The unfolded protein response sensor IRE1alpha is required at 2 distinct steps in B cell lymphopoiesis

Abstract

B lymphocyte differentiation is coordinated with the induction of high-level Ig secretion and expansion of the secretory pathway. Upon accumulation of unfolded proteins in the lumen of the ER, cells activate an intracellular signaling pathway termed the unfolded protein response (UPR). Two major proximal sensors of the UPR are inositol-requiring enzyme 1alpha (IRE1alpha), an ER transmembrane protein kinase/endoribonuclease, and ER-resident eukaryotic translation initiation factor 2alpha (eIF2alpha) kinase (PERK). To elucidate whether the UPR plays an important role in lymphopoiesis, we carried out reconstitution of recombinase-activating gene 2-deficient (rag2-/-) mice with hematopoietic cells defective in either IRE1alpha- or PERK-mediated signaling. IRE1alpha-deficient (ire1alpha-/-) HSCs can proliferate and give rise to pro-B cells that home to bone marrow. However, IRE1alpha, but not its catalytic activities, is required for Ig gene rearrangement and production of B cell receptors (BCRs). Analysis of rag2-/- mice transplanted with IRE1alpha trans-dominant-negative bone marrow cells demonstrated an additional requirement for IRE1alpha in B lymphopoiesis: both the IRE1alpha kinase and RNase catalytic activities are required to splice the mRNA encoding X-box-binding protein 1 (XBP1) for terminal differentiation of mature B cells into antibody-secreting plasma cells. Furthermore, UPR-mediated translational control through eIF2alpha phosphorylation is not required for B lymphocyte maturation and/or plasma cell differentiation. These results suggest specific requirements of the IRE1alpha-mediated UPR subpathway in the early and late stages of B lymphopoiesis.

Figure 1

Phenotype of ire1α^–/– embryos. (A) Southern blot analysis of genomic DNA from yolk sacs of ire1α^+/+, ire1α^+/–, and ire1α^–/– embryos. The WT and knockout embryos have a 3-kb WT ire1α allele and a 4-kb recombinant allele, respectively. The heterozygous embryo has both the WT and the recombinant alleles. Rec. allele, ire1α recombinant allele; WT allele, WT ire1α allele. (B) Morphology of ire1α^–/–, ire1α^+/–, and ire1α^+/+ embryos. The ire1α^–/–, ire1α^+/–, and ire1α^+/+ embryos were from the same littermates at E12.5. Magnification, ×10. (C) Histological analysis of ire1α^–/–, ire1α^+/–, and ire1α^+/+ embryos at E12.5. Paraffin-embedded sections (5 μM) of embryos were stained with H&E and visualized at magnification ×20.

Figure 2

Characterization of ire1α^–/– HSCs. (A) Phenotype of ire1α^–/– HSCs from fetal liver and AGM. FACS analysis of CD34 and c-kit in cells from ire1α^–/– and ire1α^+/+ fetal liver and AGM at E11. The ratio of the number of AGM cells to that of fetal liver cells used for FACS analysis was 2:1. Representative data from 1 of at least 3 separate experiments are shown. (B) BrdU labeling of ire1α^–/– and ire1α^+/+ fetal livers at E11. The pregnant mouse was injected with 10 mg BrdU at 2 hours before isolation of embryos. Paraffin-embedded sections (5 μM) were stained with anti-BrdU antibody and visualized at ×100 magnification. The proliferating cells in the fetal livers that incorporated BrdU can be identified by dark staining. (C) Proliferation rates of ire1α^–/– and ire1α^+/– HSCs. Fetal liver cells from WT embryos at E14.5 were first cultured for 2 days to create a hepatic stromal layer. Similar amounts of ire1α^–/– and ire1α^+/– CD34 and c-kit double-positive HSCs from embryos at E11 were overlaid on the hepatic stromal layer and were cultured for 10 days. The proliferation rates of ire1α^–/– and ire1α^+/– HSCs were determined by quantification of neomycin gene copies in the genomic DNA isolated from the proliferated ire1α^–/– and ire1α^+/– cells. The proliferation rate of the ire1α^–/– cells was defined as 1. The proliferation rates of other cells were compared with that of the ire1α^–/– cells. Experiments were performed at least 3 times, and the SD is indicated.

Figure 3

Development of B cells is blocked at the early stage in the absence of IRE1α. (A) FACS analysis of cell surface markers CD43 and B220 in bone marrow cells from rag2^–/– mice after 4 weeks of reconstitution with ire1α^–/– or ire1α^+/+ fetal liver and AGM cells. (B) Genotypes of the CD43⁺B220^– cells sorted from the reconstituted bone marrow. The upper panel shows amplification of segments from the neo gene and the murine ire1α gene. The first 2 lanes show CD43⁺B220^– cells sorted from the ire1α^–/– and ire1α^+/+ reconstituted bone marrow. The third lane shows CD43⁺B220^– cells sorted from nonirradiated rag2^–/– mice. The fourth and fifth lanes show ire1α^–/– and ire1α^+/+ MEFs, which served as controls for the genotype of ire1α^–/– and ire1α^+/+ cells, respectively. The lower panel shows amplification of the rag2 gene WT (rag2^+/+) and knockout (rag2^–/–) alleles. Through calibration of the amplified gene alleles, the ratios of donor cells to recipient cells in both ire1α^–/– and ire1α^+/+ reconstituted CD43⁺B220^– populations were found to be more than 10:1. (C) RT-PCR analysis of expression of ire1α and neo genes in the CD43⁺B220^– cells sorted from the reconstituted bone marrow. The samples are the same as described in B. (D–F) FACS analysis of cell surface markers BP-1 and HSA (D), B220 and IgM (E), and TER119 and Mac-1 (F) in ire1α^–/– and ire1α^+/+ reconstituted bone marrow cells. (G) Genotypes of TER119⁺ cells sorted from the reconstituted bone marrow. The first 2 lanes show TER119⁺ cells sorted from the ire1α^–/– and ire1α^+/+ reconstituted bone marrow. The third lane shows TER119⁺ cells from nonirradiated rag2^–/– mice. The fourth and fifth lanes show ire1α^–/– and ire1α^+/+ MEFs. Through calibration of the amplified gene alleles, the ratios of donor cells to recipient cells in both ire1α^–/– and ire1α^+/+ reconstituted TER119⁺ populations were found to be more than 8:1. For A–G, experiments were performed at least 3 times and representative data are shown.

Figure 4

Impaired VDJ rearrangements in B lymphocytes in the absence of IRE1α. (A) Analysis of Ig gene rearrangement in reconstituted B220⁺c-kit⁺ cells. DNA was isolated from B220⁺c-kit⁺ cells sorted from ire1α^+/+ and ire1α^–/– reconstituted bone marrow. B220⁺ bone marrow cells sorted from a 4-week-old WT mouse served as a positive control (Con). To control variation in input DNA or amplification efficiency, a fragment from the α-actin gene was amplified. (B) Genotypes of the B220⁺c-kit⁺ cells sorted from the reconstituted bone marrow. The B220⁺c-kit⁺ cells were the same as in A. Through calibration of the amplified gene alleles, the ratios of donor cells to recipient cells in both ire1α^–/– and ire1α^+/+ reconstituted B220⁺c-kit⁺ populations were found to be more than 15:1. (C) RT-PCR analysis of expression of ire1α and neo genes in the same B220⁺c-kit⁺ cells as in A. A fragment from β-actin transcript was amplified to control variation in input RNA and amplification efficiency. (D–F) Induction profiles of genes encoding factors required for very early stage B lymphopoiesis (D), genes encoding accessory proteins required for IgM complex (E), and genes encoding factors required for initiation of VDJ rearrangements (F). Total RNA was extracted from B220⁺ cells sorted from the ire1α^+/+ and ire1α^–/– reconstituted bone marrow. (G) Expression of VDJ-Cμ transcripts in ire1α^+/+ and ire1α^–/– reconstituted B220⁺ bone marrow cells. Total RNA was extracted from B220⁺ cells sorted from the ire1α^+/+ and ire1α^–/– reconstituted bone marrow. For A–G, experiments were performed at least 3 times and representative data are shown.

Figure 5

The IRE1α cytoplasmic domain is required for BCR formation and plasma cell differentiation. (A) Depiction of viral transduction and bone marrow cell transplantation. Lentivirus (IRE1α), lentivirus expressing IRE1α protein variants; Retrovirus (XBP1), retrovirus expressing spliced XBP1 protein. (B) FACS analysis of B220 and IgM in splenocytes from the reconstituted rag2^–/– mice. (C) Western blot analysis of expression of the IRE1α transgenes in B220⁺ splenic B cells sorted from the reconstituted rag^–/– mice as described in B. (D) Levels of serum IgM and IgG1 in the reconstituted rag2^–/– mice. Blood samples were collected from the reconstituted rag2^–/– mice at 1 month after transplantation. Levels of serum IgM and IgG1 were determined by ELISA and normalized to the number of GFP-positive B cells. (E) Western blot analysis of expression of IRE1α and XBP1 protein variants in primary splenic B cells after secondary viral transduction. (F) Levels of secreted IgM and IgG1 in LPS-stimulated B cells. The samples are labeled the same as described in E. Primary B cells after secondary viral transduction were treated with LPS (20 μg/ml) for 48 hours. Levels of secreted IgM and IgG1 in cultured medium were determined by ELISA. V/C, control virus–transduced; IRE1α °C, IRE1α cytoplasmic domain deletion; IRE1α °C + IRE1α, IRE1α °C plus WT IRE1α protein, RV, control retrovirus; XPB1s, retrovirus expressing spliced XBP1 protein. For B–F, experiments were performed at least 3 times and representative data and SD are shown.

Figure 6

IRE1α kinase and RNase activities regulate the production of Ig through spliced XBP1. (A) Depiction of IRE1α trans-dominant-negative vectors. IRE1α K599A, IRE1α kinase mutant; IRE1α K907A, IRE1α RNase mutant. (B) FACS analysis of IgD and IgM in splenocytes from reconstituted rag2^–/– mice. (C) Levels of serum IgM and IgG1 in the reconstituted rag2^–/– mice. Blood samples were collected from the reconstituted rag2^–/– mice at 1 month after transplantation. Levels of serum IgM and IgG1 were determined by ELISA and normalized to the number of GFP-positive B cells. (D) XBP1 splicing and induction of IL-6, secretory IgM, IgG2b, and IgG3 transcripts during plasma cell differentiation in the absence of the IRE1α kinase, RNase, or cytoplasmic domain. GFP and B220 double-positive splenic B cells were sorted from the reconstituted rag2^–/– mice and subsequently stimulated with LPS (20 μg/ml) for 48 hours in vitro. Total RNA was isolated from the LPS-treated B cells and subjected to semiquantitative RT-PCR analysis. IgMs, secretory IgM transcript; u-Xbp1, unspliced xbp1 transcript; s-Xbp1, spliced xbp1 transcript. (E) Western blot analysis of expression of IRE1α and XBP1 protein variants in primary B cells after secondary viral transduction. (F) Spliced XBP1 restores Ig production in the IRE1α kinase or RNase mutant B cells, but not in the IRE1α cytoplasmic domain–deleted B cells. Primary B cells after secondary viral transduction were treated with LPS (20 μg/ml) for 48 hours. Levels of secreted IgM and IgG1 in cultured supernatants of the indicated stimulated B cells were determined by ELISA. For B–F, experiments were performed at least 3 times and representative data and SD are shown.

Figure 7

PERK-eIF2α UPR signaling is not required for plasma cell differentiation. (A) IP–Western blot analysis of expression and activation of IRE1α and PERK in primary B cells upon LPS stimulation. B220⁺ primary B cells from the spleens of 2-month-old WT mice were cultured in vitro in the presence of LPS (20 μg/ml) for the indicated time intervals. To generate controls for IRE1α and PERK proteins, WT, ire1α^–/–, and perk^–/– MEFs and primary B cells were treated with thapsigargin (Thap; 0.2 μM) for 3 hours. Equivalent amounts of cellular lysates were applied for each sample. p-, phosphorylated. (B–C) Kinetics of induction of spliced XBP1 and secreted IgM transcripts (B) and EDEM and CHOP transcripts (C) in primary B cells upon LPS treatment. Primary B cells were isolated from spleens of WT adult mice and were then stimulated with LPS (20 μg/ml) in vitro for the indicated time intervals. Levels of transcripts were determined by quantitative real-time RT-PCR. (D) FACS analysis of B220 and IgD in splenocytes from rag2^–/– mice reconstituted with WT (eIF2α SS) or eIF2α phosphorylation–defective (eIF2α AA) fetal liver cells. Fetal hematopoietic liver cells (4 × 10⁶) from E13.5 embryos were used for transplantion. (E) Western blot analysis of phosphorylated and total eIF2α proteins in the reconstituted B cells. B220⁺ cells were sorted from the spleens of rag2^–/– mice reconstituted with eIF2α AA or SS fetal liver and then treated with 0.2 μM thapsigargin for 3 hours. Thapsigargin-treated eIF2α AA and SS MEFs were used as controls. (F) Levels of serum IgM and IgG1 in the rag2^–/– mice reconstituted with eIF2α SS or eIF2α AA fetal liver cells. Blood samples were collected from the reconstituted rag2^–/– mice at 1 month after transplantation. Levels of serum IgM and IgG1 were determined by ELISA. (G) The proposed model depicts that IRE1α is required for both the early and the late stages of B lymphocyte development. At the very early stage, IRE1α plays a role in the induction of the rag1, rag2, and TdT genes; thus IRE1α is required for VDJ recombination and BCR formation. At the late stage, IRE1α kinase and RNase activities regulate the terminal differentiation of B cells to plasma cells through splicing of XBP1 mRNA.