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The unfolded-protein-response sensor IRE-1α regulates the function of CD8α+ dendritic cells

Nature Immunology volume 15, pages 248–257 (2014)Cite this article

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Abstract

The role of the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress in homeostasis of the immune system is incompletely understood. Here we found that dendritic cells (DCs) constitutively activated the UPR sensor IRE-1α and its target, the transcription factor XBP-1, in the absence of ER stress. Loss of XBP-1 in CD11c+ cells led to defects in phenotype, ER homeostasis and antigen presentation by CD8α+ conventional DCs, yet the closely related CD11b+ DCs were unaffected. Whereas the dysregulated ER in XBP-1-deficient DCs resulted from loss of XBP-1 transcriptional activity, the phenotypic and functional defects resulted from regulated IRE-1α-dependent degradation (RIDD) of mRNAs, including those encoding CD18 integrins and components of the major histocompatibility complex (MHC) class I machinery. Thus, a precisely regulated feedback circuit involving IRE-1α and XBP-1 controls the homeostasis of CD8α+ conventional DCs.

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Figure 1: 'Preferential' activation of the IRE-1α pathway in CD8α+ cDCs.
Figure 2: CD8α+ cDCs from DC–XBP-1Δ mice have an altered phenotype.
Figure 3: Defects in XBP-1-deficient CD8α+ cDCs are cell intrinsic.
Figure 4: Disturbed ER in XBP-1-deficient CD8α+ cDCs.
Figure 5: Antigen-presentation defects of XBP-1-deficient CD8α+ cDCs.
Figure 6: Alterations in gene expression in XBP-1-deficient cDCs.
Figure 7: RIDD controls the phenotype and function of CD8α+ cDCs.

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Acknowledgements

We thank L. Glimcher (Cornell University) for Xbp1fl/fl mice; B. Reizis (Columbia University) for Itgax-Cre mice; B. Malissen (Aix Marseille University) for H-Y mice; J. Magalhaes (Institut Curie) for advice and protocols; D. Jankovic and A. Sher (National Institute of Allergy and Infectious Diseases) for T. gondii extracts; C. Reis e Sousa (Cancer Research UK) for bm1 T OVA cells; the VIB Microarray Facility for doing the microarray experiments; the IRC Microscopy Core Facility and, more specifically, A. Kremers, S. Lippens and C. Guerin for help with the imaging of XBP-1-deficient CD8α+ cDCs by focused ion beam–scanning electron microscopy. Supported by the European Research Council (B.N.L.), the European Union Seventh Framework Programme (B.N.L.), the Fonds Wetenschappelijk Onderzoek Vlaanderen program (B.N.L. and S.J.), Ghent University (B.N.L. and S.J.), Marie Curie Actions (F.O. and E.H.), The Federation of European Biochemical Societies (F.O.) and the Agentschap voor Innovatie door Wetenschap en Techniek (S.J.T.).

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Author notes

  1. Fabiola Osorio, Simon J Tavernier, Sophie Janssens and Bart N Lambrecht: These authors contributed equally to this work.

Authors and Affiliations

  1. Unit Immunoregulation and Mucosal Immunology, VIB Inflammation Research Center, Ghent, Belgium

    Fabiola Osorio, Simon J Tavernier, Yvan Saeys, Liesbet Martens, Jessica Vetters, Iris Delrue, Philippe Pouliot, Sophie Janssens & Bart N Lambrecht

  2. GROUP-ID Consortium, Ghent University and University Hospital, Ghent, Belgium

    Fabiola Osorio, Simon J Tavernier, Eik Hoffmann, Yvan Saeys, Liesbet Martens, Jessica Vetters, Iris Delrue, Philippe Pouliot, Sophie Janssens & Bart N Lambrecht

  3. Department of Respiratory Medicine, Ghent University, Ghent, Belgium

    Fabiola Osorio, Simon J Tavernier, Yvan Saeys, Liesbet Martens, Jessica Vetters, Iris Delrue, Philippe Pouliot, Sophie Janssens & Bart N Lambrecht

  4. Unit of Molecular Signal Transduction in Inflammation, VIB Inflammation Research Center, Ghent, Belgium

    Eik Hoffmann

  5. Microscopy Core Facility, VIB Inflammation Research Center, Ghent, Belgium

    Riet De Rycke & Eef Parthoens

  6. Advanced Scientific Research Leaders Development Unit, Gunma University, Gunma, Japan

    Takao Iwawaki

  7. Department of Pulmonary Medicine, Erasmus MC, Rotterdam, The Netherlands

    Bart N Lambrecht

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Contributions

F.O., S.J. and B.N.L. designed the research; F.O., S.J.T., E.H. and P.P. did the experiments; F.O. analyzed the results; Y.S. and L.M. helped with microarray analysis; R.D.R. did transmission electron microscopy, E.P. helped with microscopy analysis; J.V. and I.D. provided technical assistance; T.I. provided reagents; and F.O., S.J. and B.N.L. wrote the manuscript.

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Correspondence to Bart N Lambrecht.

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Integrated supplementary information

Supplementary Figure 1 Activation of the IRE-1α pathway in peripheral DCs.

Expression of VenusFP in (a) lung tissue or (b) lung draining lymph node (Lung dr. LN) from WT and ERAI reporter mice. (c and d) Histograms depict the VenusFP expression in B cells (CD19+, MHCII+) T cells (CD3e+) and DC (Lin-, MHCII+, CD11c+) in control animals (grey) and ERAI mice (blue) in lung (n=3) or lung dr. LN (n=2) and is representative of three independent experiments. (e) Schematic representation of the generation of the mixed BM chimeras.

Supplementary Figure 2 Detail of aberrant ER morphology in XBP-1-deficient CD8α+ cDCs.

Electron micrographs of FACS-sorted CD8α+ cDCs derived from DC-XBP-1Δ or control mice. Scale bars, 250nm. Data is representative of one experiment.

Supplementary Figure 3 CD8α+ cDCs from DC–XBP-1Δ mice are as efficient as CD8α+ cDCs from their control littermates in secreting IL-12.

IL-12-p70 concentration in serum (left panel) or splenic cell supernatant (right panel) from DC-XBP-1Δ mice or control littermates after injection with STAg. IL-12 concentration was quantified by ELISA. Bars represent mean ± SEM of two independent experiments. p=0,095 (Mann Whitney U test, two-sided).

Supplementary Figure 4 DCs from DC–XBP-1Δ mice sustain CD4+ T cell proliferation, and early modules of cross-presentation are normal in XBP-1-deficient CD8α+ cDCs.

FACS-sorted CD11b+ cDCs (a) or purified CD8α+ cDCs (b) from DC-XBP-1Δ mice or control littermates were cultured with CFSE-labeled OVA specific TCR Tg CD4+ OT-II cells in presence of OVA peptide and soluble OVA. Cell counts were measured on day three. Plots depict mean ± SEM of two independent experiments. (c) Phagocytosis of 1μm polystyrene microspheres conjugated to OVA after labeling of remaining microspheres with an Alexa 488-conjugated antibody. Right gate in dot plots depicts frequency of cells with surface-bound beads and left gate depicts frequency of cells with internalized particles. Data are representative of 2 independent experiments. (d) Cells were loaded with a cytosolic FRET-sensitive substrate of β-lactamase (βlac) and incubated in absence of presence of βlac. After 3 h, change in fluorescence in gated CD8α+ cDCs was measured as readout of βlac export from the endocytic pathway into the cytosol. Data are representative of 2 independent experiments.

Supplementary Figure 5 Loss of CD18 in XBP-1-deficient CD8α+ cDCs results in decreased expression of CD11c and CD11a.

(a) CD11a (LFA-1) expression in splenic CD11b+ and CD8α+ cDCs in DC-XBP-1Δ mice and control littermates. (b) Table of reported UPR target genes with fold induction (FC) below twofold in XBP-1 deficient versus sufficient CD8α+ cDCs 4,24,25,44.

Supplementary Figure 6 Schematic representation of the IRE-1α cleavage sites with mRNA secondary structure prediction.

Potential IRE-1α splicing motifs were extracted for Itgb2 (CD18), Ergic3 and Tapbp. Secondary structure prediction was performed using three different programs: Mfold (depicted), CentroidFold and RNAFold. Sequence motifs where at least two of the three programs agreed on the predicted secondary structure were retained, and further filtering was performed by retaining those motifs where the cleavage site was situated in the loop structure. Arrows indicate the splicing site.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Table 1 (PDF 4404 kb)

Aberrant ER morphology in XBP-1-deficient CD8α+ cDCs.

3D-imaging of a XBP deficient CD8α+ cDC using FIB-SEM, first showing the raw data and aberrant ER, which is partially reconstructed in green to show the increased complexity of the ER in these cells. (AVI 31681 kb)

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Osorio, F., Tavernier, S., Hoffmann, E. et al. The unfolded-protein-response sensor IRE-1α regulates the function of CD8α+ dendritic cells. Nat Immunol 15, 248–257 (2014). https://doi.org/10.1038/ni.2808

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