Fas Stabilizes and Accelerates Erythropoiesis Figure 7. Delayed response to Epo-induced stress in mice deficient in the Fas pathway. (A–C) Analysis of the ProE response to Epo injection. The same experiment and dataset as in Figure 6. (A) Spleen ProE (cells per gram body weight). Data points are individual mice, with the mean 6sem for each day marked as a horizontal line. Data pooled from 1 to 3 experiments with 3 to 18 mice per genotype. (B) The rate of change in spleen ProE between days 1 and 2 post-Epo injection in the mutant gld-Rag12/2 (indicated in red) or lpr-Rag12/2 (blue) and their matched Rag12/2 controls. Dataset as in panel (A). Each point represents the mean difference in ProE between independent experiments done on days 2 and 3, in 5 independent comparisons. (C) The rate of change in spleen ProE throughout the first 3 days of response to Epo, computed as in panel B. The rate of change between days 0 and 1, days 1 and 2, and days 2 and 3, were plotted on days 0.5, 1.5 and 2.5, respectively. Data points represent pooled mutant (lpr/gld) or control differences (mean 6sem). For all panels: *p,0.05, **p,0.005, ***p,0.0005 (t test, unequal variance). doi:10.1371/journal.pone.0021192.g007 animal. An acute decrease in erythroblast FasL by transient administration of the decoy receptor Fas:Fc resulted in an acute increase in erythropoietic rate, reducing ProE and EryA apoptosis and doubling reticulocyte number by 48–72 hours (Fig S1). Chronic loss of Fas function in the gld-Rag12/2 and lpr-Rag12/2 mice is likely to elicit compensation through the pO2/Epo-mediated negative feedback loop, which automatically adjusts Epo levels and erythropoietic rate so as to maintain a near-normal basal hematocrit and tissue pO2. Alhough lpr-Rag12/2 mice had normal reticulo- cytes and hematocrit, their plasma Epo was significantly lower, by 35%, than in matched Rag12/2 controls, evidence of compensa- tory adjustment. By contrast, gld-Rag12/2 mice were apparently unable to significantly lower their already low plasma Epo. Consequently, their reticulocyte numbers in peripheral blood, a direct measure of erythropoietic rate, more than doubled, and there was a corresponding significant 2.2% increase in hematocrit. This increase is equivalent to a third of the increase in hematocrit we observed in mice housed in 12% oxygen, equivalent to hypoxia at 14,000 feet. While many pathways have been implicated in the regulation of erythropoietic rate, a specific contribution to the stress response in vivo had been determined for very few. Of note, mice lacking ERK1, a recently described negative regulator of splenic erythropoiesis, showed a similar increase in hematocrit to that seen here for the gld-Rag12/2 mice, without a significant change in plasma Epo [49]. Taken together, both the lpr-Rag12/2 and gld-Rag12/2 strains show increased erythropoietic rate per unit plasma Epo when compared with matched controls, confirming that local negative regulation at the level of splenic erythropoietic tissue has a negative effect at the level of the whole animal. There is no indication that Fas and FasL interact with alternative receptors or ligands. Therefore, the difference in the response of the gld-Rag12/2 and lpr-Rag12/2 mice at the level of the hematocrit and serum Epo is likely attributable to their different genetic backgrounds. The response of both these mouse strains at the level of the ProE and EryA precursors, the direct sites of action of Fas and FasL, was very similar. Loss of Fas function results in a specific increase of spleen ProE and EryA pools We found a significant 1.5 to 4 fold increase in the number of splenic, but not bone-marrow, ProE and EryA in both the gld- Rag12/2 and lpr-Rag12/2 mice, consistent with the pattern of erythroid Fas and FasL expression[12]. We also identified an increase in CFU-e specific to spleen, suggesting that these cells are regulated by Fas, in agreement with their counterpart in fetal liver [31]. Bone marrow progenitor subsets were either normal or even decreased in number, possibly as a compensatory response to their increase in spleen (Fig 4C,D). PLoS ONE | www.plosone.org 10 July 2011 | Volume 6 | Issue 7 | e21192