of April 2, 2018.This information is current as Sclerosis

Regulatory T Cell Function in Multiple Function: A Novel Mechanism of Reducedbetween Regulatory T Cell and Th17 TLR2 Stimulation Regulates the Balance

Bruno GranGhaemmaghami, Cris S. Constantinescu, Amit Bar-Or and Mee, Lloyd King, Giulio Podda, Guang-Xian Zhang, AmirDumitru Constantin-Teodosiu, Sophie Drinkwater, Maureen Mukanthu H. Nyirenda, Elena Morandi, Uwe Vinkemeier,

ol.1400472http://www.jimmunol.org/content/early/2015/05/15/jimmun

published online 15 May 2015J Immunol 

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The Journal of Immunology

TLR2 Stimulation Regulates the Balance between RegulatoryT Cell and Th17 Function: A Novel Mechanism of ReducedRegulatory T Cell Function in Multiple Sclerosis

Mukanthu H. Nyirenda,*,† Elena Morandi,* Uwe Vinkemeier,‡

Dumitru Constantin-Teodosiu,‡ Sophie Drinkwater,* Maureen Mee,‡ Lloyd King,*

Giulio Podda,* Guang-Xian Zhang,x Amir Ghaemmaghami,‡ Cris S. Constantinescu,*

Amit Bar-Or,† and Bruno Gran*

CD4+CD25hi FOXP3+ regulatory T cells (Tregs) maintain tolerance to self-Ags. Their defective function is involved in the path-

ogenesis of multiple sclerosis (MS), an inflammatory demyelinating disease of the CNS. However, the mechanisms of such defective

function are poorly understood. Recently, we reported that stimulation of TLR2, which is preferentially expressed by human

Tregs, reduces their suppressive function and skews them into a Th17-like phenotype. In this study, we tested the hypothesis that

TLR2 activation is involved in reduced Treg function in MS. We found that Tregs from MS patients expressed higher levels of

TLR2 compared with healthy controls, and stimulation with the synthetic lipopeptide Pam3Cys, an agonist of TLR1/2, reduced

Treg function and induced Th17 skewing in MS patient samples more than in healthy controls. These data provide a novel

mechanism underlying diminished Treg function in MS. Infections that activate TLR2 in vivo (specifically through TLR1/2

heterodimers) could shift the Treg/Th17 balance toward a proinflammatory state in MS, thereby promoting disease activity

and progression. The Journal of Immunology, 2015, 194: 000–000.

Reduced regulatory T cell (Treg) function has been asso-ciated with a number of autoimmune diseases, includingmultiple sclerosis (MS), an inflammatory demyelinating

disease of the CNS, which is thought to be initiated by myelin-reactive T cells (1, 2). Thymus-derived natural CD4+CD25hi

FOXP3+ Tregs (nTregs) play an important role in maintainingtolerance to self-Ags and preventing autoimmune responses. De-pletion of Tregs contributes to the induction of severe autoimmunediseases in animal models, and several studies reported a defect ofTregs in various human autoimmune diseases, including MS (1, 3–5). Tregs are characterized by their expression of the transcriptionfactor FOXP3, which is a master regulator in their development

and function (6). The levels of FOXP3 in the CD4+CD25hi pop-ulation were reported to be decreased in MS (5, 7). In addition,a reduced regulatory function of peripheral blood CD4+CD25hi

Tregs was shown in patients with MS compared with healthysubjects (1–3, 5).Recent studies succeeded in dividing human Tregs into more

homogenous subsets on the basis of cell surface marker expression.The most common approach to defining human Treg subsets isbased on combining CD25 and CD127 expression with expressionof the classic markers for naive (CD45RA) and memory (CD45RO)conventional T cells (8, 9). In addition, based on the expression ofCD25 and CD45RA, we (10) and other investigators (8) classifiedhuman CD4+ T cells into six subpopulations, fractions (Fr.) I–VI.Fr. I, II, and III are FOXP3+, and the degree of FOXP3 protein ex-pression is proportional to CD25 expression. Fr. I and II are highlysuppressive when cocultured with responder T cells (Tresps)(Fr. VI), whereas Fr. III cells are nonsuppressive (8, 10, 11).Several factors may be responsible for the loss of suppression

by Tregs, including the presence of proinflammatory stimuli asa result of clinical or subclinical infections. One such proin-flammatory stimulus is the cytokine IL-6 (12), which can reduceor abolish the suppressive function of mouse (13) and human (10,14) Tregs. Stimulation of IL-6R leads to activation of severaltranscription factors, most notably STAT3 (15). IL-6 and STAT3are also required for the commitment of naive T cells toward thedifferentiation of Th17 cells (16, 17). Th17 cells produce severalproinflammatory cytokines, including IL-17, IL-6, IL-21, IL-22,and TNF-a. Although Th17 cells play a key role in the protectionagainst bacterial infections, they also may mediate pathogenicityin organ-specific autoimmune diseases (18). Indeed, it was shownthat human Th17 lymphocytes can promote blood–brain barrierdisruption and kill human neurons in vitro, suggesting a patho-genic role in MS (19). IFN-b1a, a commonly used disease-modifyingimmunotherapy for patients with relapsing-remitting MS (RRMS)

*Clinical Neurology Research Group, Division of Clinical Neuroscience, Univer-sity of Nottingham School of Medicine, Nottingham NG7 2UH, United Kingdom;†Neuroimmunology Unit, Montreal Neurological Institute, McGill University, Mon-treal, Quebec H3A 2B4, Canada; ‡School of Life Sciences, University of NottinghamSchool of Medicine, Nottingham NG7 2UH, United Kingdom; and xDepartment ofNeurology, Thomas Jefferson University, Philadelphia, PA 19107

ORCID: 0000-0001-6384-2342 (B.G.).

Received for publication February 18, 2015. Accepted for publication April 19, 2015.

This work was supported in part by grants from the Multiple Sclerosis Society ofGreat Britain and Northern Ireland (Research Grant 863/07) and by the FondazioneItaliana Sclerosi Multipla (Research Grants 2011/R/21 and 2013/R/14) to B.G. M.H.N.was supported in part by a postdoctoral fellowship from the Multiple Sclerosis Societyof Canada (EGID:1655).

Address correspondence and reprint requests to Dr. Bruno Gran, Division of ClinicalNeuroscience, University of Nottingham School of Medicine, C Floor, South Block,Queen’s Medical Centre, Nottingham NG7 2UH, U.K. E-mail address: bruno.gran@nottingham.ac.uk

The online version of this article contains supplemental material.

Abbreviations used in this article: Fr., fraction; FSL-1, fibroblast-stimulating lipo-peptide-1; HC, healthy control; MS, multiple sclerosis; nTreg, naturally occurringCD4+CD25hi FOXP3+ Treg; Pam3Cys, Pam3CysSerLys4 (Pam3CSK4); RORC,RAR-related orphan receptor C; RRMS, relapsing-remitting MS; Treg, regulatoryT cell; Tresp, responder T cell.

Copyright� 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00

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(the phase of disease characterized by clinical relapses and remis-sions), is thought to exert at least part of its ameliorating effectthrough Th17 inhibition (20). In addition, complete abrogation ofrelapsing disease (both clinically and radiologically) after bonemarrow transplantation was associated with selective reduction ofTh17, but not Th1, responses (21).TLRs are pattern recognition receptors that play a central role in

the initiation of innate immunity against invading pathogens (22).Individually, or in combination with other TLRs, they recognizea spectrum of pathogen-associated molecular patterns, includinglipids, lipoproteins, nucleic acids, and proteins (22). TLR2 formsheterodimers with either TLR1 or TLR6. The identification ofTLRs on T cells, and particularly on Tregs, was an importantdevelopment in the field of innate immune-regulation of adaptiveT cell responses (10, 13, 23, 24). We (10) and others (14, 25)demonstrated that TLRs modulate the functions of Tregs. Weshowed that stimulation of TLR2 with Pam3CysSer(Lys)4(Pam3Cys), a synthetic triacylated lipopeptide agonist (26), reducesTreg suppressive function and induces them to release IL-17A andIL-17F (10). These TLR2-mediated effects on Tregs are dependent,at least in part, on IL-6, supporting an important role for this cy-tokine in balancing Treg and Th17 functions (27). Pam3Cys isa model for the effect of bacterial lipoproteins and, more generally,it is a strong and selective TLR2 agonist of target cell functions (28).In contrast to Pam3Cys, which stimulates heterodimers composed ofTLR2 and TLR1, diacylated lipopeptides, such as PAM2Cys-Ser-(Lys)4 and FSL-1, stimulate heterodimers composed of TLR2 andTLR6 (29). Of note, the latter are not effective modulators of Tregfunction (10, 14). The effect of TLR2 stimulation on the suppressivefunctions of Tregs from MS patients has not been studied. Becauseinfections are thought to influence both susceptibility to MS (30)and the occurrence of clinical exacerbations (31, 32), studies thatwould unravel the relationship among infections, TLR2 activation,and MS pathogenesis are warranted. We hypothesized that Tregs inpatients with MS are more susceptible to TLR2-induced modulationof their function and to Th17 differentiation than in healthy controls(HC). We compared the effect of TLR2 stimulation on the sup-pressive functions of CD4+CD25hiCD127neg/low Tregs and othersubsets (including CD4+CD25++CD45RA+ naive Tregs and CD4+

CD25+++CD45RA2 effector Tregs) in patients with RRMS and HC.A T cell–suppression assay was used to measure the suppressivefunction of Tregs (10). Stimulation of TLR2 in RRMS patients re-duced Treg suppressive function more potently than in HC. In ad-dition, Treg populations isolated from RRMS patients producedmore IL-17 and IL-22 than did those from HC upon TLR2 stimu-lation. CD4+ T cells from RRMS patients also expressed higherbaseline levels of IL-6, IL-6Ra, and p-STAT3, which was furtherenhanced after stimulation with Pam3Cys.Our results demonstrate that stimulation of TLR2 modulates the

Treg/Th17 balance in RRMS patients in favor of Th17 responses.This effect is more profound in MS patients than in HC. Collec-tively, these data support the hypothesis that, in MS, infections canmodulate Treg and Th17 cells through TLR stimulation and tilt theTreg/Th17 balance toward the proinflammatory Th17 phenotypeand function, resulting in disease exacerbation. Our study alsoprovides a novel mechanism underlying the reduced Treg functionin MS, reported by several groups (1–3, 5, 7).

Materials and MethodsStudy participants

MS patients attending outpatient clinics at NottinghamUniversity Hospitalswere recruited for this study. The study included 35 adult MS patients withclinically definite MS, according to the McDonald Criteria (33), aged 23–51 y (mean: 36.5 6 6.4 SEM). Patients had not been treated with any

immunomodulatory drugs or corticosteroids within 3 mo of study entry. Inaddition, 47 HC aged 23–52 y (mean: 34.0 6 8.3 SEM) were recruited.HC had no history of autoimmune disease or recent symptomatic infec-tions. There was no significant age difference between MS patients andHC (p = 0.153). All MS patients and HC gave written informed consentprior to blood sampling. The study was approved by the Nottingham Re-search Ethics Committee and by Nottingham University Hospitals Na-tional Health Service Trust Research and Innovation Services.

Purification of CD4+CD25hiCD127neg/low Tregs andsubpopulations of CD4+ T cells by FACS sorting

PBMCs were isolated from 50 ml peripheral blood from each study par-ticipant. CD4+ T cells were then isolated from PBMCs by negative selectionusing MACS MicroBeads (Miltenyi Biotec). CD4+ T cells were stainedwith CD4-allophycocyanin-Cy7 (BD Biosciences), CD25-PE (MiltenyiBiotec), and CD127-FITC (eBioscience) and then sorted into CD4+

CD25hiCD127neg/low Tregs and CD4+CD252CD127+ Tresps (SupplementalFig. 1A). In separate experiments, CD4+ T cells were stained with CD4-allophycocyanin-Cy7, CD25-PE, and CD45RA-FITC (eBioscience) andclassified into six subpopulations of CD4+ T cells (8, 10, 34). The followingpopulations of CD4+ T cell were sorted: CD4+CD25++CD45RA+ (naive orresting Tregs; Fr. I), CD4+CD25+++CD45RA2 (effector or activated Tregs;Fr. II), CD4+CD25++CD45RA2 (memory-like non-Tregs; Fr. III), and CD4+

CD252CD45RA+ (naive T cells; Fr. VI; in our coculture experiments thesecells are designated as Tresps). The other fractions consist of memory-likeCD4+CD45RA2FOXP32 non-Tregs (Fr. IV and V together), as previouslydescribed by our group (10) and other investigators (8) (Supplemental Fig.1B). The percentage frequency of each subset in RRMS patients (n = 7) andHC (n = 7) was as follows: CD4+CD25++CD45RA+ (naive or resting Tregs;Fr. I): HC = 2.1 6 0.5, RRMS = 2.2 6 0.8, p = 0.544; CD4+CD25+++

CD45RA2 (effector or activated Tregs; Fr. II): HC = 2.16 0.5, RRMS = 1.960.5, p = 0.361; CD4+CD25++CD45RA2 (memory-like non-Tregs; Fr. III):HC = 4.1 6 2.5, RRMS = 5.6 6 3.1, p = 0.019; CD4+CD25+CD45RA2

(memory-like non-Tregs, Fr. IV): HC = 13 6 1.5, RRMS = 13.2 6 1.4, p =0.507; CD4+CD252CD45RA2 (memory-like non-Tregs, Fr. V): HC = 29.1 61.5, RRMS = 28.5 6 2.6, p = 0.298; and CD4+CD252CD45RA+ (naiveTresps, Fr. VI): HC = 48.9 6 2.2, RRMS = 48.7 6 2.0, p = 0.508. We useda MoFlo XDP cell sorter (Beckman Coulter) in all cell-sorting assays.Purity of sorted cells was always .95% (Supplemental Fig. 1C, 1D).

Suppression assays

The suppressive functions of Tregs and the effect of TLR2 stimulation werestudied in coculture suppression assays. CD4+CD25hiCD127neg/low Tregs(2.5 3 104) and CD4+CD252CD127+ Tresps (2.5 3 104) were culturedalone or cocultured in triplicate wells at 1:16, 1:8, and 1:4 Treg/Trespratios. In separate experiments, CD4+CD25++CD45RA+ (naive Tregs, Fr. I)or CD4+CD25+++CD45RA2 (effector Tregs, Fr. II) T cells (2.5 3 104) andCD4+CD252CD45RA+ (naive Tresp, Fr. VI) T cells (2.5 3 104) werecultured alone or cocultured in triplicate wells at 1:16, 1:8, and 1:4 Treg/Tresp ratios. Cultures were carried out in the absence or presence ofPam3Cys (5 mg/ml; EMC Microcollections, T€ubingen, Germany). Cellswere cultured in RPMI 1640 medium supplemented with 5% FCS. Cul-tures were set up in triplicates, incubated for 6 d at 37˚C, and then pulsedwith 1 mCi [3H]thymidine (Perkin Elmer, Beaconsfield, Buckinghamshire,U.K.) for an additional 16 h of culture. Proliferation was assessed aspreviously described (10). The 5-mg/ml concentration of Pam3Cys waschosen after titration assays (data not shown).

Flow cytometric analysis of T cells

CD4+ enriched T cells, CD4+CD25hiCD127neg/low Tregs, and subpopula-tions of CD4+ T cells (CD4+CD25++CD45RA+ cells, Fr. I; CD4+CD25+++

CD45RA2 cells, Fr. II; CD4+CD25++CD45RA2 cells, Fr. III; CD4+CD25+

CD45RA2 cells, Fr. IV; CD4+CD252CD45RA2 cells, Fr. V; and CD4+

CD252CD45RA+ naive Tresp, Fr. VI) from MS patients and HC werestained freshly or after culture for 48 or 96 h on plate-bound anti-CD3 andanti-CD28 in the absence or presence of Pam3Cys or stimulation with theTLR2/6 agonist, fibroblast-stimulating ligand-1 (FSL-1) (both at 5 mg/ml).Where indicated, neutralizing Abs to TLR2 (10 mg/ml; R&D Systems), rIL-6 (5 ng/ml), or neutralizing anti–IL-6 (1 mg/ml; R&D Systems) were addedto cultures on day 0. Cells were surface stained with anti-TLR2, anti-TLR1,anti-TLR6, and anti-CCR6 (all from eBioscience), washed with FACSbuffer (PBS containing 1% FCS), and fixed in PBS containing 2% para-formaldehyde. For intracellular staining, cells were fixed and permeabilizedusing Fix/Perm buffer and stained with anti-FOXP3 (eBioscience), anti–IL-6, anti–IL-6Ra, anti-gp130, anti–IL-17A, anti–IFN-g, anti–IL-22, anti–T-bet (all from BD Biosciences), and anti–RAR-related orphan receptor C(RORC; R&D Systems). Appropriate isotype-matched control Abs were

2 TLR2 REGULATES THE Treg/Th17 BALANCE IN MS

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used in all FACS analyses. Cells were acquired using an LSR II flowcytometer (BD Biosciences), collecting a minimum of 1 3 105 events ineach sample, and analyzed using FlowJo software (version X.0.7; TreeStar).

Flow cytometric analysis of p-STAT3 (p-Y705) and p-STAT1(p-Y701) expression

CD4+ T cells and FACS-sorted subpopulations of CD4+ T cells (CD4+

CD25++CD45RA+ [naive Tregs], CD4+CD25+++CD45RA2 [effector Tregs],CD4+CD25++CD45RA2 [memory non-Tregs], and CD4+CD252CD45RA+

[naive Tresps]) were cultured for 1 h in the presence or absence of Pam3Cys(5 mg/ml) and FSL-1 (5 mg/ml), with or without neutralizing anti-TLR2 Ab(10 mg/ml; R&D Systems). Cells were fixed with 1.5% formaldehyde (finalconcentration) for 10 min at room temperature and then permeabilized in100% ice-cold methanol for 10 min at 4˚C. Cells were then washed twicewith FACS buffer (PBS with 1% FCS) and stained with PE-conjugatedp-STAT3 (Y705) and allophycocyanin-conjugated p-STAT1 (p-Y701) Abs(all from BD Biosciences) for 1 h at room temperature. Flow cytometricanalysis was performed using an LSR II flow cytometer (BD Biosciences)and FlowJo software (version X.0.7; TreeStar).

Analysis of cytokine production in culture supernatants

Human cytokine multiplex kits (eBioscience) were used to determine IL-17A, IL-17F, IL-21, IL-22, and IL-6 in the supernatants from cultures ofCD4+ T cells, CD4+CD25hiCD127neg/low Tregs, and/or subpopulations ofCD4+ T cells, according to the manufacturer’s instructions. Where indi-cated, neutralizing Abs to TLR2 (10 mg/ml; R&D Systems) were added tocultures on day 0.

Quantitative immunoblotting

CD4+ enriched T cells were cultured or not with anti-CD3 and anti-CD28Abs and stimulated for 2 or 6 h with recombinant human IL-6 (10 ng/ml)

or Pam3Cys (5 mg/ml), with or without neutralizing anti–IL-6 Ab (10 mg/ml;eBioscience). Protein lysates were prepared using whole-cell extractionbuffer A (pH 7.4) and SDS-PAGE and were immunoblotted, as previouslydescribed (35), using Abs specific for p-Y705–STAT3 (Cell SignalingTechnology), total STAT3 (Santa Cruz Biotechnology), or b-actin (Sigma-Aldrich). p-STAT3 was visualized with IR-800 secondary Ab, whereas totalSTAT3 was visualized with IR-680 secondary Ab and infrared detected andquantified with the Odyssey system (LI-COR Biosciences), as described(36). Bound anti–p-Abs were stripped off the nitrocellulose membrane byincubation of the blots in 25 mM glycine and 2% SDS (pH 2) for 1 h at 65˚Cand reprobed for total STAT3 and actin.

Statistical analysis

The mean (6 SEM) cpm measured by thymidine uptake of triplicate cul-tures was calculated for each coculture condition. All statistical analyseswere performed using GraphPad Prism 5 (GraphPad Software, San Diego,CA). Comparisons between groups were made using the Mann–WhitneyU test. The p values , 0.05 were considered significant.

ResultsHigher density of TLR2 expression by Treg populations frompatients with RRMS compared with HC

We recently reported that the human CD4+CD25hi Treg populationexpresses higher levels of TLR2 compared with non-Treg frac-tions (10). In this study, we compared the expression of TLR2 byCD4+CD25hiCD127neg/low Tregs from HC and patients with RRMS.We FACS sorted CD4+CD25hiCD127neg/low Tregs as previouslydescribed (10). First, the comparison showed that the expressionof TLR2 density in unstimulated Tregs was higher in patients than

FIGURE 1. Tregs from RRMS patients express

higher levels of TLR2 than do those from HC.

FACS-sorted CD4+CD25hiCD127neg/low Tregs and

subpopulations of CD4+ T cells from MS patients

and HC were stained for the expression of TLR2 ex

vivo or after 48 h of culture on plate-bound anti-

CD3 and anti-CD28 (1 mg/ml) in the absence or

presence of Pam3Cys or FSL-1 (5 mg/ml). Flow

cytometry dot plots from representative HC and

RRMS patients depicting TLR2 expression by ex

vivo CD4+CD25hiCD127neg/low Tregs (A) and cul-

tured CD4+CD25hiCD127neg/low Tregs (B). A min-

imum of 1 3 105 events was acquired in each

sample. The proportion of TLR2 expression by

freshly isolated CD4+CD25hiCD127neg/low Tregs (C)

and cultured CD4+CD25hiCD127neg/low Tregs (D)

was compared between HC and RRMS patients. The

proportion of TLR2-expressing (E), TLR1-express-

ing (F), and TLR6-expressing (G) CD4+CD25++

CD45RA+ (naive Tregs, Fr. I), CD4+CD25+++

CD45RA2 (effector Tregs, Fr. II), CD4+CD25++

CD45RA2 (memory non-Tregs, Fr. III), and CD4+

CD252CD45RA+ cells (naive Tresps, Fr. VI) was

compared between HC and RRMS patients. HC,

n = 8; RRMS, n = 9. *p, 0.05, **p, 0.01, ***p,0.001. ns, not significant.

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in HC (Fig. 1A, 1C; p = 0.032). Next, these cells were stimulatedfor 48 h by plate-bound anti-CD3 and anti-CD28 (1 mg/ml) in theabsence or presence of 5 mg/ml Pam3Cys (an agonist of TLR1/2heterodimers) or FSL-1 (which stimulates TLR2/6 heterodimers).Cells from patients expressed higher levels of TLR2 than did thosefrom HC (Fig. 1B, 1D; p = 0.024). In addition, the expression ofTLR2 was enhanced in both patients and controls after stimulationwith Pam3Cys (Fig. 1B, 1D; HC: p = 0.013; RRMS: p = 0.001),with higher levels observed in the MS group (Fig. 1D; p = 0.001).In contrast, stimulation with FSL-1 did not enhance TLR2 ex-pression in either group (Fig. 1D).We then compared the expression of TLR2, TLR1, and TLR6 in

subpopulations of CD4+ T cells, as defined on the basis of CD25and CD45RA expression (8, 10), between HC and patients. Inboth groups, there was a clear pattern of higher expression ofTLR2 in naive (Fr. I) and effector (Fr. II) Tregs and in memorynon-Tregs (Fr. III) compared with other T cell populations. Inaddition, Fr. I–III from patients expressed higher levels of TLR2compared with HC (Fig. 1E). The expression of TLR1 and TLR6also was higher in Treg than in non-Treg T cell subsets in bothpatients and HC. We also found that TLR1, but not TLR6, wasexpressed more in Treg fractions from MS patients than in HC(Fig. 1F, 1G). Together, these data demonstrate that Treg pop-ulations are the preferred target of TLR2 agonists and that higherexpression of TLR2 and TLR1 on Tregs from MS patients mayrender them more responsive to TLR2 agonists than those fromHC.

TLR2 stimulation preferentially reduces the suppressivefunctions of CD4+CD25hiCD127neg/low Tregs from RRMSpatients

We compared the effect of TLR2 stimulation on the suppressivefunctions of CD4+CD25hiCD127neg/low Tregs between MS patientsand HC. We were particularly interested in this subset of Tregsbecause a previous report showed that, when CD4+CD25hi T cellsexpressing the IL-7R a-chain (CD127) were included in the Tregpopulation, the suppressive function of such Tregs was weaker inMS patients than in controls, whereas the function of CD4+CD25hi

CD127neg/low Tregs did not differ between the two groups (37).We FACS sorted highly pure CD4+CD25hiCD127neg/low Treg

and CD4+CD252 Tresp subsets. Tregs from HC and patients withRRMS did not proliferate in response to plate-bound anti-CD3/anti-CD28, with or without Pam3Cys (data not shown). By con-trast, Tresps from both groups proliferated after stimulation withplate-bound anti-CD3/anti-CD28, and the presence of Pam3Cysdid not significantly increase such proliferation in either group(data not shown).In the absence of Pam3Cys, there was no significant difference in

the suppressive capacity of Tregs between MS and HC groups(Fig. 2), whereas stimulation with Pam3Cys reduced the sup-pressive activity of Tregs in both groups (Fig. 2). However, thiseffect was more potent in patients than in controls at the testedTreg/Tresp ratios (1:16, p = 0.004; 1:8, p = 0.021; and 1:4, p =0.041) (Fig. 2). These observations are consistent with the higherexpression of TLR2 by Treg populations in MS patients (Fig. 1).Together, these data show that TLR2-induced loss of suppressivefunction is more prominent in CD4+CD25hiCD127neg/low Tregsfrom MS patients compared with HC.

Naive and effector Tregs from patients with RRMS are moresusceptible to TLR2-mediated reduction of suppressivefunction

Our next aim was to examine the effect of TLR2 stimulation ondistinct subsets of human Tregs (8, 10). To this aim, we FACS sorted

highly pure subsets of CD45RA+CD252 naive Tresp, CD45RA+

CD25++ naive Tregs, and CD45RA2CD25+++ effector Tregs fromHC and patients with RRMS. Naive Tresps from both groups pro-liferated after stimulation with plate-bound anti-CD3/anti-CD28.The addition of Pam3Cys did not induce significant proliferationin either group (data not shown). By contrast, naive and effectorTregs from both groups did not proliferate after stimulation withplate-bound anti-CD3/anti-CD28 in the absence or presence ofPam3Cys (data not shown).To measure the effect of TLR2 stimulation on Treg suppres-

sion, naive Tregs or effector Tregs were cocultured with naiveTresp at 1:16, 1:8, and 1:4 ratios on plate-bound anti-CD3/anti-CD28 in the absence or presence of Pam3Cys. Naive Tregs fromHC and RRMS patients suppressed the proliferation of naiveTresps at 1:16, 1:8, and 1:4 ratios (Fig. 3A–C, Table I). Of note,naive Tregs from RRMS patients were less potent suppressors ofTresp proliferation (p = 0.024, p = 0.042, and p = 0.031 at 1:16,1:8, and 1:4 Tregs/naive T cell ratio, respectively; Fig. 3A–C,

FIGURE 2. TLR2 stimulation reduces the suppressive functions of

CD4+CD25hiCD127neg/low Tregs from both HC and MS patients. FACS-

sorted CD4+CD252CD127+ Tresps and CD4+CD25hiCD127neg/low Tregs

from HC or RRMS patients were cocultured in triplicate wells at a ratio of

1:16 (A), 1:8 (B), or 1:4 (C) in the absence or presence of Pam3Cys

(5 mg/ml) on plate-bound anti-CD3 and anti-CD28. Cells were pulsed

with [3H]thymidine for the last 16 h of the 6-d culture. Data represent

lymphocyte proliferation expressed as cpm. CD4+CD25hiCD127neg/low

Tregs from RRMS patients (n = 5) were more responsive to TLR2-induced

loss of their suppressive functions compared with CD4+CD25hiCD127neg/low

Tregs from HC (n = 5). *p , 0.05, **p , 0.01, ***p , 0.001. ns, not

significant.

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Table I). Although stimulation with Pam3Cys led to a reductionin the suppressive function of naive Tregs from both groups, asindicated by the increased proliferation of naive Tresps (HC: p =0.013, p = 0.022, and p = 0.011; RRMS: p = 0.001, p = 0.001,and p = 0.002 at 1:16, 1:8, and 1:4 naive Treg/naive T cell ratio,respectively; Fig. 3A–C, Table I), the magnitude of Pam3Cys-induced loss of Treg suppressive function was significantlygreater in patients than in HC (Fig. 3A–C, Table I). Similarly,

effector Tregs from both HC and patients with RRMS weresuppressive (Fig. 3D–F, Table II). Unlike naive Tregs, there wasno difference in the suppressive activity of effector Tregs be-tween HC and RRMS patients (Fig. 3D–F, Table II). However,stimulation with Pam3Cys caused enhanced reduction of thesuppressive function of effector Tregs from the RRMS group(Fig. 3D–F, Table II).

TLR2 stimulation preferentially enhances Th17 differentiationof Tregs from RRMS patients

Th17 cells play a pathogenic role in several autoimmune diseases,including MS (18, 38). We recently showed that TLR2 stimulation

enhances IL-17 production and promotes a Th17 shift in human

Tregs (10). We analyzed the effect of TLR2 stimulation on the

differentiation of CD4+CD25hiCD127neg/low Tregs (10, 37) after

TLR2 activation in both study groups. Stimulation with Pam3Cys

significantly enhanced the expression of IL-17A, RORC, and

CCR6 in Tregs isolated from patients (p = 0.031, p = 0.004, p =

0.002, respectively; Fig. 4A–C) and controls (p = 0.043, p = 0.012,

p = 0.032, respectively; Fig. 4A–C). However, the magnitude of

stimulation was significantly greater in MS patients (IL-17A, p =

0.033; RORC, p = 0.041; CCR6, p = 0.031; Fig. 4A–C).

FIGURE 3. TLR2 stimulation reduces the suppressive function of CD4+CD25++CD45RA+ naive and CD4+CD25+++CD45RA2 effector Tregs from

both HC and MS patients. FACS-sorted CD4+CD252CD45RA+ cells (naive Tresps, Fr. VI) were cocultured with CD4+CD25++CD45RA+ cells (naive

Tregs, Fr. I) or CD4+CD25+++CD45RA2 cells (effector Tregs, Fr. II) from HC and RRMS patients. Cells were cultured in triplicate wells at a naive

Treg/Tresp ratio of 1:16, 1:8, and 1:4, respectively (A–C) and at an effector Treg/Tresp ratio of 1:16, 1:8, and 1:4, respectively (D–F), in the absence

or presence of Pam3Cys (5 mg/ml) on plate-bound anti-CD3 and anti-CD28 (1 mg/ml). Cells were pulsed with [3H]thymidine for the last 16 h of

the 6-d culture. Data represent lymphocyte proliferation expressed as cpm. Naive and effector Tregs from MS patients (n = 11) were more sus-

ceptible to TLR2-induced loss of suppression than were those from HC (n = 13) (Tables I, II). *p , 0.05, **p , 0.01, ***p , 0.001. ns, not

significant.

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Next, we investigated the secretion of Th17 cytokines by Tregscultured in the same conditions. Treatment with Pam3Cys en-hanced the production of IL-17A, IL-17F, IL-22, and IL-6 by Tregsin both patients and controls (Fig. 4D–G). However, the effect inpatients was stronger than in controls (IL-17A, p = 0.032; IL-17F,p = 0.043; IL-22, p = 0.021; IL-6, p = 0.031; Fig. 4D–G). TLR2stimulation did not affect the Th1 signature cytokine, IFN-g, orthe Th1 transcription factor, T-bet, in either HC (10) or RRMSpatients (data not shown). Together, these data indicate that TLR2stimulation leads to enhanced expression and secretion of Th17markers in Treg populations, with a more potent effect in MSpatients.We then assessed the expression of Th17 markers in CD4+CD25++

CD45RA+ naive Tregs, CD4+CD25+++CD45RA2 effector Tregs,CD4+CD25++CD45RA2 memory non-Tregs (Fr. III), and CD4+

CD252CD45RA+ naive Tresps in both groups. The expression ofIL-17A and IL-22 was assessed in the described subpopulationsof CD4+ T cells after culture for 72 h in the presence or absence ofPam3Cys on plate-bound anti-CD3 and anti-CD28. Effector Tregsand memory non-Tregs in the MS group had higher baseline ex-pression of IL-17A (p = 0.013 and p = 0.021, respectively;Fig. 5B, 5C) and IL-22 (p = 0.0023 and p = 0.011, respectively;Fig. 5F, 5G) than HC. Treatment with Pam3Cys enhanced theexpression of IL-17A and IL-22 by naive Tregs, effector Tregs,and memory non-Tregs in both groups (for IL-17A HC: p = 0.012,p = 0.004, p = 0.004, respectively; RRMS patients: p = 0.0021, p =0.001, p = 0.002, respectively; Fig. 5A–C, Supplemental Fig. 4;for IL-22 HC: p = 0.006, p = 0.043, p = 0.011, respectively;RRMS patients: p = 0.002, p = 0.003, p = 0.005, respectively;Fig. 5E–G, Supplemental Fig. 4). Of note, the magnitude ofTLR2-induced IL-17 expression by naive Tregs, effector Tregs,and memory non-Tregs in RRMS patients was higher than inHC (p = 0.022, p = 0.027, p = 0.013, respectively; Fig. 5A–C,

Supplemental Fig. 4). The same pattern was found for IL-22 innaive and effector Tregs (p = 0.002 and p = 0.002, respectively;Fig. 5E, 5F, Supplemental Fig. 4). TLR2 stimulation did notinduce IL-17 and IL-22 expression in naive Tresps (Fig. 5D,5H). These data are consistent with observations by Miyaraet al. (8) showing low levels of RORC and AHR transcripts inFr. VI. More recent studies also demonstrated that memorynon-Tregs (Fr. III) produce IL-17 (11).We also studied the effect of TLR2 stimulation on Th17 dif-

ferentiation by CD4+ T cells and subpopulations of T cells byanalyzing mRNA expression of Th17 transcription factors, AHR,RORgt, and STAT3, and the Th1 transcription factor, T-bet, byRT-PCR (data not shown). CD4+ T cells or the indicated subsetswere cultured for 72 h on plate-bound anti-CD3 and anti-CD28Abs in the presence or absence of Pam3Cys. Following RNAextraction and cDNA synthesis, we performed RT-PCR and foundthat TLR2 stimulation augmented the expression of STAT3, AHR,and RORgt mRNAs, but not T-bet, in CD4+ T cells. TLR2 stim-ulation upregulated the expression of STAT3 mRNA in all T cellsubsets and the expression of AHR and RORgt mRNAs in naiveand effector Tregs and memory non-Tregs. Expression of T-betmRNA was not affected in the T cell subsets (data not shown).

TLR2 stimulation preferentially enhances IL-6 expression byCD4+ T cells and Treg subsets from RRMS patients

Although it is known that TLR2 stimulation enhances the pro-duction of IL-6 by Tregs in HC (10), we extended our previousinvestigation to T cells from RRMS patients. CD4+ T cells werecultured for 24 h on plate-bound anti-CD3 and anti-CD28 in thepresence or absence of Pam3Cys. CD4+ T cells from MS patientsexpressed higher baseline levels of IL-6 and IL-6Ra, and stimu-lation with Pam3Cys increased IL-6, IL-6Ra, and gp130 expres-sion in naive Tregs and CD4+ T cells from RRMS patients to

Table I. The effect of Pam3Cys on the suppressive functions of naive Tregs (Fr. I) from MS patients and HC

HC MS Patients

Naive Tregs/Tresp (1:16)

Naive Tregs/Tresps (1:8)

Naive Tregs/Tresp (1:4)

Naive Tregs/Tresp (1:16)

Naive Tregs/Tresps (1:8)

Naive Tregs/Tresp (1:4)

% Suppression (medium) 42.20 68.32 80.17 34.87 46.13 54.95% Suppression (Pam3Cys) 23.46 55.27 69.90 12.68 16.71 32.68% Loss of suppression 18.74a 13.05b 10.27c 31.06a 25.42b 22.27c

Data are expressed as average percentage of suppression in the presence or absence of Pam3Cys. Percentage of suppression was calculated using the following formula: [12(average cpm incorporated in the coculture)/cpm of responder population alone]3 100%. Percentage loss of suppression was calculated as follows: (percentage suppression withmedium 2 percentage suppression in the presence of Pam3Cys). Significance of the difference in the percentage loss of Treg suppression between HC (n = 13) and RRMSpatients (n = 11): 1:16, p = 0.01; 1:8, p = 0.04; 1:4, p = 0.03, paired t test.

aDifference in percentage loss of suppression between HC and MS patients at 1:16 naive Treg/Tresp ratio.bDifference in percentage loss of suppression between HC and MS patients at 1:8 naive Treg/Tresp ratio.cDifference in percentage loss of suppression between HC and MS patients at 1:4 naive Treg/Tresp ratio.

Table II. The effect of Pam3Cys on the suppressive functions of effector Tregs (Fr. II) from MS patients and HC

HC MS Patients

Effector Tregs/Tresps (1:16)

Effector Tregs/Tresps (1:8)

Effector Tregs/Tresp (1:4)

Effector Tregs/Tresps (1:16)

Effector Tregs/Tresps (1:8)

Effector Tregs/Tresp (1: 4)

% Suppression (medium) 51.34 65.17 86.17 55.36 61.38 77.92% Suppression (Pam3Cys) 29.67 46.82 71.36 16.17 26.32 49.54% Loss of suppression 23.67a 18.35b 14.81c 39.19a 35.06b 28.38c

Data are expressed as average percentage of suppression in the presence or absence of Pam3Cys. Percentage of suppression was calculated using the following formula: [12(average cpm incorporated in the coculture)/cpm of Tresp population alone] 3 100%. Percent loss of suppression was calculated as follows: (percentage suppression withmedium 2 percentage suppression in the presence of Pam3Cys). Significance of the difference in the percentage loss of Treg suppression between HC (n = 13) and RRMSpatients (n = 11): 1:16, p = 0.002; 1:8, p = 0.01; 1:4, p = 0.04, paired t test.

aDifference in percentage loss of suppression between HC and MS patients at 1:16 naive Treg/Tresp ratio.bDifference in percentage loss of suppression between HC and MS patients at 1:8 naive Treg/Tresp ratio.cDifference in percentage loss of suppression between HC and MS patients at 1:4 naive Treg/Tresp ratio

6 TLR2 REGULATES THE Treg/Th17 BALANCE IN MS

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a greater extent compared with the control group (Fig. 6B,Supplemental Fig. 2). Similarly, higher expression of IL-6 wasobserved in naive Tregs, effector Tregs, and memory non-Tregs(Fig. 6B–D). Neutralizing anti-TLR2, but not anti-TLR1, blockedthe effects of TLR2 activation by Pam3Cys (Fig. 6A–D). Bothanti-TLR2– and anti-TLR1–neutralizing Abs did not significantlyblock Pam3Cys-induced IL-6 expression in the memory non-Tregsubset (Fr. III) from RRMS patients (Fig. 6D). Stimulation withPam3Cys did not enhance IL-6 expression by naive Tresps (Fig.6E). Together, these data demonstrate greater background levels ofIL-6, as well as TLR2-induced IL-6 production, in CD4+ T cellsubsets in RRMS patients.

TLR2 stimulation induces p-STAT3 expression in CD4+ T cellsubsets

Because IL-6 activates STAT3 (15), we next assessed the effectof TLR2 stimulation on the phosphorylation of STAT3 protein.STAT3 is an important transcription factor known to bind both theIL-17A and IL-17F promoters (39). CD4+ T cells and FACS-sorted subpopulations of CD4+ T cells (CD4+CD25++CD45RA+

naive Tregs, CD4+CD25+++CD45RA2 effector Tregs, CD4+

CD25++CD45RA2 memory non-Tregs, and CD4+CD252CD45RA+

naive Tresps) from patients and controls were cultured for 1 h onplate-bound anti-CD3 and anti-CD28 in the absence or presenceof Pam3Cys and neutralizing anti-TLR2 and anti-TLR1. Stim-ulation with Pam3Cys significantly enhanced STAT3 phosphor-ylation by CD4+ T cells from both groups, as determined usingflow cytometry (HC: p = 0.003; RRMS: p = 0.002, Fig. 7A).Similar to the IL-6 data, neutralizing anti-TLR2 Abs, but notanti-TLR1 Abs, blocked Pam3Cys-induced STAT3 phosphory-lation (Fig. 7A).We next evaluated the effect of TLR2 stimulation on STAT3

phosphorylation in subpopulations of CD4+ T cells. Stimulationwith Pam3Cys increased phosphorylation of STAT3 by naive andeffector Tregs and memory non-Tregs in both groups (Fig. 7B–D).However, Pam3Cys did not enhance the expression of p-STAT3 bynaive Tresps from either HC or RRMS patients (Fig. 7E). Treat-ment with neutralizing anti-TLR2, but not anti-TLR1, reducedPam3Cys-induced STAT3 phosphorylation in naive and effectorTregs in both groups (Fig. 7B, 7C). Neutralizing anti-TLR2 and

FIGURE 4. TLR2 stimulation enhances IL-17A/F,

RORC, and CCR6 expression by CD4+CD25hi

CD127neg/low Tregs from RRMS patients and HC.

FACS-sorted CD4+CD25hiCD127neg/low Tregs from

HC and RRMS patients were cultured in the absence

or presence of Pam3Cys on plate-bound anti-CD3

and anti-CD28. Cultures were incubated for 96 h,

and the cells were harvested and stained for the ex-

pression of IL-17A (A), CCR6 (B), and RORC (C).

The percentage of cells expressing IL-17A, RORC,

and CCR6 was compared between HC and RRMS

patients. Culture supernatants were analyzed by

ELISA for the production of IL-17A (D), IL-17F (E),

IL-22 (F), and IL-6 (G). Cytokine production was

compared between the two groups. Data represent

average concentrations 6 SE of the indicated cyto-

kines and markers in n = 13 HC and n =15 RRMS

patients. *p , 0.05, **p , 0.01. ns, not significant.

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anti-TLR1 Abs failed to block Pam3Cys-induced p-STAT3 ex-pression by memory non-Tregs in both groups (Fig. 7D). Neutral-izing anti–IL-6 Abs blocked Pam3Cys-induced phosphorylation ofSTAT3 in CD4+ enriched T cells, as well as in naive and effectorTreg, memory non-Treg, and naive Tresp subsets (SupplementalFig. 3).Next, we confirmed our observation that Pam3Cys-induced

phosphorylation of STAT3 is IL-6 dependent using Westernblotting experiments. Enriched CD4+ T cells from HC were ac-

tivated with anti-CD3 and anti-CD28 in the presence or absence ofPam3Cys, IL-6, and a neutralizing anti–IL-6 Ab for 2 or 6 h.Neutralization of IL-6 blocked Pam3Cys-induced phosphoryla-tion of STAT3 at both time points. At 6 h, activation of the TCRwith costimulation through CD28 was sufficient to induce IL-6–dependent phosphorylation of STAT3 (Fig. 8), suggesting thatPam3Cys-induced IL-6 accelerates phosphorylation of STAT3 butis not strictly required (Fig. 8). Of note, we observed a degree ofinterindividual variability in STAT3 phosphorylation in response

FIGURE 5. TLR2 stimulation enhances IL-17A

and IL-22 expression by subpopulations of CD4+

T cells from RRMS patients and HC. FACS-sorted

CD4+CD25++CD45RA+ (naive Tregs, Fr. I), CD4+

CD25+++CD45RA2 (effector Tregs, Fr. II), CD4+

CD25++CD45RA2 (memory non-Tregs, Fr. III),

and CD4+CD252CD45RA+ (naive Tresps, Fr. VI)

cells from HC and RRMS patients were cultured

for 96 h in the absence or presence of Pam3Cys

(5 mg/ml) on plate-bound anti-CD3 and anti-CD28.

The proportion of IL-17–expressing (A–D) and IL-

22–expressing (E–H) CD4+CD25++CD45RA+ (na-

ive Tregs, Fr. I), CD4+CD25+++CD45RA2 (effector

Tregs, Fr. II), CD4+CD25++CD45RA2 (memory

non-Tregs, Fr. III), and CD4+CD252CD45RA+

(naive Tresps, Fr. VI) cells was compared between

HC and RRMS patients. Data obtained from n = 7

HC and n = 7 RRMS patients are shown. *p ,0.05, **p, 0.01, ***p, 0.001. ns, not significant.

8 TLR2 REGULATES THE Treg/Th17 BALANCE IN MS

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to Pam3Cys in T cell subsets from different subjects (data notshown).

Neutralization of TLR2 blocks Pam3Cys-induced, but notcytokine-induced, production of Th17 cytokines

Next, we investigated the mechanism of TLR2-induced differen-tiation of Tregs into Th17 cells in our system. For this purpose,CD4+ enriched T cells were cultured on plate-bound anti-CD3 andanti-CD28 for 72 h in the absence or presence of Pam3Cys orTh17-differentiation mixture of cytokine and Abs (10 ng/ml IL-1b, 30 ng/ml IL-6, 10 ng/ml IL-23, 0.5 ng/ml TGF-b, and 10 mg/ml neutralizing anti–IL-4 and anti–IFN-g), with or without 10 mg/ml neutralizing anti-TLR2 Ab. The expression of IL-17 and IL-22was assessed using flow cytometry, and culture supernatants wereassessed for the production of Th17-associated cytokines (IL-17A,IL-17F, IL-21, and IL-22). Consistent with the FACS data(Fig. 9A–C), supernatants of cells from RRMS patients containedhigher levels of the above cytokines than did cells isolated fromcontrols (Fig. 9D–G). We next tested whether a neutralizing anti-TLR2 Ab would block Th17 differentiation induced by the stan-dard Th17-differentiation mixture of cytokines and Abs in ourcohorts. The TLR2-neutralizing Ab blocked TLR2-induced Th17

differentiation (Fig. 9); however, it did not significantly neutralizeTh17 differentiation induced by the Th17-differentiation mixtureof cytokines and Abs described above (Fig. 9). Pam3Cys inducedgreater levels of IL-17A, IL-17F, IL-21, and IL-22 in patients thanin controls. However, the cytokine/Ab mixture induced similarlevels of Th17 cytokine production in both groups (Fig. 9D–G).Together, these data indicate that different pathways are activatedin the two differentiation protocols induced by Pam3Cys and byconventional Th17-inducing cytokines and Abs, and that a numberof Th17 cytokines are more potently induced by Pam3Cys inpatients than in controls. These data also suggest that cytokine-and TLR2-induced pathways of Th17 differentiation are distinct.

DiscussionIn this study, we investigated the mechanisms of action that un-derpin TLR2-induced loss of Treg activity and shift toward Th17-like phenotype and function in MS patients. TLR expression byTregs suggests that these cells may respond directly to microbialmolecular patterns, thus linking innate signals with adaptive reg-ulatory responses. Such regulation of adaptive immune responsesby innate stimuli may explain, in part, how systemic infectionsinfluence phases of immune activation (including clinical relapses)

FIGURE 6. TLR2 stimulation

enhances IL-6 expression by CD4+

enriched T cells and FACS-sorted

subpopulations of CD4+ T cells from

RRMS patients and HC. CD4+

enriched T cells and subpopulations

of CD4+ T cells from HC and RRMS

patients were cultured on plate-

bound anti-CD3/anti-CD28 in the

absence or presence of Pam3Cys,

with or without neutralizing anti-

TLR2 and anti-TLR1 Abs. Cells

were incubated for 24 h, harvested,

and stained for IL-6 expression. The

proportion of IL-6 expression by

CD4+ enriched T cells (A), CD4+

CD25++CD45RA+ cells (naive Tregs,

Fr. I) (B), CD4+CD25+++CD45RA2

cells (effector Tregs, Fr. II) (C), CD4+

CD25++CD45RA2 cells (memory

non-Tregs, Fr. III) (D), and CD4+

CD252CD45RA+ cells (naive Tresps,

Fr. VI) (E) was compared between

HC and RRMS patients. Data ob-

tained from n = 6 HC and n = 7

RRMS patients are shown, *p, 0.05,

**p , 0.01. ns, not significant.

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in immune-mediated diseases, such as MS. Our key findings are:(1) TLR2 is expressed at higher levels by Tregs of RRMS patientsthan Tregs of HC. Consistent with this observation, CD4+CD25hi

CD127neg/low Tregs, as well as naive and effector Tregs, fromRRMS patients were more susceptible to Pam3Cys-induced re-duction of suppressive functions; similar to our previous reportin HC, it is the TLR1/2 heterodimer that mediates the effectsof TLR2 stimulation in our experimental paradigm, rather thanTLR2/6 (10). (2) Induction of IL-6, a key mediator of TLR2effects on Tregs (10, 40), was more pronounced in the MS group.(3) Activation of the STAT3 signaling pathways by Pam3Cysrequires the secretion of IL-6 by target cells, and (4) people withMS may be more susceptible than HC to the effects of TLR2-induced Th17 differentiation. These findings have implications inthe mechanisms of infection-induced inflammatory activity inRRMS.Higher expression of TLR2 by T cells in MS patients suggests

that people with MS may be more susceptible than HC to theeffects of microbial stimuli on Treg populations. This is consistentwith our functional data showing that loss of suppressive activityinduced by TLR2 agonists is more prominent in the MS group.Moreover, the ability of Pam3Cys to upregulate the expression ofTLR2 in MS patients raises the possibility that specific infectiousagents could exert such effect in vivo. Of note, higher expression ofTLR2 and greater functional responsiveness to TLR2 agonists inMS patients compared with HC could account for the reportedreduced Treg function in MS patients (1, 5, 41). TLR2 is a rela-

tively flexible innate immune receptor, with broad recognitionpotential that is due, at least in part, to the formation of hetero-dimers with TLR1 and TLR6. The clear contrast between thebiological action of Pam3Cys, a triacylated agonist of TLR1/2,and the weak or absent activity of FSL-1, a diacylated agonist ofTLR2/6, in modulating human T cells could be useful in identi-fying specific types of microbial agonists that may be exertingmodulation of Treg function in vivo and, potentially, in definingtherapeutic targets (29).We examined the role of IL-6 in mediating the effects of TLR2

stimulation. The importance of this pleiotropic cytokine in in-flammatory demyelination is underscored by its high expression inthe CNS (42) and, specifically, in MS lesions at sites of activedemyelination (43). IL-6 is also one of few cytokines uncondi-tionally required for the development of experimental autoimmuneencephalomyelitis, an animal model of MS (44). With TGF-b, itpromotes the generation of Th17 cells while inhibiting TGF-b–induced Treg differentiation (45), thereby regulating the balancebetween Th17 cells and Tregs (27). IL-6 plays a critical role inmediating the effect of TLR2 on Treg function (10, 40). Inparticular, IL-6 is required for TLR2-induced reduction ofTreg suppressive function, as demonstrated by our neutralizationexperiments. Neutralization of IL-6 also reversed TLR2-inducedexpression of IL-17 and IL-22 in human Tregs (10). In addition toits direct effects on Tregs, IL-6 can enhance the resistance of ef-fector T cells to Treg-mediated inhibition in patients with MS (46,47). Our observation of greater production of IL-6 by Tregs upon

FIGURE 7. Pam3Cys-induced p-STAT3 expres-

sion is blocked by neutralizing anti-TLR2 but not

anti-TLR1. CD4+ enriched T cells and FACS-sorted

subpopulations of CD4+ T cells from RRMS

patients and HC were cultured in the absence or

presence of Pam3Cys, with or without neutralizing

anti-TLR2 and anti-TLR1 Abs, on plate-bound

anti-CD3 and anti-CD28. Cells were incubated for

1 h, harvested, and stained for the expression of

p-STAT3 by flow cytometry. The proportion of

p-STAT3 expression by CD4+ enriched T cells (A),

CD4+CD25++CD45RA+ cells (naive Tregs, Fr. I)

(B), CD4+CD25+++CD45RA2 cells (effector Tregs,

Fr. II) (C), CD4+CD25++CD45RA2 (memory non-

Tregs, Fr. III) cells (D), and CD4+CD252CD45RA+

cells (naive Tresps, Fr. VI) (E) was compared be-

tween HC and RRMS patients. Data obtained from

n = 6 HC and n = 7 RRMS patients are shown.

*p , 0.05, **p , 0.01, ***p , 0.001. ns, not

significant.

10 TLR2 REGULATES THE Treg/Th17 BALANCE IN MS

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stimulation with Pam3Cys in subjects with MS compared withcontrols suggests that IL-6 is essential in mediating TLR2-inducedanti-regulatory and proinflammatory signals in MS. Such signalsmay be delivered in vivo by microbial stimuli, and neutralizationof IL-6 may have therapeutic potential in MS as was demonstratedin other immune-mediated diseases, including juvenile arthritisand the MS-related neuroinflammatory disease, neuromyelitis optica(48, 49).We observed increased production and secretion of Th17-

associated cytokines and increased STAT3 protein phosphoryla-tion in CD4+ T cells and subpopulations of T cells from RRMSpatients in response to TLR2. Th17 cells are involved in thepathogenesis of autoimmune inflammatory demyelination andother organ-specific autoimmune diseases, particularly when theydifferentiate in the presence of TGF-b1 and IL-6 (50). The ex-pression levels of RORC and the production of IL-17 and IL-22 inresponse to TLR2 activation by human T cells (Fig. 4) (10) sug-gest that they could have pathogenic potential in vivo (50). Inhuman tissue, Th17 cells were identified in MS lesions (51), whereIL-17 production has been associated with active inflammation(52). Our finding that, similar to observations in the mouse (53),TLR2 activation can promote human Th17 differentiation suggeststhat peripheral activation of T cells in the presence of microbialTLR1/2 stimuli could drive them to a Th17 phenotype and func-tion. IL-22 has been associated with Th22, a Th subset that isexpanded in MS and can react to the myelin autoantigen MBP(54). Th22 cells expressed lower levels of IFNAR1 and were in-sensitive to IFN-b inhibition, suggesting that expansion of Th22cells in MS could influence resistance to IFN-b therapy (54).Because naturally occurring Tregs express higher levels of TLR2and TLR1 (10, 14, 24, 55), our data indicate that they may pref-erentially respond to microbial stimuli in vivo by switching toa Th17/Th22 phenotype and function. Plasticity of human Tregsalso was reported by Hafler and colleagues, who observed that

FOXP3+ Tregs cultured in the presence of IL-12 acquired a Th1-like phenotype associated with reduced suppressive activity (41).Of relevance to MS, human Th17 cells expressing IL-17 and IL-22(Figs. 5, 9) have the capacity to cross the blood–cerebrospinalfluid barrier (19) and, therefore, could infiltrate the CNS andinitiate tissue-specific inflammatory damage.To further elucidate the mechanisms by which TLR2-induced

IL-6 modulates T cell function, we studied the phosphorylationof the transcription factor STAT3 in response to TLR2 activation.STAT3 is a key mediator of IL-6–induced signaling and of Th17-differentiation programs (50, 56). Phosphorylation of STAT3 wasinduced by TLR2 activation and found to be IL-6 dependent inneutralization experiments (Figs. 7, 8, Supplemental Fig. 3). Innaive and effector Tregs, phosphorylation was significantly greaterin MS patients compared with HC (Fig. 7A–C), suggesting thatthese cells may be particularly susceptible to TLR2 stimuli in MS.Increased STAT3 signaling was observed in experimental CNSinflammatory diseases (16, 57), with a critical role played by itsexpression in CD4+ T cells (16). In addition, STAT3 phosphory-lation in PBMCs was correlated with disease activity (58) and inmediating resistance of effector T cells to Treg-mediated inhibi-tion in MS (46). These data suggest that inhibition of IL-6/STAT3signaling can be a rational therapeutic strategy in MS. However,similar to previous reports, we observed a degree of variability inresponsiveness to TLR2 stimuli in different individuals, possiblydue to donor-dependent differences in the TLR expression pattern(10, 14) or polymorphisms in TLR2 or TLR1 (59, 60). Thissuggests that TLR-targeted treatments would need preliminarytesting of TLR expression and responsiveness in individualpatients.Pam3Cys stimulation did not significantly affect the expression

of the Th1 transcription factor T-bet and the production of IFN-g(data not shown). This is in contrast to the clear effects on Tregand Th17 phenotype and function. These data indicate that TLR2

FIGURE 8. Pam3Cys-induced p-STAT3

expression is reduced by neutralizing anti–

IL-6. CD4+ T cells were cultured or not

with anti-CD3 and anti-CD28 (1 mg/ml) and

stimulated for 2 and 6 h with recombinant

human IL-6 (10 ng/ml) or Pam3Cys (5 mg/

ml) in the presence or absence of neutral-

izing anti–IL-6. (A) Protein lysates were

prepared and probed with Abs specific for

p-Y705 (activated) STAT3, total STAT3,

and b-actin. One representative blot of four

experiments in four subjects is shown. (B)

Bar graphs show the ratio between the

quantification of p-STAT3 and total STAT3.

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signaling preferentially enhances Th17 differentiation withoutsignificantly altering IFN-g production (10, 53). This suggests

that, in MS, IFN-g is not directly involved in TLR2-induced re-duction of Treg function, an effect it can play in response to IL-12

FIGURE 9. Treatment with neutralizing anti-TLR2 reduces Pam3Cys-mediated production of Th17-associated cytokines. CD4+ enriched T cells were cultured

on plate-bound anti-CD3 and anti-CD28 for 72 h in the absence or presence of 5 mg/ml Pam3Cys or Th17 mixture of cytokine and Abs (10 ng/ml IL-1b, 30 ng/ml

IL-6, 10 ng/ml IL-23, 0.5 ng/ml TGF-b, and 10 mg/ml neutralizing anti–IL-4 and anti–IFN-g), with or without 10 mg/ml neutralizing anti-TLR2 Ab. (A) Flow

cytometry dot plots from representative HC and RRMS patients depicting IL-17 and IL-22 expression by CD4+ enriched T cells. The percent expression of IL-17

(B) and IL-22 (C) was compared between HC and RRMS patients. Culture supernatants were examined by ELISA for the production of IL-17A (D), IL-17F (E),

IL-21 (F), and IL-22 (G). Data obtained from n = 6 HC and n = 7 RRMS patients are shown. *p , 0.05, **p , 0.01, ***p , 0.001. ns, not significant.

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(41). The observation that TLR2 neutralization blocked Pam3Cys-induced, but not cytokine-induced, Th17 differentiation (Fig. 9)suggests that distinct pathways are involved in Th17 differentia-tion. We hypothesize that cytokine-induced Th17 developmentrecruits Th17 transcription factors in a combinatorial program thatis different from that induced by TLR2 activation and could befurther elucidated by transcriptional regulation studies (56).Our present findings provide a possible link between urinary and

respiratory infections and T cell dysregulation that may lead to MSclinical exacerbations (relapses) (31, 32, 61, 62), because the cellwalls or outer membranes of Gram-negative bacteria, such asEscherichia coli, as well as Staphylococcus saprophyticus andother Gram-positive bacteria, which are typically involved (61,62), contain TLR2 agonists (29, 63). In particular, most in vivorelevant lipopeptides are triacylated and, therefore, are adequatelymodeled by Pam3Cys used in our study (28, 64). Respiratoryviruses that can activate TLR2 (29, 63) also have been associatedwith MS exacerbations (31, 32, 61). In addition, EBV, which isstrongly associated with MS susceptibility (30), is able to activatethe transcription factor NF-kB and the production of the chemo-kine CCL2 in a TLR2-dependent manner (65, 66).In conclusion, our data suggest that the occurrence of relapses in

RRMS patients following specific bacterial or viral infections maybe facilitated by stimulation of TLR2 on Tregs, leading to reductionof their suppressive functions and differentiation into a pathogenicTh17 lineage that can mediate tissue damage. The observation thatbackground levels of TLR2 and its heterodimeric partner TLR1 arehigher in the MS group and that stimulation with TLR2 agonistsleads to upregulation of the receptor indicate that microbial stimulimay be driving the higher levels of TLR2 in MS patients in vivo.We propose that, during infections, TLR2 could promote auto-immune responses by inducing IL-6 and tilting the balance betweenTregs and Th17 cells toward the latter (10, 27). Thus, TLR2 effectson IL-6 and STAT3 could underpin such effects of infectionsin vivo. Therefore, acute infections could facilitate disease ex-acerbations, whereas repeated infections may contribute to thegradual disease progression that is commonly observed in MSpatients (67).

AcknowledgmentsWe thank all MS patients and healthy volunteers for blood donations, as

well as Dr. Adrian Robins and Dr. David Onion for expert advice on flow

cytometry and cell sorting. We also thank Dr. Peter J. Darlington (Depart-

ments of Exercise Science and Biology, Concordia University, Montreal,

QC, Canada) for critical reading of the manuscript.

DisclosuresThe authors have no financial conflicts of interest.

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