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A. Al-Sharea et al.
BPH/Apoe–/– mice have enhanced myelopoiesis
An overactive SNS has recently been shown to promote the mobilization of BM HSPCs to the spleen, resulting in the generation of splenic monocytes that can infiltrate into atherosclerotic lesions. Therefore, we next assessed the hematopoietic system in these mice.21 We discovered prominent monocytosis and neutrophilia in the BPH/Apoe-/- mice in the blood (Figure 3A). Next, to deter- mine if the increased monocyte and neutrophil numbers were due to activated myelopoiesis, we examined the abundance and proliferation of HSPCs and myeloid pro- genitor cells in the BM. While the levels and proliferation of HSPCs within the BM were similar (Figure 3B, C), we did observe more granulocyte-macrophage progenitors (GMPs) in the BPH/Apoe-/- mice, which were proliferating at a higher rate (Figure 3D,E). Consistent with SNS activa- tion in promoting the mobilisation of HSPCs from the BM, we detected elevated levels of circulating HSPCs and myeloid progenitors (MPCs) in the BPH/Apoe-/- mice (Figure 3F). Given the higher circulating HSPCs, we were expecting to see more HSPCs in the spleen. However, no such change in the abundance of HSPCs was detected (Figure 3G). Of note, a higher proportion were in the G2M phase of the cell cycle (Figure 3H), suggesting that the chronic activation of the SNS was influencing the HSPCs to proliferate more in the spleens of BPH/Apoe-/- mice. Indeed, monocytes and neutrophils were elevated in the spleens of the BPH/Apoe-/- mice, confirming extramedullary myelopoiesis was occurring in this chronic
sympathetic driven model (Figure 3I).
Sympathetic activation contributes to the breakdown of the HSPC bone marrow microenvironment
The Schlager mice are an established model of sympa- thetic activation-mediated hypertension.26 However, we wanted to confirm that there was evidence of increased sympathetic activation in the BM, which could account for the enhanced mobilization of HSPCs observed in Figure 3D. Firstly, to confirm an overall increase in sympa- thetic tone, we quantified plasma noradrenaline levels, which we found to be higher in the BPH/Apoe-/- mice (Figure 4A). More central to our proposed mechanism for enhanced HSPC mobilization in the BPH/Apoe-/- mice, we found enhanced expression of tyrosine hydroxylase (TH), the rate-limiting enzyme found in nerve terminals respon- sible for noradrenaline (NA) production, around the blood vessels in the BM of the BPH/Apoe-/- mice (Figure 4B). Together, these data reveal a more global increase in sym- pathetic tone in SNS-driven hypertension.
Next, we sought to determine if overactive sympathetic signaling in the BM led to changes within the BM microenvironment and whether these changes could be reversed with the use of a b-blocker. Given the impor- tance of sympathetic overdrive in mediating hyperten- sion, as expected, propranolol normalized blood pressure in the BPH/Apoe-/- mice (Figure 4C).
Having demonstrated that propranolol could reverse the systemic responsiveness of b-receptors to sympathetic activation in BPH/Apoe-/- mice, we examined key HSPC niche cells in the BM to determine if sympathetic over- drive influenced myelopoiesis via effects on the BM niche. Interestingly, we found a significant reduction in the abun- dance of CD51+ osteoblasts in the BM on the BPH/Apoe-/- mice, which were restored when these mice were treated with propranolol (Figure 4D). Consistent with this find-
ing, analysis of BM mRNA for Runx2, the transcription factor that drives osteoblast production, showed a reduc- tion in Runx2 expression in the BPH/Apoe-/- mice relative to Apoe-/- mice. Similar to our flow cytometry data, treatment with propranolol prevented the suppression of Runx2 expression (Figure 4E). When we assessed the gross mor- phological changes in the BM, it appeared that the vascu- lar structures were altered with the BPH/Apoe-/- mice showing smaller sinusoidal structures relative to the Apoe- /- mice, with propranolol reverting the sinusoids back to that seen in the Apoe-/- mice (Figure 4F). Furthermore, in examining the endothelial cell population, we noted a trend towards a decrease in the abundance of these cells, which, again, could be restored with the administration of propranolol (Figure 4G). As these niche cells are an impor- tant source of the HSPC retention factor CXCL12, we measured its mRNA expression and found that propra- nolol greatly increased Cxcl12 expression, thereby poten- tially aiding in promoting HSPC retention and reduced quiescence in the BM (Online Supplementary Figure S2, A). The changes in these two key niche cells may provide a mechanism for increased HSPC release from the BM in BPH/Apoe-/- mice. Other cells within the BM express b-adrenoreceptors, which we profiled using gene array data from Novershtern et al. and analysed using online software (BloodSpot) to generate a hierarchical differenti- ation tree.29,30 Firstly, HSPCs and myeloid progenitors did not display any enrichment for the adrenoceptors. However, neutrophils were identified as one of the cells enriched in transcripts for the b2-adrenoreceptor, but not b1- or b3-adrenoreceptors (Online Supplementary Figure S2, B-D). We pharmacologically confirmed the requirement for b2-adrenoreceptor stimulation in HSPC mobilization using the BPH mice on an Apoe+/+ background by adminis- tering the b2 specific antagonist ICI-118551 (Online Supplementary Figure S2, E). Furthermore, neutrophils have previously been shown to be responsive to NA in vitro.31,32 Mechanistically, activated neutrophils can release MMP9 which can cleave CXCR4 on HSPCs, providing another avenue to HSPC liberation from the BM.22,33 We measured levels of MMP9 in the BM extracellular fluid (BMEF) via zymography and found that both active and latent MMP9 levels increased in the BPH/Apoe-/- mice, a phenotype reversed with propranolol treatment (Figure 4H and Online Supplementary Figure S2, F). In support of this we found reduced surface CXCR4 expression on the HSPCs from the BPH/Apoe-/- mice, which was restored in mice treated with propranolol (Figure 4I). These data were fur- ther supported by BM mRNA analysis indicating that pro- pranolol treatment increases Cxcr4 expression (Online Supplementary Figure S2, G). To explore this mechanism further, we cultured HSPCs in the supernatants of neu- trophils treated with NA and examined CXCR4 cell sur- face abundance. We found significantly less CXCR4 on HSPCs cultured in supernatants from NA activated neu- trophils (isolated from wild-type mice), compared to vehi- cle treated neutrophils, which was prevented when MMP9 was inhibited (Figure 4J). When we included the b2-adrenoreceptor specific inhibitor ICI-118551 into the BM neutrophil stimulation media with NA, the harvested supernatant caused less efficient cleavage of CXCR4 (Online Supplementary Figure S2, H) thereby confirming the role for neutrophil b2-adrenoreceptors. These data sup- port the hypothesis that sympathetic activation is present in the BM of the BPH/Apoe-/- mice and responsible for the
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haematologica | 2019; 104(3)