Dynamic regulation of hypoxia-inducible factor-1α activity is essential for normal B cell development

Abstract

B lymphocyte development and selection are central to adaptive immunity and self-tolerance. These processes require B cell receptor (BCR) signaling and occur in bone marrow, an environment with variable hypoxia, but whether hypoxia-inducible factor (HIF) is involved is unknown. We show that HIF activity is high in human and murine bone marrow pro-B and pre-B cells and decreases at the immature B cell stage. This stage-specific HIF suppression is required for normal B cell development because genetic activation of HIF-1α in murine B cells led to reduced repertoire diversity, decreased BCR editing and developmental arrest of immature B cells, resulting in reduced peripheral B cell numbers. HIF-1α activation lowered surface BCR, CD19 and B cell–activating factor receptor and increased expression of proapoptotic BIM. BIM deletion rescued the developmental block. Administration of a HIF activator in clinical use markedly reduced bone marrow and transitional B cells, which has therapeutic implications. Together, our work demonstrates that dynamic regulation of HIF-1α is essential for normal B cell development.

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Fig. 1: Disruption of dynamic HIF regulation leads to severe peripheral B cell lymphopenia.
Fig. 2: Hif1a deletion in Vhl−/−Mb1-cre mice rescues peripheral B cell loss, indicating a HIF-1α-dependent effect.
Fig. 3: Regulated HIF-1α activity is required for the generation and diversification of the pre-immune B cell repertoire.
Fig. 4: Defects in development linked to BCR signaling.
Fig. 5: Developmental block linked to BIM-dependent survival.
Fig. 6: Regulated HIF-1α activity is required for BCR editing.
Fig. 7: Developing B cells are reduced in daprodustat-treated mice.

Data availability

Data are available from the corresponding author upon request. Uncropped immunoblots for Supplementary Figs. 3a–c and 8h are provided as Supplementary Data. The BCR-seq data generated in the present study are available at the SRA database (https://www.ncbi.nlm.nih.gov/sra), BioProject accession codes PRJNA574931, PRJNA574906 and PRJNA574628. The RNA-seq and microarray datasets generated in this study are available at the GEO database (https://www.ncbi.nlm.nih.gov/geo/), GEO accession codes GSE129513 and GSE152960. Data in the present study that were obtained from publically available sources can be accessed at: (1) Tabula Muris scRNA-seq data (https://tabula-muris.ds.czbiohub.org); data names: 10X_P7_2 and 10X_P7_3; and (2) Human Cell Atlas scRNA-seq data (https://data.humancellatlas.org/explore/projects/cc95ff89-2e68-4a08-a234-480eca21ce79); data name: MantonBM1.

Code availability

Customized scripts for the analysis of BCR repertoire differences are available at: Immune_receptor_NETWORK-GENERATION (https://github.com/rbr1/Immune_receptor_NETWORK-GENERATION).

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Acknowledgements

This work was funded by the Wellcome Trust (no. 19710), a Cambridge National Institute for Health Research (NIHR) senior investigator award (no. NF-SI-0514-10122) and the Rosetrees Trust (no. G102721) to P.H.M., supporting N.B., V.J.B., A.P. and J.E.G.S. R.J.M.B.-R. was supported by the Wellcome Trust (no. WT106068AIA). B.J.S. was supported by a Wellcome Clinical PhD fellowship (216366/Z/19/Z). T.M.C. was supported by a MRC Clinic Research Training Fellowship (G0802266, ID 89800). S.S. was supported by a summer studentship award from the Lister Institute of Preventive Medicine. M.G.B.T. was supported by a NIHR grant (no. CL-2006-14-006). M.R.C. was supported by a Chan-Zuckerberg Initiative Human Cell Atlas technology development grant, a Versus Arthritis Cure Challenge Research Grant (no. 21777) and an NIHR Research Professorship (no. RP-2017-08-ST2-002). The CIMR core facilities were supported by a Wellcome Trust/MRC grant (no. 097922/Z/11/Z) and a Wellcome Trust strategic award (no. 100140). We thank R. Schulte, C. Cossetti, M. Maj and G. Grondys-Kotarba (CIMR Flow Cytometry Core Facility), and M. Gratian and M. Bowen (CIMR microscopy core facility) for their help and support; Y. Umrania for bioinformatics support; M. Reth (University of Freiburg, Germany) and M. Turner (Babraham Institute, Cambridge) for providing the Mb1-cre mice; and R. Johnson (Karolinska Institutet, Sweden) for providing Hif1a−/− mice. We thank J. Sale and M. Ashcroft for useful discussions and T. Wilson, K. Scott and D. Gale for experimental and technical support.

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Contributions

N.B. led the research design, performed experiments, analyzed data, maintained the mouse lines and wrote the paper. R.J.M.B.-R. performed BCR amplification, sequencing and analysis, and wrote the paper. V.J.B. performed experiments and bioinformatics analyses. A.P., J.E.G.S. and T.M.C. performed experiments and maintained the mouse lines. J.R.F., B.J.S., L.B., G.G. and E.P. performed bioinformatics analyses. M.D.−L. helped with experimental and technical design. A.I., S.S. and M.G.B.T. performed experiments. P.A.L. helped with the design of RNA-seq experiments. M.E., K.G.C.S. and B.J.P.H. contributed to research design. R.J.C. contributed to research and experimental design and provided guidance on manuscript preparation. M.R.C. contributed to research and experimental design and wrote the paper. P.H.M. supervised the project and wrote the paper.

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Correspondence to Natalie Burrows.

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Competing interests

P.H.M. is a scientific founder and equity holder of ReOx and has received speaker honoraria from Fibrogen, which both aim to develop PHD inhibitors as therapies. R.J.M.B-R. is a cofounder and consultant for Alchemab Therapeutics Ltd, which maps antibody repertoires to develop therapeutics for patients with hard-to-treat diseases, and is a consultant for Imperial College London and VHSquared, which develops Vorabodies (oral domain antibodies). R.J.C. is a scientific founder and equity holder of MIROBIO, which aims to develop new therapies to treat a range of autoimmune diseases. All other authors declare no competing interests.

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Supplementary Figs. 1–9 and Supplementary Tables 1–3.

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Supplementary Data 1

Uncropped immunoblots.

Supplementary Data 2

Uncropped immunoblots.

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Burrows, N., Bashford-Rogers, R.J.M., Bhute, V.J. et al. Dynamic regulation of hypoxia-inducible factor-1α activity is essential for normal B cell development. Nat Immunol 21, 1408–1420 (2020). https://doi.org/10.1038/s41590-020-0772-8

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