Interferon-γ Released by Activated CD8+ T Lymphocytes Impairs the Calcium Resorption Potential of Osteoclasts in Calcified Human Aortic Valves

Abstract

In calcific aortic valve disease (CAVD), activated T lymphocytes localize with osteoclast regions; however, the functional consequences of this association remain unknown. We hypothesized that CD8⁺ T cells modulate calcification in CAVD. CAVD valves (n = 52) dissected into noncalcified and calcified portions were subjected to mRNA extraction, real-time quantitative PCR, enzyme-linked immunosorbent assay, and immunohistochemical analyses. Compared with noncalcified portions, calcified regions exhibited elevated transcripts for CD8, interferon (IFN)-γ, CXCL9, Perforin 1, Granzyme B, and heat shock protein 60. Osteoclast-associated receptor activator of NK-κB ligand (RANKL), tartrate-resistant acid phosphatase (TRAP), and osteoclast-associated receptor increased significantly. The stimulation of tissue with phorbol-12-myristate-13-acetate and ionomycin, recapitulating CAVD microenvironment, resulted in IFN-γ release. Real-time quantitative PCR detected mRNAs for CD8⁺ T-cell activation (Perforin 1, Granzyme B). In stimulated versus unstimulated organoid cultures, elevated IFN-γ reduced the mRNAs encoding for RANKL, TRAP, and Cathepsin K. Molecular imaging showed increased calcium signal intensity in stimulated versus unstimulated parts. CD14⁺ monocytes treated either with recombinant human IFN-γ or with conditioned media-derived IFN-γ exhibited low levels of Cathepsin K, TRAP, RANK, and tumor necrosis factor receptor-associated factor 6 mRNAs, whereas concentrations of the T-cell co-activators CD80 and CD86 increased in parallel with reduced osteoclast resorptive function, effects abrogated by neutralizing anti-IFN-γ antibodies. CD8⁺ cell-derived IFN-γ suppresses osteoclast function and may thus favor calcification in CAVD.

Figure 1

Calcified aortic valve cusps contain CD8⁺ T cells and interferon (IFN)-γ. A and B: Relative gene expression (normalized to noncalcified areas of stenotic valves) of CD8A (A) and IFN-γ (B). C: IFN-γ enzyme-linked immunosorbent assay (ELISA). D: Double immunofluorescence staining indicating colocalization of CD8⁺ T cells (green) and IFN-γ (red). The merged image shows colocalization and counterstaining with DAPI for visualization of the nuclei. E: Representative section of a calcified leaflet (transverse cryosection with a thickness on 6 μm), stained with hematoxylin and eosin to correlate histopathologic changes. The boxed area indicates an area adjacent to a calcified lesion, which is shown in D. n = 116 cusp portions from 52 valve donors (A–C); n = 5 (D and E). ^∗P < 0.05, ^∗∗P < 0.01. Scale bars: 50 μm (D); 5.632 mm (E). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 2

T-cell–derived and –activating cytokines in calcified aortic valve cusps. Relative gene expression (normalized to noncalcified areas of stenotic valves) of Perforin 1 (A), Granzyme B (C), heat shock protein (Hsp)60 (E), CXCL9 (G), and major histocompatibility complex class II transactivator (CIITA; I). Immunohistochemical localization of Perforin 1 (B), Granzyme B (D), Hsp60 (F), CXCL9 (H), and human leukocyte antigen (HLA)-DR (J). All sections were counterstained with hematoxylin for visualization of the nuclei. n = 116 cusp portions (A, C, E, G, and I); n = 5 (B, D, F, H, and J). ^∗P < 0.05, ^∗∗P < 0.01. Scale bars = 100 μm. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. DR, antigen-D related.

Figure 3

Osteoclast-related markers in calcified aortic valve cusps. A, D, F, and H: Relative gene expression (normalized to noncalcified areas of stenotic valves) of receptor activator of nuclear factor-κB ligand (RANKL; A), tartrate-resistant acid phosphatase (TRAP; D), osteoclast-associated receptor (OSCAR; F), and Cathepsin K (H). B: RANKL enzyme-linked immunosorbent assay (ELISA). C, E, and G: Immunohistochemistry for RANKL (C), TRAP (E), and OSCAR (G). All sections were counterstained with hematoxylin for visualization of the nuclei. n = 116 cusp portions (A, D, F, and H); n = 75 (B); n = 5 (C, E, and G). ^∗P < 0.05, ^∗∗P < 0.01. Scale bars = 100 μm. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 4

Interferon (IFN)-γ and osteoclast-related markers after T-cell activation in calcific aortic valve disease (CAVD) specimens. Relative gene expression (in stimulated calcified areas of stenotic valves, normalized to unstimulated calcified areas) of IFN-γ (A), Granzyme B (B), CD8A (C), receptor activator of nuclear factor-κB ligand (RANKL; D), tartrate-resistant acid phosphatase (TRAP; G), and Cathepsin K (H). Enzyme-linked immunosorbent assay (ELISA) for IFN-γ (E) and RANKL (F) in the conditioned medium before adding the cell activation cocktail (CAC), labeled as Before, and after 24 and 48 hours, indicated by After/1 and After/2, respectively. n = 72 cusp portions (A–D and G–H); n = 63 (E); n = 20 (F). ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 5

Molecular imaging monitoring the temporal and spatial changes in valvular calcium after T-cell activation in calcific aortic valve disease (CAVD) specimens. A–E: Data from unstimulated calcified areas of stenotic valves. F–J: Results from stimulated areas. signal intensity (SI) measurements were followed by quantification (ImageJ) within a region of interest (ROI; labeled with yellow lines, of which the position was aligned on all subsequent images). Each histogram corresponds to the distribution of SI and demonstrates the image characteristics for unstimulated (A) and stimulated (F) calcified parts. Quantification analyses show unchanged SI over time in B and significantly elevated SI in G. Near-infrared signal intensity (green) in unstimulated (C) and stimulated areas (H). Enzyme-linked immunosorbent assay (ELISA) for interferon (IFN)-γ and receptor activator of nuclear factor-κB ligand (RANKL), analyzed in unstimulated (D and E) and stimulated (I and J) supernatant fluids. n = 4 valve donors (A–J). ^∗P < 0.05, ^∗∗∗P < 0.001. Scale bars: 200 μm (C and H). Arb., arbitrary; D1, day 1 after activation; D2, day 2 after activation; D3, day 3 after activation.

Figure 6

Interferon (IFN)-γ impairs osteoclastogenesis. Human CD14⁺ monocytes were cultured in the presence of Rh IFN-γ or endogenous IFN-γ from conditioned medium (CM) IFN-γ in equivalent concentration (0.5 ng/mL). Neutralizing anti–IFN-γ Ab was added when indicated (10 μg/mL). A: Tartrate-resistant acid phosphatase (TRAP) staining. B: Morphologic changes of osteoclasts using Rhodamine Phalloidin staining, nuclei were counterstained with DAPI. C: Resorption pits (white) on black-and-white binary images (images were converted by ImageJ) in each treatment group. Representative areas of the cultures are shown for each condition. D: Numbers of osteoclasts with more than three nuclei per cell were counted and plotted in each group. E: Quantification of resorption pit areas (white), assessed as a percentage of the whole area. F–K: Relative gene expression data for TRAP (F), Cathepsin K (G), RANK (H), TRAF6 (I), CD80 (J), and CD86 (K). X-fold changes were calculated relative to M+R-treated wells. n = 6 human monocyte donors (A–K). ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001. Scale bars: 200 μm (A–C). Ab, antibody; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; M, macrophage colony-stimulating factor at 25 ng/mL; R, receptor activator of nuclear factor-κB ligand at 30 ng/mL; Rh, recombinant human; TRAF, tumor necrosis factor receptor-associated factor.