Elsevier

Journal of Autoimmunity

Volume 53, September 2014, Pages 33-45
Journal of Autoimmunity

IFN-gamma AU-rich element removal promotes chronic IFN-gamma expression and autoimmunity in mice

https://doi.org/10.1016/j.jaut.2014.02.003Get rights and content

Highlights

  • IFN-γ ARE removal stabilizes IFN-γ mRNA and results in chronic IFN-γ expression.

  • IFN-γ enhances pDC development.

  • Elevated IFN-γ coincides with MZB cell and MZM loss and lupus-like disease onset.

Abstract

We generated a mouse model with a 162 nt AU-rich element (ARE) region deletion in the 3′ untranslated region (3′UTR) of the interferon-gamma (IFN-γ) gene that results in chronic circulating serum IFN-γ levels. Mice homozygous for the ARE deletion (ARE-Del) −/− present both serologic and cellular abnormalities typical of patients with systemic lupus erythematosus (SLE). ARE-Del−/− mice display increased numbers of pDCs in bone marrow and spleen. Addition of IFN-γ to Flt3-ligand (Flt3L) treated in vitro bone marrow cultures results in a 2-fold increase in pDCs with concurrent increases in IRF8 expression. Marginal zone B (MZB) cells and marginal zone macrophages (MZMs) are absent in ARE-Del−/− mice. ARE-Del+/− mice retain both MZB cells and MZMs and develop no or mild autoimmunity. However, low dose clodronate treatment in ARE-Del+/− mice specifically eliminates MZMs and promotes anti-DNA antibody development and glomerulonephritis. Our findings demonstrate the consequences of a chronic IFN-γ milieu on B220+ cell types and in particular the impact of MZB cell loss on MZM function in autoimmunity. Furthermore, similarities between disease states in ARE-Del−/− mice and SLE patients suggest that IFN-γ may not only be a product of SLE but may be critical for disease onset and progression.

Introduction

The type II interferon, IFN-γ, is well-recognized in murine lupus models. IFN-γ transgenic mice under the control of the involucrin promoter develop a lupus-like syndrome defined by the autoantibody production, glomerular IgG deposition and glomerulonephritis [1]. Roquinsan/san mice display decreased IFN-γ mRNA decay leading to chronic circulating levels of IFN-γ and a lupus-like phenotype [2] and offspring from lupus-prone MRL/lpr mice crossed with IFN-γ−/− mice have reduced autoantibody titers and less severe end-organ disease [3]. In humans, IFNG gene polymorphisms have been identified in SLE cohorts [4], [5] and dysregulated NK cells, T cells, and PBMCs from SLE patients produce elevated amounts of IFN-γ that correlate with SLE Disease Activity Index Scores [6], [7], [8]. Mechanistically, IFN-γ induces B cell activating factor (BAFF), a protein critical for B cell differentiation, proliferation, and survival that has been identified as a therapeutic target in SLE [4], [9].

Aberrant IFN-γ expression is associated with inflammatory diseases; thus, its expression is tightly controlled. Regulation is complex and involves epigenetic modifications, IFNG promoter interactions with positive and negative regulatory factors (reviewed in [10]) and post-transcriptional control involving nuclear and cytoplasmic mRNA compartmentalization, mRNA stabilization, and miRNAs [11], [12], [13] that are partially mediated by IFN-γ 3′UTR interactions with regulatory proteins human antigen R (HuR) and tristetraprolin (TTP) [14], [15]. Approximately 50% of the mature IFN-γ mRNA is 3′UTR sequence. Little similarity between mouse and human exist with the exception of a 162 nt AU-rich fragment located in the 5′ end of the 3′UTR. AREs are best recognized as mRNA destabilizing elements; thus, we predicted that removal of the IFN-γ ARE region would eliminate inhibitory functions resulting in chronic IFN-γ mRNA and protein expression. Targeted deletion of an ARE sequence was previously achieved for the tumor necrosis factor-alpha (TNF-α) gene with subsequent elevated, stable TNF-α expression that coincided with inflammatory arthritis and Crohn's-like inflammatory bowel disease [16].

To examine the consequences of IFN-γ ARE deletion on immune function and potential disease development, we generated a mouse with a targeted substitution of the conserved 162 nt AU-rich sequence with random nucleotides. The mice, henceforth called ARE-Del mice, have low, chronic circulating IFN-γ levels, a complex phenotype consisting of neutrophilia, monocytosis, anti-nuclear antibodies (ANAs), serum hypocomplementemia, glomerular immunoglobulin and complement deposition, and mesangioproliferative glomerulonephritis; findings consistent with disease similar to human SLE. Importantly, heterozygote mice have normal blood counts, lack elevated autoreactivity, and minimal renal lesions, indicating that a threshold level of IFN-γ is critical for development and exacerbation of disease.

Section snippets

Generation of IFN-γ ARE-Del mice

The IFN-γ ARE targeting vector was obtained by recombineering technology [17]. Briefly, we generated two 200 nt PCR fragments from sequences flanking the 5′ and 3′ regions of the IFN-γ 3′UTR ARE site. PCR products were cloned into PKB644:pLTM260 vector at sites adjacent to a neomycin resistance gene cassette and flanked by loxP and Frt sites. Replacement of the 162 nt ARE-rich sequence was achieved by electroporation of the IFN-γ/neomycin cassette into DY380 bacterial strain housing a BAC

Generation of IFN-γ ARE-Del mice

The 162 nt ARE sequence containing five AUUUA elements and located in the 5′ portion of the IFN-γ 3′UTR is highly conserved with 83% identity between human and mouse (Supplemental Fig. 1A). Sequence Replacement was accomplished using recombineering technology (Supplemental Fig. 1B) and confirmed by Southern and PCR analyses (Supplemental Fig. 1C and D). Two lines of ARE-Del mice were generated from different C57BL/6-129 hybrid ES cell clones and were backcrossed more than 10 times on to a

Discussion

Our current work has focused on consequences of disrupted IFN-γ posttranscriptional control through deletion of a multi-ARE RNA regulatory region in the IFN-γ 3′UTR. AREs are associated with RNA destabilization and we predicted that removal of five AREs in the IFN-γ 3′UTR would stabilize the IFN-γ mRNA. Indeed we observed a dramatic increase in IFN-γ mRNA half-life that translated into chronic low levels of circulating IFN-γ in mice.

The syndrome in ARE-Del−/− mice develops by 16 weeks of age

Conclusions

Our findings demonstrate that small alterations in the IFN-γ signaling pathway lead to detrimental systemic outcomes in host immune physiology and response that is dependent upon crossing a “threshold” level of circulating IFN-γ. Thus, the need for multifaceted control of IFN-γ expression is imperative for proper immune function and well-being. The ARE-Del−/− mouse highlights this point demonstrating a connection between uncontrolled low IFN-γ levels and autoimmunity and suggest that a

Authorship contributions

D.L. H. and H.A.Y. designed and performed experiments and wrote manuscript.

C. B., V.C., and W. K. designed and performed experiments.

M.D. B., P. M. S., H. S., A J. S., J.J. S., M. R. A., J. R. O., F. L., D. A. R., M.E. S, performed experiments.

P. K., L. T, and D. M. K., provided intellectual input and manuscript review.

Funding

This project has been funded in part with funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E.

Disclosure

The authors declare no competing financial interests.

Acknowledgments

We thank Grace Williams, John Wine, Megan Karwan, Bill Bere and Catherine Razzook for technical assistance, animal breeding, and experiments. Dr. Daniel McVicar and Dr. Giorgio Trinchieri for helpful comments and manuscript review.

References (47)

  • T.L. McGaha et al.

    Marginal zone macrophages suppress innate and adaptive immunity to apoptotic cells in the spleen

    Blood

    (2011)
  • J.P. Seery et al.

    Antinuclear autoantibodies and lupus nephritis in transgenic mice expressing interferon gamma in the epidermis

    J Exp Med

    (1997)
  • S.L. Peng et al.

    Roles of interferon-gamma and interleukin-4 in murine lupus

    J Clin Invest

    (1997)
  • M. Harigai et al.

    Excessive production of IFN-gamma in patients with systemic lupus erythematosus and its contribution to induction of B lymphocyte stimulator/B cell-activating factor/TNF ligand superfamily-13B

    J Immunol

    (2008)
  • K. Kim et al.

    Interferon-gamma gene polymorphisms associated with susceptibility to systemic lupus erythematosus

    Ann Rheum Dis

    (2010)
  • A. Henriques et al.

    NK cells dysfunction in systemic lupus erythematosus: relation to disease activity

    Clin Rheumatol

    (2013)
  • K. Masutani et al.

    Predominance of Th1 immune response in diffuse proliferative lupus nephritis

    Arthritis Rheum

    (2001)
  • A. Davidson

    The rationale for BAFF inhibition in systemic lupus erythematosus

    Curr Rheumatol Rep

    (2012)
  • D.L. Hodge et al.

    Regulation of nuclear gamma interferon gene expression by interleukin 12 (IL-12) and IL-2 represents a novel form of posttranscriptional control

    Mol Cell Biol

    (2002)
  • F. Ma et al.

    The microRNA miR-29 controls innate and adaptive immune responses to intracellular bacterial infection by targeting interferon-gamma

    Nat Immunol

    (2011)
  • J.G. Wang et al.

    LFA-1-dependent HuR nuclear export and cytokine mRNA stabilization in T cell activation

    J Immunol

    (2006)
  • N.G. Copeland et al.

    Recombineering: a powerful new tool for mouse functional genomics

    Nat Rev Genet

    (2001)
  • L. Dong et al.

    Suppressive oligodeoxynucleotides delay the onset of glomerulonephritis and prolong survival in lupus-prone NZB x NZW mice

    Arthritis Rheum

    (2005)
  • Cited by (0)

    The publisher or recipient acknowledges right of the U.S. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article.

    View full text