Fig 1.
Imatinib treatment induces expansion of myeloid cells in mouse spleen and blood.
C57Bl/6 mice were administered imatinib at 66mg/kg/day (Imatinib) or left untreated (Control). For some experiments, mice were either injected in the tail vein with 105 CFU Mm 1218R one day after administration of drug (Mm + imatinib) or carrier (Mm). Neutrophils (CD11b+ Ly6Cint Gr-1hi SSCint), monocytes (CD11b+ Ly6Chi Gr-1int SSClow and F4/80+) and eosinophil (CD11b+ Ly6Clo Gr-1int SSChi and F4/80+) (A), or T cells (Thy1.2+), B cells (B220+ CD19+) and NK cells (NK1.1+) (B) were enumerated by flow cytometry in the blood (top panel) or spleen (bottom panel) at d7 post infection or treatment. Each symbol represents one mouse. Data shown are representative of six independent experiments. (C) Sections of spleens taken from mice left untreated or treated with imatinib at 66mg/kg/day plus or minus infection with Mm. Sections were stained with anti-CD169 to recognize marginal zone macrophages (green), anti-F4/80 to recognize red pulp myeloid cells (blue), and anti-CD11b to recognize neutrophils and monocytes (red). (D) C57Bl/6 mice were administered imatinib at 66 mg/kg/day for 28 days. On day 0, 7, 14, 21, 28 of treatment, mice were bled from the tail vein and numbers of neutrophils and monocytes enumerated by flow cytometry (three mice per time point). Data shown are from two independent experiments. For all data sets, the line in each group of data points represents the median. For A, and B, a Mann-Whitney test was used for pairwise comparisons (e.g. +/- drug or +/- infection only), and a Kruskal Wallis test for multiple comparisons across groups. For all panels, p values less than 0.05 were considered significant; p values < 0.05 are denoted by *, values <.001 by **, and values <.0001 by ***, and values <.00005 by ****.
Fig 2.
Effects of imatinib on neutrophils and monoctyes in bone marrow.
(A) Geimsa staining of femurs from C57Bl/6 mice administered imatinib at 66 mg/kg/d for seven days or left untreated as indicated. Top panels: coronal sections (10X). Middle panels: Insets (40x) derived from the area denoted by a dashed white box. Bottom panels: sagittal sections (10x). (B) C57Bl/6 mice were administered imatinib at 66 mg/kg/d for seven days or left untreated, as indicated. Beginning 24h after onset of drug, mice were either injected in the tail vein with 105 CFU Mm 1218R or left uninfected. At seven days post-treatment bone marrow was collected from femurs. Data shown are representative of three independent experiments. For all data sets, the line in each group of data points represents the median. A Mann-Whitney test was used for pairwise comparisons, and a Kruskal Wallis test for multiple comparisons.
Fig 3.
Effects of Imatinib on HSCs and multipotent progenitors in vivo.
(A-C) C57Bl/6 mice were administered imatinib at 66 or 200mg/kg/d or left untreated. Beginning 24h post-treatment mice were either injected in the tail vein with 105 CFU Mm 1218R or left uninfected. Bone marrow was collected from femurs and HSCs and MPPs enumerated by flow cytometry[41]. (A-C) Effect of imatinib or infection on Linnegsca1+CD117+ (LSK) cells (A), HSCs (CD34−CD48−CD150+CD135−) (B) or MPP1 (CD34+CD48-CD150+CD135−), MPP2 (CD34+CD48+CD150+CD135−), MPP3 (CD34+CD48+CD150−CD135−) and MPP4 (CD34+CD48+CD150−CD135+) cells (C). Each bar represents the average fold change relative to control in 2–6 independent experiments with 3–6 animals per condition per experiment. An asterisk (*) above the error bar indicates statistical significance compared to control as determined by t-test. Statistical significance was also assessed for infected (Mm) versus infected, drug treated animals (Mm + imatinib) and is indicated by a bracket over the bars. (D-E) Competitive bone marrow transplants with whole marrow (D) or LSK cells (E) from control animals or animals treated with 66mg/kg/d imatinib for 7d. (D) Congenic CD45.1+ C57Bl/6 mice were lethally irradiated and transplanted with 2 x 106 whole bone marrow cells from untreated GFP+ C57Bl/6 donors mixed with 2 x 106 whole bone marrow cells from C57Bl/6 (CD45.2) donors that were either treated with imatinib for seven days or left untreated. Numbers of leukocytes derived from the different donor sources or the recipient mice were determined by flow cytometry at 4 months post transplant (n = 5 recipient mice per condition). (E) GFP+ C57Bl/6 marrow (3x105 cells) was mixed with 5x103 sorted LSK cells before transplantation, and donor chimerism was determined using blood drawn 4 months post-transplantation (n = 5 recipient mice per condition). A Mann-Whitney nonparametric test was used to determine significance.
Fig 4.
Effects of imatinib on progenitor differentiation in culture, and effects of anti-c-Kit neutralizing antibody.
(A) Colony forming cell (CFC) assays with bone marrow derived from mice treated with imatinib at 66 or 200mg/kg/d for seven days or left untreated (Control). Bone marrow was cultured in the absence of imatinib, CFU-GM colonies were counted after 10 days. (B) CFC assays with bone marrow derived from naïve mice and cultured in the presence of increasing concentrations of imatinib. CFU-GM colonies were counted after 10 days. (C) CFC assays with human donor bone marrow cultured in the absence or presence of increasing concentrations of imatinib. CFU-GM colonies were counted after 14 days. All CFU results are presented as a percentage of untreated controls. Results represent combined data from three independent experiments with three replicates per experiment. A Kruskal-Wallis nonparametric test was used to determine significance of each concentration compared to control. (D) Effect of anti-c-Kit neutralizing antibody. 3μg of either anti-c-Kit neutralizing antibody (ACK2) or the IgG2b isotype control was administered for five to seven days, and the number of myeloid cells, including neutrophils, monocytes, and eosinophils, and lymphoid cells, including B cells and T cells and NK cells, in the blood determined by flow cytometry. Average fold changes compared to the isotype control on combined myeloid or lymphoid cell populations are presented. (E) Mice were treated with imatinib at 66 or 200mg/kg/day or with pumps filled with water for 28 days, the vehicle for imatinib. On day 7, mice were bled from the tail vein and numbers of neutrophils determined by flow cytometry with 6 mice per condition. For all data sets, the line in each group of data points represents the median. (F) Effects of dosage on neutrophil and monocyte numbers in bone marrow. Gleevec was administered at 66 or 200 mg/kg/d for 7 days without infection, and neutrophils were enumerated by flow cytometry. A Mann-Whitney nonparametric test was used to determine significance. Data shown are representative of three independent experiments.
Fig 5.
Effects of Imatinib on CXCR2 expression, activation, and apoptosis in neutrophils.
C57Bl/6 mice were administered imatinib at 66 mg/kg/d or left untreated. Beginning 24h post-treatment mice were either injected in the tail vein with 105 CFU Mm 1218R or left uninfected. At 7d post-treatment, bone marrow was collected from both femurs. (A) Representative flow cytometry plots of CXCR4 and CXCR2 expression on neutrophils from bone marrow using flow cytometry. CXCR2hiCXCR4lo and CXCR4hiCXCR2lo subsets were identified, and frequencies of total neutrophils are displayed in boxes. (B) Median fluorescence intensity (MFI) of CXCR2 and CXCR4 on neutrophils from bone marrow. Cumulative data from two independent experiments are presented with six mice per condition. The line in each data set represents the median. (C) C57Bl/6 mice were treated as in A. Activation status was assessed by surface expression of CD66b (secondary granules), CD63 (primary granules) and apoptosis by intracellular staining for caspase 3 and 7 activity of the total neutrophils (Ly6G+Ly6C+) from spleen. Cumulative data from two independent experiments are presented with 6 mice per condition. (D) C57Bl/6 mice were administered imatinib at 66mg/kg/d or left untreated. On d7, splenic neutrophils were isolated using Ly6G+ microbeads and 4 x106 cells were injected via the left tail vein into each naïve recipient mice, and infected via the right tail vein with 105 CFU Mm. Forty eight hours later, spleens were harvested and CFU/gram were determined. CFU/g spleen from mice injected with carrier, or with neutrophils from imatinib-treated mice or mice treated with carrier (water). Cumulative data from three independent experiments are presented with 15–25 animals per condition. The line represents the median. A Mann-Whitney test was used for pairwise comparisons, and a Kruskal Wallis test for multiple comparisons.
Fig 6.
Imatinib decreases bacterial load of pathogenic Francisella spp. in vivo.
(A-B) C57Bl/6 mice were treated with imatinib at 66 mg/kg/d or water for 7d prior to infection and for the duration of the experiment, and then injected subcutaneously with either ~6x106 Fn (A) or ~2x105 LVS (B). After 48h for Fn or 5d for LVS, skin and spleens were collected and CFU/gram of tissue was determined. The limit of detection of the assay was 100 CFUs. (C) Images of lesions on control or imatinib-treated mice infected with LVS. Hair was removed from injection site. The lesions are indicated by white arrows. (D) Quantitation of lesions from control or imatinib-treated mice. Lesions were scored on a scale of one to three, with a score of one denoting no visible lesion, two a visible lesion but without a break in the skin, and three an open wound. 15 animals were scored per condition. (E) Four days following infection with strain Ft Schu S4, CFU/gram of tissue was determined. The limit of detection of the assay was 100 CFU/gm for Ft. The line in each data set represents the median. A Mann-Whitney nonparametric test was used to determine significance. Combined data from three independent experiments are shown. (F) Effects of imatinib at 66mg/kg/d on neutrophils and monocytes upon infection with LVS. Data from a representative experiment are shown. (G) Summary of effects of Imatinib on myelopoiesis and migration of mature myeloid cells to the blood and organs. Imatinib at doses of 66 or 200 mg/kg/day induces activation, expansion, and maturation of hematopoietic stem cells (HSCs) and multipotent hematopoietic progenitors 1–4 (MPP1-4) in the bone marrow. Imatinib at these doses also stimulates a lineage determination step that induces MPPs to differentiatiate into myeloid precursors and then mature cells (green arrow), but not into cells of the lymphoid lineage (red arrow). Data with low doses of neutralizing antibodies against the c-Kit tyrosine kinase support the hypothesis that effects on HSCs and MPPs are mediated by partial inhibition of c-Kit, whereas effects of the drug on lineage determination appears to be independent of c-Kit. Ultimately, imatinib increases the numbers of mature myeloid cells in the bone marrow, an effect seen with drug alone; the accumulation of mature cells is not evident with the drug in context of infection, which itself appears to mobilize migration of mature cells from the bone marrow to the periphery. Notably, increased numbers of mature cells are only evident in the blood and tissues at lower doses (66mg/kg/d), suggesting that at low doses, Imatinib induces migration of mature cells out of the bone marrow; alternatively, the drug may inhibit migration at higher doses (200mg/kg/d). Importantly, Imatinib mimics the “emergency response” to infection, which likewise induces HSC activation, MPP expansion, myelopoiesis, and migration of mature myeloid cells from the bone marrow to the periphery (green arrows).