Endothelial cells are a critical component of the bone marrow (BM) stromal network
that maintains and regulates hematopoietic cells
. Vascular regeneration precedes, and is necessary for, successful hematopoietic stem
cell (HSC) transplantation, the only cure for most hematopoietic diseases
. Recent data suggest that mature hematopoietic cells can regulate BM stromal cells
. Whether a similar crosstalk regulates the BM vasculature is not known. Here we find
that donor hematopoietic cells act on sinusoidal endothelial cells to induce host
blood vessel and hematopoietic regeneration after BM transplantation in mice. Adoptive
transfer of BM, but not peripheral, granulocytes prevented the death of mice transplanted
with limited HSC numbers and accelerated recovery of host vessels and hematopoietic
cells. Moreover, selective granulocyte ablation in vivo impaired vascular and hematopoietic
regeneration after BM transplantation. Gene expression analyses indicated that granulocytes
are the main source of the cytokine TNFα, whereas its receptor, TNFR1, is selectively
upregulated in regenerating blood vessels. In adoptive transfer experiments, wild-type,
but not Tnfa−/−
, granulocytes induced vascular recovery, and wild-type granulocyte transfer did not
prevent death or promote vascular regeneration in Tnfr1−/−:Tnfr2−/−
mice. Thus, by delivering TNFα to endothelial cells, granulocytes promote blood vessel
growth and hematopoietic regeneration. Manipulation of the crosstalk between granulocytes
and endothelial cells may lead to new therapeutic approaches to improve blood vessel
regeneration and increase survival and hematopoietic recovery after HSC transplantation.
Blood cell production takes place in the bone marrow (BM), where a network of non-hematopoietic
stromal cells provides a unique microenvironment that maintains and regulates differentiating
. The endothelial cells that form the blood vessels of the BM are key constituents
of this stromal network, producing angiocrine factors such as CXCL12, SCF, Pleiotrophin,
and Notch ligands that regulate hematopoietic stem cells and progenitors
and other stromal cells
. Endothelial cells also play a critical role after HSC transplantation by producing
angiocrine factors that promote hematopoietic regeneration
. The myeloablative treatments used to eliminate host hematopoietic cells severely
damage BM blood vessels
, which must regenerate before they can support hematopoiesis
. Therapies that promote vessel survival or regeneration also promote hematopoietic
. Recent studies have shown that mature hematopoietic cells like macrophages
and peripheral blood neutrophils
can regulate the function of BM osteoblastic stromal cells, but it is not known whether
mature blood cells also regulate the BM vasculature.
To test whether hematopoietic cells crosstalk with the vasculature in the context
of BM transplantation and regeneration, we transplanted different amounts of CD45.2+
bone marrow nucleated cells (BMNCs) into lethally irradiated CD45.1+ recipient mice.
Fourteen days later the transplanted mice showed a dose-dependent increase in the
numbers of hematopoietic and CD45−Ter119−CD31+CD105+ endothelial cells in the BM (Fig.
1a,b and Supplementary Fig. 1a). To analyze the vascular network of these mice, we
injected αCD31 and αCD144 antibodies intravenously, followed by dissection and 3D
whole-mount imaging of the sternal vasculature
. This allowed us to distinguish between true vessels—those with a perfused lumen—and
vascular sheets that appear after vascular injury (Supplementary videos 1 and 2).
Mice transplanted with a higher number of BMNCs showed a denser vascular network with
more numerous and longer vessels, as compared to mice transplanted with a lower number
of BMNCs (Fig. 1c,d and Supplementary videos 1 and 2). In agreement with these results,
we found reduced vascular permeability (as measured by Evans Blue extravasation
) in mice transplanted with a higher number of donor BMNCs (Fig. 1e). To test whether
the endothelial cells observed in the sternal vasculature after transplantation were
host or donor derived, we transplanted WT mice with 20×106 BMNCs purified from Ubc-gfp
mice, which constitutively express GFP in all cells. We did not detect GFP expression
in the endothelial cells from the recipients , indicating that virtually all endothelial
cells were host derived (Fig. 1f). Transplantation of a higher number of BMNCs led
to increased endothelial cell recovery early after transplantation (Supplementary
Fig. 1b) and reductions in the fractions of necrotic endothelial cells (Supplementary
Fig. 1c), but had no effect on the cycling of endothelial cells (Supplementary Fig.
1d). These data demonstrate that donor hematopoietic cells promote endothelial cell
survival and drive the recovery of a functional, host-derived, vascular network in
the recipient mice.
These results were surprising because hematopoietic progenitors have been reported
to inhibit vascular regeneration via angiopoietin-1
. We thus investigated whether the observed vascular recovery was mediated by hematopoietic
cells that are more mature than the progenitors previously investigated. After FACS-purifying
CD45.2+ hematopoietic cells into subpopulations based on the expression of mature
lineage markers, we adoptively transferred each of these subpopulations, together
with 105 CD45.1+ BMNC (as a source of donor HSCs), into γ-irradiated CD45.2+ recipients;
each subpopulation was transplanted at the same ratio as it is present in vivo. Since
adoptively-transferred cells are thought to have limited self-renewal capacity in
vivo, we adoptively transferred the same populations every 2–3 days for 2 weeks (Fig.
2a). Of the mature cell types tested , only Gr1+CD115− granulocytes, which contain
all BM neutrophils and late neutrophil progenitors
, were capable of inducing rapid recovery of endothelial cells (Fig. 2b). Notably,
granulocyte transfer also promoted survival of the recipient mice (Fig. 2c) and led
to faster recovery of hematopoietic cells in the periphery (Fig. 2d). In agreement
with previous studies
that demonstrated a strong correlation between endothelial cell recovery and survival,
we found that moribund mice showed less vascular recovery (Supplementary Fig. 2a).
Imaging of the sternal vasculature showed that granulocyte-treated mice had more blood
vessels than PBS-treated controls or mice transferred with monocyte/macrophages, lymphocytes
or erythroid-lineage cells (Fig. 2e,f). Granulocyte transfer also rescued vascular
leakiness in irradiated mice, but only when transferred at very high doses (Fig. 2g).
Granulocyte-induced recovery was not mediated by induction of systemic inflammation,
as we did not detect significant increases in inflammatory cytokine levels in BM or
plasma (Supplementary Fig. 2b); injury to peripheral tissues, as assessed by pathology
analyses (Supplementary Fig. 2c); or increased granulocyte recruitment to peripheral
tissues (Supplementary Fig. 2d). We also did not find increased endothelial cell recovery
in peripheral tissues in granulocyte-treated mice (Supplementary Fig. 2e).
Transfer of common myeloid progenitors can protect lethally irradiated mice from death
until the few host HSC that survive irradiation can restore hematopoiesis
. To test if granulocytes induce survival through a similar mechanism, we adoptively
transferred granulocytes without accompanying BMNCs into lethally irradiated mice.
In the absence of donor hematopoietic stem and progenitor cells (HSPCs), granulocytes
were not capable of rescuing irradiation-induced death (Supplementary Fig. 2f).
Next, we performed granulocyte transfer together with BMNCs and followed vascular
and hematopoietic recovery up to 4 weeks after transplantation (Fig. 2h and Supplementary
Fig. 3a,b). These analyses revealed that compared to BMNCs alone, granulocyte transfer
promoted endothelial cell survival early after transplantation, as evidenced by reduced
numbers of apoptotic and necrotic endothelial cells (Fig. 2i); this endothelial protection
was comparable to that induced by transfer of a high number of BMNCs (Supplementary
Fig. 1c). Moreover, we found that granulocytes induced expression of Cflar (also known
as cFlip), which encodes an antiapoptotic prosurvival factor
, in BM endothelial cells in vivo (Supplementary Fig. 3c). In contrast granulocytes
did not induce expression of hematopoietic supportive molecules such as Cxcl12, Scf,
or Notch ligands, or lead to activation of Hif1a or its downstream targets (Supplementary
Fig. 3d-f). These results indicate that granulocytes promote endothelial cell survival.
To further analyze the effect of granulocytes on hematopoietic recovery, we performed
lineage tracing experiments (Supplementary Fig. 4a). In granulocyte-treated mice,
the increase in peripheral blood and BM hematopoietic recovery was associated with
persistence of the transferred BM granulocytes (Supplementary Fig. 4d,g) and increased
production of host cells (Supplementary Fig. 4c-h). Notably, adoptive transfer of
peripheral blood neutrophils, which home to the bone marrow and crosstalk with CXCL12
abundant reticular stromal cells to regulate HSC trafficking
, did not induce vascular or hematopoietic recovery (Supplementary Fig. 4b-h), and,
in contrast to BM granulocytes, did not persist in the recipient mice (Supplementary
Fig. 4d,g). We hypothesized that increased vascular recovery might facilitate engraftment
of donor-derived HSPCs. To test this concept we performed competitive transplants.
We FACS-purified donor CD45.1+ cells from primary recipients which had been transplanted
with or without granulocytes and transplanted the CD45.1+ cells, together with competitor
CD45.1+:CD45.2+ BMNCs, into lethally irradiated CD45.2+ secondary recipients (Fig.
2a). Mice transplanted with CD45.1+ BMNCs from granulocyte-treated mice displayed
increased short-term T- and myeloid cell, but not long-term (16 weeks) multilineage,
CD45.1+ cell contribution to the peripheral blood (Fig. 2j). These indicate that granulocyte-induced
vascular recovery promotes engraftment of short-lived, lineage-committed, hematopoietic
progenitors but not HSC. Taken together, these results demonstrate that adoptive transfer
of BM granulocytes is sufficient to promote survival and drive vascular and hematopoietic
cell recovery after myeloablation, without exhausting the donor HSC pool.
Next, to test whether granulocytes produced by BMNC transplantation promote vascular
regeneration, we bred Mrp8-cre-IRES-GFP mice, in which Cre expression is largely restricted
to granulocytes and a fraction of granulocyte monocyte progenitors (GMP), with iDTR
mice, in which the diphtheria toxin receptor (DTR) is induced by Cre-mediated recombination.
We transplanted lethally irradiated recipients with 5×106 BMNCs purified from iDTR
or Mrp8-cre-IRES-GFP:iDTR mice, followed by DT treatment. This strategy led to efficient
granulocyte ablation in the recipient mice transplanted with Mrp8-cre-IRES-GFP:iDTR,
but not control iDTR, BMNCs (Fig. 3a,b). In the mice transplanted with Mrp8-cre-IRES-GFP:iDTR
BMNCs, granulocyte ablation led to impaired vascular regeneration and increased vascular
leakage (Fig. 3c-e) and impaired peripheral blood cell recovery (Fig. 3f), as well
as reduced numbers of monocytes and macrophages in the BM (Fig. 3g-i). To confirm
that the monocyte/macrophage loss was not responsible for the reduced number of blood
vessels, we ablated granulocytes in vivo by injection of α-Ly6G, an antibody that
ablates granulocytes exclusively
. Treatment with α-Ly6G, but not an isotype control antibody, depleted granulocytes
(Fig. 3j) and impaired vascular regeneration (Fig. 3k), and also caused loss of BM
monocytes and lymphocytes (Supplementary Fig. 5a,b). In a complementary approach we
transplanted recipient mice with a single graft of total BMNCs or a graft in which
the granulocytes were removed prior to transplantation via FACS. As expected, mice
transplanted with a granulocyte-depleted graft showed reduced granulocyte numbers
(Fig. 3l) and impaired endothelial and hematopoietic regeneration 6 days after transplantation
(Fig. 3m and Supplementary Fig. 5c,d). These experiments demonstrate that donor-derived
granulocytes are necessary for efficient vascular regeneration.
To investigate the mechanisms through which granulocytes promote vascular regeneration,
we profiled BM granulocytes for the expression of multiple angiogenic factors, as
assessed by qPCR. We detected expression of fibroblast growth factor 1 (Fgf1), progranulin
(Prgn), pleiotrophin (Ptn), vascular endothelial growth factor A (Vegfa) and TNFα
(Tnfa, Supplementary Fig. 6a). We focused on TNFα because Tnfa was enriched in BM
granulocytes (Supplementary Fig. 6b), Tnfr1 (Tnfrsf1a, encoding a TNFα receptor) was
enriched in endothelial cells as compared to other BM stromal cells (Supplementary
Fig. 6c), Tnfr1 was upregulated in regenerating endothelial cells (Supplementary Fig.
6c) and TNFα induces cFLIP expression
. TNFα is a powerful angiogenic factor in mice
and neutrophil-derived TNFα drives blood vessel formation during zebrafish development
. Analyses of Tnfa−/−
mice revealed fewer endothelial cells in the steady-state bone marrow (Supplementary
Fig. 7), indicating that TNFα regulates vascular homeostasis.
To test whether TNFα also plays a role in regeneration, we myeloablated WT or Tnfa−/−
mice with a single injection of 5-fluorouracil. As compared to WT mice, Tnfa−/−
mice displayed reduced numbers of BM endothelial cells and rapidly succumbed to this
treatment (Fig. 4a,b). The reduced survival of Tnfa−/−
mice suggests a defect in endothelial regeneration, but might also be due to the reduced
number of BM endothelial cells in the steady-state. Moreover, injection of recombinant
TNFα induced faster vascular recovery in 5-fluorouracil-treated WT and Tnfa−/−
mice and delayed death in Tnfa−/−
mice (Fig. 4a,b). These results indicate that TNFα is needed for normal BM endothelial
cell numbers during homeostasis and that TNFα can directly promote endothelial regeneration.
BM granulocytes expressed the membrane-bound form of TNFα, as assessed by flow cytometry
(Supplementary Fig. 8a). To test whether granulocytes promote vascular regeneration
specifically via TNFα production, we performed adoptive transfer experiments using
granulocytes purified from WT and Tnfa−/−
mice. To distinguish between the regenerative effects of granulocytes on sinusoids
and arterioles, we used Nestin-gfp mice; in this model, Nestin-GFPbright cells label
. WT but not Tnfa−/−
granulocytes induced sinusoidal vessel recovery, as demonstrated by quantification
of endothelial cells by FACS (Fig. 4c) and sinusoidal and arteriolar vessel numbers
by 3D whole-mount immunofluorescence (Fig 4d,e). In addition, WT but not Tnfa−/−
granulocytes rescued survival of the recipient mice and led to faster white and red
blood cell recovery in the peripheral blood (Fig. 4f,g). Imaging analyses showed that,
during regeneration, granulocytes preferentially localized to sinusoids: 76% of granulocytes
were found within 5µm of a sinusoid, whereas a much smaller fraction (14%) localized
to Nestin-GFPbright arterioles (Fig. 4h,i). B lymphocytes, which did not induce regeneration
(Fig. 2b), showed fewer interactions with BM vessels than did granulocytes (Supplementary
Fig. 8b,c). Granulocyte recruitment to regenerating vessels was independent of CXCR4
and CXCR2 (Supplementary Fig. 8d-f), the major receptors that regulate neutrophil
trafficking during homeostasis
, indicating that other mechanisms regulate granulocyte recruitment to injured vessels.
Adoptive granulocyte transfer failed to promote survival and vascular regeneration
mice, as compared to control mice, after transplantation (Fig. 4j,k), suggesting that
the crosstalk between granulocytes and endothelial cells is direct.
To confirm that granulocyte-derived TNFα does not promote regeneration via effects
on hematopoietic cells , we transplanted WT recipients with an initial graft of 105
WT or Tnfr1−/−:Tnfr2−/−
BMNCs followed by adoptive transfer of WT granulocytes. We found reduced peripheral
blood and BM hematopoietic recovery in mice transplanted with Tnfr1−/−:Tnfr2−/−
as compared to WT BMNCs (Supplementary Fig. 9a,b), consistent with previous work showing
that, during regeneration, TNFα acts on HSPC to promote engraftment
. Mice transplanted with Tnfr1−/−:Tnfr2−/−
BMNCs also showed delayed vascular regeneration (Supplementary Fig. 9c), associated
with a deficit in granulocyte production (Supplementary Fig. 9a). Adoptive transfer
of WT granulocytes rescued death (Supplementary Fig. 9d) and dramatically increased
vascular and hematopoietic recovery (Supplementary Fig. 9a,b) in mice transplanted
with either WT or Tnfr1−/−:Tnfr2−/−
BMNCs. These demonstrate that granulocyte-derived TNFα does not act on hematopoietic
cells to promote vascular regeneration
Myeloid cells are key players in vascular remodeling and regeneration in many tissues
. Our data demonstrate that granulocytes, the most abundant cells in the BM, interact
with the BM microenvironment to drive vascular regeneration specifically via TNFα.
Since endothelial cell-derived factors (e.g. Notch ligands, EGF, Pleiotrophin)
and vessel regeneration
are necessary for hematopoietic recovery, these results suggest the existence of a
positive feedback loop after BM injury. In this loop, endothelial cell regeneration
drives hematopoietic progenitor proliferation and generation of granulocytes, which
in turn support further vessel regeneration. Our results also indicate that granulocytes
overcome the inhibition of vascular regeneration induced by hematopoietic progenitors
. After transplantation, HSCs and multipotent progenitors preferentially generate
, a preference believed to satisfy the physiological demand for peripheral neutrophils
to prevent infections. Our results suggest that this myeloid bias, by generating granulocytes,
might also contribute to recovery by promoting vessel regeneration.
The effects of TNFα on HSPC function are controversial. Some reports suggest that
TNFα acts on HSPCs to promote their maintenance in the steady state and engraftment
. Other reports suggest that TNFα acts on HSPCs to inhibit their function during homeostasis
and that TNFα-receptor blockade prior to transplantation promotes HSPC survival
. Our results support the notion that TNFα has pro-regenerative effects on HSPCs.
Beyond its effects on hematopoietic cells, our results indicate that granulocytes
employ TNFα to drive vascular regeneration. Granulocytes also produce other pro-regenerative
factors, such as VEGF, which is necessary for BM vascular regeneration
and can recruit angiogenic myeloid cells to the heart
, and pleiotrophin, which promotes hematopoietic recovery by inducing Ras activation
. Future studies are needed to determine whether these factors cooperate with TNFα
in granulocyte-driven regeneration.
Our data suggest that the composition and cellular output of the initial graft affect
the recovery of host vessels and hematopoietic cells which may have implications in
the context of clinical HSC transplantation. In patients especially vulnerable to
myeloablation, such as those with DNA repair defects or those in which the BM stroma
has been damaged by prior chemotherapy treatment
, treatment with BM granulocytes could potentially be utilized to ameliorate vascular
injury and promote hematopoietic recovery.
C57BL/6J (CD45.2+) and B6.SJL-Ptprca Pepcb
/BoyJ (B6.SJL, CD45.1+) were purchased from the Jackson laboratory and bred in-house.
C57BL/6j:B6.SJL hybrids (CD45.2+:CD45.1+) were generated by breeding C57BL/6J with
B6.SJL mice. Ubc-gfp mice
, Mrp8-cre-IRES-gfp (Mrp8-cre)
mice were originally purchased from the Jackson laboratory. Tnfr1−/−
mice were also purchased from the Jackson laboratory and then backcrossed for four
additional generations into the C57BL/6J background and then bred in-house. Nestin-gfp
were a gift from Paul S. Frenette. All experiments were performed in 8–14 week old
male mice. All mice were housed at the SPF facility managed by the Unit for Laboratory
Animal Medicine (ULAM) at the University of Michigan. Experiments complied with all
relevant ethical regulations and were approved by the Institutional Animal Care and
Use Committee at the University of Michigan.
Bone marrow isolation
Mice were euthanized by isoflurane overdose. Bone marrow was harvested by flushing
mouse long bones with 1 ml of ice-cold PEB (2mM EDTA 0.5% Bovine serum albumin in
PBS). Red blood cells were lysed once by adding 1 mL of RBC Lysis Buffer (NH4Cl 150mM,
NaCO3 10mM, EDTA 0.1mM). Cells were immediately decanted by centrifugation, resuspended
in ice-cold PEB and used in subsequent assays. Due to the known effect of circadian
rhythm effects in granulocytes and HSCs
, we performed BM harvest at zeitgeber times 3–6 in mice under standard (12h light:12h
Peripheral blood analyses
Blood was collected from the facial vein in tubes containing EDTA. White blood cell
(WBC), red blood cell (RBC) and platelet counts were obtained using an Advia Counter
(Siemens) or a Hemavet 950 (Drew Scientific). Prior to flow cytometry staining and
analyses, red blood cells in peripheral blood were lysed once by adding 1 mL of RBC
Lysis Buffer (NH4Cl 150mM, NaCO3 10mM, EDTA 0.1mM). Cells were immediately decanted
by centrifugation, resuspended in ice-cold PEB and used in subsequent assays.
To purify the stromal cell fraction of the bone marrow (including endothelial cells),
we used a modified version of the serial digestion protocol developed by the Simmons
. Digestion buffer was made using 2 mg/ml Collagenase Type IV (Gibco, 17104-019) and
3 mg/ml Dispase (Gibco, 17105-041) dissolved in room temperature PBS. We harvested
the BM by flushing a tibia with 1 ml of digestion buffer into a 5 ml polypropylene
snap-cap tube containing another 1 ml of digestion buffer. We mixed the tubes vigorously
by hand and incubated at 37°C for 5–7 minutes. Following the first incubation, the
tubes were mixed vigorously by hand and then placed back at 37°C for another 5–7 minutes.
After this second incubation we collected the supernatant, taking care to leave any
macroscopic clumps in the tube. We transferred the digested cells to a tube containing
5 ml of ice-cold PEB. Then we added one ml of digestion buffer to the snap-cap tubes
and the process above was repeated until all macroscopic pieces of bone marrow had
been digested. The red blood cells were lysed once using RBC Lysis Buffer, filtered
through a 100 µm filter (Greiner Bio-one, 542-000), and then immediately spun down
in the centrifuge. The cells were resuspended in 1 ml of ice-cold PEB and used for
Cells were stained for 30 minutes in PEB buffer with the indicated antibodies and
analyzed in a BD LSRFortessa (BD Biosciences) or FACS-purified using a BD FACS Aria
II or a Synergy SY3200 Cell sorter (Sony). Dead cells and doublets were excluded based
on FSC and SSC distribution and DAPI (Sigma) exclusion. Data was analyzed using FACS
Diva software 8.0 (BDBiosciences). Antibodies used were against B220 (clone RA3-6B2,
Cat No: 103224), CD3 (clone 145-2C11, Cat No:100304), CD4 (clone GK1.5, Cat No:100422),
CD8 (clone 53-6.7, Cat No:100722), CD11b (clone M1/70, Cat No:101216 or 101204), CD16/32
(clone 93, Cat No:101328), CD19 (clone 6D5, Cat No: 115508), CD31 (clone A20, Cat
No:110724), CD41 (clone MWReg30, Cat No:133921 or clone D7, Cat No: 108104), CD45
(clone 30-F11, Cat No:103116), CD45.1 (clone A20, Cat No:110723 or 110708), CD45.2
(clone 104, Cat No:109845, 109823 or 109814), CD105 (clone MJ7/18, Cat No:120410),
CD115 (clone AFS98, Cat No:135506 or 135513), CD144 (clone BV13, Cat No: 138006),
CD150 (clone TC15-12F12.2, Cat No:115904), F4/80 (clone BM8, Cat No:123122), Gr1 (clone
RB6-8C5, Cat No: 108406 or 108404), Ly6-G (clone 1A8, Cat No: 127625), Sca-1 (clone
D7, Cat No:108106), Secondary antibody (Goat anti-rat IgG, clone Poly4054, Cat No:
405418), and Ter119 (clone TER-119, Cat No:116220), all from Biolegend, CD117 (clone
2B8, Cat No:105828 and 105833 from Biolegend or 562417 from BD Biosciences), Ki67
(clone SolA15, Cat No:50-5698-82 from ThermoFisher Scientific), Ly6G (clone 1A8, or
Cat No: BP0075-1 from Bioxcell) or isotype control (clone 2A3, Cat No: BP0089 from
Bioxcell), TNFα (clone MP6-XT22. Cat No: 506303). For cell cycle analyses, cells were
first fixed with 4% paraformaldehyde for 20 minutes at 4°C followed by permeabilization
of the plasma membrane using 0.1% Triton X-100 for 20 minutes at 4°C. The cells were
then stained for 30 minutes in PEB buffer containing 0.1% Triton X-100 with Ki67 (cat
No:50-5698-82). Lastly, the cells were incubated in PEB buffer containing DAPI for
one hour at room temperature before being analyzed as described above. Apoptotic cells
were detected using the CellEvent™ Caspase-3/7 Green Flow Cytometry Assay Kit from
Life Technologies (Cat No:C10427). Apoptotic cells were analyzed as described above.
Primary and secondary bone marrow transplantation and adoptive transfer experiments
Recipient male mice were conditioned with two doses of 600 rads using a 137Cs source
, 3 hours apart, and immediately transplanted with the indicated amount of donor BMNC
(retroorbital injection). For adoptive transfer experiments, the indicated doses of
FACS-purified hematopoietic cells were resuspended in 200µl of PBS and injected retroorbitally
into the recipient mice. Due to the known effect of circadian rhythm effects in granulocytes
, we performed adoptive transfers at zeitgeber times 10–12 in mice under standard
(12h light:12h dark) cycles.
In vivo granulocyte ablation
For diphtheria toxin-mediated granulocyte ablation, we treated mice with daily intraperitoneal
injections of diphtheria toxin (D0564, Sigma) (0.25µg/mouse/day) for one week starting
one week after transplantation. For αLy6G-mediated depletion experiments, we treated
mice with 100µg (intraperitoneal) of αLy6G (clone 1A8, BP0075-1, Bioxcell) or isotype
control (clone 2A3, BP0089, Bioxcell) at days 1, 3 and 5 after transplantation. Binding
of αLy6G prevents staining with αGr1. This prevented the use of Gr1 for detection
of granulocytes in αLy6G-injected mice. Thus, in these experiments, Ly6G+ granulocytes
were detected by staining BM cells with isotype or αLy6G antibodies: 1µg/ml of αLy6G
or isotype antibody was used , followed by staining with a secondary antibody (405418,
We injected mice (retroorbitally) with a single dose of 250mg of 5-fluorouracil (Sigma)
per kg of body weight in PBS.
Recombinant TNFα injection
We injected the mice (0.5µg/mouse, intraperitoneally) with recombinant mouse TNFα
(718004, Biolegend) daily.
AMD3100 and SBD
AMD3100 (5mg/kg s.c) and SBD (0.3mg/kg, i.p. SB225002, Sigma) were injected intraperitoneally
at days 1, 3, and 5 after transplantation and 1 hour before harvest.
Quantification of inflammatory cytokines in plasma and bone marrow extracellular fluid
Inflammatory cytokines were detected using the Legendplex Mouse inflammation panel
(740446, Biolegend), following the manufacturer’s instructions.
RNA isolation and qPCR analyses
RNA isolation was performed using the Dynabeads mRNA direct kit (Life Technologies)
following the manufacturer’s instructions. cDNA synthesis was performed using the
RNA to cDNA EcoDry Premix (Clontech) following the manufacturer’s instructions. qPCR
was performed using the SYBR Green Supermix Quanta Biosciences using a ABI PRISM 7900HT
Sequence Detection System (Appliedbiosystems). Results were analyzed using SDS 2.4
software (Appliedbiosystems). Oligonucleotides used for amplification are enumerated
in Supplementary Table 1.
Whole-mount immunofluorescence analyses
Imaging of the mouse BM sternal vasculature has been described previously
. Briefly, mice were injected (retroorbitally) with 5µg of Alexa Fluor 647 anti-mouse
CD31 (110724, Biolegend) and 5µg of Alexa Fluor 647 anti-mouse CD144 (138006, Biolegend).
Twenty minutes later, mice were euthanized by isoflurane overdose and the sterna retrieved.
For each sternum we removed muscle and connective tissue using a scalpel. The sternum
was then divided into segments and each segment cut sagitally to expose the BM cavity.
Segments were fixed in 4% paraformaldehyde (Sigma) in PBS for 20 minutes at room temperature,
washed thrice in PBS and imaged immediately or incubated in blocking solution (20%
Goat Serum (Sigma) in PBS) for two hours prior staining with antibodies. Granulocytes
or B cells were detected by staining in a solution containing 2.5µg/ml Alexa Fluor
488 anti-mouse Ly6-G (Clone 1A8, Biolegend) or Phycoerythrin conjugated anti-mouse
CD19 (Clone 6F5, Biolegend), 5% Goat Serum (Sigma) in PBS for 3 days. Prior to imaging,
each segment was glued to 35mm dishes and imaged in a Leica SP5 upright confocal microscope
using a 20X, long-distance, water immersion objective, Hybrid detectors, and Leica
Application Suite Advanced Fluorescence software. We acquired z-stacks (2.98µm between
slices) 20–200µm , depending on the position of the bone. Each slice measured 434.17
in the×axis and 434.17µm in the y axis and was 1024×1024 pixels. All images were acquired
at room temperature. For Alexa Fluor 647, the wavelength of the excitation laser was
647 nm and emitted light detected between 660 to 720nm. For Alexa Fluor 488, the wavelength
of the excitation laser was 488nm and emitted light detected between 500 to 540nm.
For Phycoerythrin, the wavelength of the excitation laser was 560nm and detection
from 570 to 600nm. We used Fiji
software to generate 3D reconstructions and maximum projections from each image. Blood
vessels numbers and length were counted manually in 3D reconstructions in order to
be able to distinguish true vessels (with lumen) from vascular sheets (lacking lumen).
Composite images of each sternum were assembled by stitching together the maximum
projection images in Power Point (Microsoft).
Mice received 200 µl of 0.5% Evans Blue Dye (Sigma Aldrich, E2129) in PBS via retro-orbital
injection. Thirty minutes later mice were euthanized and perfused with 5 ml of PBS
to remove excess Evans Blue from the vessels. To harvest the bone marrow, one femur
was flushed with 1 ml of ice-cold PEB. The cells were centrifuged and the supernatant
containing the extravasated Evans Blue was removed and placed in a clean 1.5 ml tube.
To analyze the amount of Evans Blue in the supernatant, samples were placed in a 96-well
plate and read using a Spectramax 340PC from Molecular Devices with the absorbance
measured set to 610 nm.
Mice were euthanized by isoflurane overdose. Liver, lung, and intestine were removed,
processed, embedded in paraffin, sectioned, and stained with hematoxylin/eosin by
a necropsy technician in the In-vivo Animal Core (IVAC) at the University of Michigan
Medical School. We received the sections and did imaging analysis using an Olympus
BX51 with a 20X dry objective lens. Images were captured using a DP-70 digital camera
and the associated software. In-depth pathological analysis was performed by the veterinary
pathologist of the IVAC.
In most graphs of data, the actual values for each mouse are plotted and the means
indicated. In others the means and standard errors are plotted. Sample size was not
predetermined and all mice were included in the analyses. Mice were randomly allocated
to the different groups based on cage and litter size. For all experiments we aimed
to have the same number of mice in the control and experimental groups. For all experiments,
at the time of analyses, the investigator was blinded to the group allocation. The
only exception was the blood vessel quantification in 3D reconstructions of sternal
segments. Statistical differences were calculated using two tailed, Two-sample, T-tests
(for experiments with two groups), ANOVA (3 or more groups) on log transformed data,
or Log-Rank tests for survival analysis.
The primary data that support the results described here are available upon reasonable
request. A Life Sciences Reporting Summary is also available.