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systemic sclerosis ( ssc ) is a complex autoimmune disease of unknown aetiology characterized by excessive fibrosis of the skin and internal organs , presence of autoantibodies to nuclear antigens , and vascular damage [ 14 ] .
several genetic and environmental agents have been proposed to be responsible for causing ssc . among these agents
are infections , toxin exposure , single nucleotide polymorphisms ( snps ) , and interferon treatment [ 57 ] .
regardless of the origin , the immune system on ssc patients shows evidence of homeostatic alterations , including increased levels of chemokines in blood serum and different populations of lymphocytes in peripheral blood mononuclear cells ( pbmcs ) .
in addition to these alterations , there is increased evidence that the interferon ( ifn ) system is modified in patients with ssc [ 815 ] .  
ifns are immunomodulatory cytokines that act as an important link between the innate and adaptive immune system in vertebrates .
ifns bind to distinct cellular receptors , and their biological activities are mediated by the regulation of interferon stimulated genes ( isgs ) .
type i and type iii ifns are important regulators of innate immunity and are produced after stimulation of pattern recognition receptors in order to initiate and regulate the immune response .
one of the main pathways leading to type i and iii ifns induction depends on the induction and activation of irf7 .
almost every cell type is able to produce these ifns after stimulation , but its main producers are cells from the immune system such as plasmacytoid dentritic cells .
type i and iii ifns have redundant biological activities , even though they bind to different cellular receptors and have distinct structures .
ifn gamma , the only known type ii ifn , has distinct biological activities and is important for the regulation of adaptative immunity [ 16 , 17 ] .
the innate immune system and type i ifns have been proposed as important factors in the initiation and maintenance of some autoimmune diseases , and the influence of type i ifns in ssc has not been studied in detail [ 1820 ] .
several activities of type i and type iii ifns are redundant , and until now there has been no description of type iii ifns participation in ssc or other autoimmune diseases . an snp in one chain of the type iii ifn receptor ( il10r2 ) has been associated with ssc , indicating that at least responsivity to these molecules may be important for the disease .  
previous studies have shown an increase of several isgs in pbmcs of patients with autoimmune diseases such as systemic lupus erythematosus ( sle ) and type i diabetes [ 10 , 22 ] .
differential induction of isgs in blood cells and fibroblasts from patients has already been described [ 19 , 2325 ] . when pbmc gene expression was compared by microarray between healthy donors and ssc patients ,
some of these genes were also found in patients with sle , which is an autoimmune disease marked by several alterations in the type i ifn system [ 4 , 24 ] .
levels of 25 oligoadenylate synthetase ( 25oas ) and double - stranded rna - activated protein kinase ( pkr ) , two isgs , are found at higher levels in fibroblasts from ssc patients when compared to controls . to date
, there is no direct evidence of abnormal induction of type i ifns in ssc , even thought cells treated with sera from ssc patients produce more ifn alpha and other cytokines than cells treated with sera from controls .
these findings suggest that ifns may also be important in ssc pathology .   in this paper ,
we show that pbmcs from ssc patients basally expressed more type i ifns ( alpha and beta ) than pbmcs from healthy donors .
in addition , there was no detectable basal induction of type iii ifns in patients or healthy donors . when isgs were measured , we observed increased 25oas basal levels , consistent with previous studies .
these findings suggest that , similar to other autoimmune diseases , type i ifns play an important role in ssc .
ten patients fulfilling the american college of rheumatology preliminary criteria for diagnosis of diffuse ssc and four healthy subjects were chosen as blood donors ( table 1 ) . before donating blood , each subject read and signed an informed consent previously approved by the ethics committee of the universidade federal de minas gerais , brazil .  
vero cells ( african green monkey kidney cell line ) were obtained from the atcc and grown at 37c in dulbecco 's modified eagle 's medium ( dmem ) supplemented with 2  mm glutamine , 5% fetal calf serum ( cultilab , campinas , sp , brazil ) , and antibiotics .
pbmcs from healthy donors or ssc patients were purified using the ficoll - hypaque purification technique .
briefly , fresh blood collected in vacuum tubes containing heparin was diluted in an equal volume of 1x phosphate buffered saline ( pbs ) .
twenty milliliters of the diluted blood were carefully added over 10  ml of ficoll and centrifuged at 400    g for 30  min .
the layer containing pbmcs was collected and washed once in 1x pbs , and the cells were counted .
after the extraction , one microgram of rna was treated with 1  u of dnase i enzyme ( biolabs ) , and after treatment the rna was used as template in reverse transcriptions carried out using mmlv reverse transcriptase ( promega ) .
real time pcrs were performed in a step - one real - time pcr machine ( applied biosystem ) using the relative quantification methodology .
the results were analyzed using stepone software v2.2 , and all data were expressed as a ratio relative to the beta - actin level .
pcr primers used for human genes are listed below : ifn alpha2 forward 5-ttgacctttgctttactggt-3 and reverse 5-cacaagggctgtatttcttc-3. ifn beta forward 5-cctgtggcaattgaatgggaggc-3 and reverse 5-caggtagatggtatagcgtgg-3. ifn lambda1 forward 5-cttccaagcccaccccaact-3 and reverse 5-ggcctccaggaccttcagc-3. ifn lambda2/3 forward 5-tttaagagggccaaagatgc-3 and reverse 5-tgggctgaggctggatacag-3. irf-7 forward 5-caagtgcaaggtgtactgg-3 and reverse 5-caggtagatggtatagcgtgg-3 .
25oas forward 5-aactgcttccgacaatcaac-3 and reverse 5-cctccttctccctccaaaa-3. mxa forward 5-atcctgggattttggggctt-3 and reverse 5-ccgcttgtcgctggtgtcg-3. 616 forward 5-catgcggcagaaggcggtat-3 and reverse 5-cgacggccatgaaggtcagg-3. beta - actin forward 5-ccaaccgcgagaagatga-3 and reverse 5-ccagaggcgtacagggatag-3. after cell fractionation by the ficoll - hypaque technique , one milliliter of sera was collected from each donor and frozen at 70c until use . at the time of titration , each serum sample was serially diluted from 1  :  2 to 1  :  4096 and used to treat 96-well plates of vero cells with 90% confluency .
eighteen hours after treatment , the medium was discarded and the cells were infected with 10  tcid50/ml of encephalomyocarditis virus ( emcv ) . the infection was monitored for 48 hours , at which time the plates were fixed with 3.7% formaldehyde before being stained with 1% crystal violet ( adapted from ) . alongside the samples , the following controls were used : 600  u / ml of recombinant human ifn alpha 2a ( roche ) and a negative serum sample to which 600  u / ml of recombinant human ifn alpha 2a ( roche ) was added .
student 's t - test and nonparametric mann - whitney test were used to analyze the results .
the expression profile of type i and type iii ifns was measured by qpcr using rna from pbmcs freshly collected from ssc patients or controls .
we observed that the basal levels of ifn alpha2 and ifn beta were increased in patients while there was little or no expression of these genes on healthy donors ( figures 1(a ) and 1(b ) ) .
when type iii ifns were measured , we observed that the basal level of ifn lambda1 and lambda2/3 was the same in both groups ( figures 1(c ) and 1(d ) ) .
these results were obtained from cells without any treatment or culture , indicating that these genes might be normally induced in patients .
we also attempted to detect ifn proteins in sera from patients and controls through a biological assay commonly used for ifn titration .
however we were unable to detect any activity higher than the detection limit of the assay ( 20  iu / ml ) .
our controls worked perfectly , indicating that the negative results obtained were not due to sera toxicity or experimental artifacts ( data not shown ) .  
in addition to ifn basal levels , we also measured irf7 , 25oas , mxa , and 616 basal levels in pbmcs from patients and controls
. irf7 is an important factor for type i and type iii ifn induction , whereas the other genes are isgs that are commonly induced by type i and type iii ifns .
there was no statistically significant difference in the levels of irf7 ( figure 2(a ) ) between patients and controls .
similarly , there was no difference between levels of mxa ( figure 2(c ) ) and 616 ( figure 2(d ) ) .
however a difference on 25oas levels was detected , and this gene basal expression level was increased in patients but not in controls ( figure 2(b ) ) .
however , the influence of type iii ifns in these diseases is not yet known , despite the similarities in the biological activities of these ifn types .
ssc is an autoimmune disease with unknown etiology in which the immune system homeostasis is severely compromised . to date
there has been little information about the influence of ifns in ssc patients in the literature and no direct evidence of type i or type iii ifn basal induction in patients . in this paper , we show that some components of the ifn system can be found at higher levels in freshly purified pbmcs from ssc patients , compared with cells from controls .
increased basal expression of ifn alpha2 and ifn beta ( type i ifns ) was detected in ssc patients , while there was no difference between the basal levels of ifn lambda1 and ifn lambda2/3 ( type iii ifns ) in patients and controls ( figure 1 ) .
these results show that , at least for mrna levels , there is a higher expression of type i ifns by pbmcs from ssc patients than from controls .
this abnormal expression , especially in the case of ifn alpha2 , is consistent with the status of autoimmunity in several other diseases and can have a major role in initiating and maintaining the disease .
high levels of ifn beta can be even more dramatic for an autoimmune status , since this ifn has similar biological activities to ifns alpha but is even more potent .
we also tried to titrate ifn protein levels on sera obtained from patients and controls , but the levels were below our limit of detection ( 20  iu / ml ) . considering that this titration method is able to detect any mixture of ifns capable of inducing antiviral activity in host cells , we can hypothesize that even though ifn levels are increased on ssc , they are not found in high levels
. methodologies with greater sensitivity must be used in order to detect and measure type i ifn proteins in ssc patients sera .
expression levels of irf7 , an inducible transcription factor responsible for type i and type iii ifn gene induction , were not altered between patients and controls ( figure 2(a ) ) .
this finding leads us to conclude that ifn alpha2 and beta are being induced by another stimulus in the disease , which is specific to their induction and is not common to type iii ifns induction .
25oas basal expression levels were higher in patients than in controls ( figure 2(b ) ) .
differential induction of 25oas is consistent with a previous study that used fibroblasts from ssc patients , in which higher basal levels of 25oas were detected when compared to control fibroblasts .
it also indicates that there is , at least to some degree , some similarity between alterations in the skin and blood cells of ssc patients .
other measured isgs , 616 , and mxa were not differentially expressed between patients and controls ( figures 2(c ) and 2(d ) ) .
the factors leading to this differential induction on isgs are unknown , but could be due to cell exposure to various ifn subtypes abnormally expressed during the disease .
the capacity of ssc sera to induce cytokines in treated cells   supports this observation , since this activation probably occurs due to cytokines and other molecules in the plasma .  
based on these findings , we hypothesize that the ifn system is modified in ssc . by still unknown mechanisms , at least ifns alpha2 and ifn beta are induced in patients by a pathway that does not induce type iii ifns .
type i ifns produced by patients ' cells circulate and through activation of certain subsets of isgs maintain the autoimmune status of ssc .
these molecules are important links between innate and adaptative immune responses and can , among other actions , activate immune cells to improve autoantigens detection and autoantibodies production .
it has been already described that ifn alpha2 can induce tlr3 activation in ssc fibroblasts in culture .
this activation can also be happening in vivo by type i ifns naturally produced by pbmcs , corroborating our hypothesis .
ifn beta activation is a novel finding on the disease and can be of great importance to ssc altered immune homeostasis .
even though there is some redundancy between the activity of type i and type iii ifns , we could not link type iii ifns to ssc , at least when pbmcs are involved .
future experiments with higher sample size and using distinct cell populations isolated from total pbmcs are essential to further investigate these results .  
these findings are of great importance to our understanding of the pathogenesis and pathology of ssc .
increased basal levels of type i ifns can lead to abnormal activation of isgs and undesirable side effects on the patients , even without interference by type iii ifns .
the differential expression profile of isgs detected in pbmcs is consistent with the profile found previously in fibroblasts   and could be a signature related to the disease .
these unique modifications in the ifn response may be considered as candidates for ssc biomarkers and can be considered for further studies as diagnostic tools for ssc .
objective . systemic sclerosis ( ssc ) 
 is a complex autoimmune disease in which interferons ( ifns ) may play an essential role . 
 we hypothesized that type i and iii ifns may be found in increased levels in patients and be responsible for ssc autoimmune status . methods . 
 type i and iii ifn and isg basal expression profiles were measured by qpcr using rna from pbmcs of patients and controls . results . 
 type i ifns are increased in ssc patients , while no induction of type iii ifns was detected . 
 this induction can not be related to irf7 , since no upregulation of this gene was seen on patients . of 
 the isgs tested , 25oas levels were increased in patients , while 616 and mxa levels were not . conclusions . 
 while there is no indication of type iii ifn induction , increased levels of type i ifns may lead to abnormal regulation of isgs that can be responsible for immune system alterations described for ssc .