ARIH2 is essential for embryogenesis, and its hematopoietic deficiency causes lethal activation of the immune system

Journal name:
Nature Immunology
Volume:
14,
Pages:
27–33
Year published:
DOI:
doi:10.1038/ni.2478
Received
Accepted
Published online

Abstract

The E3 ligase ARIH2 has an unusual structure and mechanism of elongating ubiquitin chains. To understand its physiological role, we generated gene-targeted mice deficient in ARIH2. ARIH2 deficiency resulted in the embryonic death of C57BL/6 mice. On a mixed genetic background, the lethality was attenuated, with some mice surviving beyond weaning and then succumbing to an aggressive multiorgan inflammatory response. We found that in dendritic cells (DCs), ARIH2 caused degradation of the inhibitor IκBβ in the nucleus, which abrogated its ability to sequester, protect and transcriptionally coactivate the transcription factor subunit p65 in the nucleus. Loss of ARIH2 caused dysregulated activation of the transcription factor NF-κB in DCs, which led to lethal activation of the immune system in ARIH2-sufficent mice reconstituted with ARIH2-deficient hematopoietic stem cells. Our data have therapeutic implications for targeting ARIH2 function.

At a glance

Figures

  1. ARIH2 deficiency causes an overwhelming inflammatory response.
    Figure 1: ARIH2 deficiency causes an overwhelming inflammatory response.

    (a) Survival of Arih2+/− and Arih2−/− mice (n = 15 per genotype) after weaning. P < 0.0001 (log-rank (Mantel-Cox) test). (b) Hematoxylin-and-eosin staining of Arih2+/− and Arih2−/− liver, heart and lung, showing cellular infiltrates. Scale bar, 100 μm. (c) Weight of Arih2+/− and Arih2−/− mouse pups at postnatal day 1 (PND1) and adult mice >5 weeks of age (n = 8–13 per genotype). *P < 0.001 and **P = 0.002 (Student's t-test). (d) Total viable cells and frequency of apoptotic cells (positive for annexin V and the DNA-intercalating dye 7-AAD) in Arih2+/− and Arih2−/− fetal livers at E14.5 and E16.5 (n = 3–20 per genotype). *P < 0.0001 and **P = 0.031 (Student's t-test). (e) Hematoxylin-and-eosin staining (H+E) and in situ end labeling of fragment DNA (ISEL) of sections of Arih2+/− and Arih2−/− fetal liver at E16.5 (insets, enlargement of main images). Scale bars, 100 μm. Data represent at least three independent experiments (mean ± s.d. in c).

  2. Chimeras generated with Arih2-/- cells develop lethal systemic inflammatory responses.
    Figure 2: Chimeras generated with Arih2−/− cells develop lethal systemic inflammatory responses.

    (a) Weight change over time in chimeras generated with Arih2+/− or Arih2−/− cells (n = 7 per genotype). *P = 0.003 and **P = 0.005 (Student's t-test). (b) Disease onset in chimeras generated with Arih2+/+, Arih2+/− or Arih2−/− cells (n = 6–30 per genotype). P < 0.0001 (Log-rank (Mantel-Cox) test). (c) Serum immunoglobulin concentrations in chimeras generated with Arih2+/− or Arih2−/− cells (n = 6–18 per genotype). P < 0.05 (Student's t-test). (d) Serum cytokine concentrations in chimeras as in c (n = 7–20 mice per genotype). P < 0.05 (Student's t-test). (e) Hematoxylin-and-eosin staining of heart, liver, lung, small intestine (GI) and colon from chimeras as in c. Sale bar, 100 μm. Each symbol (c,d) represents an individual mouse; small horizontal lines indicate the mean. Data represent two to three independent experiments (mean ± s.d. in a).

  3. Arih2-/- T and B cells do not have intrinsic signaling defects.
    Figure 3: Arih2−/− T and B cells do not have intrinsic signaling defects.

    (a) Proliferation of Arih2+/− and Arih2−/− B cells among cells collected 6 weeks after reconstitution of chimeras generated with Arih2+/− or Arih2−/− cells (n = 3–8 recipient mice per genotype) and then stimulated for 48 h with medium alone (Med), anti-IgM alone, anti-IgM plus antibody to the costimulatory molecule CD40 (anti-IgM + anti-CD40), LPS, CpG or poly(I:C). (b) Immunoblot analysis of phosphorylated (p-) and total downstream signaling molecules in T cells among cells collected as in a and then stimulated for 0–60 min (above lanes) with anti-CD3 plus anti-CD28 or with PMA plus ionomycin (P+I); β-actin serves as a loading control. (c) Proliferation of Arih2+/− and Arih2−/− T cells among cells collected as in a and then stimulated for 24 or 48 h with plate-bound anti-CD3 plus anti-CD28 or with PMA plus ionomycin, assessed as dilution of the cytosolic dye CFSE. *P = 0.001 and **P = 0.0005 (Student's t-test). Data represent two to three independent experiments (mean and s.d. in a,c).

  4. ARIH2 does not affect the function of Treg or Teff cells.
    Figure 4: ARIH2 does not affect the function of Treg or Teff cells.

    (a) Quantification of Foxp3+ Treg cells isolated from chimeras generated with Arih2+/− or Arih2−/− cells (n = 6 recipient mice per genotype). *P = 0.01 (Student's t-test). (b) Weight change of Rag1−/− recipient mice (n = 3–4 per genotype) over 14 weeks after injection of various combinations of Arih2+/− or Arih2−/− CD4+CD25+ Treg cells or CD4+CD25 Teff cells (key). P ≤ 0.0001 (Student's t-test). (c) Transverse colon sections in mice as in b at 14 weeks after cell injection, assessed by staining with hematoxylin and eosin (top row) and immunohistology of CD3 (bottom row). Scale bar, 100 μm. (d) Histological grading of colitis in mice as in b, corresponding to extent of disease23. Each symbol represents an individual mouse; small horizontal lines indicate the mean. Data represent two to three independent experiments (mean and s.d. in a,b).

  5. Loss of ARIH2 in DCs leads to spurious activation, proinflammatory responses and tissue destruction.
    Figure 5: Loss of ARIH2 in DCs leads to spurious activation, proinflammatory responses and tissue destruction.

    (a) Quantification of cells with expression of DC activation and maturation markers, plus staining with isotype-matched control antibody, among Arih2+/− and Arih2−/− fetal liver–derived DCs incubated with medium alone, LPS or CpG, assessed by flow cytometry. MHCII, major histocompatibility complex class II. (b) Cytokine production by Arih2+/− and Arih2−/− DCs incubated with medium alone (n = 3–5 cells per genotype). P = 0.034 (Student's t-test). (c) Blood glucose (Glu) of RIP-GP mice given an injection of Arih2+/− or Arih2−/− DCs (n = 5–10 recipient mice per genotype and treatment condition) incubated with medium alone or CpG and pulsed with LCMV gp33, gp61 and gp276 (left; each line represents a mouse), and frequency of disease-free mice (vertical axes, right) and number (above lines, right) of those mice with a blood glucose concentration of ≥15 mmol/l (measured at random times). P < 0.04 (Student's t-test). Data represent three independent experiments (mean and s.d. in b).

  6. Loss of ARIH2 leads to hyperactive and sustained NF-[kappa]B signaling.
    Figure 6: Loss of ARIH2 leads to hyperactive and sustained NF-κB signaling.

    (a) Immunoblot analysis of total MyD88 and total and phosphorylated Akt and IκBα in the cytosol (top) and total and phosphorylated p65 and total IκBα in the nuclear fraction (bottom) of Arih2+/− and Arih2−/− fetal liver–derived DCs stimulated with CpG. H2AX and β-actin serve as loading controls throughout. (b) Gel mobility-shift assay of the nuclear fraction of Arih2+/− and Arih2−/− fetal liver–derived DCs stimulated with CpG, assessed with NF-κB probes. (c) Immunoassay of nuclear extracts of fetal liver–derived DCs left untreated or incubated for 30 min with LPS and then collected or treated for a further 2–4 h with 25 μM MG132 (above lanes), assessed by immunoprecipitation with anti-IκBβ (IP IκBβ) or without immunoprecipitation (IP input), followed by immunoblot analysis (Nuclear; top group), and immunoblot analysis of cytosolic extracts without immunoprecipitation (Cytosol; below). (d) Immunoblot analysis of lysates of MG132-treated 3T3 cells expressing Flag-tagged full-length IκBβ (IκBβ-WT) or IκBβ-ΔPEST together with histidine-tagged ARIH2, left untreated (−) or immunoprecipitated with anti-ARIH2 (+) and probed for tagged IκBβ and ARIH2. (e) Immunoblot analysis of total lysates of 3T3 cells expressing Flag-tagged full-length IκBβ or IκBβ-ΔPEST and treated with scrambled short hairpin RNA (shRNA Ctrl) or specific shRNA that silences Arih2 expression. Data represent three independent experiments.

References

  1. Marín, I., Lucas, J.I., Gradilla, A.C. & Ferrus, A. Parkin and relatives: the RBR family of ubiquitin ligases. Physiol. Genomics 17, 253263 (2004).
  2. van der Reijden, B.A., Erpelinck-Verschueren, C.A., Lowenberg, B. & Jansen, J.H. TRIADs: a new class of proteins with a novel cysteine-rich signature. Protein Sci. 8, 15571561 (1999).
  3. Tan, N.G. et al. Characterisation of the human and mouse orthologues of the Drosophila ariadne gene. Cytogenet. Cell Genet. 90, 242245 (2000).
  4. Aguilera, M., Oliveros, M., Martinez-Padron, M., Barbas, J.A. & Ferrus, A. Ariadne-1: a vital Drosophila gene is required in development and defines a new conserved family of ring-finger proteins. Genetics 155, 12311244 (2000).
  5. Wenzel, D.M., Lissounov, A., Brzovic, P.S. & Klevit, R.E. UBCH7 reactivity profile reveals parkin and HHARI to be RING/HECT hybrids. Nature 474, 105108 (2011).
  6. Chuang, T.H. & Ulevitch, R.J. Triad3A, an E3 ubiquitin-protein ligase regulating Toll-like receptors. Nat. Immunol. 5, 495502 (2004).
  7. Fearns, C., Pan, Q., Mathison, J.C. & Chuang, T.H. Triad3A regulates ubiquitination and proteasomal degradation of RIP1 following disruption of Hsp90 binding. J. Biol. Chem. 281, 3459234600 (2006).
  8. Nakhaei, P. et al. The E3 ubiquitin ligase Triad3A negatively regulates the RIG-I/MAVS signaling pathway by targeting TRAF3 for degradation. PLoS Pathog. 5, e1000650 (2009).
  9. Ghosh, S. & Hayden, M.S. New regulators of NF-κB in inflammation. Nat. Rev. Immunol. 8, 837848 (2008).
  10. Vallabhapurapu, S. & Karin, M. Regulation and function of NF-κB transcription factors in the immune system. Annu. Rev. Immunol. 27, 693733 (2009).
  11. Mladek, C., Guger, K. & Hauser, M.T. Identification and characterization of the ARIADNE gene family in Arabidopsis. A group of putative E3 ligases. Plant Physiol. 131, 2740 (2003).
  12. Marteijn, J.A. et al. The ubiquitin ligase Triad1 inhibits myelopoiesis through UbcH7 and Ubc13 interacting domains. Leukemia 23, 14801489 (2009).
  13. Wang, H., Bei, L., Shah, C.A., Horvath, E. & Eklund, E.A. HoxA10 influences protein ubiquitination by activating transcription of ARIH2, the gene encoding Triad1. J. Biol. Chem. 286, 1683216845 (2011).
  14. Pietschmann, K. et al. Differential regulation of PML-RAR α stability by the ubiquitin ligases SIAH1/SIAH2 and TRIAD1. Int. J. Biochem. Cell Biol. 44, 132138 (2012).
  15. Lee, E.G. et al. Failure to regulate TNF-induced NF-κB and cell death responses in A20-deficient mice. Science 289, 23502354 (2000).
  16. Starr, R. et al. Liver degeneration and lymphoid deficiencies in mice lacking suppressor of cytokine signaling-1. Proc. Natl. Acad. Sci. USA 95, 1439514399 (1998).
  17. Yeh, W.C. et al. Early lethality, functional NF-κB activation, and increased sensitivity to TNF-induced cell death in TRAF2-deficient mice. Immunity 7, 715725 (1997).
  18. Chiffoleau, E. et al. TNF receptor-associated factor 6 deficiency during hemopoiesis induces Th2-polarized inflammatory disease. J. Immunol. 171, 57515759 (2003).
  19. Turer, E.E. et al. Homeostatic MyD88-dependent signals cause lethal inflamMation in the absence of A20. J. Exp. Med. 205, 451464 (2008).
  20. Nguyen, L.T. et al. TRAF2 deficiency results in hyperactivity of certain TNFR1 signals and impairment of CD40-mediated responses. Immunity 11, 379389 (1999).
  21. Metcalf, D., Di Rago, L., Mifsud, S., Hartley, L. & Alexander, W.S. The development of fatal myocarditis and polymyositis in mice heterozygous for IFN-γ and lacking the SOCS-1 gene. Proc. Natl. Acad. Sci. USA 97, 91749179 (2000).
  22. Alexander, W.S. et al. SOCS1 is a critical inhibitor of interferon γ signaling and prevents the potentially fatal neonatal actions of this cytokine. Cell 98, 597608 (1999).
  23. Asseman, C., Mauze, S., Leach, M.W., Coffman, R.L. & Powrie, F. An essential role for interleukin 10 in the function of regulatory T cells that inhibit intestinal inflammation. J. Exp. Med. 190, 9951004 (1999).
  24. Lutz, M.B. et al. An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J. Immunol. Methods 223, 7792 (1999).
  25. Naik, S.H. et al. Development of plasmacytoid and conventional dendritic cell subtypes from single precursor cells derived in vitro and in vivo. Nat. Immunol. 8, 12171226 (2007).
  26. Ohashi, P.S. et al. Ablation of “tolerance” and induction of diabetes by virus infection in viral antigen transgenic mice. Cell 65, 305317 (1991).
  27. Oldstone, M.B., Nerenberg, M., Southern, P., Price, J. & Lewicki, H. Virus infection triggers insulin-dependent diabetes mellitus in a transgenic model: role of anti-self (virus) immune response. Cell 65, 319331 (1991).
  28. Dissanayake, D. et al. Nuclear factor-κB1 controls the functional maturation of dendritic cells and prevents the activation of autoreactive T cells. Nat. Med. 17, 16631667 (2011).
  29. Lin, A.C., Dissanayake, D., Dhanji, S., Elford, A.R. & Ohashi, P.S. Different toll-like receptor stimuli have a profound impact on cytokines required to break tolerance and induce autoimmunity. PLoS ONE 6, e23940 (2011).
  30. Garza, K.M. et al. Role of antigen-presenting cells in mediating tolerance and autoimmunity. J. Exp. Med. 191, 20212027 (2000).
  31. Suyang, H., Phillips, R., Douglas, I. & Ghosh, S. Role of unphosphorylated, newly synthesized IκBβ in persistent activation of NF-κB. Mol. Cell. Biol. 16, 54445449 (1996).
  32. Rao, P. et al. IκBβ acts to inhibit and activate gene expression during the inflammatory response. Nature 466, 11151119 (2010).
  33. Scheibel, M. et al. IκBβ is an essential co-activator for LPS-induced IL-1β transcription in vivo. J. Exp. Med. 207, 26212630 (2010).
  34. McKinsey, T.A., Chu, Z.L. & Ballard, D.W. Phosphorylation of the PEST domain of IκBβ regulates the function of NF-κB/IκBβ complexes. J. Biol. Chem. 272, 2237722380 (1997).
  35. Herold, M.J., van den Brandt, J., Seibler, J. & Reichardt, H.M. Inducible and reversible gene silencing by stable integration of an shRNA-encoding lentivirus in transgenic rats. Proc. Natl. Acad. Sci. USA 105, 1850718512 (2008).
  36. Geng, H., Wittwer, T., Dittrich-Breiholz, O., Kracht, M. & Schmitz, M.L. Phosphorylation of NF-κB p65 at Ser468 controls its COMMD1-dependent ubiquitination and target gene-specific proteasomal elimination. EMBO Rep. 10, 381386 (2009).
  37. Schwabe, R.F. & Sakurai, H. IKKβ phosphorylates p65 at S468 in transactivaton domain 2. FASEB J. 19, 17581760 (2005).
  38. Tanaka, T., Grusby, M.J. & Kaisho, T. PDLIM2-mediated termination of transcription factor NF-κB activation by intranuclear sequestration and degradation of the p65 subunit. Nat. Immunol. 8, 584591 (2007).
  39. Saccani, S., Marazzi, I., Beg, A.A. & Natoli, G. Degradation of promoter-bound p65/RelA is essential for the prompt termination of the nuclear factor κB response. J. Exp. Med. 200, 107113 (2004).
  40. Ryo, A. et al. Regulation of NF-κB signaling by Pin1-dependent prolyl isomerization and ubiquitin-mediated proteolysis of p65/RelA. Mol. Cell 12, 14131426 (2003).
  41. Maine, G.N., Mao, X., Komarck, C.M. & Burstein, E. COMMD1 promotes the ubiquitination of NF-κB subunits through a cullin-containing ubiquitin ligase. EMBO J. 26, 436447 (2007).
  42. Ruland, J. et al. Bcl10 is a positive regulator of antigen receptor-induced activation of NF-κB and neural tube closure. Cell 104, 3342 (2001).
  43. Pasparakis, M., Alexopoulou, L., Episkopou, V. & Kollias, G. Immune and inflammatory responses in TNFα-deficient mice: a critical requirement for TNFα in the formation of primary B cell follicles, follicular dendritic cell networks and germinal centers, and in the maturation of the humoral immune response. J. Exp. Med. 184, 13971411 (1996).
  44. Dalton, D.K. et al. Multiple defects of immune cell function in mice with disrupted interferon-γ genes. Science 259, 17391742 (1993).
  45. Hou, B., Reizis, B. & DeFranco, A.L. Toll-like receptors activate innate and adaptive immunity by using dendritic cell-intrinsic and -extrinsic mechanisms. Immunity 29, 272282 (2008).
  46. Wijsman, J.H. et al. A new method to detect apoptosis in paraffin sections: in situ end-labeling of fragmented DNA. J. Histochem. Cytochem. 41, 712 (1993).
  47. Bonnard, M. et al. Deficiency of T2K leads to apoptotic liver degeneration and impaired NF-κB-dependent gene transcription. EMBO J. 19, 49764985 (2000).
  48. Calzascia, T. et al. TNF-α is critical for antitumor but not antiviral T cell immunity in mice. J. Clin. Invest. 117, 38333845 (2007).
  49. Esashi, E. et al. The signal transducer STAT5 inhibits plasmacytoid dendritic cell development by suppressing transcription factor IRF8. Immunity 28, 509520 (2008).
  50. Pellegrini, M. et al. Adjuvant IL-7 antagonizes multiple cellular and molecular inhibitory networks to enhance immunotherapies. Nat Med 15, 528536 (2009).

Download references

Author information

  1. These authors contributed equally to this work.

    • Tak W Mak &
    • Marc Pellegrini

Affiliations

  1. Department of Medical Biophysics, University of Toronto, Toronto, Canada.

    • Amy E Lin,
    • Raymond H Kim,
    • Pamela S Ohashi &
    • Tak W Mak
  2. Campbell Family Institute for Breast Cancer Research, Ontario Cancer Institute, University Health Network, Toronto, Canada.

    • Amy E Lin,
    • Yongkai Ow,
    • Masato Sasaki,
    • Samuel D Saibil,
    • Dilan Dissanayake,
    • Raymond H Kim,
    • Andrew Wakeham,
    • Annick You-Ten,
    • Arda Shahinian,
    • Gordon Duncan,
    • Jennifer Silvester,
    • Pamela S Ohashi &
    • Tak W Mak
  3. Department of Medicine, University of Toronto, Toronto, Canada.

    • Amy E Lin,
    • Samuel D Saibil &
    • Raymond H Kim
  4. Walter and Eliza Hall Institute of Medical Research, Parkville, Australia.

    • Gregor Ebert,
    • Simon P Preston,
    • Jesse G Toe,
    • James P Cooney,
    • Hamish W Scott &
    • Marc Pellegrini
  5. Department of Medical Biology, University of Melbourne, Melbourne, Australia.

    • Simon P Preston,
    • Jesse G Toe,
    • Samuel D Saibil,
    • Dilan Dissanayake &
    • Marc Pellegrini
  6. Department of Immunology, University of Toronto, Toronto, Canada.

    • Pamela S Ohashi &
    • Tak W Mak

Contributions

A.E.L., M.P., G.E., Y.O., M.S., P.S.O. and T.W.M. designed and did all of the research with assistance from S.P.P., J.G.T., J.P.C., H.W.S., S.D.S., D.D. and R.H.K. and technical assistance from A.W., A.S., G.D., A.Y.-T., and J.S.; A.E.L. wrote the manuscript; and T.W.M. and M.P. directed the project and contributed to the preparation of the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary Text and Figures (500K)

    Supplementary Figures 1–4 and Tables 1–5

Additional data