Molecular mechanisms of autoimmunity triggered by microbial infection.

Abstract

Autoimmunity can be triggered by microbial infection. In this context, the discovery of Toll-like receptors (TLRs) provides new insights and research perspectives. TLRs induce innate and adaptive antimicrobial immune responses upon exposure to common pathogen-associated molecules, including lipopeptides, lipopolysaccharides, and nucleic acids. They also have the potential, however, to trigger autoimmune disease, as has been revealed by an increasing number of experimental reports. This review summarizes important facts about TLR biology, available data on their role in autoimmunity, and potential consequences for the management of patients with autoimmune disease.

Full text

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CpG = unmethylated cytosine-guanosine; ds = double stranded; IFN = interferon; IL = interleukin; LFA = lymphocyte function associated antigen;
LPS = lipopolysaccharide; MHC = major histocompatibility complex; MyD88 = myeloid differentiation (primary-response) factor 88; NF = nuclear
factor; ODN = oligodeoxyribonucleotide; PAMP = pathogen-associated molecular pattern; ss = single stranded; Th = helper T cell; TIR = Toll-IL-1
receptor; TLR = Toll-like receptor.
Available online http://arthritis-research.com/content/7/5/215
Abstract
Autoimmunity can be triggered by microbial infection. In this
context, the discovery of Toll-like receptors (TLRs) provides new
insights and research perspectives. TLRs induce innate and
adaptive antimicrobial immune responses upon exposure to
common pathogen-associated molecules, including lipopeptides,
lipopolysaccharides, and nucleic acids. They also have the
potential, however, to trigger autoimmune disease, as has been
revealed by an increasing number of experimental reports. This
review summarizes important facts about TLR biology, available
data on their role in autoimmunity, and potential consequences for
the management of patients with autoimmune disease.
Introduction
Autoimmunity is believed to develop from genetic
predispositions while the onset of autoimmune tissue injury or
disease flare is often triggered by microbial infection.
Diseases such as type I diabetes mellitus, lupus erythema-
tosus, myocarditis, rheumatoid arthritis, and multiple sclerosis
often manifest themselves in association with microbial
infection. Patients with chronic forms of autoimmunity may
experience symptomatic disease flares following infections.
These clinical observations raise a set of questions: what
classes of receptors recognize microbes or vaccine adjuvants
in the host? What molecular mechanisms induce immune
activation upon recognition of the pathogen? And how does
antimicrobial immunity modulate tolerance? Answers to these
questions are expected from the recent discovery of Toll-like
receptors (TLRs). TLRs have been identified as a new family
of innate receptors that recognize a set of microbial
molecules known as pathogen-associated molecular patterns
(PAMPs). The role of TLRs in antimicrobial immunity
continues to be extensively studied. However, an increasing
number of reports provide evidence that TLR ligation can
trigger autoimmune tissue injury. In this review, we summarize
important facts on TLR biology and available data on their
role in autoimmunity. Furthermore, we provide future research
perspectives that could influence the management of patients
with autoimmune disease.
TLRs recognize pathogen-associated
molecular patterns
To date, 11 proteins related to the Drosophila gene Toll have
been characterized in vertebrates [1] (Figure 1; Table 1).
TLR2, TLR1 and TLR6
TLR2 recognizes several different ligands (Table 1). It
responds to lipoproteins, which are the main cell wall
components of Gram-positive bacteria [2,3], and hetero-
dimerizes with TLR1 and TLR6, which enables discrimination
between diacetylated and triacetylated lipopeptides in human
monocytes [4]. TLR1-TLR2 heterodimerization activates
dendritic cells, B cells, NK cells, mast cells, and keratinocytes
[1]. TLR2 and TLR6 collaborate in detecting yeast zymosan
[1]. In addition, components of necrotic, but not apoptotic,
cells activate fibroblasts and macrophages via TLR2 [5].
Such endogenous ligands may play a role in both bacterial as
well as aseptic arthritis [6-8]. Studies that suggest TLR
recognition of self proteins such as heat shock protein-70
have been questioned, however, as such preparations may
have been contaminated by other TLR ligands [9].
TLR3
TLR3 is a detector of double-stranded (ds)RNA that may
originate from single-stranded (ss)RNA or dsRNA viruses
[10,11]. It is believed that TLR3 recognizes secondary RNA
structures as synthetic RNAs, mRNA, and siRNA similarly
induce the production of type I IFNs and pro-inflammatory
Review
Molecular mechanisms of autoimmunity triggered by microbial
infection
Hans-Joachim Anders, Daniel Zecher, Rahul D Pawar and Prashant S Patole
Medical Policlinic, Ludwig-Maximilians-University, Munich, Germany
Corresponding author: Hans-Joachim Anders, hjanders@med.uni-muenchen.de
Published: 30 August 2005
This article is online at http://arthritis-research.com/content/7/5/215
© 2005 BioMed Central Ltd
Arthritis Research & Therapy 2005, 7:215-224 (DOI 10.1186/ar1818)

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Arthritis Research & Therapy October 2005 Vol 7 No 5Anders et al.
cytokines. Viral dsRNA induces dendritic cell maturation
through TLR3 [10]. Apparently, viral RNA can act as a natural
adjuvant promoting loss of tolerance against presented
endogenous or exogenous antigens and that modulates the T
helper cell (Th)1/Th2 balance of the subsequent T cell
response. Among the monocytic immune cell subsets, TLR3
is expressed on murine macrophages; whereas in humans,
TLR3 is exclusively expressed on myeloid dendritic cells
[10,12,13]. In addition, TLR3 is reported to be expressed on
an increasing number of non-immune cells, including
glomerular mesangial cells [14], astrocytes [15], uterine
epithelial cells [16], and fibroblasts [17]. These cells express
TLR3 constitutively at low levels and upregulate TLR3 upon
exposure to dsRNA or other TLR ligands. Most cell types
express TLR3 in an endosomal compartment, which supports
the idea that viruses need to be processed before their RNA
can be exposed to TLR3. Fibroblasts have been reported,
however, to express TLR3 also on their outer surface
membrane [17].
TLR4
TLR4 is a critical component of the lipopolysaccharide (LPS)
receptor complex that activates cells upon exposure to Gram-
negative bacteria. However, it also responds to other ligands
(Table 1). Endotoxic shock is mediated by TLR4, which
induces the release of pro-inflammatory cytokines and
chemokines from immune and non-immune cells. For
example, lack of appropriate TLR4 signalling can predispose
to septicemia in patients with rheumatoid arthritis treated with
an anti-tumour necrosis factor regimen [18], but functional
TLR4 does not predispose to rheumatoid arthritis per se [19].
TLR4 is required for neutrophil sequestration during
endotoxin-induced lung injury [20]. In 1998, a point mutation
in the gene encoding TLR4 was found to be the molecular
basis of LPS hyporesponsiveness in C3H/HeJ and
C57BL/10ScCr mice, the latter having a chromosomal defect
that leads to a loss of the Tlr4 gene [21]. Mice belonging to
these strains show a similar phenotype to mice with target
deletions of the Tlr4 gene. In humans, TLR4 mutations are
Figure 1
Toll-like receptor (TLR) signalling. TLRs recognize pathogen-associated molecules through homophilic or heterophilic interactions. Nucleic-acid-
specific TLRs localize in intracellular endosomes. Specificity of TLR signalling is provided by a group of cytosolic adaptor molecules redistributing
to the intracellular Toll-IL-1 receptor (TIR) domain upon activation. CD14, cluster of differentation (Cd) 14 antigen, a surface protein preferentially
expressed on monocytes/macrophages that binds to LPS-binding protein; CpG, unmethylated cytosine-guanosine; ds, double stranded; LPS,
lipopolysaccharide; MD-2, myeloid differentiation protein-2; MyD88, myeloid differentiation (primary-response) factor 88; ss, single stranded;
TIRAP, TIR-domain-containing adaptor protein; TRAM, TRIF-related adaptor molecule; TRIF, TIR domain-containing adaptor protein-inducing-
interferon beta; UPEC, uropathogenic Escherichia coli.

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also associated with impaired responsiveness to LPS, but
lack of TLR4 in humans does not affect the outcome of
bacterial sepsis [22,23]. Reports showing that endogenous
molecules act through TLR4 have been questioned as
studies using low-endotoxin preparations of such molecules
failed to confirm these data (reviewed in [9]).
TLR5
The only known ligand for TLR5 is bacterial flagellin present
in both Gram-positive and Gram-negative bacteria [24]. TLR5
induces maturation of human but not murine dendritic cells
[25]. In addition to immune cells, TLR5 is also expressed on
the basolateral, but not on the apical, membrane of human
intestinal epithelial cells. This finding supports the hypothesis
that TLR5 recognizes flagellin on the surface of entero-
pathogenic bacteria, such as Salmonella, that can translocate
across epithelia in contrast to commensual bacteria such as
Escherichia coli [26].
TLR7 and TLR8
Similar to TLR3, TLR7, TLR8, and TLR9 are located
intracellularly in endosomes and recognize phagocytosed
ligands [27,28]. TLR7 and TLR8 both recognize viral ssRNA
as well as distinct synthetic guanosine analogs [1,29]. Both
can activate dendritic cells to mature and produce pro-
inflammatory cytokines [29].
TLR9
Unmethylated
important ligand for TLR9 [30]. The CpG dinucleotide is the
stimulatory motif of bacterial and viral DNA [31]. CpG-DNA is
a B cell mitogen and a strong activator of plasmacytoid
dendritic cells in humans [28]. In complex with other proteins
it induces an enhanced antigen-specific humoral and cellular
immune response of the Th1 type [32]. TLR9 resides in the
endoplasmic reticulum but redistributes to late endosomes
for the interaction with ingested CpG-DNA [27]. Various
synthetically manufactured CpG-oligodeoxynucleotides repre-
sent powerful tools for research in this field. A recent study
described plasmodia-derived hemozoin as another natural
ligand for TLR9, which raises doubt about the concept that
TLR9 recognizes specific nucleic acid sequences [33,34].
TLR9 might recognize particle-related secondary structures
rather than specific DNA sequences. This is supported by the
observation that the formation of DNA nanoparticles can
modulate TLR9 signalling towards production of high levels
of type I IFNs [35].
cytosine-guanosine (CpG)-DNA is an
TLR10 and TLR11
TLR10 is expressed on human B cells, but the ligands for
human TLR10 as well as the effects of its activation remain to
be identified [36]. TLR11 was only recently described [37].
TLR11 recognizes a yet undefined molecule of uropathogenic
Available online http://arthritis-research.com/content/7/5/215
Table 1
Important microbial ligands of Toll-like receptors
Receptor LigandPathogen
TLR1Triacyl lipopeptides Bacteria, mycobacteria
TLR2Peptidoglycan
Lipoteichoic acid
Lipoprotein, lipopetides
Atypical lipopolysaccharide
Glycoinositolphospholipids
Zymosan
Gram-positive bacteria
Gram-positive bacteria
Different pathogens
Leptospira interrogans
Trypanozoma cruzii
Fungi
TLR3Double-stranded RNAViruses
TLR4Lipopolysaccharide
Fusion protein
Taxol
Gram-negative bacteria
Respiratory syncytial virus
Plants
TLR5Flagellin Bacteria
TLR6Diacyl lipopeptides
Mycoplasma
TLR7Single-stranded RNA
Imidazoquinoline, Loxoribine, Bropirimine
Viruses
Synthetic compounds
TLR8 Single-stranded RNA
Imidazoquinoline
Viruses
Synthetic compounds
TLR9 CpG-DNA
Hemozoin
Bacteria, viruses
Plasmodium falciparum
TLR10NdNd
TLR11 Nd
Profilin-like molecule
Uropathogenic E. coli
Toxoplasma gondii
CpG, unmethylated cytosine-guanosine; nd, not determined; TLR, Toll-like receptor.

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E. coli and a profilin-like molecule of Toxoplasma gondii
[37,38]. Whether humans express TLR11 protein is un-
certain, as published TLR11 sequences include a stop codon
within the coding sequence [37].
Species-specific expression of TLRs on immune cell
subsets
Species-specific differences in TLR expression are important
when data derived from rodents are to be interpreted in the
human context. Differences between mice and humans
include: the expression of TLR3 by murine but not human
macrophages (Table 2); the expression of TLR8 by human
but not murine myeloid dendritic cells; and the expression of
TLR9 by murine but not human myeloid dendritic cells.
Specificity of TLR signalling
The specificity of TLR signalling depends on their cell type-
specific expression, the potential for heterodimerization of
certain TLRs, and on a group of cytoplasmic adaptor
molecules [1] (Figure 1). Myeloid differentiation (primary-
response) factor 88 (MyD88) was the first adaptor identified
[39]. Studies with MyD88-deficient mice revealed other
MyD88-independent signalling pathways involving the
Toll-IL-1 receptor (TIR) domain-containing adaptor molecules
TRIF (TIR domain-containing adaptor protein-inducing-
interferon beta) and TRAM (TRIF-related adaptor molecule).
MyD88 is the only adaptor molecule for TLR9, but TLR2,
TLR4, and TLR6 can use TIR domain-containing adaptor
proteins or MyD88, and TLR3 and TLR4 activation can
involve MyD88 or TRAM [39] (Figure 1). Downstream of the
adaptor molecules, TLR signalling involves members of the
IL-1 receptor-associated kinase family and IFN regulatory
factors that ultimately activate the transcription factor nuclear
factor (NF)-κB or members of the IFN-regulatory factor family
[39]. A detailed description of TLR signalling is beyond the
scope of this article, so the reader is referred to one of the
excellent in depth reviews about this issue [39]. Genetic
defects in TLR signalling cause immunodeficiency rather than
autoimmune syndromes [40], although a recent linkage
analysis study of 44 single nucleotide polymorphisms in 13
genes from the type I IFN pathway identified two poly-
morphisms in the tyrosine kinase 2 and IFN regulatory factor 5
genes that are associated with systemic lupus erythematosus
[41].
Ligating TLRs induces innate and adaptive
antimicrobial immunity
PAMPs that ligate TLRs induce potent mechanisms of innate
antimicrobial immunity. TLRs that signal through MyD88
induce NF-κB-dependent expression of pro-inflammatory
cytokines and chemokines, which trigger local and systemic
inflammation including arthritis [42]. For example, both CpG
DNA and LPS can cause a massive release of tumour
necrosis factor-α and other pro-inflammatory mediators in
mice, while TLR4-deficient and TLR9-deficient mice fail to
respond to the respective ligands [21,30]. In macrophages
and antigen-presenting cells, TLR ligation induces the
phagocytic capacity as an important component of pathogen
control and antigen-processing [43]. Viral nucleic acids ligate
a selected set of TLRs. For example, TLR3 signals through
the adaptor TRIF, which induces expression of type I IFNs, a
major component of antiviral immunity [44]. TLR9 signalling
through MyD88 by plasmacytoid dendritic cells can also
produce high amounts of type I IFNs upon recognition of
bound viral CpG-DNA [35]. Antimicrobial immunity involves
adaptive immunity, however, which also is readily activated by
TLRs [45,46]. Upon TLR ligation, antigen-presenting cells
upregulate expression of co-stimulatory molecules and
secrete modulatory cytokines. TLR ligation mostly induces
secretion of Th1 cytokines, which drives subsequent T cell
functions towards Th1-type immunity [47]. Thus, TLR ligands
are being increasingly explored as vaccine adjuvants [48,49].
Arthritis Research & Therapy October 2005 Vol 7 No 5 Anders et al.
Table 2
Toll-like receptor expression on human and mouse immune cell subsets
Monocyte/
macrophages
Plasmacytoid
dendritic cells
Myeloid
dendritic cellsReceptor B cells T cells
TLR1m, h– m, h m, h?
TLR2m, h– m, hm, h–
TLR3m–m, h––
TLR4 m, h–m, hm, h–
TLR5 m, h–m, h–?
TLR6m, h–m, h m, h–
TLR7m, h m, hm, hm, h–
TLR8––h–
TLR9– m, hm m, h–
m, mouse; h, human; TLR, Toll-like receptor.

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Many other aspects of TLR biology in antimicrobial immunity
have been described in great detail in several excellent
reviews [1,45,46,50,51]. In the following part of this review
we focus on those mechanisms that link TLR-induced
immunity to mechanisms clearly involved in autoimmunity.
TLR ligation and autoimmunity
Autoimmunity is obviously caused by several coincident
mechanisms that relate to the presence of autoreactive
immune cell subsets and loss of tolerance. Tolerance is
maintained by controlling autoreactive T and B cells as well
as by tolerogenic stimuli provided by dendritic cells that
constantly process autoantigens [52]. It is thought that
autoimmunity develops upon uncontrolled proliferation of
autoreactive immune cell subsets and non-tolerogenic
cytokine signalling by antigen-presenting cells in a host with
defects in tolerance control. Consistent with the observation
that microbial infections are common triggers of auto-
immunity, several lines of evidence show that TLR ligation can
cause autoimmune tissue injury.
Exposure to TLR ligands can induce local inflammation
Intra-articular injection of bacterial CpG-DNA causes aseptic
arthritis characterized by macrophage infiltrates in healthy
mice [53]. Similar findings were reported after intraventricular
CpG-DNA injections as a model of meningitis [54].
Macrophages produce large amounts of CC-chemokines and
express chemokine receptors on their surface upon exposure
to bacterial CpG-DNA to promote further leukocyte recruit-
ment to the site of injection [55]. Thus, aggravation of local
inflammatory tissue injury during microbial infection can be a
consequence of circulating TLR ligands activating tissue
macrophages. We traced CpG-DNA and viral dsRNA by
fluorescence labelling and detected their uptake by
glomerular macrophages present in nephritic kidneys of
MRLlpr mice with lupus nephritis [56]. As predicted, CpG-
DNA-treated mice developed a marked aggravation of lupus
nephritis characterized by strong CCL2 and CCL5
expression in inflammatory cell infiltrates. Similar results have
been obtained in other models of autoimmune tissue injury,
including collagen-induced arthritis [57] or experimental
encephalomyelitis [58]. Thus, pre-existing autoimmune tissue
injury can be exacerbated by exposure to TLR ligands via
activation of TLRs on parenchymal or infiltrating immune cells,
which leads to increased local production of pro-inflammatory
mediators. Studies with human synovial fibroblasts suggest
similar mechanisms occur in human arthritis [59].
TLR ligation modulates dendritic cell functions
Dendritic cells play a central role in coordinating both
adaptive antimicrobial immunity and control of tolerance [60].
Immature dendritic cells process microbial antigens as well
as autoantigens. In the absence of stimulatory signals,
dendritic cells remain immature, providing tolerogenic signals
to T cells [60]. In the presence of additional signals, dendritic
cells mature, upregulate co-stimulatory molecules, and
secrete cytokines, which all provide mitogenic signals to
T cells with appropriate antigen-specificity. PAMPs that ligate
TLRs on dendritic cells provide such stimulatory signals to
dendritic cells. As TLR ligands are usually in complex with
other microbial antigens, TLRs are potential adjuvant
receptors for microbial antigens. Furthermore, exposure to
microbial TLR ligands can break tolerance by activating
dendritic cells that present autoantigens and thereby induce
autoimmunity. For example, Eriksson et al. [61] showed that
dendritic cells loaded with a heart-specific self peptide
induce CD4+ T-cell-mediated myocarditis in non-transgenic
mice after transfer of dendritic cells that had been pulsed
with LPS or CpG-DNA. Similar mechanisms may contribute
to loss of tolerance to self DNA after exposure to bacterial
CpG-DNA. For example, bacterial DNA induces production of
cross-reactive DNA autoantibodies
NZB/NZW mice [62]. The latter argues for a role of TLR9
ligation by exogenous CpG-DNA in lupus induction in
genetically predisposed individuals.
in autoimmune
TLRs on dendritic cells and control of regulatory T cells
In vertebrates, autoreactive T cells are kept under tight
control by a number of mechanisms, including the
suppressive effect of regulatory T cells. Autoimmunity is
commonly associated with uncontrolled proliferation of
autoreactive T cells, which can, in part, be attributed to a
modified function of regulatory T cells. Recently, it was shown
that dendritic cells play a major role in determining the
functional state of regulatory T cells. Pasare et al. [63]
attempted to identify the critical factor among the many
cytokines that were produced by LPS or CpG-DNA-pulsed
dendritic cells that determines the functional state of
regulatory T cells. They identified IL-6 to be this factor and
showed that IL-6 can induce proliferation of autoreactive
T cells through functional blockade of CD4+/CD25+
regulatory T cells. Thus, dendritic cells exposed to microbial
PAMPs signal for proliferation of T cells specific for microbial
antigens as well as for autoreactive T cells, which may link
host defence to loss of tolerance or autoimmunity.
TLR-induced interferon production
IFN producing plasmacytoid dendritic cells play a dominant
role in antimicrobial immunity as well as in various types of
autoimmunity, including lupus erythematosus [64-66]. Among
the type I IFNs, IFN-α is a dominant mediator of autoimmune
disease activity, and is known to drive tolerance towards
autoimmunity. Nuclear particles released from ultraviolet light-
treated cells induce IFN-α production by plasmacytoid
dendritic cells [67]. Most interestingly, this response was
abolished by DNAse or RNA digestion, which indicates that
endogenous nucleic acids are major modulators of type I IFN
production [67]. Using a genetic model of type I diabetes,
Lang et al. [68] showed that upregulation of major
histocompatibility complex (MHC) I on beta islet cells but not
on exocrine pancreatic cells was dependent on interaction of
circulating IFN-α with beta cell IFN type I receptors. Ligands
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for TLR3, TLR7, and TLR8 were found to induce high levels
of IFN-α and beta cell MHC I expression in the host, leading
to diabetes, which could be neutralization of IFN-α [68].
Ligation of TLR4 or TLR9 that failed to induce IFN-α did not
cause diabetes. It was concluded that organ-specific effects
secondary to the immunostimulatory effect of systemic
exposure to microbial TLR ligands can convert T cell
autoreactivity into overt autoimmune disease. Furthermore,
IFN-α released by plasmacytoid dendritic cells modulates the
sensitivity of TLR7 on B cells to respond to TLR7 ligands,
which remains unresponsive in the absence of IFN-α [69].
TLR expression by non-immune cells can modulate
autoimmune tissue injury
From the findings discussed above, it becomes clear that
autoimmune tissue injury also relates to tissue specific
responses during exposure to microbial molecules. We have
recently identified that from all known TLRs, glomerular
mesangial cells express TLR3 at high levels in vitro and in
vivo in an endosomal compartment [14]. In cultured
glomerular mesangial cells, TLR3 ligation with viral dsRNA
induces the production of pro-inflammatory cytokines and
chemokines. Labelled dsRNA injected into MRLlpr/lprmice
with lupus nephritis was found to localize to endosomes of
glomerular mesangial cells, consistent with immunostaining
for TLR3. A course of repetitive injections with viral dsRNA
caused severe aggravation of lupus nephritis in autoimmune
MRLlpr/lpr
mice associated with increased glomerular
production of CCL2 and CCL5 and subsequent leukocytic
cell infiltrates. By contrast, no changes in serum DNA auto-
antibody levels were detected as viral dsRNA does not
induce B cell stimulation. These data indicate that TLR3 on
glomerular mesangial cells recognizes circulating viral
dsRNA, causing local inflammation in pre-existing lupus
nephritis, which may correspond to renal disease flares in
lupus patients that experience intercurrent viral infections. As
TLR3 is expressed by other non-immune cell types, including
astrocytes [15], uterine epithelial cells [16], and fibroblasts
[17], similar mechanisms may account for autoimmune tissue
injury of organs that harbour these cell types.
TLR ligands are B cell mitogens
B cells are professional antigen-presenting cells that
express TLR7 and TLR9. B cells isolated from MRLlpr/lpr
mice with lupus-like disease produce large amounts of
autoantibodies when exposed to immune complexes that
contain CpG-DNA [70]. Autoreactive B cells from these
mice recognize the IgG part of the immune complex by their
surface B cell receptor, then internalize immune complexes,
which exposes CpG-DNA to TLR9 in the endosomal
compartment [32]. These mechanisms may also apply in
vivo, because autoimmune MRLlpr/lprmice injected with
bacterial CpG-DNA produce large amounts of DNA
autoantibodies [56] in association with increased MHC II
expression on B cells isolated from spleens of these mice
[14]. The avidity of DNA autoantibodies present in MRLlpr/lpr
mice and those induced by unmethylated CpG-DNA is
comparable [71]. Systemic exposure to bacterial CpG-DNA
can induce the production of DNA autoantibodies in non-
immune mice [72]. Furthermore, CpG-DNA has a strong
adjuvant effect on DNA autoantibody production in mice
that have been exposed to vertebrate DNA [72]. These data
support the hypothesis that exposure to bacterial DNA, for
example, during bacterial infection, provides a strong signal
for enhanced DNA autoantibody production in systemic
lupus erythematosus.
Together, the cell type- and tissue-specific expression of TLR
contribute to the ligand-specific immune effects of microbes.
Viral dsRNA can promote autoimmune tissue injury through
TLR3 expressed on intrinsic parenchymal cells. Bacterial
CpG-DNA and possibly ssRNA of viral origin induce B cell
proliferation, including autoreactive B cell subsets. Further-
more, TLR ligands induce dendritic cell maturation towards a
non-tolerogenic phenotype that promotes antimicrobial
immunity as well as autoimmunity through secretion of
selected cytokines that modulate subsequent immune
responses, including the blockade of the suppressive effect
of regulatory T cells.
TLR9 and DNA recognition in lupus
As DNA particles are important autoantigens in lupus,
recognition of CpG motifs in self DNA through TLR9 might
be involved in the pathogenesis of systemic lupus erythema-
tosus. In fact, immune complexes isolated from lupus patients
can activate plasmacytoid dendritic cells to produce
cytokines, chemokines, and IFN-α through TLR9 [73].
Whether such patient-derived immune complexes contain
self-DNA or microbial DNA remains elusive [74]. CpG motifs
in self DNA need to be protected from activating TLR9 to
prevent autoimmunity. In vertebrates at least three such
mechanisms exist.
Methylation of CpG-DNA
Methylation reduces the immunostimulatory effects of
unmethylated bacterial or synthetic DNA [31,75], but in
vertebrates only 70% to 80% of CpG motifs are methylated
[76]. Hypomethylation of human DNA is associated with
autoimmunity [77]. Interestingly, ultraviolet light, hydralazine,
and procainamide, all known triggers of lupus-like syndromes,
inhibit the activity of DNA methyltransferases and induce
autoreactive T cell subsets [78]. Experimentally, DNA methy-
lation inhibitors induce lymphocyte function associated
antigen (LFA)-1 positive autoreactive T cells that mediate
DNA autoantibody production [78]. Individuals with active
systemic lupus erythematosus show decreased DNA
methyltransferase activity, lower rates of genomic methylated
cytosine nucleotides, increased levels of circulating
hypomethylated DNA, and increased numbers of autoreactive
T-cells that overexpress LFA-1 [78]. Thus, DNA methylation
maintains tolerance to self DNA, which can lead to impaired
DNA methylation associated with lupus disease activity.
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Number of CpG-motifs
Demethylating vertebrate DNA does not result in equivalent
stimulatory activity when compared to bacterial DNA [75].
Thus, additional factors exist that block the activation of
immunity by self DNA. Comparative genome analysis for the
frequency of CpG-motifs in different species revealed that
CpG motifs are present in vertebrate genomes at only 20%
of random frequency [76], but are over-represented in E. coli
DNA [75]. Possibly, during evolution, stimulatory CpG motifs
were negatively selected in vertebrate genomes and
positively selected in bacterial DNA.
Suppressive DNA sequence elements
In search of oligodeoxyribonucleotide (ODN) with optimal
stimulatory activity, several groups have detected sequence
motifs that can suppress CpG-DNA-induced immunity, for
example, in arthritis [79]. Vertebrates and bacteria show
considerably different frequencies of suppressive DNA
sequences. In mice, such suppressive DNA sequence
elements are present at a high frequency whereas these
elements are underrepresented in the E. coli genome [75].
Thus, the ratio of stimulatory and suppressive sequence
elements may determine the immunomodulatory potential of
self DNA. This would imply that human DNA has a high
number of suppressive sequence elements that neutralize a
small number of unmethylated CpG motifs, representing a
mechanism to discriminate self DNA from bacterial DNA via
TLR9.
If recognition of self DNA is involved in the pathogenesis of
lupus, additional amounts of suppressive DNA should reduce
autoantibody production and autoimmune tissue injury in
experimental lupus [80]. Dong et al. [81] used synthetic ODN
expressing TTAGGG motifs in the spontaneous lupus model
of NZB/NZW mice. Treatment with suppressive ODN
improved survival of these mice associated with improved
lupus nephritis, proteinuria, and lower serum dsDNA auto-
antibody levels [81]. This finding was confirmed in MRLlpr/lpr
mice, another model of progressive lupus nephritis [82]. As in
these studies NZB/NZW mice were not exposed to
exogenous CpG-DNA, these data suggest that the
suppressive ODN blocked endogenous CpG-DNA-induced
immunity. Thus, CpG motifs in self DNA appear to be a
pathogenic factor in the progression of established tissue
injury in autoimmune mouse strains and possibly in human
lupus [83,84]. In order to elucidate the role of TLR9 in the
development of lupus, TLR9 deficient mice have to be
backcrossed into the appropriate lupus mouse strain. This
approach was used in a recent study in which, overt lupus
nephritis was reported, despite decreased dsDNA auto-
antibody production, in TLR9-deficient MRLlpr/lprmice after
two backcrosses [85]. The role of TLR9 in the development
of lupus remains unclear and its determination could require
data from TLR9-deficient lupus mice that have appropriately
backcrossed for at least five generations into their specific
genetic background.
Pharmacological blockade of TLR9 signalling
Specific small molecule TLR antagonists are not yet available,
although drugs currently in use for the treatment of
autoimmune diseases interfere with TLR signalling. For
example, chloroquine, an antimalarial drug used to treat
milder forms of lupus, inhibits CpG-DNA-induced immunity
[86-88]. Chloroquine is a strong base that inhibits endosomal
acidification, which is required for the interaction of CpG-
DNA with TLR9 [89]. In fact, treatment with chloroquine
somewhat reduces mRNA expression of IFN-α-related genes
in patients with active lupus [65], which argues for a role of
the aforementioned pathways for lupus disease activity.
Chloroquine is no TLR9 specific antagonist, however, and
may interfere with other endosome-dependent disease
mechanisms, for example,
recognition [90,91]
TLR7-dependent ssRNA
Clinical implications and future perspectives
In view of the aforementioned potential of microbial molecules
to trigger or modulate autoimmunity, new hypotheses arise
that may influence the management of patients with
autoimmune disease.
Concerns about therapeutic use of TLR agonists in
patients with autoimmune disease
Mycobacterial vaccine adjuvants have now been identified to
ligate TLRs. Experimental studies that exposed rodents with
lupus to TLR agonists, for example, experimental autoimmune
encephalomyelitis, collagen-induced
complex glomerulonephritis and other types of autoimmune
tissue injury, raise considerable concern about the safety of
TLR agonists in patients with autoimmune disease
[55-57,92]. These studies reported disease aggravation after
repeated injections with, for example, CpG-ODN, but side
effects of TLR ligands may relate to the dose, treatment
intervals, and route of administration [29,93]. Topical
application or a single vaccination regimen might have less
effects on pre-existing autoimmunity. CpG-DNA is currently in
clinical trials for the treatment of cancer, atopy and as vaccine
adjuvant [48,93,94]. So far, clinical or serological signs of
drug-induced lupus or flares of autoimmunity have not been
reported in recently reported trials that applied CpG-DNA as
a vaccine adjuvant for vaccination against influenza or
hepatitis B virus [93,95,96].
arthritis, immune
Therapeutic use of TLR antagonists in patients with
autoimmune disease
As discussed above, chloroquine already has an established
role in the treatment of milder forms of lupus. In addition,
suppressive DNA elements may modulate the stimulatory
effects of CpG DNA. In vitro studies suggest that CpG-DNA-
induced activation of B cells, macrophages, or dendritic cells
can be blocked with ODN containing suppressive DNA
motifs [75,80]. Thus, suppressive ODN may represent a
functional antagonist for TLR9 signalling induced by CpG-
DNA, a hypothesis supported by our studies with MRLlpr/lpr
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mice. These findings support a role for TLR9 signalling in the
pathogenesis of lupus. Thus, developing specific small
molecule TLR9 antagonists may represent a new approach
as a preventive therapy for systemic lupus erythematosus.
Conclusion
TLRs are critical receptors for innate pathogen recognition.
Their specific role in modulating innate and adaptive immunity
also interferes with the mechanisms that maintain tolerance in
the host. Thus, TLR ligation can contribute to loss of
tolerance by multiple mechanisms. The specific roles of
individual signalling pathways
autoimmune conditions remain a future challenge in this field.
A role for TLR9 in the pathogenesis of lupus is suggested for
both infection-induced disease flares as well as for the
recognition of CpG motifs in self DNA. Preliminary studies
with functional antagonists of TLR signalling suggest that
TLRs may provide a new set of potential targets for the
treatment of autoimmune diseases.
for several different
Competing interests
The author(s) declare that they have no competing interests.
Acknowledgements
The work was supported by a grant from the Deutsche Forschungsge-
meinschaft (AN372/4-1 and GRK 1201) and the Fritz Thyssen Foun-
dation to HJA.
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