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Non-catalytic ubiquitin binding by A20 prevents psoriatic arthritis–like disease and inflammation

Nature Immunology volume 21, pages 422–433 (2020)Cite this article

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Abstract

A20 is an anti-inflammatory protein that is strongly linked to human disease. Here, we find that mice expressing three distinct targeted mutations of A20’s zinc finger 7 (ZF7) ubiquitin-binding motif uniformly developed digit arthritis with features common to psoriatic arthritis, while mice expressing point mutations in A20’s OTU or ZF4 motifs did not exhibit this phenotype. Arthritis in A20ZF7 mice required T cells and MyD88, was exquisitely sensitive to tumor necrosis factor and interleukin-17A, and persisted in germ-free conditions. A20ZF7 cells exhibited prolonged IκB kinase activity that drove exaggerated transcription of late-phase nuclear factor-κB response genes in vitro and in prediseased mouse paws in vivo. In addition, mice expressing double-mutant A20 proteins in A20’s ZF4 and ZF7 motifs died perinatally with multi-organ inflammation. Therefore, A20’s ZF4 and ZF7 motifs synergistically prevent inflammatory disease in a non-catalytic manner.

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Fig. 1: A20’s ZF7 motif prevents arthritis of DIP joints.
Fig. 2: Spontaneous immune activation in ZF7 knock-in mice.
Fig. 3: Arthritis in A20ZF7/ZF7 mice requires T cells but not B cells, and requires MyD88- but not TRIF-dependent signals.
Fig. 4: A20’s ZF7 motif prevents IL-17- and TNF-dependent arthritis.
Fig. 5: A20’s ZF7 motif (versus C103 or ZF4 motifs) selectively restricts IKK activity.
Fig. 6: A20’s ZF7 motif synergizes with A20’s ZF4 motif to restrict inflammation in vitro and in vivo.

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Data availability

The data that support the findings of this study are available from the corresponding authors upon reasonable request. Source data for Figs. 5 and 6 and Extended Data Fig. 3 are provided with this paper.

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Acknowledgements

We thank J. Ashouri Sinha, J. Zikherman, H.-E. Liang and J. Zhang for helpful discussions and advice and N. Szeto for the micro-CT reconstructions. This work was supported by NIH R01 AI135198 and R01 AI117908 (to A.M.), AR070155 (to M.C.N.) and a Dermatology Foundation Fellowship (to B.R.). M.C.N. is also supported by the Russell/Engleman Arthritis Center at UCSF and the Department of Veterans Affairs Health Care System. B.R. is also supported by the Department of Veterans Affairs Health Care System. Micro-CT analyses were performed by the UCSF Core Center for Musculoskeletal Biology and Medicine, supported by P30AR066262 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases. The FACS analyses were supported by UCSF Liver Center grant P30DK026743 from the National Institute of Diabetes and Digestive and Kidney Diseases.

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Author notes

  1. These authors contributed equally: Bahram Razani, Michael I. Whang.

Authors and Affiliations

  1. Department of Medicine, University of California, San Francisco, San Francisco, CA, USA

    Bahram Razani, Michael I. Whang, Francis S. Kim, Mary C. Nakamura, Xiaofei Sun, Rommel Advincula, Mihir Pendse, Priscilia Tanbun, Philip Achacoso, Barbara A. Malynn & Averil Ma

  2. Department of Dermatology, University of California, San Francisco, San Francisco, CA, USA

    Bahram Razani

  3. Department of Pediatrics, University of California, San Francisco, San Francisco, CA, USA

    Francis S. Kim

  4. Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA

    Jessie A. Turnbaugh & Peter J. Turnbaugh

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Contributions

B.R. and M.I.W. helped to conceptualize the project, conducted experiments, analyzed data and helped to write the manuscript. F.S.K. and X.S. performed experiments and analyzed data. M.C.N. analyzed and interpreted gross and histological data. R.A., P.T., P.A. and M.P. helped to conduct experiments. J.A.T. conducted experiments with germ-free mice under the supervision of P.J.T. B.A.M. and A.M. conceptualized the overall project, acquired funding for the project, generated the gene-targeted mice, supervised experiments and wrote the manuscript.

Corresponding authors

Correspondence to Barbara A. Malynn or Averil Ma.

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The authors declare no competing interests.

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Peer review information L. A. Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Arthritis in A20ZF7/ZF7 mice.

a, Quantitation of live born pups at 12 days of age. b, Longitudinal quantitation of digital disease of A20ZF7/ZF7 (ZF7) mice by numbers of digits with dactylitis (distal digit swelling) and nail loss in male and female mice at the indicated ages (from 4-12 weeks of age, Mean ± SD for independent mice, n = 10 for each genotype). c, Gross photos (left) and H&E histology (right) of forepaws of A20ZF7-ΔFGN/ZF7-ΔFGN and A20ZF7-FG/ZF7-FG mice at 3 months of age. Scale bars = 1 mm. Representative of three independent mice.

Extended Data Fig. 2 Mild systemic inflammation in A20ZF7/ZF7 mice.

Representative histology (H&E) from Kidney (a), Liver (b), Colon (c), and Lung (d) of wild-type and ZF7 mice at 3months of age. Scale bars are 100 μm for Kidney and Liver, 200 μm for Colon, and 1 mm for Lung. Representative of three independent mice.

Extended Data Fig. 3 WT and ZF7 T cell signaling and T cell profiling.

a, Flow cytometry analysis of TCRβ+ and TCRδ+ T cells in lymph nodes (axillary, brachial and inguinal combined, left) and total quantitation of TCRδ+ cells in lymph nodes (right). b, (left) Representative flow cytometry analysis of naive CD4+ T cells after separation from spleen and lymph nodes (EasySep Mouse Naive CD4+ T cell Isolation Kit). (right) Immunoblot analysis of TCR signaling from purified naive CD4+ T cells shown at left. Purified naive CD4+ T cells were stimulated with anti-CD3 mAb (145-2C11; Tonbo) followed by crosslinking with goat anti-hamster IgG (Jackson Immuno) for the indicated times at 37 °C with gentle shaking. Data are representative of three independent experiments.

Source data

Extended Data Fig. 4 Myeloid activation status in wild-type and A20ZF7/ZF7 mice.

Representative flow cytometry analysis of I-A/E cell surface expression on CD11b+ Gr1+ a, and CD11chi b, cells in the spleen of 3-month old WT and ZF7 mice. Cells were gated on lineage negative population (TCRβ, CD19). Data are representative of 5 biological replicates per genotype.

Extended Data Fig. 5 Quantitation of T cell subsets in 3-month old wild-type and A20ZF7/ZF7 mice.

a, Flow cytometric analysis of CD25 vs FoxP3 expression on CD4+ T cells. b, Flow cytometric analysis of intracellular IFNγ on ex vivo CD4+ T cells stimulated with PMA and ionomycin. Data are representative of two independent experiments.

Extended Data Fig. 6 Expression of early NF-κB -induced genes.

(a) Quantitative qPCR analyses of indicated early NF-κB-induced genes in mouse embryonic fibroblasts (MEFs) transiently stimulated with TNF for 15 minutes. Mean ± SD for technical replicates. Results are representative from three independent experiments. (b) Quantitative PCR analyses of indicated genes in paws from pre-diseased 3-week-old mice of the indicated genotypes. Data are representative of paws from multiple independent mice (WT n = 5, ZF7, ZF4, OTU n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. One-way ANOVA with Tukey’s multiple comparison test.

Extended Data Fig. 7 Liver inflammation in A20ZF4ZF7/ZF4ZF7 mice.

Gross (a) and hepatic H&E histology (b) of A20ZF4ZF7/ZF4ZF7 and WT littermate mice. Scale bar = 100 μM. Representative of three independent mice.

Extended Data Fig. 8 Gating strategy for flow cytometric analysis.

Representative gating strategy for flow cytometric analysis of intracellular IL-17A staining is shown.

Supplementary information

Source data

Source Data Fig. 5

Unprocessed western blots.

Source Data Fig. 6

Unprocessed western blots.

Source Data Extended Data Fig. 3

Unprocessed western blots.

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Razani, B., Whang, M.I., Kim, F.S. et al. Non-catalytic ubiquitin binding by A20 prevents psoriatic arthritis–like disease and inflammation. Nat Immunol 21, 422–433 (2020). https://doi.org/10.1038/s41590-020-0634-4

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