multiple sclerosis ( ms ) is a chronic , demyelinating disease of the central nervous system ( cns ) , affecting young adults with a female preponderance . at an individual level , ms is extremely heterogeneous in its initial presentation , rate and severity of relapses , pattern of disease progression , underlying immunopathology , radiological appearance , and response to the disease modifying treatments ( dmts ) . in the last 20 years the initial paucity of available , approved drugs for ms prophylaxis has been replaced by a considerable number of treatment options . the first dmts approved for relapsing - remitting ms ( rr - ms ) prophylaxis were the interferons - beta ( ifn ) and glatiramer acetate ( ga ) , which remain first - line drugs , followed by other agents like natalizumab , mitoxantrone , and fingolimod , assigned as second - line treatments . the availability of multiple dmts and the need for early initiation of prophylactic treatment have rendered prognostic markers of treatment - response highly desirable . patients with ms receiving dmts can be classified as good responders ( slower disease progression or lower relapse rate ) , partial responders , and nonresponders ( no effect on disease activity ) . although there are many ways to assess disease activity and treatment response , including clinical , radiological , neurophysiological , and molecular , the concept of treatment response is mainly based on the clinical expression of ms , that is , relapses and disease progression . clinical criteria for treatment response are still uncertain and are usually extrapolated from natural history studies and placebo cohorts of clinical trials of drugs . in clinical trials , ifn has demonstrated efficacy in rr - ms patients by reducing relapse rate , lesion formation in the cns , and progression of sustained disability [ 57 ] . however , ifn therapy is not efficacious in a significant proportion of patients . in two studies comparing three ifn preparations ( ifn-1 30  g  qw versus ifn-1 250  g  qod and ifn-1 44  g  tiw versus ifn-1 30  g  qw ) , the percentage of patients that remained relapse - free in the 2-year follow - up ranged between 36 and 51% [ 8 , 9 ] . the percentage of active t2 lesion - free patients ranged between 26 and 58% and of edss progression - free patients between 70 and 87% [ 8 , 9 ] . proposed markers of poor prognosis include ongoing mri activity for 1 - 2 years after treatment initiation , with relapses and/or disease progression , or the development of neutralizing antibodies against ifn [ 1012 ] . nevertheless , these markers require that ifn is initiated and taken for a considerable time period before they can be assessed . the advantages of using cytokines as biological markers of ms are that they reflect the underlying immunopathology in the periphery ( blood ) and/or the cns ( csf ) , are easily measured , and are most likely to be affected by dmts that target the immune component of ms pathogenesis . several studies have investigated whether interleukin-17 ( il-17 ) can be used as a biological marker of response to ifn , with conflicting results [ 1316 ] . the focus on il-17 as a potential biomarker followed the discovery of il-17 producing t - cells ( th17 ) as key players in the pathogenesis of ms [ 17 , 18 ] . however , the immune pathogenesis of ms is far from straightforward with many components of the innate and adaptive immune system participating and interacting in a complex manner [ 19 , 20 ] . the aim of our study was to expand the search for prognostic biomarkers of treatment response to ifn among pro- and anti - inflammatory cytokines in the serum and csf of rr - ms patients who were prospectively followed up for a considerable time period ( mean 5.3 years ) . the patients included in this study were recruited from a single ms center ( neurology clinic of patras university hospital ) . they were hospitalized with symptoms suggestive of ms and underwent a thorough diagnostic workup , which included brain and spinal cord mri , lumbar puncture for csf analysis ( including detection of oligoclonal bands ( ocb ) and/or igg index calculation ) , visual evoked potentials ( vep ) , and other laboratory tests necessary for the exclusion of other diagnoses ( e.g. , systemic lupus erythematosus , vasculitis , infections , vitamin b12 deficiency , etc . ) . a detailed history was taken , for information about symptoms in the past that could be attributed to ms , estimation of disease duration , concomitant illnesses , and family history , and a detailed neurological examination was performed to determine the patients ' level of disability according to the expanded disability status scale ( edss ) . exclusion criteria included diagnoses other than ms , active infection or inflammation of any kind , and current or recent treatment with immunosuppressive or immunomodulatory drugs . a total of 43 patients , who were eventually diagnosed as having rr - ms , according to the 2005 mcdonald diagnostic criteria , were included in the study . the study protocol was noninterventional , meaning that all patient - related decisions such as the time and type of treatment , follow - up schedule , and repeat mris were made by the patients ' treating physicians . all patients received a 3-day corticosteroid treatment ( 1  g / day of methylprednisolone , iv ) during their hospitalization . blood and csf samples were collected prior to corticosteroid treatment and before initiation of ifn treatment . patients were prospectively followed up with visits twice a year ( approximately every 6 months ) , except for the first visit after enrollment , which was performed as soon as the patients entered remission ( usually 1 - 2 months after baseline ) . in each follow - up visit , information about relapses since the last visit , current medication , and results of repeat mris were collected , and a neurological examination was performed for determination of the edss score . at the end of the follow - up period , patients were characterized as poor responders ( pr ) to dmts if they had a sustained increase in edss score of 1 point in a 2-year period and/or an annualized relapse rate ( arr ) of 1 . patients who did not fulfill these criteria were characterized as good responders ( gr ) . the treatment response criteria were applied during the period in which the patients were under ifn treatment ( table 3 ) . the study started on january 2005 ( first patient , first visit ) and ended on february 2013 ( last patient , last visit ) . the study protocol was approved by the patras university hospital ethics ( re : 296 ) and scientific ( re : 451 ) committees as part of a general application to collect biological samples from patients attending the neurology clinic to study in vitro the role of t helper cell populations and cytokines in the pathogenesis , prognosis , and natural course of multiple sclerosis . the hospital abides by the helsinki declaration on ethical principles for medical research involving human subjects . serum and csf samples from patients and controls were collected and stored at 75c until processing . determination of serum and csf levels of the cytokines ifn- , tnf- , il-2 , il-4 , il-6 , il-10 , and il-17a was performed on a bd facsarray bioanalyzer using the cytometric bead array ( cba ) assay ( human th1/th2/th17 cytokine kit , bd biosciences , san diego , usa ) . serum and csf levels of tgf-1 were measured by elisa ( r&d systems quantikine tm , minneapolis , mn , usa ) . cytokine ratios ( th1/th2 , th1/th17 , th17/th2 , type-1/type-2 , ifn-/il-10 , and il-17a / il-10 ) were also calculated ( table 1 ) . cytokine levels in serum and csf , the various cytokine ratios , and patient data were compared between the patient groups using the kruskal - wallis nonparametric statistical hypothesis test because the application of shapiro - wilk normality test showed that in most cases of the dataset the normality assumption does not hold . when the null hypothesis of the kruskal - wallis test was rejected , the mann - whitney test with bonferroni corrections was employed for the pairwise comparisons of the groups . data were analyzed using the graphpad prism v. 5.03 ( san diego , ca , usa ) . table 2 shows the data of the study subjects , including their clinical and laboratory parameters . eight out of the 43 patients who completed the follow - up period did not receive any dmts ( norx ) . the remaining 35 patients received treatment with at least one of the ifn formulations ( ifn-1 i m , avonex , n = 11 , ifn-1 sc , rebif , n = 12 , ifn-1 sc , betaferon , n = 12 ) at standard doses . those who , according to the assessment of their treating physician , did not respond well to ifn switched to ga or a second - line therapy ( fingolimod or natalizumab ) . accordingly , 3 patient groups were formed : the 1st included patients with good response to ifn ( gr , n = 19 ) , the 2nd included patients with poor response to ifn ( pr , n = 16 ) , and the 3rd included patients who received no treatment ( norx , n = 8) ( table 3 ) . the norx patients had a significantly milder disease compared to the other groups ( lower edss scores from baseline to the end of the follow - up period , no or rare relapses ) and significantly fewer gd - enhancing lesions on baseline mri and cells in the csf . the main reasons these patients did not receive a prophylactic treatment included milder disease at presentation with a more benign course and the patients ' own preference . gr patients , compared to pr , had lower edss score , which became statistically significant early on , since the initial remission ( p = 0.01 ) . the main criteria of treatment response , that is final edss score and arr under ifn treatment , were , as expected , much higher in the pr patients compared to gr ( p = 0.001 and p < 0.0001 , resp . ) . all other parameters , like age at diagnosis , disease duration , pre - ifn arr , igg index and cells in the csf , lesions at baseline mri , and total follow - up duration , did not differ significantly between the two groups . in the pr group , the numbers of female and male patients were equal , whereas in the gr and norx groups there was a higher female to male ratio , although the difference did not reach statistical significance . the duration of ifn treatment was significantly shorter in the pr group ( p = 0.05 ) , a finding that was expected since pr patients were more likely to switch earlier to an alternative dmt ( table 3 ) . the proportion of relapse - free patients at the end of the follow - up period was 87.5% for norx , 58.8% for gr , and 0% for pr patients . similarly , the proportion of patients free of edss progression ( > 1 point ) was 100% for norx , 94.1% for gr , and 33.3% for pr patients gr patients had significantly higher serum levels of il-17a compared to pr and norx patients ( p = 0.03 and p = 0.05 , resp . ) and significantly higher serum levels of ifn- ( p = 0.03 ) , tnf- ( p = 0.05 ) , and il-2 ( p = 0.05 ) compared to norx patients ( figure 1 ) . pr patients had significantly higher ifn- serum levels than norx patients ( p = 0.05 ) ( figure 1 ) . no significant differences were observed in serum levels of il-6 , il-4 , il-10 , or tgf-1 between the 3 groups of patients ( figure 1 ) . csf cytokine levels did not show statistically significant differences between the 3 groups of patients ( see tables  s1 and s2 in supplementary materials available online at http://dx.doi.org/10.1155/2014/436764 ) . gr patients had significantly higher il-17a / il-10 ( p = 0.01 ) and th17/th2 ratios ( p = 0.03 ) and a lower th1/th17 ratio ( p = 0.05 ) of serum cytokines than pr patients , reflecting their higher serum levels of il-17a ( figure 2 ) . gr patients had also a significantly higher ifn-/il-10 ratio of serum cytokines than pr patients ( p = 0.02 ) , whereas the th1/th2 and type 1/type 2 serum cytokine ratios were not statistically different between the two groups ( figure 2 ) . gr patients had significantly higher th1/th2 ( p = 0.05 ) , type 1/type 2 ( p = 0.01 ) , il-17a / il-10 ( p = 0.05 ) , and ifn-/il-10 ( p = 0.01 ) serum cytokine ratios than norx patients ( figure 2 ) . the differences in serum cytokine ratios between pr and norx patients were not statistically significant ( figure 2 ) . differences in cytokine ratios in the csf between the 3 groups were not statistically significant ( tables  s3 and s4 ) . the calculations of type 1/type 2 cytokine ratios were performed with or without the inclusion of il-2 values in the nominator , with no significant alterations in the outcome ( cf . results from clinical trials have shown that ifn preparations slow down disease progression and reduce relapse rates in rr - ms patients by an average of 30% [ 57 , 23 ] . it has been suggested that they may be attributed to the development of neutralizing antibodies , or , as it can be inferred from a recent study , to failure of induction of a new regulatory t - cell population , the foxa1 + regulatory t - cells , that are induced by ifn and are found in ms patients that responded to ifn therapy . in our study , we searched for prognostic biomarkers of treatment response to ifn among pro- and anti - inflammatory cytokines in the serum and csf of rr - ms patients who were prospectively followed up for a considerable time period ( mean 5.3 years ) . we also calculated various cytokine ratios , to assess the relative concentrations of antagonizing cytokines , as expressed with calculated ratios that reflect the profiles of t helper cells ( th1/th2 , th1/th17 , th17/th2 , ifn-/il-10 , and il-17a / il-10 ) , or an overall picture of the immune response ( type-1/type-2 ) since it includes cytokines the expression of which is not restricted to specific cell populations [ 25 , 26 ] . type 1/type 2 ratios were calculated with and without the values for il-2 concentrations because il-2 is a pleiotropic cytokine that , in humans , is secreted by naive th cells when activated , stimulates proliferation and effector functions of th , cytotoxic t - cells , b - cells , and nk cells , promotes activation - induced cell death , but it also suppresses th17 differentiation and is an essential growth factor of regulatory t - cells [ 27 , 28 ] . serum il-17a was the main cytokine that distinguished gr from pr or norx patients , and this difference was confirmed by the cytokine ratios th1/th17 , il-17a / il-10 , and th17/th2 . serum ifn- was the only cytokine that distinguished pr patients from norx patients who , otherwise , presented with the lowest levels of proinflammatory cytokines . we found no significant differences in the levels of anti - inflammatory cytokines in serum or in the levels of all cytokines measured in the csf of gr , pr , or norx patients . several studies have shown that the beneficial effects of ifn are , at least partially , mediated through reduction of il-17 [ 13 , 2931 ] , which could explain the favorable response of patients with higher baseline il-17a observed in our study . .   showed that rr - ms patients with active disease have a significantly higher percentage of th17 cells than patients with inactive disease and that ifn decreases th17 cells , but not th1 cells . the effect of ifn was attributed to increased expression of ifn-r1 on th17 cells causing a stronger ifn-dependent stat1 phosphorylation , leading to apoptosis . ramgolam et al .   showed that ifn downregulates il-1 and il-23p19 gene expression whereas it upregulates il-12p35 and il-27p28 in ms patients ' dendritic cells , leading to suppression of th17 differentiation . they also found a direct effect of ifn on t - cells , mediated by inhibition of rorc , il-17a , and il-23r gene expression and by upregulation of il-10 gene expression . on the other hand , axtell et al .   found that a subset of nonresponders to ifn had high pretreatment serum levels of il-17f and endogenous ifn , compared to responders . this , according to the authors , could be explained by the hypothesis that this subset of patients had aggressive th17 disease , which the immune system tried to counteract by upregulating endogenous ifn ; thus , the addition of exogenous ifn could not be effective . the notion that high pretreatment levels of il-17 are a prognostic biomarker of poor response to ifn could not be confirmed by subsequent studies [ 16 , 32 , 33 ] . in our study blood and csf samples were collected during ms exacerbation ( ongoing symptoms , evidence of active lesions on mri ) , as opposed to most relevant studies [ 14 , 16 , 32 , 33 ] . since cytokines are short - lived molecules and the immunological reactions are most evident during the inflammatory , acute phase of the disease , measuring cytokines at this phase depicts more accurately the ongoing immune response . a potential pitfall is that cytokine levels measured in blood samples collected from ms patients during exacerbations may be influenced by a concurrent systemic infection , a phenomenon quite common , as shown by buljevac et al . . in addition , the follow - up period of the patients is the longest in any other study that addressed treatment response , giving the opportunity for a better assessment of disability progression and relapse rates in the long - term . placebo cohorts of clinical trials have been very diverse regarding arr , ranging from 1.28 in the earlier ifn pivotal trials [ 57 ] to as low as 0.39 in the more recent laquinimod or bg-12 clinical trials [ 36 , 37 ] . natural history studies , performed before the advent of dmts , provide a clearer picture of the long - term course of the disease in terms of relapse rates and progression . such studies indicate an average arr of around 0.5 , which is even higher when longitudinal , prospective assessments are considered . the application of the treatment response criteria set in our study ( an arr of 1 and a sustained increase in edss of 1 over a period of 2 years indicating poor response ) provided a clear - cut difference between gr and pr patients . gr patients had a mean arr under ifn of 0.18 and a progression of disability of only 0.23 points in the edss during the follow - up period ( mean 5.07 years ) , whereas pr patients had a mean arr of 1.42 and an increase in edss of 1.78 ( mean 5.39 years ) ( table 3 ) . since our study was not interventional , mri scans were performed at a frequency defined by each treating physician . these mri scan results were made available to us and were taken into consideration when the patients were characterized as having had a relapse or not since their previous visit . benign ms is another controversial issue , with many definitions and criteria , the most common being an edss of 3 after 10 years of disease duration [ 4 , 38 ] . the subgroup of patients with no treatment ( norx ) of our study fits this definition , because their total mean disease duration ( from onset of symptoms until the end of the follow - up period ) exceeded 10 years and the mean edss score at the end of the study was 1.12 . they also had almost no relapses during the follow - up period ( only one patient had a relapse , with no residual deficit , mean arr 0.03 ) ( table 3 ) . this group of patients had the lowest levels of nearly all serum proinflammatory cytokines and of the corresponding cytokine ratios ( tables  s1s4 ) , and the fewest gadolinium - enhancing lesions on mri and inflammatory cells in the csf . in the new era of multiple available treatment options for rr - ms , a reliable , easily measured biomarker of treatment response we searched for biomarkers for the prediction of response to ifn and of a benign disease course , among serum and csf pro- and anti - inflammatory cytokines . our results indicate that ( i ) baseline serum il-17a levels distinguish gr from pr patients , ( ii ) serum ifn- levels distinguish pr from norx patients , and ( iii ) norx patients ( with minimal disability and a benign course ) also exhibit minimal inflammation . the cytokine profiles of gr patients versus pr / norx patients discriminate the latter groups as those who should obviate treatment with ifn. our results need , of course , to be validated in a larger cohort of patients and for other dmts .
background . ms patients show a remarkable heterogeneity in their response to disease modifying treatments . given the need for early treatment initiation and the diversity of available options , a predictive marker that indicates good or poor response to treatment is highly desirable . objective . to find a biomarker for treatment response to ifn among pro- and anti - inflammatory cytokines . 
 materials and methods . 
 ifn- , tnf- , il-2 , il-4 , il-6 , il-10 , il-17a , and tgf-1 levels were measured in serum and csf of 43 patients with rr - ms who were followed up for a mean period of 5.3 years . 
 thirty - five patients received ifn treatment and were divided into good responders ( gr , n = 19 ) and poor responders ( pr , n = 16 ) . 
 the remaining 8 patients showed a very favorable outcome and remained untreated ( norx ) . 
 results . 
 gr had significantly higher serum baseline levels of il-17a than pr and significantly higher serum levels of il-17a , ifn- , tnf- , and il-2 than norx . 
 pr had significantly higher ifn- serum levels than norx . 
 no significant differences were observed in serum levels of il-6 , il-4 , il-10 , and tgf-1 or the levels of all cytokines measured in csf between the 3 groups of patients . 
 conclusions . 
 baseline serum levels of il-17a can be used as a biomarker of ifn treatment response .