Fas Stabilizes and Accelerates Erythropoiesis Figure 2. Fas and FasL-mediated negative autoregulation of the EryA pool. (A) Wild-type Balb/C mice were injected with a single Epo injection subcutaneously, of 1, 3, 6, 10, 30, 100 or 300 U/ 25 g body weight. Spleen EryA were examined on day 3 post injection. Left panel shows EryA cell frequency relative to basal frequency, plotted against the number of EryA cells that express Fas (Fas+ EryA, expressed as a ratio to basal levels). Data points represent individual mice. Blue = mice in the basal state (n = 15), red = mice injected with Epo (n = 38). For clarity only mice injected with 30 U (which maximally suppress Fas) or less are included in the left panel. Data is fitted with a curve derived from the mathematical model described in panel (B) and in the Text S2. Right panel shows the dependence of Fas+ EryA on the dose of injected Epo, in the same dataset as in the left panel; all mice injected with a given Epo dose were pooled into one data point, mean 6 sem. (B) Schematic of the factors that regulate the size of the EryA pool, ‘A’, measured as the fraction of all Ter119+ cells that are EryA. F = fraction of EryA cells that express Fas. ß = input into the EryA pool from earlier progenitor stages. aA = output from the EryA pool into later erythroid subsets (EryB). A2F = Fas-mediated cell loss. See mathematical model described in Text S2. In panel (A) of this figure, A is plotted against F, expressed as a ratio to the A and F values in the basal state, respectively. doi:10.1371/journal.pone.0021192.g002 The inverse relationship between EryA and Fas in Fig 2A is fitted well by a hyperbolic curve that represents the (positive) steady-state solutions for EryA (‘A’) in this equation, at different steady-state levels of Fas (‘F’) (R2 = 0.89, Text S2). This goodness of fit supports the model’s key assumption, that Fas-mediated cell loss is a result of negative autoregulation within the EryA pool, and is proportional to the square of the frequency of EryA within the erythroblastic island. Further, it suggests that Epo concentration, which sets the desired steady-state EryA pool size, does so in part by regulating the level of Fas expression in the EryA population (Fig 1A). Of note, using a different modeling approach, we found a similar relationship in fetal liver, where Fas-mediated loss of early erythroblasts was proportional to the square of the frequency of early erythroblasts in the tissue [31]. Generation of Fas and FasL-deficient mice on an immune-deficient background We made use of the lpr and gld mouse strains that carry naturally-occurring loss-of-function mutations of Fas and FasL, respectively [40]. The lpr mutation consists of an insertion of an early transposable element, carrying a polyadenylation signal, in the second intron of the Fas gene. This causes premature termination of the transcript and a drastic decrease in Fas transcription, though it does not fully eliminate it. In the gld strain, there is a point mutation at the C-terminus of FasL that abolishes its ability to bind Fas. Both the lpr and gld mice develop a lymphoid proliferative autoimmune syndrome [41]. In order to avoid this complication, we bred these mouse strains onto an immune- deficient, Rag12/2 background that lacks T and B cells [42]. Erythroid parameters, such as basal hemoglobin concentration, though consistent within a given inbred strain, differ somewhat between mice of different genetic backgrounds, likely reflecting quantitative differences in the control of erythropoiesis [43]. We therefore chose to generate two distinct genetic background strains. The gld-Rag12/2 mice were bred on the Balb/C background, and the lpr-Rag12/2 mice on the C57BL/6 background. As controls, we used age and strain-matched Rag12/2 mice, which on either the Balb/C or the C57BL/6 backgrounds, have normal erythropoietic parameters when compared with wild-type mice of the same background strain. Further, the double homozygous mutant strains, gld-Rag12/2 and lpr-Rag12/2, showed no sign of autoimmunity, as evident from their small spleen size, absence of immune cells from the blood and spleen, and lack of anemia (Table S1 and data not shown). Basal Erythropoiesis in lpr-Rag12/2 and gld-Rag12/2 mice The chronic absence of an erythropoietic regulator may not be apparent from simple inspection of the hematocrit in the steady state [20]. This is due to a vast erythropoietic reserve, coupled with the pO2/Epo negative feedback loop which automatically adjusts Epo levels and erythropoietic rate so as to maintain a near- normal tissue pO2. Therefore, to assess how the loss of a regulator affects steady state erythropoiesis requires analysis at all levels of the pO2/Epo negative feedback loop, including Epo concentration PLoS ONE | www.plosone.org 4 July 2011 | Volume 6 | Issue 7 | e21192