there is ample evidence showing that 711% of hcv - infected children and adult individuals have thyroid dysfunction , mainly subclinical hypothyroidism , prior to the initiation of treatment [ 1 , 2 ] .
the association between autoimmune thyroiditis in hcv - infected patients , in particular in subjects with hcv - related mixed cryoglobulinemia , has also been confirmed .
once combined pegifnalpha2b + ribavirin therapy is initiated , the percentage of patients with thyroid dysfunction goes up to 1520% .
immunological mechanisms at the origin of this thyroid dysfunction are presumed but the precise mechanism still is unknown .  
it is involved in some organ - specific autoimmune diseases such as rheumatoid arthritis , asthma , inflammatory bowel disease , multiple sclerosis , and even organ allograft rejection [ 69 ] , among many others .
the main function of il-17-secreting t cells is to mediate inflammation , by stimulating production of inflammatory cytokines , such as tnf - a , il-1b , il-6 , and inflammatory chemokines that promote the recruitment of neutrophils and macrophages .
initial evidence suggested that human th17 cells were regulated by cd4 + 25+foxp3 + regulatory t cells , but recent evidence suggests an alternative regulatory mechanism mediated by il-10 and tgf - b [ 11 , 12 ] .
ag - specific th17 cells are induced in hcv - infected patients , but the hcv - ns4 protein also induces the secretion of il-10 and tgf - b by monocytes .
neutralization of both anti - inflammatory cytokines significantly enhances ns4-specific il-17 and ifn - gamma production by t cells from hcv - infected donors .
although this may represent a novel immune subversion mechanism by the virus to evade host protective immune responses , it could also be responsible for the thyroid dysfunction
an initial evaluation of the thyroid function in chronically infected hcv patients referred to the gastroenterology service at mexico 's h.r .
issste , showed that a high percentage of patients had subclinical hypothyroidism prior to the initiation of combined treatment .
therefore , our aim was to evaluate the serological status of il-10 , tgf - b and il-17 in chronically non - interferon treated hcv - infected patients with or without subclinical hypothyroidism .
all patients tested positive for antibodies to hcv using an enzyme immunoassay ( abbott diagnostics ) .
they were all infected as determined by consistently positive hcv rna serum detection results using a qualitative rt - pcr ( amplicor ; roche diagnostic systems ) .
none of the patients ( 25 males and 62 females ) had ever received treatment with pegylated interferon and/or ribavirin .
all the patients were catalogued as a class a child - pugh score and were selected to initiate pegylated interferon treatment .
the control group was conformed by 40 individuals ( 20 males , 20 females ; with a mean age of 48 years old ) with no history of liver disease , negative to hcv antibodies and normal liver function tests .
blood samples , freshly obtained from each patient one week before treatment was initiated , stood for 5 minutes at room temperature before being centrifuged for 10 minutes at 1800  rpm at 4c . plasma and sera was obtained from each patient and aliquots of 0.1  ml were kept at 70c until use .
plasma levels of il-2 , il-10 , il-12 , il-17 , and tgf - b were determined using commercially available kits purchased from genzyme and r & d systems ( quantikine ) carefully following the manufacturer instructions .
the r value for the linear regression of the standard curve had to be 0.95 or higher , a lower value was considered inadequate and the data obtained of such plates was eliminated . in order to activate latent tgf - b to immunoreactive tgf - b platelet - poor plasma samples was incubated for 10  min at room temperature with 1 n hcl and then the sample was neutralized with a solution made of 1.2 n naoh/0.5 m hepes for another 10 minutes before the determination was performed .
commercially available kits , also from r & d systems were used to determine antithyroglobulin ( tg ) , antithyroid peroxidase ( tpo ) , anticardiolipin , antiglomerular basement membrane ( gbm ) and anticyclic citrullinated peptide ( ccp ) antibodies .
the plates were read in a multiskan ascent ( thermolab systems , maltham , ma , usa ) microplate reader using a 450 and 490  nm filters .
hormone determination ( tsh , t3 , and free t4 ) and liver function tests ( alt , ast , albumin , immunoglobulin ) were performed in the laboratory of the hospital .
subclinical hypothyroidism was defined as a tsh value of 4 pg / ml with normal t3 and t4 values .  
the results of all these evaluations in the control group were within the established normal values determined in our laboratory ( 0.254.0  ui / ml for tsh ; 0.61.9  ng / ml for t3 ; 0.71.8  ng / dl for free t4 ; 240  ui / ml for ast and 240  ui / ml for alt ) . the results obtained in the control samples for the different cytokines were 6.8  2.7  pg / ml for il-2 ; 4.9  1.4  pg / ml for il-10 ; 52.7  8.1  pg / ml for il-12 ; 6.75  1.14  pg / ml for il-17 , and 398  271  pg / ml for tgf - b1 ) .
the normal value for anti - tg is 034  iu / ml , 0 - 12  iu / ml for anti - tpo , > 10  iu / ml for anti - cardiolipin , and 07  iu / ml for anti - gbm and anti - ccp antibodies .
differences between groups were evaluated by student 's t test , confidence interval at the 95% value , tukey test and kruskal - wallis one way analysis of variance on ranks , and holm sidak method .
the mean age of male patients was 45.7 years old whereas that of the females was 51.8 years old .
the predominant hcv genotype was 1b ( 42 patients ) followed by 1a ( 18 patients ) , 2b ( 17 patients ) , 2a ( 9 patients ) , and one patient with genotype 4 .  
seventy percent of the patients with elevated tsh ( 6.45  2.21  iu / ml ) ( group a ) were females ; the percentage of female patients in the group with normal tsh values ( 2.58  0.95  ui / ml ) ( group b ) was similar ( 71% ) . the difference in tsh values between both groups was highly significant ( p < .0001 ) as well as between group a and control group ( p < .001 ; according to the all pairwise multiple comparison procedures - holm - sidak method ) . there were no statistical differences in tsh values related to gender ( 5.82  1.73  ui / ml for males and 6.71  2.40  ui / ml for females in group a versus 2.26  0.76  ui / ml for males and 2.68  0.99  ui / ml for females , in group b ) .
similarly there were no statistical significant differences between both groups in relation to viral load .
although the values of the following markers were statistically different from those of the control group , there were no statistical significant differences between group a and group b results in relation to ast , alt , t3 , and free t4 .  
one patient with normal anti - tg , anti - tpo , and anti - ccp values had elevated anti - gbm and anti - cardiolipin antibodies .
this patient had consistently low platelet counts but had no history of miscarriage or deep vein thrombosis .
none of the anti - tpo nor anti - tg positive patients in group a had signs or symptoms of hashimoto 's thyroiditis .
the percentage of autoantibodies in group b was 33% for anti - tg antibodies , and 7% for anti - tpo .
none of the patients in group b had elevated values of anti - gbm or anti - ccp , and only one had elevated anti - cardiolipin antibodies .
interestingly , the latter patient was also positive for anti - tpo antibodies and had deep vein thrombosis of the lower extremities .   in relation to the cytokines it was interesting to observe that although the concentrations in hcv+ patients were significantly higher than those found in the control group for all the cytokines , the intergroup differences ( group a individuals versus group b ) for il-10 ( 14.83  2.98  pg / ml versus 15.27  1.70  pg / ml ) and il-12 ( 271.59  196.43  pg / ml versus 221.13  101.60 
the cytokines that showed a highly statistically significant difference between group a and group b patients were : il-2 , il-17 , and tgf - b ( table 1 ) .
the kruskal - wallis analysis of variance at the 0.05 level of significance for il-2 showed a median value of 611  pg / ml for group a , 195  pg / ml for group b , and 7.36  pg / ml for the control group ( p < .001 ) ; the significance of these differences between groups a and b with the control was confirmed by the tukey test ( p < .05 ) . as far as il-17 was concerned
, the kruskal - wallis analysis of variance showed a median value of 19.4  pg / ml for group a , 18.5  pg / ml for group b , and 8.9  pg / ml for the control group ( p < .001 ) ; similarly to il-2 , il-17 results were also confirmed by the tukey test .
there were no differences in il-17 concentration in both groups in relation to the viral genotype .
finally , tgf - b values passed the normality test and the one way analysis of variance demonstrated a significant difference between groups a and b with the control group ( p < .001 ) but not between group a and group b. it was interesting to observe that tgf - b concentration in group a patients was 38% below that of group b patients .
hcv infection has been related to various autoimmune disorders including thyroid dysfunction [ 14 ] and papillary thyroid cancer .
the presence of hypothyroidism in chronically hcv - infected individuals , either under treatment or not , has been well documented [ 1 , 16 , 17 ] .
one interesting observation was the high percentage of hcv - infected patients having hypothyroidism ( 23% ) versus an average 12% cited in the literature ; this could be secondary to the significantly higher frequency of thyroid microsomal antibodies in mexican women in comparison with caucasian or black women   thus representing a higher predisposition to autoimmunity against thyroid gland .
we found autoimmune thyroid involvement as judged from serum auto - antibodies and tsh values in 20 of the 87 patients that we analyzed .
the percentage of individuals in the subclinical hypothyroidism group with autoantibodies was significantly higher than the percentage found in normothyroid hcv+ patients and control .
none of our patients had manifestations of hashimoto 's disease and only one of the female patients in the hypothyroid group , had a pattern compatible with an antiphospholipids syndrome , an association found in 20% of hcv - infected patients .
the percentage of females with autoimmune disease in our study ( 70% ) is identical to that found in a multicenter international study [ 20 , 21 ] . nevertheless , it has recently been suggested that the presence of autoantibodies in hcv - infected patients do not appear to be of clinical importance .
one patient in each group had elevated anticardiolipin antibodies but none of them suffered stroke , although the patient with deep vein thrombosis is a strong candidate .
the thyroid dysfunction in hcv - infected patients has been associated to interleukin 2 [ 2426 ] , a cytokine secreted primarily by th1 cells .
patients with non - hcv - related diseases being treated with high doses of il-2 also develop thyroid dysfunction [ 27 , 28 ] .
the increased interleukin 2 serum concentration determined in our non - il-2 or ifn - treated hcv+ hypothyroid patients , corroborates the above mentioned findings .
interleukin 2 is known to expand th17 cells in some autoimmune diseases . a key cytokine for il-2 production and th1 cell differentiation is il-12 [ 30 , 31 ] .
although the core protein of hcv seems to have a suppressive action on il-12 production at the transcriptional level , our results showed that the concentration of il-12 was similar in both of our hcv - infected groups , but considerably higher than the control group .
this suggests that hypothyroidism was not related to interleukin-2 despite the fact that transgenic mice with il-12 serum concentrations in the 150 pg / ml range develop a moderate primary hypothyroidism .
the th17 response is characterized by a serum increase in interleukin-17a , commonly known as il-17 .
this cytokine , secreted by the cd4 + t cells known as th17 , is also secreted by some cd8 + t cells , nkt , alpha - beta or gamma - delta t cells , eosinophils , neutrophils , and monocytes [ 3537 ] .
th17 cells are mainly linked to autoimmune diseases , namely , multiple sclerosis , inflammatory bowel disease , rheumatoid arthritis , lyme disease , psoriasis , and uveitis [ 29 , 38 ] .
of the five different il-17 receptors ( a to e ) , il-17 only bind to il-17ra and c [ 35 , 39 ] . the pituitary gland and the thyroid ,
the regulation of th17 cells is basically mediated by cd4+cd25+foxp3 + t reg cells which are capable of inducing anergy towards self- and alloantigens , thus playing an important role in autoimmunity [ 4143 ] .
nevertheless , a new regulatory mechanism mediated by il-10 and tgf - b , both of which suppress il-17 production [ 11 , 12 ] , has been suggested .
our hypothyroid patients had similar amounts of il-10 than the normo thyroid group but lower tgf - b serum concentration .
therefore , the only abnormality that could explain the high il-17 serum concentration and the thyroid dysfunction was related to alterations in the foxp3 + treg cells , probably through enhanced stat3 phosphorylation [ 36 , 45 ] . nevertheless , recent evidence shows that tgf - beta orchestrates th17 cell differentiation in a concentration - dependent manner .
low tgf - b concentrations favors th17 cell differentiation whereas high tgf - b concentrations favors foxp3 + treg cell differentiation .
the former is accompanied by high levels of il-17 , as we observed in our hypothyroid patients , whereas the latter is accompanied by a tighter regulation of il-17 secretion , as we observed in our normothyroid patients . despite tgf - b apparent importance
it has recently been shown that the proportion of peripheral th17 cells in patients with autoimmune thyroid disease is higher than in control subjects .
although there is evidence showing that il-2 can inhibit human th17 cell differentiation through a stat-5 mediated pathway and enhanced tgf - b - induced foxp3 expression [ 48 , 49 ] , a recent report by deknuydt et al .   show that cd4+cd25 + foxp3 + t regulatory cells in the presence of il-2 , in an inflammatory microenvironment , can be converted into proinflammatory th17 cells .
this could explain the link between inflammation , high il-2 and il-17 concentrations , and autoimmunity in our patients .
interleukin 17 is also involved in human alcoholic liver disease , an entity that shares some features with autoimmune diseases .  
the viral load in our patients could be definitively considered low and such viral loads have been linked with the development of auto antibodies due to the lowering of the b cell activation threshold or by inducing self - reactivity through a mechanism of molecular mimicry [ 52 , 53 ] .
we believe that our results point towards an abnormality in the t regulatory cells , induced by the hepatitis c virus . the fact that not all the hcv - infected patients develop an full blown autoimmune disease suggests that there are many individual variations that go beyond the viral load , viral genotype , presence of auto antibodies or liver biochemical alterations
. we must not forget that the hcv does replicate within the thyroid tissue   and that the evolution of the hcv infection is related to distinct immune cell cytokine expression profiles .
recently it has been postulated that a th1cell mediated immune response underpins the association of chronic hcv infection with endocrine disease , our results suggest that a th17 immune response should also be considered .
 hcv - ag - specific th17 cells secrete il17 , a cytokine involved in autoimmune diseases and regulated by il10 and tgf - b . 
 512% of patients with chronic hcv infection have hypothyroidism . 
 we evaluated the role of these cytokines in this patients by determining serum concentration of tsh , t3 , free t4 , il2 , il10 , il12 , il17 , tgf - b , anti - tg , tpo , ccp , gbm , and cardiolipin antibodies in 87 chronically noninterferon treated hcv - infected patients . 
 20 patients ( group a ) had elevated tsh values ( > 5  ui / ml ) whereas the remaining 67 ( group b ) had normal values . 
 the percentage of anti - tpo , tg , gbm , and cardiolipin antibodies in group a patients ( 33% , 41% , 5% and 5% , resp . ) as well as il17 , il2 and tgf - b concentrations ( 25  23 pg / ml , 643  572 pg / ml , and 618  221 pg / ml , resp . ) 
 were significantly higher than group b. abnormal th17 regulation mediated by il-2 and low tgf - b concentrations is associated with hypothyroidism in chronically - infected hcv patients . 
1. Introduction
2. Material and Methods
3. Results
4. Discussion
5. Conclusions