2012 TLRs en Células de Infiltrados de Miositis
2012 TLRs en Células de Infiltrados de Miositis
2012 TLRs en Células de Infiltrados de Miositis
10
Copyright Ó 2012 by the American Association of Neuropathologists, Inc. October 2012
pp. 855Y867
ORIGINAL ARTICLE
and dendritic cells (2). All TLRs, except for TLR3, signal via
Abstract myeloid differentiation response gene 88 (MyD88), the central
The roles of Toll-like receptors (TLRs) and their myeloid differen- adaptor protein, finally resulting in activation of the tran-
tiation response gene 88 (MyD88)Ydependent and MyD88-independent scription factor nuclear factor-JB (NF-JB) pathway, leading
signaling cascade particularly with regard to the pathogenesis and regu- to an increased production of interferon-F (IFN-F) and inter-
J Neuropathol Exp Neurol Volume 71, Number 10, October 2012 855
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Brunn et al J Neuropathol Exp Neurol Volume 71, Number 10, October 2012
Copyright © 2012 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
J Neuropathol Exp Neurol Volume 71, Number 10, October 2012 Toll-Like Receptors in Inflammatory Myopathies
DM (Figure, Supplemental Digital Content 1, part C, volume of 20 KL. All reactions were performed in triplicate.
http://links.lww.com/NEN/A384), IMV (Figure, Supplemental For normalization, the human genes of TATA-box binding
Digital Content 1, part D, http://links.lww.com/NEN/A384), protein and RNA polymerase II were chosen as reference
and neurogenic muscular atrophy throughout serial sections. In genes (28). Normalized data were calibrated by comparison
addition, formalin-fixed, paraffin-embedded specimens of each with expression values of control muscles using the $$Ct
muscle biopsy were prepared. method (29). The PCR was realized on an Applied Bio-
systems 7300 Real-Time PCR System with SDS software
Quantitative Real-Time Polymerase version 1.4.
Chain Reaction
Transcription of TLR2 (assay identification number, Immunohistochemistry
Hs01872448_s1), TLR4 (Hs00152939_m1), TLR9 (Hs00370913_ Formalin-fixed, paraffin-embedded muscle specimen sec-
s1), MyD88 (Hs01573837_g1), IFN-F (Hs00989291_m1), IL-4 tions (4 Km thick) were stained using the avidin-biotin complex
(Hs00174122_m1), IL-6 (Hs00985639_m1), IL-12p40 technique, with 3,3¶-diaminobenzidine (Sigma, Deisenhofen,
(Hs01011518_m1), IL-17 (Hs00174383_m1), RelA (Hs01042010_ Germany) as chromogen and H2O2 as cosubstrate, as previously
m1), Rel (Hs00968440_m1), IRF3 (Hs01547283_m1), and described (30). Tonsillar tissue served as a positive control.
IFN-A1 (Hs01077958_s1) mRNA was analyzed by quantitative Antibodies used for immunohistochemistry are listed in Table 2.
real-time polymerase chain reaction (RT-PCR) using Taqman
gene expression assays (Applied Biosystems, Darmstadt,
Germany), as previously described (27). In brief, RNA was Immunofluorescence
extracted from cryopreserved muscle specimen using the For double immunofluorescence, incubation steps were
TRIzol/chloroform method (Invitrogen, Karlsruhe, Germany) as follows: first primary antibody, species-specific biotinylated
according to the manufacturer’s instructions. RNA concen- immunoglobulin, extravidinYfluorescein isothiocyanate (FITC)
trations were measured fluorometrically. First-strand cDNA or extravidin-Cy3, respectively (1:80/Sigma 1:100; Dianova,
was synthesized using the High Capacity cDNA Reverse Hamburg, Germany), second primary antibody, species-specific
Transcription Kit (Applied Biosystems). Water was used secondary antibody, that is, goat anti-rabbit immunoglobulin
instead of cDNA as negative control. The PCR reactions were coupled with DyLight 549 red and goat anti-mouse immuno-
prepared with a cDNA equivalent of 5 ng RNA in a final globulin coupled with FITC, respectively. Primary antibodies
Copyright © 2012 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
Brunn et al J Neuropathol Exp Neurol Volume 71, Number 10, October 2012
Copyright © 2012 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
J Neuropathol Exp Neurol Volume 71, Number 10, October 2012 Toll-Like Receptors in Inflammatory Myopathies
Copyright © 2012 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
Brunn et al J Neuropathol Exp Neurol Volume 71, Number 10, October 2012
whereas CD80 and CD86 were totally absent from neurogenic mRNA transcripts were not significantly increased in inflam-
muscular atrophy biopsies (data not shown). matory myopathies versus controls (Fig. 8AYD).
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J Neuropathol Exp Neurol Volume 71, Number 10, October 2012 Toll-Like Receptors in Inflammatory Myopathies
FIGURE 4. CD68-positive macrophages express TLR4 and MyD88 in acute inflammatory myopathies. Coexpression of TLR4 (A)
and MyD88 (E) on CD68-positive macrophages in a case of PM (Case 4). Coexpression of TLR4 (B) and MyD88 (F) on CD68-
positive macrophages in a case of IBM (Case 10). Coexpression of TLR4 (C) and MyD88 (G) on CD68-positive macrophages in a
case of DMa (Case 14). Coexpression of TLR4 (D) and MyD88 (H) on CD68-positive macrophages in a case of IMV (Case 18).
Double immunofluorescence of rabbit anti-human TLR4 Dylight 549 with mouse anti-human CD68 fluorescein isothiocyanate and
goat anti-human MyD88 Cy3 with mouse anti-human CD68, respectively. Original magnification: 400.
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Brunn et al J Neuropathol Exp Neurol Volume 71, Number 10, October 2012
FIGURE 5. Expression of TLR4 and MyD88 on CD8-positive T cells in PM, IBM, and on CD20-positive B cells in DMa, respectively.
(AYF) Coexpression of TLR4 (A) and MyD88 (B) on CD8-positive T cells in a case of PM (Case 4, arrows). Coexpression of
TLR4 (C) and MyD88 (D) on CD8-positive T cells in a case of IBM (Case 10, arrows). Coexpression of TLR4 on CD20-positive B cells
(E, arrows) and of MyD88 on CD4-positive T cells (F, arrow) in a case of DMa (Case 14). Double immunofluorescence of rabbit
anti-human TLR4 Dylight 549 with mouse anti-human CD8 FITC (A, C) and mouse anti-human CD20 (E), respectively, and double
immunofluorescence of goat anti-human MyD88 Cy3 with mouse anti-human CD8 (B, D) and mouse anti-human CD3 (F),
respectively. Original magnification: 400.
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J Neuropathol Exp Neurol Volume 71, Number 10, October 2012 Toll-Like Receptors in Inflammatory Myopathies
numbers of immune cells. Thus, together with the identi- respective inflammatory myopathy. Our results extend data of
fication of the pathogenetically relevant cell populations as a previous study in which TLR4 expression was detected
cellular sources of TLR4, that is, CD8 T cells in PM and IBM in PM and DM by immunohistochemistry and quantitative
and B cells and CD4 T cells in DMa, respectively, this RT-PCR. The authors suggested that TLR4 is involved in the
observation may indicate a potential role for TLR4 in the immunopathogenesis of PM and DM because of a possible
pathogenesis of these inflammatory myopathies. Furthermore, connection with CD4-positive T cell activation (37); however,
TLR4 correlated with disease activity in inflammatory myo- its cellular sources still remained to be determined. Toll-like
pathies and may, in this regard, be similar to rheumatoid receptor 4 activation leads to activation of 2 different signaling
arthritis, in which TLR4 levels in the synovial tissue corre- pathways: the MyD88-dependent and the TRIF-dependent
lated with disease activity (33). pathway (38, 39). Transcription of MyD88 mRNA and ex-
The TLRs are also involved in driving autoimmune in- pression of MyD88 protein, the central adapter protein in TLR
flammation via acting directly on T cells to induce autoimmunity signaling (40), were upregulated in all inflammatory myo-
(34Y36). Interestingly, cell populations expressing TLR4 pathies, most prominently in PM, IBM, and DMa, suggesting
depend on the type of the underlying IIM, that is, CD8-- activation of this signaling pathway in all IIMs. Furthermore,
positive T cells in PM and IBM and CD20-positive B cells inflammatory infiltrates in PM, IBM, DMa, and IMV ex-
and CD4-positive T cells in DMa. Therefore, TLR4 is pressed the costimulatory molecules CD80 and CD86. This
expressed by pathogenetically relevant cell populations in the observation provides indirect evidence for activation of the
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Brunn et al J Neuropathol Exp Neurol Volume 71, Number 10, October 2012
FIGURE 7. Expression of the costimulatory molecules CD80 and CD86 in inflammatory infiltrates of PM, IBM, DMa, and IMV.
(AYH) The vast majority of inflammatory cells express CD80 (AYD) and CD86 (EYH) in PM (Case 4, A, E), IBM (Case 10, B, F),
DMa (Case 14, C, G), and IMV (Case 18, D, H). Immunohistochemistry with anti-CD80 and anti-CD86 and slight counterstain
with hemalum. Original magnification: 400.
TRIF-dependent pathway, which results from upregulation of increased transcription of IFN-F, IL-12p40, and IL-17 mRNA
costimulatory molecules (e.g. CD80 and CD86 or CD40) in in PM, IBM, and DMa, which may result from MyD88-
response to TLR4 activation (38). Functional activation of dependent, as well as MyD88-independent, TLR signaling,
the NF-JB pathway is further evidenced by a significantly ultimately contributing to a proinflammatory milieu characterized
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J Neuropathol Exp Neurol Volume 71, Number 10, October 2012 Toll-Like Receptors in Inflammatory Myopathies
by increased IL-12p40 expression associated with increased IFN- impact on T-helper cell differentiation. A study on human blood
F expression (7, 9, 10). Although early NF-JB activation via the mononuclear cells revealed high levels of the CXC chemokine
MyD88-dependent and late NF-JB activation via the TRIF- IP-10, a chemoattractant for TH1 cells, after TLR4 stimulation,
dependent pathway are suggested to underlie increased pro- resulting in an increased recruitment of Th1 cells, therefore,
duction of IFN-F, IL-12p40, and IL-17, however, the alternative indicating that TLR signaling contributes to a polarization of the
MyD88-independent signaling cascade via IRF3 resulting in adaptive immune response toward a TH1 milieu (42). Our data
increased production of IFN-A seemed not to be activated suggest a central role for TLR, in particular, TLR4, in the
because mRNA transcripts of IRF3 and IFN-A were not sig- orchestration of the immune response, fostering a TH1 response
nificantly increased. Interestingly, a previous study revealed with IL-12 and IFN-F production. Thus, inflammatory diseases
significant upregulation of type I IFNYdependent transcripts by of skeletal muscle share similarities with cardiac muscle, as
gene expression profiling, especially in juvenile DM muscle Eriksson et al (43) and Gonella et al (44) have defined a central
biopsies (41); however, the present study was confined to role for TLR4 in the development of autoimmune myocarditis
patients with adult DM. Although these data are strong indica- by activation of NF-JB via directing a TH1 response with pro-
tors of an active and efficient NF-JB signaling, this conclusion duction of IFN-F preceded by increased IL-12 production. These
formally requires electrophoretic nuclear shift assays to demon- data may encourage efforts to develop therapeutic strategies
strate nuclear translocation of NF-JB gene products, which toward modulation of TLRs and their downstream signaling
unfortunately was precluded in this retrospective study because molecules. Toll-like receptor 4, an antagonistic antibody (N1-
of limitation of material. Toll-like receptor 4 is known for its 0101) that interferes with TLR4 dimerization, has been tested for
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Brunn et al J Neuropathol Exp Neurol Volume 71, Number 10, October 2012
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