Ischemic stroke activates hematopoietic bone marrow stem cells

Abstract

Rationale: The mechanisms leading to an expanded neutrophil and monocyte supply after stroke are incompletely understood.

Objective: To test the hypothesis that transient middle cerebral artery occlusion (tMCAO) in mice leads to activation of hematopoietic bone marrow stem cells.

Methods and results: Serial in vivo bioluminescence reporter gene imaging in mice with tMCAO revealed that bone marrow cell cycling peaked 4 days after stroke (P<0.05 versus pre tMCAO). Flow cytometry and cell cycle analysis showed activation of the entire hematopoietic tree, including myeloid progenitors. The cycling fraction of the most upstream hematopoietic stem cells increased from 3.34%±0.19% to 7.32%±0.52% after tMCAO (P<0.05). In vivo microscopy corroborated proliferation of adoptively transferred hematopoietic progenitors in the bone marrow of mice with stroke. The hematopoietic system's myeloid bias was reflected by increased expression of myeloid transcription factors, including PU.1 (P<0.05), and by a decline in lymphocyte precursors. In mice after tMCAO, tyrosine hydroxylase levels in sympathetic fibers and bone marrow noradrenaline levels rose (P<0.05, respectively), associated with a decrease of hematopoietic niche factors that promote stem cell quiescence. In mice with genetic deficiency of the β3 adrenergic receptor, hematopoietic stem cells did not enter the cell cycle in increased numbers after tMCAO (naive control, 3.23±0.22; tMCAO, 3.74±0.33, P=0.51).

Conclusions: Ischemic stroke activates hematopoietic stem cells via increased sympathetic tone, leading to a myeloid bias of hematopoiesis and higher bone marrow output of inflammatory Ly6C(high) monocytes and neutrophils.

Keywords: bone marrow; hematopoietic stem cells; monocyte; stroke.

Figure 1. Stroke increases bone marrow progenitor activity

A, Infarct size from TTC-stained brain sections on day 1 and day 3 after stroke induced by tMCAO in C57BL/6 mice (n= 4–6 mice per group). Flow cytometric-based enumeration of (B) CD11b⁺ myeloid cells, (C) neutrophils and (D) monocyte subsets per femur following tMCAO (n= 4–6 mice per group, one-way ANOVA). E, Representative bioluminescence images of MITO-Luc mice monitored one day before (baseline) and after tMCAO over a 2-week period. Quantification of luciferase activity in MITO-Luc mice following tMCAO at indicated time points is shown on the right (n= 4 mice per group, one-way ANOVA). F, Bone marrow (BM) colony-forming unit (CFU) assay on day 4 after tMCAO (n = 8 mice per group, Mann-Whitney test). G, Representative gating strategy and FACS dot plots for GMP (Lin⁻ c-kit⁺ Sca-1⁻ CD16/32⁺ CD34⁺) and MDP (Lin⁻ c-kit⁺ Sca-1⁻ CD16/32⁺ CD34⁺ CD115⁺) in the bone marrow on day 3 after tMCAO. Bar graphs display both frequencies in whole bone marrow and absolute numbers of cells per femur (n = 6–10 mice per group, Mann-Whitney test). H, On day 3 after tMCAO, mice were given BrdU i.p (1mg) and BrdU incorporation in myeloid progenitors was analyzed 24 hours later. Representative FACS dot plots show BrdU⁺ GMP (upper panels) and MDP (lower panels). Bar graphs on the right display percentages of BrdU⁺ GMP and MDP progenitors. I, Absolute numbers of neutrophil (upper panels) and monocytes per femur (lower panels). J, Analysis of BrdU incorporation in bone marrow neutrophils and monocytes following a single injection of BrdU (n= 4–6 mice per group, Mann-Whitney test). Mean ± s.e.m., *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2. Stroke decreases activity of lymphoid progenitors in the bone marrow

A, Representative flow cytometry staining of CLP (Lin⁻ IL7Rα⁺ c-kit^int Sca-^int) from bone marrow cells on day 3 after tMCAO. B, Representative staining of immature B cells (Lin⁻ B220^int CD93⁺) from bone marrow cell suspensions. Bar graphs show both frequencies of lymphoid progenitors in total bone marrow cells and total cell numbers per femur. n= 4–6 mice per group, one-way ANOVA. Mean ± s.e.m., *P < 0.05, **P < 0.01.

Figure 3. Bone marrow LKS cells exhibit a myeloid bias after stroke

Experimental stroke was induced or not in C57BL/6 mice by tMCAO and LKS from the bone marrow were sorted by FACS three days later. Changes in gene expression levels were evaluated by RT-qPCR. n = 4–5 mice per group, Mann-Whitney test. Mean ± s.e.m., *P < 0.05, **P < 0.01.

Figure 4. The bone marrow response after stroke occurs at the most upstream hematopoietic stem cell level and correlates with injury size

Bone marrow cell suspensions were stained for LKS and HSC on days 1 and 3 after tMCAO in C57BL/6 mice. A, Representative staining, frequencies and numbers of LKS (Lin⁻ c-kit ⁺ Sca-1⁺; upper panels) and HSC (Lin⁻ c-kit ⁺ Sca-1⁺ CD48 ⁻ CD150⁺; lower panels) per femur. B, Correlation between frequencies and numbers of SLAM HSC with infarct sizes was evaluated from different cohorts of mice on day 3 after tMCAO. C, FACS analysis of Ki-67⁺ LKS and HSC cells at indicated time points after experimental stroke. n= 6 mice per group, one-way ANOVA. Mean ± s.e.m., *P < 0.05.

Figure 5. Confocal imaging of LKS progenitor expansion in bone marrow of mice with stroke

Mice were injected intravenously with a mixture of FACS-sorted Lin⁻ c-kit ⁺ Sca-1⁺ labeled ex-vivo with red CM-Dil and green SP-DiOC18(3) fluorescent dyes prior to stroke induction. Imaging of whole mount sternal bone marrow preparations was performed three days after tMCAO. Fluorescent signal from the bisphosphonate imaging agent Osteosense-750 is depicted in blue and outlines bone. Vascular endothelial cells were stained by intravenous injection of fluorescently labeled antibodies targeting CD31, Ve-Cadh and Sca1. Scale bar represents 200 μm (low magnification) and 50μm (high magnification). The bar graph on the right displays the number of either green (SP-DiOC18) or red (CM-Dil) clusters with ≥2 cells per sternal preparation (n=3 mice per group). Neighboring cells of mixed color were not detected (ND). Mean ± s.e.m., *P < 0.05.

Figure 6. Serial intravital microscopy reports increased HSC expansion in the bone marrow of mice with stroke

Mice with and without stroke were transplanted with 20, 000 FACS-sorted donor Lin⁻ c-kit ⁺ Sca-1⁺ CD48 ⁻ CD150⁺ HSC labeled ex-vivo with DiD fluorescent dye. Intravital microscopy of the mouse calvarium was performed serially, one day before and again 3 days after tMCAO. Blue color represents the fluorescence signal produced by the bone imaging agent Osteosense-750; the fluorescence lectin signal stained blood vessels in red. DiD labeled hematopoietic stem cells (HSC) are shown in white. Scale bar represents 50 μm. Bar graph displays the fold increase of HSC per field of view between the two imaging sessions in both groups (n=3 mice per group). Mean ± s.e.m., *P < 0.05.

Figure 7. Stroke increases the sympathetic nervous activity in the bone marrow

A, One day following tMCAO, bone marrow norepinephrine content was measured by ELISA. B, Whole mount immunofluorescence staining of tyrosine hydroxylase rich nerve fibers of the sternal BM on day 1 after tMCAO. C, Bar graph shows quantification of tyrosine hydroxylase positive area per field of view. D, Quantitative real-time PCR of HSC retention/maintenance related genes in the bone marrow on day 1 after tMCAO. n= 4–6 mice per group, Mann-Whitney test. Mean ± s.e.m., *P < 0.05.

Figure 8. Adrenergic signaling regulates hematopoietic stem cell activation after stroke via the β3 adrenergic receptor

tMCAO was induced in wild type (A,B) and Adrb3^−/− mice (C,D) and cell cycle was analyzed in LKS progenitors and hematopoietic stem cells (HSC) one day later. A,C Representative FACS dot plots of cell cycle staining in LKS (upper panels) and HSC (lower panels). B, D, Bar graphs show flow cytometric analysis of LKS and HSC in both G1 and S/G2/M phases of the cell cycle. In wild type mice, HSC and LKS entered the active cell cycle phases, whereas Adrb3^−/− mice showed a lack of HSC activation after stroke. n= 4–8 mice per group, Mann-Whitney test. Mean ± s.e.m., **P < 0.01 versus naive controls of same genotype.