Infection perturbs Bach2- and Bach1-dependent erythroid lineage ‘choice’ to cause anemia

Abstract

Elucidation of how the differentiation of hematopoietic stem and progenitor cells (HSPCs) is reconfigured in response to the environment is critical for understanding the biology and disorder of hematopoiesis. Here we found that the transcription factors (TFs) Bach2 and Bach1 promoted erythropoiesis by regulating heme metabolism in committed erythroid cells to sustain erythroblast maturation and by reinforcing erythroid commitment at the erythro–myeloid bifurcation step. Bach TFs repressed expression of the gene encoding the transcription factor C/EBPβ, as well as that of its target genes encoding molecules important for myelopoiesis and inflammation; they achieved the latter by binding to their regulatory regions also bound by C/EBPβ. Lipopolysaccharide diminished the expression of Bach TFs in progenitor cells and promoted myeloid differentiation. Overexpression of Bach2 in HSPCs promoted erythroid development and inhibited myelopoiesis. Knockdown of BACH1 or BACH2 in human CD34+ HSPCs impaired erythroid differentiation in vitro. Thus, Bach TFs accelerate erythroid commitment by suppressing the myeloid program at steady state. Anemia of inflammation and myelodysplastic syndrome might involve reduced activity of Bach TFs.

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Fig. 1: Ineffective erythropoiesis occurs in DKO mice.
Fig. 2: Erythroblast-maturation disorder is induced by de-repression of Hmox1 in DKO mice.
Fig. 3: Erythropoiesis disorder at the erythro–myeloid bifurcation in DKO mice.
Fig. 4: Bach TFs are necessary for appropriate regeneration of the blood system.
Fig. 5: Stimulation with LPS induces myelopoiesis by reducing the expression of Bach TFs.
Fig. 6: Bach1 and Bach2 repress myeloid genes and induce erythroid genes by competitively working with C/EBPβ.
Fig. 7: Bach2 has the potential to induce erythropoiesis.
Fig. 8: Involvement of Bach TFs in human erythropoiesis and pathophysiology of MDS.

Data availability

The microarray data have been deposited to Expression Omnibus (GEO) under the accession codes GSE86253 and GSE79697. The data that support the findings of this study are available from the corresponding author upon request.

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Acknowledgements

We thank members of the Departments of Biochemistry and of Hematology and Rheumatology in Tohoku University Graduate School of Medicine for discussions and support; J Sun (Hiroshima University) for initial analysis of DKO mice; the Biomedical Research Core of Tohoku University Graduate School of Medicine for technical support; and S Shibahara (Tohoku University School of Medicine) for HO1 cDNA. Supported by Grants-in-Aid from the Japan Society for the Promotion of Science (17H06527 to H.K.; 25860203 to M.W.-M.; 22134006, 26221308 and 15H05909 to S.O.; and 15H02506, 24390066, 21249014 and 18H04021 to K.I.), Agency for Medical Research and Development-Core Research for Evolutional Science and Technology (AMED-CREST, JP16gm0510001 to K.I.), research grants from Takeda Science Foundation (S.O.), Associazione Italiana per la Ricerca sul Cancro (Individual Grant 20125 to L.M.; and Special Program Molecular Clinical Oncology 5 per Mille (project 1005) to M.C.), and Fondazione Regionale Ricerca Biomedica (FRRB, project no. 2015-0042 to M.C.).

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Writing of original draft, H.K.; conceptualization, and methodology, H.K., A.I.-N. and K.I.; major investigation, H.K. and A.I.-N.; bioinformatics analysis, H.K., A.I.-N. and M.M.; supportive investigation, Y.I., M.I., R.E.-S., Y.S., M.K., H.N., K.S., Y.N., L.M. and M.C.; investigation of BM macrophages, M.W.-M. and A.M.; review and editing, H.K., A.I.-N., A.M., T.F. and K.I.; collection and analysis of clinical samples, Y.N., L.M., M.C. and S.O.; supervision, H.H. and K.I.

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Correspondence to Kazuhiko Igarashi.

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Supplementary Figure 1 Ineffective erythropoiesis occurs in DKO mice.

a, Colors of BM suspensions from indicated genotypes. BM fluids obtained from bilateral femurs and tibias of a body from each genotype were suspended in 5 ml buffer. b, Representative flow cytometry plots showing lineage committed mature cells in BM. c, PB WBC and platelets concentrations (per μl) of each genotype (wild-type = 9 mice, Bach1–/– = 9 mice, Bach2–/– = 7 mice, DKO = 8 mice; each dot represents an individual mouse). d, Representative flow cytometry plots showing erythroblasts in BM. e, Total spleen cell numbers of each genotype (n = 10 mice per genotype, each dot represents an individual mouse). f, Mature cell numbers in the spleen of each genotype. B cell (B220+Ter119Gr-1CD4), CD4+ T cell (CD4+Ter119Gr-1B220), Erythroid cell (Ter119+Gr-1B220CD4), Myeloid cell (Gr-1+Ter119B220CD4) (n = 10 mice per genotype, each dot represents an individual mouse). g, Immature (subset I+subset II) and mature (subset III+subset IV) erythroblast numbers in the spleen of each genotype (n = 10 mice per genotype, each dot represents an individual mouse). h, Erythroblast subset III to II ratio in BM of each genotype (n = 6 mice per genotype, each dot represents an individual mouse). i, Erythroblast subset III to II ratio in the spleen of each genotype (n = 10 mice per genotype, each dot represents an individual mouse). Box-and-whisker plot show median, interquartile range, minimum and maximum values (c,e). Bar plots show mean ± s.e.m (f-i). Data are representative of or pooled from two or more experiments (a-i). Statistical analysis was done with two-tailed Student’s t test or Welch’s t test (e-i). No adjustments were made for multiple comparisons. *P < .05, **P < .01, ***P < .001.

Supplementary Figure 2 Investigations about the cause of erythroblast maturation disorder in DKO mice.

a, A flow cytometry analysis of BM macrophages (LinDAPISSC-AlowGr-1lowCD115F4/80+, Lineage; B220 and CD3e) in Bach2-RFP mice. Data were representative from two or more experiments. b,c, Gene expression results of the microarray analysis of BM macrophages from Bach2-RFP mice (n = 3 mice). Amounts of Bach2 mRNA (normalized expression; log2) in RFP-negative and -positive cells were shown in (b). A heat map of the expression of genes related to heme- and iron-metabolism was shown in (c). d, Expression of genes related to heme- and iron-metabolism in wild-type or DKO BM macrophages. Relative gene expression normalized by Actb expression was shown (n = 3 mice per genotype, data were obtained from one experiment, each dot represents an individual mouse). e-h, Effects of HO-1 overexpression in MEL cells. Heme concentrations (e), representative flow cytometry plots showing the percentages of CD71+DCFDA+ (f), the percentages of CD71+DCFDA+ (g) and CD71 MFI values (h) in each condition after 1.5% DMSO treatment for 96 h were shown (n = 6 individual cell cultures per condition, data were obtained from or representative from one (f-h) or two (e) experiments, each dot represents an individual sample). Bar plots show mean ± s.e.m (d,e,g,h). Statistical analysis was done with two-tailed Student’s t test or Welch’s t test (b,d,e,g,h). No adjustments were made for multiple comparisons. *P < .05, **P < .01, ***P < .001.

Supplementary Figure 3 Erythropoiesis disorder also occurred in erythro-myeloid bifurcation in DKO mice.

a-c, Erythro-myeloid progenitor cell populations identified with conventional method in BM. Representative flow cytometry plots of wild-type and DKO mice (a), erythro-myeloid progenitor cell numbers of each genotype (b), relative cell populations of erythro-myeloid progenitor cell of each genotype (c) were shown (n = 10 mice per genotype, each dot represents an individual mouse). d, Numbers of HSC, MPP1, MPP2, MPP3 and MPP4 of each genotype (n = 10 mice per genotype, each dot represents an individual mouse). Bar plots show mean ± s.e.m (c,d). Data are representative of or pooled from two or more experiments (a-d). Statistical analysis was done with two-tailed Student’s t test or Welch’s t test. No adjustments were made for multiple comparisons. *P < .05, **P < .01.

Supplementary Figure 4 Effects of in vitro or in vivo treatment of heme or LPS.

a, Expression of indicated gene in M1 cells stimulated by ctlr or heme (5 μM) for 6 h. Relative gene expression normalized by Actb expression was shown (n = 3 individual cell cultures per condition). b, Expression of indicated gene in sorted CMPs stimulated by heme (5 μM) for 6 h. Relative gene expressions normalized by Actb expression were shown (n = 6 mice derived individual primary cell cultures per condition) c, Western blotting result of M1 cell with or without treatment of heme (5 mM) for 6 h. d, Heme treatment in wild-type or DKO mice. Heme (500 μg/body) were intraperitoneally injected at day1, 3, 5. Mice BMs were analysis at day 8. Then, mature cells in BM were analyzed by flow cytometry. (n = 6 mice per condition, each dot represents an individual mouse). e, Expression of indicated gene in M1 cells stimulated by PBS or LPS (0.1, 1.0, 10 μg/ml) for 48 h. Relative gene expression normalized by Actb expression was shown (n = 3 individual cell cultures per condition). f,g, Results of the in vivo LPS stimulations to wild-type mice. Representative flow cytometry plots of mature cells (f) and mature cell proportions (g) (PBS, n = 3 mice; LPS, n = 4 mice). h, Erythro-myeloid progenitor cells in wild-type or DKO with LPS treatment (50 μg/body) (wild-type, n = 8 mice; DKO, n = 6 mice) i, Expression of indicated gene in differentiated M1 cells by Pam3CS4 (1 μg/ml) or IL6 (0.1 μg/ml) for 48 h. For the control, unstimulated M1 cells were used (n = 3 individual cell cultures per condition). Bar plots show mean ± s.e.m (a,b,d,e,g-i).Data were representative of or pooled from one (d-g), two (a-c,i) or four (h) experiments. Mature cells were identified as follows; Erythroid cell (Ter119+Gr-1B220CD4), Myeloid cell (Gr-1+Ter119B220CD4), B cell (B220+Ter119Gr-1CD4). Statistical analysis was done with two-tailed Student’s t test or Welch’s t test. No adjustments were made for multiple comparisons. *P < .05, **P < .01, **P < .001.

Supplementary Figure 5 Bach1 and Bach2 repress myeloid genes and induce erythroid genes by competitively working with C/EBPβ.

a-c, Results of motif analysis identified by HOMER. Motifs of C/EBPβ single targets (a), C/EBPβ and Bach1 targets (b), C/EBPβ and Bach2 targets (c). d, ChIP-qPCR analysis of indicated transcription factors in M1 cells with or without LPS (1 μg/ml, 48 h) stimulation. Values of precipitated DNA relative to input DNA were adjusted using those of negative control region (MCM5) (n = 2 experiments, each dot represent the value obtained from each experiment).

Supplementary Figure 6 Bach TFs functions in the differentiation of HSPCs.

a,b, Results of the LSK transplantations with or without Bach1 overexpression. Recipient mice were analyzed after 2 weeks. Representative flow cytometry plots of mature cells (a) and proportions of mature erythroid and myeloid cells (b) (n = 4 mice per condition, each dot represents an individual mouse, data were obtained from one experiment with individual measurement). c, Relative expression levels normalized by Actb expression of indicated genes in Gr1+ cells from wild-type or DKO derived HSPCs in the competitive transplantation analysis (n = 4 mice per genotype, each dot represents an individual mouse, samples were obtained individually and RT-qPCR was performed as in one experiment). d, Flow cytometry analysis in the competitive transplantation to determine wild-type HSPCs or DKO HSPCs derived HSC/MPP/LMPP percentages. Data were representative from one experiment using two mice with similar results. Bar plots show mean ± s.e.m (b,c). Statistical analysis was done with two-tailed Student’s t test or Welch’s t test (b,c). No adjustments were made for multiple comparisons. **P < .01, **P < .001.

Supplementary Figure 7 Bach TFs may contribute to the development of MDS phenotype.

a, The erythroid differentiation of human cord blood CD34+ cells with Ctlr-shRNA or shRNA targeting BACH1 or BACH2 were analyzed by flow cytometry. CD71+GlyA+ cells were considered as differentiated erythroid cells. Three different shRNAs targeting BACH1 or BACH2 were used as shown in the dot plot (Ctlr n = 1 vector, Bach1 n = 3 vectors, Bach2 n = 3 vectors, Data obtained from three experiments were shown). b, RNA-based next-generation sequeincing analysis of indicated genes in CD34+ cells from healthy control (HC, n = 3) or from MDS patients (MDS, n = 100). Results were shown in violin plot. c, Scatter plot of Bach2 and Rag2 expressions in RNA-based next-generation sequeincing data. The correlation coefficient r = 0.73. d, Enrichments of the Bach1 and Bach2 on Ccl2. e, Results of the serum cytokines (IL-6 and CCL2) analysis (wild-type, n = 6 mice; Bach1—/—, n = 6 mice; Bach2—/—, n = 5 mice; each dot represents individual mice. Analysis was done using the samples obtained from two experiments). f, GSEA results of indicated gene set using RNA-based next-generation sequeincing data of MDS patients were shown. MDS patients were divided into two groups depending on their BACH2 expression levels (BACH2_high; higher than median expression levels of BACH2, Bach2 _low; lower than median expression levels of BACH2). ES: enrichment score, P-val: nominal p-value as implemented in GSEA. g,h, Subtype analysis of BACH2 expression levels in MDS. Subtype analysis of the data shown in Figure 8b (g). Subtype analysis of the data shown in Supplementary figure 7b (h). Violin plot and Box-and-whisker plot show median, interquartile range, minimum and maximum values (b,e,g,h). Statistical analysis was done with two-tailed Student’s t test or Welch’s t test (b) and two-sided Jonckheere-Terpstra test (g). No adjustments were made for multiple comparisons.

Supplementary Figure 8 Schematic model of the Bach TFs functions in erythro-myeloid bifurcation and their deregulation in MDS.

a, Bach TFs function in erythro-myeloid bifurcations. Bach TFs repress myelopoiesis and induce erythropoiesis under steady state (left panel), whereas their reduced activities upon infection lead to enhanced myelopoiesis by the expense of erythropoiesis (right panel). b, Deregulation of Bach TFs contributes to MDS phenotypes such as anemia, auto-immune reactions and AML transformations.

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Kato, H., Itoh-Nakadai, A., Matsumoto, M. et al. Infection perturbs Bach2- and Bach1-dependent erythroid lineage ‘choice’ to cause anemia. Nat Immunol 19, 1059–1070 (2018). https://doi.org/10.1038/s41590-018-0202-3

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