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Expression of toll-like receptors by human muscle cells in vitro and in vivo: TLR3 is highly expressed in inflammatory and HIV myopathies, mediates IL-8 release and up-regulation of NKG2D-ligands

Bettina Schreiner^*, Joachim Voss, Jörg Wischhusen^*,¶, Yvonne Dombrowski^¶, Alexander Steinle, Hanns Lochmüller, Marinos Dalakas, Arthur Melms^* and Heinz Wiendl^*,||,1

^* Department of General Neurology, Hertie-Institute for Clinical Brain Research, Eberhard-Karls-University Tuebingen, Tuebingen, Germany;
National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA;
Department of Immunology, Institute for Cell Biology, Eberhard-Karls-University Tuebingen, Tuebingen, Germany;
Genzentrum, Friedrich-Baur Institut, and Department of Neurology, Ludwig-Maximilians University, Munich, Germany; and Departments of
^|| Neurology and
^¶ Gynecology, University of Wuerzburg, Julius-Maximilians-University Wuerzburg, Wuerzburg, Germany

¹Correspondence: Department of Neurology, Julius-Maximilians-University Wuerzburg, Josef-Schneider-Strasse 11, Wuerzburg D-97080, Germany. E-mail: heinz.wiendl{at}klinik.uni-wuerzburg.de

SPECIFIC AIMS

Our study aimed at investigating the expression and function of toll-like receptors (TLRs), essential components of the innate arm of immune responses, in human skeletal muscle.

PRINCIPAL FINDINGS

1. TLR1-7 and TLR9 mRNA are found in cultured human myoblasts and TE671 cells
Myoblasts and human rhabdomyosarcoma cells (TE671) were screened for mRNA expression of members of the TLR family (TLR1-9). Muscle cells constitutively expressed low levels of TLR1-7 and 9 mRNA as shown by agarose gel electrophoresis and QRT-PCR. TLR8 mRNA was undetectable in muscle cells under any conditions.

2. TLR3 mRNA and protein are expressed in human muscle cells and are strongly up-regulated by poly (I:C) and IFN-
As shown in Fig. 1 A, myoblasts constitutively expressed TLR3 mRNA. Incubation with IFN- or the synthetic dsRNA analog poly (I:C) induced TLR3 mRNA, in contrast to TNF- or the TLR4 ligand LPS.

View larger version (18K): [in this window] [in a new window]   Figure 1. Expression and regulation of TLR3 mRNA and protein in human myoblasts. A) Primary human myoblasts cultured in the absence or in the presence of poly (I:C) (25 µg/mL), IFN- (500 U/mL), TNF- (1000 U/mL), or LPS (5 µg/mL) were harvested after 48 h of culture and analyzed for the expression of TLR3 mRNA by QRT-PCR. Bars represent the relative gene expression of TLR3 calculated in relation to unstimulated myoblasts. Monocytes were used as negative controls. B) Primary human myoblasts were cultured as described under (A) and expression of TLR3 protein was assessed by flow cytometry using the specific monoclonal antibody TLR3.7 (10 µg/mL). Cell surface and intracellular staining are shown. TLR3 protein was located mainly intracellularly. Open histograms represent staining with the specific antibody underlaid with the isotype-matched control antibody (shaded histograms). Numbers in the upper corner indicate the SFI values specific for the TLR3 mAb. Immature and LPS-matured DC served as positive controls (A–B).

Human myoblasts constitutively expressed TLR3 protein, which was up-regulated upon challenge with IFN- and poly (I:C) (Fig. 1B ). To distinguish between extracellular and intracellular TLR3 protein expression, flow cytometry was performed using untreated muscle cells or muscle cells subjected to permeabilization of the cell membrane. As demonstrated in Fig. 1B , TLR3 protein could be detected mainly intracellularly. IFN- and poly (I:C) up-regulated intracellular TLR3 protein, while extracellular levels were unaffected.

3. Ligation of TLR3 induces NF-B activation, IL-8 secretion and up-regulation of NKG2D-ligands in human muscle cells
To analyze whether dsRNA is capable of initiating the classical TLR signal transduction pathway leading to the activation of NF-B, we transiently transfected human muscle cells (TE671 and primary human myoblasts) with a NF-B luciferase reporter construct. Poly (I:C) was able to activate NF-B in TE671 and human myoblasts, demonstrating the functional signaling pathway after TLR3 ligation in muscle cells (Fig. 2 A). We included the stimulus TNF- as a positive control and achieved a maximum poly (I:C) induction between 8.6 and 11.6 (depending on the donor), 22% of the TNF--stimulated control myoblasts (Fig. 2A ).

View larger version (29K): [in this window] [in a new window]   Figure 2. Functional consequences of TLR 3 ligation in human muscle cells. A) Primary human myoblasts were transfected transiently with firefly luciferase-linked NF-B reporter plasmids (NF-B-Luc) and renilla luciferase-linked control plasmids (pRL-CMV). Cells were stimulated with different concentrations of poly (I:C) and anti-TLR3 mAb (TLR3.7; 10 µg/mL) or isotype control Ab (10 µg/mL), IFN- (1000 U/mL), or TNF- (5 ng/mL) for 24 h, lysed, and assayed for firefly and renilla luciferase activities. Results were normalized to renilla activities (internal control). Each experiment was performed in triplicates. Means and standard deviations of one representative experiment are shown (n=4). Two-tailed t test against unstimulated controls (**P<0.01, *P<0.05). B) Primary human myoblasts cultured in the absence (left) or in the presence of poly (I:C) (25 µg/mL, right) for 48 h. Secretion of different inflammatory cytokines into the culture supernatants was assayed by a human inflammation antibody array. IL-8 and TIMP-2, both produced into the supernatant of unstimulated myoblasts, are up-regulated by poly (I:C). C) Primary human myoblasts and TE671 were cultured in the absence (bright bars) or in the presence of IFN- (1000 U/mL, dark bars) for 72 h and subsequently stimulated with different concentrations of poly (I:C) for 48 h in serum-containing medium. Supernatants were taken and IL-8 was measured by ELISA. Data show the mean and standard deviations of one representative experiment done in duplicates (n=4). Two-tailed t test against unstimulated controls D) Myoblasts were pretreated with 1000 U/mL IFN- and stimulated with poly (I:C) 25 µg/mL for 24 and 48 h. Human MICA, MICB, and ULBP1, 2, 3 mRNA levels were quantified by QRT-PCR. Results were normalized to the 18S housekeeping gene. Bars represent the relative gene expression of the NKG2D-ligands calculated in relation to unstimulated myoblasts. Means and standard deviation of two representative experiments done in duplicates are shown. One-sample t test (theoretical mean=1, **P<0.01, *P<0.05).

We next examined the production of inflammatory proteins by human muscle cells and the influence of TLR3 ligation (e.g., stimulation with poly (I:C)). We first used a human inflammation antibody array. Primary human myoblasts constitutively release the chemokine IL-8 and tissue inhibitor of metalloproteinase-2 (TIMP-2) into the culture supernatants (Fig. 2B ), both up-regulated by the stimulation of TLR3 via poly (I:C). Similar results were obtained for the muscle cell line TE671. Subsequently, the influence of TLR3 ligation on the expression of IL-8 was investigated in more detail using an IL-8 ELISA. We found that incubation with poly (I:C) increased IL-8 protein in the supernatant of cultured primary human myoblasts as well as of TE671 cells. This effect was most impressive when muscle cells were pretreated with IFN- (Fig. 2C ).

We next investigated whether pathogenic stimulation of muscle cells would alter the expression of NKG2D ligands. Treatment of IFN- prestimulated primary myoblasts with poly (I:C) resulted in a significant mRNA up-regulation of the NKG2D ligands MHC class I-chain related molecule A (MICA), MICB, and the UL16 binding proteins (ULBP) 2 and 3 as determined by QRT-PCR (Fig. 2D ). Treatment of muscle cells with IFN- alone did not induce transcription of any NKG2D ligand.

NKG2D, the receptor for NKG2D ligands, is found on cytotoxic cells. To demonstrate in principle that stimulation of muscle cells by a TLR3 agonist can increase their vulnerability to cell-mediated lysis, we performed in vitro lysis assays with TE671 targets and NK effector cells from polyclonal cultures. Poly (I:C) pretreated TE671 were lysed better than untreated targets (relative increase between 26 and 86% depending on the effector:target ratio). The relevance of the NKG2D pathway for NK cell-mediated lysis of muscle cells was confirmed by addition of a blocking anti-NKG2D antibody that reduced lysis in unstimulated TE671 cells expressing basal levels of NKG2DL and, even more so, in poly (I:C) treated target cells in which NKG2DL were further induced.

4. TLR3 is up-regulated in muscle biopsy specimens of inflammatory myopathies
To validate our data achieved with muscle cells in vitro, we analyzed muscle biopsy specimens of 6 HIV patients with inclusion body myositis/polymyositis (HIV/IBM/PM), 7 patients with sporadic IBM (IBM), 5 nonmyopathic controls (control), and 4 nonmyopathic controls from HIV patients (HIV) for TLR3 mRNA expression by QRT-PCR. TLR3 mRNA was detectable in muscle biopsy specimens of inflammatory myopathies (HIV/IBM/PM and IBM). Levels of TLR3 mRNA were elevated in biopsy specimens from patients with IBM- and HIV-myopathies in comparison to the control specimens. We found highly significant differences between IBM vs. control and HIV/IBM/PM vs. control samples (P<0.01; Mann-Whitney test).

We performed immunohistochemistry for TLR3 in specimens with IBM (n=5) and controls (n=5). TLR3 was not detected on muscle biopsy specimens from nonmyopathic controls or noninflammatory myopathies (n=5). In contrast, TLR3 expression was detectable in sporadic IBM (IBM: 4 of 5). TLR3 was mainly localized in areas where inflammatory cells were in close contact to damaged or non-necrotic muscle fibers.

CONCLUSIONS AND SIGNIFICANCE

Since muscle is one of the few body compartments that lack MHC expression under physiological conditions, immune reactions triggered by or directed against muscle cells proceed along specific pathways. The present study was aimed at elucidating the role of TLR in skeletal muscle and thereby improve our understanding of muscular responses to infectious or inflammatory stimuli as they occur in immune mediated disorders of the skeletal muscle (e.g., the myositis syndromes) or during vaccinations.

We found that muscle cells express TLR3 mRNA and protein. TLR3 protein in muscle cells was located primarily intracellularly.

Ligation of TLR3 induces NF-B activation found that IL-8, already present in culture supernatants of muscle cells under basal conditions, is up-regulated by TLR3 ligation. Our data suggest that dsRNA generated by viruses can up-regulate the production of chemokines like IL-8 by muscle cells, thereby contributing to the attraction of leukocytes to the site of muscle inflammation. We could further show that myoblasts stimulated with poly (I:C) express ligands for the NKG2D receptor, which is found on NK cells, activated CD8 T cells and macrophages. NKG2D is one of the best characterized receptors associated with responses to cellular distress such as transformation, infection, or cell stress. Up-regulation of its ligands could be a mechanism by which infected muscle cells communicate directly to cells that can kill them and clear up. We could show that treatment with dsRNA renders muscle cells more susceptible to NK cell-mediated cytotoxicity.

Our study suggests an important role of TLR3 in the immunobiology of muscle and has substantial implications for the understanding of the pathogenesis of inflammatory myopathies or therapeutic interventions like vaccinations or gene transfer strategies.

View larger version (45K): [in this window] [in a new window]   Figure 3. Postulated TLR3-mediated mechanisms in human muscle. 1) Inflammatory cytokines such as IFN-; 2) viral dsRNA / infectious "stranger" signals; or 3) mRNA/endogenous "danger" signals up-regulate TLR3 in muscle cells. Ligation of TLR3 a) activates NF-B leading to the production of inflammatory cytokines; b) releases chemokines such as IL-8-attracting leukocytes to the site of inflammation; and c) induces NKG2D ligands, stimulating infiltrating NK cells and/or cytotoxic CD8 T cells that lyse damaged muscle cells and clear up.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4342fje; doi: 10.1096/fj.05-4342fje

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