Chronic TNF in the aging microenvironment exacerbates Tet2 loss-of-function myeloid expansion

Abstract

Somatic mutations in the TET2 gene occur more frequently with age, imparting an intrinsic hematopoietic stem cells (HSCs) advantage and contributing to a phenomenon termed clonal hematopoiesis of indeterminate potential (CHIP). Individuals with TET2-mutant CHIP have a higher risk of developing myeloid neoplasms and other aging-related conditions. Despite its role in unhealthy aging, the extrinsic mechanisms driving TET2-mutant CHIP clonal expansion remain unclear. We previously showed an environment containing tumor necrosis factor (TNF) favors TET2-mutant HSC expansion in vitro. We therefore postulated that age-related increases in TNF also provide an advantage to HSCs with TET2 mutations in vivo. To test this hypothesis, we generated mixed bone marrow chimeric mice of old wild-type (WT) and TNF-/- genotypes reconstituted with WT CD45.1+ and Tet2-/- CD45.2+ HSCs. We show that age-associated increases in TNF dramatically increased the expansion of Tet2-/- cells in old WT recipient mice, with strong skewing toward the myeloid lineage. This aberrant myelomonocytic advantage was mitigated in old TNF-/- recipient mice, suggesting that TNF signaling is essential for the expansion Tet2-mutant myeloid clones. Examination of human patients with rheumatoid arthritis with clonal hematopoiesis revealed that hematopoietic cells carrying certain mutations, including in TET2, may be sensitive to reduced TNF bioactivity following blockade with adalimumab. This suggests that targeting TNF may reduce the burden of some forms of CHIP. To our knowledge, this is the first evidence to demonstrate that TNF has a causal role in driving TET2-mutant CHIP in vivo. These findings highlight TNF as a candidate therapeutic target to control TET2-mutant CHIP.

Graphical abstract

Figure 1.

Age-associated TNF inflammation increases myeloid progenitor differentiation in female mice 8 weeks after BMT with WT and Tet2^–/– HSPCs. (A) Experimental design and representative flow cytometric data showing CD45⁺ T-cell–depleted (TCD) BM donor cells transplanted in mice conditioned with busulfan. Young (6 months) and old (18-22 months) C57Bl/6-J WT and old TNF^–/– animals received 80 mg/kg busulfan and received transplantation with 2 × 10⁶ TCD-BM cells from young CD45.1⁺ WT and CD45.2⁺Tet2^–/– mice. (B) Histogram showing percentage of CD45.1⁺ WT and CD45.2 Tet2^–/– donor cells transplanted into recipient mice. (C) Ratio of CD45.2⁺Tet2^–/– to CD45.1⁺ WT leukocytes in the BM of recipient mice 8 weeks after BMT. (D-I) Flow cytometric analyses showing absolute counts of HSPC (D), common myeloid progenitor (CMP) cells (E), monocyte-dendritic progenitor (MDP) cells (F), granulocyte-monocyte progenitor (GMP) cells (G), common monocyte progenitor (cMoP) cells (H), and mature monocytes (I). (J) Absolute count of Ly6C^high inflammatory monocytes. (K) Geometric mean of CX₃ chemokine receptor 1 (CX3CR1) in BM monocytes. (L) Absolute counts of circulatory Ly6C^high monocytes. Statistical significance determined by 1-way analysis of variance (ANOVA). ∗P ≤ .05; ∗∗P ≤ .01; ∗∗∗P ≤ .001; ∗∗∗∗P ≤ .0001. ELISA, enzyme-linked immunosorbent assay; MFI, geometric mean fluorescence intensity.

Figure 2.

Tet2 mutations increase circulating myeloid cells of old WT but not TNF^–/– recipient mice. Circulating myeloid immune populations were compared in young WT, old WT, and old TNF^–/– recipient mice. (A-C) Total counts of circulating monocytes (A), Ly6C^high monocytes (B), and neutrophils (C). (D-F) Total circulating monocytes, neutrophils, and Ly6C^high monocytes as a proportion of total leukocytes (CD45⁺) cells, respectively. (G) Surface expression of F4/80 on Ly6C^high monocyte. (H) Intracellular expression of TNF in monocytes after stimulation with lipopolysaccharides. Data are shown as a stacked bar plot, in which orange represents gated CD45.2 Tet2^–/– cells and blue represents CD45.1 WT cells. Statistical significance determined by 2-way ANOVA with Tukey multiple comparisons test. Letters in the orange and blue columns denote significant differences (P ≤ .05) in the group means of CD45.2 or CD45.1 alleles, respectively. For all variables with the same letter, the difference between the means is significantly different. If 2 variables have different letters or no letters, they are not significantly different. Black bars with asterisks denote group differences, inclusive of both CD45.1 and CD45.2 alleles. ∗P ≤ .05; ∗∗P ≤ .01; ∗∗∗P ≤ .001; ∗∗∗∗P ≤ .0001.

Figure 3.

TNF exacerbates Tet2–mutant T-cell remodeling with age. Peripheral whole blood of recipient mice was collected 8 weeks after BMT and analyzed by flow cytometry for the absolute and relative counts of circulating lymphocytes. (A-C) Absolute and relative (as a percentage of total CD45⁺ leukocytes) counts of total T cells (A), CD8⁺ T cells (B), and CD4⁺ T cells (C). (D-E) Absolute and relative counts of CD8⁺ (D) and CD4⁺ naïve T cells (E) as a percentage of total CD8⁺ and CD4⁺ T cells, respectively. (F) Absolute counts of circulating CD8+ naïve T cells expressing CD183. (G-I) Relative count of CD8⁺ T_EM (G), T_VM (H), and T_CM cells (I). (J-M) Relative count of CD4⁺ T_VM (J), T_EM (K), and T_CM cells (L). (M) Myeloid-to-lymphoid ratio. (N) Leukocyte summary. Significance was assessed by a 2-way ANOVA with Tukey multiple comparisons test. Letters in the orange and blue columns denote significant differences (P ≤ .05) in the group means of CD45.2 or CD45.1 alleles, respectively. For all variables with the same letter, the difference between the means is significantly different. If 2 variables have different letters or no letters, they are not significantly different. Black bars with asterisks denote group differences, inclusive of both CD45.1 and CD45.2 alleles. ∗P ≤ .05; ∗∗P ≤ .01; ∗∗∗P ≤ .001; ∗P ≤.0001. Abbreviations: T_EM, effector memory T cells; T_VM, virtual memory T cells; T_CM, central memory T cells; NK, natural killer; NKT, natural killer T cells.

Figure 4.

CH clones, including mutant TET2, become undetectable in the peripheral blood of older adults administered the TNF blocker, HUMIRA (adalimumab). Mutant-TET2, ASXL1, TP53, CBL, PPM1D, PHF6, and STAG2 clones identified in the peripheral whole blood of patients with RA before any immunomodulatory treatment (baseline) and at 3 and 6 months after treatment with the adalimumab (V1 and V2, respectively), show a significant reduction in CH clones after treatment. (A-B) Mean ± standard error of the mean (A) and detected mutant clones (B) shown. nd, none detected; V1, visit 1; V2, visit 2.