IFN-gamma AU-rich element removal promotes chronic IFN-gamma expression and autoimmunity in mice☆
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)
- et al.
Interferon-gamma excess leads to pathogenic accumulation of follicular helper T cells and germinal centers
Immunity
(2012) - et al.
Oxidative stress in systemic lupus erythematosus: relationship to Th1 cytokine and disease activity
Immunol Lett
(2010) - et al.
Regulation of interferon-gamma during innate and adaptive immune responses
Adv Immunol
(2007) - et al.
MicroRNA-29 regulates T-box transcription factors and interferon-gamma production in helper T cells
Immunity
(2011) - et al.
Tristetraprolin mediates interferon-gamma mRNA decay
J Biol Chem
(2009) - et al.
Impaired on/off regulation of TNF biosynthesis in mice lacking TNF AU-rich elements: implications for joint and gut-associated immunopathologies
Immunity
(1999) - et al.
A spatially-organized multicellular innate immune response in lymph nodes limits systemic pathogen spread
Cell
(2012) - et al.
Interferon-gamma is required for lupus nephritis in mice treated with the hydrocarbon oil pristane
Kidney Int
(2001) - et al.
IFN-gamma enhances secretion of IgG2a from IgG2a-committed LPS-stimulated murine B cells: implications for the role of IFN-gamma in class switching
Cell Immunol
(1991) - et al.
Transcription factor E2-2 is an essential and specific regulator of plasmacytoid dendritic cell development
Cell
(2008)
Marginal zone macrophages suppress innate and adaptive immunity to apoptotic cells in the spleen
Blood
Antinuclear autoantibodies and lupus nephritis in transgenic mice expressing interferon gamma in the epidermis
J Exp Med
Roles of interferon-gamma and interleukin-4 in murine lupus
J Clin Invest
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
Interferon-gamma gene polymorphisms associated with susceptibility to systemic lupus erythematosus
Ann Rheum Dis
NK cells dysfunction in systemic lupus erythematosus: relation to disease activity
Clin Rheumatol
Predominance of Th1 immune response in diffuse proliferative lupus nephritis
Arthritis Rheum
The rationale for BAFF inhibition in systemic lupus erythematosus
Curr Rheumatol Rep
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
The microRNA miR-29 controls innate and adaptive immune responses to intracellular bacterial infection by targeting interferon-gamma
Nat Immunol
LFA-1-dependent HuR nuclear export and cytokine mRNA stabilization in T cell activation
J Immunol
Recombineering: a powerful new tool for mouse functional genomics
Nat Rev Genet
Suppressive oligodeoxynucleotides delay the onset of glomerulonephritis and prolong survival in lupus-prone NZB x NZW mice
Arthritis Rheum
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