Clearance of Senescent Neutrophils Maintains Homeostatic Levels of Hematopoietic Progenitor Cells in the Circulation

Abstract 863

Neutrophils (PMN) are short-lived cells generated in the bone marrow (BM). After just a few hours in the circulation, aged PMN upregulate the expression of the chemokine receptor CXCR4 and acquire tropism to the BM, where they are cleared by resident macrophages. We have found that a population of “senescent” PMN (sPMN), identified by low expression of L-selectin and high expression of CXCR4, is detectable in the blood of wild-type (WT) mice and that their levels fluctuate circadianally in a way similar to that reported for hematopoietic progenitor cells (HPC). In addition, clearance of sPMN by resident phagocytes has been reported to alter the levels of cytokines involved in HSPC trafficking. In this study, we have asked whether clearance of sPMN modulates the hematopoietic niche and thus controls the physiological trafficking of HSPC.

We initially found that mice deficient in endothelial P- and E- selectins (PE mice) or selectin ligands (deficient in Fucosyltransferase 7, Fut7−/−) present granulocytosis and elevated HPC in blood, as detected by clonogenic assays and flow cytometry. To determine whether this phenotype could be corrected by components present in WT blood, we generated parabiotic pairs of these mice with fluorescently-tagged WT partners. Neutrophilia and HSPC levels in the blood of Fut7−/− mice were corrected in WT:Fut7−/− chimeras, whereas in WT:PE parabiotic chimeras we observed neutrophilia and increased circulating levels of HPC derived from the WT partner. To determine whether sPMN were the blood component responsible for modulating HPC trafficking, we prepared and injected BM-derived sPMN into WT mice. This treatment resulted in a 2.5-fold increase in circulating HPC, and this correlated with reduced levels of the chemokine CXCL12 in BM. sPMN treated with pertussis toxin or deficient in Fut7−/− were unable to efficiently home to the BM and completely lost their capacity to modulate HPC trafficking. We next investigated whether sPMN clearance by resident macrophages was required for the modulation of the hematopoietic niche and HPC egress. For this purpose, we treated mice with clodronate-loaded liposomes and injected sPMN 10 days later, a time in which macrophages but not monocytes or PMN were still depleted in the BM. Under these conditions, sPMN lost their capacity to reduce CXCL12 levels in BM and to induce HPC egress. When analyzing transcriptional alterations in the BM of Fut7−/− mice, where sPMN trafficking to BM is impeded, we found reduced expression of genes directly regulated by the nuclear liver × receptors (LXR) α and β, two receptors involved in signalling after phagocytosis. We thus speculated that ingestion of sPMN by BM-macrophages induced signals through LXRαβ which might be important for modulating the hematopoietic niche and triggering HPC egress. Indeed, in vivo administration of the LXR agonist GW3965 in WT mice mimicked sPMN treatment by inducing a 3-fold increase in circulating HPC levels and a reduction of CXCL12 expression in BM. Together, these results demonstrate that sPMN extravasation and clearance by tissue-resident macrophages trigger signals that modulate the hematopoietic niche and induce the egress of HPC, and suggest that LXR-mediated signals are required for this process. Given that the relatively modest changes in HPC levels in blood induced by sPMN were comparable to those found during normal light-dark cycles, we reasoned that this phenomenon might be important to maintain these physiological fluctuations. Indeed, normal HSPC fluctuations in blood were lost in mice in which PMN were depleted with an anti-Ly6G antibody or in mice deficient in LXRαβ.

In summary, we uncover a mechanism that links the homeostasis of the immune and hematopoietic systems and that may have important implications for understanding the behavior and function of circulating HPC.