Clonal hematopoiesis associated with TET2 deficiency accelerates atherosclerosis development in mice

Abstract

Human aging is associated with an increased frequency of somatic mutations in hematopoietic cells. Several of these recurrent mutations, including those in the gene encoding the epigenetic modifier enzyme TET2, promote expansion of the mutant blood cells. This clonal hematopoiesis correlates with an increased risk of atherosclerotic cardiovascular disease. We studied the effects of the expansion of Tet2-mutant cells in atherosclerosis-prone, low-density lipoprotein receptor-deficient (Ldlr^-/-) mice. We found that partial bone marrow reconstitution with TET2-deficient cells was sufficient for their clonal expansion and led to a marked increase in atherosclerotic plaque size. TET2-deficient macrophages exhibited an increase in NLRP3 inflammasome-mediated interleukin-1β secretion. An NLRP3 inhibitor showed greater atheroprotective activity in chimeric mice reconstituted with TET2-deficient cells than in nonchimeric mice. These results support the hypothesis that somatic TET2 mutations in blood cells play a causal role in atherosclerosis.

Fig. 1. Clonal expansion of TET2-deficient cells accelerates atherosclerosis in Ldlr^−/− mice

10% KO-BMT mice and 10% WT-BMT controls were fed a high-fat/high-cholesterol (HFHC) diet for 9 weeks, starting 4 weeks after BMT. (A) Percentage of CD45.2⁺ WBCs in blood, evaluated by flow cytometry (n = 9 mice per genotype). (B) Representative images of CD45.1/CD45.2 flow cytometry analysis of WBC populations. (C) qRT-PCR analysis of TET2 transcript levels in CD45.2⁺ WBCs from 10% WT-BMT (n = 14 mice) and 10% KO-BMT mice (n = 15 mice). (D) Percentage of CD45.2⁺ cells within main blood cell lineages 13 weeks after BMT, measured by flow cytometry (n = 11 10% WT-BMT mice per genotype; n = 14 10% KO-BMT mice per genotype). (E) Aortic root plaque size. Representative images of hematoxylin and eosin (H&E)–stained sections are shown; atherosclerotic plaques are delineated by dashed lines. Scale bars, 100 µm. (F) Percentage of CD45.2⁺ cells within the CD45⁺ immune cell population, F4/80⁺ macrophages (Macs), and CD3⁺ T cells in the aortic arch (n = 4 pools of two aortic arches per genotype). (G) Representative images of CD45.1/CD45.2 flow cytometry analysis of aortic macrophages and T cells. Statistical significance was evaluated by two-way analysis of variance (ANOVA) with Sidak multiple comparison tests (*P < 0.05, ***P < 0.001) [(A), (C), (D), and (F)] and by two-tailed unpaired Student’s t test (E). n.d., not detected. Error bars indicate SEM.

Fig. 2. TET2 deficiency in macrophages promotes inflammation and aggravates atherosclerosis

(A and B) Ldlr^−/− Mye-Tet2-KO mice (LysM-Cre⁺ Tet2^flox/flox BMT) and WT controls (LysM-Cre⁻ Tet2^flox/flox BMT) were fed a HFHC diet for 10 weeks. (A) qRT-PCR analysis of TET2 transcript levels in BM-derived macrophages isolated from Mye-Tet2-KO mice and WT controls (n = 6 mice per genotype). (B) Aortic root plaque size. Representative images of H&E-stained sections are shown; atherosclerotic plaques are delineated by dashed lines. Scale bars, 100 µm. (C to F) Peritoneal macrophages were isolated from Tet2^−/− mice or WT controls [n = 3 mice per genotype in (C) to (E); n = 4 mice per genotype in (F)] and treated with 10 ng/ml LPS and 2 ng/ml IFN-γ to induce proinflammatory activation. (C) Heat map of genes with expression change exceeding a factor of 1.5 (q < 0.05) after 10 hours of LPS/IFN-γ stimulation, from a genome-wide expression profiling by microarray. (D) PANTHER analysis of genome-wide expression profiling by microarray. Three overrepresented classes were identified in Tet2^−/− macrophages compared with all genes in Mus musculus (Bonferroni correction P < 0.05). (E) Heat map of selected genes up-regulated in Tet2^−/− macrophages with expression change exceeding a factor of 1.5 (q < 0.05) from the genome-wide expression profiling by microarray. (F) qRT-PCR analysis of transcript levels of proinflammatory cytokines (IL-6 and IL-1β). Unt, untreated. Statistical significance was evaluated by two-tailed unpaired Student’s t test with Welsh’s correction (A), by two-tailed unpaired Student’s t test (B), and by two-way ANOVA (P value for effect of genotype shown in graph) with Sidak multiple comparison tests (*P < 0.05, **P < 0.01, ***P < 0.001) (F). Error bars indicate SEM.

Fig. 3. TET2 regulates IL-1β expression in macrophages

(A and B) qRT-PCR analysis of IL-1β expression in Tet2^−/− and Tet2^+/+ peritoneal macrophages treated with 25 µg/ml oxidized LDL (oxLDL), 5 ng/ml TNF, and 2 ng/ml IFN-γ [(A), n = 6 mice per genotype] or in aortic arch samples (B) obtained from ND- or HFHC-fed 10% WT-BMT mice (n = 10 ND, 9 HFHC) and 10% KO-BMT mice (n = 8 ND, 8 HFHC). (C and D) IL-1β immunofluorescent staining in aortic root plaques (C) or CD68⁺ macrophage-rich areas (D) of 10% KO-BMT mice and 10% WT-BMT controls (n = 6 mice per genotype), quantified as integrated fluorescence intensity normalized to plaque or macrophage area. Representative images of IL-1β–stained plaques are shown. Dashed lines indicate plaques. Scale bars, 30 µm. (E) qRT-PCR analysis of IL-1β expression in Tet2^−/− and Tet2^+/+ peritoneal macrophages (n = 3 mice per genotype) transiently overexpressing WT TET2, catalytically inactive mutant TET2, or GFP as control. Macrophages were treated for 6 hours with 10 ng/ml LPS and 2 ng/ml IFN-γ. (F) qRT-PCR analysis of IL-1β expression in macrophages isolated from Tet2^−/− or Tet2^+/+ mice (n = 3 mice per genotype) and treated for 8 hours with LPS/IFN-γ in the absence or presence of 0.5 µM trichostatin A (TSA). (G) ChIP-qPCR analysis of H3 acetylation in the IL-1β promoter of macrophages isolated from Tet2^−/− or Tet2^+/+ mice (n = 6 to 8 mice per genotype and condition). Statistical significance was evaluated by two-way ANOVA with Sidak multiple comparison test [nonsignificant (ns) P > 0.4, *P < 0.05, **P < 0.01, ***P < 0.001] [(A), (B), and (E) to (G)] and by two-tailed unpaired Student’s t test [(C) and (D)]. Error bars indicate SEM.

Fig. 4. The NLRP3 inflammasome is essential for the exacerbated atherosclerosis associated with clonal expansion of TET2-deficient hematopoietic cells

(A and B) Western blot analysis of intracellular IL-1β in peritoneal macrophages isolated from Tet2^−/− mice and Tet2^+/+ controls (n = 3 mice per genotype) after 6 hours of treatment with 10 ng LPS and 2 ng IFN-γ (A) or after the same treatment combined with a final 15-min incubation with 5 mM ATP (B). Data were normalized to β-actin levels. (C) Western blot analysis of IL-1β in the supernatant of Tet2^−/− and Tet2^+/+ macrophages (n = 3 mice per genotype) after 6 hours of LPS/IFN-γ treatment combined with a final 30-min incubation with ATP. (D) ELISA analysis of IL-1β in the supernatant of Tet2^−/− and Tet2^+/+ macrophages (n = 3 mice per genotype) after 6 hours of LPS/IFN-γ treatment combined with a final 30-min incubation with 5 mM ATP in the presence or absence of 10 µM MCC950. (E) ELISA analysis of IL-1β in the supernatant of Tet2^−/− and Tet2^+/+ macrophages (n = 3 mice per genotype) after 8 hours of oxLDL/TNF/IFN-γ stimulation in the presence of 1 mg/ml cholesterol crystals (CC). (F) Caspase 1 activity detected by fluorescent staining with a FAM-YVAD-FMK FLICA reagent in CD68⁺ macrophage-rich areas of HFHC-fed 10% KO-BMT mice and 10% WT-BMT controls (n = 6 mice per genotype), quantified as integrated fluorescence intensity normalized to macrophage area. Representative images are shown. DAPI, 4′,6-diamidino-2-phenylindole. Scale bars, 25 µm. (G) Aortic root plaque size in HFHC-fed 10% KO-BMT and 10% WT-BMT mice. Mice received a continuous infusion of MCC950 (5 mg per kg per day) or phosphate-buffered saline vehicle via subcutaneous osmotic pumps. Representative images of H&E-stained sections are shown; plaques are delineated by dashed lines. Scale bars, 100 µm. Statistical significance was evaluated by two-tailed unpaired Student’s t tests [(A) to (C), (E), and (F)], by two-way ANOVA with Sidak multiple comparison test (D), and by two-way ANOVA with Tukey multiple comparison test (G) (*P < 0.05, ***P < 0.001, ****P < 0.001). Error bars indicate SEM.