Unexpected regulatory roles of TLR4 and TLR9 in experimental autoimmune encephalomyelitis

Unexpected regulatory roles of TLR4 and TLR9 inexperimental autoimmune encephalomyelitis

Monica Marta1, �sa Andersson2,3, Magnus Isaksson4, Olle K�mpe4 andAnna Lobell1,4

1 Neuroimmunology Unit, Department of Clinical Neuroscience, Karolinska Institute,

Stockholm, Sweden2 Applied Immunology Unit, Department of Clinical Neuroscience, Karolinska Institute,

Stockholm, Sweden3 Department of Medicine, Division of Pulmonary and Critical Care Medicine,

University of California Los Angeles School of Medicine, Los Angeles, CA, USA4 Department of Medical Sciences, Uppsala University, Uppsala, Sweden

Innate immune mechanisms essential for priming encephalitogenic T cells in

autoimmune neuroinflammation are poorly understood. Experimental autoimmune

encephalomyelitis (EAE) is a IL-17-producing Th (Th17) cell-mediated autoimmune

disease and an animal model of multiple sclerosis. To investigate how upstream TLR

signals influence autoimmune T cell responses, we studied the role of individual TLR

andMyD88, the commonTLR adaptor molecule, in the initiation of innate and adaptive

immune responses in EAE. Wild type (WT) C57BL/6, TLR-deficient and MyD88-

deficient mice were immunized with myelin oligodendrocyte glycoprotein (MOG) in

CFA. MyD88–/–mice were completely EAE resistant. Purified splenic myeloid DC (mDC)

from MyD88–/– mice expressed much less IL-6 and IL-23, and serum and T cell IL-17

were absent. TLR4–/– and TLR9–/– mice surprisingly exhibited more severe EAE

symptoms than WT mice. IL-6 and IL-23 expression by mDC and Th17 responses were

higher in TLR4–/– mice, suggesting a regulatory role of TLR4 in priming Th17 cells. IL-6

expression by splenocytes was higher in TLR9–/– mice. Our data suggest that MyD88

mediates the induction of mDC IL-6 and IL-23 responses after MOG immunization,

which in turn drives IL-17-producing encephalitogenic Th17 cell activation.

Importantly, we demonstrate that TLR4 and TLR9 regulate disease severity in MOG-

induced EAE.

Introduction

DC migration to peripheral LN and subsequent matura-

tion is essential for initiation of adaptive immune

responses. DC maturation leads to dramatic changes in

cellular shape and function, and upregulated expression

of MHC, CD80, CD86 and cytokines as well as increased

ability to prime na�ve T cells [1]. A given DC population

will only respond to pathogen-associated molecular

patterns (PAMP) for which it has appropriate pattern-

recognition receptors (PRR) such as TLR [2]. The

recognition of PAMP by TLR triggers intracellular

signaling pathways resulting in the induction of

Clinical immunology

Correspondence: Dr. Anna Lobell, Department of MedicalSciences, Uppsala University, Lab 21, Forskningsavd 2,Ingang 70, 3 tr., University Hospital, 751 85 Uppsala, SwedenFax: +46-18-553-601e-mail: [email protected]

Received 17/2/07Revised 9/11/07

Accepted 13/12/07

[DOI 10.1002/eji.200737187]

Key words:Antigen-presenting

cells � Autoimmunity� Cytokines � Innateimmunity � T helper

cells

Abbreviations: ANA: anti-nuclear antibody � IRF-7: IFNregulatory factor-7 � mDC: myeloid DC � MOG: myelinoligodendrocyte glycoprotein � MyD88–/–: MyD88-deficient �PAMP: pathogen-associated molecular pattern � pDC: plas-macytoid DC � PRR: pattern-recognition receptor � Q-PCR: real-time quantitative RT-PCR � Th17: IL-17-producing Th cell � TIR:Toll-IL-1R � TLR–/–: TLR-deficient � TRIF: TIR domain-containingadaptor-inducing beta interferon

Eur. J. Immunol. 2008. 38: 565–575 Clinical immunology 565

f 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

proinflammatory cytokines, type I IFN, chemokines and

DC maturation that leads to activation of adaptive

immunity. Each TLR recognizes different PAMP: TLR4

recognizes LPS and TLR2 collaborates with TLR1 or

TLR6 in the recognition of bacterial lipopeptides. TLR3

and TLR9 are expressed in endosomes and are involved

in the recognition of double-stranded RNA and

unmethylated CpG DNA motifs, respectively [3]. TLR

recognition of PAMP triggers the TLR cytoplasmic Toll-

IL-1R (TIR) domain to associate with adaptors such as

MyD88 or TIR domain-containing adaptor-inducing beta

interferon (TRIF). All identified TLR utilize the MyD88-

dependent signaling pathway, except TLR3, which uses

theMyD88-independent TRIF pathway. TLR4 signals via

both pathways. TheMyD88-dependent pathway induces

genes encoding proinflammatory cytokines via activa-

tion of NF-jB, except in plasmacytoid DC (pDC), in

which MyD88 binds IFN regulatory factor-7 (IRF-7)

directly and induces genes encoding type I IFN [4]. The

MyD88-independent TRIF pathway mainly induces

genes encoding type I IFN [3, 5].

Little is known about priming of autoaggressive

T cells and subsequent human organ-specific autoim-

mune disease. EAE is an animal model for the human

autoimmune demyelinating disease MS [6]. Murine EAE

is induced by injection of myelin autoantigens in CFA

containing killed Mycobacterium tuberculosis and per-

tussis toxin. Mycobacterial PAMP are recognized by

TLR2/6 and TLR4/CD14 [7, 8], mycobacterial DNA is

rich in the TLR9 ligand CpGDNA [9], and pertussis toxin

is recognized by TLR4 [10]. MyD88 controls myeloid DC

(mDC) maturation and generation of Th1 responses

upon CFA immunization [11]. TLR promote Th1

responses and EAE was previously thought to be a

Th1-mediated autoimmune disease. Langrish et al. [12]

demonstrated, however, that IL-23-driven proinflam-

matory IL-17-, IL-17F-, TNF- and IL-6-producing Th cells

designated Th17 mediate EAE. These Th17 cells

represent a lineage distinct from the Th1 and Th2 cells

[13, 14]. Importantly, naive CD4 Tcells differentiate into

Th17 cells in the presence of soluble TGF-b or

CD4+CD25+ Treg and DC-derived IL-6 [15–17]. In

addition, IL-23 is essential for effective antibacterial

Th17 responses [15] and for the survival but not

differentiation of Th17 cells [17]. IL-1b acts directly on

Tcells to further promote Th17 differentiation [18]. Th2

responses are normal after CFA immunization in

MyD88-deficient (MyD88–/–) mice [11] and it is not

known whether MyD88 promotes Th17 cell responses.

It was recently reported that MyD88–/– mice are

resistant to myelin oligodendrocyte glycoprotein (MOG)

peptide-induced EAE and that TLR9 modulates patho-

genicity in this model [19], but the precise role of

MyD88 during the early, peripheral induction of

encephalitogenic immune responses in EAE has never

been investigated and little is known about the role of

individual TLR in EAE [10, 19–21]. Therefore, we

immunized WT C57BL/6, MyD88–/– and TLR-deficient

(TLR–/–)mice withMOGprotein in CFA and investigated

the role of MyD88 and individual TLR in the initiation of

innate and adaptive immune responses. Proinflamma-

tory cytokine expression was measured in purified mDC,

pDC, T cells and sera from individual mice to link

upstream TLR-based signals to disease-inducing me-

chanisms. MyD88–/– mice were EAE resistant. Splenic

mDC from MyD88–/– mice failed to express IL-6 and

IL-23, and Th17 responses and serum IL-17 were much

lower compared to WT mice. In contrast to the results of

Prinz et al [19], disease was exacerbated in TLR4–/– and

TLR9–/–mice in MOG protein-induced EAE, demonstrat-

ing an unexpected regulatory role for these TLR in the

induction of autoimmune neuroinflammation. IL-6 and

IL-23 expression by mDC and Th17 responses were

higher in TLR4–/– mice, while IL-6 expression by

splenocytes was higher in TLR9–/– mice compared to

WT mice. Thus, our data show that MyD88 mediates

peripheral mDC IL-6 and IL-23 expression and Th17

responses with the ensuing development of EAE,

whereas TLR4 and TLR9 modulate EAE symptoms.

Results

Induction of EAE requires MyD88

To study the role of MyD88-dependent signaling path-

ways during EAE development we immunized mice

deficient in MyD88 with MOG in CFA [22]. MyD88–/–

mice were resistant to EAE while WT mice exhibited

chronic symptoms of EAE with disease onset between

days 12–16. The incidence of EAE was 0% for MyD88–/–

mice, whereas for WT mice incidence was 50% with a

maximum mean EAE score of 1.3 (p <0.05) and a mean

accumulated EAE score of 14 � 7 (p <0.03) (Fig. 1A).

Due to the low incidence of EAE in WT mice in the first

experiment, we next induced a more severe disease by

increasing the dose of MOG from 200 to 250 lg. Even

with the higher dose of MOG, 0% of MyD88–/– mice

exhibited EAE, while 100% WT mice exhibited very

severe symptoms of EAE and had to be sacrificed on

day 13 with a maximum mean EAE score of 4.1

(p <0.0006) (Fig. 1B). Thus, MyD88-dependent signal-

ing pathways are required for initiation of EAE.

MyD88 mediates proinflammatory cytokineexpression

Next, we investigated how MyD88-dependent signaling

pathways affect initial, peripheral immune activation

following MOG immunization. During the initiation of

Monica Marta et al. Eur. J. Immunol. 2008. 38: 565–575566


EAE it is unclear which Ag-presenting cell primes

encephalitogenic T cells in the periphery. Because TLR

are important for DC activation and maturation, we

purified CD11chiB220–Gr-1– mDC and CD11cloB220+

Gr-1+ pDC (depicted in Fig. 2A) from spleens 4 and

10 days after induction of EAE. The expression of IL-6

and IL-23 by mDC was very low on day 4 after MOG

immunization and increased between days 4 and 10 in

all mouse strains tested (Fig. 2B). On day 10 after MOG

immunization, expression of mRNA encoding IL-6

(p <0.05) and one subunit of the proinflammatory

cytokine IL-23, IL-23p40 (p <0.05), was much lower in

mDC from individual MyD88–/– mice compared to WT

mice (Fig. 2B), whereas IL-23p19 expression by mDC

was similar in MyD88–/– andWTmDC, suggesting that it

was induced by MyD88-independent mechanisms

(Fig. 2B). The mRNA expression profiles of inflamma-

tory mediators differed greatly between mDC and pDC.

In contrast to mDC, IL-6 and IL-23p40 was not induced

in pDC (Fig. 2B).

T cells from MyD88–/– mice fail to express IL-17

In response to MOG immunization mDC expression of

IL-6 and IL-23 was dependent on MyD88 (Fig. 2B). IL-6

is essential for – whereas IFN-c inhibits – priming of

encephalitogenic Th17 cells and these cells are main-

tained by IL-23 [12–14, 17]. We hypothesized that Th17

cells were not primed in the absence of MyD88. The

splenic Th17 responses and gene expression profile of

purified splenic T cells from MOG-immunized WT or

MyD88–/– mice was compared.

To assess Th17 responses, the frequency of IL-17+ of

total CD3+ T cells was measured by flow cytometry in

MOG-stimulated splenocytes from MOG-immunized

mice. The mean frequency of Th17 cells was only

1.4% of total CD3+ in WT mice, and the frequency of

Th17 cells from MyD88–/– mice spleens was lower

(p <0.05) compared to WT mice (Fig. 3A and B).

Thy1.2+ T cells from MyD88–/– mice spleens

expressed dramatically less IL-17 mRNA (p <0.05) than

WT mice (Fig. 3C). Thy1.2+ T cells from draining LN

exhibited a similar expression profile to splenic T cells

(data not shown). In contrast, T cells from spleens of

MyD88–/– or WT mice expressed similar levels of IFN-c

Figure 1.MyD88–/–mice are resistant toMOG-induced EAE,while TLR4–/– andTLR9–/–mice exhibitmore severe EAE symptoms thanWTmice. (A and B) Mean EAE scores � SEM for MyD88–/– (squares) andWT (triangles) mice (n = 5–8/group). (C) Mean EAE scores �SEM for TLR6–/– (crosses), MyD88–/– (squares) and WT (triangles) mice (n = 2–7/group). (D) Mean EAE scores � SEM for TLR4–/–

(squares) and WT (triangles) mice (n = 8–12/group). (E) Mean EAE scores � SEM for TLR9–/– (circles) and WT (triangles) mice (n =12–14/group). (D) and (E) depict the EAE score for mice from the same experiment. * p <0.05, **p <0.008 relative to WT mice.



(Fig. 3C), IL-21, IL-22, TNF and IL-10 mRNA (data not

shown). Even after 48 h MOG stimulation, nearly no

IL-17 mRNA was expressed in splenocytes from

MyD88–/– mice (data not shown). Importantly, these

data demonstrate that Th17 cells are not primed in

MyD88–/– mice.

IL-23 and IL-12 receptors use a common IL-12RB1

subunit combined with unique subunits IL-23R or

IL-12RB2, respectively. IL-23R ligation is required for

T cell IL-17 production, whereas IL-12R ligation is

required for T cell IFN-c production [13, 23]. Because of

the absent IL-17 and sustained IFN-c production in

MyD88–/– Tcells we asked whether IL-23R and IL-12RB2

were expressed. IL-23R mRNA expression was much

lower in MyD88–/– mice (p <0.01) than in WT mice

(Fig. 3C), but IL-12RB2mRNA expressionwas expressed

at similar levels in the two strains (data not shown).

These data suggest that IL-23R, but not IL-12R, is

directly or indirectly upregulated through MyD88

signaling. Mangan et al. [15] demonstrated that

TGF-b up-regulates IL-23R expression on naive T cells

upon priming of Th17 cells, suggesting that TGF-b may

Figure 2. Impaired IL-6 and IL-23 mRNA expression in splenic mDC from MyD88–/– mice. (A) CD11c+ splenic cells were gated andanalyzed for the expression of B220 and Gr-1. CD11chiB220–Gr-1– mDC and CD11cloB220+Gr-1+ pDC were isolated 4 days (n = 4/group) and 10 days (n = 6/group) after MOG immunization. (B) Q-PCR analysis of the mRNA expression of proinflammatorycytokines inmDC and pDC fromWT (black bars), MyD88–/– (white bars), TLR4–/– (light grey bars) or TLR9–/– (dark grey bars)mice. (C)Q-PCR analysis of the mRNA expression of proinflammatory cytokines in mDC from unimmunized WT (black bars), MyD88–/–

(white bars), TLR4–/– (light grey bars) or TLR9–/– (dark grey bars) mice after 24 h stimulation with killed Mycobacterium tuberculosis.Bars represent mean � SEM. All values are normalized to 18 S RNA. *p <0.05, **p <0.02 relative to WT mice.



be induced by MyD88-dependent mechanisms. How-

ever, T cells from MyD88–/– and WT mice expressed

similar levels of TGF-b mRNA (Fig. 3C), which suggests

that TGF-b expression by T cells is induced by MyD88-

independent mechanisms.

Serum IL-6 and IL-17 are absent inMyD88–/– mice

To investigate whether protein levels of IL-6 and IL-17

correlated with the observed mRNA levels, we measured

these cytokines in sera on days 2, 4 and 10 after MOG

immunization. IL-6 was absent in sera from unimmu-

nized mice, while on day 2 after MOG immunization

serum IL-6 levels were high in all mouse strains tested

(Fig. 4A). On day 10 after MOG immunization no IL-6

was detected in sera from MyD88–/– mice, whereas sera

fromWTmice exhibited high levels of IL-6 (Fig. 4A). The

experiment was repeated with similar results. Thus,

early serum IL-6 responses on day 2 after MOG/CFA

immunization are induced by MyD88-independent

mechanisms, whereas the IL-6 responses on day 10

after immunization are mediated by MyD88.

Measurement of IL-17 levels in sera 2, 4 or 10 days

after MOG immunization revealed low levels of IL-17 on

days 2 and 4 after immunization in all mouse strains

tested. However, on day 10 after immunization sera

from WT mice exhibited high levels of IL-17 whereas

IL-17 in sera from MyD88–/– mice was much lower than

WT mice (p <0.01) (Fig. 4B), which correlated with the

observed T cell IL-17 mRNA levels (Fig. 3C).

Figure 3. Lack of IL-17 expression in T cells fromMyD88–/–mice.(A) Percentage of IL-17+ Th17 cells of total CD3+ T cells in spleen10 days after MOG immunization. (B) Mean frequency (%) ofIL-17+CD3+ Th17 cells in spleen fromWT (black bars), MyD88–/–

(white bars), TLR4–/– (light grey bars), TLR9–/– (dark grey bars)mice respectively (n = 6/group). Bars representmean� SEM. (C)Thy1.2+ T cells isolated from mouse spleen 10 days after MOGimmunization. Q-PCR analysis of the mRNA expression ofrelevant cytokines and cytokine receptors in T cells from WT(black bars), MyD88–/– (white bars), TLR4–/– (light grey bars),TLR9–/– (dark grey bars) mice respectively (n = 5–8/group). Dataare representative of two separate experiments with similarresults. All values are normalized to 18S RNA. Bars representmean � SEM. *p <0.05, **p <0.01 relative to WT mice.

Figure 4. Higher IL-17 levels in sera from TLR4–/– and TLR9–/–

mice. (A) Serum IL-6 and (B) serum IL-17 levels from WT (blackbars), MyD88–/– (white bars), TLR4–/– (light grey bars), TLR9–/–

(dark grey bars) mice, respectively, 2 days (n = 4/group), 4 days(n = 4/group) and 10 days (n = 6–14/group) after MOGimmunization. Bars represent mean � SEM. *p <0.05, **p<0.01, ***p <0.001 relative to WT mice.



Mice deficient in TLR6 are susceptible to EAE

The role of individual TLR during induction of EAE was

then studied. Recognition of mycobacterial PAMP by

TLR2/TLR6 is needed to elicit an efficient adaptive

immune response against mycobacteria [7]. Conse-

quently, the role of TLR2/TLR6 in the induction of EAE

in mice deficient in TLR6 was examined. TLR6–/– mice

attained a maximummean EAE score comparable to WT

mice. The incidence of EAE was 86% and the maximum

mean EAE score of 1.4 for TLR6–/– mice, compared to

100% incidence and a maximum mean EAE score of 1.4

for WT mice (NS) (Fig. 1C). These data indicate that

TLR2/TLR6 have a redundant or limited role in the

induction of EAE.

Lack of TLR4 exacerbates EAE

Mycobacterial PAMP and pertussis toxin are also

recognized by TLR4 [7, 10]. Thus, EAE in TLR4–/– mice

was compared to WT mice. The dose of MOG was

carefully titrated to induce EAE in all mice and with

moderate severity (data not shown). TLR4–/–mice had a

100% incidence and more severe EAE with a maximum

mean EAE score of 3.4 � 0.3 (p <0.03), compared to

100% incidence and maximum mean EAE score of 2.3 �

0.3 for WT mice (Fig. 1D). The mean accumulated EAE

score was 35 � 0.7 (p <0.01) for TLR4–/– mice,

compared to 25 � 2.5 for WT mice. Similar results were

obtained in a separate experiment (data not shown).

This shows that TLR4 signaling regulates EAE, rather

than promotes the disease.

IL-23 expression is enhanced in TLR4–/– mDC

TLR4 is one of several TLR expressed on murine DC, and

signals via both MyD88 and TRIF pathways. Thus, the

cytokine expression by mDC and pDC in MyD88–/– mice

does not only reflect loss of TLR4 signaling. Conse-

quently, we investigated mDC and pDC cytokine

expression in individual TLR4–/– mice. Interestingly,

the splenic mDC mRNA expression of IL-6 (p <0.05),

IL-23p40 (p<0.05) and IL-23p19 (p<0.01) was several-

fold higher in mDC from TLR4–/– than from WT mice

(Fig. 2B). Our data indicate that TLR4 signals regulate

IL-6 and IL-23 production by mDC during initiation of

EAE.

To determine if the observed induction of IL-6 and

IL-23 in TLR4–/– mDC from MOG immunized mice was

an effect of mycobacterial stimulation on mDC, we

sorted mDC from unimmunized WT, MyD88–/– and

TLR–/– mice followed by 24-h stimulation with myco-

bacteria. Protein levels of IL-6 and IL-23 in the

supernatants were undetectable (data not shown).

There was a tendency towards a higher IL-6 expression

by TLR4–/– mDC (NS) after stimulation with mycobac-

teria, which may suggest that TLR4 regulates IL-6

expression in mDC (Fig. 2C). The levels of IL-23p40

mRNA were similar (NS) in TLR4–/– and WT mDC

(Fig. 2C). This indicates that the enhanced IL-23 mRNA

in mDC from MOG immunized TLR4–/– mice is not a

direct effect of mycobacterial stimulation of TLR4–/–

mDC.

T cells from TLR4–/– mice express more IL-17 butless IFN-c

The exacerbated disease in TLR4–/– mice suggested that

the Th17 responses in TLR4–/– mice were enhanced.

Therefore we assessed the Th17 responses in individual

TLR4–/– mice 10 days after MOG immunization. The

frequency of Th17 cells from TLR4–/– mice spleens was

much higher (p <0.01) than WT mice (Fig. 3A and B).

Moreover, we investigated the cytokine expression

profile of purified T cells from TLR4–/– mice 10 days

after immunization. Indeed, the T cell IL-17 (p <0.05)

mRNA expression was more than two times higher in

TLR4–/– mice than in WT mice, while the expression of

IL-21, IL-22 (data not shown) and IL-23R was unaltered

(Fig. 3C). In contrast, IFN-c (p<0.05) mRNA expression

was strongly reduced in T cells from TLR4–/– mice

compared to WT mice (Fig. 3C). Moreover, the mRNA

expression of IFN-c was strongly reduced in splenocytes

from TLR4–/–mice compared toWTmice (p<0.05) after

48-h restimulation with MOG (data not shown). The

experiments were repeated with similar results.

Importantly, 10 days after MOG immunization

serum IL-17 levels were strongly increased in TLR4–/–

mice, compared to WT mice (p<0.001) thus correlating

with the enhanced Th17 responses (Fig. 4B). Thus, in

the absence of TLR4 there was increased Th17 cell

generation and IL-17 secretion.

Lack of TLR9 exacerbates EAE

DNA from Mycobacterium tuberculosis is rich in hypo-

methylated CpG DNA [24]. The role of TLR9 in the

induction of EAE in TLR9–/– mice compared to WT mice

was therefore examined. All TLR9–/– mice developed

EAE and displayed a more severe EAE with a higher

maximum mean EAE score 3.4 � 0.2 (p = 0.008),

compared to 100% incidence and maximum mean EAE

score of 2.3 � 0.3 for WT mice (Fig. 1E). The mean

accumulated EAE score was 36 � 1.3 (p <0.002) in

TLR9–/– mice, compared to 25 � 2.5 for WT mice.

Similar results were obtained in two additional experi-

ments (data not shown). Thus, TLR9 like TLR4 regulates

EAE, rather than promotes it.



DC cytokine expression is unaffected inTLR9–/– mice

TLR9 is expressed in lysosomes in murine mDC but in

endosomes in pDC and other cells [25]. As a

consequence, TLR9 stimulation may induce pDC type

I IFN production that inhibits the generation of Th17

cells [13], whereas in mDC and macrophages it may

induce IL-6 and/or IL-23 production. pDC and mDC

gene expression profiles were studied 10 days after

MOG immunization in individual TLR9–/– and WTmice.

Surprisingly, pDC from TLR9–/– and WT mice expressed

similar levels of type I IFN and mDC from TLR9–/– mice

produced equal amounts of proinflammatory cytokines

as WT mice did (Fig. 2B). Thus, we failed to observe any

differences in mDC and pDC cytokine production in

these mice compared to WT mice.

Serum IL-17 levels in TLR9–/– mice

IL-6 or IL-23 expression by mDC was not enhanced in

TLR9–/– mice, even though TLR9–/– mice exhibited an

aggravated EAE phenotype. We therefore asked whether

the Th17 responses and cytokine expression profile was

altered in purified T cells from TLR9–/– mice. The Th17

responses were similar in T cells from TLR9–/– and WT

mice (Fig. 3A and B). IL-17, IFN-c (Fig. 3C), IL-21, IL-22

and IL-10 (data not shown) expressionwere all similar in

T cells from TLR9–/– and WT mice. The experiment was

repeated with similar results. Serum IL-17 levels were

similar in TLR9–/– and WT mice (Fig. 4B). Another

possible mechanism for the observed aggravated disease

in TLR9–/– mice could be a higher expression of IL-23R

or lower expression of IL-12RB2. The expression of these

cytokine receptors on T cells from these mice were then

examined. The expression of IL-23R (Fig. 3C) and

IL-12RB2 (data not shown) was, however, not altered in

TLR9–/– mice relative to WT mice. Our data could

indicate that T cells present outside of the spleen are

important for disease exacerbation in TLR9–/– mice, or

that the MOG-specific T cells have left the spleen at

10 days after immunization.

Given the exacerbated disease in TLR9–/– mice, yet

similar cytokine patterns in T cells, we suspected that

other cell types (e.g. macrophages) might express

increased proinflammatory cytokines. When comparing

TLR9–/– and WT gene expression profiles in splenocytes

following MOG-specific restimulation ex vivo, spleno-

cytes from MOG-immunized TLR9–/– mice exhibited

enhanced IL-6 (p<0.05) mRNA expression compared to

WTmice (Fig. 5). The experiment was repeated with the

same results. The enhanced IL-6 expression in TLR9–/–

mice was, however, limited to the spleen, as serum IL-6

levels were similar in TLR9–/– mice and WT mice

(Fig. 4A). The experiment was repeated with the same

results.

Discussion

We here show that the induction of EAE is dependent on

MyD88. This explains why adjuvants such as mycobac-

teria have to be included when EAE is induced. In fact,

immunization with myelin autoantigens without adju-

vants leads to tolerance to the autoantigen [26, 27].

MyD88 is an adaptor molecule for multiple receptors,

which complicates the understanding of TLR role in

EAE. We have individually examined different TLR that

bind mycobacterial PAMP, but no TLR alone could

account for theMyD88–/– phenotype. Because IL-1R and

IL-18R signal via MyD88 [22], the observed EAE

resistance in MyD88–/– mice could be due to failure to

signal via these receptors. However, it is very unlikely

that IL-1 or IL-18 is the sole inducer of pathogenic Th17

cells, because de novo differentiation of naive CD4 cells

into Th17 cells requires DC-derived IL-6, and IL-6

production is induced via TLR ligation [17, 28]. Our data

are in agreement with a recent report, in which

MyD88–/– mice were EAE resistant and TLR2–/– mice

were EAE susceptible [19].

Importantly, we observed novel regulatory roles for

TLR4 and TLR9 in EAE. This is in sharp contrast to other

autoimmune disorders where TLR are disease-promot-

ing. Vallin et al. [29] first showed that immune

complexes consisting of immune stimulatory DNA and

anti-nuclear antibodies (ANA) induce IFN-a production

in human pDC that mimic the IFN-a inducer in human

lupus. Sera from patients with primary Sj�gren's

syndrome containing autoantibodies to RNA-binding

proteins, combined with material released from apop-

totic or necrotic cells, triggers IFN-a production by pDC

[30]. Likewise, TLR9 and TLR7 ligation activates

rheumatoid factor-producing B cells [31, 32] and

TLR9 mediates the generation of ANA in murine lupus

Figure 5. Enhanced IL-6 expression in splenocytes from TLR9–/–

mice. IL-6 mRNA expression in splenocytes after 48 h culturewithMOG isolated fromWT (black bars), MyD88–/– (white bars),TLR4–/– (light grey bars), TLR9–/– (dark grey bars) mice,respectively, 10 days after MOG immunization (n = 5–8/group).Data are representative of two separate experiments. All valuesare normalized to 18s RNA. Bars represent mean � SEM. * p

<0.05 relative to WT mice.



[33]. In contrast, TLR9–/– lupus-prone mice developed

more severe disease than TLR9+/+ lupus-prone mice

[33, 34] and we have previously demonstrated an

essential role for CpG DNA in the plasmid backbone of

an EAE-suppressive DNA vaccine [35, 36]. In agreement

with our results, these latter studies indicate a

regulatory role for TLR9 in certain autoimmune disease

models.

We investigated howMyD88-dependent mechanisms

influence the initial, peripheral immune reactivity

towards MOG. The production of the proinflammatory

cytokines IL-6 and IL-23 by splenic mDC as well as their

maturation (data not shown) was dependent onMyD88.

Interestingly, activation of proinflammatory cytokine

IL-6 and IL-23 expression by splenic WT mDC was not

observed before day 10 after MOG immunization, which

is later than expected. Most importantly, Th17 cells were

missing and sera did not contain any detectable IL-17 in

MyD88–/– mice, which demonstrates that Th17 cells are

not primed in the absence of MyD88. Consistent with

our data, Prinz et al. [19] showed that MyD88–/– T cells

fromMOG peptide-immunizedmice transferred intoWT

mice did not induce EAE, but a role for Th17 cells was

not examined in their study. Threemechanisms that may

all contribute to the lack of Th17 cell priming in

MyD88–/– mice were observed: (i) reduced mDC IL-6

production, because naive CD4 T cells are de novo

differentiated into Th17 cells after exposure to TGF-b

and mDC-derived IL-6 [16, 17], (ii) reduced mDC IL-23

production, because IL-23 maintains Th17 cells [17] and

(iii) reduced T cell IL-23R expression. Interestingly,

TGF-b induces T cell IL-23R expression [15]. This could

indicate that TGF-b expression is induced by MyD88-

dependent mechanisms after MOG immunization.

However, in our hands TGF-b expression by T cells

was similar in WTand MyD88–/– mice, which argues for

MyD88-independent mechanisms in vivo.

TLR4 and TLR9 regulate EAE by distinct mechan-

isms. These mechanisms may differ because the

signaling pathways or the ligands differ. We tried to

link the more severe EAE pathogenesis to cytokine

secretion in different cell subsets in TLR4–/– and TLR9–/–

mice. The mDC expression of IL-6 and IL-23 was higher,

and peripheral T cells expressed more IL-17 in TLR4–/–

compared to WT mice. Mycobacterial stimulation of

TLR4–/– mDC suggested that TLR4 may directly regulate

IL-6 - but not IL-23 expression - by mDC. The IL-6 data

are not contradictory because several cell types beside

mDC such as macrophages, Th17 cells and B cells can

produce IL-6. The enhanced IL-6 expression was limited

to mDC, as serum IL-6 and IL-6 expression by

splenocytes were not enhanced inTLR4–/–mice. Because

mDC are rare in the spleen, less than 0.3% (data not

shown), the observed serum IL-6 levels and IL-6

production by splenocytes likely reflects the production

by a more abundant cell type, e.g. macrophages or

B cells. IFN-c inhibits the differentiation of Th17 cells

[13] and disruption of the IFN-cR gene is sufficient to

convert an EAE-resistant into susceptible mouse strain

[37], which argues for the fact that low Tcell IL-12R and

IFN-c expression in TLR4–/–mice most likely contributes

to the enhanced IL-17 production and EAE severity. To

date, it is not known whether IFN-c also inhibits the

generation of Th17 cells at the DC level. We speculate

that TLR4 promotes IFN-c production that regulates the

activation of mDC and/or IL-17 production by T cells.

Consistent with this, tolerization with LPS in vivo

suppressed subsequently induced EAE [20]. In contrast,

mice developed EAE after immunization with LPS and

MOG peptide in IFA [20].

We originally hypothesized that type I IFN expression

by pDC would inhibit the development of EAE in

response to TLR9 ligation, because these cytokines

inhibit Th17 cell differentiation [13]. We failed to

observe any altered mDC, pDC or T cell cytokine

expression in TLR9–/– mice. Nevertheless, we observed

significantly higher IL-6 mRNA expression by TLR9–/–

splenocytes, that may create an enhanced inflammatory

mileu that increases the disease severity [15–17]. Since

the spleen includes less than 0.3% mDC (data not

shown), the enhanced IL-6 levels likely reflect produc-

tion by a more abundant cell type such as macrophages

or B cells.

Our data demonstrating exacerbated EAE in TLR4–/–

and TLR9–/– mice conflict with other studies in which

TLR9–/– mice were less affected by EAE [19], mice

developed EAE after immunization with CpG DNA [38,

39] and TLR4-mutated mice were less affected or as

affected by EAE as WT mice [10]. A critical difference is

that we immunized once with MOG protein, which was

sufficient to prime encephalitogenic Th17 cells, whereas

the MOG peptide regimen used by Prinz et al. and

Kerfoot et al. [10, 19] needed a boost in order to induce

EAE. This boost may have obscured the regulatory role

of TLR4 and TLR9 that is clearly seen with a more

traditional induction procedure. The use ofMOG protein

rather than MOG peptide may also be the reason for the

observed differences, because in contrast to MOG

peptide, immunization with MOG protein induces

pathogenic B cell responses and demyelinating anti-

bodies similar to human MS [40, 41]. Furthermore,

CD8 T cell responses are elicited after MOG protein

immunization. MOG35–55 only contains one CD8 T cell

epitope, whereas MOG protein contains several

CD8 T cell epitopes [42]. TLR4 and TLR9 signaling

may dampen these disease processes, although the

observed enhanced serum IL-17 argues for a regulatory

role of TLR4 during activation of Th17 cells.

Our study links upstream TLR-based signals to the

disease-inducing mechanism of Th17 cells. We propose a



model for initiation of the peripheral, adaptive immune

response in EAE that integrates these MyD88-dependent

TLR signals. Stimulatory TLR induce peripheral mDC

IL-6 and IL-23 expression and maturation viaMyD88. In

order to generate effective Th1 responses, IL-12 has to

be produced by the antigen-presenting DC [43]. If

priming of Th17 cells operates in a similar way to Th1

cells, mDC most likely prime encephalitogenic T cells in

EAE via production of IL-6 in the presence of TGF-b [16,

17]. Consistent with this, we found that mDC, and not

pDC, expressed IL-6. Th17 cells would then be primed by

activated mDC that secrete the critical cytokine IL-6 and

maintained by IL-23.

The requirement for MyD88 signaling for induction

of EAE supports the case for a microbial etiology of

human MS, because TLR are primarily ligated by

microbial components. Molecular mimicry has been

proposed as a mechanism for initiation of MS. Microbial

antigens, together with certain TLR ligands, could

activate mDC to prime Th17 cells directed against

microbial antigens, and subsequently Th17 cells could

recognize cross-reacting myelin antigens and become

encephalitogenic after reactivation within the CNS by

DC, as described [44, 45]. As additional endogenous

ligands for TLR are being described [3, 33, 46], there is

also support for the notion that encephalitogenic Th17

cells may be primed by DC activated by endogenous TLR

ligands.

The present study suggests possible new therapeutic

approaches. First, the MyD88 pathway may represent a

novel target for therapeutic intervention of human MS,

although more studies on the regulatory roles of TLR are

warranted. Secondly, treatment with ligands for reg-

ulatory TLR may provide a more specific therapy.

Materials and methods

Antigens and mice

Escherichia coli-derived rat rMOG was produced as previously

described [47]. Mice were originally obtained from

Dr. S. Akira, Osaka, Japan and bred at Karolinska Institute.

TLR9–/– mice were first backcrossed at least four generations

with Japanese C57BL/6 mice, and subsequently backcrossed

for seven generations with C57BL/6 mice of the same local

colony of C57BL/6 mice we used as WT mice in all

experiments. MyD88–/–, TLR6–/– and TLR4–/– mice were first

backcrossed four generations with Japanese C57BL/6 mice,

and subsequently backcrossed for at least three generations

with C57BL/6 mice of the same local colony of C57BL/6 mice

we used as WT mice in all experiments. All studies have been

reviewed and approved by the local ethics committee.

EAE

Age-matched 8–12-week-old female mice were immunized

with 190–250 lg of MOG in CFA containing 0.5 mg Myco-

bacterium tuberculosis H37RA (Difco, BD Diagnostic systems,

Sparks, MD) in IFA (Sigma-Aldrich, St. Louis, MO) s.c. At the

day of immunization and 2 days after, mice were injected with

200 ng of pertussis toxin (Sigma-Aldrich) i.p. Clinical

symptoms of EAE were scored daily as follows: 1, tail weakness

or tail paralysis; 2, hind leg paraparesis; 3, partial hind leg

paralysis; 4, complete hind leg paralysis; 5, tetraplegia or

moribund state. In Fig. 1A and C, clinical symptoms of EAE

were scored as follows: 1, tail weakness or tail paralysis; 2, hind

leg paraparesis; 3, hind leg paraparalysis; 4, tetraplegia or

moribund state.

Isolation of splenocytes and LN cells and sorting of cells

Splenocytes and LN cells were isolated 4 and 10 days after

immunization as previously described [35]. Freshly isolated

splenocytes were stained with the following antibodies for

fluoroscence-activated cell sorting analysis: anti-CD11c-PE,

anti-B220-CyChrome and anti-Gr-1-FITC (BD Biosciences, San

Jose, CA). Sorting of mDC and pDC was performed as

previously described [48, 49] with a modification:

CD11chiB220–Gr-1– mDC and CD11cloB220+Gr-1+ pDC were

sorted to greater than 92% purity using a Moflo cytometer

(Dakocytomation, Glostrup, Denmark). Thy1.2+ T cells from

spleens or regional LN were purified to greated than 95%

purity using Thy1.2-MACS magnetic beads according to the

manufacturer0s instructions (Miltenyi Biotec, Bergisch Glad-

bach, Germany).

Cell cultures

Splenocytes were cultured in DMEM supplemented with 10%

FCS, 100 U/mL penicillin, 100 lg/mL streptomycin, 292 lg/

mL L-glutamine (DMEM complete) (all from Invitrogen,

Carlsbad, CA) with or without 10lg/mL MOG and 20lg/mL

polymyxin B sulfate (Sigma-Aldrich) for 48 h at 37�C.

Stimulation of mDC with mycobacteria

The mDC were sorted from spleens from unimmunized WT,

MyD88–/–, TLR4–/– or TLR9–/– mice. They were subsequently

cultured in DMEM supplemented with 10% FCS, 100 U/mL

penicillin, 100 lg/mL streptomycin, 292 lg/mL L-glutamine

(DMEM complete) (all from Invitrogen) at a concentration of

40 000 cells per well with or without 10 lg/mL Mycobacteria

tuberculosis H37RA (Difco) for 24 h at 37�C.

Intracellular staining of IL-17 in T cells

Splenocytes were cultured with rMOG for 48 h as described

above. Cells were stimulated and stained as previously

described [17]. Briefly, cells were subsequently stimulated

with 500 ng/mL phorbol dibutyrate (Sigma-Aldrich),

500 ng/mL ionomycin (Sigma-Aldrich) and brefeldin A (BD

Biosciences) for 4 h at 37�C. Cells were fixed, permeabilized

and incubated with rat anti-mouse CD16/CD32 (BD PharMin-



gen) to prevent unspecific binding of antibodies to Fc

receptors. The following antibodies were used for staining:

anti-CD3-FITC, anti-IL17-PE or rat isotype control IgG1-PE (all

from BD Biosciences). Cells were analyzed on a FACSCaliburTM

flow cytometer (BD Biosciences) using Cellquest software (BD

Biosciences).

Isolation of total RNA and cDNA synthesis

Total RNA was isolated from splenocytes and LN cells using

RNeasy mini or micro kit according to manufacturer0s

instructions (Qiagen, Hilden, Germany). RT-PCR was per-

formed using Superscript II (Invitrogen) as previously

described [50].

Gene expression analysis

Real-time quantitative RT-PCR (Q-PCR) was performed using

Quantitect SYBR green PCR kit (Qiagen) as previously

described [16, 51]. Amplification was performed using a

ABI7700 Sequence Detection System (Applied biosystems,

Norwalk, CT) or MyiQ Real-time PCR Detection System (Bio-

Rad Laboratories, Hercules, CA).

Serum IL-6 and IL-17 ELISA

Sera were collected 2, 4 or 10 days after immunization. IL-6

and IL-17 were measured by ELISA according to manufac-

turer's instructions (R & D Systems, UK). The detection limit

was 5 pg/mL for IL-17 and 1.6 pg/mL for IL-6.

Statistical analysis

Differences between mean daily EAE scores for individual

transgene vs. WTgroups were analyzed with Mann Whitney U

test. The p values lower than 5% were considered significant.

Because TLR9 is not an exclusive receptor for signals via

MyD88, and MyD88 is not an exclusive adaptor molecule for

TLR4 signaling, no direct comparisons between the transgenics

were performed. To measure differences between gene

expression and cytokine levels for individual transgene vs.

WT groups, we first tested if the groups were normally

distributed. If they were, we analyzed differences using

unpaired t-test. If the groups were not normally distributed,

we analyzed the differences with Mann-Whitney U test. To

measure differences between IL-17 levels in sera on day 10

after immunization, we pooled the data from two independent

experiments. Samples belonging to experiment “1” or “2” were

used as a factor in a two-way ANOVA analysis. The total

variability between the independent experiments was less than

2% (NS). Thus, we could pool the individual samples from the

two experiments in the statistical analysis. All analyses were

performed using Graphpad PrismTM software.

Acknowledgements: We thank D. J. Cua for helpfuladvice and generously sharing Q-PCR primer se-quences; L. Good for critical reading of the manuscript;S. Akira for providing the mice; R. A. Harris for valuablecritical comments; A. van Vollenhoven, H. Tian and A.

Norling for excellent technical assistance. This workwas supported by a grant from The Montel Williams MSfoundation, Petrus and Augusta Hedlund foundation,�ke Wiberg foundation, Tore Nilson foundation, Social-styrelsen funds, Center for Autoimmunity and Inflam-mation at Uppsala University and Swedish ResearchCouncil. M. Marta is supported by a fellowship from thePortuguese Funda�ao para a CiÞncia e Tecnologia,Programa Operacional CiÞncia, Tecnologia e Inova�ao-Formar e Qualificar-Medida 1.1.

Conflict of interest: The authors declare no financial orcommercial conflict of interest.

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Unexpected regulatory roles of TLR4 and TLR9 in experimental autoimmune encephalomyelitis

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