Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Aug;16(8):829-37.
doi: 10.1038/ni.3225. Epub 2015 Jul 6.

The transcription factor XBP1 is selectively required for eosinophil differentiation

Sarah E Bettigole  1 , Raphael Lis  2 , Stanley Adoro  3 , Ann-Hwee Lee  4 , Lisa A Spencer  5 , Peter F Weller  5 , Laurie H Glimcher  3
Affiliations

The transcription factor XBP1 is selectively required for eosinophil differentiation

Sarah E Bettigole et al. Nat Immunol. 2015 Aug.

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.

PubMed Disclaimer

Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors declare competing financial interests. L.H.G. is on the Board of Directors and holds equity in Bristol Myers Squibb Pharmaceutical Company.

Figures

Figure 1
Figure 1
XBP1 is required for eosinophil differentiation. (a) Flow cytometry of cells from the bone marrow (BM), spleen and blood of Xbp1f/f and Xbp1Vav1 mice, gated on non-B, non-T cells (spleen and blood only). Numbers adjacent to outlined areas indicate percent Siglec-F+CCR3+ mature eosinophils (top right) or Siglec-F+CCR3− immature bone marrow eosinophils (top left, far left column). (b) Frequency of mature (Siglec-F+CCR3+) eosinophils (eos) in the bone marrow, spleen and blood of Xbp1f/f and Xbp1Vav1 mice (n = 3 per genotype). (c) Absolute number of mature eosinophils in the bone marrow (two tibias and two femurs per mouse), spleen and blood of Xbp1f/f and Xbp1Vav1 mice (n = 3 per genotype). (d) Flow cytometry of cells from the blood of Ern1f/f and Ern1Vav1 mice. Numbers adjacent to outlined areas indicate percent Siglec-F+CCR3+ mature eosinophils. (e) Frequency of mature eosinophils in blood of Ern1f/f mice (n = 5) and Ern1Vav1 mice (n = 4). Each symbol (b,c,e) represents an individual mouse; small horizontal lines indicate the mean (± s.e.m.). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 (Student’s t-test). Data are representative of at least three independent experiments with at least three mice per group in each.
Figure 2
Figure 2
XBP1 is potently activated during eosinophil differentiation in vivo and is required upon commitment to the eosinophil lineage. (a) PCR analysis of spliced (Xbp1s) and unspliced (Xbp1us) Xbp1 mRNA in LSK cells, CMPs, GMPs, EoPs and CCR3− or CCR3+ eosinophils purified by flow cytometry. (b) Frequency of Xbp1s mRNA among total Xbp1 mRNA in sorted LSK cells, CMPs, GMPs, EoPs, CCR3− eosinophils, and CCR3+ eosinophils (n = 3 mice per cell type). (c) Quantitative PCR analysis of the Xbp1s isoform in cells as in a (n = 3 mice per cell type); results were normalized to those of Actb. (d) Quantitative PCR analysis of Sec24d, P4hb, Edem1 and Ddit3 in cells as in a (n = 3 mice per cell type); results (normalized as in c) are presented relative to those of LSK cells. (e) Gating strategy for flow cytometry of bone marrow GMPs from Xbp1f/f and Xbp1Vav1 mice. Numbers adjacent to outlined areas indicate percent cells with leukocyte side scatter (SSC-A) or forward scatter (FSC-A) (far left), c-Kit+Lin− cells (middle left) or c-Kit+Sca-1− cells (middle right); numbers in quadrants indicate percent GMPs in each (far right). (f) Frequency of GMPs in the bone marrow of Xbp1f/f and Xbp1Vav1 mice (n = 3 per genotype). (g) Gating strategy for flow cytometry of bone marrow EoPs from Xbp1f/f and Xbp1Vav1 mice. Numbers adjacent to outlined areas indicate percent cells with leukocyte side or forward scatter (far left), CD34+Lin− cells (middle left), c-Kitpos–negSca-1− cells (middle right) or c-KitintIL-5Rα+ cells (far right). (h) Frequency of EoPs in bone marrow from Xbp1f/f and Xbp1Vav1 mice (n = 3 per genotype). (i) Frequency of EoPs exhibiting caspase activity (Casp+), assessed by flow cytometry (n = 3 mice per genotype). (j) Flow cytometry of eosinophils in bone marrow from Xbp1f/f and Xbp1eoCRE mice. Numbers adjacent to outlined areas indicate percent CCR3pos–negSiglec-F− cells (left), CCR3+Siglec-F+ cells (top right) or CCR3−Siglec-F+ cells (bottom right). (k) Frequency of eosinophils in bone marrow from Xbp1f/f and Xbp1eoCRE mice (n = 5 mice per genotype). Each symbol (f,h,i,k) represents an individual mouse; small horizontal lines indicate the mean (± s.e.m.). NS, not significant (P > 0.05); *P < 0.001 and **P < 0.0001 (Student’s t-test). Data are from one experiment representative of three experiments with more than three independent biological replicates (a) or are representative of at least three independent experiments with at least three mice per group in each (b–k; mean and s.e.m. in b–d,f,h,i,k).
Figure 3
Figure 3
XBP1 is a cell-intrinsic requirement for eosinophil development. (a) Flow cytometry assessing expression of the congenic markers CD45.2 (Xbp1f/f or Xbp1Vav1) and CD45.1 (wild type) on peripheral blood eosinophils from chimeras generated with a mixture of wild-type bone marrow plus either Xbp1f/f bone marrow (WT + Xbp1f/f) or Xbp1Vav1 bone marrow (WT + Xbp1Vav1). Numbers in quadrants indicate percent cells in each. (b) Reconstitution efficiency of eosinophils, neutrophils (Neut) and basophils (Baso) in blood from mixed-bone marrow chimeras as in a (n = 6 host mice per chimera type). (c) Flow cytometry analyzing Siglec-F expression Xbp1f/f and Xbp1Vav1 BMDEs at day 8 of culture, gated on DAPI− singlets. Numbers adjacent to outlined areas indicate percent Siglec-F+ cells. (d) Frequency of Siglec-F+ cells among Xbp1f/f and Xbp1Vav1 BMDEs at days 0, 4, 8 and 10 of culture (n = 3 independent cultures per genotype). (e) Microscopy of hematopoietic colonies from Xbp1f/f and Xbp1Vav1 BMDEs at day 8 of culture. Scale bars, 100 μm. (f) Microscopy of cytospin analysis of DAPI−Siglec-F+ singlets sorted from Xbp1f/f and Xbp1Vav1 BMDEs at day 8 of culture. Scale bars, 5 μm. *P < 0.0001 (two-way analysis of variance (ANOVA) with the Šidák correction for multiple comparisons). Data are from two independent experiments with six mice per group (a,b; mean and s.e.m. in b) or more than three independent experiments with at least three mice per group (c–f; mean and s.e.m. in d).
Figure 4
Figure 4
Xbp1s can restore eosinophil development in Xbp1-deficient bone marrow cultures, but Xbp1u cannot. (a) Flow cytometry analyzing the expression of GFP and Siglec-F in Xbp1f/f and Xbp1Vav1 BMDEs transduced with retrovirus expressing Xbp1u mRNA (XBP1U-RV), Xbp1us mRNA (XBP1US-RV) or Xbp1s mRNA (XBP1S-RV) assessed at day 8 of culture. Numbers in quadrants indicate percent cells in each. (b) Frequency of Siglec-F+ eosinophils among total GFP− and GFP+ cells from Xbp1f/f and Xbp1Vav1 BMDE cultures transduced as in a. Each symbol represents an individual mouse (n = 3 per genotype); small horizontal lines indicate the mean (± s.e.m.). (c) Microscopy of cytospin analyses of GFP+Siglec-F+ cells sorted from Xbp1f/f and Xbp1Vav1 BMDEs transduced as in a (below images), assessed at day 8 of culture. Scale bars, 5 μm. *P < 0.01, **P < 0.001 and ***P < 0.0001 (one-way ANOVA with the Holm-Šidák correction for multiple comparisons). Data are representative of three independent experiments with three biological replicates in each.
Figure 5
Figure 5
The GMP-differentiation capacity is not affected by Xbp1 deficiency. (a) Quantitative PCR analysis of genes encoding products known to regulate eosinophil differentiation, in sorted GMPs from Xbp1f/f and Xbp1Vav1 mice (n = 3 per genotype); results were normalized to those of Actb and are presented relative to those of Xbp1f/f GMPs. (b) RNA-seq analysis of selected genes encoding eosinophil markers, in Xbp1f/f and Xbp1Vav1 GMPs (n = 3 mice per genotype). (c) Pathway analysis of the ten molecular and cellular gene-ontology functions most significantly dysregulated in Xbp1Vav1 GMPs relative to their regulation in Xbp1f/f GMPs, ranked by P value from most significant (left) to least significant (right); results are presented as raw P values. Data are representative of three independent experiments (a; mean and s.e.m.) or one experiment with three independent biological replicates per group (b,c).
Figure 6
Figure 6
Loss of XBP1-mediated protein-quality-control mechanisms interferes with eosinophil transcriptional identity. (a,b) RNA-seq analysis of genes encoding eosinophil markers (a) or regulators of eosinophil development (b), in GATA-2-transduced Xbp1f/f and Xbp1Vav1 GMPs (n = 3 independent cultures per genotype). (c) Quantitative PCR analysis of Gata1 and Gata2 in GATA-2-transduced Xbp1f/f and Xbp1Vav1 GMPs (n = 3 independent cultures per genotype) (presented as in Fig. 5a). (d) Pathway analysis–predicted top five activated or inhibited upstream transcriptional regulators (ranked by activation z-score) most responsible for the transcriptional differences between GATA-2-transduced (GATA-2 TD) Xbp1f/f GMPs and GATA-2-transduced Xbp1Vav1 GMPs, showing functional repression (blue) or activation (red) in Xbp1Vav1 cells compared with Xbp1f/f cells, and predicted influence on freshly sorted GMPs (Fresh). (e) Pathway analysis–predicted ten most significantly dysregulated molecular and cellular gene-ontology functions in GATA-2-transduced Xbp1f/f and Xbp1Vav1 GMPs (presented as in Fig. 5b). (f) Quantitative PCR analysis of Ddit3, Asns, Trib3 and Hspa5 in GATA-2-transduced Xbp1f/f and Xbp1Vav1 GMPs (n = 3 independent cultures per genotype) (presented as in Fig. 5a). *P < 0.05, **P < 0.01 and ***P < 0.0001 (Student’s t-test (c) or one-way ANOVA with the Holm-Šidák correction for multiple comparisons (f)). Data are representative of one experiment (a,b,d,e) or two independent experiments with three independent biological replicates per group (c,f; mean and s.e.m.).
Figure 7
Figure 7
Xbp1 deficiency causes defects in granule-protein maturation and secretory-pathway ultrastructure. (a) Immunoblot analysis of EPX maturation (left margin) in GATA-2-transduced Xbp1f/f (f/f) and Xbp1Vav1 (Vav1) GMPs. (b) Frequency of mature EPX in GATA-2-transduced Xbp1f/f and Xbp1Vav1 GMPs (n = 3 independent cultures per genotype). (c) Immunoblot analysis of the maturation of pre-pro-PRG2 in GATA-2-transduced Xbp1f/f and Xbp1Vav1 GMPs; below (Ctrl), nonspecific background band (loading control). (d) Abundance of immature pro-PRG2 (pre-pro-PRG2) in GATA-2-transduced Xbp1f/f and Xbp1Vav1 GMPs (n = 3 independent cultures per genotype); results were normalized to those of the nonspecific background band and are presented relative those of Xbp1f/f GMPs. (e) Transmission electron microscopy of GATA-2-transduced Xbp1f/f and Xbp1Vav1 GMPs: blue arrows indicate developing secretory granules; black arrows indicate the ER. Scale bars, 500 nm. *P < 0.05 and **P < 0.01 (Student’s t-test). Data are representative of (a,c,e) or from (b,d) one experiment with three independent biological replicates per group (mean and s.e.m. in b,d).
Figure 8
Figure 8
Xbp1 deficiency prevents the accumulation of GATA-1 in eosinophils through an indirect mechanism. (a) Quantitative PCR analysis of Gata1, Prg2 and Epx in sorted DAPI−Siglec-F+ BMDEs from Xbp1f/f and Xbp1Vav1 mice (n = 3 independent cultures per genotype), assessed at day 8 of culture; results were normalized to those of Actb. (b) Immunoblot analysis of GATA-1 and β-actin (loading control) in Xbp1f/f and Xbp1Vav1 BMDEs at day 8 of culture. (c) Immunofluorescence microscopy of developing eosinophils among Xbp1f/f and Xbp1Vav1 BMDEs at day 8 of culture, stained for PRG2, GATA-1 and DAPI. (d) Scatter plots of PRG2 fluorescence versus GATA-1 fluorescence in Xbp1f/f BMDEs (n = 50) and Xbp1Vav1 BMDEs (n = 56) at day 8 of culture. (e) Staining intensity of GATA-1 in developing PRG2+ eosinophils among Xbp1f/f BMDEs (n = 47) and Xbp1Vav1 BMDEs (n = 30) at day 8 of culture. (f) Quantitative PCR analysis of the canonical eosinophil genes Gata1, Prg2 and Epx, as well as Xbp1s and the XBP1 target genes Sec24d, P4hb, Sec61a1 and Dnajb9, in BMDE cultures treated for 8 h with the vehicle dimethyl sulfoxide (DMSO) or 4μ8C (20 μM) (n = 3 independent cultures per condition); results were normalized to those of Actb and are presented relative to those of vehicle-treated cells. *P < 0.05 and **P < 0.01 and ***P < 0.0001 (Student’s t-test). Data are representative of at least two independent experiments (a,f; mean and s.e.m.), two independent experiments (b) or two experiments with three independent biological replicates per genotype (c) or are from one experiment with three biological replicates pooled per genotype (d,e; mean and s.e.m. in e).
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Calfon M, et al. IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA. Nature. 2002;415:92–96. - PubMed
    1. Harding HP, Zhang Y, Ron D. Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature. 1999;397:271–274. - PubMed
    1. Lu PD, Harding HP, Ron D. Translation reinitiation at alternative open reading frames regulates gene expression in an integrated stress response. J Cell Biol. 2004;167:27–33. - PMC - PubMed
    1. Lee AH, Iwakoshi NN, Glimcher LH. XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response. Mol Cell Biol. 2003;23:7448–7459. - PMC - PubMed
    1. Shoulders MD, et al. Stress-independent activation of XBP1s and/or ATF6 reveals three functionally diverse ER proteostasis environments. Cell Rep. 2013;3:1279–1292. - PMC - PubMed

Publication types

MeSH terms

Associated data