Bone marrow cell recruitment to the brain in the absence of irradiation or parabiosis bias - PubMed (original) (raw)

Bone marrow cell recruitment to the brain in the absence of irradiation or parabiosis bias

Katrin Kierdorf et al. PLoS One. 2013.

Abstract

The engraftment of bone marrow-derived cells has been described not only during diseases of the central nervous system (CNS) but also under healthy conditions. However, previous studies pointing to an ample bone marrow cell engraftment used irradiation-induced bone marrow chimeras that evoked severe alterations of the CNS micromilieu including disturbances of the blood brain barrier (BBB), damage of endothelial cells and local induction of proinflammatory cytokines. On the other hand, parabiosis experiments using temporarily joined circulatory systems generally yielded low levels of myeloid cell chimerism thereby potentially underestimating bone marrow cell turnover with the CNS. To avoid these drawbacks we established a protocol using the alkylating agent busulfan prior to allogenic bone marrow transplantation from CX3CR1(GFP/+) donors. This regimen resulted in a stable and high peripheral myeloid chimerism, significantly reduced cytokine induction and preserved BBB integrity. Importantly, bone marrow cell recruitment to the CNS was strongly diminished under these conditions and only weakly enhanced during local neurodegeneration induced by facial nerve axotomy. These results underscore the requirement of local CNS conditioning for efficient recruitment of bone marrow cells, establish busulfan as an alternative treatment for studying bone marrow chimeras and suggest a critical re-evaluation of earlier chimeric studies involving irradiation or parabiosis regimens.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Reduced conditioning of the CNS after busulfan treatment.

A) Strongly diminished induction of proinflammatory cytokines and chemokines in busulfan-treated chimeras compared to irradiation protocols. Quantitative real-time PCR analysis of cytokine and chemokine induction in brains of busulfan-treated (grey columns) and whole-body irradiated (white columns) chimeras 24 hours (h), 7 days (d), 14 d and 16 weeks (w) after treatment. The mRNA expression was normalized to GAPDH and compared to untreated mice, indicated by the grey line. Data are shown as mean ± SEM. One out of two experiments is shown with three to six animals per group. Statistical significance is marked with asterisks (p<0.05 = *; p<0.01 = **; p<0.001 = ***). B) Largely preserved blood-brain-barrier (BBB) integrity after busulfan challenge. Direct fluorescence microscopic visualization depicting CD31+ endothelial cells (green), extravasal albumin (red) and nuclei (DAPI, blue). Arrow heads point to extravasated albumin. Representative pictures of cortices are shown. Scale bar = 100 µm. C) Albumin staining of the CNS parenchym is shown for both treated groups 24 hours (h), 7 days (d) and 14 d after treatment. Arrow heads point to extravasated albumin in superficial and deeper brain regions. Representative pictures are shown. Scale bar = 400 µm. Insert: Albumin staining of an untreated animal. Scale bar = 400 µm.

Figure 2

Figure 2. High peripheral blood myeloid chimerisms in busulfan-treated chimeras.

A) Representative FACS dot plots showing the expression of GFP in blood granulocytes, monocytes, B cells and T cells of busulfan-treated and irradiated CX3CR1GFP/+ → CX3CR1+/+ chimeras and untreated mice four weeks after bone marrow transfer. Percentages of the respective cell populations are indicated. CX3CR1+/+ mice served as untreated controls. B) Quantitative assessment of GFP+ expression in overall monocytes (SSCloCD11b+) and the two monocyte subsets SSCloCD11b+Ly6Chi and SSCloCD11b+Ly6Clo out of all SSCloCD11b+ cells. Data depict high reconstitution efficiencies and comparable chimerisms in both groups. One symbol represents one mouse (Busulfan-treated: filled circle; irradiated: filled squares). Mean ± SEM are shown. Six to seven animals per group were analysed. C) Photograph showing change of fur color 16 weeks after treatment. Irradiation leads to a loss of fur color, whereas busulfan-treated animals remain unaffected. Top: untreated animal, middle: whole-body irradiated animal, bottom: busulfan-treated mouse.

Figure 3

Figure 3. Engraftment of donor-derived GFP+ bone marrow cells into the brain strongly reduced in busulfan-treated animals.

The number of engrafted GFP+Iba-1+ donor-derived phagocytes is strongly decreased in the busulfan-treated animals compared to irradiated mice. A) Immunohistochemistry of chimeric brains shows GFP+Iba-1+ cells in the cortex, hippocampus, thalamus and choroid plexus of busulfan-treated and irradiated chimeric mice 16 weeks after reconstitution. Arrow heads indicate representative ramified GFP+ (green) and Iba-1+ (red) cells of donor origin in the CNS. Nuclei are counterstained with DAPI (blue). Bars = 200 µm. B) High magnification images of donor-derived GFP+ Iba1+ reveal ramified morphology of engrafted cells in both groups. Sections are stained for Iba-1 (red), CX3CR1 (GFP) and DAPI (blue). Scale bars = 50 µm. C) Quantification of ramified GFP+Iba-1+ cells in cortex, hippocampus, thalamus and choroid plexus. Each symbol represents one mouse. Data show significant differences in the number of engrafted GFP+Iba-1+ donor-derived cells in all investigated brain areas between the busulfan-treated (filled circle) and irradiated (filled squares) animals 16 weeks after bone marrow cell transfer. Asterisks indicate statistical significance (p<0.05 = *; p<0.01 = **; p<0.001 = ***). All graphs show mean ± SEM.

Figure 4

Figure 4. Recruitment of donor-derived GFP+ bone marrow cells into the lesioned brain depends on irradiation.

Following facial nerve axotomy, the recruitment of GFP+Iba-1+ donor-derived bone marrow cells to the lesioned N. facialis is strongly diminished in busulfan-treated mice compared to irradiated animals. A) Representative FACS dot plots of peripheral blood in the SSCloCD11b+ monocyte compartment in untreated, busulfan-treated and irradiated mice. B) Quantification of the peripheral blood chimerism shows sufficient and comparable reconstitution levels in busulfan-treated (filled circles) and irradiated mice (filled squares). One symbol represents one mouse. Chimerism was analyzed nine weeks after reconstitution. Six animals per group were analysed. All graphs show mean ± SEM. C) Immunohistochemistry of recruited GFP+Iba-1+ phagocytes in the lesioned facial nucleus two weeks following axotomy. Elevated numbers of ramified donor-derived GFP+ cells were found in the degenerating neurons (NeuN, red) of irradiated CX3CR1GFP/+ → CX3CR1+/+ mice compared to busulfan-treated CX3CR1GFP/+ → CX3CR1+/+ animals. GFP+ cells were in close proximity to NeuN-immunoreactive neurons in the facial nucleus. No GFP expressing cells were found on the control site in busulfan-treated mice, in contrast to irradiated mice where a few GFP+ cells could be found. Scale bars: overview = 200 µm; detail = 50 µm. Nuclei were stained with DAPI (blue). D) Quantification of engrafted GFP+ Iba-1+ cells 14 days after axotomy. A robust engraftment of donor-derived GFP+ cells was found in irradiated chimeric mice (filled squares), whereas only few GFP+ cells were detectable in busulfan-treated chimeric mice (filled circle). Symbols indicate individual mice. Data are expressed as mean ± SEM. Asterisks indicate statistical differences (p<0.001 = ***).

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MP is supported by the Deutsche Forschungsgemeinschaft (DFG) funded research unit (FOR) 1336 “From monocytes to brain macrophagesconditions influencing the fate of myeloid cells in the brain”. MP is further granted by the BMBF-funded competence network of multiple sclerosis (KKNMS), the competence network of neurodegenerative disorders (DZNE), the centre of chronic immunodeficiency (CCI), and the DFG (SFB 620, PR 577/8-2). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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