Mitochondrial complex I activity suppresses inflammation and enhances bone resorption by shifting macrophage-osteoclast polarization

Abstract

Mitochondrial complex I (CI) deficiency is associated with multiple neurological and metabolic disorders. However, its effect on innate immunity and bone remodeling is unclear. Using deletion of the essential CI subunit Ndufs4 as a model for mitochondrial dysfunction, we report that mitochondria suppress macrophage activation and inflammation while promoting osteoclast differentiation and bone resorption via both cell-autonomous and systemic regulation. Global Ndufs4 deletion causes systemic inflammation and osteopetrosis. Hematopoietic Ndufs4 deletion causes an intrinsic lineage shift from osteoclast to macrophage. Liver Ndufs4 deletion causes a metabolic shift from fatty acid oxidation to glycolysis, accumulating fatty acids and lactate (FA/LAC) in the circulation. FA/LAC further activates Ndufs4(-/-) macrophages via reactive oxygen species induction and diminishes osteoclast lineage commitment in Ndufs4(-/-) progenitors; both inflammation and osteopetrosis in Ndufs4(-/-) mice are attenuated by TLR4/2 deletion. Together, these findings reveal mitochondrial CI as a critical rheostat of innate immunity and skeletal homeostasis.

Copyright © 2014 Elsevier Inc. All rights reserved.

Figures

Figure 1. Global Ndufs4 deletion causes systemic inflammation manifested as alopecia

(A-B) Ndufs4−/− pups exhibit alopecia. (A) A representative image on postnatal day 22 (P22). (B) Alopecia could not be rescued by WT foster dams. (C) Skin of Ndufs4−/− pups were infiltrated with leukocytes such as macrophages (detected by anti-CD11b, anti-F4/80 and anti-Gr-1) but not dendritic cells (detected by anti-CD11c) on P22. Scale bars, 25 μm. (D-E) Expression of inflammatory genes in the skin (D) and liver (E) on P22 (n=6). (F) Cytokine levels in the serum on P22 (n=6). (G-H) Percentage of CD11b+Ly6Chi pro-inflammatory macrophages in bone marrow (BM) and spleen on P22. (G) FACS 2D dot plots. (H) Quantification (n=5). (I) Expression of inflammatory genes in the primary bone marrow cells or splenocytes on P22 (n=5). (J-K) Percentage of CD11b+Ly6Chi pro-inflammatory macrophages in BM-Mf cultures on day 6. (J) FACS 2D dot plots. (K) Quantification (n=5). (L) Expression of inflammatory genes in BM-Mf cultures on day 6 (n=5). Error bars, SD.

Figure 2. Global Ndufs4 deletion decreases bone resorption and increases bone mass

(A-G) μCT analyses of tibiae on P22 (male, n=8). (A) Representative images of the trabecular bone of the tibial metaphysis (top) (scale bar, 10 μm) and the entire proximal tibia (bottom) (scale bar, 1 mm). (B-F) Quantification of trabecular bone volume and architecture. (B) BV/TV, bone volume/tissue volume ratio. (C) BS, bone surface. (D) Tb.N, trabecular number. (E) Tb.Sp, trabecular separation. (F) Conn.D., Connectivity Density. (G) Cortical BV/TV. (H-I) Bone histomorphometry (P22, male, n=8). (H) Osteoclast number (Oc.N/B.Ar) and osteoclast surface (Oc.S/B.S) (n=8). (I) Osteoblast number (Ob.N/B.Ar) and osteoblast surface (Ob.S/B.S) (n=8). B.Ar, bone area. (J) Serum CTX-1 (P22, male, n=8) (K) Serum P1NP (P22, male, n=8) (L-Q) Bone marrow osteoclast differentiation assay. (L) Representative images of differentiation cultures on day 12. Mature osteoclasts were multinucleated (>3 nuclei) TRAP+ (purple) cells. Scale bar, 25 μm. (M) Osteoclast activity (n=8). (N) Osteoclast precursor proliferation (n=8). (O) Osteoclast apoptosis (n=8). (P-Q) Expression of osteoclast differentiation markers (P) and osteoclast function genes (Q) (n=4). P-Q, * compares each treatment with vehicle (Veh) control, + compares Ndufs4−/− with WT control in the same treatment group. Error bars, SD.

Figure 3. Ndufs4 deletion shifts metabolism to potentiate macrophage activation

(A) Representative image showing that Tie2-Ndufs4 KO mice had no alopecia. (B) Expression of inflammatory genes in Tie2-Ndufs4 KO BM-Mfs was increased on day 6 (n=3). (C-D) Percentage of CD11b+Ly6Chi pro-inflammatory macrophages in the bone marrow (BM) and spleen was normal in Tie2-Ndufs4 KO pups on P22. (C) FACS 2D dot plots. (D) Quantification (n=5). (E-G) Serum triglycerides (E), non-esterified fatty acid (NEFA) (F) and lactate (G) were increased in Ndufs4−/− pups (n=8). (H) Ndufs4−/− macrophages secrets more lactic acid. Left, images showing the medium of Ndufs4−/− BM-Mf cultures was more acidic (yellow). Right, quantification of lactate in culture medium (n=6). (I) Serum triglycerides (left) and lactate (right) (n=8). (J) Expression of Hif1α and Pfkfb was higher in the liver of Ndufs4−/− mice (n=5). (K) Serum triglycerides (left) and lactate (right) in Alb-Ndufs4 KO mice and controls (n=8). (L-M) Fatty acids (L) and lactate (M) potentiate the activation of inflammatory genes in Ndufs4−/− macrophages (n=5). BM-Mfs were treated with palmitic acid (PA) or linoleic acid (LA) (400 μM) or lactate (15 mM) for 15 hr. (N) Percentage of CD11b+Ly6Chi pro-inflammatory macrophages in the bone marrow (BM) was increased in Alb-Ndufs4 KO pups on P22 (n=5). (O) Expression of inflammatory genes in the spleen was increased in Tie2-Ndufs4 KO and Alb-Ndufs4 KO, and further potentiated in Alb+Tie2-Ndufs4 DKO mice on P22 (n=5). (P) Alb-Ndufs4 and Alb+Tie2-Ndufs4 DKO mice had no alopecia. Error bars, SD.

Figure 4. ROS level is elevated in Ndufs4−/− macrophages and exacerbated by fatty acids

(A-B) Representative images of Mito-SOX staining of skin on P22. Scale bars, 25 μm. (B) FACS quantification of Mito-SOX levels in bone marrow and spleen on P22 (n=5). (C) Mito-SOX levels in BM-Mfs treated with PA or LA (400 μM). Left, FACS 2D dot plots. Right, Quantification (n=5). (D) Mitochondrial membrane potential was decreased in Ndufs4−/− macrophages and further reduced by PA, but not LA (n=5), quantified by percentage of MitoTracker Deep Red via FACS. C-D, * and n.s. compare treatment with vehicle control in the same genotype; + compares Ndufs4−/− with WT in the same treatment group. (E-F) Expression of the stress sensor genes Gadd45α/β (E) and apoptosis (F) were higher in Ndufs4−/− macrophages and further potentiated by PA (n=3). * compares Ndufs4−/− with WT under the same treatment; + compares PA with vehicle in the same genotype. (G) PA induction of mitochondrial ROS in macrophages, measured by Mito-SOX, was abolished by ROS inhibitors 2R,4R-APDC (50 μM) or Mito-TEMPO (50 μM). (H-I) ROS inhibitors 2R,4R-APDC (H) or Mito-TEMPO (I) partially rescued the PA-activated inflammatory gene expression in WT and Ndufs4−/− macrophages. (J) Mito-TEMPO partially rescued the PA-activated stress sensor gene expression in WT and Ndufs4−/− macrophages. BM-Mfs were treated with PA (400 μM), with or without APDC or Mito-TEMPO (50 μM). Error bars, SD.

Figure 5. Inflammation and alopecia in Ndufs4−/− pups can be rescued by TLR2/4 deletion

(A-F) Comparison of Ndufs4/TLR4/TLR2 triple KO, Ndufs4−/− and WT controls on P22 (n=4). (A) Representative images showing alopecia phenotype. (B) CD11b immuno-staining of skin. Scale bars, 25 μm. (C) Expression of inflammatory genes in the skin. (D) Expression of inflammatory genes in BM-Mfs treated with PA or vehicle (Veh). (E-F) Mito-SOX levels in BM-Mfs treated with PA (400 μM). (E) FACS 2D dot plots. (F) Quantification (n=5). (G) TLR2/4 deletion did not alter the metabolic defects in Ndufs4−/− mice (n=5). (H) Alopecia in Ndufs4−/− pups was rescued by TLR4 deletion but not TLR2 deletion (P22). (I) Alopecia in Ndufs4−/− pups was rescued by TLR4 inhibitor TAK-242 (P22). Ndufs4−/− pups were gavaged with TAK-242 (10mg/kg/day) or vehicle control starting P1. Error bars, SD.

Figure 6. Both hematopoietic and liver Ndufs4 deletion inhibits bone resorption

(A-D) Ex vivo bone marrow osteoclast differentiation assay of Tie2-Ndufs4 KO mice. (A) Expression of osteoclast differentiation markers (n=4). (B) Expression of osteoclast function genes (n=4). (C) Images of differentiation cultures on day 12. Scale bar, 25 μm. (D) Osteoclast activity (n=8). (E) Serum CTX-1 (3 month, male, n=6). (F-J) Comparison of Alb-Ndufs4 KO, Tie2-Ndufs4 KO, Alb+Tie2-Ndufs4 DKO pups or controls (P22, male, n=6). (F-G) μCT. (F) Images of the trabecular bone of the tibial metaphysis (scale bar, 10 μm). (G) Trabecular bone volume and architecture. (H) Osteoclast surface. (I) Serum CTX-1. (J) Serum P1NP. Error bars, SD.

Figure 7. Fatty acids and lactate exacerbate osteoclastogenic defects in Ndufs4−/− cells

(A-C) Bone marrow osteoclast differentiation cultures from WT or Ndufs4−/− mice were treated with PA or LA (400 μM). (A-B) Expression of osteoclast differentiation markers (n=3). + compares with WT cells treated with vehicle (Veh). (C) Images of differentiation cultures on day 12. Scale bar, 25 μm. (D-E) Percentage of FMS+RANK+ osteoclast precursors in the bone marrow (D) (n=6) and osteoclast differentiation cultures 48 hr after RANKL treatment, with or without PA, LA (400 μM) or lactate (15 mM) (E) (n=6). (F-H) TLR2/4 deletion partially restored the osteoclastogenic defect in Ndufs4−/− mice (P22, male, n=4). (F) Serum CTX-1. (G) Serum P1NP. (H) TRAP expression in osteoclast differentiation cultures treated with PA, LA (400 μM) or lactate (15 mM). (I) A simplified model for how Ndufs4 and mitochondrial CI shift macrophage-osteoclast polarization to inhibit inflammation and stimulate bone resorption. FAO, fatty acid oxidation; Inflam, inflammation; Diff, differentiation; Mito Bio, mitochondria biogenesis. Error bars, SD.