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A. McCabe et al.
whether IFNγ was necessary for preservation of Mfs dur- ing SAA. Using transgenic mice in which Mf-lineage cells are insensitive to IFNγ (referred to as MIIG mice) due to a CD68-driven dominant-negative IFNγ receptor,20 we noted improved cellularity upon induction of SAA (Figure 2A and B). MIIG and littermate control (LC) mice showed no significant difference in response to sublethal radiation; however, CD11blo/- and CD11b+ Mfs were significantly reduced in MIIG mice relative to LC counterparts 8 d.p.s.t, when HSC loss is first noted (Figure 2C). Thus, we define a novel role for IFNγ in maintaining and increasing BM Mfs during SAA.
MIIG mice exhibit increased CD41hi HSCs and megakaryocytes during SAA
Inflammation-induced megakaryopoiesis reportedly relies on the emergence of a CD41hi stem-like Mk progen- itor cell type (SL-MkP) within the phenotypic HSC gate.15 SL-MkPs self-renew and rapidly produce Mks and platelets, while CD41lo/int HSCs contain multi-lineage potential.15,16 CD41 expression increased robustly on HSCs in MIIG relative to LC mice at day 15 p.s.t. (Figure 3A), suggesting that IFNγ-sensing Mfs limit CD41hi HSC emergence in response to SAA-induced inflammation. MIIG and LC radiation-control mice exhibited similar numbers of CD41lo/int and CD41hi at days 8 and 15 p.s.t. (Figure 3B). In SAA conditions, however, MIIG mice exhibited significantly more CD41hi HSCs (Figure 3C), and increased CD41lo/int on day 15 p.s.t. than LC. Consistent with increased phenotypic SL-MkPs we observed increased BM Mks in MIIG SAA mice, relative to LC (Figure 3D and E). Our data indicate that IFNγ signaling in Mfs during SAA is associated with rapid loss of both CD41lo/int and CD41hi HSCs, which correlates with Mk depletion and severe thrombocytopenia. Moreover, SAA- induced mortality was significantly reduced in MIIG com- pared to LC mice (Figure 3F). Thus, Mfs are key sensors of IFNγ, and our data strongly suggest that Mfs drive disease and death by reducing platelet-biased CD41hi HSCs.
Figure 2. IFNγ sensing by macrophages is required for bone marrow (BM) macrophage maintenance and hematopoietic stem cell (HSC) loss in aplastic anemia. (A) Severe aplastic anemia (SAA) was induced in MIIG and littermate control (LC) F1 hybrids. (B) Hematoxylin and eosin-stained BM of LC and MIIG mice 15 days post induction. Scale bar=50 μm. (C) Frequencies and absolute numbers of CD11blo/- Mfs and CD11b+ Mfs in LC (D) and MIIG (▲) mice 8 days post induction. Shading represents ranges of each Mf population in radiation control LC and MIIG mice. (D) CD150 and CD48 expression on BM Lin- c-Kit+ (LK+) cells. Numbers rep- resent mean HSC (LK+ CD150+ CD48–) frequency±Standard Error of Mean (SEM). (E) HSC numbers in MIIG (D) and LC (▲) mice 8 days post induction. Shading rep- resents ranges of radiation control LC and MIIG mice. (F) Red blood cells (RBCs), (F) hemoglobin, and (H) platelets in the blood 15 days post induction. Shading rep- resents ranges of radiation control LC and MIIG mice. Data represent one experiment repeated at least two times, n=5-7 mice/group. Mean±SEM is shown. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
Mfs drive IFNγ-induced HSC depletion, we predicted that the BM HSC pool would be preserved in MIIG mice during SAA. Indeed, HSCs were preserved in MIIG mice, relative to LCs (Figure 2D and E), demonstrating IFNγ- sensing by Mf-lineage cells drives HSC loss in SAA. Anemia was slightly, but significantly, ameliorated (Figure 2F and G) whereas thrombocytopenia was strikingly res- cued in MIIG relative to LC controls (Figure 2H). In fact, platelet levels were higher in MIIG mice with SAA than in radiation-control mice. The robust platelet rescue suggest- ed that IFNγ-stimulated Mfs contribute specifically to thrombocytopenia in SAA.
Based on our prior findings in bacterial infection, where 21
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haematologica | 2018; 103(9)