Rps14 haploinsufficiency causes a block in erythroid differentiation mediated by S100A8 and S100A9

Abstract

Impaired erythropoiesis in the deletion 5q (del(5q)) subtype of myelodysplastic syndrome (MDS) has been linked to heterozygous deletion of RPS14, which encodes the ribosomal protein small subunit 14. We generated mice with conditional inactivation of Rps14 and demonstrated an erythroid differentiation defect that is dependent on the tumor suppressor protein p53 (encoded by Trp53 in mice) and is characterized by apoptosis at the transition from polychromatic to orthochromatic erythroblasts. This defect resulted in age-dependent progressive anemia, megakaryocyte dysplasia and loss of hematopoietic stem cell (HSC) quiescence. As assessed by quantitative proteomics, mutant erythroblasts expressed higher levels of proteins involved in innate immune signaling, notably the heterodimeric S100 calcium-binding proteins S100a8 and S100a9. S100a8--whose expression was increased in mutant erythroblasts, monocytes and macrophages--is functionally involved in the erythroid defect caused by the Rps14 deletion, as addition of recombinant S100a8 was sufficient to induce a differentiation defect in wild-type erythroid cells, and genetic inactivation of S100a8 expression rescued the erythroid differentiation defect of Rps14-haploinsufficient HSCs. Our data link Rps14 haploinsufficiency in del(5q) MDS to activation of the innate immune system and induction of S100A8-S100A9 expression, leading to a p53-dependent erythroid differentiation defect.

Figure 1. Rps14 haploinsufficiency results in a p53-mediated erythroid differentiation defect

(a) Hemoglobin levels (Hb) and % of reticulocytes in the peripheral blood from Rps14^−/+Mx1Cre⁺ mice in comparison to Mx1Cre⁺ wild-type controls. (mean±SD, n=10; *p<0.05). (b) Kaplan-Meier survival curve of Rps14^−/+Mx1Cre⁺ (n=10) and Mx1Cre⁺ control mice (n=10). Time point 0 is the day of the first of three poly(I:C) inductions. (c) Frequency of RI-RIV erythroid progenitor populations (RI: CD71^highTer119; RII: CD71^highTer119⁺; RIII: CD71^intermediateTer119⁺; RIV: CD71⁻Ter119⁺) among viable bone marrow cells in Mx1Cre⁺ and Rps14^−/+Mx1Cre⁺ mice 18 months after poly(I:C); (mean±SD, n=5; **p<0.001). (d) Relative spleen to body weight [%] of Mx1Cre⁺ and Rps14^−/+Mx1Cre⁺ mice 18 months after poly(I:C); (mean±SD, n=5; **p<0.001). (e) Hb level and reticulocyte counts in the peripheral time blood at serial time points before and after 25mg/kg Phenylhydrazine injection (mean±SD, n=8; *p<0.05). (f) Cell pellets of lineage-negative HSPCs subjected to erythroid differentiation in vitro for 5 days. (g) Quantification of different erythroid differentiation stages 5 days after induction of erythroid differentiation (mean±SD; n=3 biological replicates; **p<0.001). (h) Quantification of p21 and Rps14 transcript levels by quantitative real-time PCR in cells exposed for 5 days to erythroid differentiation. Data are normalized to expression in Mx1Cre⁺ control cells (n=5 biological replicates; mean±SD). (i) Kaplan-Meier survival curve after treatment with 35mg/kg Phenylhydrazine on two consecutive days (day 0 and day 1) of Rps14^−/+Mx1Cre⁺ (n=10), p53^−/+Mx1Cre⁺ (n=10), Rps14^−/+p53^−/+Mx1Cre⁺ (n=10) and Mx1Cre⁺ control mice (n=10). (j) Frequency of RIII and RIV erythroid progenitor populations among viable bone marrow cells in 10–12 week old Rps14^−/+Mx1Cre⁺ (n=14), p53^−/+Mx1Cre⁺ (n=5), Rps14^−/+p53^−/+Mx1Cre⁺ (n=5) and Mx1Cre⁺ control mice (n=8) characterized by differential CD71 and Ter119 expression 9 days after the first treatment with 25mg/kg Phenylhydrazine (mean±SD; **p<0.001). Unpaired two-sided t-test (a–i) or multiple group comparison (i, j) using analysis of variance with posthoc Tukey correction were applied for statistical analysis.

Figure 2. Rps14 haploinsufficiency alters hematopoietic stem and progenitor cells

(a) Bone marrow histopathology in HE-stained bone marrow sections (40x magnification; scale bar 50µm) 18 months after the first poly(I:C) injection in Rps14^−/+Mx1Cre⁺ (n=5) and Mx1Cre⁺ mice (n=4), representative pictures are shown. (b) Frequency of MPPs (lineage^lowckit⁺Sca1⁺CD48⁺CD150⁻), ST-HSCs (lineage^lowckit⁺Sca1⁺CD48⁻CD150⁻) and LT-HSCs (lineage^lowckit⁺Sca1⁺CD48⁻CD150⁺) 18 months after the first poly(I:C) injection in Rps14^−/+Mx1Cre⁺ (n=5) and Mx1Cre⁺ mice (n=4); (mean±SD; *p<0.05). (c) Cell cycle was analyzed by combined proliferation (Ki67) and cell cycle (Hoechst33342) staining in permeabilized LT-HSCs from bone marrow (GO: Ki67⁻Hoechst; G1: Ki67⁺Hoechst; S-G2-M: Ki67⁺Hoechst⁺); (mean±SD; n=5; **p<0.001). (d) Whole bone marrow cells from primary 18 months old mice were transplanted in 6–8 week old SJL/CD45.1 recipient mice (n=5). Hemoglobin levels (Hb) in the peripheral blood from chimeric Rps14^−/+Mx1Cre⁺ mice in comparison to Mx1Cre⁺ wild-type controls 6 weeks after transplantation and frequency of the RI-RIV erythroid progenitor populations (RI: CD71^highTer119⁻; RII: CD71^highTer119⁺; RIII/IV: CD71^{intermediate/low}Ter119⁺) in CD45.1 mice transplanted with aged Mx1Cre⁺ and Rps14^−/+Mx1Cre⁺ bone marrow cells 6 weeks after transplantation. (e) Representative flow plots of the HSC compartment of populations defined as MPPs, ST-HSCs and LT-HSCs in CD45.1 mice transplanted with old Mx1Cre⁺ (n=4) and Rps14^−/+Mx1Cre⁺ (n=5) bone marrow cells 6 weeks after transplantation. ((f) Identical numbers of Rps14^−/+Mx1Cre⁺ and Mx1Cre⁺ controls were mixed in equal ratios (approximately 50:50) and transplanted in lethally irradiated CD45.1 recipients. Time point 0 reflects the first bleeding 4 weeks after transplantation (engraftment) before inducing the excision of Rps14 by poly(I:C) injections. After 24 weeks, bone marrow was harvested and transplanted for secondary transplants. (mean±SD, n=5; *p<0.05). (g) Donor chimerism (CD45.2) of the HSC (MPP, ST-HSC, LT-HSC) compartment (mean±SD, n=5; *p<0.05). (h) Hemoglobin levels (Hb) in the peripheral blood from chimeric Rps14^−/+Mx1Cre⁺ or Mx1Cre⁺ mice 64 weeks after engraftment (40 weeks after secondary transplantation). (mean±SD, n=5; *p<0.05). Unpaired two-sided t-test was applied for statistical analysis.

Figure 3. Reduced protein synthesis in Rps14 haploinsufficient cells

(a) OP-Puro incorporation in bone marrow (BM) cells in vivo 1 h after administration in 20 week old Mx1Cre⁺ control cells or Rps14^−/+Mx1Cre⁺ cells [(16 weeks after the first injection of poly(I:C)]. Quantification of OP-Puro fluorescence reflecting protein synthesis rate in hematopoietic stem and progenitor cells relative to unfractionated bone marrow. Relative protein synthesis and quantification of OP-Puro fluorescence in erythroid RI-RIV progenitor populations relative to unfractionated bone marrow (mean±SD, n=5; **p<0.001; Unpaired two-sided t-test was applied for statistical analysis). (b) Polysome profiles from sort-purified lineage^lowCD71^highTer119⁺ erythroid progenitor cells in Mx1Cre⁺ control cells or Rps14^−/+Mx1Cre⁺ cells. The x-axis shows the distance along the gradient. The arbitrary Y-axis shows the relative absorbance. Data are representative of 3 independent experiments (each n=3 biological replicates). (c) Proteomic analysis of induced changes in protein expression of Rps14 haploinsufficient sort-purified lineage^lowCD71^highTer119⁺ erythroid progenitor cells relative to Mx1Cre⁺ cells (300µg protein for each technical replicate=4 biological replicates). Log2 ratios and scatter plot for individual proteins for replicate 1 and 2, where each dot represents a unique protein. The upper right quadrant represents proteins that are significantly up-regulated by Rps14 haploinsufficiency in replicate 1 and 2 (Rep 1 and 2) relative to Mx1Cre⁺ control cells. Detailed statistical methods for the proteomic analysis are described in the methods section.

Figure 4. S100a8 is significantly up-regulated in Rps14 haploinsufficient bone marrows, is regulated by p53 induction, and is necessary for the erythroid differentiation defect

(a) Immunohistochemical staining of S100a8 in bone marrows from Mx1Cre⁺ and Rps14^−/+Mx1Cre⁺ mice 8 weeks after the induction of the Rps14 excision with poly(I:C); representative pictures are shown (n=5). Scale bar 100µm. (b) Quantification of S100a8 in lineage-negative bone marrow cells by quantitative real-time PCR (mean±SD, n=5; *p<0.05). Data are normalized to expression in Mx1Cre⁺ control cells 8 weeks post poly(I:C). Western blot on protein lysates from Mx1Cre⁺ and Rps14^−/+Mx1Cre⁺ lineage-negative bone marrow cells. Data are representative of 3 independent experiments. (c) Mean fluorescence intensity (MFI) in CD11b⁻Gr1⁻ erythroid progenitor populations characterized by CD71 and Ter119 expression (RI-RIV). (mean±SD, n=5; *p<0.05). Mean fluorescence intensity (MFI) of S100a8 expression in Gr1⁺CD11⁺ granulocytes, Gr1⁻CD11b⁺ monocytes and F4/80⁺ macrophages (mean±SD, n=5; *p<0.05). (d) Co-immunofluorescent staining of F4/80 (green), p53 (dark blue) and S100a8 (red) on cytospins of Rps14^−/+Mx1Cre⁺ and Mx1Cre⁺ bone marrow cells 12 weeks after the induction of the Rps14 excision with poly(I:C). Inserts highlight areas of magnification shown in the lower panel. Scale bar: 20µm. Data are representative of 3 independent experiments. (e) Percentage of p53 and S100a8 co-expressing cells in RI-III erythroid progenitor cells from Rps14^−/+Mx1Cre⁺ and Mx1Cre⁺ mice 12 weeks after the induction of the Rps14 excision with poly(I:C), (mean±SD, n=5; *p<0.05). (f) Representative histograms showing S100a8 expression in the RIII erythroid progenitor cell population and mean fluorescence intensity (MFI) of S100a8 expression in F4/80⁺ macrophages and RIII erythroblasts in Mx1Cre⁺, p53^−/−, Rps14^−/+Mx1Cre⁺ and Rps14^−/+p53^−/−Mx1Cre⁺ mice 6 days after induction of hemolysis with Phenylhydrazine (mean±SD, n=5; *p<0.05; *p*<0.001). (g) Fluorescence intensity normalized to background signals of inflammatory cytokines in bone marrows from Mx1Cre⁺ and Rps14^−/+Mx1Cre⁺ mice 12 weeks after the induction of the Rps14 excision with poly(I:C). Log10 scale. (mean±SD, n=4; **p<0.001). (h) Ckit⁺ HSPCs were transduced with lentiviral shRNAs (n=5 each) targeting Tnfα, S100a8 and Tlr4 and a luc control. Transduced cells were selected with puromycin and induced to undergo erythroid differentiation for 5 days in vitro. The frequency of RIV erythroid progenitor populations in the culture is shown (mean±SD, **p<0.001). Circles represent the median of three replicates for each individual shRNA (n=5). The mean of all shRNAs targeting a given gene is shown with a grey bar or red bar. (i) Representative histogram presentation and mean fluorescence intensity (MFI) of p53 expression in the RIII population of HSPCs transduced with shRNA targeting Tnfα, S100a8 and Tlr4 and luc control after 5 days of erythroid differentiation in vitro. (mean±SD, **p<0.001, n=5). Circles represent the median of three replicates for each individual shRNA (n=5). The mean of all shRNAs targeting a given gene is shown with a grey bar or red bar. (j) Representative histograms in F4/80⁺ macrophages and in the RIII population and mean fluorescence intensity (MFI) of S100a8 expression in macrophages of HSPCs transduced with shRNA targeting Tnfα, S100a8 and Tlr4 and luc control after 5 days of erythroid differentiation in vitro. (mean±SD, **p<0.05, **p<0.001, n=5). Circles represent the median of three replicates for each individual shRNA (n=5). The mean of all shRNAs targeting a given gene is shown with a grey bar or red bar. Unpaired two-sided t-test (b, c, e; h-j) or multiple group comparison by analysis of variance with posthoc Tukey correction (f) were applied for statistical analysis.

Figure 5. S100a8 is essential for the erythroid differentiation defect due to Rps14 haploinsufficiency

(a) Lineage negative wild-type HSPCs were subjected to erythroid differentiation for 5 days in the presence of vehicle or S100a8 recombinant protein (rS100a8). Cell pellets 5 days after induction of differentiation (representative picture of 5 biological replicates is shown) and frequency of RI-RIV erythroid progenitor populations in the culture. (mean±SD, n=5 biological replicates; **p<0.001). (b) Intracellular flow cytometry for p53 expression within the RII/RIII populations in erythroid differentiation culture in vitro in presence or absence of recombinant S100a8. Representative histograms of p53 expression and mean fluorescence intensity of p53 within this population (mean±SD, n=5 biological replicates; **p<0.001). (c) Cell pellets of ckit⁺ HSPCs from Rps14^−/+Mx1Cre⁺ or Mx1Cre⁺ mice expressing S100a8 or control sgRNAs following erythroid differentiation in vitro. Representative flow plots of the erythroid progenitor populations characterized by CD71 and Ter119 expression (RI-RIV); (data representative from n=3 biological replicates are shown). (d) Representative histogram presentation of S100a8 expression in Gr1^lowCD11b⁺ monocytes in the erythroid differentiation culture in vitro after 5 days from Rps14^−/+Mx1Cre⁺ or Mx1Cre⁺ cells which were transduced with either S100a8 or control sgRNAs. The pie chart represents the mutations introduced by the S100a8 sgRNA:Cas9 transduced cells after sequencing of S100a8 high, intermediate, and low fractions. (e) ckit⁺ HSPCs were transduced with a lentiviral vector expressing Cas9 and an sgRNA targeting S100a8 or control sgRNAs (non-targeting guide, NTG) and were transplanted 24 hours after infection into lethally irradiated wild-type recipients. 6 weeks after transplantation, recovered hematopoiesis in the transplanted mice was confirmed and PH was injected to induce hemolysis. 6 days after the first dose of PH, the bone marrow was harvested. Representative flow plots of the RI - RIV erythroid progenitor populations (mean±SD, n=5; **p<0.001; *p<0.05). (f) Representative histograms depicting GFP expression, representing cells transduced with control- or S100a8 sgRNA:Cas9 in the RIV population (mean±SD, n=5; **p<0.001). (g) p53 immunohistochemistry in bone marrows from Rps14^−/+Mx1Cre⁺ or Mx1Cre⁺ mice transduced with either control control or S100a8 sgRNA:Cas9. Scale bar: 50µm. (h) Quantification of the mean fluorescence intensity (MFI) of intracellular staining with p53 and S100a8 in erythroid progenitor populations (RIII and IV) in bone marrows from Rps14^−/+Mx1Cre⁺ or Mx1Cre⁺ mice transduced with either control or S100a8 sgRNA:Cas9. (mean±SD, n=5; *p<0.05; **p<0.001). (i) Quantification of the mean fluorescence intensity (MFI) of intracellular staining with Tlr4 in macrophages (CD11b⁺F4/80⁺), monocytes (CD11b⁺F4/80⁻) and in erythroid progenitor populations (RIII) in bone marrows from Rps14^−/+Mx1Cre⁺ or Mx1Cre⁺ mice transduced with either control or S100a8 sgRNA:Cas9. (mean±SD, n=5; *p<0.05; **p<0.001). Unpaired two-sided t-test (a–b) or multiple group comparison (e–i) using analysis of variance with posthoc Tukey correction were applied for statistical analysis.

Figure 6. S100A8-frequency is significantly increased in del(5q) MDS human bone marrows compared to non-MDS controls

(a) Representative images of co-immunoflurescent staining of Glycophorin A (GlyA), S100A8 and DAPI as a nuclear staining in healthy (non-MDS; n=15), del(5q) MDS patients (n=21) and normal karyotype MDS (non del(5q), n=9). DAPI (blue), S100A8 (green), GlyA (magenta). Insert depicts S100A8 and GlyA expressing cells. Scale bars: 100µm. Immunofluorescence for all patients is shown in Supplementary Figure 6. (b) Quantification of S100A8⁺ cells as a percentage of DAPI-positive nucleated bone marrow cells (S100A8 frequency in the nucleated marrow). (mean±SD, p<0.001). Unpaired two-sided t-test was applied for statistical analysis.