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The microRNA miR-148a functions as a critical regulator of B cell tolerance and autoimmunity

Nature Immunology volume 17, pages 433–440 (2016)Cite this article

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Abstract

Autoreactive B cells have critical roles in a large diversity of autoimmune diseases, but the molecular pathways that control these cells remain poorly understood. We performed an in vivo functional screen of a lymphocyte-expressed microRNA library and identified miR-148a as a potent regulator of B cell tolerance. Elevated miR-148a expression impaired B cell tolerance by promoting the survival of immature B cells after engagement of the B cell antigen receptor by suppressing the expression of the autoimmune suppressor Gadd45α, the tumor suppressor PTEN and the pro-apoptotic protein Bim. Furthermore, increased expression of miR-148a, which occurs frequently in patients with lupus and lupus-prone mice, facilitated the development of lethal autoimmune disease in a mouse model of lupus. Our studies demonstrate a function for miR-148a as a regulator of B cell tolerance and autoimmunity.

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Figure 1: In vivo functional screen of miRNAs regulating B cell tolerance.
Figure 2: miR-148a promotes the escape of B cells from the central tolerance checkpoint.
Figure 3: miR-148a protects immature B cells from apoptosis induced by engagement of the BCR.
Figure 4: miR-148a suppresses the expression of Bim, PTEN and Gadd45α.
Figure 5: Deletion of Gadd45a, Bcl2l11 or Pten impairs B cell tolerance.
Figure 6: Increased miR-148a expression facilitates the development of lethal autoimmunity.

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Acknowledgements

We thank D. Kono for advice on the analysis of MRL-lpr mice; L. Sherman (The Scripps Research Institute, La Jolla) for Bcl2l11−/− mice; members of the Xiao and Nemazee laboratories for advice and technical assistance; and the TSRI Flow Cytometry and Genomics Core Facilities for support. Supported by the Pew Charitable Trusts (C.X.), the Cancer Research Institute, the Lupus Research Institute and the US National Institutes of Health (R01 AI089854 to C.X.; R01 AI59714 to D.N.; and RC4 AI092763 to C.X. and D.N.).

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Authors and Affiliations

  1. Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, USA

    Alicia Gonzalez-Martin, Maoyi Lai, Jovan Shepherd, David Nemazee & Changchun Xiao

  2. Department of Genetics, Yale Stem Cell Center, Yale Cancer Center, and Yale Center for RNA Science and Medicine, Yale University, New Haven, Connecticut, USA

    Brian D Adams & Jun Lu

  3. Department of Immunology and Oncology, National Biotechnology Center, Madrid, Spain

    Maria Salvador-Bernaldez & Jesus M Salvador

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  1. Alicia Gonzalez-Martin
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Contributions

A.G.-M., D.N. and C.X. conceptualized and designed the project, and wrote the manuscript with contributions from all authors; A.G.-M. performed most of the experiments; B.D.A. and J.L. provided the retroviral miRNA-expression library for pool screen and retroviral vectors encoding individual miRNAs for validation of positive 'hits', and performed miRNA barcode analysis to identify miRNAs that broke B cell tolerance; M.L. performed bone marrow–reconstitution experiments with Bim- or PTEN-deficient mice; J.S. managed the mouse colony and provided technical support; M.S.-B. and J.M.S. bred Gadd45α-deficient mice, harvested bones and shipped these for bone marrow–reconstitution experiments; and D.N. and C.X. supervised the project.

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Correspondence to David Nemazee or Changchun Xiao.

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Integrated supplementary information

Supplementary Figure 1 Barcode analysis for the identification of miRNAs that break B cell tolerance.

(a) Barcode identification method. Genomic DNA samples from purified splenic B cells that escaped tolerance in the IgMb-macroself recipient mice and bone marrow B cell precursors from the same mice were subjected to a universal PCR that amplified the miRNA cassette of this retroviral library. PCR products were hybridized with barcode probes coupled to specific beadsets and subsequently incubated with SA-PE. Samples were analyzed by flow cytometry for barcode identification. Adapted from reference 29. (b,c) Log2 signal distribution of a water control (b) and experimental samples (c). The background noise is represented by values lower than 7 in the x-axis. Values greater than 7 represent miRNAs that were encoded by retroviruses integrated into the genomes of B cells and drove their escape from the central tolerance checkpoint imposed by the IgMb-macroself superantigen.

Supplementary Figure 2 No synergistic effect between miRNAs that break B cell tolerance in IgMb-macroself mice.

(a) Representative FACS plots showing splenic B cells (CD19+IgM+) in IgMb-macroself mice reconstituted with HSPCs infected with control, miR-148a or a mixture of 6 different retroviruses encoding miRNAs identified in the screen (miR-148a, -26a, -26b, -342, -423, -511) at terminal analysis. (b,c) Percentages (b) and numbers (c) of splenic B cells in mice analyzed in a. Data are pooled from four individual experiments (a-c; mean ± s.e.m. in b-c): n=3 mice in control and retroviral miR-148a, -26a, -26b, -342, -423, -511 mixture and n=6 mice in miR-148a group.

Source data

Supplementary Figure 3 miR-148a expression during the development and activation of B cells.

(a) Small RNA deep sequencing analysis showing miR-148a expression in murine B lineage cells at various development and activation stages. The graph was generated based on previously published data10. (b) Taqman microRNA assay showing miR-148a expression in purified B cells in the absence of stimulation or stimulated with 10μg/ml anti-IgM, 10μg/ml LPS or 10μg/ml anti-IgM + 10μg/ml LPS as indicated. Data are representative of two independent experiments (b; mean ± s.d. of technical triplicates).

Source data

Supplementary Figure 4 TNFR2 does not have a major role in controlling B cell central tolerance.

(a) FACS analysis showing TNFR2 surface expression of non-stimulated and stimulated (2μg/ml anti-IgM for 14h) WEHI-control, WEHI-miR-148a, WEHI-miR-26a and WEHI-miR-182 cells. (b) Representative FACS plots showing splenic B cells (CD19+IgM+) at terminal analysis of IgMb-macroself recipient mice reconstituted with bone marrow cells from mice of the indicated genotypes. (c,d) Percentages (c) and numbers (d) of splenic B cells in mice analyzed in b. Data are representative of two independent experiments (a) or pooled from two individual experiments (b-d; mean ± s.e.m. in c,d): n=5 mice in control and n=6 mice in Tnfrsf1b-/- group. Statistical analysis was performed with a two-tailed Student’s T test. ** P<0.01.

Source data

Supplementary Figure 5 miR-148a, but not miR-26a or miR-182, regulates the expression of Bim, PTEN and Gadd45 simultaneously.

(a) qRT-PCR analysis showing target gene mRNA expression of WEHI-control, WEHI-miR-148a, WEHI-miR-26a and WEHI-miR-182 stimulated with anti-IgM (2μg/ml) for 14 h. (b) Western blot analysis showing miR-148a target gene expression in stimulated (2μg/ml anti-IgM for 14 h) WEHI-control, WEHI-miR-148a, WEHI-miR-26a and WEHI-miR-182. The target gene protein/β-actin ratio in the control samples was arbitrarily set as 1. Data are pooled from two individual experiments (a; mean ± s.d.) or pooled from two or three individual experiments for Bim and PTEN respectively (b; mean ± s.d.). Statistical analysis was performed with a two-tailed Student’s T test. *P<0.05, ** P<0.01.

Source data

Supplementary Figure 6 B cell–activation status and miR-148a expression in young MRL-lpr mice, and overexpression of miR-148a achieved by a retroviral vector.

(a) FACS analysis showing B cell activation marker CD69 staining of splenic B cells of 6 week-old control and MRL-lpr mice. LPS-activated B cells (10μg/ml for 20h) were included as a positive control. (b) Taqman microRNA assay showing miR-148a expression in splenic B cells from the control and MRL-lpr mice. Data are representative of two independent experiments (a) or pooled from two independent experiments (b; mean ± s.e.m.): n=3 mice/group. (c) Taqman microRNA assay showing miR-148a expression in splenic B cells purified from MRL-lpr recipient mice in experiments described in Figure 6. The average miR-148a expression level of the control group was arbitrarily set as 1. Data are pooled from two independent experiments: n=6 mice in control and n=7 mice in miR-148a group. Statistical analysis was performed with a two-tailed Student’s T test. ** P<0.01.

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Gonzalez-Martin, A., Adams, B., Lai, M. et al. The microRNA miR-148a functions as a critical regulator of B cell tolerance and autoimmunity. Nat Immunol 17, 433–440 (2016). https://doi.org/10.1038/ni.3385

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