O) Kinetic arthritis score (N) and measurements of grip strength (O) in hTNFtg−/−, hTNFtg+/−, hTNFtg−/−/L-Arginine and hTNFtg+/−/L-Arginine groups (n=5–10/group) during the course of spontaneous arthritis; (P, Q) Representative tibial μCT images (P) and quantification of tibial BV/TV, Tb. N, Tb. Th and Tb. Sp (Q) in hTNFtg−/−, hTNFtg+/−, hTNFtg−/−/L-arginine and hTNFtg+/−/L-arginine groups (n=5–10/group). Scale bars: 1 mm; (R, S) Representative images of tartrate-resistant acid phosphatase (TRAP) staining (R) and quantification of tibial Oc.S/BS, N.Oc/T.Ar and N.Oc./B.Pm. (S) in hTNFtg−/−, hTNFtg+/−, hTNFtg−/−/L-arginine and hTNFtg+/−/L-arginine groups (n=5–10/group). Scale bars: 500 µm. Graph points indicate individual mice. Data are shown as mean±SE.e.m. Asterisks mark statistically significant difference (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001), Student’s t-test in (H) (AUC result) and one-way ANOVA in (B) (AUC test), (D, F, J, L, Q, S), two-way repeated measures ANOVA in (B, H, N, O). See also in online supplemental figures S1 and S2. ANOVA, analysis of variance.
L-arginine reduces TNFα-induced osteoclastogenesis and resorption activity. (A) Schematic diagram of osteoclastogenesis from the bone marrow cells of C57BL/6 J mice; supplementation of L-arginine (10 mM) was done during the whole procedure (days 0–3) or only at the later phase (days 2–3), with or without stimulation with 40 ng/mL TNFα; (B) Cell viability on day 2 of WT osteoclasts exposed to different doses of L-arginine supplementation (0.5 mM, 1 mM, 3 mM, 5 mM, 10 mM) (n=3/group, representative of three independent experiments); (C, D) Quantification of TRAP+ osteoclasts (C) and representative TRAP staining images (D) of mature osteoclasts at day 3 from different treatments (n=5–10/group, representative of three independent experiments). Scale bars: 100 µm; (E) Representative F-actin staining images of mature osteoclasts at day 3, supplementation of L-arginine (10 mM) was done at the later phase, with or without stimulation with 40 ng/mL TNFα. Scale bars: 50 µm; (F, G) Representative images of pit formation assay (F) and quantification of bone resorption area (G) at day 3, supplementation of L-arginine (10 mM) was done at the later phase, with or without stimulation with 40 ng/mL TNFα (n=3/group, representative of three independent experiments). Scale bars: 100 µm. (H) Expression of osteoclast-associated genes at day 0, day 1, day 2 and day 3 of culture; supplementation of L-arginine (10 mM) was done at the later phase, with or without stimulation with 40 ng/mL TNFα (n=3/group, representative of three independent experiments) (I, J) KEGG pathway enrichment results from RNA sequencing data in preosteoclasts supplemented or not with 10 mM L-arginine at the later phase, with or without the stimulation with 40 ng/mL TNFα (n=3/group), comparing L-arginine versus control (I) and TNFα/L-arginine versus TNFα (J); (K) Gene set enrichment analysis (GSEA) results indicating significant difference in oxidative phosphorylation (OXPHOS), comparing TNFα/L-arginine versus TNFα; (L) Heatmap showing the differentially expressed genes linked to OXPHOS pathway in control, L-arginine, TNFα and TNFα/L-arginine groups. Genes with an adjusted p value (p-adj) less than 0.05 are assigned as differentially expressed. Pathways with p-adj value less than 0.05 are considered significantly enriched. Data are shown as mean±SE. Asterisks mark statistically significant difference (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001), one-way ANOVA in (B) and two-way ANOVA in (C, F). See also in online supplemental figures S3–S6. ANOVA, analysis of variance.
L-arginine boosts oxidative phosphorylation (OXPHOS) and ATP production of osteoclast under inflammatory conditions. (A) Extracellular acidification rate (ECAR) analysed by extracellular flux assay in preosteoclasts from WT cells; supplementation with L-arginine (10 mM) was done at the later phase, with or without stimulation with 40 ng/mL TNFα (n=9–14/group, representative of three independent experiments); (B, C) Oxygen consumption rate (OCR) (B) and associated mitochondrial ATP production (C) analysed by extracellular flux assay in preosteoclasts from WT cells; supplementation of L-arginine (10 mM) was done at the later phase, with or without stimulation with 40 ng/mL TNFα (n=9–14/group, representative of three independent experiments); (D, E) ATP rate (D) and percentage of ATP produced by glycolysis or OXPHOS (E) analysed by real-time ATP rate assay in preosteoclasts from WT cells; supplementation of L-arginine (10 mM) was done at the later phase, with or without stimulation with 40 ng/mL TNFα (n=6–12/group, representative of three independent experiments); (F, G) Representative histogram of flow cytometry analysis with the treatment of DMSO, 2-DG, Oligomycin and 2-DG/Oligomycin (F) and percentage of mitochondria dependence (G) via Single Cell ENergetIc metabolism by profilIng Translation inHibition (SCENITH) in preosteoclasts (CD45+CD11b+Ly6G- Mcsfr+RANK+ cells) from WT cells; supplementation of L-arginine (10 mM) was done at the later phase, with or without 40 ng/mL TNFα stimulation (n=3–6/group, representative of three independent experiments); (H) Glucose consumption in the cell supernatant of mature osteoclasts at day 3; supplementation of L-arginine (10 mM) was done at the later phase, with or without 40 ng/mL TNFα stimulation (n=2–3/group, pooled from three independent experiments); (I, J) Representative images of JC-1 staining (I) and ratio of JC-1 Aggregate (red) / JC-1 monomeric (green) fluorescence (J) in mature osteoclasts at day 3; supplementation of L-arginine (10 mM) was done at the later phase, with or without 40 ng/mL TNFα stimulation (n=2–3/group, pooled from two independent experiments). Scale bars: 50 µm; (K–N) Quantification of TRAP+ osteoclasts (K, M) and representative TRAP staining images (L, N) of mature osteoclasts at day 3 in control versus L-arginine treated groups (K, L) and TNFα versus TNFα/L-arginine groups (M, N), combined with different concentrations (5 nM, 10 nM, 20 nM) of OXPHOS inhibitors Rotenone and Antimycin. Scale bars: 100 µm. Data are shown as mean±SEM. Asterisks mark statistically significant difference (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001), one-way ANOVA in (C, E, K, M) and two-way ANOVA in (A, B, G, H, I). For A and B, black asterisk means group TNFα versus control and purple asterisk means group TNFα/L-arginine versus TNFα. See also in online supplemental figures S7 and S8. ANOVA, analysis of variance.
Induction of Arginase-1 is mediating osteoclast inhibition by L-arginine. (A) Transcription factors enriched from three databases with the differentially expressed genes in RNA-seq data comparing TNFα/L-arginine versus TNFα treated groups; (B) Protein levels of c-Jun in mature osteoclasts at day 3 (normalised to b-actin levels), with supplementation of L-arginine (10 mM) during the whole culture or only at the later phase, with or without 40 ng/mL TNFα stimulation; (C, D) Schematic diagram of the osteoclastogenesis with bone marrow cells from cJun
∆LysM mice or littermates (C, above); representative TRAP staining images (C, bottom) and quantification of TRAP+ osteoclasts (D) from c-Jun-deficient cells or littermate controls at day 3, with or without 40 ng/mL TNFα stimulation (n=3–5/group, representative of three independent experiments). Scale bars: 100 µm; (E) Arginase (Arg)-1 expression in mature osteoclasts from c-Jun-deficient cells or littermate controls at day 3, with or without 40 ng/mL TNFα stimulation (n=3–6/group, representative of three independent experiments); (F) Normalised arginase activity in the paw lysate of mice from controls, control/L-arginine, CIA and CIA/L-arginine groups (n=4–9/group); (G) Putative binding site of c-Jun on the promoter sites of Arg-1 predicted by JASPAR; Binding of c-Jun or IgG on the promoter sites of Arg-1 in preosteoclasts at day 2, stimulated with TNFα for 4 hours (n=4/group); (H) Binding of H3me3K4, H3me3K27 or IgG on the promoter sites of Arg-1 in preosteoclasts at day 2, stimulated with TNFα for 4 hours (n=6/group); (I–J) Schematic diagram of the osteoclastogenesis with bone marrow cells from Arg-1
∆Tie2 mice or littermates, representative TRAP staining images (I) and quantification of TRAP+ osteoclasts (J) from Arg-1-deficient cells or littermate controls at day 3; supplementation of L-arginine (10 mM) was done at the later phase, with or without 40 ng/mL TNFα stimulation (n=3/group, representative of three independent experiments). Scale bars: 100 µm; (K, L) Schematic diagram of the osteoclastogenesis procedure with bone marrow cells from Arg-1
∆Ctsk mice or littermates, representative TRAP staining images (K) and quantification of TRAP+ osteoclasts (L) from Arg-1-deficient cells or littermate controls at day 3, supplementation of L-arginine (10 mM) was done at the later phase, with or without 40 ng/mL TNFα stimulation (n=4/group, representative of three independent experiments). Scale bars: 100 µm. Data are shown as mean±SE. Asterisks mark statistically significant difference (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001), one-way ANOVA in (F, G, H) and two-way ANOVA in (D, E, J, L). See also in online supplemental figure S9. ANOVA, analysis of variance; CIA, collagen-induced arthritis.
L-arginine perturbates purine metabolism in TNFα stimulated cells. Mature osteoclasts were harvested at day 3; supplementation of L-arginine (10 mM) or 13C6-L-arginine (10 mM) was done at the later phase, with or without 40 ng/mL TNFα stimulation. Untargeted metabolic tracing of 13C-isotopologue or untargeted mass spectrometry (MS) of L-arginine was performed within the following four groups: control, L-arginine, TNFα and TNFα/L-arginine (three replicates per group). (A) Schematic diagram delineating metabolic flux of 13C-isotopologue labelled L-arginine and the isotopologue distribution chart of the detected metabolites (Created with BioRender.com); (B) Metabolic pathway enrichment results showing affected pathways, comparing the groups L-arginine versus control and TNFα/L-arginine versus TNFα; (C) Schematic diagram combining the differentially expressed metabolites from MS data and expression of enzymes from RNA-seq results associated with purine metabolism pathway, comparing TNFα/L-arginine versus TNFα groups; (D) Expression of metabolites from MS results affected in purine metabolism among the four groups; (E) GO pathway enrichment results indicating significant difference in purine metabolism, comparing TNFα/L-arginine versus TNFα groups; (F) Expression of enzymes from RNA-seq results connected to purine metabolism among the four groups. Genes with an adjusted p value (p-adj) less than 0.05 were assigned as differentially expressed. Data are shown as mean±SE. Asterisks mark statistically significant difference (*p<0.05; **p<0.01), two-way ANOVA in (D). See also in online supplemental figure S10. ANOVA, analysis of variance.
L-arginine attenuates inflammatory bone loss through controlling adenosine deaminase. (A) Schematic diagram of osteoclastogenesis with WT bone marrow cells, supplemented with 20 mM inosine at the later phase, with or without 40 ng/mL TNFα stimulation (above); Representative TRAP staining images (bottom left) and quantification of TRAP+ osteoclasts (bottom right) at day 3 (n=6/group, representative of three independent experiments). Scale bars: 100 µm; (B) Schematic diagram of osteoclastogenesis with WT bone marrow cells, supplemented with 1 mM hypoxanthine at the later phase, with or without 40 ng/mL TNFα stimulation (above); Representative TRAP staining images (bottom left) and quantification of TRAP+ osteoclasts (bottom right) at day 3 (n=5/group, representative of three independent experiments). Scale bars: 100 µm; (C–E) Schematic diagram of osteoclastogenesis with WT bone marrow cells (C), quantification of TRAP+ osteoclasts (D) and representative TRAP staining images (E) at day 3, supplemented with ADA inhibitors (20/40 µM Pentostatin or 2.5/5 µM erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA)) at the later phase, with or without the treatment of 10 mM L-arginine at the later phase, stimulated by 40 ng/mL TNFα (n=3–6/group, representative of three independent experiments). Scale bars: 100 µm; (F) Arthritis induced by immunisation with an emulsion of complete Freund’s adjuvant and type II collagen (CII) into DBA/1 J mice, supplemented with 40 g/L L-arginine in drinking water after onset of arthritis (day 23). ADA inhibitor pentostatin (2 mg/g) was administrated intraperitoneally into the mice at an interval of 24 hours for successive 3 days every 2 weeks starting from day 23. Mice were sacrificed at day 43 after first immunisation; (G) Arthritis score in controls, CIA, CIA/L-arginine, pentostatin, CIA/pentostatin and CIA/L-arginine/pentostatin groups (n=4–5/group); (H, I) Representative images of TRAP staining (H) and quantification of tibial BV/TV, Tb. N, Tb. Th, Tb. Sp, Oc.S/BS and N.Oc./B.Pm. (I) in controls, CIA, CIA/L-arginine, pentostatin, CIA/pentostatin and CIA/L-arginine/pentostatin groups (n=4–5/group). Scale bars: 500 µm; Data are shown as mean±SEM. Asterisks mark statistically significant difference (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001), one-way ANOVA in (D), two-way repeated measures ANOVA in (G), two-way ANOVA in (A, B, I). ADA, adenosine deaminase; ANOVA, analysis of variance; CIA, collagen-induced arthritis.
Altered arginine metabolism in rheumatoid arthritis (RA) patients and L-arginine inhibits human osteoclastogenesis. (A) Levels of arginine, ornithine, proline and citrulline in the serum collected from RA patients and healthy individuals (n=23 for RA; n=29 for healthy controls); (B) Correlation between each amino acid and DAS28 scores in mild to moderate RA patients (2.6
In inflammatory condition, TNFα promoted c-Jun expression, enhanced glycolysis and blocked oxidative phosphorylation, leading to accelerated osteoclastogenesis. In L-arginine supplementation, L-arginine inhibited c-Jun expression, restored oxidative phosphorylation and promoted ATP production. L-arginine also elevated hypoxanthine and inosine levels by reprogramming purine metabolism, which further mediate the inhibitory role of L-arginine in inflammatory osteoclastogenesis. ADA, adenosine deaminase.
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