hepatitis c virus ( hcv ) is one of the major causes of chronic liver disease in the world .
more importantly , many of those patients with chronic hepatitis c eventually develop cirrhosis and hepatocellular carcinoma . following infection with hcv ,
only about 15% of patients can clear the virus , while 6080% of patients develop persistent chronic infection [ 3 , 4 ] .
previous studies have revealed that the persistence of viral infection and chronic inflammation are dependent on the interaction among the virus , hepatocyte , and the host immune system [ 4 , 5 ] .
the viral infection and related hepatocyte injuries are known to suppress the immune system [ 6 , 7 ] .
although experimental evidence suggests that antigen - specific th1 immunity and proinflammatory cytokines play an important role in the hcv - related liver injury and clearance of viruses [ 810 ] , the pathogenesis of chronic hcv infection has not been fully understood .
interleukin-33 ( il-33 ) is one of the newly described members in the il-1 family and can be produced by epithelial tissues and vascular endothelial cells .
il-33 binds to its heterodimer receptors composed of il-1 receptor - related protein st2 and il-1 receptor accessory protein ( il-1rap ) and can activate the myd88 and nf-b - related signal pathway [ 11 , 12 ] .
st2 has transmembrane form of st2 ( st2 or st2l ) and soluble form of st2 ( sst2 ) .
st2 is expressed on th2 and mast cells and functions as a mediator of il-33 bioactivities , while sst2 acts as a decoy receptor for il-33 .
biologically , il-33 induces th2 cell differentiation and activates mast cells , leading to th2 cytokine production and th2 response as well as pulmonary and mucosal th2 inflammation .
furthermore , il-33 can antagonize the lps - induced mortality in a model of septic shock , and the levels of serum il-33 were elevated in sle and ra patients .
recently , il-33 has been found to be an important factor of the pathogenesis of hiv infection and dengue virus infection [ 14 , 15 ] .
however , little is known on whether il-33 could participate in the pathogenic process of hcv infection . in the current study , we examined the concentrations of serum il-33 and sst2 in patients with chronic hepatitis c ( chc ) , individuals with spontaneously resolved hcv infection ( sr - hcv ) , and healthy controls ( hc ) to evaluate the potential role of il-33/st2 axis in the pathogenic process of chc .
furthermore , we determined the concentrations of serum il-33 before and after antivirus therapies in patients with chc .
we found that il-33 response appeared to be an important factor of the pathogenesis of chc , associated with the severity of liver injury in chc patients .
a total of 154 patients with chc , 24 cases with sr - hcv , were recruited sequentially at the outpatient service of first hospital .
another 20 gender- , age- and ethnic - matched hc were recruited , and they had no historical liver diseases and no evidence of hbv , hcv , and hdv infection .
individuals with positive anti - hcv antibodies and serum hcv rna for at least six months were diagnosed with chc .
individuals with sr - hcv were defined as those subjects who had prior hcv rna detection ( hcv - ab ) but lacked hcv rna detection for 12 weeks after enrollment in the absence of treatment .
genotyping of hcv showed that 22 chc patients had genotype 2a , 45 had genotype 1b , and 14 had unclassified genotype .
individuals with historical and current hepatitis b , d virus or hiv infection , autoimmune hepatitis , or metabolic liver disease , who had received immunosuppressive therapy or antiviral therapy during the past 12 months before entry , were excluded .
fifty patients with chc were treated subcutaneously with 500 million units of a short - acting interferon ( sinogen , jinan , china ) every other day for 12 weeks .
the concentrations of serum il-33 , sst2 , hcv - rna , alt , and ast were measured before and after treatment .
individuals with 100-fold reduced serum virus load were defined as drug - responsive patients ; otherwise , individuals were defined as drug - nonresponsive patients .
written informed consent was obtained from individual participants , and the study was approved by the first hospital ethical committee of jilin university .
peripheral blood samples were obtained from individual subjects , and their sera were prepared and then stored at 80c till needed .
the levels of serum il-33 were measured at the entry of individual participants , before and after treatment of patients with chc , and the concentrations of serum ifn- , tnf- , il-2 , il-4 , il-10 , and il-6 in individual chc patients were also determined .
the concentrations of serum il-33 and sst2 in individual patients and healthy controls were determined by elisa using human il-33 and sst2 elisa kits , according to the manufacturers ' instruction ( roche diagnostics , lewes , uk ) .
briefly , individual sera at 1  :  4 dilutions were subjected to elisa analysis , and the concentrations of serum il-33 and sst2 in individual samples were calculated , according to the standard curve established using the recombinant il-33 and sst2 provided .
the concentrations of serum cytokine levels ( ifn- , tnf- , il-2 , il-4 , il-10 , and il-6 ) were determined by cytometric bead array ( cba ) , according to the manufacturer 's protocol ( bd biosciences , san joes , usa ) with minor modification .
briefly , 25  l of individual sera was used in duplicate for analysis , as described previously .
the concentrations of serum cytokines were quantified using the cellquestpro and cba software ( becton dickinson ) on a facscalibur cytometry ( bd biosciences ) .
the concentrations of serum antibodies against hcv were detected by elisa ii ( abbott laboratories , abbott park , usa ) .
the levels of serum alt and ast were detected using a biochemistry automatic analyzer ( roche diagnostics , branchburg , usa ) .
the amounts of serum hcv rna were measured by quantitative pcr assay using a luciferase quantization detection kit , following the protocols ( roche amplicor , basel , switzerland ) .
the detection limit of viral rna was 300  copies / ml . the data are expressed as median and range unless specified .
the differences between the groups were analyzed by wilcoxon - rank sum test and chi - square test using the spss 14.0 software .
to determine the potential role of il-33 in the pathogenic process of chc , a total 154 patients with chc , 24 with sr - hcv , and 20 with hc were sequentially recruited .
as expected , there was no significant difference in the distribution of age and gender among these groups of subjects , but the concentrations of serum alt and ast in patients with chc were significantly higher than those in the hc and those with sr - hcv ( table 1 ) . while high levels of virus rna were detected in chc patients , there was no detectable viremia in both hc and sr - hcv .
in addition , the anti - hcv antibody was detected in chc patients and individuals with sr - hcv , but not in hc .
analysis of serum cytokines indicated that there was no significant difference in the concentrations of serum il-33 between individuals with sr - hcv and hc , while the concentrations of serum il-33 in patients with chc were significantly higher than those in individuals with sr - hcv and hc ( p < 0.001 , figure 1(a ) ) .  
furthermore , stratification of patients with chc revealed that the concentrations of serum il-33 in chc patients with abnormal levels of serum alt ( > 50  units / l ) or ast ( >
40  units / l ) were significantly higher than those in chc patients with normal levels of alt ( < 50  units / l ) or ast ( < 40  units / l ) , respectively ( p < 0.001 , p < 0.001 , resp . , figures 1(b ) and 1(c ) ) .
the concentrations of serum il-33 in chc patients were correlated positively with the levels of serum alt and ast ( r = 0.388 , p < 0.001 ; r = 0.400 , p < 0.001 , resp .
, figures 1(d ) and 1(e ) ) . apparently , il-33 is a pathogenic factor , associated with the damage of the liver in chc patients .
further analysis revealed that the levels of serum sst2 were significantly higher in chc and sr - chc patients than that in hc ( p = 0.004 , p = 0.041 , resp .
, figure 2 ) , but there was no significant correlation between the levels of serum il-33 and sst2 in those subjects ( r = 0.050 , p = 0.678 ) .
moreover , the concentrations of serum ifn- and il-6 , but not tnf- , il-2 , il-10 , and il-4 , in patients with chc , were significantly lower than those in individuals with sr - hcv ( figures 3(a ) and 3(b ) ) .
following treatment of chc patients with ifn for 12 weeks , 45 out of 50 chc patients displayed dramatically reduced levels of serum hcv virions .
notably , treatment with ifn remarkably reduced il-33 responses in those patients because the levels of serum il-33 in patients with chc after treatment with ifn were significantly lower than those before treatment .
however , treatment with ifn for 12 weeks did not change significantly in the levels of serum sst2 in chc patients ( p = 0.641 , figure 4(b ) ) .
il-33 , through the receptor complex composed of st2 and il-1rap , can activate the map kinase and nf-b signal pathways and promote th2 response and cytokine production .
indeed , intranasal administration of il-33 triggered an immediate allergic response in the airway , and endogenous il-33 contributes to airway inflammation .
a recent study revealed that the levels of serum il-33 were elevated in sle and ra patients and correlated with the levels of serum esr and crp , two inflammation markers , indicating that il-33 may participate in the acute - phase response of sle .
in addition , il-33 can protect against septic shock by enhancing neutrophils infiltration at the site of inflammation .
other studies suggest that il-33 participates in the pathogenic process of acute hepatitis induced by con - a [ 25 , 26 ] , and il-33 overexpression is associated with the development of hbv / hcv - related liver fibrosis .
to investigate the role of il-33 in the pathogenic process of chc , we determined the levels of serum il-33 and sst2 in 154 chc patients .
we found that the levels of serum il-33 in chc patients were significantly higher than in those with sr - hcv and hc .
furthermore , the levels of serum il-33 in chc patients with abnormal concentrations of alt or ast were significantly higher than in those with normal levels of alt and ast in this population .
in addition , treatment with ifn to inhibit the replication of hcv dramatically decreased the levels of serum il-33 in chc patients .
more importantly , the concentrations of serum il-33 were correlated positively with the levels of serum alt and ast in chc patients .
given that abnormal levels of alt and ast are indicative of abnormal liver function and injuries , our data suggest that il-33 may be a pathogenic factor of the pathogenic process of chc in chinese patients .
therefore , if the levels of serum il-33 are also correlated with pathogenic degrees of the liver in chc patients , the levels of serum il-33 may be used as a new biomarker for the diagnosis of liver damages in chc patients .
furthermore , we found that the levels of serum sst2 were significantly higher in chc and sr - chc patients than those in hc , but were not correlated with the levels of il-33 in patients .
treatment with ifn for 12 weeks did not significantly change the levels of serum sst2 .
a previous study has shown that the levels of serum sst2 in patients with acute liver failure are higher than in those with chronic liver failure and healthy controls .
it is possible that high levels of serum sst2 are an early biomarker of liver injury , while high levels of serum il-33 may be associated with the development and progression of liver fibrosis and damage .
we are interested in further examining the mechanisms underlying the action of il-33/sst2 axis in hcv - related liver injury .
we found that the levels of serum ifn- and il-6 in chc patients were significantly lower than those in sr - hcv , supporting the notion that proinflammatory cytokines , such as ifn- and il-6 , are not only important factors for the clearance of infected hcv , but also for liver injury [ 2931 ] .
the lower levels of ifn- and il-6 in chc patients were unlikely to have come from the antagonization of il-33-induced th2 responses in those patients , because we failed to detect significant difference in the levels of serum il-4 and il-10 between those chc patients and sr - hcv and hc . given that il-6 is a critical factor of the functional development of th17 cells   and that ifn- is an effector of th1 response , the lower levels of serum ifn- and il-6 in those chc patients indicated continual viral replication and pathogenic progression . although il-33 has been shown to promote ifn- production by invariant nkt and nk cells , il-33 may , through an unknown pathway , downregulate the functional development of hcv - related th1 response and inhibit ifn- production .
our data indicate , for the first time , that the concentrations of serum il-33 are significantly higher in those with sr - hcv and hc and are significantly correlated with the levels of serum alt and ast , suggesting that il-33 may be a pathogenic factor of hcv - related liver injury in chc patients .
we recognized that the current study has limitations , including no source for il-33 and no histopathological examination of liver tissues .
although more detailed studies are necessary to determine the role and mechanisms of il-33 in regulating the pathogenic process of chc , our novel findings may provide new insights into understanding the pathogenesis of chc .
 interleukin-33 ( il-33 ) is associated with the development of th2 responses . 
 this study examined the potential role of il-33 in the pathogenic process of chronic hepatitis c ( chc ) in chinese patients . 
 the levels of serum il-33 and sst2 in 154 patients with chc , 24 with spontaneously resolved hcv ( sr - hcv ) infection and 20 healthy controls ( hc ) , were analyzed by elisa . 
 the concentrations of serum il-2 , ifn- , tnf- , il-4 , il-6 , and il-10 , hcv loads , alt , ast , and hcv - ab were measured . 
 we found that the levels of serum il-33 in chc patients were significantly higher than those of sr - hcv and hc but decreased after treatment with interferon for 12 weeks . 
 more importantly , the levels of serum il-33 were correlated with the concentrations of alt and ast in chc patients . 
 the levels of serum sst2 , as a decoy receptor of il-33 , were significantly higher in chc and sr - chc patients than those in hc , and there was no correlation between the levels of serum sst2 and il-33 . 
 the concentrations of serum ifn- and il-6 in chc patients were significantly lower than those of sr - hcv . 
 these data suggest that il-33 may be a pathogenic factor contributing to chc - related liver injury . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussions