Fas Stabilizes and Accelerates Erythropoiesis Figure 1. Epo regulation of erythropoiesis through Fas-mediated apoptosis. (A) Epo-dependent erythroblastic island precursors CFU-e, ProE and EryA (in blue) co-express Fas and FasL, and mature into Epo-independent EryB, EryC and red blood cells (RBC, in red). ‘F’ = Fas expressing cells, shown undergoing cell death as a result of interaction with FasL-expressing cells within the Epo- dependent (blue) compartment (black flat- headed arrow). A negative feedback loop driven by tissue pO2 regulates Epo levels in blood, which in turn enhance erythroblast survival, by either suppressing Fas and FasL expression, or by non-Fas dependent pathways. HSC = hematopoietic stem cells. (B) Flow-cytometric identification of Epo- dependent ProE and EryA subsets (in blue) and Epo-independent EryB and EryC (in red), in adult Balb/C mouse spleen, in basal conditions (top panels) or 48 hours following Epo injection (300 U/ mouse, lower panels). ProE are defined as Ter119medCD71high cells; Ter119high cells are further subdivided based on forward scatter (FSC) and CD71 expression into EryA (CD71highTer119highFSChigh), EryB (CD71highTer119highFSClow) and EryC (CD71lowTer119highFSClow). (C) The erythropoietic response of mice to a hypoxic environment. Mice (Balb/C) were examined either in the basal state (‘a’, 21% atmospheric oxygen), when housed in 11% oxygen for 8 days (assay times ‘b’ and ‘c’ at 13 hours and 3 days, respectively), and when placed back in normoxia (21%; assay times ‘d’ and ‘e’, at 1 and 2 days post-hypoxia). Top panels show representative flow-cytometric histograms of Fas expression and Annexin V binding at the indicated assay times. Gates refer to the Fas+ and Annexin V+ populations, determined with reference to staining controls in which either the anti-Fas antibody (left panel) or Annexin V (right panel) were omitted. The fraction of cells positive for Fas or Annexin V at each time point is noted. Middle panel shows a summary of similar data, 2 to 9 mice per time point. Lower panels show corresponding serum Epo levels and EryA cell number in spleen (expressed as total EryA cells /gram body weight). *p,0.05, **p,0.002, ***p,0.0001 (two-tailed t test, unequal variance). doi:10.1371/journal.pone.0021192.g001 EpoR activates several survival pathways, including Stat5- mediated induction of bcl-xL [17,18,19,20], other Stat5 targets [21,22,23,24] and the EpoR-activated phosphoinositide 3-kinase (PI3K) -AKT pathway [25,26]. The death receptor Fas, and its ligand, FasL, were first proposed to contribute to erythroid homeostasis based on their expression in human bone-marrow [27]. We found that, in vivo in the mouse, splenic ProE and EryA, but not their bone-marrow counterparts, co-express Fas and FasL. During stress, high Epo suppresses their Fas expression, strongly correlating with their decreased apoptosis [12]. These findings suggested the hypothesis that, in the basal state, splenic ProE and EryA negatively regulate their own survival through Fas and FasL- mediated inter-cellular interactions; and that Epo-mediated Fas suppression is a key mechanism regulating erythropoietic expan- sion during stress (Fig 1A) [12]. Negative autoregulation through co-expression of Fas and FasL was previously implicated in terminating the clonal expansion of activated T cells [28,29,30]. Similarly, we recently found that Fas and FasL co-expression in fetal liver erythroid progenitors terminates their initial wave of expansion at the onset of fetal erythropoiesis [31]. Here we set out to assess the contribution of Fas and FasL to erythropoiesis in the adult. We bred lpr or gld mice, mutant in Fas and FasL respectively, onto the Rag12/2 immune-deficient background, in order to avoid an autoimmune syndrome that may impact erythropoiesis. Our findings in both the lpr-Rag12/2 and gld-Rag12/2 mice were similar, showing that Fas-mediated autoregulation in spleen negatively regulates eryth- ropoiesis at the whole animal level. Strikingly, these experiments also revealed that Fas-mediated autoregulation imparted key dynamic properties to the erythropoietic system. We found that it PLoS ONE | www.plosone.org 2 July 2011 | Volume 6 | Issue 7 | e21192