Smoking worsens multiple sclerosis prognosis: Two different pathways are involved

Journal of Neuroimmunology 281 (2015) 23–34

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Smoking worsens multiple sclerosis prognosis: Two different pathways are involved Jorge Correale ⁎, Mauricio F. Farez Department of Neurology, Institute for Neurological Research Dr. Raúl Carrea, FLENI, Argentina

a r t i c l e

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Article history: Received 9 January 2015 Received in revised form 3 March 2015 Accepted 4 March 2015 Keywords: Multiple sclerosis Smoking Indoleamine 2,3-dioxygenase Renin–angiotensin-system

a b s t r a c t Smoking worsens multiple sclerosis (MS) prognosis. Our study provides evidence that indoleamine 2,3dioxygenase activity is reduced in MS patients who smoke, leading to increased production of IL-6 and IL-13. Additionally, both degree of expression and renin–angiotensin system activity levels were increased in MS patients who smoked, inducing increase in IL-17 and IL-22-producing cell numbers as well as significantly greater production of CCL2, CCL3 and CXCL10 chemokines by monocytes. Finally, both pathways contributed to a significant decrease in the number of CD4+CD25+FoxP3+ regulatory T cells in MS patients who smoked. Both pathways could be responsible for the association between smoking and MS risk. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Multiple sclerosis (MS) is a chronic inflammatory disorder causing demyelination and axon injury within the central nervous system (CNS). Considerable evidence indicates that autoimmunity plays an important role in its etiology (McFarland and Martin, 2007). Autoimmune diseases like MS arise from complex interactions between individual genetic susceptibility and environmental factors, which influence both incidence and clinical course of the disease. Because immunomodulatory treatments may partially decrease the number of patients with clinically isolated syndrome (CIS) undergoing progression, or may slow down, although not entirely prevent MS progression, identifying these environmental factors and elucidating how they increase autoimmune disease risk or aggravate its course, could help develop new treatment strategies. Possible environmental risk factors contributing to both increased disease frequency as well as accelerated progression include: low exposure to ultraviolet radiation (Dobson et al., 2013), vitamin D deficiency Abbreviations: 3-HAA, 3-hydroxyanthranilic acid; ACE, angiotensin-converting enzyme; APCs, antigen presenting cells; Ang, angiotensin; ATR1, angiotensin type 1 receptor; CIS, clinically isolated syndrome; CDMS, clinically definite multiple sclerosis; CNS, central nervous system; DCs, dendritic cells; EAE, experimental autoimmune encephalomyelitis; EDSS, expanded disability status scale; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IDO, indoleamine 2,3-dioxygenase; Kyn, kynurenine; LPS, lipopolysaccharide; MBP, myelin basic protein; MS, multiple sclerosis; NAT1, N-acetyltransferase 1; NO, nitric oxide; PBMCs, peripheral blood mononuclear cells; PHA, phytohemagglutinin; poly (I:C), polyinosinic–polycytidylic acid; RAS, renin–angiotensin system; RIA, radio immune assay; ROS, reactive oxygen species; RRMS, relapsing remitting multiple sclerosis; SPMS, secondary progressive multiple sclerosis; TCLs, T cell lines; TLR, Toll-like receptor; Try, tryptophan. ⁎ Corresponding author at: Raúl Carrea Institute for Neurological Research, FLENI, Montañeses 2325, 1428 Buenos Aires, Argentina. E-mail addresses: jcorreale@fleni.org.ar, [email protected] (J. Correale).

http://dx.doi.org/10.1016/j.jneuroim.2015.03.006 0165-5728/© 2015 Elsevier B.V. All rights reserved.

(Munger et al., 2006), cigarette smoking (Wingerchuck, 2012), high sodium intake (Farez et al., 2015) and early life Epstein–Barr virus infection (Levin et al., 2010). Cigarette smoking has been linked to an estimated 40–80% increase in risk of MS in case–control studies (Hernán et al., 2001; Handel et al., 2011). This risk has recently been associated with the N-acetyltransferase 1 (NAT1) 73688368 genotype, supporting a genetic/environmental interaction in disease susceptibility (Briggs et al., 2014). Furthermore, a dose dependent relationship between cumulative exposure to smoking and MS has been shown. Cigarette smoke increased not only the risk of developing MS, but also accelerated disease progression (Hedström et al., 2009; Healy et al., 2009). However, molecular mechanisms underlying these effects remain unclear. Indoleamine 2,3-dioxygenase (IDO) is the initial rate-limiting enzyme catalyzing tryptophan (Try) to kynurenine (Kyn) and other metabolites. An increasing body of data shows that IDO plays a critical role in immune tolerance induction as well as in suppression of immunity during pregnancy, after transplantation, and in autoimmune diseases (Mellor and Munn, 2004). Indeed, systemic inhibition of IDO by 1-methyl tryptophan in pregnant mice leads to breakdown of immune tolerance, and subsequent rejection of semiallogeneic fetuses (Munn et al., 1998). It also exacerbates experimental autoimmune encephalomyelitis (EAE) disease scores (Kwidzinski et al., 2005). Furthermore, IDO activity is decreased in smokers, inversely correlating with serum cotinine concentration, a biomarker of exposure to tobacco smoke (Pertovaara et al., 2006). The renin–angiotensin system (RAS) is a major endocrine system regulating blood pressure, and body fluid homeostasis. However, beyond controlling circulatory homeostasis, recent studies have also shown that Angiotensin (Ang) II promotes inflammation and tissue

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injury, mediating key events in mouse models of autoimmune disease (Sagawa et al., 2005; Marchesi et al., 2008; Okunuki et al., 2009; Platten et al., 2009; Stegbauer et al, 2009). Renin cleaves off Ang I from the protein angiotensinogen. Angiotensin-converting enzyme (ACE) then breaks off 2 additional amino acids, generating the main effector molecule Ang II, which acts on target cells via Ang II type 1 (AT1R), and Ang II type 2 receptors. AT1R is responsible for most known effects of Ang II (Benigni et al., 2010). Interestingly, different studies have demonstrated that serum ACE activity increases significantly after smoking (Kitamura, 1987), and chronic smoking results in enhanced RAS activation in monozygotic twins, discordant for smoking (Laustiola et al., 1988). Based on these observations we set out to evaluate whether IDO and RAS were involved in immunomodulatory effects of smoking on MS progression. 2. Material and methods 2.1. Study design and patient selection Impact of smoking on the course of MS was evaluated in two different groups of patients. In the first group we determined whether smoking was a risk factor for early conversion to definitive MS after a CIS. One-hundred and twenty-five patients presenting CIS were included. CIS was defined as the first neurologic event suggestive of MS, lasting for at least 24 h, with signs and symptoms indicating either a single CNS lesion (monofocal) or more than one lesion (multifocal). Patients had to have at least two clinically silent lesions on T2-weighted brain MRI scans, of at least 3 mm, one of which was ovoid, periventricular or infratentorial. Patients were subjected to comprehensive neurological examination every 3 months, including physical assessment of disease activity and estimation of expanded disability status scale (EDSS) score. Brain magnetic resonance imaging was performed at 6-month intervals on a 1.5-T Signa unit (General Electric, Milwaukee, Wisconsin). Axial 5-mm-thick slices were obtained with T2-weighted, proton density, fast spin-echo, fluid attenuated inversion recovery, and T1weighted sequences before and after administration of gadolinium diethylenetriamine penta-acetic acid (0.1 mmol/kg). Ninety-two percent of CIS-presenting patients received treatment with intravenous methylprednisolone (1000 mg/day for 3 days) during attacks, followed by 2 weeks of tapered oral prednisone. After CIS diagnosis patients received no other treatment except IFN-β. None of the patients of this group was under treatment with either losartan or enalapril. Conversion to definite MS was defined by: 1) Poser criteria (Poser et al., 1983): new relapse with clinical evidence of at least one CNS lesion, and in cases with initial monofocal presentation, new lesion different from that responsible for CIS or 2) conversion to MS fell within McDonald criteria (Polman et al., 2011; McDonald MS).

Demographic and clinical characteristics of patients are summarized in Table 1. In the second group, effects of smoking on disability progression were investigated in a cohort of 203 RRMS patients (defined by Poser or McDonald criteria). Clinical disability assessments were conducted at 3 to 6 month intervals, using EDSS scores. Patient evaluations were carried out by the same clinician to avoid inter-rater variability (JC). Median follow-up was 88 months. Conversion from RRMS to SPMS was defined following Lublin and Reingold criteria (Lublin and Reingold, 1996). None of the patients of this group was under treatment with either losartan or enalapril. Demographic and clinical characteristics of the second patient cohort are described in Table 2. In both patient groups, smoking history at time of CIS or RRMS diagnosis was obtained from medical records or, when not documented, through telephone interviews or email questionnaires. “Ever smoking” was defined following European Community Respiratory Health Survey III criteria (Burney et al., 1994). Sixty individuals selected to match patients with respect to race/ ethnicity, age and gender served as controls. Forty-two subjects (70%) were females (age range 24–36 years), and 18 (30%) were males (age range 26–34 years). Healthy individuals were recruited among hospital staff members. Thorough clinical and neurological examination, as well as standard chemical and hematological laboratory examinations ruled out the presence of underlying disorders in these subjects. Control individuals were not receiving any medication or dietary supplement. Study protocol was approved by the Institutional Ethics Committee, and all subjects signed informed consent forms. 2.2. Cells Blood samples were obtained from RRMS patients in remission for ex vivo/in vitro analysis in all cases. All MS patients had been off steroids for at least 6 months prior to any blood draws conducted for this study. CD4+ T cells, and CD14+ monocytes, were selected from peripheral blood mononuclear cells (PBMCs) using anti-CD4 and anti-CD14coated magnetic beads (Invitrogen, Carlsbad, CA), following the manufacturer's instructions. Separation was monitored using flowcytometry analysis, demonstrating N97% purity. Monocyte-derived dendritic cells (DCs) and myelin basic protein (MBP)-peptide specific T-cell lines (TCLs) expanded from PBMCs were generated as previously described (Correale et al., 1995; Correale and Farez, 2007). 2.3. Antigen preparation MBP83–102, and MBP143–168 peptides were synthesized with an automated peptide synthesizer using FAST-MOC chemistry, and expected peptide amino acid composition confirmed by HPLC. Both peptides

Table 1 Demographic and clinical data of smoker and non-smoker patients with CIS at baseline.a Smokers (n = 68)

Non-smokers (n = 57)

p value

Number of women (%) Age at CIS (years) EDSS at CIS

47 (69) 31.2 ± 5.8 2.1 ± 1.1

38 (66) 29.8 ± 5.3 1.9 ± 1.3

ns ns ns

Clinical presentation of first event Monofocal (%) Multifocal (%)

37 (54%) 31 (46%)

30 (53%) 27 (47%)

ns ns

MRI at CIS Number of lesions on T2-weighted images Number of Gadolinium-enhanced T1-weighted images Interferon-β therapy after CIS diagnosis (%)

6.3 ± 2.8 2.1 ± 0.5 51 (75%)

6.6 ± 3.2 1.9 ± 0.7 40 (70%)

ns ns ns

a

Values represent number (%) or mean ± SD; CIS: clinically isolated syndrome; EDSS: expanded disability status scale score; ns: not significant.

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Table 2 Baseline characteristics of relapsing remitting multiple sclerosis patients.a

Number of women (%) Age at the diagnosis of RRMS (years) EDSS at the diagnosis of RRMS Disease duration (first symptom-last EDSS score in years) Immunomodulatory treatment (%) a

Smokers (n = 118)

Non-smokers (n = 85)

p value

85 (72) 30.5 ± 5.8 2.1 ± 0.6 9.8 ± 5.3 107 (91)

59 (69) 32.8 ± 6.1 2.3 ± 0.8 10.8 ± 6.1 75 (88)

ns ns ns ns ns

Values represent n (%) or mean ± SD; EDSS: expanded disability status scale score; ns: not significant.

were selected because they have been shown to be immunodominant epitopes in MS patients. 2.4. Real-time quantitative RT-PCR analysis For quantitative assessment of relative mRNA levels, total RNA was prepared using TRIzol LS reagent (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. RNA was reverse transcribed using a M-MLV RT reverse transcription kit with random hexamer primers (Invitrogen). Relative levels of IDO, and AT1R mRNAs were determined by real-time PCR using an ABI 7000 sequence detection system (Applied Biosystems, Foster City, CA). Values obtained were normalized to GAPDH amount. Primer sequences used were as follows: GAPDH: forward 5′-GAAGGTGAAGTCGGAGTC-3′, reverse 5′-GAAGATGGTGATGG GATTTC-3′; IDO: forward 5′-ACTGGAGGCACTGATTTA-3′, reverse 5′ATTAGTTTGTGGCTCTGTTA-3′; AT1R: forward 5′-CAGATGACGGCTGC TCGAAG-3′, reverse 5′-TGGAAACTGGACAGAACAATCTGG-3′. 2.5. Measurement of IDO activity For IDO activity evaluation, CD4+ T cells as well as MBP-peptidespecific TCLs were cultured in medium supplemented with additional tryptophan and stimulated with 1 μg/ml of phytohemagglutinin (PHA; Sigma-Aldrich), or the cognate antigen (Ag; 10 μg/ml). After 48 h in culture, supernatants were collected and Kyn concentrations measured with HPLC using a reverse-phase column as previously described (Laich et al., 2002). Optimal timing and concentration of stimuli were established in preliminary experiments. 2.6. Serum cotinine determination Cotinine is a nicotine metabolite, recognized as a biochemical marker indicating tobacco use. Cotinine serum concentration was determined using commercially available ELISA kits, following the manufacturer's instructions (Abnova, Walnut CA).

Aldrich), and MBP peptide-specific T cell lines with the cognate Ag (10 μg/ml). After 48 h in culture, supernatants were collected and concentrations of both molecules assessed using commercially available ELISA kits (R&D Systems) following the manufacturer's instructions. Optimal timing and concentration of stimuli were established in preliminary experiments. To measure biological activity of renin, angiotensin I concentration was measured using a commercially available RIA kit (IBL, Hamburg, Germany) according to the manufacturer's instructions. In vitro ACE activity was determined following the Sentandreu and Toldrá protocol (Sentandreu and Toldrá, 2006). 2.10. Chemokine assessment Monocytes were stimulated as previously described, and CCL2/MCP1, CCL3/MIP-1, CCL5/RANTES, and CXCL10/IP-10 cell culture supernatant concentrations were measured after 72 h, using commercially available ELISA kits according to the manufacturer's instructions (R&D Systems). 2.11. Statistical analysis Clinical and demographic data were compared between MS patients who were smokers and MS patients who were non-smokers using the Mann–Whitney U-test. Kaplan–Meier curves were calculated to establish definite risk of MS development and progression. Hazard ratios were estimated using Cox proportional hazards under multivariable analysis with adjustment for potential confounding variables (age at onset, gender, disease duration, neurological symptoms at CIS diagnosis, number of lesions on T2-weighted and on Gadolinium-enhanced T1weighted MRI, interferon-β treatment after CIS diagnosis, and immunomodulatory treatment in RRMS patients). Differences in immunological variables were tested for significance using a Mann–Whitney U-test. Statistical significance was considered present for two sided p values under 0.05.

2.7. Quantification of secreted cytokines 3. Results PBMCs and MBP-peptide specific T cells secreting IL-6, IL-10, IL-13, IL17, IL-22, TGF-β, and IFN-γ were evaluated using commercially available kits for single-cell resolution enzyme-linked immunospot (ELISPOT) assays following the manufacturer's instructions (R&D Systems). 2.8. Evaluation of CD4+CD25+FoxP3+ regulatory T cells CD4+CD25+FoxP3+ T cell number was evaluated by flow cytometry, using commercially available regulatory T cell staining kits, following the manufacturer's instructions (eBioscience, San Diego, CA). 2.9. Renin–angiotensin system assessment To determine renin and ACE production and activity, isolated CD14 + monocytes were stimulated with LPS (5 μg/ml; Sigma-

3.1. Smoking is a risk factor for early conversion to definite MS as well as for worse prognosis One-hundred and twenty-five patients with CIS and known smoking status were followed for at least 36 months. During the period between CIS and definite MS diagnosis (according to McDonald criteria) no nonsmoker patient took up the habit of smoking. Demographic, clinical and MRI characteristics were similar between groups (Table 1). Association between smoking status and risk of conversion to definite MS during a 36 month-period following CIS diagnosis is shown in Fig. 1A. By the end of follow-up, 77% of smokers but only 43% of nonsmokers developed definite MS. Furthermore, CIS patients who smoked presented significantly shorter interval to first clinical relapse (350 days vs. 730 days; p = 0.001).

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Fig. 1. Smoking worsens the MS prognosis. (A) Kaplan–Meier estimates for probability of definite MS (McDonald criteria) over 3 years in smokers (n = 68) and non-smokers (n = 57) CIS patients. (B) Kaplan–Meier plots of time to secondary progressive disease, comparison between smokers (n = 118) and non-smokers (n = 85) with RRMS. (C–D) Kaplan–Meier estimates from the time of relapsing–remitting MS onset to assignment of total EDSS scores of 4 and 6 among smoker (n = 118) and non-smoker (n = 85) MS patients.

To evaluate the role of smoking on MS prognosis, 203 RRMS patients with known smoking status were included in this study. Demographic and clinical characteristics were similar between groups (Table 2). After a median of 88 month follow-up, patients who smoked were more likely to convert to progressive disease compared to non-smokers (p = 0.001; Fig. 1B). Likewise, ever-smokers were more likely to reach EDSS scores 4 and 6 than never-smokers (p = 0.001, and p = 0.03, respectively; Fig. 1C and D). Furthermore, individuals who began smoking at an early age (≤15 years) were more likely to present progressive disease and to progress sooner compared to those who started smoking later in life (N 15 years; p = 0.002 and p = 0.003 respectively). In subsequent experiments the impact of IDO and RAS pathways on the course of MS in smoker patients was examined.

3.2. IDO activity is decreased in MS patients who smoke RT-PCR analysis indicated constitutive expression of IDO mRNA in DCs and CD4 + T cells isolated from MS patients who smoked, from non-smoker MS patients and control subjects (Fig. 2A). However, levels of expression were significantly lower in MS patients and controls who smoked compared to levels in non-smokers (p = 0.001). Likewise, IDO activity in both ex vivo isolated CD4+ T cells and MBP-peptide specific T cells, measured by the Kyn/Try ratio was significantly lower in smokers than in non-smokers (both MS patients and controls; p b 0.0001; Fig. 2B). Furthermore, when subjects were divided into groups according to serum cotinine levels ≥100 μg/l (active smokers) and b100 μg/l (non-smokers), Kyn/Try was significantly lower in active

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smokers. Thus, IDO activity correlated inversely with serum cotinine concentration in active smokers (r2 = −0.52, p = 0.01; Fig. 2C). 3.3. Smoking modifies immunological profiles in MS patients. The role of IDO Impact of smoking on cytokine production was assessed using MBP83–102, and MBP143–168 specific T cell lines stimulated with the cognate peptide. Fig. 3A shows differences in cytokine-secreting cell numbers between MS patients who smoked and those who did not. Smoking led to increased number of IL-6, IL-13, IL-17 and IL-22 producing cells (p = 0.003 to p b 0.0001). No differences between MS patient groups were observed in numbers of MBP peptide-specific cells producing IL-10, IFN-γ or TGF-β. Similar patterns of cytokine secretion were seen for PBMCs stimulated with MBP83–102 or MBP143–168 peptides, as well as for MBP-peptide specific T cell lines isolated from smokers and non-smokers stimulated with immobilized anti-CD3 mAb (Supplementary Table 1). These findings were similar in both MS patients and healthy control subjects. In addition, impact of smoking on CD4+CD25+FoxP3+ regulatory T cell development was analyzed. As illustrated in Fig. 3B, percentage of CD4+CD25+FoxP3+ regulatory T cells was significantly lower in PBMCs collected from MS patients who smoked compared to non-smokers with MS (p b 0.0001). We then studied the immunological profile of MS patients who smoked, after IDO induction. To evaluate IDO induction, MBP peptidespecific T cell lines were pre-incubated before stimulation with polyinosinic–polycytidylic acid [poly (I:C)], a TLR-3 ligand which induces IDO mRNA and protein expression (Wang et al., 2011). RT-PCR results revealed that IDO mRNA expression was induced after 12 h treatment with poly (I:C), in a dose-dependent manner (Fig. 3C). Given the induction of IDO by poly (I:C), we next assessed IDO enzymatic activity on MBP83–102-, and MBP143–168-specific T cell lines by measuring concentration of Kyn in culture media. As illustrated in Fig. 3D, Kyn concentration in media from poly (I:C) treated mononuclear cells significantly increased over time compared to untreated cells, indicating that poly (I:C) induced IDO enzymatic activity in mononuclear cells, catabolizing Try. IDO induction led to decreased number of IL-6 and IL-13 secreting cells in MS patients who smoked (p b 0.0001). In contrast, no effects were observed in number of T cells producing IL-17 or IL-22 (Fig. 3E). In addition, IDO induction significantly increased CD4+CD25+FoxP3+ regulatory T cell percentages (p b 0.0001; Fig. 3F), suggesting that the IDO pathway plays an important role in immune modulation in MS patients who smoke. 3.4. Expression and activity of RAS components are increased in MS patients who smoke

Fig. 2. IDO activity in MS patients who smoked. (A) Dendritic cells and CD4+ T cells from both relapsing remitting MS patients (n = 22) and control subjects (n = 20) constitutively expressed IDO. (B) MBP83–102 specific T cells were cultured in medium supplemented with additional tryptophan and stimulated with specific Ag (10 μg/ml) for 48 h. IDO activity, measured by Kyn/Try ratio was significantly lower in both MS patients (n = 40) and controls (n = 33) who smoked, compared to non-smokers with MS (n = 35) and non-smoker controls (n = 30). (C) Correlation between serum Kyn/Try ratio and serum cotinine concentration in smokers. Although total patient numbers differed for different assays, patients participating in study experiments corresponded mostly to the same individuals. In some cases the required number of cells was not enough to perform all assays reported.

As illustrated in Fig. 4A, both monocytes and autoreactive T cells derived from MS patients who smoked, produced significantly higher amounts of renin and ACE compared to levels in non-smoker MS patients (p = 0.02 to p = 0.001). Likewise, activity of both molecules was significantly increased in both cell populations isolated from MS patients who smoked (p = 0.01 to p b 0.0001; Fig. 4B). We next analyzed AT1R expression in different immune cell subsets. As shown in Fig. 4C, RT-PCR analyses of monocytes, ex vivo CD4 + T cells and MBP peptide-specific T cells revealed significantly increased AT1R expression (up to 6-fold) in cells derived from MS patients who smoked, compared to cells isolated from non-smokers with MS or from healthy control subjects, demonstrating that the RAS system is activated in peripheral immune cells of individuals who smoke. 3.5. ACE inhibition and blockade of AT1R impair IL-17 and IL-22 production as well as chemokine production, in MS patients who smoke As shown in Fig. 5A, inhibition of ACE using enalapril and AT1R blockade by losartan down-regulated IL-17 and IL-22 secreting cell

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numbers in MS patients who smoked, suggesting that the RAS pathway modulated Th-17-mediated autoimmunity (p b 0.0001). In contrast, neither enalapril nor losartan showed any effect on IL-6 or IL-13 producing T cell numbers. In addition, monocytes from MS patients who smoked showed significantly higher production of chemokines CCL2, CCL3, and CXCL10 compared to non-smokers with MS (p = 0.001 to p b 0.0001; Fig. 5B). Inhibition of ACE and AT1R blockade abolished these effects, also demonstrating an active role of the RAS pathway in chemokine

secretion modulation. Smoking had no effect on CCL5 secretion. Similar results were observed in both MS patients and healthy control subjects. 3.6. RAS pathway reduces regulatory T cell frequency in MS patients who smoke We then studied whether RAS pathways were involved in CD4+CD25+FoxP3 + regulatory T cell development. To this end,

J. Correale, M.F. Farez / Journal of Neuroimmunology 281 (2015) 23–34

peripheral blood mononuclear cells were cultured in the presence of either losartan or enalapril, and resulting numbers of CD4+CD25+ FoxP3 + regulatory T cells were evaluated by flow cytometry. As shown in Fig. 5C, inhibition of RAS pathways by losartan or enalapril significantly increased CD4+CD25+FoxP3+ regulatory T cell percentages (p b 0.001), indicating that the RAS pathways inhibited regulatory T cell development. 3.7. Blockade of ATR1 by losartan improves CIS course in smoker MS patients To establish a direct link between RAS pathway and CIS course we studied a second group of CIS patients receiving losartan for hypertension (25–50 mg daily) who were also smokers (n = 18). Patients were followed for at least 36 months, and clinical course was compared to 18 CIS smoker patients not receiving losartan, and 18 CIS nonsmokers' subjects. Demographic, clinical and MRI characteristics were similar between groups (see Supplementary Table 2). As illustrated in Fig. 6A, losartan treatment was associated with significant decrease in risk of conversion to definite MS (under McDonald criteria). By the end of follow-up, 68% of untreated smokers had developed definite MS, but only 42% of non-smokers, and 50% of losartan treated smokers, indicating that RAS pathway blockade improved CIS prognosis in smokers' patients. Moreover, as illustrated in Fig. 6B–D, losartan treatment resulted in significant decrease in IL-17 and IL-22 secreting cell numbers, together with considerable increase in CD4+CD25+FoxP3+ regulatory T cell percentage, compared to untreated CIS smoker patients. In contrast, no effects were observed on IL-6 and IL-13 producing cell numbers (data not shown). Overall, these results replicated those previously observed in “in vitro” experiments. 4. Discussion In this study, we found that cigarette smoking was a risk factor for early conversion to clinically definite MS (CDMS) among patients with CIS and disease progression was more rapid in ever-smoker patients compared to non-smokers. Moreover, MS patients who smoked had a significantly higher risk of attaining EDSS score milestones (4 and 6) compared to non-smoker MS patients. Our study also provides evidence that IDO activity is reduced in MS patients who smoke leading to increased production of IL-6 and IL-13. Additionally, both expression and activity of RAS components were increased in MS patients who smoked, inducing robust increase in IL-17 and IL-22-producing cell numbers as well as significantly greater production of CCL2, CCL3 and CXCL10 chemokines by monocytes, compared to non-smoker MS patients. Finally, both pathways contributed to a significant decrease in the number of CD4+CD25+FoxP3+ regulatory T cells in MS patients who smoked. Observational studies suggest that cigarette smoking may exert a negative influence on the course of established MS (Casetta et al., 1994; Hernán et al., 2001; Hedström et al., 2009, 2011; Jafari et al., 2009; Simon et al., 2010; Salzer et al., 2012). Our results are in agreement with previous data on smoking and disease prognosis, where smoking was associated with an increased risk of early conversion to

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CDMS after a CIS (Di Pauli et al., 2008). Likewise, studies evaluating the association of smoking and MS progression or disability have also typically, although not always, detected a significant relationship, in which smoking was associated with greater risk of conversion from relapsing–remitting to secondary progressive MS, and worse degree of disability in established progressive forms of MS (Hernan et al., 2005; Koch et al., 2007; Sundström and Nystrom, 2008; Healy et al., 2009; Manouchehrinia et al., 2013). Interestingly, beneficial effects of smoking cessation on disability progression have been described in patients with MS (Hedström et al., 2009; Manouchehrinia et al., 2013). Thus, adding smoking prevention or cessation to other treatment strategies could be a reliable and effective way to improve MS outcomes. It is not clear whether increased impairment and disability in MS patients who smoke is due to direct influence of tobacco on MS, or to increased co-morbidities. For example, cigarette smoking diminished anti-microbial activity relevant to airway infection clearance, while simultaneously modulating mucosal functions resulting in increased frequency of respiratory infections. The latter are known to be important triggers of MS relapse (Correale et al, 2006). Of interest, relapses associated with infections seem to result in more permanent disability than non-infection related exacerbations (Buljevac et al., 2002). Interestingly, recent observations in experimental autoimmune encephalomyelitis (EAE) showed that the lung can serve as site for autoreactive T cell reactivation and competence development. These cells can then access the CNS (Odoardi et al., 2012). Direct contact between myelin-specific T cells and tobacco smoke may elicit a pathogenic response in these cells. A variety of mechanisms have been suggested to explain the association between smoking and disability accumulation in MS. Exposure to tobacco smoke has been shown to alter both innate and adaptive immunity (Arnson et al., 2010), increasing the risk of different autoimmune diseases (Arnson et al., 2010). Cigarette smoke acts on cellular and humoral components of the immune system, having both pro- and antiinflammatory effects that may be reversible once exposure ceases (Sopori, 2002; Arnson et al., 2010). Other hypothesized mechanisms demonstrating deleterious effects of smoking on MS include direct toxicity of tobacco components on neurons and oligodendrocytes. In animal experiments for example, demyelinating lesions have been produced in the CNS following chronic low dose exposure to cyanide (Philbrick et al, 1979). Likewise, exposure to NO or its byproducts such as peroxynitrite, has been shown to cause axonal degeneration or block axonal conduction (Rejdak et al., 2004). Interestingly, although nicotine has been shown to increase blood–brain barrier permeability (Hawkins et al., 2004), an early event in MS development, other evidence suggests that nicotine may exert systemic effects on the immune system by inhibiting both innate and adaptive immune responses (Sopori, 2002). In EAE, nicotine significantly ameliorated symptoms, and microglia activation (Gao et al., 2014). Furthermore, the use of oral tobacco in the form of moist snuff was not associated with elevated risk of MS, suggesting that nicotine alone is not a risk factor for MS (Hedström et al., 2009). In contrast, nonnicotine components of cigarette smoke, particularly acrolein, had a detrimental effect on EAE progression, mostly at early stages of the disease, associated with microglial activation (Gao et al., 2014), which may play a critical role in MS progression (Correale, 2014).

Fig. 3. IDO influences the immune profile of MS patients who smoked. (A) Number of cytokine-secreting cells specific for MBP83–102 per 105 cells as determined by ELISPOT assays, from smoker and non-smoker patients with MS. Cytokine secreting cell numbers were calculated by subtracting the number of spots obtained in control cultures without Ag stimulation, from the number of spots obtained in cultures exposed to stimulating Ag. (B) CD4+CD25+FoxP3+ regulatory T cell frequency was evaluated using flow cytometry with commercially available regulatory T cell-staining kits, in the same patient groups. (C) IDO induction by poly (I:C). MBP83–102 T cell lines were cultured in the absence or presence of different concentrations of poly (I:C) for 12 h. Results correspond to mean values ± SEM of IDO mRNA relative to GAPDH, obtained from 32 T cell lines from 15 MS patients who also smoked. (D) IDO activity. MBP83–102 T cell lines were cultured with or without 10 μg/ml of poly (I:C), tissue culture medium was collected at time points indicated and concentrations of kynurenine determined by HPLC. Data represent mean values of 7 different experiments. (E) Number of cytokine-secreting cell numbers specific for MBP83–102 per 105 cells as determined by ELISPOT assay, as described above, from both smoker and non-smoker MS patients. Induction of IDO using poly (I:C) down-regulated IL-6 and IL-13 secreting cell numbers in MS patients who smoked. (F) Culture of PBMCs from MS patients who smoked, in the presence of poly (I:C), significantly increased CD4+CD25+FoxP3+ regulatory T cell percentage measured by flow cytometry. In figures A and E, results correspond to mean values ± SEM, from 62 MBP peptide-specific T cell lines, obtained from 35 MS patients who smoked, and 60 MBP peptide-specific T cell lines obtained from 32 non-smokers with MS. In figures B and F, results correspond to mean values ± SEM obtained from 35 MS patients who smoked, and 32 non-smokers with MS. Although total patient numbers differed for different assays, patients participating in study experiments corresponded mostly to the same individuals. In some cases the required number of cells was not enough to perform all assays reported.

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Fig. 4. Expression and activity of RAS components on immune cells. (A) Production of renin and ACE by monocytes and MBP83–102 autoreactive T cells isolated from smoker and nonsmoker patients with MS. (B) Analysis of renin and ACE activity in monocytes, and MBP83–102 autoreactive T cells, isolated from the same groups of patients. For figures A and B, results represent mean values ± SEM obtained from 35 MS patients who smoked, and 32 non-smokers with MS. (C) AT1R receptor expression is upregulated in monocytes, ex vivo CD4+ T cells, and MBP83–102 autoreactive T cells isolated from smoker MS patients. Total RNA was extracted from the different cell populations, and gene expression analyzed using RT-PCR. Results correspond to mean values ± SEM of AT1R mRNA relative to GAPDH, in monocytes, ex vivo CD4+ T cells, and MBP83–102 autoreactive T cells, isolated from 25 smokers with MS and 22 non-smokers with MS. Although total patient numbers differed for different assays, patients participating in study experiments corresponded mostly to the same individuals. In some cases the required number of cells was not enough to perform all assays reported.

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Fig. 5. The RAS pathway influences the immune profile of MS patients who smoked. (A) Number of cytokine-secreting cells specific for MBP83–102 per 105 peripheral blood mononuclear cells as determined by ELISPOT assays, from smoker and non-smoker MS patients. Inhibition of ACE using enalapril (100 μM), and blocking AT1R using losartan (1 μM) down-regulated IL17 and IL-22 secreting cell numbers in MS patients who smoked. (B) Smoking increased production of CCL2, CCL3, and CXCL10 by monocytes, but did not influence CCL5 production. ACE inhibition and AT1R blockade as described above impaired chemokine production measured by ELISA. In both figures, results correspond to mean values ± SEM obtained from 25 MS patients who smoked, and 22 non-smokers with MS. (C) Culture of PBMCs in the presence of losartan (1 μM) or enalapril (100 μM) significantly increased CD4+CD25+FoxP3+ regulatory T cell percentage measured by flow cytometry. Data represent mean ± SEM values from 25 MS patients who smoked, and 22 non-smoker MS patients. Although total patient numbers differed for different assays, patients participating in study experiments corresponded mostly to the same individuals. In some cases the required number of cells was not enough to perform all assays reported.

Experimental data have shown that IDO has important immunosuppressive properties involved in immune tolerance. Different mechanisms are thought to underlie IDO-mediated regulation of immune responses. Normally expressed at low levels, IDO is induced by IFN-γ in antigen presenting cells (APCs), which in turn hinder T cell

proliferation (Kwidzinski et al., 2005). Moreover, T cells become more susceptible to CD95L-induced apoptosis (Lee et al., 2002). This death ligand is functionally expressed on astrocytes in intact and damaged CNS, where it is induced also by IFN-γ, possibly providing a self-limiting loop initiated by Th1 cells within the CNS, capable of down-regulating

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Fig. 6. Blockade of RAS pathway improves prognosis of CIS patients who smoke. (A) Kaplan–Meier estimates for probability of definite MS (McDonald criteria) over 36 months in CIS patients who were treated with losartan and smokers (n = 18), untreated smokers (n = 18), and non-smokers (n = 18). (B–C) Longitudinal follow-up of IL-17 and IL-22 secreting cell numbers specific for MBP83–102 peptide per 105 PBMC, determined by ELISPOT assays, from CIS patients treated with losartan who smoked, CIS patients who smoked, CIS patients who were non-smokers and healthy controls (18 subjects in each group). Cytokine-secreting cell numbers were calculated as described in Fig. 3. (D) CD4+CD25+FoxP3+ regulatory T cell percentages in the same patient groups.

immune responses to minimize immune-mediated tissue damage. Several studies have confirmed the ability of DCs expressing IDO to induce differentiation of CD4+CD25+FoxP3 + Treg cells from CD4+CD25− precursors in both murine and human experimental systems (Chen et al., 2008). Beyond these effects on lymphocytes, IDO deficiency is associated with decreased downstream immunosuppressive Try metabolites such as Kyn and 3-hydroxyanthranilic acid (3-HAA). IDO-deficient mice develop exacerbated EAE with enhanced encephalitogenic Th1 and Th17 responses, and reduced Treg cell responses. In contrast, administration of 3-HAA enhanced Treg percentage, inhibited Th1 and Th17 cells, and ameliorated EAE (Yan et al., 2010). Interestingly, immunocytochemistry revealed activated microglia expressing IDO during the course of EAE and in vitro experiments confirmed IDO induction in microglia after IFN-γ treatment (Kwidzinski et al., 2005). Taken

together, these data support the notion that decreased IDO activity observed in MS patients who smoke could explain, at least in part, immunostimulatory effects of tobacco smoke. Recent studies have found molecular components of RAS in different cells including those from the immune system (Benigni et al, 2010). The discovery of local and intracellular RAS highlights several prominent non-hemodynamic effects of Ang II including pro-inflammatory, proliferative and fibrotic activities. The recent observation that Ang II modulated T cell responses and DC activity suggests a possible role of this peptide in autoimmune diseases. In EAE, peripheral CD4 + T cells showed increased levels of Ang II which acting through the AT1R promoted Th1 and Th17 cytokine synthesis. Drugs limiting Ang II activity either by inhibiting ACE or blocking AT1R result in suppression of Th1 and Th17 cytokine release, and promote antigen-specific

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CD4+CD25+Foxp3 + Treg cells (Platten et al., 2009). Additionally, AT1R blockade impaired expression of chemokines CCL2, CCL3 and CXCL10, reducing CCL2-induced APC migration (Stegbauer et al., 2009). Furthermore, Ang II via AT1R receptor stimulation can activate NAD(P)H oxidase to produce ROS, resulting in oxidative stress damage (Hoch et al., 2009). Interestingly, during the course of EAE AT1R is expressed in astrocytes, microglia and neurons and Ang II acts as a paracrine mediator sustaining inflammation in the CNS via TGF-β. This increase in disease activity is abrogated by AT1R inhibitors leading to delay and amelioration of chronic progressive EAE (Lanz et al., 2010). Overall, these observations suggest that during the course of MS extensive cross-talk is induced in smoker patients among: resident CNS cells, infiltrating T cells and RAS, contributing to disease progression. Recent epidemiological studies have identified new environmental factors associated not only with increased risk of MS, but also with worse disease course. While some of these risk factors, such age and gender are not modifiable, newly identified factors such as smoking and vitamin D levels are. Public health measures to counter these factors might facilitate disease control. Additionally, if MS epidemiology and basic laboratory research work together, our overall understanding of disease processes occurring in MS will increase, further contributing to the development of new therapeutic and preventive strategies. Acknowledgments This study was supported by an internal grant from the Institute for Neurological Research Dr. Raúl Carrea, FLENI, to JC. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jneuroim.2015.03.006. References Arnson, Y., Shoenfeld, Y., Amital, H., 2010. Effects of tobacco smoke on immunity, inflammation and autoimmunity. J. Autoimmum. 34, J258–J265. Benigni, A., Cassis, P., Remuzzi, G., 2010. Angiotensin II revisited: new roles in inflammation, immunology and aging. EMBO Mol. Med. 2, 247–257. Briggs, F.B., Acuna, B., Shen, L., Ramsay, P., Quach, H., Bernstein, A., Bellesis, K.H., Kockum, I.S., Hedström, A.K., Alfredsson, L., Olsson, T., Schaefer, C., Barcellos, L.F., 2014. Smoking and risk of multiple sclerosis. Evidence of modification by NAT1 variants. Epidemiology 25, 605–614. Buljevac, D., Flach, H.Z., Hop, W.C., Hijdra, D., Laman, J.D., Savelkoul, H.F., van Der Meché, F.G., van Doorn, P.A., Hintzen, R.Q., 2002. Prospective study on the relationship between infections and multiple sclerosis exacerbations. Brain 125, 952–960. Burney, P.G., Luczynska, C., Chinn, S., Jarvis, D., 1994. The European Community Respiratory Health Survey. Eur. Respir. J. 7, 954–960. Casetta, I., Granieri, E., Malagu, S., Tola, M.R., Paolino, E., Caniatti, L.M., Govoni, V., Monetti, V.C., Fainardi, E., 1994. Environmental risk factors and multiple sclerosis: a community-based, case–control study in the province of Ferrara, Italy. Neuroepidemiology 13, 120–128. Chen, W., Liang, X., Peterson, A.J., Munn, D.H., Blazar, B.R., 2008. The indoleamine 2,3dioxygenase pathway is essential for human plasmacytoid dendritic cell-induced adaptive T regulatory cell generation. J. Immunol. 181, 5396–5404. Correale, J., 2014. The role of microglial activation in disease progression. Mult. Scler. J. 20, 1288–1295. Correale, J., Farez, M., 2007. Monocyte-derived dendritic cells in multiple sclerosis: the effect of bacterial infection. J. Neuroimmunol. 190, 177–189. Correale, J., McMillan, M., McCarthy, K., Le, T., Weiner, L.P., 1995. Isolation and characterization of autoreactive proteolipid protein-peptide specific-T cell clones from multiple sclerosis patients. Neurology 45, 1370–1378. Correale, J., Fiol, M., Gilmore, W., 2006. The risk of relapses in multiple sclerosis during systemic infections. Neurology 67, 652–659. Di Pauli, F., Reindl, M., Ehling, R., Schautzer, F., Gneiss, C., Lutterotti, A., O'Reilly, E., Munger, K., Deisenhammer, F., Ascherio, A., Berger, T., 2008. Smoking is a risk factor for early conversion to clinically definite multiple sclerosis. Mult. Scler. 14, 1026–1030. Dobson, R., Giovannoni, G., Ramagopalan, S., 2013. The month of birth effect in multiple sclerosis: systematic review, meta-analysis and effect of latitude. J. Neurol. Neurosurg. Psychiatry 84, 427–432. Farez, M.F., Fiol, M.P., Gaitán, M.I., Quintana, F.J., Correale, J., 2015. Sodium intake is associated with increased disease activity in multiple sclerosis. J. Neurol. Neurosurg. Psychiatry 86, 26–31. Gao, Z., Nissen, J.C., Ji, K., Tsirka, S.E., 2014. The experimental autoimmune encephalomyelitis disease course is modulated by nicotine and other cigarette smoke components. PLoS One 9, e107979.

33

Handel, A.E., Williamson, A.J., Disanto, G., Disanto, G., Dobson, R., Giovannoni, G., Ramagopalan, S.V., 2011. Smoking and multiple sclerosis: an updated metaanalysis. PLoS One 6, e16149. Hawkins, B.T., Abbruscato, T.J., Egelton, R.D., Brown, R.C., Huber, J.D., Campos, C.R., Davis, T.P., 2004. Nicotine increases in vivo blood–brain barrier permeability and alters cerebral microvascualr tight junction protein distribution. Brain Res. 1027, 48–58. Healy, B.C., Ali, E.N., Guttmann, C.R., Chitnis, T., Glanz, B.I., Buckle, G., Houtchens, M., Stazzone, L., Moodie, J., Berger, A.M., Duan, Y., Bakshi, R., Khoury, S., Weiner, H., Ascherio, A., 2009. Smoking and disease progression in multiple sclerosis. Arch. Neurol. 66, 858–864. Hedström, A.K., Bäärnhielm, M., Olsson, T., Alfredsson, L., 2009. Tobacco smoking, but not Swedish snuff use, increases the risk of multiple sclerosis. Neurology 73, 696–701. Hedström, A.K., Bäärnhielm, M., Olsson, T., Alfredsson, L., 2011. Exposure to environmental tobacco smoke is associated with increased risk for multiple sclerosis. Mult. Scler. 17, 788–793. Hernán, M.A., Olek, M.J., Ascherio, A., 2001. Cigarette smoking and incidence of multiple sclerosis. Am. J. Epidemiol. 154, 69–74. Hernan, M.A., Jick, S.S., Logroscino, C., Olek, M.J., Ascherio, A., Jick, H., 2005. Cigarette smoking and the progression of multiple sclerosis. Brain 128, 1461–1465. Hoch, N.E., Guzik, T.J., Chen, W., Deans, T., Maalouf, S.A., Gratze, P., Weyand, C., Harrison, D.G., 2009. Regulation of T-cell function by endogenously produced angiotensin II. Am. J. Physiol. Regul. Integr. Comp. Physiol. 296, R208–R216. Jafari, N., Hoppenbrouwers, I.A., Hop, W.C., Breteler, M.M., Hintzen, R.Q., 2009. Cigarette smoking and risk of MS in multiplex family. Mult. Scler. 15, 1363–1367. Kitamura, S., 1987. Effects of cigarette smoking on metabolic events in the lung. Environ. Health Perspect. 72, 283–296. Koch, M., van harten, A., Uyttenboogaart, M., De Keyser, J., 2007. Cigarette smoking and progression in multiple sclerosis. Neurology 69, 1515–1520. Kwidzinski, E., Bunse, J., Aktas, O., Richter, D., Mutlu, L., Zipp, F., Nitsch, R., Bechmann, I., 2005. Indolamina 2,3-dioxygenase is expressed in the CNS and down-regulates autoimmune inflammation. FASEB J. 19, 1347–1349. Laich, A., Neurauter, G., Widner, B., Fuchs, D., 2002. More rapid method for simultaneous measurement of tryptophan and kynurenine by HPLC. Clin. Chem. 48, 579–581. Lanz, T.V., Ding, Z., Ho, P.P., Luo, J., Agrawal, A.N., Srinagesh, H., Axtell, R., Zhang, H., Platten, M., Wyss-Coray, T., Steinman, L., 2010. Angiotensin II sustains brain inflammation in mice via TGF-β. J. Clin. Invest. 120, 2782–2794. Laustiola, K.E., Lassila, R., Nurmi, A.K., 1988. Enhanced activation of the renin–angiotensin–aldosterone system in chronic cigarette smokers: a study of monozygotic twin pairs discordant for smoking. Clin. Pharmacol. Ther. 44, 426–430. Lee, G.K., Park, H.J., Macleod, M., Chandler, P., Munn, D.H., Mellor, A.L., 2002. Tryptophan deprivation sensitizes activated T cells to apoptosis prior to cell division. Immunology 107, 452–460. Levin, L., Munger, K.L., O'Reilly, E.J., Falk, K.I., Ascherio, A., 2010. Primary infection with the Epstein–Barr virus and risk of multiple sclerosis. Ann. Neurol. 67, 824–830. Lublin, F.D., Reingold, S.C., 1996. Defining the clinical course of multiple sclerosis: results of an international survey. Neurology 46, 907–911. Manouchehrinia, A., Tench, C.R., Maxted, J., Bibani, R.H., Britton, J., Constantinescu, C.S., 2013. Tobacco smoking and disability progression in multiple sclerosis: United Kingdom cohort study. Brain 136, 2298–2304. Marchesi, C., Paradis, P., Schffrin, E.L., 2008. Role of the renin–angiotensin system in vascular inflammation. Trends Pharmacol. Sci. 29, 367–374. McFarland, H.F., Martin, R., 2007. Multiple sclerosis: a complicated picture of autoimmunity. Nat. Immunol. 8, 913–919. Mellor, A.L., Munn, D.H., 2004. IDO expression by dendritic cells: tolerance and tryptophan catabolism. Nat. Rev. Immunol. 4, 762–774. Munger, K.L., Levin, L.I., Hollis, B.W., Howard, N.S., Ascherio, A., 2006. Serum 25hydroxyvitamin D levels and risk of multiple sclerosis. JAMA 296, 2832–2838. Munn, D.H., Zhou, M., Attwood, J.T., Bondarev, I., Conway, S.J., Marshall, B., Brown, C., Mellor, A.L., 1998. Prevention of allogenic fetal rejection by tryptophan catabolism. Science 281, 1122–1124. Odoardi, F., Sie, C., Streyl, K., Ulaganathan, V.K., Schläger, C., Lodygin, D., Heckelsmiller, K., Nietfeld, W., Ellwart, J., Klinkert, W.E., Lottaz, C., Nosov, M., Brinkmann, V., Spang, R., Lehrach, H., Vingron, M., Wekerle, H., Flügel-Koch, C., Flügel, A., 2012. T cells become licensed in the lung to enter the central nervous system. Nature 488, 675–679. Okunuki, Y., Usui, Y., Nagai, N., Kezuka, T., Ishida, S., Takeuchi, M., Goto, H., 2009. Suppression of experimental autoimmune uveitis by angiotensin II type 1 receptors blocker telmisartan. Invest. Ophthalmol. Vis. Sci. 50, 2255–2261. Pertovaara, M., Heliövaara, M., Raitala, A., Oja, S.S., Knekt, P., Hurme, M., 2006. The activity of the immunoregualtory enzyme indolamine 2,3-dioxygenase is decreased in smokers. Clin. Exp. Immunol. 145, 469–473. Philbrick, D.J., Hopkins, J.B., Hill, D.C., Alexander, J.C., Thomson, R.G., 1979. Effects of prolonged cyanide and thyocianate feeding in rats. J. Toxicol. Environ. Health 5, 579–592. Platten, M., Youssef, S., Hur, E.M., Ho, P.P., Han, M.H., Lanz, T.V., Phillips, L.K., Goldstein, M.J., Bhat, R., Raine, C.S., Sobel, R.A., Steinman, L., 2009. Blocking angiotensinconverting enzyme induces potent regulatory T cells and modulates TH1- and TH17-mediated autoimmunity. Proc. Natl. Acad. Sci. 106, 14948–14953. Polman, C.H., Reingold, S.C., Banwell, B., Clanet, M., Cohen, J.A., Filippi, M., Fujihara, K., Havrdova, E., Hutchinson, M., Kappos, L., Lublin, F.D., Montalban, X., O'Connor, P., Sandberg-Wollheim, M., Thompson, A.J., Waubant, E., Weinshenker, B., Wolinsky, J.S., 2011. Diagnostic criteria for multiple sclerosis: 2010 revision to the McDonald criteria. Ann. Neurol. 69, 292–302. Poser, C.M., Paty, D.W., Scheinberg, L., McDonald, W.I., Davis, F.A., Ebers, G.C., Johnson, K.P., Sibley, W.A., Silberberg, D.H., Tourtellotte, W.W., 1983. New diagnostic criteria for multiple sclerosis: guidelines for research protocols. Ann. Neurol. 13, 227–231.

34

J. Correale, M.F. Farez / Journal of Neuroimmunology 281 (2015) 23–34

Rejdak, K., Eikelenboom, M.J., Petzold, A., Thompson, E.J., Stelmasiak, Z., Lazeron, R.H., Barkhof, F., Polman, C.H., Uitdehaag, B.M., Giovannoni, G., 2004. CSF nitric oxide metabolites are associated with activity and progression of multiple sclerosis. Neurology 63, 1439–1445. Sagawa, K., Nagatani, K., Komagata, Y., Yamamoto, K., 2005. Angiotensin receptor blockers suppress antigen-specific T cell responses and ameliorate collagen-induced arthritis in mice. Arthritis Rheum. 52, 1920–1928. Salzer, J., Hallmans, G., Nyström, M., Stenlund, H., Wadell, G., Sundström, P., 2012. Smoking as a risk factor for multiple sclerosis. Mult. Scler. J. 19, 1022–1027. Sentandreu, M.A., Toldrá, F., 2006. A fluorescence-based protocol for quantifying angiotensing-converting enzyme activity. Nat. Protoc. 1, 2423–2427. Simon, K.C., van der Mei, I.A., Munger, K.L., Ponsonby, A., Dickinson, J., Dwyer, T., Sundström, P., Ascherio, A., 2010. Combined effects of smoking, anti-EBNA antibodies and HLA-DRB1*1501 on multiple sclerosis risk. Neurology 74, 1365–1371. Sopori, M., 2002. Effects of cigarette smoke on the immune system. Nat. Rev. Immunol. 2, 372–377. Stegbauer, J., De-Hyung, L., Seubert, S., Ellrichmann, G., Manzel, A., Kvakan, H., Muller, D.N., Gaupp, S., Rump, L.C., Gold, R., Linker, R.A., 2009. Role of the renin–angiotensin

system in autoimmune inflammation of the central nervous system. Proc. Natl. Acad. Sci. 106, 14942–14947. Sundström, P., Nystrom, L., 2008. Smoking worsens the prognosis in multiple sclerosis. Mult. Scler. 14, 1031–1035. Wang, B., Koga, K., Osuga, Y., Cardenas, I., Izumi, G., Takamura, M., Hirata, T., Yoshino, O., Hirota, Y., Harada, M., Mor, G., Taketani, Y., 2011. Toll-like receptor-3 ligation induced indoleamine 2,3-dioxygenase expression in human trophoblasts. Endocrinology 152, 4984–4992. Wingerchuck, D.M., 2012. Smoking:effects on multiple sclerosis susceptibility and disease progression. Ther. Adv. Neurol. Disord. 5, 13–22. Yan, Y., Zhang, G.X., Gran, B., Fallarino, F., Yu, S., Li, H., Cullimori, M.L., Rostami, A., Xu, H., 2010. IDO regulates regulatory T cells via tryptophan catabolite and suppresses encephalitogenic T cell responses in experimental autoimmune encephalomyelitis. J. Immunol. 185, 5953–5961.