Abstract
The transcription factor XBP1 has been linked to the development of highly secretory tissues such as plasma cells and Paneth cells, yet its function in granulocyte maturation has remained unknown. Here we discovered an unexpectedly selective and absolute requirement for XBP1 in eosinophil differentiation without an effect on the survival of basophils or neutrophils. Progenitors of myeloid cells and eosinophils selectively activated the endoribonuclease IRE1α and spliced Xbp1 mRNA without inducing parallel endoplasmic reticulum (ER) stress signaling pathways. Without XBP1, nascent eosinophils exhibited massive defects in the post-translational maturation of key granule proteins required for survival, and these unresolvable structural defects fed back to suppress critical aspects of the transcriptional developmental program. Hence, we present evidence that granulocyte subsets can be distinguished by their differential reliance on secretory-pathway homeostasis.
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Acknowledgements
We thank L. Cohen-Gould for assistance with electron microscopy; J. McCormick for assistance with cell sorting; the Starr Foundation Tri-Institutional Stem Cell Derivation Laboratory and Flow Cytometry and Microscopy Core facility for technical assistance, the Weill Cornell Epigenomics Core facility for library preparation and RNA sequencing; J. Cubillos-Ruiz and C. Tan for technical assistance; A. Espinosa, S. Hai and members of the Glimcher laboratory for suggestions and critical reading of this manuscript; A. Doyle for conversations; J.J. Lee and N.A. Lee (Mayo Clinic) for eoCRE mice, antibody to PRG2 and antibody to EPX; C. Gerard (Children's Hospital Medical, Boston) for Il5-transgenic BALB/c mice; and T. Iwawaki (Gunma University) for Ern1f/f mice. Supported by the US National Institutes of Health (R01DK082448 to L.H.G., R01HL095699 to L.A.S. and R37AI020241 to P.F.W.).
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S.E.B. and L.H.G. designed and analyzed the experiments; S.E.B. conducted experiments and wrote the manuscript; R.L. performed high-resolution immunofluorescence microscopy imaging. S.A. contributed to the design of certain experiments; A.-H.L. provided reagents; and L.A.S., P.F.W. and L.H.G., supervised the research and edited the manuscript.
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L.H.G. is on the board of directors of and holds equity in Bristol Myers Squibb Pharmaceutical Company.
Integrated supplementary information
Supplementary Figure 1 Xbp1 deficiency does not alter hematopoietic cellularity.
Absolute number of leukocytes obtained from bone marrow (BM, 2 tibias and 2 femurs per mouse) of Xbp1f/f and Xbp1Vav1 mice (n = 3 per genotype). Each symbol represents an individual mouse; small horizontal lines indicate the mean (± s.e.m.). NS, not significant (P > 0.05, Student’s t-test). Data are representative of at least three independent experiments with at least three mice per genotype per experiment.
Supplementary Figure 2 Xbp1 deficiency minimally influences most leukocyte subsets.
(a) Flow cytometry of splenic CD3+ T cells and B220+ B cells in Xbp1f/f and Xbp1Vav1 mice. Numbers adjacent to outlined areas indicate percent CD3+B220− T cells (top left) or CD3−B220+ B cells (bottom right). (b,c) Frequency of T cells (b) and B cells (c) in spleens of Xbp1f/f and Xbp1Vav1 mice (n = 3 per genotype). (d) Flow cytometry of splenic FcɛRI+ basophils in Xbp1f/f and Xbp1Vav1 mice. Numbers adjacent to outlined areas indicate percent SSClowFcɛRI+ basophils gated on non-B and non-T cells. (e) Splenic basophil frequency in Xbp1f/f and Xbp1Vav1 mice (n = 3 per genotype). (f) Flow cytometry of splenic CD11b+Ly6G+ neutrophils in Xbp1f/f and Xbp1Vav1 mice. Numbers adjacent to outlined areas indicate percent CD11b+Ly6G+ neutrophils. (g) Splenic neutrophil frequency in Xbp1f/f and Xbp1Vav1 mice (n = 3 per genotype). (h) Flow cytometry of splenic CD11b+F4/80+ macrophages in Xbp1f/f and Xbp1Vav1 mice. Numbers adjacent to outlined areas indicate percent CD11b+F4/80+ macrophages. (i) Splenic macrophage frequency in Xbp1f/f and Xbp1Vav1 mice (n = 3 per genotype). (j) Flow cytometry of splenic CD11c+ dendritic cells (DCs) in Xbp1f/f and Xbp1Vav1 mice. Numbers adjacent to outlined areas indicate percent B220−CD11c+ macrophages. (k) Splenic DC frequency in Xbp1f/f and Xbp1Vav1 mice (n = 3 per genotype). Each symbol (b,c,e,g,i,k) represents an individual mouse; small horizontal lines indicate the mean (± s.e.m.). NS, not significant (P > 0.05); *P < 0.05 (Student’s t-test). Data are representative of at least three independent experiments with at least three mice per group in each.
Supplementary Figure 3 The Vav1-Cre transgene does not affect eosinophil differentiation.
Flow cytometry of CCR3+Siglec-F+ eosinophils from the blood of Xbp1f/f, Xbp1WT/WT Vav1-Cre+, and Xbp1Vav1 mice (n = 2 mice per group). Numbers adjacent to outlined areas indicate percent CCR3+Siglec-F+ eosinophils. Data are representative of at least 2 independent experiments with at least two mice per group in each.
Supplementary Figure 4 Cre expressed from the Vav1-Cre transgene results in efficient deletion of Xbp1 in basophils, neutrophils and GMPs.
(a,b,c) Quantitative PCR analysis of the floxed Xbp1 exon 2 in bone marrow basophils (a), neutrophils (b), and GMPs (c) sorted from Xbp1f/f and Xbp1Vav1 mice (n = 3 mice per genotype). Results were normalized to those of Actb and are presented relative to those of Xbp1f/f cells. *P < 0.05 and **P < 0.01 (Student’s t-test). Data are from one experiment (a,b; mean and s.e.m.) or are representative of more than three experiments with three mice per genotype (c; mean and s.e.m.).
Supplementary Figure 5 Inhibition of IRE1α does not affect the survival of mature eosinophils.
(a) Flow cytometry of annexin-V and propidium iodide (PI) staining of Siglec-F+SSChi bone marrow cells after 24 h in vitro incubation with vehicle dimethyl sulfoxide (DMSO) or 20 μM of the IRE1α inhibitor 4μ8C. Numbers adjacent to outlined areas indicate percent PI+Annexin-V+ late apoptotic eosinophils (top right), PI−Annexin-V+ early apoptotic eosinophils (bottom right), or PI−Annexin-V− live eosinophils. (b) Frequency of eosinophils (eos) of total bone marrow cells after 24 h in vitro incubation with DMSO or 20 μM 4μ8C (n = 3 cultures per condition). (c) Percent viability of eosinophils from bone marrow cultures after 24 h in vitro incubation with DMSO or 20 μM 4μ8C (n = 3 cultures per condition). Each symbol (b,c) represents an individual mouse; small horizontal lines indicate the mean (± s.e.m.). NS, not significant (P > 0.05, Student’s t-test). Data are representative of two independent experiments with three independent biological replicates per condition.
Supplementary Figure 6 Overexpression of GATA-2 in GMPs drives commitment to the eosinophil lineage.
(a) Microscopy of GMPs transduced with GFP control (GFP-RV, ‘Control’) or GATA-2 expressing (GATA-2-RV, ‘GATA-2 TD’) retroviruses after indicated timepoints in culture. Scale bars, 5 μm. (b) Quantitative PCR analysis of Prg2 and Epx in GMPs transduced as in a after 96 h in culture; results were normalized to those of Actb (n = 4 cultures per virus). (c) Microscopy of GATA-2-transduced Xbp1f/f GMPs and GATA-2-transduced Xbp1Vav1 GMPs after 96 h in culture. Scale bars, 5 μm. ***P < 0.0001 (Student’s t-test). Data are representative of two independent experiments with at least three independent biological replicates per condition (a–c; mean and s.e.m. in b).
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–6 (PDF 2634 kb)
Supplementary Table 1
Full DESeq transcriptome statistical analysis results comparing Xbp1f/f and Xbp1Vav1 GMPs (XLSX 1895 kb)
Supplementary Table 2
Enriched gene ontology categories and predicted upstream transcriptional regulators from IPA analyses comparing freshly sorted Xbp1f/f GMPs to Xbp1Vav1 GMPs or GATA2-transduced Xbp1f/f GMPs to GATA2-transduced Xbp1Vav1 GMPs 48 hours after infection (XLSX 64 kb)
Supplementary Table 3
Full DESeq transcriptome statistical analysis results comparing GATA2-transduced Xbp1f/f and Xbp1Vav1 GMPs 48 hours after infection. (XLSX 2186 kb)
Supplementary Table 4
Table of RT-qPCR primers used in this study (XLSX 54 kb)
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Bettigole, S., Lis, R., Adoro, S. et al. The transcription factor XBP1 is selectively required for eosinophil differentiation. Nat Immunol 16, 829–837 (2015). https://doi.org/10.1038/ni.3225
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DOI: https://doi.org/10.1038/ni.3225
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