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Aging-elevated inflammation promotes DNMT3A R878H-driven clonal hematopoiesis

Liao, Min, Chen, Ruiqing, Yang, Yang, He, Hanqing, Xu, Liqian, Jiang, Yuxuan, Guo, Zhenxing, He, Wei, Jiang, Hong, Wang, Jianwei·2022·Acta Pharmaceutica Sinica B
DOI 10.1016/j.apsb.2021.09.015PMC PMC8897035
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Abstract

Aging-elevated DNMT3A R882H-driven clonal hematopoiesis (CH) is a risk factor for myeloid malignancies remission and overall survival. Although some studies were conducted to investigate this phenomenon, the exact mechanism is still under debate. In this study, we observed that DNMT3A R878H bone marrow cells (human allele: DNMT3A R882H) displayed enhanced reconstitution capacity in aged bone marrow milieu and upon inflammatory insult. DNMT3A R878H protects hematopoietic stem and progenitor cells from the damage induced by chronic inflammation, especially TNFα insults. Mechanistically, we identified that RIPK1–RIPK3–MLKL-mediated necroptosis signaling was compromised in R878H cells in response to proliferation stress and TNFα insults. Briefly, we elucidated the molecular mechanism driving DNMT3A R878H-based clonal hematopoiesis, which raises clinical value for treating DNMT3A R882H-driven clonal hematopoiesis and myeloid malignancies with aging.

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Graphical abstract Graphical abstract With aging, hematopoietic stem cells accumulate DNMT3A mutation with necroptosis resistance which results in clonal hematopoiesis, and this process can be aggravated by inflammation challenge.
Graphical abstract Graphical abstract With aging, hematopoietic stem cells accumulate DNMT3A mutation with necroptosis resistance which results in clonal hematopoiesis, and this process can be aggravated by inflammation challenge.CC BY-NC-ND · PubMed Central
Figure 1 DNMT3A R878H mutation expands the HSC compartment. (A) The schematic diagram depicts the targeting strategy to generate the DNMT3A R878H conditional knockin mouse. Asterisk representing guanine at the 2633 site in the exon 23 of Dnmt3a was replaced by an adenine to encode R878H mutation. (B) The histogram shows the count of white blood cell (WBC), lymphocyte (LYM), neutrophil (NEUT), red blood cell (RBC) and platelet (PLT) between WT (Dnmt3afl-R878H/+, Vav-icre–) and R878H mice (Dnmt3afl-R878H/+, Vav-icre+; 4 months). n = 6 mice per group from two independent experiments. (C) Six 4-month old R878H mice and six age-matched WT mice were analyzed for myeloid, B and T cells. The histogram displays the frequency of B cells (B220+), T cells (CD3+) and myeloid (CD11b+) in the peripheral blood (PB). (D) Representative plots depict the gating strategies for HSC (Lineage– SCA-1+ c-KIT+ CD150+ CD34–), CMP (Lineage– SCA-1– c-KIT+ CD16/32– CD34+), GMP (Lineage– SCA-1– c-KIT+ CD16/32+ CD34+), MEP (Lineage– SCA-1– c-KIT+ CD16/32– CD34–), CLP (Lineage– SCA-1low c-KITlow CD135+ CD127+), ST-HSC (Lineage– SCA-1+ c-KIT+ CD135– CD34+) and MPP (Lineage– SCA-1+ c-KIT+ CD135+ CD34+) cells analysis in the bone marrow (BM) of WT and R878H mice. (E)–(G) 4 months old R878H mice and age-matched WT mice were analyzed for HSC and progenitors. (E) The histogram exhibits the absolute number of CMPs, GMPs, MEPs, CLPs, ST-HSCs and MPPs in the BM of WT and R878H mice. The histograms depict the frequency (F) and absolute number (G) of HSCs in the BM of WT and R878H mice. n = 5 mice per group from two independent experiments. (H)–(I) Cell cycle analysis of HSCs in six-month-old WT and R878H mice using Ki67 and DAPI. Representative flow cytometry plots (H) and the frequency of cells in each phase (I) are shown. WT, n = 3; R878H, n = 4. All data above are shown as mean ± SD.
Figure 1 DNMT3A R878H mutation expands the HSC compartment. (A) The schematic diagram depicts the targeting strategy to generate the DNMT3A R878H conditional knockin mouse. Asterisk representing guanine at the 2633 site in the exon 23 of Dnmt3a was replaced by an adenine to encode R878H mutation. (B) The histogram shows the count of white blood cell (WBC), lymphocyte (LYM), neutrophil (NEUT), red blood cell (RBC) and platelet (PLT) between WT (Dnmt3afl-R878H/+, Vav-icre–) and R878H mice (Dnmt3afl-R878H/+, Vav-icre+; 4 months). n = 6 mice per group from two independent experiments. (C) Six 4-month old R878H mice and six age-matched WT mice were analyzed for myeloid, B and T cells. The histogram displays the frequency of B cells (B220+), T cells (CD3+) and myeloid (CD11b+) in the peripheral blood (PB). (D) Representative plots depict the gating strategies for HSC (Lineage– SCA-1+ c-KIT+ CD150+ CD34–), CMP (Lineage– SCA-1– c-KIT+ CD16/32– CD34+), GMP (Lineage– SCA-1– c-KIT+ CD16/32+ CD34+), MEP (Lineage– SCA-1– c-KIT+ CD16/32– CD34–), CLP (Lineage– SCA-1low c-KITlow CD135+ CD127+), ST-HSC (Lineage– SCA-1+ c-KIT+ CD135– CD34+) and MPP (Lineage– SCA-1+ c-KIT+ CD135+ CD34+) cells analysis in the bone marrow (BM) of WT and R878H mice. (E)–(G) 4 months old R878H mice and age-matched WT mice were analyzed for HSC and progenitors. (E) The histogram exhibits the absolute number of CMPs, GMPs, MEPs, CLPs, ST-HSCs and MPPs in the BM of WT and R878H mice. The histograms depict the frequency (F) and absolute number (G) of HSCs in the BM of WT and R878H mice. n = 5 mice per group from two independent experiments. (H)–(I) Cell cycle analysis of HSCs in six-month-old WT and R878H mice using Ki67 and DAPI. Representative flow cytometry plots (H) and the frequency of cells in each phase (I) are shown. WT, n = 3; R878H, n = 4. All data above are shown as mean ± SD.CC BY-NC-ND · PubMed Central
Figure 2 Aged bone marrow microenvironment promotes DNMT3A R878H-based clonal hematopoiesis. (A)–(F) Freshly isolated 1 × 105 BM cells (CD45.2) from 2 months old R878H mice or age-matched WT mice were transplanted into lethally irradiated young (2 months) or aged (15 months) recipients (CD45.1) together with 5 × 105 competitor cells (CD45.1/2). Chimera in the peripheral blood was evaluated every month until the fourth month. (A) Experimental design. (B) Representative flow cytometry plots displaying the frequencies of donor-derived cells in the PB of young or aged recipient mice transplanted with the indicated bone marrow cells at the 4th month after transplantation (n = 4–5 mice per group). (C) and (D) These line plots depict the percentage of indicated donor-derived cells in the PB of young or aged recipients carrying R878H (C) or WT (D) BM cells. (E) and (F) The histograms show the lineage distribution of myeloid, T cells and B cells among donor-derived cells in the PB of young or aged recipients carrying WT (E) or R878H (F) BM cells at the 4th month after transplantation. n = 4–5 mice per group from two independent experiments. (G) The histogram displays the percentage of WT/R878H-derived HSCs in young recipients (black) and aged recipients (red) (n = 4–5 mice per group). All data above are shown as mean ± SD; ∗P < 0.05, ∗∗P < 0.01, NS represents no significance.
Figure 2 Aged bone marrow microenvironment promotes DNMT3A R878H-based clonal hematopoiesis. (A)–(F) Freshly isolated 1 × 105 BM cells (CD45.2) from 2 months old R878H mice or age-matched WT mice were transplanted into lethally irradiated young (2 months) or aged (15 months) recipients (CD45.1) together with 5 × 105 competitor cells (CD45.1/2). Chimera in the peripheral blood was evaluated every month until the fourth month. (A) Experimental design. (B) Representative flow cytometry plots displaying the frequencies of donor-derived cells in the PB of young or aged recipient mice transplanted with the indicated bone marrow cells at the 4th month after transplantation (n = 4–5 mice per group). (C) and (D) These line plots depict the percentage of indicated donor-derived cells in the PB of young or aged recipients carrying R878H (C) or WT (D) BM cells. (E) and (F) The histograms show the lineage distribution of myeloid, T cells and B cells among donor-derived cells in the PB of young or aged recipients carrying WT (E) or R878H (F) BM cells at the 4th month after transplantation. n = 4–5 mice per group from two independent experiments. (G) The histogram displays the percentage of WT/R878H-derived HSCs in young recipients (black) and aged recipients (red) (n = 4–5 mice per group). All data above are shown as mean ± SD; ∗P < 0.05, ∗∗P < 0.01, NS represents no significance.CC BY-NC-ND · PubMed Central
Figure 3 DNMT3A R878H blunts the response of hematopoietic stem cells upon inflammation challenge. (A) A model was used to evaluate the response of R878H mice to acute inflammatory challenges. The schematic diagram showing the experimental design: WT and R878H mice were challenged by LPS (0.8 mg/kg) or an equal volume of PBS intraperitoneally and these mice were analyzed 48 h later. 5 × 105 LPS/PBS-treated WT or R878H BM cells (CD45.2) together with 5 × 105 competitor cells (CD45.1) were transplanted into lethally irradiated recipients (CD45.1/2). Chimerism in the PB was analyzed monthly. (B)–(F) The histograms show the cell number (left) of BM cells (B), WBC (C), PLT (D), LYM (E), NEUT (F), and the fold change (LPS/PBS, right) of WT and R878H mice upon LPS (red) or PBS (black) treatment (n = 6 mice per group from 4 independent experiments). (G)–(I) The left histograms display the absolute cell number of ST-HSC (G), MPP (H), HSC (I) in the BM of the indicated mice treated with a single dose (0.8 mg/kg) of LPS or PBS. The right histograms depict a ratio of the absolute cell number between LPS and PBS groups in the indicated mice from different populations of hematopoietic cells (n = 6 mice/group from 4 independent experiments). (J) These line plots show the percentage of donor-derived overall and B cells, T cells, myeloid within PB cells of the indicated recipients at each time point (n = 4–6 mice per group from 2 independent experiments). All data above are shown as mean ± SD; NS represents no significance.
Figure 3 DNMT3A R878H blunts the response of hematopoietic stem cells upon inflammation challenge. (A) A model was used to evaluate the response of R878H mice to acute inflammatory challenges. The schematic diagram showing the experimental design: WT and R878H mice were challenged by LPS (0.8 mg/kg) or an equal volume of PBS intraperitoneally and these mice were analyzed 48 h later. 5 × 105 LPS/PBS-treated WT or R878H BM cells (CD45.2) together with 5 × 105 competitor cells (CD45.1) were transplanted into lethally irradiated recipients (CD45.1/2). Chimerism in the PB was analyzed monthly. (B)–(F) The histograms show the cell number (left) of BM cells (B), WBC (C), PLT (D), LYM (E), NEUT (F), and the fold change (LPS/PBS, right) of WT and R878H mice upon LPS (red) or PBS (black) treatment (n = 6 mice per group from 4 independent experiments). (G)–(I) The left histograms display the absolute cell number of ST-HSC (G), MPP (H), HSC (I) in the BM of the indicated mice treated with a single dose (0.8 mg/kg) of LPS or PBS. The right histograms depict a ratio of the absolute cell number between LPS and PBS groups in the indicated mice from different populations of hematopoietic cells (n = 6 mice/group from 4 independent experiments). (J) These line plots show the percentage of donor-derived overall and B cells, T cells, myeloid within PB cells of the indicated recipients at each time point (n = 4–6 mice per group from 2 independent experiments). All data above are shown as mean ± SD; NS represents no significance.CC BY-NC-ND · PubMed Central
Figure 4 LPS-induced chronic inflammation promotes DNMT3A R878H-driven clonal hematopoiesis. (A) Schematic diagram of the experimental procedure is designed for generating chimeric mice simulating clonal expansion of Dnmt3aR878H/+ cells upon inflammation challenges. WT or R878H BM cells (CD45.2) together with competitor cells (CD45.1) at a ratio of 1 to 5 (1 × 105:5 × 105) were transplanted into lethally irradiated recipients (CD45.1/2). One month post-transplantation, the recipients were subjected to sustaining treatment with LPS (0.8 mg/kg injected i.p. every other day for 15 doses) or an equal volume of PBS. Chimerism in the PB was analyzed monthly until the fifth month. (B) These line plots show the frequencies of R878H-derived overall and B cells, T cells, myeloid within PB cells at the indicated time point in response to LPS (red) and PBS (black) treatment. (C) The histogram displays the lineage distribution of R878H-derived T, B and myeloid in peripheral blood at the 5th month post-transplantation. (D) These line plots depict the percentage of WT-derived overall and B cells, T cells, myeloid within PB cells at indicated the time point in response to LPS (red) and PBS (black) treatment. (E) The histogram exhibits the lineage distribution of WT-derived T, B and myeloid in peripheral blood at the 5th month post-transplantation. (F) Representative flow cytometry plots depict the gating strategy and the percentage of donor-derived HSC (CD34– CD150+ LSK) in the indicated recipients at the fifth month after transplantation. (G) The histograms show the frequency of WT/R878H-derived HSCs in response to LPS (red) and PBS (black) treatment; the right histogram shows the fold change of HSCs (WT: black; R878H: red) in response to LPS treatment. n = 5–7 mice per group from two independent experiments, the data are shown as mean ± SD; NS represents no significance.
Figure 4 LPS-induced chronic inflammation promotes DNMT3A R878H-driven clonal hematopoiesis. (A) Schematic diagram of the experimental procedure is designed for generating chimeric mice simulating clonal expansion of Dnmt3aR878H/+ cells upon inflammation challenges. WT or R878H BM cells (CD45.2) together with competitor cells (CD45.1) at a ratio of 1 to 5 (1 × 105:5 × 105) were transplanted into lethally irradiated recipients (CD45.1/2). One month post-transplantation, the recipients were subjected to sustaining treatment with LPS (0.8 mg/kg injected i.p. every other day for 15 doses) or an equal volume of PBS. Chimerism in the PB was analyzed monthly until the fifth month. (B) These line plots show the frequencies of R878H-derived overall and B cells, T cells, myeloid within PB cells at the indicated time point in response to LPS (red) and PBS (black) treatment. (C) The histogram displays the lineage distribution of R878H-derived T, B and myeloid in peripheral blood at the 5th month post-transplantation. (D) These line plots depict the percentage of WT-derived overall and B cells, T cells, myeloid within PB cells at indicated the time point in response to LPS (red) and PBS (black) treatment. (E) The histogram exhibits the lineage distribution of WT-derived T, B and myeloid in peripheral blood at the 5th month post-transplantation. (F) Representative flow cytometry plots depict the gating strategy and the percentage of donor-derived HSC (CD34– CD150+ LSK) in the indicated recipients at the fifth month after transplantation. (G) The histograms show the frequency of WT/R878H-derived HSCs in response to LPS (red) and PBS (black) treatment; the right histogram shows the fold change of HSCs (WT: black; R878H: red) in response to LPS treatment. n = 5–7 mice per group from two independent experiments, the data are shown as mean ± SD; NS represents no significance.CC BY-NC-ND · PubMed Central
Figure 5 Elevated-TNFα in aged bone marrow promotes DNMT3A R878H-based clonal hematopoiesis. (A) The pooled bone marrow aspirates of young recipients and aged recipients reconstituted by WT cells (Fig. 2A) were subjected to a mouse cytokine antibody array. Cytokines were identified as upregulated candidates in aged recipients with the criteria that fold change between aged recipients and young recipients are greater than 1.2, while fold change lower than 0.83 is considered as downregulated cytokines. The histogram depicts the top 10 cytokines upregulated in the bone marrow aspirates of aged recipients which are ranked by the expression levels. Each sample was pooled from 3 mice for each group. (B)–(H) 100 HSCs (CD34– CD150+ LSK) were freshly isolated from either WT or R878H mice (CD45.2), and the HSCs were cultured in vitro in the presence or absence of TNFα (200 ng/mL) for 3 or 7 days (all HSCs were cultured in SFEM medium supplemented with 30 ng/mL SCF, 30 ng/mL TPO and 100 U/mL Penicillin–Streptomycin). Three days later, each culture was mixed with 3 × 105 competitor cells (CD45.1) and transplanted into lethally irradiated recipients (CD45.1/2). Chimera in the peripheral blood was evaluated for 4 months. (B) Experimental design. (C) On the 7th day, cell growth was monitored by a light microscopy, and images were acquired under an inverted microscope (Olympus CKX41). Scale bar, 0.5 mm. (D) and (E) The number of live cells (D, DAPI–) and HSCs (E, CD150+ CD48– c-KIT+ SCA-1+) was analyzed on days 7 (n = 5). (F) These line plots show the percentage of donor-derived cells within PB cells at the indicated time point after transplantation. (G) The histogram depicts the frequency of donor-derived HSC (CD34– CD150+ LSK) in the indicated recipients at the fourth month post-transplantation. (H) The histograms exhibit the lineage distribution of WT-derived or R878H-derived T, B and myeloid cells in peripheral blood at the 4th month post-transplantation. n = 5–7 mice per group from two independent experiments, the data are shown as mean ± SD, ∗P < 0.05, NS represents no significance. (I) and (J) Experiment design for TNFα treatment in vivo. 1 × 105 freshly isolated total bone marrow cells from either 2–4 months old WT or R878H mice (CD45.2) together with 5 × 105 competitor cells from age-matched WT mice (CD45.1) were transplanted into lethally irradiated recipients (CD45.1/2). One month later, the recipients were challenged with TNFα (100 μg/kg) every other day intravenously for one month, and donor-derived HSCs were evaluated at the 5th month. (I) Schematic diagram of the experimental procedure. (J) The left histogram shows the number of WT/R878H-derived HSCs in response to TNFα (red) and PBS (black) treatment; the right histogram shows the fold change of HSCs (WT: black; R878H: red) in response to TNFα treatment. n = 5–7 mice per group, the data are shown as mean ± SD, NS represents no significance.
Figure 5 Elevated-TNFα in aged bone marrow promotes DNMT3A R878H-based clonal hematopoiesis. (A) The pooled bone marrow aspirates of young recipients and aged recipients reconstituted by WT cells (Fig. 2A) were subjected to a mouse cytokine antibody array. Cytokines were identified as upregulated candidates in aged recipients with the criteria that fold change between aged recipients and young recipients are greater than 1.2, while fold change lower than 0.83 is considered as downregulated cytokines. The histogram depicts the top 10 cytokines upregulated in the bone marrow aspirates of aged recipients which are ranked by the expression levels. Each sample was pooled from 3 mice for each group. (B)–(H) 100 HSCs (CD34– CD150+ LSK) were freshly isolated from either WT or R878H mice (CD45.2), and the HSCs were cultured in vitro in the presence or absence of TNFα (200 ng/mL) for 3 or 7 days (all HSCs were cultured in SFEM medium supplemented with 30 ng/mL SCF, 30 ng/mL TPO and 100 U/mL Penicillin–Streptomycin). Three days later, each culture was mixed with 3 × 105 competitor cells (CD45.1) and transplanted into lethally irradiated recipients (CD45.1/2). Chimera in the peripheral blood was evaluated for 4 months. (B) Experimental design. (C) On the 7th day, cell growth was monitored by a light microscopy, and images were acquired under an inverted microscope (Olympus CKX41). Scale bar, 0.5 mm. (D) and (E) The number of live cells (D, DAPI–) and HSCs (E, CD150+ CD48– c-KIT+ SCA-1+) was analyzed on days 7 (n = 5). (F) These line plots show the percentage of donor-derived cells within PB cells at the indicated time point after transplantation. (G) The histogram depicts the frequency of donor-derived HSC (CD34– CD150+ LSK) in the indicated recipients at the fourth month post-transplantation. (H) The histograms exhibit the lineage distribution of WT-derived or R878H-derived T, B and myeloid cells in peripheral blood at the 4th month post-transplantation. n = 5–7 mice per group from two independent experiments, the data are shown as mean ± SD, ∗P < 0.05, NS represents no significance. (I) and (J) Experiment design for TNFα treatment in vivo. 1 × 105 freshly isolated total bone marrow cells from either 2–4 months old WT or R878H mice (CD45.2) together with 5 × 105 competitor cells from age-matched WT mice (CD45.1) were transplanted into lethally irradiated recipients (CD45.1/2). One month later, the recipients were challenged with TNFα (100 μg/kg) every other day intravenously for one month, and donor-derived HSCs were evaluated at the 5th month. (I) Schematic diagram of the experimental procedure. (J) The left histogram shows the number of WT/R878H-derived HSCs in response to TNFα (red) and PBS (black) treatment; the right histogram shows the fold change of HSCs (WT: black; R878H: red) in response to TNFα treatment. n = 5–7 mice per group, the data are shown as mean ± SD, NS represents no significance.CC BY-NC-ND · PubMed Central
Figure 6 DNMT3A R878H prevents the activation of necroptosis in HSPCs upon TNFα challenge. (A) Experimental procedure to evaluate the expression of TNF signaling genes: 1 × 105 bone marrow cells freshly isolated from either R878H mice or wild-type mice (CD45.2) were transplanted into lethally irradiated aged recipients (CD45.1) together with 5 × 105 young competitor cells (CD45.1/2). Four months after transplantation, 2 × 104 donor-derived LSK cells (CD45+ LIN– c-KIT+ SCA-1+) were isolated for qRT-PCR analysis. (B) and (C) The histogram showing the relative expression of apoptosis-related genes (B) and necroptosis-associated genes (C) in the indicated cells. (D) and (E) Experiments were designed to evaluate the response of R878H HSPCs upon apoptotic and necroptotic stress. (D) The schematic diagram showing the procedure for in vitro TSZ or TS treatment in the indicated experiment (S, 100 nmol/L SMAC mimetic; T, 50 ng/mL TNFα; Z, 20 μmol/L z-VAD). To determine the percentage of cell death upon TS or TSZ treatment, LSK cells were isolated freshly from WT or R878H mice and cultured with the indicated chemicals for 6 h, and the resulting cells were stained with DAPI and the dead cells were determined by flow cytometry. (E) The histogram depicts the percentage of cell death in LSK cells upon TS, TSZ and TSZ plus RIPA56 (RIPA56: 20 μmol/L) treatment (n = 3–4 per group from 2 independent experiments). (F) and (G) c-KIT+ bone marrow cells were freshly isolated from WT or R878H mice and cultured in SFEM medium overnight. Then half of the cultures were treated with TSZ for 1 h, and the resulting cells were harvested for Western blotting analysis. (F) Experimental design. (G) Representative Western blot showing the expression of RIPK1, RIPK3, MLKL and phosphorylated MLKL in c-KIT+ cells upon indicated treatment (three biological replicates). (H) and (I) 100 HSCs (CD34– CD150+ LSK) were freshly isolated from either WT or R878H mice, and the HSCs were cultured in vitro with the indicated treatment for 7 days (all HSCs were cultured in SFEM medium supplemented with 30 ng/mL SCF, 30 ng/mL TPO and 100 U/mL penicillin–streptomycin; TNFα: 200 ng/mL, RIPA56: 20 μmol/L, RES: 20 μmol/L). (H) Experimental design. (I) The histogram shows the number of HSCs (CD150+ CD48– c-KIT+ SCA-1+) analyzed on days 7. (J) and (K) An in vivo assay was applied to evaluate the role of necroptosis in R878H mice. (J) The cartoon shows the procedure for repeated in vivo TNFα injections in WT, Dnmt3aR878H/+, Ripk1K45A, Ripk3Δ/Δ and Mlkl–/– mice (TNFα: 5 μg/mouse), intravenously. All of these mice were analyzed 48 h later. (K) The histogram depicts the absolute number of HSC (CD34– CD150+ LSK) in the bone marrow of indicated genotypic mice treated with TNFα (n = 3 mice per group from 2 independent experiments). All data above are shown as mean ± SD, NS represents no significance.
Figure 6 DNMT3A R878H prevents the activation of necroptosis in HSPCs upon TNFα challenge. (A) Experimental procedure to evaluate the expression of TNF signaling genes: 1 × 105 bone marrow cells freshly isolated from either R878H mice or wild-type mice (CD45.2) were transplanted into lethally irradiated aged recipients (CD45.1) together with 5 × 105 young competitor cells (CD45.1/2). Four months after transplantation, 2 × 104 donor-derived LSK cells (CD45+ LIN– c-KIT+ SCA-1+) were isolated for qRT-PCR analysis. (B) and (C) The histogram showing the relative expression of apoptosis-related genes (B) and necroptosis-associated genes (C) in the indicated cells. (D) and (E) Experiments were designed to evaluate the response of R878H HSPCs upon apoptotic and necroptotic stress. (D) The schematic diagram showing the procedure for in vitro TSZ or TS treatment in the indicated experiment (S, 100 nmol/L SMAC mimetic; T, 50 ng/mL TNFα; Z, 20 μmol/L z-VAD). To determine the percentage of cell death upon TS or TSZ treatment, LSK cells were isolated freshly from WT or R878H mice and cultured with the indicated chemicals for 6 h, and the resulting cells were stained with DAPI and the dead cells were determined by flow cytometry. (E) The histogram depicts the percentage of cell death in LSK cells upon TS, TSZ and TSZ plus RIPA56 (RIPA56: 20 μmol/L) treatment (n = 3–4 per group from 2 independent experiments). (F) and (G) c-KIT+ bone marrow cells were freshly isolated from WT or R878H mice and cultured in SFEM medium overnight. Then half of the cultures were treated with TSZ for 1 h, and the resulting cells were harvested for Western blotting analysis. (F) Experimental design. (G) Representative Western blot showing the expression of RIPK1, RIPK3, MLKL and phosphorylated MLKL in c-KIT+ cells upon indicated treatment (three biological replicates). (H) and (I) 100 HSCs (CD34– CD150+ LSK) were freshly isolated from either WT or R878H mice, and the HSCs were cultured in vitro with the indicated treatment for 7 days (all HSCs were cultured in SFEM medium supplemented with 30 ng/mL SCF, 30 ng/mL TPO and 100 U/mL penicillin–streptomycin; TNFα: 200 ng/mL, RIPA56: 20 μmol/L, RES: 20 μmol/L). (H) Experimental design. (I) The histogram shows the number of HSCs (CD150+ CD48– c-KIT+ SCA-1+) analyzed on days 7. (J) and (K) An in vivo assay was applied to evaluate the role of necroptosis in R878H mice. (J) The cartoon shows the procedure for repeated in vivo TNFα injections in WT, Dnmt3aR878H/+, Ripk1K45A, Ripk3Δ/Δ and Mlkl–/– mice (TNFα: 5 μg/mouse), intravenously. All of these mice were analyzed 48 h later. (K) The histogram depicts the absolute number of HSC (CD34– CD150+ LSK) in the bone marrow of indicated genotypic mice treated with TNFα (n = 3 mice per group from 2 independent experiments). All data above are shown as mean ± SD, NS represents no significance.CC BY-NC-ND · PubMed Central

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Metadata

Authors
Liao, M., Chen, R., Yang, Y., He, H., Xu, L., Jiang, Y., Guo, Z., He, W., Jiang, H., & Wang, J.
Venue
Acta Pharmaceutica Sinica B
Publisher
Elsevier BV
Published
2022-02
Volume
12
Issue
2
Pages
678-691
ISSN
2211-3835
DOI
10.1016/j.apsb.2021.09.015
PMC
PMC8897035
Cited by
67
References
48
Language
en
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