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. 2016 Nov;68(11):2686-2696.
doi: 10.1002/art.39795.

Expression of Long Interspersed Nuclear Element 1 Retroelements and Induction of Type I Interferon in Patients With Systemic Autoimmune Disease

Clio P Mavragani  1 , Irina Sagalovskiy  2 , Qiu Guo  2 , Adrianos Nezos  3 , Efstathia K Kapsogeorgou  3 , Pin Lu  2 , Jun Liang Zhou  2 , Kyriakos A Kirou  2 , Surya V Seshan  4 , Haralampos M Moutsopoulos  3 , Mary K Crow  5
Affiliations

Expression of Long Interspersed Nuclear Element 1 Retroelements and Induction of Type I Interferon in Patients With Systemic Autoimmune Disease

Clio P Mavragani et al. Arthritis Rheumatol. 2016 Nov.

Abstract

Objective: Increased expression of type I interferon (IFN) and a broad signature of type I IFN-induced gene transcripts are observed in patients with systemic lupus erythematosus (SLE) and other systemic autoimmune diseases. To identify disease-relevant triggers of the type I IFN pathway, this study sought to investigate whether endogenous virus-like genomic repeat elements, normally silent, are expressed in patients with systemic autoimmune disease, and whether these retroelements could activate an innate immune response and induce type I IFN.

Methods: Expression of type I IFN and long interspersed nuclear element 1 (LINE-1; L1) was studied by polymerase chain reaction, Western blotting, and immunohistochemistry in samples of kidney tissue from patients with lupus nephritis and minor salivary gland (MSG) tissue from patients with primary Sjögren's syndrome (SS). Induction of type I IFN by L1 was investigated by transfection of plasmacytoid dendritic cells (PDCs) or monocytes with an L1-encoding plasmid or L1 RNA. Involvement of innate immune pathways and altered L1 methylation were assessed.

Results: Levels of L1 messenger RNA transcripts were increased in lupus nephritis kidneys and in MSG tissue from patients with SS. Transcript expression correlated with the expression of type I IFN and L1 DNA demethylation. L1 open-reading frame 1/p40 protein and IFNβ were expressed in MSG ductal epithelial cells and in lupus nephritis kidneys, and IFNα was detected in infiltrating PDCs. Transfection of PDCs or monocytes with L1-encoding DNA or RNA induced type I IFN. Inhibition of Toll-like receptor 7 (TLR-7)/TLR-8 reduced the induction of IFNα by L1 in PDCs, and an inhibitor of IKKε/TANK-binding kinase 1 abrogated the induction of type I IFN by L1 RNA in monocytes.

Conclusion: L1 genomic repeat elements represent endogenous nucleic acid triggers of the type I IFN pathway in SLE and SS and may contribute to initiation or amplification of autoimmune disease.

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Figures

Figure 1
Figure 1. Expression of L1 mRNA in involved tissues from SLE and SS patients
(A) Total mRNA from kidney tissue was extracted and relative expression of L1 mRNA quantified by RT-PCR, using a primer set that amplifies the 5’UTR of human LRE2. Kidney biopsy tissue was obtained from either healthy individuals (HD, healthy donors) or lupus nephritis patients with different classes (IV, III, V) of renal pathology. (B) Relative expression of L1 mRNA was quantified in human MSG from 31 patients with primary SS; 12 patients with IFN-I-associated autoimmune disease (AD) and sicca symptoms; 5 non-autoimmune sicca controls (SC); and pooled mRNA from MSG of 24 healthy individuals (Clontech laboratories, Inc.) (HD).
Figure 2
Figure 2. Immunohistochemical and western blot detection of L1 in salivary gland tissue and kidney tissue
(A–O) Representative examples of immunohistochemical data are demonstrated. MSG tissue from a SS patient and a SC patient and renal tissue from a patient with class IV lupus nephritis were stained with antibodies specific for ORF1/p40 (L1), BDCA-2 (a specific marker for pDCs), IFNα, IFNβ or an IgG control. L1 and IFNβ staining were particularly strong in SS salivary ductal epithelial cells and lupus renal tubular epithelial cells (A, K, D, N), while positive IFNα stained cells (representative cells indicated by arrow) coincided with anti-BDCA-2-stained cells (pDCs) (B, C, L, M). (P, Q) Western blot analysis of protein extracts from 5 SS and 3SC MSG samples was performed with rabbit anti-ORF1/p40 and mouse anti-GAPDH antibodies. ORF-1/p40 levels were significantly higher in the SS than the SC samples. Representative gels from 2 SS patients and 1 SC are shown.
Figure 3
Figure 3. Coordinate expression of L1 and type I IFN mRNA in affected tissues from patients with systemic autoimmune disease
IFNα2 (A, C, E) and IFNβ (B, D) mRNA expression were measured by RT-PCR in MSG tissue from patients with SS (A,B), in lupus nephritis renal tissues (C,D), and in MSG tissue from patients with IFN-I-associated AD and sicca symptoms (E) and related to expression of L1 mRNA. Spearman correlation is shown for L1 with IFNα2 or IFNβ.
Figure 4
Figure 4. L1 induces IFNα in human plasmacytoid dendritic cells and monocytes
(A) Expression of L1 mRNA in pDC 6 hours post transfection with an L1-containing plasmid (pDONR221-L1) or empty vector (pDONR221) as measured by RT-PCR. (B) Expression of IFNα2 by pDC 6 hours post-transfection with an L1-containing or control plasmid. (C) Human pDC were transfected directly with a purified L1 RNA fragment, with U1 or hY3 RNA, or with unrelated control RNA using DOTAP transfection reagent. Relative expression of IFNα2 was determined after 6 hours. (D) IFN-I protein activity was assessed in supernatants from the experiments shown in (C) using a reporter cell (WISH) assay. Relative expression of interferon inducible gene MX1 in WISH cells was measured by RT-PCR after 4 hours of incubation with 50% supernatants. (E) Expression of IFNα2 mRNA in human CD14+ monocytes 6 hours after electroporation of an L1-containing or control plasmid or purified L1 RNA.
Figure 5
Figure 5. Inhibition of TLR7/8 and TBK1/IKKε pathways
(A) Human pDC were pre-treated for 1 hour with either TLR9 inhibitor IRS 894 or TLR7/8 inhibitor IRS 661 followed by stimulation for 4 hours with either TLR9 ligand CpG or with L1 RNA. Relative expression of IFNα2 was measured by RT-PCR, and the percent of the level induced by either CpG or L1 RNA is shown. (B) Human CD14+ monocytes were pre-treated for 30 min with TBK1/IKKε inhibitor Bx795 that blocks signaling downstream of the RIG-I-like receptor (RLR) pathway followed by transfection with either positive or negative RNA controls (+RNA/−RNA) or L1 RNA. After 4 hours of incubation relative expression of interferon inducible gene IFIT3 was measured by RT-PCR. (C) Supernatants from the CD14+ cells cultured as shown in (B) were incubated with reporter (WISH) cells to assess IFN-I protein activity. The level of interferon inducible gene MX1 in WISH cells after 4 hours of stimulation with 50% supernatants is shown.
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References

    1. Crow MK. Type I interferon in the pathogenesis of lupus. J Immunol. 2014;192:5459–5468. - PMC - PubMed
    1. Hua J, Kirou K, Lee C, Crow MK. Functional assay of type I interferon in systemic lupus erythematosus plasma and association with anti-RNA binding protein autoantibodies. Arthritis Rheum. 2006;54:1906–1916. - PubMed
    1. Bennett L, Palucka AK, Arce E, Cantrell V, Borvak J, Banchereau J, et al. Interferon and granulopoiesis signatures in systemic lupus erythematosus blood. J Exp Med. 2003;197:711–723. - PMC - PubMed
    1. Crow MK, Wohlgemuth J. Microarray analysis of gene expression in lupus. Arthritis Res Ther. 2003;5:279–287. - PMC - PubMed
    1. Baechler EC, Batliwalla FM, Karypis G, Gaffney PM, Ortmann WA, Espe KJ, et al. Interferon-inducible gene expression signature in peripheral blood cells of patients with severe lupus. Proc Natl Acad Sci U S A. 2003;100:2610–2615. - PMC - PubMed

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