Published August 23, 2013, doi:10.4049/jimmunol.1301110 The Journal of Immunology

Parasite Infections in Multiple Sclerosis Modulate Immune Responses through a –Dependent Pathway

Jorge Correale and Mauricio F. Farez

We recently demonstrated better outcomes in helminth-infected multiple sclerosis (MS) patients, compared with uninfected ones. The present study evaluates the role of TLR2 and retinoic acid (RA) in parasite-driven protection in MS patients. RA serum levels were significantly higher in helminth-infected MS patients than in uninfected MS subjects or healthy controls. Genes involved in RA biosynthesis and metabolism, such as Adh1 and Raldh2, as well as RA receptors and IL-10, were induced in dendritic cells (DCs) via TLR2-dependent ERK signaling. This programmed DCs to induce FOXP3+ T regulatory cells and suppressed production of proinflammatory cytokines (IL-6, IL-12, IL-23, and TNF-a) via induction of suppressor of cytokine signaling 3 (SOCS3), an effect mediated by soluble egg Ag (SEA) obtained from Schistosoma mansoni, and by RA. SEA-activated DCs also inhibited IL-17 and IFN-g production through autoreactive T cells. These inhibitory effects were abrogated when SOCS3 gene expression was silenced, indicating that SEA-mediated signaling inhibited production of these cytokines by T cells, through a SOCS3-dependent pathway. Overall, helminth-related immunomodulation observed in MS patients was mediated by TLR2- and RA-dependent path- ways, through two different mechanisms, as follows: 1) induction of IL-10 and FOXP3+ T regulatory cells, and 2) suppression of proinflammatory cytokine production mediated by SOCS3. The Journal of Immunology, 2013, 191: 000–000.

ultiple sclerosis (MS) is an inflammatory demyelin- once a critical threshold of T. trichiura prevalence (∼10%) is ating disease affecting the CNS. Although its etiology exceeded (6). In line with these observations, pre-established M remains unknown, several lines of evidence indicate Schistosoma mansoni infection in mice or pretreatment with S. autoimmunity plays a major role in disease susceptibility and mansoni OVA significantly reduces incidence, delays onset, and development (1). Autoimmune diseases are currently considered attenuates clinical course of experimental autoimmune encepha- to be the result of both genetic susceptibility and environmental lomyelitis (EAE) in mice (7, 8). Likewise, we recently demon- factors (2, 3). The genetic component of MS is thought to derive strated that helminth-infected MS patients showed significantly from the presence of common allelic variants in several genes (2). lower number of relapses, minimal changes in disability scores, However, discordance of MS between monozygotic twins suggests and significantly lower lesion activity on magnetic resonance additional factors, such as environmental modulators, are also imaging (MRI) compared with uninfected individuals with MS involved (4). (9). Extended follow-up also showed that, after antihelminth drug Epidemiological data provide strong evidence of a steady rise in administration, clinical and radiological activity increased to levels MS incidence in developed countries in recent decades, concomitant observed in uninfected MS subjects (10). with decreased incidence of many infectious diseases resulting Retinoic acid (RA) is an active metabolite of (also from antibiotic use, vaccination, or improved hygiene and socio- known as ), which has multiple isoforms; however, the all- economic conditions (5). Supporting these findings, recent inves- trans isoform predominates in most tissues and is known to be tigations demonstrate a dichotomous relationship between regional important in a variety of physiological processes, including im- distribution of MS and that of the parasite Trichuris trichiura, munological responses (11). RA mediates these activities by a common human pathogen. MS prevalence appears to fall steeply binding to nuclear receptors, members of the steroid superfamily, and altering transcription activity. Two families of receptors have been identified, RA receptors Department of Neurology, Dr. Rau´l Carrea Institute for Neurological Research, Foundation for the Fight against Infant Neurological Illnesses, 1428 Buenos Aires, (RAR) and retinoid X receptors. Each receptor family has at least Argentina three subtypes (a, b, and g), which act as activated ligand tran- Received for publication April 25, 2013. Accepted for publication July 23, 2013. scription factors controlling the expression of a number of target This work was supported by an internal grant from the Dr. Rau´l Carrea Institute for genes (12, 13). In recent years, RA has received particular at- Neurological Research, Foundation for the Fight against Infant Neurological Illnesses tention as it has been shown to influence multiple immune cell (to J.C.). lineages and to modulate a broad range of immune processes, such Address correspondence and reprint requests to Dr. Jorge Correale, Rau´l Carrea as lymphocyte activation and proliferation, Th cell differentiation, Institute for Neurological Research, Foundation for the Fight against Infant Neuro- logical Illnesses, Montan˜eses 2325, 1428 Buenos Aires, Argentina. E-mail address: tissue-specific lymphocyte homing, and production of specific Ab jcorreale@fleni.org.ar isotypes (11, 14, 15). The online version of this article contains supplemental material. Different combinations of TLRs are expressed on many cells of Abbreviations used in this article: Adh, alcohol dehydrogenase; CSF, cerebrospinal the immune system. Helminth parasite infections may alter the fluid; DC, dendritic cell; EAE, experimental autoimmune encephalomyelitis; MBP, regulation, function, and levels of expression of these receptors. myelin basic protein; MOG, myelin oligodendrocyte glycoprotein; MRI, magnetic resonance imaging; MS, multiple sclerosis; OIND, other inflammatory neurologic Our group has provided evidence indicating that surface expression disease; RA, retinoic acid; Raldh, retinol dehydrogenase; RAR, RA receptor; rhIL-2, of TLR2 on both B cells and dendritic cells (DCs) is significantly human rIL-2; SEA, soluble egg Ag; siRNA, small interfering RNA; SOCS, suppres- higher in helminth-infected MS patients. Exposure of either cell sor of cytokine signaling; TCL, T cell line; Treg, T regulatory. population to soluble egg Ag (SEA) obtained from S. mansoni Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 resulted in significant upregulation of TLR2 in helminth-infected

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301110 2 PARASITE INFECTIONS MODULATE MS THROUGH RA

MS patients, but not in uninfected individuals (16). In different RA serum level assay experimental settings, some authors have found similar results, RA serum levels were measured using commercially available ELISA kits indicating higher expression of TLR2 on PBMCs and enhanced (MyBioSource, San Diego, CA), according to manufacturer instructions. response to TLR2 ligands in parasite-infected individuals, com- Assay sensitivity level was 0.4 ng/ml. Intra- and interassay variation coeffi- pared with uninfected counterparts (17, 18). However, recent studies cients were 5.1 and 4.8%, respectively. using DCs from TLR22/2 and TLR42/2 mice demonstrated that Ag preparation TLR2 and TLR4 were not required for SEA-pulsed DCs to induce anti-inflammatory responses in naive mice, suggesting other re- Myelin basic protein (MBP)83–102, MBP143–168, and myelin oligodendro- ceptors, such as C-type lectins, might be implicated in DC response cyte glycoprotein (MOG)63–87 peptides were synthesized with an auto- mated peptide synthesizer using FAST-MOC chemistry, and expected peptide to SEA (19). Discrepancies between these investigations may be amino acid composition confirmed by HPLC. due to the presence of different moieties in SEA preparations or, The SEA was prepared aseptically, as previously described elsewhere alternatively, to differences in parasite Ag recognition mechanisms (21), and used at a concentration of 50 mg/ml. between human and mouse TLR2 (20). We present results from several studies specifically conducted to Isolation of DCs and generation of monocyte-derived DCs evaluate the role of RA on modulation of various immunological PBMCs were isolated from heparinized venous blood through Ficoll- pathways, under helminth-driven protection observed in MS patients. Hypaque (Pharmacia LKB Biotechnology, Piscataway, NJ) density gradi- ent centrifugation, and for some experiments DCs were purified with a blood isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany), in accor- Materials and Methods dance with manufacturer instructions to a purity of 95–98%. Because of the Patients large number of DCs required to perform the experiments described in this investigation (quantities not readily obtainable through venipuncture or Fifteen patients (10 females and 5 males) with clinical diagnosis of MS leukapheresis), in vitro DC cultures were established from monocytes, according to Poser’s criteria and presenting eosinophilia were assessed in generating sufficient cell numbers. Monocyte-derived DCs were therefore a prospective double-cohort study, as previously reported elsewhere (9). generated from PBMCs, as previously described (22). Briefly, monocytes Intestinal parasites were present in stool samples from all patients and were selected from PBMCs using anti-CD14–coated magnetic beads identified as the cause of eosinophilia. Three patients were infected with (Invitrogen, Carlsbad, CA) and cultured in the presence of 100 ng/ml GM- Hymenolepis nana, three with T. trichiura, three with Ascaris lumbricoides, CSF and 50 ng/ml IL-4 (R&D Systems, Minneapolis, MN). On days 2 and three with S. mansoni, two with Strongyloides stercolaris, and one with 4, half the culture medium was replaced, maintaining the same IL-4 and Enterobius vermicularis. Presence of other endemic parasitoses, including GM-CSF concentrations. After 5 d in culture, 100 ng/ml LPS was added to trypanosomiasis, leishmaniasis, amebiasis, giardiasis, and toxoplasmosis, stimulate maturation. In some experiments, maturation was achieved using was ruled out in this study population using microscopic stool examination 2 mg/ml CD40L (Amgen, Thousand Oaks, CA). Cells were collected on 6 and serological tests. Mean patients’ age was 32.9 7.4 y, Kurtzke ex- day 7, and, prior to use in experiments, extensively washed in PBS to 6 panded disability status scale score was 2.5 0.6, and mean disease du- remove exogenously added cytokines or residual LPS. CD3+ T lymphocyte 6 ration 8.7 1.9 y. MS diagnosis preceded parasitic intestinal infection contamination was ,0.3%. Although monocyte-derived DCs may not be 6 diagnosis by 28.3 6.1 mo (range 20–41 months). Fifteen healthy sub- fully representative of their ex vivo isolated counterparts, control experi- jects, 15 MS subjects in remission without helminth, and 12 helminth- ments performed using both cell populations showed monocyte-derived infected subjects without MS, matched for age, sex, and disease dura- DCs maintained specific imprinting capacity and characteristics similar tion, served as controls. Healthy individuals were recruited among family to ex vivo isolated DCs, leading us to believe that results observed were members of helminth-infected MS patients. Prior clinical and neurologic not the product of in vitro culture manipulation during DC isolation. examination as well as standard and hematological laboratory tests ruled out presence of any other underlying conditions. Uninfected MS control Generation of MBP and MOG peptide-reactive T cell lines subjects included in this study presented clinical course profiles similar to those of other MS patients at our center. Both eosinophil counts and stool MBP and MOG peptide-specific T cell lines (TCLs) were expanded from examination performed in uninfected MS subjects and healthy controls peripheral blood, as previously described elsewhere (23). Briefly, 5 3 106 were negative at study entry, and remained so throughout the duration of PBMCs were stimulated with optimal concentrations of MBP or MOG the study, indicating these individuals were not asymptomatically infected. peptides (10 mg/ml). After 5–7 d, cells were recultured in fresh medium Helminths infecting subjects without MS included the following: T. tri- containing 50 U/ml human rIL-2 (rhIL-2; R&D Systems, Minneapolis, chiura, three cases; A. lumbricoides, three cases; S. stercolaris, two cases; MN) for an additional week. Restimulation cycles were repeated weekly and S. mansoni, four cases. using autologous irradiated PBMCs (3000 rads) as APCs, plus peptide, No patients had received steroids for at least 3 mo prior to study entry, or followed by expansion with rhIL-2. After four cycles of restimulation and other immunomodulatory, immunosuppressive drugs or dietary supple- expansion, TCLs were evaluated using standard proliferation assays. ments. Because none of the patients developed clinical disease as a result of Cutoff values for positive response were set at a stimulation index .3. All the parasite infection, no antiparasite therapy had been prescribed at study MBP- and MOG-reactive TCLs were .93% CD4+. entry. After 60–78 mo of follow-up, six patients required antiparasite treatment for helminth symptom exacerbation (fever, abdominal pain, Expression of alcohol dehydrogenase 1, retinol dehydrogenase anorexia, weight loss, general malaise, diarrhea, or anemia). None of the 2, and retinoic receptors on DCs patients had clinical evidence of extraintestinal disease (e.g., pulmonary involvement). Two patients infected with A. lumbricoides and two patients For quantitative assessment of relative mRNA levels, total RNAwas isolated infected with T. trichiura received a single dose of albendazole (400 mg), from DCs using TRIzol LS reagent (Invitrogen) following manufacturer whereas two patients infected with S. stercolaris were treated with two instructions. RNAwas reverse transcribed using a Moloney murine leukemia doses of ivermectine (0.2 mg/kg) separated by 2-wk interval. virus reverse-transcription kit with random hexamer primers (Invitrogen). Cerebrospinal fluid (CSF) samples were available from eight helminth- Relative levels of alcohol dehydrogenase (Adh1), retinol dehydrogenase infected MS patients. None had received antiparasite treatment prior to CSF (Raldh2), retinoic receptor a, and retinoic receptor g mRNA were deter- collection. Ten individuals (7 women and 3 men, mean age 34.7 6 6.8 y) mined by real-time PCR, on an ABI 7000 sequence detection system admitted for extra spinal orthopedic surgery served as controls. In this (Applied Biosystems, Foster City, CA). Values obtained were normalized to group, CSF samples were obtained through lumbar tap draw prior to spinal the amount of GAPDH. Primer sequences used were as follows: GAPDH, anesthesia injection. A second control group consisting of 10 patients (7 sense 59-GAAGGTGAAGTCGGAGTC-39, antisense 59-GAAGATGGT- women and 3 men, mean age 37.5 6 7.9 y) suffering from other inflam- GATGGGATTTC-39; Adh1, sense 59-AACTCCTCTCACTGCTCTCCAC- matory neurologic diseases (OIND) is as follows: 5 cases of viral en- 39, antisense 59-GTCACCCTCTTCAGATTGCTTTTCC-39; Raldh2, sense cephalitis, 2 cases of aseptic meningitis, and 3 cases of CNS vasculitis 59-TACTTCAGAACGGGAATGACAAAC-39, antisense 59-CCAGTGAT- were also included. GTAGGTAAATAAGATAGAGGG-39; RARa, sense 59- GGTCGGCGA- This study was approved by the Institutional Ethics Committee of the TGGTGAGGGT-39, antisense 59-TGGGCAAATACACTACGAACAACA- Rau´l Carrea Institute for Neurological Research, and participants gave G-39; RARg, sense 59-CGCCGAAGCATCCAGAAGAAC-39, antisense written informed consent. 59-GGTGACAGGGTCGTTCGGCG-39. The Journal of Immunology 3

Induction of CD4+CD25+FOXP3+ regulatory T cells for TLR2, TLR5, TLR9, SOCS1, and SOCS3, or with scrambled non-

+ + + silencing control oligonucleotides using TransIT-TKO siRNA transfection To determine the role of SEA and RA in CD4 CD25 FOXP3 T regulatory reagent (Mirus Bio, Madison, WI), according to manufacturer instructions. (Treg) induction from naive cells, CD4+ T cells were isolated from PBMCs + 2 The sequence of siRNA strands used to silence the TLR2 gene was sense 59- using a CD4 T cell isolation kit (Miltenyi Biotec). CD25 cells were then GCACUUUAUAUUCACUUACtt-39, antisense 59-GUAAGUGAAUAUA- isolated by negative selection with CD25 microbeads (Miltenyi Biotec). AAGUGCtc-39; for TLR5 was sense 59-GGAGCAAUUUCCAACUUA- . + 2 3 3 Purity was determined to be 98% for CD4 CD25 T cells. Next, 5 10 Utt-39, antisense 59-AUAAGUUGGAAAUUGCUCCtt-39; for TLR9 was DCs were stimulated with SEA (50 mg/ml), and 12 h later, washed and sense 59-GACGGCAACUGUUAUUACAtt-39, antisense 59-UGUAAUA- cocultured at 1:10 ratio (DCs to T cells) together with 5 3 104 autologous + 2 ACAGUUGCCGUCtt-39; for SOCS3 was sense, 59-CCAAGAACCUG- naive CD4 CD25 T cells. Cultures were stimulated with soluble anti- CGCAUCCAdTdT-39,antisense,59-UGGAUGCGCAGGUUCUUGGdT- human CD3 mAb and soluble anti-CD28 Abs (BD Biosciences, San Jose, dT-39; and for SOCS1 was sense 59-GCAUCCGCGUGCACUUUCAUU- CA), both at 5 mg/ml, in the presence of rhIL-2 (400 U/ml; R&D Systems) 39, antisense 59-AAUGAAAGUGCACGCGGAUGC-39. and human rTGF-b (5 ng/ml; R&D Systems), in the presence and absence of retinol (500 nM; Sigma-Aldrich, St. Louis, MO). After 5 d, the number Evaluation of MAPK signaling of CD4+CD25+FOXP3+ Treg was evaluated by flow cytometry, using commercially available regulatory T cell staining kits, following manu- p38, ERK1/2, and JNK1/2 activities were evaluated using commercially 3 6 facturer instructions (eBioscience, San Diego, CA). available ELISA kits (Invitrogen). Briefly, DCs (1.5–2 10 ) were col- lected on day 7 and cultured for pre-established periods of time with Measurement of cytokine production different stimuli. ELISA tests were performed following the manufacturer instructions. For inhibition studies, DCs were incubated with the specific MBP and MOG peptide-specific TCLs were stimulated with the cognate ERK1/2 inhibitor, U0126 (2 mM; Calbiochem), for 1 h before stimulation. Ag, and with DCs previously incubated with SEA, or SEA plus retinol during 12 h. Forty-eight hours later, supernatants were collected, and IL-17 and Statistical analysis IFN-g production was assessed by ELISA using commercially available kits Differences observed between groups were evaluated using the Mann- (R&D Systems) and following manufacturer instructions. To measure LPS- , or SEA-stimulated DC cytokine production, cells were cultured in the Whitney U test. The p values 0.05 were considered statistically sig- presence and in the absence of retinol. Supernatants were collected 48 h nificant. poststimulation, and IL-6, IL-10, IL-12 p70, IL-23, IL-27, and TNF-a were measured using commercially available ELISA kits, purchased from Results R&D Systems following manufacturer instructions. RA serum levels are increased in parasite-infected MS patients Small interfering RNA technique As illustrated in Fig. 1, RA serum levels were significantly higher TLR, SOCS1, and SOCS3 expression were silenced using the small in- in parasite-infected MS patients (11.2 6 0.7 ng/ml), compared terfering RNA (siRNA) technique. DCs were transfected with 25 nM siRNA with levels in healthy controls (3.7 6 0.3 ng/ml; p , 0.0001), in

FIGURE 1. Parasite-infected MS patients showed higher serum levels of RA compared with healthy controls, uninfected MS patients, and treated parasite- infected MS patients (p , 0.001). Each circle repre- sents serum levels of RA from a single individual. Horizontal lines indicate mean group values. In six parasite-infected MS patients, serum samples were obtained before and after antiparasite treatment. 4 PARASITE INFECTIONS MODULATE MS THROUGH RA uninfected MS patients (3.3 6 0.3 ng/ml; p , 0.0001), or in Because cell exposure to SEA resulted in significant TLR2 treated parasite-infected MS patients (3.7 6 0.4 ng/ml; p , upregulation, we next examined the possible role of TLR2 in the 0.0001). induction of Adh1, Raldh2, or RARs in DCs. Silencing of TLR2 The limited number of patients infected with each different type using the siRNA technique led to significant inhibition of Adh1, of parasite precludes appropriate statistical analysis regarding Raldh2, and RAR expression induced by SEA in DCs (p , impact of specific helminth infections on RA serum levels and 0.0001). Furthermore, neither the TLR9 ligand CpG 1668 nor the immunological responses. TLR5 ligand flagellin induced the enzymes. In contrast, Pam3Cys, another TLR2-specific ligand, did induce substantial expression of SEA induces expression of Adh1, Raldh2, and RARs on DCs Adh1 and Raldh2, as well as RARa and RARg (p = 0.001; Fig. Previous studies have reported expression in DCs of genes involved 2A–D). in the biosynthesis and metabolism of RA, such as Adh1, Raldh2, To gain insight into potential signaling mechanisms mediating Adh5, and Raldh1 (24, 25). Locally produced RA can act on Adh1, Raldh2, and RAR induction in DCs by SEA, we investi- immune cells in autocrine or paracrine fashion by binding to nu- gated the role of MAPK signaling pathways by assessing p38, clear receptors of RAR or families. RAR pro- ERK1/2, and JNK1/2 phosphorylation. As shown in Fig. 2E, SEA teins are ubiquitously expressed on immune cells (26). We there- induced ERK phosphorylation in DCs to levels 8–10 times above fore studied expression and regulation of Adh1, Raldh2mRNAby baseline. ERK phosphorylation activity was abrogated when cells DCs, and examined RARa and RARg expression on these cells. were preincubated with U0126, a specific inhibitor of ERK1/2 RT-PCR analysis demonstrated that DCs constitutively expressed activation. In contrast, SEA had no measurable effect on p38 or low levels of both enzymes, and that levels of expression were JNK1/2 phosphorylation (Fig. 2E). As positive control, LPS stim- significantly upregulated (9- to 12-fold) following stimulation ulation of DCs led to phosphorylation of all three MAPK species. with SEA (p , 0.0001; Fig. 2A, 2B). Likewise, both ex vivo In line with these observations, induction of Adh1 and Raldh2, isolated as well as monocyte-derived DCs expressed low levels of as well as RAR mRNA expression, was largely abrogated by RARa and RARg mRNA. After SEA activation, an 8- to 10-fold inhibitors against ERK (Fig. 2A–D). In summary, SEA induced increase in DC expression of both RARs was observed (p , Adh1, Raldh2, RARa, and RARg on DCs via TLR2-mediated 0.0001; Fig. 2C, 2D). activation of ERK. SEA effects did not differ between infected

FIGURE 2. SEA induced retinol-metabolizing en- zymes and RARs in DCs. Expression of Adh1 (A), Raldh2 (B), RA a (C), and RA g (D) mRNA in DCs cultured in vitro for 24 h in the presence of SEA (50 mg/ml), Pam3Cys (ligand for TLR2; 5 mg/ml), flagellin (ligand for TLR5; 100 ng/ml), or CpG 1668 (ligand for TLR9; 10 mg/ml). Expression of different mRNAs relative to expression of mRNA encoding GAPDH was analyzed using RT-PCR in all figures. In some experi- ments, DCs were incubated with U0126, a specific ERK1/2 inhibitor (2 mM), for 1 h before stimulation. Data represent mean values 6 SEM from 14 indepen- dent experiments. (E) SEA differentially stimulated MAPK on DCs. Ex vivo isolated DCs from uninfected MS patients (2 3 105) were treated with SEA (50 mg/ ml) for 0, 15, 30, 60, 90, 120, and 180 min. Amounts of phosphorylated p38, ERK1/2, and JNK 1/2 were eval- uated by ELISA for each time point. In some experi- ments, DCs were treated before stimulation with ERK1/2 inhibitor U0126 (2 mM), or with a drug vehicle (control) for 1 h. Positive controls (dotted lines) con- sisted of DCs stimulated with LPS (10 mg/ml). Data represent one of nine independent experiments. The Journal of Immunology 5

MS patients and helminth-infected subjects without MS (Supple- to levels observed prior to SEA treatment. Overall, these results mental Fig. 1). indicate that SEA induced DCs to express RA synthesis enzymes, + + stimulating the development of FOXP3 Treg cells. These results SEA and retinol induce FOXP3 Treg cells are also consistent with the effects of TLR2-mediated ERK sig- Because SEA induced enzymes involved in RA synthesis, we set naling, inducing Raldh2 in DCs. out to determine whether these DCs metabolized retinol to RA and Th cell differentiation depends on a unique combination of whether they induced CD4+CD25+FOXP3+ Treg cells. DCs were stimulation and subsequent activation of diverse transcription stimulated with SEA, and 12 h later washed and cocultured with factors. Cooperative activation of NFAT and Smad3 leads to CD4+ autologous naive CD4+CD252 T cells, as described in Materials CD25+FOXP3+ Treg cell induction (27), and cooperation between and Methods. As shown in Fig. 3A, SEA-treated DCs significantly STAT3 and Smad3 switches on Th17 cell induction (28). We there- increased CD4+CD25+FOXP3+ Treg cell percentages (p = 0.0001). fore set out to investigate whether parasite Ag induced Smad3 In contrast, CpG 1668–stimulated DCs had no effect. Treg cell phosphorylation in T cells, through RA-mediated effects. After numbers induced by SEA-treated DCs were further increased in coculture of CD4+CD252 with SEA-treated DCs, as well as with the presence of retinol. To determine whether this effect was untreated DCs, Smad3 phosphorylation was measured using mediated through RA synthesis, DCs were stimulated with SEA ELISA kits in nuclear extracts of CD4+ T cells. Smad3 phos- both in the presence and in the absence of the Raldh inhibitor phorylation was significantly increased after coculture of CD4+ disulfiram. After 12 h, DCs were washed and cocultured with CD252 T cells with SEA-treated DCs (Fig. 3B). In contrast, co- CD4+CD252 T cells in the presence of SEA plus retinol. Inhibition culture of CD4+CD252 T cells with untreated DCs did not affect of de novo RA synthesis in DCs suppressed SEA-induced Treg cells. phospho-Smad3 levels. This effect was reproduced by culturing Furthermore, SEA-stimulated DCs treated with the ERK1/2 in- naive T cells with RA, or with retinol blocked either by the RAR hibitor U0126 restored CD4+CD25+FOXP3+ Treg cell percentages antagonist LE135, or by Raldh inhibitor disulfiram, respectively

FIGURE 3. SEA stimulated DCs to induce CD4+ CD25+FOXP3+ Treg cells. (A) DCs were treated with SEA and cocultured with CD4+CD252 T cells, as de- scribed in Materials and Methods, and a significant increase in CD4+CD25+FOXP3+ Treg cell percentage was observed (p , 0.001). This increase was dependent of RA, because it was inhibited by stimulation of DCs with SEA plus the Raldh inhibitor disulfiram (100 nM). In line with these results, DCs treated with the ERK1/2 inhibitor U0126 (2 mM) were unable to induce CD4+ CD25+FOXP3+ Treg, demonstrating the effect of TLR2- mediated ERK signaling, that is, inducing Raldh2 in DCs. (B) CD4+CD252 T cells were cocultured at 1:10 ratio (DCs to T cells) with SEA-treated as well as untreated DCs, and stimulated with soluble anti-human CD3 mAb and soluble anti-CD28 Abs, both at 5 mg/ml, in the presence of rhIL-2 (400 U/ml) and human rTGF-b (5 ng/ml); in some experiments, naive T cells were cultured in the presence of RA (15 nM) or retinol (500 nM). CD4+ T cells were sorted after being exposed to the different culture conditions described. T cell nu- clear extracts were then obtained, and phospho-Smad3 was measured using an ELISA kit (eBioscience). (C) Naive CD4+ T cells were cultured, as previously de- scribed, and phosphorylation of STAT3 was similarly assessed using an ELISA kit (R&D Systems). Data represent mean 6 SEM values from 14 uninfected MS patients. (D) Percentage of CD4+CD25+FOXP3+ Treg in CSF from healthy controls, uninfected MS patients, helminth-infected MS subjects, and patients with OIND was evaluated by flow cytometry. Each circle represents the percentage of cells from a single individual. Hori- zontal lines indicate mean group values. (E) Percentage of CD4+CD25+FOXP3+ Treg from each helminth-infected MS patient in peripheral blood and CSF. 6 PARASITE INFECTIONS MODULATE MS THROUGH RA

(p , 0.0001; Fig. 3B). Neither SEA, RA, nor retinol affected this effect, resulting in significant STAT3 phosphorylation. Over- Smad2 phosphorylation under these conditions (data not shown). all, these findings demonstrate that SEA, through RA-dependent We subsequently examined whether SEA and RA influenced pathways, may play an important role in modulating the balance STAT3 phosphorylation. As shown in Fig. 3C, STAT3 phosphor- between CD4+CD25+FOXP3+ Treg cells and Th17 cell differen- ylation was significantly decreased in naive T cells after coculture tiation. with SEA-treated DCs (p , 0.0001). Conversely, untreated DCs Notably, as illustrated in Fig. 3D, significantly higher levels (control) did not affect phospho-STAT3 levels. Similar results of CD4+CD25+FOXP3+ Treg cells were detected in CSF from were observed when naive T cells were cultured in the presence of helminth-infected MS patients (15.2 6 1.02%), compared with RA or of retinol. Addition of LE135 or of disulfiram abrogated levels observed in healthy controls (5.7 6 0.43%; p , 0.0001),

FIGURE 4. DCs stimulated with SEA produced lower amounts of pro- inflammatory cytokines and lower amounts of IL-10. DCs from hel- minth-infected MS patients were stimulated with SEA (50 mg/ml) or LPS (100 ng/ml), in the presence as well as in the absence of retinol (500 nM). Supernatants were collected 48 h after activation, and production of IL-6 (A), IL-12 (B), TNF-a (C), IL-23 (D), or IL-10 (E) was mea- sured by ELISA. In some experi- ments, a blocking anti–IL-10R mAb (10 m/ml), a control isotype (IgG1) murine mAb, or the RAR antagonist LE135 (1 mM) was added to the cul- ture medium at time 0. Data represent mean values 6 SEM from 10–12 independent experiments. The Journal of Immunology 7 uninfected MS patients (5.4 6 0.39; p , 0.0001), or patients with OIND (5.5 6 0.5%; p , 0.0001). Values were even higher than peripheral blood levels (p = 0.009; Fig. 3E). CD4+CD25+FOXP3+ Treg cell function induced by SEA-treated DCs was further investigated by testing ability of these cells to suppress proliferative responses and IFN-g secretion in CD4+ CD252 cells. To this end, CD4+CD252 T cells were stimulated with anti-CD3 and anti-CD28 mAb, whereas increasing numbers of autologous induced CD4+CD25+FOXP3+ Treg cells were added. The CD4+CD25+FOXP3+ Treg cells suppressed prolifera- tion of CD4+CD252 T cells in response to PHA, and to plate- bound anti-CD3 stimulation (63 6 15% at ratio 1:10), as well as the production of IFN-g (53 6 25% at ratio 1:10) by indicator CD4+CD252 T cells. Collectively, these results suggest that SEA- treated DCs induce CD4+CD25+FOXP3+ Treg cells, which retain immunosuppressive activity and may thus be considered bona fide Treg cells. Because of the low number of CD4+CD25+FOXP3+ Treg cells in CSF, it was not possible to investigate suppressive capacity of these cells. DCs stimulated with SEA produce lower amounts of IL-6, IL-12, IL-23, and TNF-a and higher amounts of IL-10 We previously demonstrated that SEA significantly suppressed LPS-induced IL-6, IL-12, and TNF-a production, and enhanced that of IL-10 and TGF-b by DCs (16). To determine whether RA contributes to these effects, we stimulated DCs with SEA in the presence as well as in the absence of retinol, and assessed pro- duction of IL-6, IL-10, IL-12, IL-23, IL-27, and TNF-a. SEA- treated DCs produced lower amounts of IL-6, IL-12, IL-23, and TNF-a (p = 0.01). Addition of retinol further reduced levels of these cytokines (p = 0.01; Fig. 4A–D). Inhibition of RARs using the antagonist LE135 was associated with significant increase in A production of all of these cytokines (p , 0.0001), and specific FIGURE 5. SEA and retinol induced SOCS3. ( ) DCs treated for 12 h with SEA (50 mg/ml) or SEA plus retinol (500 nM) were lysed, and 20 mg neutralization of IL-10Rs produced significant enhancement of , cell extract was used for SOCS1 and SOCS3 concentration measurement, proinflammatory cytokines in response to SEA (p 0.0001; Fig. using a capture ELISA kit, according to manufacturer instructions 4A–D). In contrast, SEA-exposed DCs significantly enhanced IL- (MyBioSource). In some experiments, the Raldh inhibitor disulfiram (100 10 production, and addition of retinol further increased IL-10 nM), or the RAR antagonist LE135 (1 mM), was added at time 0. Data levels (p , 0.01; Fig. 4E). Inhibition of RARs was also associ- represent one of seven independent experiments. (B) In another set of ated with a significant reduction of IL-10 production (p , experiments, TLR2, TLR5, and TLR9 were silenced before stimulation 0.0001), suggesting that enhanced secretion of IL-10 by DCs using the siRNA technique, as described in Materials and Methods. Data exposed to SEA is mediated by RA. Notably, stimulation of DCs represent mean values 6 SEM from 12 independent experiments. with SEA or SEA plus retinol did not result in significant differ- ences in IL-27 secretion compared with unstimulated DCs (data SOCS1 expression under identical culture conditions (data not not shown). shown). To assess the role of different TLRs on SOCS3 expression Similar results were observed in infected MS patients and during SEA stimulation, TLR2, TLR5, and TLR9 genes were si- helminth-infected subjects without MS (Supplemental Fig. 2). lenced before stimulation using the siRNA technique. Enhance- ment of SOCS3 expression mediated by SEA was not present SEA and retinol induce SOCS3 expression when TLR2 expression was silenced (p , 0.001; Fig. 5B). In Next, we investigated the mechanisms by which RA suppressed contrast, no effects were observed when TLR5 and TLR9 were proinflammatory cytokines. There is accumulating evidence that silenced. These results indicate that SOCS3 expression induced by suppressor of SOCS, particularly SOCS1 and SOCS3, may sup- SEA in DCs is TLR2 dependent. press inflammatory reactions under pathological conditions in Similar findings were observed in both infected MS patients and which proinflammatory cytokines play an important role (29). helminth-infected subjects without MS (Supplemental Fig. 3). Moreover, previous investigations have demonstrated that reduc- tion in DC proinflammatory cytokine production mediated by RA SEA inhibits proinflammatory cytokine production through was associated with increase in SOCS3 expression (25). Similarly, a SOCS3-dependent pathway in our system, SOCS3 expression levels were significantly up- To better understand the role of SOCS3 in SEA suppression of regulated following SEA stimulation (p , 0.0001; Fig. 5A). proinflammatory cytokines by DCs, we used the siRNA technique Addition of retinol to the culture system further increased SOCS3 to inhibit its expression. Suppression of IL-23, IL-6, IL-12, and expression 10- to 15-fold. In contrast, addition of Raldh inhibitor TNF-a disappeared when SOCS3 expression was silenced (p , disulfiram, or of the RAR antagonist LE135, significantly reduced 0.001). In contrast, silencing of SOCS1 had no effect (Fig. 6A–D). SOCS3 expression (p , 0.001; Fig. 5A). Overall, these results To further assess the role of SOCS3 on T cell activation during suggest that SEA-induced reduction of proinflammatory cytokines helminth infection, DCs from infected MS patients were stimu- is, at least in part, mediated by RA. No changes were observed in lated for 12 h with SEA, then cocultured with MBP or MOG 8 PARASITE INFECTIONS MODULATE MS THROUGH RA

FIGURE 6. Inhibition of proin- flammatory cytokines in SEA-treated DCs was SOCS3 dependent. DCs from helminth-infected MS patients were stimulated with LPS or SEA for 48 h, and IL-6 (C), IL-12 p70 (B), IL-23 (A), and TNF-a (D) super- natant concentrations were measured using ELISA. In parallel cultures, DCs were previously transfected with SOCS1-specific, SOCS3-specific siRNA, or scrambled nonsilencing control oligonucleotides, and then stimulated for 48 h, as described previously. To evaluate T cell acti- vation suppression by SEA, DCs from helminth-infected MS patients were stimulated with SEA for 12 h and then cocultured at a ratio of 1:5, with MBP peptide- or MOG peptide- specific TCLs, stimulated with spe- cific Ags during 48 h. IL-17 (F) and IFN-g (E) secretion were measured by ELISA. As described for DCs, ex- periments included DCs previously transfected with SOCS1-, SOCS3- specific siRNA or scrambled non- silencing control oligonucleotides. Data represent mean values 6 SEM from 11 independent experiments.

peptide-specific TCLs, and stimulated with specific Ags. IL-17 SEA effects did not differ between infected MS patients and and IFN-g secretion was measured after 48 h using ELISA. helminth-infected subjects without MS (Supplemental Fig. 4). SEA-activated DCs significantly inhibited IL-17 and IFN-g pro- duction (p , 0.001). As observed for DCs, inhibitory effects on Discussion production of both cytokines were abrogated when SOCS3 (but The long life span of helminths is evidence enough of just how not SOCS1) gene expression was silenced using the siRNA accomplished these organisms are at immune evasion. Several technique (Fig. 6E, 6F), indicating that SEA-mediated signaling studies in humans and in animal models have shown that chronic inhibited IL-17 and IFN-g production by T cells, also through helminth infections trigger prominent anti-inflammatory net- a SOCS3-dependent pathway (Fig. 7). work development, leading to attenuation of Ag-specific immune The Journal of Immunology 9

noninflammatory neurologic diseases and with healthy controls has already been reported (32, 33). Furthermore, a recent study described inverse association between increasing levels of s-retinol and MRI lesion activity in MS patients, finding s-retinol levels predicted MRI outcomes during subsequent months of follow-up (34). This is consistent with observations in different animal models. Oral administration of retinoid prevented development of EAE and improved clinical course, even when given after the onset of disease (35). Likewise, RA prolonged survival in mice with lupus nephritis (36) and reduced murine collagen-induced arthritis development (37). Although recent studies have highlighted an important role for RA in the induction of Treg cells in the gut, its role in systemic immune responses is poorly understood. The present data dem- FIGURE 7. Helminth immunomodulation in MS patients: proposed mechanisms for RA- induced regulatory pathways. The helminth Ag SEA onstrate that helminth-mediated immunomodulation observed in is recognized by DCs and induces the enzymes Adh1 and Raldh2, both MS patients is, at least in part, exerted by TLR2- and RA-dependent involved in RA synthesis, via TLR2-mediated activation of ERK. These pathways, through two different mechanisms, as follows: 1) in- enzymes convert retinol to RA, which has both an autocrine and paracrine duction of IL-10 and of FOXP3+ Treg cells, and 2) suppression of effect, acting on RARs also induced by SEA on DCs. TGF-b and induced proinflammatory cytokine production, mediated mainly by SOCS3 RA synergistically increase Smad3 phosphorylation, whereas RA reduces (Fig. 7). STAT3 phosphorylation. Both these processes, together with IL-10 and Recent insights into the role of RA in the promotion and TGF-b production enhancement, induce differentiation of CD4+CD25+ + regulation of multiple immunological pathways draw attention to FOXP3 Treg cells. In addition, SEA and RA induce SOCS3 expression, the influence of RA on immunity. For example, mucosal CD103+ which mediates suppression of the proinflammatory cytokines TNF-a, IL- DCs express RA synthesizing enzymes, and are able to induce 6, IL-12, and IL-23. Decreased levels of IL-12 inhibit IFN-g–secreting cell development. Likewise, decreased IL-23 and IL-6 levels, together with molecules such as CCR9 and a4b7 on conventional T cells in- increased TGF-b levels, and changes in Smad3 and STAT3 phosphoryla- volved in directing gut trophism (38). In addition, RA produced tion dampen differentiation of Th17. In summary, parasite-induced im- by DCs from the GALT, in synergy with TGF-b, induces strong munomodulation observed in MS patients is mediated by TLR2 and extrathymic FOXP3+ Treg cell differentiation, and inhibits RA-dependent pathways through two different mechanisms: 1) induction Th17 differentiation (39, 40). These effects can be reversed by of IL-10, TGF-b, and CD4+CD25+FOXP3+ Treg cells, and 2) suppression blocking RARs, indicating the capacity of the gut to induce of proinflammatory cytokines. tolerogenic outcomes through a RA-dependent pathway. Like- wise, recent studies have demonstrated that other tissues that constitute environmental interfaces (e.g., skin, lungs) resemble responses to both the parasite and unrelated pathogens. These the gut in that they also contain DCs that constitutively produce findings suggest parasites can not only suppress host immune RA to induce FOXP3+ Treg cells (41). RA has also been im- responses directed against them, but can also exert bystander plicated in the generation of IgA-secreting B cells, adding fur- suppression against third-party Ags. Following this premise, we ther evidence to support a multifactorial role for RA in mucosal recently demonstrated that helminth-infected MS patients expe- immunity (42). rienced milder disease course compared with noninfected MS Vitamin A metabolites, particularly RA, also modulate more subjects (9). Parasite-driven protection was associated with in- specific functional aspects of immune responses, such as the Th1- duction of Treg cells secreting suppressive cytokines IL-10 and Th2 balance. Thus, vitamin A deficiency correlates with decreased TGF-b, as well as of CD4+CD25+FOXP3+ T cells displaying Th2 cell responses (43) and, conversely, vitamin A supplemen- significant suppressive function. In addition to the development of tation blocks the production of Th1 cell cytokines (44). These Treg cells, helminth infections also induced regulatory B cells in effects of vitamin A on Th1 and Th2 cell differentiation are me- MS patients, capable of dampening the immune response through diated by RA. In fact, RA promotes Th2 cell differentiation by production of IL-10 (30). These findings provide evidence to sup- inducing IL-4 gene expression (45), as well as Th2 cell–promoting port autoimmune downregulation secondary to parasite infections in transcription factors, such as GATA3 and STAT6 (44, 46). More- MS patients, through regulatory T and B cell action, with effects over, RA blocks the expression of the Th1 cell master regulator extending beyond simple response to an infectious agent. Evidence T-bet (44, 46). In contrast, recent experiments using Th1-driving of regulatory mechanisms present during helminth infections is now intracellular parasite T. gondii have shown that RA is required for emerging, offering potential explanations as to why infected hosts the generation of Th1 and Th17 responses in the gut (47). To exhibit altered immune responses to bystander Ags. Helminths may reconcile these conflicting observations, it has been proposed that thus increase regulatory cell numbers or activity, either by gener- RA may act to aid T cell activation during early stages of the ating new cells or by activating or expanding existing populations. immune response, but downregulate effector responses at later The last few decades have witnessed a major effort to identify stages. Although a mechanism for this process has not yet been factors associated with MS onset and progression (3). Among elucidated, it could be due to changes in expression of either potentially relevant environmental factors investigated to date, it Raldh or RAR during inflammatory responses, which could in turn has been suggested that lipophilic vitamin metabolites, such as lead to activation of distinct signaling pathways (47). those of vitamins A and D, may have immunomodulatory prop- Recently, both in vitro and in vivo experiments have demon- erties, and therefore influence the course and severity of MS (11, strated that RA suppresses Th17 cell differentiation, reciprocally 14). The immunological and neurotrophic effects of vitamin A promoting generation of Treg cells expressing FOXP3 through metabolites (31) make a causal relationship between vitamin A RARa binding, associated to downregulation of ROR-gt, a key levels and MS disease activity biologically plausible. A reduc- for Th17 cell development (40, 48). In tion of retinol levels in MS patients compared with patients with agreement with our results, possible mechanisms to explain these 10 PARASITE INFECTIONS MODULATE MS THROUGH RA observations include the following: suppression of IL-6– and IL- Disclosures 23–driven signaling, as well as Smad3-dependent enhanced TGF- Jorge Correale is a board member of Merck–Serono Argentina, Biogen– b signaling (49). Previous investigations have demonstrated that Idec Latin America, Genzyme Latin America, and Merck–Serono Latin RA was insufficient to induce FOXP3 in the absence of TGF-b, America. Dr. Correale has received reimbursement for developing educa- indicating that responsiveness to TGF-b is a prerequisite for RA to tional presentations for Merck–Serono Argentina, Merck–Serono Latin induce Treg cell differentiation (49). Likewise, blockade of TGF- America, Biogen–Idec Argentina, and Teva–Tuteur Argentina, Teva–Ivax, b–induced Smad2/3 phosphorylation diminished TGF-b–medi- as well as professional travel/accommodations stipends. Mauricio F. Farez ated Treg cell generation and abrogated the additive effect of RA has received professional travel and accommodations stipends from Merck–Serono Argentina. (50). In contrast, RA increased Smad3 expression in activated CD4+ T cells (49, 51). Overall, these findings indicate that RA and TGF-b synergistically promote enhanced Smad3 activity, in- References creasing mutual signaling to further enhance FOXP3 expression. 1. McFarland, H. F., and R. Martin. 2007. Multiple sclerosis: a complicated picture Alternatively, RA concentration may be a factor in determining of autoimmunity. Nat. Immunol. 8: 913–919. 2. Oksenberg, J. R., S. E. Baranzini, S. Sawcer, and S. L. Hauser. 2008. The ge- RA signaling outcome. 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