Edison Biotechnology Institute (N.H., C.J.L., K.M., U.B.-P., X.S., L.D.K.), the Department of Biomedical Sciences (L.D.K.), College of Osteopathic Medicine, and the Department of Chemical Engineering (D.J.G.), Ohio University, Athens, Ohio 45701; Medstar Research Institute (V.V., M.D.R., M.S.), Washington Hospital Center, Washington, D.C. 20010; The Ohio State University (V.V., M.D.R., M.S.), Arthur G. James Cancer Center and Richard J. Solove Research Institute, Columbus, Ohio 43210; and Section of Endocrinology (C.G., G.N.), Department of Science of Aging, Università degli Studi "G.D. Annunzio," Faculty of Medicine and Surgery, 66100 Chieti, Italy
Address all correspondence and requests for reprints to: L. D. Kohn, Edison Biotechnology Institute, Ohio University, Athens, Ohio 45701.
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ABSTRACT |
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INTRODUCTION |
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Infectious agents have been implicated in the induction of autoimmune disease (8, 9, 10, 11, 12). One example is the association of foamy virus infections and DeQuervains thyroiditis (13). In the 1990s, it was suggested that viral triggering of autoimmunity might result from local infection of tissues, induction of abnormal or increased expression of MHC genes, presentation of self-antigens to immune cells, and bystander activation of T cells (10, 11, 12). This concept is now updated by the description of Toll-like receptors (TLRs), their signaling, and their link to autoimmune/inflammatory disorders (14, 15).
TLRs are a family of 10 known cell surface receptors related to IL-1 receptors (14, 15, 16). They protect mammals from pathogenic organisms, such as viruses, by generating an innate immune response to products of the pathogenic organism (14, 15, 16). The innate immune response increases genes for several inflammatory cytokines, and costimulatory molecules; it is critical for the development of antigen-specific adaptive immunity, both humoral and cell-mediated (14, 16). TLRs are present in most monocytes, macrophages, or immune cells; TLR3, which mediates a potent antiviral response (17), is present primarily on dendritic cells in humans, i.e. antigen-presenting cells that process, then present antigenic peptides to lymphoid cells in lymphoid organs (18). Recently, TLR3 and TLR4, which have common features in their signal pathways, were noted on nonimmune cells in association with autoimmune/inflammatory diseases. TLR3 on pancreatic ß-islet cells was implicated in the pathogenesis of insulinitis and type 1 diabetes (19, 20). TLR4 on intestinal and arterial endothelial cells was implicated in inflammatory bowel disease (21) and atherosclerosis, respectively (14, 22, 23). TLR3/4 are not described on thyrocytes nor implicated in thyroid autoimmune/inflammatory disease.
TLR3 recognizes double-stranded (ds) RNA, assumed to be released by viral killing of cells (16, 24). The dsRNA binding to TLR3, mimicked by incubation with polyinosine-polycytidylic acid [Poly (I:C)] in vitro, activates two distinct pathways (16, 24, 25, 26, 27). One signals through nuclear factor B (NF-
B), involves MAPK, and produces various cytokines, e.g. TNF-
(14, 16, 24, 28). The second activates IFN regulatory factor (IRF)-3 and causes the synthesis and release of type I IFNs (
or ß). Coupling to each path involves different sites on an adapter molecule Toll-like IL-1 receptor (TIR) domain-containing molecule adapter-inducing IFN-ß (TRIF) (14, 16, 24, 25, 26, 27) The type I IFN further up-regulates TLR3 in an autocrine/paracrine manner, a phenomenon linked to its antiviral gene defense action (17).
Using Fisher rat thyroid cell line-5 (FRTL-5) thyrocytes (29), we had noted that dsRNA transfection into the cytoplasm increased type I (IFN-ß) gene expression, whereas dsDNA transfection did not. Both dsRNA and dsDNA transfection were associated with increased or abnormal MHC expression, increased expression of genes necessary for antigen presentation/processing, including dsRNA-dependent kinase (PKR), as well as increases in the activation of signal transducers and activation of transcription (STAT), MAPK, and NF-B. The observations 1) that dsRNA increased IFN-ß (29), and 2) that TLR3 recognized dsRNA (14, 15, 18, 19, 20, 21, 22) led us to the following questions concerning mechanism. Was dsRNA-increased type I (IFN-ß) gene expression in FRTL-5 cells related to the presence of functional TLR3, could TLR3 activation induce an innate immune response in thyrocytes, and, if so, were the same signal mechanisms involved as in immune cells? Could TLR3/TLR3 signaling be overexpressed in thyrocytes in the absence of immune cells, how, and could overexpression be associated with an autoimmune/inflammatory disease, e.g. Hashimotos autoimmune thyroiditis? Might TLR3 overexpression/signaling be sensitive to the immunomodulatory actions of methimazole (MMI) or a more potent derivative, phenylmethimazole (C10) (30, 31, 32, 33, 34) and how?
We answer these questions in this report and conclude that Hashimotos thyroiditis may be grouped with insulinitis and type 1 diabetes, colitis, and atherosclerosis as an autoimmune/inflammatory disease associated with TLR3/4 overexpression and signaling in nonimmune cells by an induction process involving molecular signatures of environmental pathogens (14, 15, 17, 19, 20, 22, 23, 35, 36). We suggest that C10 inhibits TLR3 overexpression and signaling because it acts on the IRF-3/ of IFN-stimulated response element (ISRE)/IFN-ß/STAT arm of the signal path, not the NF-B arm and discuss its potential relationship to our recent report that C10 reduces proinflammatory cytokine (TNF-
)-induced vascular cell adhesion molecule (VCAM)-1 and leukocyte adhesion in human arterial endothelial cells by suppressing IRF-1 gene overexpression, not by altering NF-
B gene expression or activity (37).
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RESULTS |
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TLR3 Is Functional in FRTL-5 Cells
Poly (I:C), a chemically synthesized dsRNA that is a specific ligand for TLR3 (16, 24, 28), was added to the culture medium to stimulate TLR3 signaling. Extracellular dsRNA is known to be specifically recognized by TLR3 as evidenced by the lack of response to extracellular dsRNA in TLR3/ mouse-derived fibroblasts (28). TLR3 activation of the NF-B/p38MAPK and IRF-3/IFN-ß signals bifurcate at TRIF (14, 16, 24, 25, 26, 27, 38). We asked whether both of these pathways were activated in FRTL-5 thyroid cells.
The presence of the NF-B pathway was evaluated by incubating extracellular dsRNA, [Poly (I:C)] with pNF-
B-luc-transfected FRTL-5 cells and measuring reporter gene activity (Fig. 2A
) and by EMSA in nontransfected cells, using an NF-
B consensus oligo probe (Fig. 2B
). Poly (I:C) increased NF-
B Luc activity by comparison to nontreated cells or cells incubated with Escherichia coli dsDNA (Fig. 2A
). Poly (I:C) incubation also increased formation of a p65/p50 NF-
B complex as evidenced by the appearance of a major complex whose formation was inhibited by incubations containing the nuclear extracts from treated cells with anti-p50 or anti-p65 (Fig. 2B
), but not by incubations with anti c-rel, anti-p52, or anti-relB, which served as negative antibody controls.
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Poly (I:C) incubation was also able to activate ERK1/2 MAPK within 15 min as detected by measuring phosphorylated ERK1/2 in immunoblots (Fig. 2C, third lane). Lysates from insulin-treated FRTL-5 cells were used as a positive control (Fig. 2C
). Transcription factor Elk1 was also transactivated by Poly (I:C) treatment of reporter gene-coupled ELK-1-FRTL-5 cell transfectants, as measured by luciferase activity (Fig. 2D
). IL-1ß treatment, which can activate ELK-1 (16), served as a positive control (Fig. 2D
).
We next determined whether there was functional expression of the TLR3-IRF-3/IFN-ß-coupled signal system in FRTL-5 cells. We first measured the ability of Poly (I:C) incubations to increase IFN-ß promoter activity (Fig. 3A) and IFN-ß mRNA levels (Fig. 3B
). IFN-ß promoter activity was measured by incubating Poly (I:C) with FRTL-5 cells transfected with pIFN-ß-luc constructs. Poly (I:C) incubation strongly increased IFN-ß promoter activity, whereas E. coli dsDNA had no effect (Fig. 3A
, left panel). As a control, we show that pIFN-ß-luc-transfected HEK293 cells, which do not have endogenous TLR3 (see Fig. 1A
, right panel), failed to respond to Poly (I:C) incubation unless they were first transfected with a human TLR3 expression plasmid (Fig. 3A
, right panel). Although Northern analysis did not detect significant levels of IFN-ß mRNA (data not shown), RT-PCR analysis using gene-specific primers (41) demonstrated that IFN-ß mRNA was increased in FRTL-5 cells by the addition of Poly (I:C) (Fig. 3B
).
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To see whether the TRIF adapter protein could couple TLR3 and signal generation in FRTL-5 cells, we asked whether cotransfection of wild-type TIR domain-containing molecule adapter inducing IFN-ß/TIR-containing adapter molecule (TRIF/TICAM)-1 would enhance Poly (I:C)-induced IFN-ß gene activation. Exogenous expression of TRIF/TICAM-1 in FRTL-5 cells enhanced the Poly (I:C)-induced IFN-ß promoter activity in a dose-dependent manner but did not enhance IL-1ß-increased IFN-ß promoter activity (Fig. 2D). IL-1ß (negative control) does not activate IRF-3 and IFN-ß by a TRIF coupling mechanism (14, 18, 19, 20, 21, 38). Overexpression of wild-type or dominant-negative MyD88 (negative controls) did not result in any significant Poly (I:C)-induced IFN-ß promoter activation or inhibition (Fig. 3D
), nor did they significantly activate or inhibit Poly (I:C)-increased NF-
B luciferase activity (data not shown).
In sum, FRTL-5 cells not only express the TLR3 receptor, they seem to signal through both the NF-B and IRF-3/IFN-ß pathways when incubated with extracellular Poly (I:C), similar to immune cells. Their dual activation results in gene responses characteristic of an innate immune response (14, 15, 16, 24).
Overexpression of TLR3 and TLR Signaling in FRTL-5 Cells
dsRNA Transfection and IFN-ß Induce Overexpression of TLR and TLR3 Signaling.
Incubating FRTL-5 cells with Poly (I:C) did not increase TLR3, PKR, or MHC class I mRNA levels over a 24-h period, although Poly (I:C) incubation significantly increased IP-10 mRNA and slightly increased ICAM-1 mRNA levels, demonstrating the functional activity of the Poly (I:C) in this experiment (Fig. 4A). IL-1ß (the positive control) also caused an increase in IP-10 and ICAM-1 mRNA levels but did not change TLR3, class I, or PKR mRNA levels (Fig. 4A
). This suggested that dsRNA incubation with TLR3 was not an effective means of increasing TLR3 expression in FRTL-5 cells.
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As reported (29), dsRNA transfection and dsDNA transfection differ primarily in the induction of IFN-ß but not PKR. Nevertheless, to evaluate a possible role of PKR activation in TLR3 overexpression by transfected dsRNA, we treated cells with 2-aminopurine (2-AP) (Fig. 4C), a PKR inhibitor (42). TLR3 mRNA was still increased by dsRNA transfection by comparison to control cells (C) or a mock transfection (L) in the presence of 2-AP (Fig. 4C
, lane 7 vs. 3). Like the case for TLR3 expression, 2-AP did not inhibit the dsRNA-transfection-induced increase in IFN-ß mRNA levels (Fig. 4C
, lane 7 vs. 3); however, 2-AP strongly inhibited the ability of dsRNA-transfection to increase NF-
B p65/p50 complex formation in EMSA (Fig. 4C
, bottom). Moreover, whereas the dsRNA transfection-induced increase in PKR and MHC class I was only slightly decreased by 10 mM 2-AP (Fig. 4C
, lane 7 vs. 3), the dsDNA transfection-induced increase in PKR was eliminated and the increase in MHC I was reduced to near control levels (Fig. 4C
, lane 8 vs. 4). This suggested a different mechanism of up-regulation of PKR and MHC class I by the two transfecting agents, the dsDNA effect possibly linked to NF-
B activation, whereas the dsRNA transfection effect potentially more linked to IRF-3/IFN-ß signaling.
Like mouse macrophages (27), exogenously added type I IFN, in our case IFN-ß, increased TLR3 mRNA levels in FRTL-5 thyrocytes in a time- (Fig. 5A) and dose-dependent (Fig. 5B
) manner. The increases were not duplicated by a type II IFN, IFN-
, (Fig. 5
, A and B), even if a high dose of IFN-
was used (Fig. 5B
). IFN-ß also increased MHC I, PKR, and IP-10 mRNA levels, concurrent with the increase in TLR3 mRNA levels (Fig. 5A
).
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Influenza A Virus Mimics the Action of dsRNA Transfection and IFN-ß to Induce Overexpression of TLR and TLR3 Signaling.
The ability of dsRNA transfection, but not dsRNA incubation, to increase TLR3 levels is presumed to mimic the action of a virus to inject RNA into the cell as previously suggested (8, 9, 10, 11, 12, 13, 26, 27, 28, 29, 38, 47, 48, 49, 50). To test this possibility, we infected FRTL-5 cells with influenza A virus, a single-strand RNA virus. Use of a single strand virus rather than a dsRNA virus was suggested by our previous work (51), wherein we showed that replication of a single-strand virus RNA in cultured cells could induce MHC class I.
Treatment of FRTL-5 cells with influenza A for 24 h mimicked the ability of dsRNA transfection to overexpress TLR3mRNA as measured by Northern analysis (Fig. 6A) and increase IFN-ß mRNA as measured by PCR (Fig. 6B
). Of note, both dsRNA transfection and influenza A infection also caused increases in IRF-1 and MHC class II mRNA levels (Fig. 6A
). The less impressive MHC II complex induced by dsRNA transfection is consistent with our previous results indicating a greater response of MHC I than II (29). The data were obtained at a multiplicity of infection of 1 and were not duplicated by coxacki or Herpes simplex infection at the same or 10-fold higher multiplicity of infection (data not shown). Viral specificity remains to be further investigated as will be discussed below.
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C10 Inhibits TLR3 Signaling through the IRF-3/ISRE/IFN-ß/STAT Pathway in FRTL-5 Thyrocytes
MMI is used to treat autoimmune Graves disease and is effective, in part, because it inhibits thyroid hormone formation (53). However, MMI contributes to long term remission of autoimmune/inflammatory diseases by functioning as a broadly active immunomodulator. Thus, MMI has been used as an immunosuppressive in treating psoriasis in humans (54) and in treating murine models of systemic lupus, autoimmune blepharitis, autoimmune uveitis, thyroiditis, and diabetes (34, 55, 56, 57). It is a transcriptional inhibitor of abnormally increased MHC class I and II gene expression in FRTL-5 cells and has been suggested to mimic the effect of a class I knockout in preventing autoimmune disease (30, 31, 32, 33, 34). C10 is a derivative that is 50- to 100-fold more potent in suppressing MHC gene expression (34).
We evaluated the ability of C10 and MMI to inhibit TLR3 expression and signaling. C10 prevented the ability of dsRNA transfection and incubation with IFN-ß to increase TLR3 RNA levels in FRTL-5 cells, as measured by PCR, the same as will be shown for human thyrocytes (see Fig. 11C). Additionally it prevented the ability of both IFN-ß (Fig. 7
) and dsRNA transfection (data not shown) to increase TLR3 by Northern analysis and was significantly better than MMI in this respect, even at 10-fold lower concentrations (0.5 vs. 5 mM). Dimethylsulfoxide (DMSO) is the vehicle for C10 because of its poor solubility; it had no significant effect on basal activity and was used as the control value in all experiments described herein.
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C10, at 0.5 mM, inhibited the ability of Poly (I:C) to increase IFN-ß promoter activity (luciferase luminescence; P < 0.01) when incubated with FRTL-5 thyrocytes transfected with pIFN-ß-luc constructs [Fig. 8A, Poly (I:C) C10 vs. untreated ()]. Even at the concentrations used, which are maximal for MMI (data not shown), the C10 was significantly better than MMI (P < 0.05 or better) [Fig. 8A
, Poly (I:C) treated, MMI vs. C10 and untreated ()]. Lipopolysaccharide (LPS) incubation as well as treatment with IL-1ß also increased IFN-ß luciferase activity in FRTL-5 cells (Fig. 8A
) and in both cases 0.5 mM C10 and 5 mM MMI significantly (P < 0.05 or better) inhibited the increase [Fig. 8A
, LPS or IL-1ß, C10 or MMI vs. untreated ()]. Again, C10 was significantly better than MMI (P < 0.05 or better) (Fig. 8A
), bringing values to basal levels.
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We measured the ability of C10 to inhibit IRF-3 transactivation activity in FRTL-5 thyrocytes, using the IRF-3 cis reporter system (Fig. 8, bottom). We could show that incubation with 0.5 mM C10 significantly (P < 0.05 or better) inhibited IRF-3 transactivation by Poly (I:C), LPS, or IL-1ß (Fig. 8B
). MMI was significantly less effective (data not sown).
A key to activation of other IFN-inducible genes by the autocrine/paracrine action of IFN-ß is its action to regulate downstream genes with ISREs, in part by phosphorylation of STATs, which are important activators ISRE and IFN--activated sites (58, 59, 60). Using an ISRE sequence coupled to luciferase as a reporter gene (ISRE-Luc, Fig. 9B
) we could show that C10 was an effective inhibitor of ISRE activation by Poly (I:C), LPS, IL-1ß, TNF-
, IFN-ß, and IFN-
(Fig. 9A
). Despite the similarity in sequence between ISRE and NF-
B (Fig. 9
, B vs. C), and despite the ability of Poly (I:C) to activate NF-
B-luc in FRTL-5 cells (Fig. 2
), both 0.5 mM C10 or 5 mM MMI had a minimal effect on Poly (I:C)-increased NF-
B-luciferase activity (data not shown). Additionally, they did not have any significant effect on Poly (I:C)- or LPS-increased p65/p50 complex formation (Fig. 10A
).
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In sum, these data suggest that C10, significantly better than MMI, acts by inhibition of the TLR3 regulated IRF-3/IFN-ß/ISRE/STAT signal path not the NF-B signal path. The clear separation of these paths in TLR3 signaling as opposed to TLR4, despite a common adapter molecule (TRIF), has been reported (61). We provide for the first time a potential mechanism for the immunomodulatory actions of MMI, i.e. inhibition of pathogen activated innate immunity, which is critical for the onset of adaptive, antigen-specific adaptive immunity. We show, however, C10 is significantly better in this regard.
TLR3 Expression in Human Thyroid
TLR3 expression and regulation in humans can be very different from that in rats. To address this, we first asked whether TLR3 RNA was expressed in human thyroid tissue using commercial tissue blots from CLONTECH (Palo Alto, CA). We detected TLR3 RNA expression in thyroid tissue by comparison with the spleen positive control (data not shown), results were similar to our observations in mouse tissues (see Fig. 1A).
To confirm the presence and functionality of TLR3 in human thyrocytes, we evaluated TLR3 expression in cultured NPA human thyrocytes, a papillary thyroid cancer cell line. NPA thyrocytes are from a papillary carcinoma but are known to retain some functional properties of normal thyrocytes. Transfection with dsRNA [Poly (I:C)], but not transfection by dsDNA (Fig. 11A) or incubation with Poly (I:C) (data not shown), was able to increase TLR3 mRNA levels in Northern blots. The dsRNA but not the dsDNA transfection could also increase IFN-ß mRNA levels, measured as before with PCR (Fig. 11B
). Additionally, as was the case for FRTL-5 cells and dsRNA transfection, IFN-ß increased PKR mRNA levels as well as TLR3 RNA levels (Fig. 11A
). Finally, as was again the case for FRTL-5 cells, we could show that 0.5 mM C10 decreased the ability of dsRNA transfection or IFN-ß to increase TLR3 mRNA levels (Fig. 11C
).
A fundamental question we posed was the issue of TLR3 overexpression in autoimmune/inflammatory disease. We evaluated TLR3 protein levels in human thyroid tissues by immunohistochemistry. We compared thyroid sections from 10 patients with Hashimotos disease with 10 patients with Graves disease and 10 normal individuals. As described (1), thyroid tissues from patients with Hashimotos thyroiditis exhibited lymphoid and plasma-cell infiltration of the gland (Fig. 12C), fibrosis, and oxyphilic metaplasia of the follicular epithelium Graves disease tissue showed hyperplasia of the follicular epithelial cells with resultant papillary infoldings into preexisting follicles (Fig. 12B
).
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DISCUSSION |
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We asked whether TLR3/TLR3 signaling could be overexpressed in thyrocytes in the absence of immune cells, how it could occur, and could overexpression be associated with an autoimmune/inflammatory disease? We show that overexpression of TLR3 and its signal system in thyrocytes can be induced by transfection with dsRNA or infection with Influenza A virus, a single-strand RNA virus whose replication and activity after infection is likely to be mimicked by the dsRNA transfection. A recent report in lung epithelial cells also links influenza A virus infection, dsRNA transfection, and TLR3 overexpression (62), suggesting this linkage may be applicable to nonimmune cells in multiple tissues.
We show that TLR3 and IFN-ß protein are expressed in situ in thyrocytes from patients with Hashimotos thyroiditis, which are surrounded by immune cells but not in thyrocytes from normal individuals or Graves autoimmune hyperthyroidism, a novel finding never previously demonstrated. The results from human thyrocytes in culture indicate that TLR3 activation and increases can occur in a human as well as rat thyrocyte in culture, and this can occur in the absence of lymphocytes or a lymphocyte-produced IFN because lymphocytes primarily produce type II IFN (63). Consistent with this, the immunocytochemistry study in Fig. 12 shows that the intense brown stain for IFN-ß is localized in the thyrocytes and is not significant in the immune cells. The results thus raise the possibility that thyrocytes are affected by a primary insult that activates the TLR3 system to produce an innate immune response mimicking that of a dendritic cell. The resultant cytokine and costimulatory molecule changes in the thyrocyte may then contribute to attracting lymphocytes to the gland because, unlike dendritic cells, the thyrocytes cannot migrate to the lymphoid organ.
The results herein are startlingly similar to studies of another disease with TLR3 involvement and overexpression, a role for pathogen induction and dsRNA, involvement of a type 1 IFN as an apparent autocrine/paracrine factor, immune cell infiltrates, and cell-specific destruction causing hypofunction: insulinitis and type 1 diabetes (19, 20). Wen et al. (19) show that dsRNA could induce insulinitis and type 1 diabetes in animals, consistent with the known animal model wherein coxsacki virus induces type 1 diabetes in nonobese diabetic mice. Devendra and Eisenbarth (20) emphasize human relevance and note that enteroviruses have been the focus of many research studies as a potential agent in the pathogenesis of type 1 diabetes. They note that the mechanism of viral infection leading to ß-cell destruction involves IFN- [a type I IFN like IFNß]. They hypothesize that activation of TLR by dsRNA or Poly (I:C) (a viral mimic), through induction of IFN-
, may activate or accelerate immune-mediated ß-cell destruction. They note that numerous clinical case reports have implicated IFN-
therapy with autoimmune diseases [thyroiditis, in particular (see Discussion regarding hepatitis treated with IFN-
)] and that elevated serum IFN-
levels have been associated with type 1 diabetes as well as thyroid autoimmune/inflammatory disease (64). Taken together with data in the present report, the possibility is raised of an important mechanistic association relevant to disease pathogenesis. Hashimotos and type 1 diabetes may have immune cell infiltrates and destructive thyrocyte or ß-cell changes because of a primary insult to the specific tissue cell that activates TLR3 and an innate immune response in the tissue cells; this may be an early event in the pathogenic mechanism (14, 15, 19, 20).
Devendra and Eisenbarth suggest (20) that therapeutic agents targeting IFN- (overproduction or activity) may potentially be beneficial in the prevention of type 1 diabetes and autoimmunity. We had asked whether TLR3 overexpression/signaling might be sensitive to the immunomodulatory actions of methimazole (MMI) or its more potent derivative, C10 (30, 31, 32, 33, 34) and how? Our data indicate that C10, to a significantly greater extent than MMI, blocks overexpression of TLR/TLR signaling by inhibition of the TLR3 regulated IRF-3/IFN-ß/ISRE/STAT signal path not the NF-
B signal path. It acts more broadly than just inhibition of IRF-3 transactivation and, therefore, may inhibit activation of a broad range of ISRE sequences on other genes. In this respect, it is notable that, in addition to a NF-
B site, IRF-1 has an IFN-
-activated site that binds STAT-1. It is reasonable to suggest that the ability of C10 to block IRF-1 gene expression, both herein and in our studies of C10 inhibition of TNF-
-induced VCAM-1 and leukocyte adhesion, is related to its action on components of the TLR3 regulated IRF-3/IFN-ß/ISRE/STAT signal path. In short, C10 may be an example of an agent that meets the new therapeutic paradigm requested by Davendra and Eisenbath in their review (20).
TLR signaling remains complex with many unknowns. The role of C10 in these processes will, therefore, be an interesting series of continuing future mechanistic studies. The role of PI3 kinase and Akt involvement in phosphorylation of IRF-3 has recently emerged (65); full phosphorylation of IRF-3 requires TBK-1 and Akt. Reactive oxygen species involvement in virus-induced activation of STATs is recognized (66). The P38MAPK pathway is important in downstream effectors that participate in type I IFN-dependent gene transcription and involvement (67). Transcriptional activation of the IFN-ß gene requires assembly of an enhanceosome containing transcription factors ATF-2/c-Jun, IRF-3/IRF-7, NF-
B, and HMGI(Y) (68) and thus indicates the two signal paths are intertwined both at the earliest level of IRF-3/IFN-ß activation as well as at downstream molecules such as VCAM-1 gene expression. In short, our data are important mechanistic steps but by no means a final answer given the complexity of the pathways emerging in reports this year. At the very least, C10 is a novel agent to help dissect the complexity of the TLR3/4 signal pathway.
Our previous description of C10 efficacy in inhibiting TNF--induced VCAM-1 gene expression and leukocyte adhesion is highly relevant to atherosclerosis and colitis, two other diseases where TLR4 overexpression or signaling in nonimmune cells is linked to autoimmune/inflammatory disease (14, 15, 22, 23, 35, 36, 37). It is reasonable to conclude that Hashimotos may, therefore, not only be grouped with insulinitis and type 1 diabetes, but also with colitis, and atherosclerosis as autoimmune/inflammatory diseases associated with TLR3/4 overexpression and signaling in nonimmune cells, whose overexpression involves induction by molecular signatures of environmental pathogens (14, 15, 19, 20, 22, 23, 35, 36). We show in the in vitro studies of this report that C10 can be used to suppress dsRNA and virally induced signals that have been implicated in the above mentioned autoimmune/inflammatory diseases.
We have separately shown that C10 is efficacious in an in vivo dextran sodium sulfate (DSS) colitis model where C10 improves survival, attenuates pathology, and suppresses increases in TLR4, VCAM-1 and IP-10 expression (Ref.69 and manuscript in preparation). Moreover, C10 reduced TLR4 expression in colonic epithelial cells and attenuated disease more effectively than prednisolone, a drug commonly used to treat inflammatory bowel disease. The DSS model is used to study ulcerative colitis and Crohns disease. Recent work indicates that TLR4 is strongly up-regulated in both (21) and that enterocolitis is significantly improved in TLR4-deficient mice in another mouse model of human inflammatory bowel disease (70). These data indicate the importance of innate immunity and TLR4 in Th1-dependent enterocolitis (70) and thus the importance of C10 in blocking TLR4 overexpression in vitro and in colonic epithelial cells in the DSS model.
It is reasonable to speculate that Hashimotos and type 1 diabetes may be prototypes of each other and that studies in FRTL-5 cells may well be a relevant model for studies in pancreatic ß islet cells and diabetes. We have already reported that high glucose levels could transcriptionally increase MHC I expression and amplify IFN- (action in FRTL-5 thyroid cells (71). In retrospect, this might be applicable to the islet cell changes induced by high glucose levels.
We as well as others (14, 15, 19, 20, 22, 23, 35, 36, 62) show that type I IFN (IFN- or ß) is an important factor in the innate viral immune response. We suggest that an increase in type I IFN gene expression in nonimmune cells can result in an autocrine/paracrine manner to further up-regulate TLR3 by activation of IRFs. Type I IFNs act as potent extracellular mediators of host defense and homeostasis and lead to the synthesis of proteins that mediate antiviral, growth inhibitory, and immunomodulatory responses. The secreted type I IFN can sensitize the same or adjacent cells to dsRNA by increasing expression of dsRNA recognition molecules such as TLR3 and PKR. A similar model invoking TLR3 and type I IFN in the innate immune response of nonimmune cells has been invoked in Influenza A-infected lung tissue (62).
Because it is a protective cytokine, type I IFNs have been used in the clinical setting to treat hepatitis C and B, chronic myelogenous leukemias, melanoma, and renal cancer (72). One side effect of type I IFN therapy is, however, a higher incidence of thyroid autoimmune disease. The risk of Hashimotos thyroiditis is known to be increased with type I IFN treatment in human cytomegalic virus hepatitis patients. Thyroid autoantibodies are found in up to 20% of patients who receive treatment with type I IFNs and approximately 5% of these patients develop clinical hypothyroidism (73, 74, 75). Treatment with IFN- does not induce chronic autoimmune thyroiditis (76). Additionally, 1) administration of IFN-
or its overexpression in an autoimmunity-prone target tissue such as the thyroid does not induce typical autoimmune disease (77, 78) and 2) increased MHC gene expression has been reported after tissue damage in vivo even in IFN-
or IFN-
receptor knockout mice (79). Consistent with the possible autoimmune-inducing activity of type I IFNs, up-regulation of Type I IFNs was observed in some patients with psoriasis, systemic lupus erythematosus, and insulin-dependent diabetes mellitus (80, 81, 82, 83). It will be interesting to see in future studies whether administration of type I IFN is associated with TLR overexpression in nonimmune cells of patients prone to the autoimmune responses.
What might be an inciting agent or mechanism to activate the TLR3-innate immune response in the thyrocyte. The ability of a virus to induce experimental autoimmune lymphocytic thyroiditis in mouse models, with elevated thyroglobulin autoantibody levels, was demonstrated by infection with Reovirus, an RNA virus (84, 85, 86). In humans, an association of foamy virus infections, another RNA virus, with DeQuervains thyroiditis is described (13). Serologic evidence of recent viral and bacterial infection has been reported in patients with chronic autoimmune thyroiditis (13, 87). Also, human T-lymphotropic virus type I (HTLV-I), a RNA retrovirus causing adult T-cell leukemia lymphoma and HTLV-I-associated myelopathy/tropical spastic paraparesis, is associated with Hashimotos thyroiditis, as well as some forms of pulmonary alveolitis, chronic arthropathy, polymyositis, and uveitis (88, 89, 90, 91). A high prevalence of thyroid peroxidase autoantibodies and/or thyroglobulin autoantibodies, as well as a high prevalence of hypothyroidism, has been demonstrated in adult T-cell leukemia patients and HTLV-I carriers (88, 89, 90, 91). Kawai et al. (92) published a case report that HTLV-1 virus envelope protein and signals for the mRNA were detected in many of the follicular epithelial cells of the thyroid tissue from one patient of Hashimotos thyroiditis by immunohistochemistry and in situ hybridization. PCR-Southern blotting revealed the presence of HTLV-I DNA in the thyroid tissue, in which the viral protein and mRNA were detected, although no virus particles were found in the epithelial cells by electron microscopy (92).
Nevertheless, no unequivocal reports have clearly correlated virus infections with Hashimotos thyroiditis, nor is it likely that unequivocal evidence relating viral infection and Hashimotos thyroiditis will emerge. It is difficult to detect the exact time of onset of the disease or viral infection of thyrocytes in these patients; additionally, there is the obvious problem of trying to fulfill Kochs (93) postulate in humans by infecting an individual with a virus that causes autoimmune destruction of the thyroid gland, even if we discover one. At this point, therefore, we are faced with the need to pursue indirect evidence in humans and use animals and cells to better define the likely disease mechanism, after the lead of TLR4 studies in colitis (21) and the Influenza A data herein and in lung cells (62).
Two last points are worth noting. The dsRNA transfection was used to activate PKR-dependent NF-B activation or a separate kinase system leading to IFN-ß gene expression through IRF-3 activation. The upstream mechanism resulting in IRF-3 activation after dsRNA transfection or viral infection in vitro is being clarified. Pharmacological and molecular studies suggested that a novel viral-activated serine/threonine kinase, instead of PKR, might activate IRF-3 in response to cytosolic dsRNA (43, 44, 45, 46). We now know this is a complex phenomenon involving PI3 kinase/Akt and I
B-related kinases (IKK)-IKK
/TANK binding kinase 1 (43, 44, 45, 46). Consistent with these observations, PKR/ mice are physically normal and the induction of type I IFNs by Poly (I:C) and virus is unimpaired (94), despite the evidence that PKR is a major intracellular RNA-recognition molecule, leading to an antiviral cellular response.
Second, the sum of data suggests that the presence of TLR3 in FRTL-5 cells, and its up-regulation and signaling by dsRNA transfection, can account for the data in our previous study (29), which showed that viral infection, plasmid transfection, transfection of dsDNA, or transfection of dsRNA into the cytoplasm of the cell could increase expression of MHC class I, cause aberrant expression of MHC class II, and cause the expression of other genes necessary for antigen presentation (antigen-presenting cell generation). The action of the dsRNA or dsDNA transfection appeared to involve NF-B activation, but only the dsRNA transfection increased IFN-ß RNA levels (29). These phenomena were evidenced in other cells including monocytes and macrophages and were associated with an immune response in animals (95).
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MATERIALS AND METHODS |
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Cells
The F1 subclone of FRTL-5 thyrocytes [Interthyr Research Foundation, Baltimore, MD (ATCC CRL 8305)] was grown in Coons modified Hams F-12 medium supplemented with 5% calf serum, 2 mM glutamine, and 1 mM nonessential amino acids, plus a six-hormone mixture (6H medium), containing bovine TSH (1 x 1010 M), insulin (10 µg/ml), cortisol (0.4 ng/ml), transferrin (5 µg/ml), glycyl-L-histidyl-L-lysine acetate (10 ng/ml), and somatostatin (10 ng/ml). HEK293H cells (Invitrogen, Carlsbad, CA) were maintained in DMEM with 10% fetal calf serum. CHO-K1 cells (ATCC CCL-61) were maintained in Hams F-12 medium with 10% fetal calf serum.
NPA-87 cells are a continuous line of human thyrocytes derived from papillary carcinoma cells. They retain several functional responses included TSH-increased cAMP signaling (96). They were kindly provided by Dr. Guy Juillard (University of California, Los Angeles, CA) and grown in RPMI 1640 medium supplemented with 2 g/liter sodium bicarbonate, 0.14 mM nonessential amino acids, 1.4 mM sodium pyruvate, and 10% fetal bovine serum (pH 7.2).
RNA Isolation and Northern Analysis
RNA was prepared using the RNeasy Mini Kit (QIAGEN Inc., Valencia, CA) and the method described by the manufacturer. For Northern, 1520 µg total RNA were run on denatured agarose gels, capillary blotted on Nytran membranes (Schleicher & Schuell, Keene, NH), UV cross-linked, and subjected to hybridization. Probes were labeled with [-32P] deoxy-CTP using a Ladderman Labeling Kit (Takara, Madison, WI). The probes for MHC class I, ICAM-1, IRF-1, PKR, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been described (29). The probes for IFN-ß used gene-specific primers that have also been described (41). The probes for TLR3 were the mouse or human whole cDNAs obtained from the Invivogen expression vectors. The IP-10 probe was a partial mouse IP-10 cDNA (469 bp) prepared by RT-PCR from mouse macrophage total RNA with the following primers: mIP-10(5') 5'-CCATCAGCACCATGAACCCAAGTCCTGCCG-3' and mIP-10(3') 5'-GGACGTCCTCCTCATCGTCGACTACACTGG-3'. Membranes were hybridized and washed as described previously (29).
RT-PCR
DNA was removed from total RNA using the DNA-free Kit (Ambion) according to the manufacturers instructions. One microgram of RNA was used to synthesize cDNA using the Advantage RT-for-PCR Kit (BD Biosciences, Palo Alto, CA) according to the manufacturers protocol. Fifty nanograms of cDNA were subsequently used for PCR of TLR-3, and ß-actin; 250 ng of cDNA was used for PCR of IFN-ß. The primers used for amplification of human TLR-3 and ß-actin have been previously described (97). The gene-specific primers for rat IFN-ß and GAPDH and PCR conditions have been described (29, 41) Human IFN-ß primers are as follows: (5' primer) 5'-TGGCAATTGAATGGGAGGCTTG-3' and (3' primer) 5'-TCCTTGGCCTTCAGGTAATGCAGA-3'. PCR conditions for humanTLR-3 and ß-actin are as follows: 94 C for 5 min followed by 35 cycles of 94 C for 30 sec, 55 C for 30 sec, 72 C for 1 min, and a final cycle of 72 C for 7 min. Human IFN-ß PCR conditions are: 94 C for 3 min, followed by 35 cycles of 94 C for 10 sec, 58 C for 30 sec, 72 C for 1 min, and a final cycle of 72 C for 10 min.
Plasmids for Reporter Gene Assays
Human IRF-3 was amplified from human cDNA and cloned into pCR 2.1 by the TOPO/TA (Invitrogen) cloning method, and then sequenced. IRF-3 was then excised by EcoRI digestion and subcloned into pCMV-BD (Stratagene, La Jolla, CA) for use in transactivation assays. To construct IFNß-luc the human IFN-ß promoter sequence was amplified from human genomic DNA (CLONTECH) using Ex Taq polymerase (Takara, Madison, WI). The PCR fragment contained human IFN-ß promoter sequence from 125 to +34 relative to the transcription start site (+1) and incorporated KpnI and XhoI restriction sites at the 5' and 3' ends, respectively. The primers were as follows: hIFN-ß (125) KpnI (5'-CAG GGT ACC GAG TTT TAG AAA CTA CTA AAA TG-3') and hIFN-ß (+34) XhoI (5'-GTA CTC GAG CAA AGG CTT CGA AAG G-3'). The fragment was digested with KpnI and XhoI and then ligated into a similarly digested pGL3 Basic (Promega, Madison, WI) vector. The human MyD88 wild and dominant-negative expression vectors were kindly donated by Dr. P. E. Auron (Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA). pFR-luc (5x Gal4 DNA binding domains and minimal TATA box), ISRE-Luc, NF-B-luc, and the Elk1 trans-Reporting System were purchased from Stratagene. pRL TK-Int was purchased from Promega.
Transient Expression Analysis
A diethylaminoethyl (DEAE) procedure was used to transfect promoter-luciferase gene constructs and expression plasmids into FRTL-5 cells. Briefly, FRTL-5 cells were grown in 24-well plates to about 70% confluence, washed with 0.5 ml of serum-free culture medium (6H0 medium), then exposed to 125 µl of premade plasmid-DEAE mixture per well for 15 min at room temperature. The plasmid-DEAE mixture was prepared by incubating 100 ng of plasmid DNA, unless otherwise noted in individual experiments, with 3.125 µl of DEAE-dextran (10 mg/ml) (Promega). FRTL-5 cells were incubated with this mixture for 2 h at 37 C in a CO2 incubator, before 2 ml of 6H5 medium was added. CHO-K1 and FRTL-5 cells for transfecting expression vectors were subjected to the lipofection method. Cells were grown in 10-cm dishes to about 80% confluence and then exposed to the plasmid-Lipofectamine 2000 mixture as described by the manufacturer (Invitrogen).
Immunoprecipitation and Western Blot Analysis
Whole cell lysates were prepared in lysis buffer [10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40] containing protease inhibitors. Nuclear extracts were prepared using the NE-PER extraction reagents with protease inhibitors stated below (Pierce Chemical Co., Rockford, IL). Twenty-five micrograms of either whole cell lysate or nuclear extract was resolved on denaturing gels using the Nu-PAGE System (Invitrogen). All proteins were transferred to nitrocellulose membranes and subsequent antibody binding was revealed using ECL Plus reagents (Amersham Pharmacia Biotech, Piscataway, NJ). For immunoprecipitation, lysates were incubated with anti-TLR3 antibody (Imgenex, San Diego, CA) (10 µg/ml) at 4 C for 6 h, followed by adsorption to protein G-Sepharose beads (Amersham Pharmacia Biotech). Precipitates were washed and resolved as stated above. CHO-K1 cells were transiently transfected with 20 µg of expression vector.
Nuclear Extracts and DNA Mobility Shift Assays (EMSA)
FRTL-5 cells were harvested by scraping into PBS (pH 7.4) and washing twice with PBS. Nuclear extracts were then prepared using NE-PER extraction reagents (Pierce Chemical Co., Rockford, IL). The protocol was performed in accordance with manufacturers instructions and involved the presence of protease inhibitor cocktail III [4-(2-aminoethyl)benzenesulfonylfluoride hydrochloride, aprotinin, bestatin, E-64 protease inhibitor, leupeptin, pepstatin] (Calbiochem, San Diego, CA). Oligonucleotides (NF-B sense 5'-AGT TGA GGG GAC TTT CCC AGG C-3'; NF-
B antisense 5'-GCC TGG GAA AGT CCC CTC AAC T-3') were annealed and labeled with [
32P]-ATP using T4 polynucleotide kinase. EMSA was performed using 3 µg of nuclear extracts. In competition studies, 50-fold molar excess of unlabeled oligonucleotide or 2 µg of antibody was added to the mixtures. A 32P-labeled oligonucleotide probe (100,000 cpm) was added and the incubation was continued for 20 min at room temperature. Mixtures were analyzed on 5% native polyacrylamide gels and autoradiographed.
Virus Infections
Influenza A A/Victoria/3/75 was obtained from Diagnostic Hybrids Inc. (Athens, OH). FRTL-5 cells were grown in 6H growth media until 60% confluence, then maintained in 5H (TSH) media for 7 d before infections. Ten-centimeter dishes were 95100% confluent at the time of infection. Seven million viral particles were added to each 10-cm dish of cells in 5H media. Fresh 5H media were added 24 h before infection. Cells were incubated with virus for 24 h and then C10 was added directly to the media and incubated for 6 h before cells were harvested.
Patients and Tissue Samples
This project has been approved by the Institutional Research Committee at individual institutions and informed consent was obtained. Tissue specimens were obtained from 30 individuals treated at the Ukrainian Center of Endocrine Surgery in Kiev. Thyroid lesions were classified as Hashimotos thyroiditis in 10 cases, hyperplasia associated with Graves disease in 10 cases. Normal thyroid tissue was from the contralateral glands of 10 patients undergoing thyroid surgery for adenomas or tumors. After fixation in 10% formalin and embedding in paraffin, 5 µm-thick serial sections were made for each specimen. The 5-µm sections were stained with hematoxylin and eosin.
Immunohistochemical Staining
Sections were dewaxed, soaked in alcohol, and after microwave treatment in antigen unmasking solution for 10 min, incubated in 3% hydrogen peroxide for 15 min to inactivate endogenous peroxidase activity. Then sections were incubated at 4 C overnight with anti-TLR3 antibody (1:100 dilution). Immunostaining was performed by use of the Vectastain Universal Quick kit according to the manufactured instruction. Peroxidase staining was revealed in 3,3-diaminobenzidine. Negative control was applied by omission of antiserum.
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FOOTNOTES |
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Abbreviations: 2-AP, 2-Aminopurine; C10, phenylmethimazole; CHO, Chinese hamster ovary; DEAE, diethylaminoethyl; DMSO, dimethylsulfoxide; ds, double-stranded; DSS, dextran sodium sulfate; FRTL-5, Fisher rat thyroid cell line-5; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HEK, human embryonic kidney; HTLV-I, human T-lymphotropic virus type I; ICAM, intercellular adhesion molecule; IFN, interferon; IKK, IB-related kinase; IP, inducible protein; IRF, IFN regulatory factor; ISRE, IFN-stimulated response element; LPS, lipopolysaccharide; MHC, major histocompatibility complex; MMI, methimazole; NF-
B, nuclear factor
B; PKR, dsRNA-dependent protein kinase; Poly (I:C), polyinosine-polycytidylic acid; STAT, signal transducers and activation of transcription; TIR, Toll-like IL-1 receptor; TLR3, Toll-like receptor 3; TRIF, TIR domain-containing molecule adapter-inducing IFN-ß; VCAM, vascular cell adhesion molecule.
Received for publication March 9, 2004. Accepted for publication January 10, 2005.
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REFERENCES |
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