2012 TLRs en Células de Infiltrados de Miositis

J Neuropathol Exp Neurol Vol. 71, No.
10
Copyright Ó 2012 by the American Association of Neuropathologists, Inc. October 2012
pp. 855Y867
ORIGINAL ARTICLE
Toll-Like Receptors Promote Inflammation in Idiopathic

Inflammatory Myopathies
Anna Brunn, MD, Katja Zornbach, BSc, Volkmar H. Hans, MD, Walter F. Haupt, MD,
and Martina Deckert, MD
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-
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lation of immune responses in idiopathic inflammatory myopathies are leukin 12p40 (IL-12p40) (3). Toll-like receptor 4 also signals
unclear. We investigated these pathways in muscle biopsies from 5 cases via a second pathway with Toll-IL-1 receptor (TIR) domainY
each of polymyositis, inclusion body myositis, dermatomyositis, vasculi- containing adaptorYinducing IFN-F (TRIF) as a central adaptor
tis-associated interstitial myositis, and noninflammatory neurogenic atro- molecule. Its activation may result in NF-JB activation or, via
phy. Toll-like receptor 2, TLR4, TLR9, and MyD88 mRNA transcripts IRF3, in activation of type I interferons (4Y6). The NF-JB
and protein expression were increased in all subtypes of idiopathic signaling pathway is the major regulator of innate and adap-
inflammatory myopathies. Upregulation of MyD88 was associated with tive immunity, proliferation, and cell death (7). Whereas the
increased mRNA levels of interferon-F, interleukin 12p40, and inter- MyD88-dependent pathway exclusively induces the expres-
leukin 17, suggesting NF-JB activation via the MyD88-dependent sion of proinflammatory cytokines by activation of NF-JB, the
pathway in early stages. The costimulatory molecules CD80 and CD86 MyD88-independent, TRIF-dependent pathway induces expres-
were expressed on inflammatory infiltrates in idiopathic inflammatory sion of IFN-A1 via activation of IRF3. A further TIR domainY
myopathies and may additionally contribute to activation of the MyD88- containing adaptor, TRAM, which is TRIF associated and
independent pathway, leading to nuclear factor-JB activation in late specific for the TLR4-mediated MyD88-independent pathway,
stages. Our data suggest that nuclear factor-JB activation via both the results in late NF-JB activation, with increased expression of
MyD88-dependent and the MyD88-independent pathways contributes proinflammatory cytokines (8). Translocation of NF-JB from
to the proinflammatory milieu in idiopathic inflammatory myopathies. the cytoplasm into the nucleus is a hallmark of NF-JB activa-
tion. Nuclear factor-JB is a ubiquitous transcription factor
Key Words: Dermatomyositis, Inclusion body myositis, MyD88,
formed by different proteins of the Rel family, including the
NF-JB, Polymyositis, Toll-like receptors.
Rel-like domainYcontaining protein REL/p65 (RelA) and REL
(c-Rel). The RelA is one of the most abundant Rel proteins.
The c-Rel is required for the expression of the p40 subunit
INTRODUCTION of IL-12 by activated macrophages (9) and might, therefore,
Toll-like receptors (TLRs) are pattern-recognition recep- additionally contribute to an enhanced expression of IFN-F
tors of the innate immune system that recognize conserved (10) (Fig. 1).
microbial motifs, that is, peptidoglycan (recognized by TLR2), In the central nervous system, microglia, astrocytes,
lipopolysaccharide (recognized by TLR4), CpG DNA (recog- oligodendrocytes, and neurons express TLRs (11Y14); their
nized by TLR9), and viral RNA (recognized by TLR3, TLR7, function has been studied in both infections and autoimmune
and TLR8) (Fig. 1); they also play a role in adaptive immunity inflammation (13, 15Y18), revealing their essential function
(1). They are expressed by effector cells of the innate and of induction, development, and maintenance of autoimmune
adaptive immune system, that is, macrophages, granulocytes, inflammation (18). In the peripheral nervous system, TLRs are
expressed on Schwann cells (19Y22). However, their expres-
From the Department of Neuropathology (AB, KZ, MD), University of sion pattern in skeletal muscle and their roles particularly with
Cologne, Cologne; Department of Neuropathology (VHH), Evangelisches respect to their contributions to autoimmune inflammation in
Krankenhaus Bielefeld, Bielefeld; and Department of Neurology (WFH),
University of Cologne, Cologne, Germany. idiopathic inflammatory myopathies (IIMs) are largely un-
Supported by Else Kröner-Fresenius-Stiftung (2010/A139). known. Understanding their functional roles may, accordingly,
Send correspondence and reprint requests to: Anna Brunn, MD, Department help establish specific therapeutic strategies.
of Neuropathology, University Hospital of Cologne, Kerpener Str. 62, The aim of this study was to investigate the cell type-
D-50924 Köln, Germany; E-mail: [email protected]
Supplemental digital content is available for this article. Direct URL citations
specific expression pattern of TLR2, TLR4, and TLR9 in the
appear in the printed text and are provided in the HTML and PDF versions 3 most common IIMs, polymyositis (PM), inclusion body
of this article on the journal’s Web site (www.jneuropath.com). myositis (IBM), and dermatomyositis (DM). These are severe,
J Neuropathol Exp Neurol Volume 71, Number 10, October 2012 855
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
Here, we show that TLR4 and MyD88 are upregulated

in cases of PM, IBM, and DM. Together with increased levels
of RelA, Rel, IL-12p40, and IFN-F mRNA, these data suggest
a role for TLR in activation of the NF-JB signaling pathway
in IIM.
MATERIALS AND METHODS

Patients
Archival muscle biopsy samples were obtained from
15 patients with acute IIM, that is, 5 patients each with PM,
IBM, and DM. Acute IIM was clinically evident by a rapid
onset of disease with severe myalgia and increased C-reactive
protein and/or erythrocyte sedimentation rate. In addition,
muscle specimens were obtained from 5 patients with inter-
stitial myositis (IM) as part of systemic vasculitis (vasculitis
associated with IM [IMV]). Morphologically, all muscle speci-
mens harbored at least 10 high-power fields (HPFs) in which

at least 20 leukocytes were present. Five muscle specimens
from patients with mild neurogenic muscular atrophy served
as controls. The diagnosis in all IIM cases was based on
neurological and electrophysiologic examination, with typical
electromyographic alterations, including low amplitude, patho-
logic spontaneous activity like fibrillations, and/or repetitive
discharge indicative of myositis. The diagnoses were confirmed
by morphologic analysis. Additional criteria for inclusion in
the present study were a comprehensive documentation of
the clinical course and the findings of comparable numbers
of inflammatory infiltrates within the biopsy samples. Clinical
data are presented in Table 1.
FIGURE 1. Toll-like receptor 2, TLR4, and TLR9 signaling path- In DM, 2 stages were distinguished, that is, an active
ways. Toll-like receptor 2, TLR4, and TLR9 signal via the adaptor (DMa) and an inactive stage (DMi), defined as DM with or
protein MyD88. In addition, TLR2 and TLR4 signal through the without B cells, respectively, which is in accordance with
MyD88 adaptorYlike/TIR-associated protein (MAL/TIRAP) pathway.
other groups (24Y26). Thus, 3 of the 5 DM cases (i.e. No. 11,
Both signaling pathways share the protein kinases IRAK4 and
TRAF6 ultimately activating the IKK complex (IJB). The IJB pro- No. 13, and No. 14; 60%) were classified as DMa; 2 of the
teolysis leads to activation and nuclear translocation of NF-JB. 5 (No. 12 and No. 15; 40%) were classified as DMi. Three
Toll-like receptor 4 also signals via the adaptor proteins TRIF and patients (No. 2 PM, No. 10 IBM, and No. 14 DMa) were
TRAM. The TRAM pathway requires costimulatory molecules, treated with corticosteroids before biopsy. Two patients (cases
such as CD80 and CD86, to stimulate NF-JB during late stages 2 and 10) were treated with oral corticosteroids for 1 day, that
of activation. Simultaneously, TRIF upregulates IRF3, resulting in is, on the day before biopsy. In patient 14, oral corticosteroids
increased expression of IFN-A1. CpG, CpG-oligodeoxynucleotides were administered for the last 3 days preceding biopsy.
recognized by TLR9; PGN, peptidoglycan recognized by TLR2; Muscle specimens obtained from these patients also fulfilled
LPS, lipopolysaccharide recognized by TLR4. all criteria required for inclusion in this study. Other anti-
inflammatory agents were not administered.
disabling, often acute diseases characterized predominantly Neuropathology

by proximal weakness, pain, and increased levels of serum Muscle specimens were snap-frozen in isopentane
creatine kinase. The diagnosis of these IIMs is based on (Fluka, Neu-Ulm, Germany), precooled in liquid N2 immedi-
clinical and electrophysiological criteria, combined with his- ately after biopsy and stored at -80-C. Nine-micrometer-thick
topathologic and immunohistochemical analysis of muscle serial frozen sections were fixed in acetone and chloroform.
biopsies (23). In PM and IBM, CD8-positive cytotoxic T cells First and last sections of every case were stained with hem-
invade non-necrotic muscle fibers that express major histo- atoxylin and eosin to ensure the presence of inflammatory
compatibility complex class I antigens on their surface. In infiltrates in all cases (Table 1), as well as the presence of
DM, CD4-positive T cells, B cells, and plasmacytoid den- typical morphological hallmarks of PM (Figure, Supplemental
dritic cells form predominantly perivascular infiltrates, and Digital Content 1, part A, http://links.lww.com/NEN/A384),
damaged endomysial capillaries and perifascicular atrophy are IBM http://links.lww.com/NEN/A384), (Figure, Supplemental
characteristic. Digital Content 1, part B, http://links.lww.com/NEN/A384),
856 Ó 2012 American Association of Neuropathologists, Inc.
J Neuropathol Exp Neurol Volume 71, Number 10, October 2012 Toll-Like Receptors in Inflammatory Myopathies
TABLE 1. Clinical Data

Case No. Diagnosis Sex Age at Onset, years Muscle Corticosteroids Before Biopsy CK No.
1 PM F 82 Quadriceps No n.a.
2 PM* F 86 Quadriceps Yes n.a.
3 PM F 79 Deltoid n.a. n.a.
4 PM F 61 Quadriceps No n.a.
5 PM F 72 Biceps brachii No n.a.
6 IBM M 84 Deltoid No G170 U/L
7 IBM F 63 Quadriceps No 957 U/L
8 IBM M 72 Biceps brachii No G150 U/L
9 IBM M 64 Quadriceps No 2,787 U/L
10 IBM* F 69 Anterior tibial Yes 2,830 U/L
11 DMa F 71 n.a. n.a. 1,200 U/L
12 DMi F 54 Quadriceps n.a. n.a.
13 DMa F 62 Quadriceps No 1,000 U/L
14 DMa* F 37 Biceps brachii Yes 1,500 U/L
15 DMi F 68 Biceps brachii n.a. 229 U/L
16 IMV M 71 Quadriceps n.a. 11,900 U/L

17 IMV M 32 Quadriceps No G170 U/L
18 IMV M 76 Anterior tibial No G170 U/L
19 IMV M 73 Biceps brachii No G170 U/L
20 IMV M 68 Deltoid No 6,490 U/L
21 Neurogenic MA M 68 Quadriceps No G150 U/L
22 Neurogenic MA F 59 Quadriceps No G170 U/L
23 Neurogenic MA F 40 Quadriceps No G170 U/L
24 Neurogenic MA M 68 Quadriceps No 365 U/L
25 Neurogenic MA M 62 Quadriceps No G150 U/L
*Patients treated with corticosteroids; Case 2 and Case 10 for 1 day on the day before biopsy; Case 14 for 3 days before biopsy.
CK, creatine kinase; n.a., information not available; neurogenic MA, neurogenic muscular atrophy.
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
Ó 2012 American Association of Neuropathologists, Inc. 857
Whitney U test was applied. Values of p G 0.05 were considered

TABLE 2. Primary Antibodies Used for Immunohistochemistry significant. Statistics was calculated with the software GraphPad
and Immunofluorescence
Prism 5.04 (GraphPad Software, Inc, CA).
Antibody To Clone Species Dilution Company
CD80 2B11 Mouse 1:100 Antikoerper-online* RESULTS
CD86 EP1158Y Rabbit 1:100 Abcam†
CD45 2B11 + PD7/26 Mouse 1:100 Dako‡
TLRs and MyD88 Are Induced in Inflammatory
CD3 F7.2.38 Mouse 1:100 Dako Myopathies
CD4 4B12 Mouse 1:20 BioGenex§ Toll-like receptor 2, TLR4, TLR9, and MyD88 mRNA
CD8 C8/144B Mouse 1:20 Covancek was induced in all IIMs (Fig. 2). Toll-like receptor 4 and MyD88
CD20 L26 Mouse 1:250 Dako mRNA levels in PM, DMa, and IMV differed significantly from
CD68 KP1 Mouse 1:100 Dako neurogenic muscular atrophy in which TLR2, TLR4, TLR9, and
MHC class I W6/32 Mouse 1:100 Dako MyD88 mRNA was not transcribed (Figure, Supplemental Dig-
C5b-9 aE11 Mouse 1:50 Dako ital Content 1, http://links.lww.com/NEN/A384, and Figure, Sup-
TLR2 Polyclonal Rabbit 1:500 Sigma plemental Digital Content 2, http://links.lww.com/NEN/A386;
TLR4 Polyclonal Rabbit 1:500 Sigma p G 0.05). In addition, TLR2, TLR4, TLR9, and MyD88 protein
TLR9 Polyclonal Rabbit 1:150 Sigma was expressed in IIM (Table 4; Fig. 3AYP). Even though 1
MyD88 Polyclonal Goat 1:250 Sigma patient of each group (i.e. PM, IBM, and DMa) was treated for
brief durations with corticosteroids before biopsy, mRNA
*Antikoerper-online, Aachen, Germany.
expression levels of TLR4 and MyD88 in the individual cases

†Abcam, Cambridge, UK.
‡Dako, Glostrup, Denmark. showed differences of p G 0.05 and mean values with low SDs,
§Biogenex, San Ramon, CA. which indicate only minor variation among individual patients
kCovance, Princeton, NJ.
C5b-9, terminal complement complex C5b-9; MHC, major histocompatibility (Fig. 2). The TLR4 and MyD88 mRNA transcripts were sig-
complex. nificantly greater in PM, IBM, and DMa versus DMi (p G 0.05).
In IMV, TLR4 mRNA was significantly lower as compared
with PM, IBM, and DMa (p G 0.05). The cellular sources of
TLR2, TLR4, TLR9, and MyD88 included CD8-positive and
CD4-positive T cells, CD20-positive B cells, and CD68-pos-
used for double labeling immunofluorescence and protocols are itive macrophages, but in all IIMs, CD68-positive macrophages
detailed in Tables 2 and 3. seemed to be the major cellular sources (Fig. 4AYH). In PM
(Fig. 5AYC) and IBM (Fig. 5D-F), endomysial and occasion-
Statistical Analysis ally sarcoplasmic CD8-positive T cells in non-necrotic muscle
To compare the expression pattern of the different TLRs fibers also expressed TLR4 and MyD88, whereas in DMa,
and MyD88 on lymphocytes and macrophages, as well as CD20-positive B cells and CD4-positive T cells also con-
the extent of inflammatory infiltrates between the various tributed to TLR4 and MyD88 protein expression (Fig. 5G-I).
inflammatory myopathies, a semiquantitative grading system Therefore, PM, IBM, and DMa are characterized by a differ-
was used as follows: 0, absence of leukocytes and TLR/MyD88 ential expression of TLR4 and MyD88 mRNA and protein.
protein expression; +, less than 5 CD45-positive cells in 1 HPF
in less than 10 HPFs with TLR/MyD88 expression; ++, less Increased Transcription of IFN-F, IL-12p40, and
than 20 CD45-positive cells in 1 HPF in more than 5 HPFs with IL-17 mRNA in Inflammatory Myopathies
TLR/MyD88 expression; +++, more than 20 CD45-positive cells To investigate the effect of TLR2, TLR4, TLR9, and
in more than 10 HPFs with TLR/MyD88 expression. To test for MyD88 on the proinflammatory milieu in inflammatory myo-
differences in the expression level of mRNA transcripts between pathies, mRNA transcripts of IFN-F, IL-6, IL-12p40, and
the inflammatory myopathies and the control samples, a Mann- IL-17, proinflammatory cytokines known to be involved in
TABLE 3. Double Immunofluorescence Staining Protocol

1. Primary Antibody to 1. Secondary Antibody Fluorescence 2. Primary Antibody 2. Secondary Antibody
CD45 Horse anti-mouse BSP Extravidin- FITC TLR2, TLR4, TLR9 Goat anti-rabbit DyLight 549 red
MHC class I Horse anti-mouse BSP Extravidin- FITC TLR2, TLR4, TLR9 Goat anti-rabbit DyLight 549 red
C5b-9 Horse anti-mouse BSP Extravidin- FITC TLR2, TLR4, TLR9 Goat anti-rabbit DyLight 549 red
MyD88 Rabbit anti-goat BSP Extravidin Cy3 LCA, CD3, CD4, CD8, CD20, CD68, Goat anti-mouse FITC
MHC class I, C5b-9
BSP, biotinylation sequence peptide; LCA, leukocyte common antigen; MHC, major histocompatibility complex; C5b-9, terminal complement sequence C5b-9.

FIGURE 2. Messenger RNA transcripts (log10-transformed) of TLR2, TLR4, TLR9, and MyD88 in muscle biopsies of PM (A), IBM
(B), DMa and DMi (C), and IMV (D). Muscle biopsies of 5 cases of neurogenic muscular atrophy served as control (value
log10-transformed = 0). Mean T SD of 5 muscle biopsies each of PM, IBM, and IMV, 3 cases of DMa, and 2 cases of DMi are shown.
* p G 0.05 versus controls.
the activation pathway of TLRs during inflammation (31, 32),

were determined in the muscle biopsies. In PM, IBM, and
DMa, levels of IFN-F, IL-12p40, and IL-17 mRNA tran- TABLE 4. Expression of TLR2, TLR4, TLR9, and MyD88
scripts were significantly induced versus controls (Fig. 6AYC, in Myositis and Neurogenic Muscular Atrophy
p G 0.05), as well as versus DMi and IMV (Fig. 6AYD, p G TLR2 TLR4 TLR9 MyD88
0.05). Correspondingly, in DMi and IMV, IFN-F, IL-12p40, PM ++ +++ ++ +++
and IL-17 mRNA transcripts were only slightly increased IBM + +++ + +++
versus controls (Fig. 6C, D). The IL-6 mRNA was sig- DMa ++ +++ ++ +++
nificantly induced in DMa but not in PM, IBM, DMi, and DMi + + + +
IMV versus controls (Fig. 6AYD). The mRNA of the anti- IMV ++ +++ + +++
inflammatory cytokine IL-4 was not significantly induced in Neurogenic MA 0 0 0 0
any of the IIMs (Fig. 6AYD).
0, absence of leukocytes and TLR/MyD88 protein expression; +, less than 5 CD45-
positive cells in 1 HPF in less than 10 HPFs with TLR/MyD88 expression; ++, less than
In Inflammatory Myopathies, Leukocytes 20 CD45-positive cells in 1 HPF in more than 5 HPFs with TLR/MyD88 expression;
Express CD80 and CD86 +++, more than 20 CD45-positive cells in more than 10 HPFs with TLR/MyD88
expression.
Most of the inflammatory infiltrates in IIMs expressed Neurogenic MA, neurogenic muscular atrophy.
the costimulatory molecules CD80 and CD86 (Fig. 7AYH),

FIGURE 3. Expression of TLR2, TLR4, TLR9, and MyD88 in PM, IBM, DMa, and IMV. (AYP) In PM (Case 4, AYD), IBM (Case 10,
EYH), DMa (Case 14, IYL), and IMV (Case 18, MYP), all TLRs and MyD88 are strongly expressed on leukocytic infiltrates (arrows).
Immunofluorescence with rabbit anti-human TLR2 with goat anti-rabbit DyLight 549 red (A, E, I, M), rabbit anti-human TLR4 with
goat anti-rabbit DyLight 549 red (B, F, J, N), rabbit anti-human TLR9 with goat anti-rabbit DyLight 549 red (C, G, K, O), and goat
anti-human MyD88 with rabbit anti-goat biotinylation sequence peptide (BSP) and extravidin Cy3 (D, H, L, P), respectively.
Original magnification: 400.
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).
Increased Transcription of RelA and Rel DISCUSSION

Suggests Activation of NF-JB The innate immune system and, especially, TLRs and
To investigate the effect of activation of the TLR4 sig- the components of their signaling pathways play an impor-
naling cascade via the MyD88-dependent or the MyD88- tant role in autoimmune responses. This study demonstrates
independent pathway, respectively, mRNA transcripts of RelA differential expression patterns of TLR2, TLR4, TLR9, and
and Rel were determined. In PM, IBM, and DMa, levels of MyD88 in PM, IBM, DM, and IMV. In particular, TLR4
RelA and Rel mRNA transcripts were significantly induced mRNA transcripts were prominently induced and, accord-
versus controls (Fig. 8AYC, p G 0.05). In contrast, in DMi ingly, TLR4 protein was expressed in IIM. Interestingly,
and IMV, RelA and Rel mRNA transcripts were only slightly TLR4 mRNA levels were significantly increased in PM, IBM,
increased versus controls (Fig. 8C, D). The IRF3 and IFN-A and DMa versus DMi and IMV, albeit harboring comparable
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.
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.

FIGURE 6. (AYD) Messenger RNA transcripts (log10-transformed) of IFN-F, IL-6, and IL-12p40 in muscle biopsies of PM (A),
IBM (B), DMa and DMi (C), and IMV (D). Muscle biopsies of 5 neurogenic muscular atrophy cases served as controls (value
log10-transformed = 0). Mean and SD in PM, IBM, and IMV are shown. * p G 0.05 versus controls.
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
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

FIGURE 8. (AYD) Messenger RNA transcripts (log10-transformed) of RelA, Rel, IRF3, and IFN-A1 in muscle biopsies of PM (A),
IBM (B), DMa and DMi (C), and IMV (D). Muscle biopsies of 5 neurogenic muscular atrophy cases served as control (value
log10-transformed = 0). Data show mean and SD in PM, IBM, and IMV. * p G 0.05 versus controls.
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
treatment of inflammatory bowel disease and seems to reduce 16. Prinz M, Garbe F, Schmidt H, et al. Innate immunity mediated by TLR9
inflammatory activity (45). This is in line with observations in a modulates pathogenicity in an animal model of multiple sclerosis. J Clin
Invest 2006;116:456Y64
murine model of dextran sulfate sodiumYinduced colitis in 17. Marta M, Andersson A, Isaksson M, et al. Unexpected regulatory roles of
which treatment with a TLR4 monoclonal antibody significantly TLR4 and TLR9 in experimental autoimmune encephalomyelitis. Eur J
decreased disease activity as well as severity of inflammation as Immunol 2008;38:565Y75
assessed with morphology (46). In addition, therapeutic modu- 18. Miranda-Hernandez S, Gerlach N, Fletcher JM, et al. Role for MyD88,
TLR2 and TLR9 but not TLR1, TLR4 or TLR6 in experimental auto-
lation of the TLR4 response in murine models of RA sig- immune encephalomyelitis. J Immunol 2011;187:791Y804
nificantly abated disease activity by reducing activation of 19. Cheng C, Qin Y, Shao X, et al. Induction of TNF-alpha by LPS in
inflammatory cells, especially macrophages, during the time Schwann cell is regulated by MAPK activation signals. Cell Mol Neu-
window when TLR4 ligands activate inflammatory macro- robiol 2007;27:909Y21
phages downstream of TLR activation (47, 48). 20. Lee H, Jo EK, Choi SY, et al. Necrotic neuronal cells induce inflamma-
tory Schwann cell activation via TLR2 and TLR3: Implication in Wal-
In summary, our results suggest that TLR4 is a cru- lerian degeneration. Biochem Biophys Res Commun 2006;350:742Y47
cial receptor of the innate immune system in the immunopa- 21. Oliveira RB, Ochoa MT, Sieling PA, et al. Expression of Toll-like
thogenesis of PM, IBM, and DM via activation of both receptor 2 on human Schwann cells: A mechanism of nerve damage in
the classical MyD88Ydependent signaling pathway and the leprosy. Infect Immun 2003;71:1427Y33
22. Goethals S, Ydens E, Timmerman V, et al. Toll-like receptor expression
MyD88-independent, TRIF/TRAM-dependent signaling path- in the peripheral nerve. Glia 2010;58:1701Y9
way, thereby activating the NF-JB pathway with increased 23. Dalakas MC. Polymyositis, dermatomyositis and inclusion-body myosi-
production of IL-12p40, and finally resulting in enhanced pro- tis. N Engl J Med 1991;325:1487Y98
duction of the potent proinflammatory cytokine IFN-F. 24. Grundtman C, Malmstrom V, Lundberg IE. Immune mechanisms in the

pathogenesis of idiopathic inflammatory myopathies. Arthritis Res Ther
2007;9:208
ACKNOWLEDGMENTS 25. Ishii W, Matsuda M, Shimojima Y, et al. Flow cytometric analysis of
The authors thank Mariana Carstov and Elena Fischer lymphocyte subpopulations and TH1/TH2 balance in patients with pol-
ymyositis and dermatomyositis. Intern Med 2008;47:1593Y99
for expert technical assistance. 26. Aleksza M, Szegedi A, Antal-Szalmas P, et al. Altered cytokine expres-
sion of peripheral blood lymphocytes in polymyositis and dermatomyo-
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2012 TLRs en Células de Infiltrados de Miositis

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J Neuropathol Exp Neurol Vol. 71, No.

Toll-Like Receptors Promote Inflammation in Idiopathic

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Here, we show that TLR4 and MyD88 are upregulated

MATERIALS AND METHODS

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disabling, often acute diseases characterized predominantly Neuropathology

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TABLE 1. Clinical Data

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Ó 2012 American Association of Neuropathologists, Inc. 857

Whitney U test was applied. Values of p G 0.05 were considered

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TABLE 3. Double Immunoﬂuorescence Staining Protocol

858 Ó 2012 American Association of Neuropathologists, Inc.

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the activation pathway of TLRs during inﬂammation (31, 32),

Ó 2012 American Association of Neuropathologists, Inc. 859

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Increased Transcription of RelA and Rel DISCUSSION

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862 Ó 2012 American Association of Neuropathologists, Inc.

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864 Ó 2012 American Association of Neuropathologists, Inc.

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Ó 2012 American Association of Neuropathologists, Inc. 865

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