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A. McCabe et al.
underlying mechanisms whereby IFNγ drives SAA are still unknown.
Bone-associated cell analysis
Bones were crushed using a mortar and pestle in HBSS, washed and twice digested in 2 mg/mL collagenase type 2 and 1X trypsin enzyme solution at 37°C (with rocking, 30 min).
Complete blood cell count
Complete blood counts (CBCs) were determined using an auto- mated hematology analyzer (Cell Dyn 3700, Abott Laboratories).
Tissue preparation for protein quantification
Bone marrow cell lysates were homogenized with a pestle in a buffer containing IGEPAL CA-630 and proteinase inhibitors for protein analysis.
Transplantation
150 HSCs (Lin– cKit+ CD48- CD150+) were sorted from PBS- and clod-lip-treated ACTB-tdTomato F1 mice 8 d.p.s.t. and transplant- ed separately into lethally irradiated F1 recipients with 2.5x105 protective F1 whole BM cells.
Thrombocytopenia causes substantial morbidity and mortality in SAA patients.11 Megakaryocytes (Mks) not only produce platelets via thrombopoiesis, they serve as critical niches for HSCs.12,13 Thrombopoiesis is regulated by soluble factors, vascular integrity, and extracellular matrix composi- tion, and requires adequate numbers and location of Mks.14 Platelet-biased HSCs, including HSCs that highly express CD41, have been observed in settings of inflammation and aging, where increased platelet output may be necessary to maintain vascular function.15,16 Inflammation can impact megakaryopoiesis and thrombopoiesis, though it is unclear whether this process is modulated in SAA.
Bone marrow macrophages (Mfs) support stromal niche cell function at steady-state,17-19 however, little is known about the impact of inflammation on BM Mfs. Herein, we demonstrate that Mfs are essential for IFNγ-dependent HSC loss in murine SAA. IFNγ signaling in Mfs was nec- essary for the selective maintenance of BM resident Mfs, whereas all other myeloid cells were diminished. Targeting Mfs during SAA, via depletion or blocking their ability to respond to IFNγ, rescued HSCs and markedly improved survival. We demonstrate a key role for BM Mfs in sensing IFNγ, and our findings suggest that dys- functional megakaryopoiesis and thrombopoiesis underlie hematopoietic collapse in SAA.
Macrophage depletion and antagonist delivery
Methods
Mice
C57BL/6 (Hb/b) and BALB/c (Hd/d) mice were from Taconic (Albany, NY, USA). C57BL/6-TG(UBC-GFP)30Scha/J mice and ACTB-tdTomato mice were from Jackson Laboratory (Bar Harbor, ME, USA). MIIG (Mf-insensitive to IFNγ) mice20 were a gift from Dr. Michael Jordan. Hybrid B6 F1 (Hb/d) were generated by cross- ing C57BL/6 with BALB/c mice. To generate MIIG F1 (Hb/d) mice, MIIG (C57BL/6 background) and BALB/c mice were crossed. Hybrid F1 progeny were screened by PCR to identify mice carry- ing the MIIG transgene, and MIIG-negative mice were included as littermate controls (LC). Mice were bred and housed in the Animal Research Facility at Albany Medical College (AMC) under microisolator conditions. Protocols were approved by the AMC Institutional Animal Care and Use Committee.
SAA induction
Age- (6-8 weeks) and sex-matched B6 F1 mice were sublethally irradiated (300 RADs) using a 137Cs source four hours prior to intraperitoneal (i.p.) transfer of 5x107 C57BL/6 splenocytes.4 Mice were euthanized by CO2 inhalation at the indicated day post splenocyte transfer (d.p.s.t.). For survival studies, mice were exam- ined twice daily and humanely euthanized upon 20% loss of ini- tial body weight or if found moribund. Surviving mice were euth- anized approximately ten days after the last mouse succumbed to disease.
Cell preparation and flow cytometry
Bone marrow was flushed from femurs and tibias, and spleens were homogenized. After red blood cell (RBC) lysis single-cell sus- pensions were plated and stained. Surface-stained cells underwent nuclear or cytoplasmic permeabilization (BD Pharmingen) prior to T-bet (4B10) and IFNγ staining, respectively. Data were collected on an LSR II (BD Biosciences) and analyzed using FlowJo software (TreeStar, Ashland, OR, USA).
Fluorescently-tagged anti-Gp1bβ antibodies (Emfret Analytics) were administered to mice 5 or 10 d.p.s.t., according to the man- ufacturer’s instructions.
250 μL of PBS- or clodronate-liposomes (ClodronateLiposomes. com) was administered intravenous (i.v.) at 1 d.p.s.t. (day 7 analy- sis) or 1 and 7 d.p.s.t. (day 15 analysis). Anti-podoplanin (PDPN) antibody (clone 8.1.1) or Syrian Hamster IgG (both from BioXcell) was administered i.v. 3, 7, and 10 d.p.s.t. at 125 μg/dose.
Platelet analysis
Histology
Sternums were fixed in 10% buffered formalin, decalcified in 14% EDTA, and paraffin-embedded. Megakaryocytes were stained with anti-rat GP1bβ (Emfret) and nuclear fast red (Poly Scientific R&D) counterstaining. Images were taken on an Olympus SC30 light microscope using CellSens software.
Statistical analysis
Analysis was performed using Prism software. Two-tailed Student t-test was used to compare between indicated groups, unless otherwise reported.
Results
BM macrophages are maintained during SAA
Hematopoietic stem cell loss and BM destruction are key features of SAA and are associated with cytokine produc- tion by T cells.6-8 It is unclear, however, if inflammation depletes HSCs directly or through the microenvironment. To examine resident Mfs in SAA, we used an established murine model of BM failure involving histocompatibility mismatched recipients and splenocyte infusion.4 SAA was induced via sublethal irradiation of F1 hybrids (C57BL/6 x BALB/c), followed by adoptive transfer of C57BL/6 spleno- cytes (Online Supplementary Figure S1A). Significant cytope- nias were observed in SAA mice at 8 and 15 d.p.s.t. com- pared to radiation controls (Rad) (Online Supplementary Figure S1B-D). Thrombocytopenia was evident 8 d.p.s.t in control and SAA mice, and progressed in SAA mice (Online Supplementary Figure S1D). SAA-associated cytopenias coincided with BM hypocellularity (Figure 1A) and HSC loss (Online Supplementary Figure S1E-G).
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haematologica | 2018; 103(9)