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Du et al. Virology Journal 2014, 11:51http://www.virologyj.com/content/11/1/51

RESEARCH Open Access

Correlation between the suppressor of cytokinesignaling-1 and 3 and hepatitis B virus: possibleroles in the resistance to interferon treatmentLing-yao Du1,2, Yao-li Cui1,2, En-qiang Chen1,2, Xing Cheng1,2, Li Liu1,2 and Hong Tang1,2*

Abstract

Background: The suppressor of cytokine signaling family (SOCS) is an important negative regulator in the JAK-STATsignaling pathway. This study was designed to explore the correlation between SOCS-1, 2 and 3, Hepatitis B Virus(HBV) and interferon (IFN), and the relationship between SOCS and IFN therapeutic efficacy.

Methods: Four types of mouse models were established. Mice were administered with HBV replicative plasmidpHBV4.1 and IFN inducer Poly IC (Group A), pHBV4.1 (Group B), Poly IC (Group C) and saline (Group D), respectively.Liver tissues were harvested from the mice and SOCS expression was determined. Meanwhile, patients with chronichepatitis B (CHB) were treated with pegylated interferon α-2b for 24-48 weeks. Liver biopsy was collected andthe baseline SOCS expression was determined. Serum assay was performed for efficacy evaluation and correlationanalysis.

Results: In animal studies, the expression level of SOCS-1 and 3 was found in the descending order of B, A, C and D.The difference between Group B and D suggested that HBV could induce SOCS. The difference between Group A andC suggested that HBV could still induce SOCS with up-regulated endogenous IFN. The difference between Group Cand D suggested that ploy IC could induce SOCS, while the difference between Group B and A suggested that Poly ICmight have a stronger inhibition effect for SOCS. There was no difference in SOCS-2 expression. In clinical studies, eightof twenty-four enrolled patients achieved either complete or partial therapeutic response. The expression of bothSOCS-1 and 3 was higher in CHB patients than in normal controls. The baseline HBV-DNA level was positively correlatedwith SOCS-1 and 3. The age, viral genotype, HBVDNA, SOCS-1 and SOCS-3 were found to be related to IFN efficacy.

Conclusion: HBV could induce both SOCS-1 and 3 expression regardless of endogenous IFN level. Elevated IFN coulddirectly up-regulate SOCS-1 and 3 expression, but it could also indirectly down-regulate SOCS-1 and 3 expression byinhibiting HBV replication. HBV might play a more important role in the SOCS up-regulation than IFN, a possible reasonwhy patients with high HBV viral load encounter poor efficacy of IFN treatment.

Keywords: SOCS, Chronic Hepatitis B, PEG-IFN α-2b

BackgroundThe antiviral therapy, immunomodulatory therapy andanti-inflammatory therapy are the most common treat-ments for patients with Chronic Hepatitis B (CHB) [1],among which the antiviral therapy with interferon (IFN)or nucleoside analogues (NA) is the key treatment [2,3].INF-α has been widely accepted by the public for its use

* Correspondence: [email protected] of Infectious Diseases, West China Hospital of Sichuan University,Chengdu 610041, China2Division of Infectious Diseases, State Key Laboratory of Biotherapy, SichuanUniversity, Chengdu 610041, China

© 2014 Du et al.; licensee BioMed Central Ltd.Commons Attribution License (http://creativecreproduction in any medium, provided the or

as an antiviral drug. However, there are still many un-known impact factors that affect its efficacy [4,5]. Thetherapeutic efficacy of INF is mainly achieved throughthe complicated “IFN System” where several signalingtransduction pathways are activated by the binding ofINF-α to INF-α receptor (INFAR). The pathway of Januskinase-the signal transducer and activator of transcrip-tion (JAK-STAT) is one of the typical pathways [6]. Inthis pathway, INF-α binds to INFAR-I on the membraneto form a dimer, which activates Jak-1 and Tyk2, the sig-nal transducers in the cytoplasm, to phosphorylate and

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activate STAT1 and STAT2. Activated STAT dimertranslocates into the nucleus to bind the interferon-sensitive response elements (IFNSRE). Antiviral proteinproducts are then induced to inhibit HBV-DNA tran-scription, promote HBV mRNA degradation and sup-press HBV protein translation [7,8].The suppressor of cytokine signaling (SOCS) is in-

duced by cytokines and is an important negative regula-tory factor in the JAK-STAT signaling pathway. So far,eight members of the SOCS family have been found,which include SOCS 1-7 and cytokine inducible SrcHomology 2 (SH2) containing protein (CIS). They allconsist of an amino-terminus, a SH2 domain in the mid-dle, and a SOCS box in carboxy-terminus [9-11]. It hasbeen demonstrated that SOCS protein could inhibit theactivity of JAKs and compete with STAT2 to bind tothe phosphorylated tyrosine residues in cytokine re-ceptors through its SH2 domain. As a result, the phos-phorylation of STATs is reduced. SOCS protein couldalso mediate the degradation pathways of the activatedsignaling proteins or bind to the cytoplasmic proteintyrosine phosphatase SHP2, thus inhibiting the signaltransduction [12].Recent studies have suggested that SOCS-1 and SOCS-3

are the negative regulators in the IFN signal transductionpathway in patients with chronic hepatitis C (HCV) infec-tion. HCV core protein can induce SOCS-3 protein expres-sion to reduce the therapeutic effect of IFN-α [13-15].However, in CHB patients with IFN treatment, it’s unclearwhether SOCS related elements impact INF efficacy inaddition to HBV genotype and viral load. Therefore, fur-ther investigation is required to determine the correlationbetween SOCS expression, HBV and IFN therapeutic effi-cacy in CHB patients. In this study, mouse models of HBVreplication were established to explore the interactions be-tween SOCS expression and HBV. The expression ofSOCS family in CHB patients during IFN treatment wasalso investigated to explore the correlation between thetherapeutic efficacy of IFN and the expression of SOCS.

MethodsAnimal studiesStudy designTwenty-four healthy BALB/c male mice at 6-9 weeks ofage and 18-20 g of weight were purchased from Experi-mental Animal Center, Sichuan University, and dividedinto four groups. Mice in Group A were administeredwith the replicative HBV plasmid DNA pHBV4.1-DNA(10 μg pHBV4.1-DNA dissolved in saline equivalent to8% mouse body weight) via the caudal vein using high-pressure injection. 24 hours after the injection, the micewere injected intraperitoneally with Poly IC at 200 μL/dayfrom day 0 to day 3. Mice in Group B were administeredwith an equal dose of pHBV4.1-DNA via the caudal vein

as in Group A, followed by intraperitoneal injection of sa-line at 200 μL/day from day 0 to day 3. Mice in Group Creceived the caudal vein high-pressure injection of anequal volume of saline and the intraperitoneal injection ofPoly IC at 200 μL/day. Mice in Group D received both thecaudal vein high-pressure and intraperitoneal injection ofsaline. Six hours after the last injection, these mice wereeuthanized and the liver tissues were harvested.The plasmid DNA pHBV4.1-DNA we used here is an

HBV replication competent plasmid. It contains 1.3 cop-ies of the HBV genome of serotype ayw and is capable ofHBV transcription, replication and expression both in vitroand vivo [16,17]. It’s kindly given by the Scripps ResearchInstitute, La Jolla, CA, USA as a gift. The mouse model weestablished here was verified effective in our previous stud-ies. It’s a transient HBV transfection model with viral repli-cation and expression lasting for 10 days and peaking atday 3 and day 4. It fulfilled the applicable requirement inthe study of HBV and related host factors [18].

Tissue sample analysisPart of the mouse liver was fixed by formalin for immu-nohistochemical staining to detect the expression ofSOCS proteins. Primary antibodies included anti-SOCS-1, anti-SOCS-2 (Santa Cruz) and anti-SOCS-3 (Abcam).PBS was used as the negative control for primary anti-bodies. Samples with known SOCS expression were usedas the positive controls. As the SOCS proteins would bestained brown, positive sample was defined as those withstained particles present in the cytoplasm, or withstained cytoplasm. Two pathologists with no priorknowledge of the study were asked to score the tissuesections according the Axiotis score standard. The per-centage of positive cells and intensity of positive stainingscored as follows:

Percentage score

0 = 0 ~ 10% positive cells;1 = 11 ~ 25% positive cells;2 = 26 ~ 50% positive cells;3 = 51 ~ 75% positive cells;4 = 76 ~ 100% positive cells.

Intensity score

0 = no colour;1 = yellow;2 = brown;3 = tan.

The sum of the percentage score and intensity scoreequaled the final score if there was no difference in two


pathologists’ opinions. Or the mean of two sum scoreswould equal the final score [19].The remaining liver tissue was put in liquid nitrogen

immediately after the harvest and stored at -70°C. Tis-sues were ground into powder in liquid nitrogen. TotalRNAs were extracted from the ground liver tissue and re-versely transcribed into total cDNA by RT-PCR amplifica-tion. The sequences of mouse SOCS-1, SOCS-2, SOCS-3and GAPDH genes were amplified from cDNA for

Figure 1 The expression of SOCS-1 in mouse liver tissues. A. Representissues (×400. HE) and statistical diagram of score; B. Representative RT-Pof three times results quantified and expressed as a ratio of SOCS-1/GAPDH; C.mouse liver tissues and statistical diagram of three times results quantified and

semiquantitative analysis. Protein samples extractedfrom liver powder were analyzed using western blot-ting to evaluate the expression of SOCS proteins.

Clinical studiesStudy designAll human subjects enrolled should meet the criteria in-cluding qualified age from eighteen to sixty-five yearsold, free of antiviral and immunomodulating treatment

tative IHC images showing the expression of SOCS-1 in mouse liverCR image of SOCS-1 mRNA in mouse liver tissues and statistical diagramRepresentative western blotting image of SOCS-1 proteins expression inexpressed as the ratio of SOCS-1/GAPDH.


in the past six months, negative pregnancy test in femalein the past 24 hours and effective contraception in bothsexes during the study. They should also have positiveserum HBsAg for more than 24 weeks, elevated serumHBV-DNA at more than 5 × 105copies/ml and twice ele-vated ALT between 2-10 ULN in the past 6 months. Allsubjects were provided with written informed consent.The exclusion criteria included lactating women, de-

creased albumin at less than 3.5 g/L, extended prothrom-bin time for more than 4 s, elevated serum bilirubin atmore than 34umol/L, decreased neutrophile granulocytesat less than 1.5 × 109/L, decreased platelets at less than9.0 × 1012/L, coinfection with other hepatitis viruses andaccompaniment of diabetes, thyroid dysfunction, auto-immune diseases and psychological issues.After the enrollment, all human subjects were sub-

cutaneously administered with pegylated interferon α-2b(Peg-Intron™, Schering Plough, USA), with the dosageand duration varied based on the individual weight, andboth personal choice and therapeutic response, respect-ively. The observation lasted for 48 weeks and had littleimpact on the conventional medical course. All subjectsaccepted physical examination, serum assay and liver bi-opsy before the treatment. At Week 12, 24 and 48 dur-ing the treatment only physical examination and serumassay were performed for therapeutic response evaluation.

Sample analysisThe liver tissue from the human subjects was examinedby immunohistochemistry to evaluate the expression ofSOCS family. Normal liver tissue sample was obtainedfrom the tissue bank of West China Hospital, SCU. Asmentioned before, the SOCS proteins would be stainedbrown as well. Samples with stained particles in the

Figure 2 The expression of SOCS-2 in mouse liver tissues. A. Represenstatistical diagram of three times results quantified and expressed as a ratiproteins expression in mouse liver tissues and statistical diagram of three time

cytoplasm were defined as positive samples. The expres-sion level of SOCS were also scored by the two patholo-gists mentioned before.The serum samples collected at each medical assess-

ment were detected for HBV-DNA quantification (Light-cycler-480, Roche, Switzerland), serum makers of HBVinfection (Alisei Quality System, RADIM, Italy), liverand kidney function (Modular EVO, Roche, Switzerland)and prothrombin time (Sysmex CA-7000 Systems, Sys-mex, Japan). The human subjects also received routineblood and urine examination and electrocardiogram. Ac-cording to the therapeutic efficacy, those with completeor partial virological, serological or biochemical re-sponses were assigned into the response group, whilethe rest were assigned into the non-response group.

Statistical analysisFive different areas of each liver tissue section after im-munohistochemical staining were scored and the RT-PCR and western blotting were repeated three times.The result of RT-PCR amplification was semi-quantifiedby a professional image analysis software Image Pro Plus4.5 (Media Cybernetics Inc, Silver Spring, MD,USA).The outcome of western blotting was semi-quantified byImage J, a public domain Java image processing programinspired by NIH Image for the Macintosh. All data wereused for statistical analysis. Measurement data weretested for normality and presented as mean ± standarddeviation. Statistical analysis was performed by t test, χ2

test, and Wilcoxon and Spearman’s rank correlation testfor measurement data, enumeration data, and rankeddata, respectively. Logistic regression was used to deter-mine the factors that would affect the efficacy of PEG-IFN treatment in the CHB patients. All statistical analysis

tative RT-PCR image of SOCS-2 mRNA in mouse liver tissues ando of SOCS-2/GAPDH; B. Representative western blotting image of SOCS-2s results quantified and expressed as the ratio of SOCS-2/GAPDH.


was performed using Stata 10.0 and p < 0.05 was definedas significant difference.

Ethics statementThis study was approved by both the Laboratory Animalethics committee of Sichuan University and the WestChina Hospital Ethics Committee. Written informed con-sents were obtained from all patients before we carriedout the liver biopsy.

Figure 3 The expression of SOCS-3 in mouse liver tissues. A. Represen(×400. HE) and statistical diagram of score; B. Representative RT-PCR image of Sresults quantified and expressed as a ratio of SOCS-3/GAPDH; C. Representativetissues and statistical diagram of three times results quantified and expr

ResultsAnimal studiesExpression of SOCS-1 in liver tissuesIn this study, four groups of mice were administeredwith HBV replicative plasmid pHBV4.1 followed withIFN inducer Poly IC (Group A), pHBV4.1 (Group B),Poly IC (Group C) and saline (Group D), respectively.SOCS-1 expression was found in all four groups withthe SOCS-1 expression level in descending order from

tative IHC images showing the expression of SOCS-3 in mouse liver tissuesOCS-3 mRNA in mouse liver tissues and statistical diagram of three timeswestern blotting image of SOCS-3 proteins expression in mouse liver

essed as the ratio of SOCS-3/GAPDH.

Table 1 General information of the enrolled 24 CHBpatients

Patients (n = 24)

Age (years) (Mean ± SD) 25.54 ± 8.08

Gender (Male/Female) 17 / 7

Gene Type (A/B/C) 1 / 14 / 9

HBeAg (+/–) 24 / 0

HBeAb (+/–) 0 / 24

ALT (IU/L) 224.79 ± 95.82

AST (IU/L) 107.00 ± 62.45

ALB (g/L) 36.98 ± 2.21

TBIL (mg/dL) 18.50 ± 11.57

CHE (U/L) 5857.50 ± 623.08

INR for PT 2.26 ± 0.57

HBV DNA level (lg IU/ml) 17.17 ± 1.84


groups B, A, C, to D as shown by immunohistochemis-try, RT-PCR, and western blotting (Figure 1). The ex-pression of SOCS-1 in Group B was significantly higherthan that in Group D, suggesting that HBV could inducethe expression of SOCS-1. The expression difference be-tween Group A and C suggested that HBV could still in-duce SOCS with up-regulated endogenous IFN. Theexpression in Group C was significantly higher than thatin Group D, suggesting that after the direct up-regulationof endogenous IFN, SOCS-1 expression might be up-regulated via some negative feedback pathway. The ex-pression in Group B was significantly higher than that inGroup A, suggesting that Poly IC might not only mediatethe up-regulation of SOCS-1 via increasing endogenousIFN level to inhibit HBV replication, but also have a stron-ger inhibiting effect than its inducing ability.

Expression of SOCS-2 in liver tissuesThere was no significant difference in SOCS-2 expres-sion levels in all groups as determined by RT-PCR andwestern blotting (Figure 2), suggesting that the expres-sion of SOSC-2 in mouse liver was affected by neitherHBV nor Poly IC.

Expression of SOCS-3 in liver tissuesConsistent with the expression of SOCS-1, SOCS-3positive cells were found in all groups using immunohis-tochemistry. The expression level of SOCS-3 was in thedescending order of B > A > C > D (Figure 3), same as

Table 2 The therapeutic response in CHB patients after treatm

Treatmentcourse

Responders (N = 8)

HBeAg seroconversion Non-HBeAg seroconvers

24 weeks 3 2

48 weeks 3 0

that of SOCS-1. These results suggested that the expres-sion of SOCS-3 and SOCS-1 was similarly mediated byHBV and Poly IC.

Clinical studiesSubjectsTwenty-four subjects, including 17 female and 7 male,were enrolled for clinical observation. The average agewas 25.54 years old. Detailed patient information waslisted (Table 1).

Therapeutic response to PEGINF α-2b in CHB patientsFourteen patients (14/24) received 24 weeks’ treatment,and among them 5 achieved either complete or partialtherapeutic response according to the judgmental cri-teria of the therapeutic responses in subjects. Amongthese 5 patients, 3 achieved HBeAg seroconversion. An-other ten patients (10/24) received 48 weeks’ treatment,and among them 3 achieved HBeAg seroconversion. The8 patients who achieved either complete or partial thera-peutic response were assigned into the responder group,while the remaining 16 patients were assigned into thenon-responder group (Table 2).

Expression of SOCS-1, 2 and 3 in the liver tissue of normalcontrols and CHB patients before treatmentBoth SOCS-1-positive and SOCS-3 positive cells wereobserved in the liver tissues of the CHB patients fromboth the responder and non-responder groups. SOCS-1and SOCS-3 were mainly found in the cytoplasm of thehepatocytes. In contrast, only a trace of SCOS-3 wasfound in the liver tissues from the normal controls(Figure 4). In addition, there was a significant differ-ence in the expression level of both SOCS-1 andSOCS-3 between the responder and non-respondergroups before the treatment. The expression of SOCS-1 and SOCS-3 was higher in the non-responder groupthan in the responder group. SOCS-2 was not detectedin any of the liver tissues examined.

Factors affecting the therapeutic efficacyFour serum assays representing the hepatic functionwere performed before the treatment. Results were ana-lyzed for correlation with SOCS-1 and SOCS-3 usingSpearman Rank Correlation Test. Only the baselineHBV-DNA level was positively correlated with SOCS-1and SOCS-3 (p < 0.05), while none of the ALT, AST or

ent.

Non-responders (N = 16)

ion HBeAg seroconversion Non-HBeAg seroconversion

0 9

0 7

Figure 4 The expression of SOCS-1, 2 and 3 proteins in patients before treatment. The representative IHC images showing expression ofSOCS-1, SOCS-2 and SOCS-3 proteins in all CHB patients before treatment and normal controls as well as the statistical diagram of score.


TBIL levels showed significant correlation with SOCS-1or SOCS-3 (Table 3).To investigate the factors which might affect the thera-

peutic efficacy of PEGINF α-2b, 15 possible factors in-cluding SOCS-1 and SOCS-3 expression level werechosen for the univariate analysis. Five factors showedsignificant differences between the responder and non-responder groups, suggesting that SOCS-1 and SOCS-3,in addition to the gender, viral genotype and HBV-DNA,might impact the therapeutic response (Table 4).Logistic regression was used to determine the correl-

ation between the 15 factors. Five variables includinggender, viral genotype, HBV-DNA, SOCS-1 and SOCS-3were admitted into the regression model (Table 5). Toprove the sensitivity and specificity of the model, the re-ceiver operating characteristic curve (ROC curve) andcut-off point were calculated. The area under the ROCcurve in this logistic regression model was 82.68% andthe cut-off point was 0.50, suggesting that the modelwas valid.

DiscussionCHB patients usually encounter poor therapeutic responsewith IFN treatment. Therefore, we set to determine

Table 3 The correlation between SOCS family, liver function a

S

Item Mean ± SD Correlation

ALT (IU/L) 224.79 ± 95.82 −0.29

AST (IU/L) 107.00 ± 62.45 −0.17

TBIL (mg/dL) 20.10 ± 11.24 0.07

HBV DNA level 17.17 ± 1.84 0.49

whether HBV might influence the efficacy via some nega-tive cytokine regulators. The JAK/STAT pathway is themost important antiviral signaling transduction pathwayof IFN, and SOCS proteins induced by cytokines are thekey negative regulating proteins in the JAK/STAT path-way. By inhibiting the activity of JAK and competitivelybinding to the phosphorylated tyrosine residues in cyto-kine receptors, SOCS proteins reduce the phosphorylationof STATs. SOCS proteins also act as adaptor molecules toguide the active cell signals to biodegradation pathway[10]. Recently the SOCS-3 protein induced by HCVcore protein has been shown to be important for HCVabsconding from host INF [20,21]. However, there areonly a few studies on the correlation between HBVand SOCS proteins, which focus on single HBV pro-tein such as HBx protein. HBx has been shown toaffect the JAK/STAT pathway in the transfected hepa-tocytes [22,23]. The subcellular mislocalization of themutant HBx has been found to up-regulate STAT3 ac-tivation, resulting in STAT1 inhibition and SOCS-1and SOCS-3 expression silencing. In the HBV-relatedhepatocellular carcinoma, the dysregulation of STAT/SOCS signaling is involved in the hepatocarcinogen-esis [24-26].

nd HBVDNA

OCS-1 SOCS-3

P value Correlation P value

0.17 −0.33 0.11

0.42 −0.20 0.36

0.74 −0.17 0.44

0.03 0.59 0.002

Table 4 Univariate analysis of the factors affecting IFNα-2b therapeutic efficacy

Responders(n = 8)

Non-responders(n = 16)

χ2/t/U P values

Age (years)(Mean ± SD)

23.75 ± 5.60 25.50 ± 9.45 −0.48 0.64

Gender (Male/Female)

7/1 6/10 – 0.03

Genotype(A/B/C)

0/6/2 1/3/12 7.29 0.03

HBeAg (+/–) 8/0 16/0 – –

HBeAb (+/–) 0/8 0/16 – –

ALT (IU/L) 251.50 ± 108.03 205.19 ± 94.44 −1.03 0.31

AST (IU/L) 106.63 ± 36.92 107.19 ± 73.09 −0.02 0.98

ALB (g/L) 37.95 ± 1.48 36.49 ± 2.39 1.57 0.13

TBIL (mg/dL) 24.27 ± 13.63 25.62 ± 9.59 −0.28 0.78

CHE (U/L) 6218.50 ± 477.86 6677.25 ± 620.29 −1.83 0.08

CREA (mg/dL) 72.11 ± 62.02 67.49 ± 9.55 0.30 0.77

INR for PT 2.20 ± 0.41 2.02 ± 0.50 0.88 0.39

HBV DNA level 15.34 ± 1.29 18.09 ± 1.32 −4.85 0.001

SOCS-1 5.13 ± 1.89 15.38 ± 3.65 −7.40 0.001

SOCS-3 6.75 ± 1.58 20.63 ± 3.24 −11.37 0.001


It was found that SOCS-3 expression increased in liverspecimens from patients with CHB and was positivelycorrelated with the severity of inflammation. Same resultwas also demonstrated in the cell culture. However, thechange of SOCS-1 level was not observed [27]. In ouranimal studies, the change of the two SOCS proteinswas observed. The expression level of SOCS-1 and

Table 5 Multivariate analysis of the factors affecting IFNα-2bPrognostic factors Regression coefficent Standard error

Age 1.03 0.77

Gender 0.32 0.55

Genotype (A/B/C) 0.96 0.27

HBeAg (+/–) 0.86 0.31

HBeAb (+/–) −0.80 0.27

ALT (IU/L) −0.58 0.32

AST (IU/L) −1.93 0.34

ALB (g/L) 0.17 0.77

TBIL (mg/dL) 0.001 0.004

CHE (U/L) 2.18 1.23

CREA (mg/dL) −0.04 0.03

INR for PT −0.003 0.001

HBV-DNA 0.003 0.001

SOCS-1 −3.67 1.49

SOCS-3 −2.50 1.24

Cons −0.11 4.07

SOCS-3 proteins varied in different groups. The higherexpression in Group B (pHBV4.1) as compared to GroupD (NS) suggested that HBV induced the expression ofSOCS-1 and SOCS-3 proteins. Poly IC is a well-knowninducer of the endogenous IFN. Therefore, the higherexpression of SOCS in Group A (pHBV4.1 + Poly IC)than in Group C (Poly IC) suggested that HBV couldstill induce SOCS expression with up-regulated en-dogenous IFN. Moreover, the expression level in GroupC was higher than in Group D, suggesting that Poly ICmight induce the up-regulation of the endogenous IFN,and then as a negative cytokine in IFN pathway, inducethe expression of SOCS-1 and SCOS-3 via some nega-tive feedback pathway. As HBV and Poly IC were bothinducers, the expression level in Group A was supposedto be higher. However, IFN is an inhibitor for HBV.HBV inhibition-mediated decrease in SOCS-1 and SOCS-3 expression might be stronger than IFN up-regulationmediated increase in SOCS-1 and SOCS-3 expression,leading to the higher expression level in Group B. The ex-pression of SOCS-2 protein was not significantly differentamong all groups, suggesting that HBV and Poly ICcouldn’t affect SOCS-2 expression.In previous clinical studies on the IFN treatment in

CHB patients, the genotype and viral load of HBV wereconsidered as the key indicators for INF therapeutic effi-cacy. Although many studies have focused on HBV, only afew focus on the host factors that might affect the INFtherapeutic efficacy [4,28,29]. It has been reported previ-ously that SOCS-1 and SOCS-3 proteins could down-regulate the INF therapeutic efficacy as negative regulatorsin the IFN signaling pathway in CHC patients, and the

therapeutic efficacy

Z value P value OR value 95% confidence interval

1.33 0.18 2.80 [-0.48~2.54]

0.58 0.03 1.37 [0.75~1.39]

3.51 0.04 2.62 [0.73~1.50]

2.80 0.75 2.37 [-0.26~1.47]

−2.92 0.63 0.45 [-1.34~1.27]

−1.82 0.07 0.56 [-1.21~0.05]

−5.65 0.96 0.14 [-2.60~1.26]

0.22 0.83 1.62 [-1.35~1.68]

1.40 0.16 1.00 [1.00~1.24]

1.78 0.08 8.91 [0.80-99.22]

−1.55 0.12 0.96 [0.91~1.01]

−1.94 0.05 0.99 [0.99~1.00]

2.03 0.04 1.00 [1.00~1.01]

−2.47 0.03 0.03 [0.01~0.47]

−2.02 0.04 0.08 [0.01~0.93]

−0.03 0.98 — —


overexpression of SOCS-3 protein could be used for theprognosis of CHC patients with INF treatment. Therefore,it is possible that SOCS-1 and SOCS-3 proteins couldsimilarly affect the therapeutic efficacy in CHB patients bynegatively regulating IFN signaling transduction. Recently,it has been shown that plasmacytoid dendritic cells(pDCs) treated with HBsAg present a defect in INF-α se-cretion due to HBsAg-mediated upregulation of SOCS-1expression [30], which might partially explain the HBVimmune escape and persistent infection. In our clinicalobservation, under a natural HBV infection status, sameresults were obtained regarding the change of SOCS-1and SOCS-3 as in the animal models. The higher expres-sion of SOCS-1 and SOCS-3 in liver tissues of CHB patientscompared to the normal controls again demonstrated thepotential ability of HBV to induce SOCS-1 and SOCS-3. Inaddition, we also found higher expression of the two SOCSproteins in liver tissues in the responders as compared tothe non-responders, suggesting that the efficacy of INFtreatment might be impacted by the expression level ofSOCS-1 and SOCS-3 proteins before the treatment. Nega-tive correlation between the expression of SOCS proteinsbefore treatment and the INF therapeutic efficacy was foundusing both one-way ANOVA and multivariate analysis.These results might explain why the patients with higherexpression of SOCS-1 and SOCS-3 proteins in liver beforetreatment showed INF resistance, and the severity wasconsistent with the expression level.HBx and HBsAg are able to change the expression of

some SOCS proteins [24,30]. We showed that the mostimportant medical indicator, HBVDNA, did interact withSOCS expression in CHB patients. In addition to the ex-pression differences between the CHB patients and thenormal controls, the expression of SOCS proteins alsovaried in patients with different viral load, which waspositively correlated with the expression level of SOCS-1and SOCS-3 proteins. The higher the viral load, themore expression of SOCS-1 and SOCS-3 in the liver tis-sues, and the more effective in their ability to block IFN-induced signaling pathway. Together with the resultsfrom animal studies, our data explained why the viralload is the most important indicator for IFN therapeuticefficacy predication. HBV induced the expression of SOCSproteins, which then inhibited IFN signaling transductionpathway. As a result, IFN antiviral efficacy was decreased.Thus, these results provided the theoretical foundation forstudying the relationship between SOCS proteins inCHB patients before treatment and IFN therapeuticefficacy, and explained why HBV appeared to play amore important role than IFN in SOCS up-regulation.However, since the transfection efficacy of hydro-dynamic delivery in this mouse model in vivo was toolow, our findings need to be confirmed with furtherinvestigations.

In conclusion, HBV could induce SOCS-1 and SOCS-3expression regardless of the level of the endogenous IFN.The elevated IFN would directly up-regulate SOCS-1 andSOCS-3 via some negative feedback pathway, and in-directly down-regulate SOCS-1 and SOCS-3 by inhibitingHBV. However, HBV played a more important role thanIFN in SOCS up-regulation, a possible reason whypatients with high HBV viral load would encounter poorefficacy in IFN treatment. HBV induced SOCS proteins,which could negatively regulate IFN signaling to decreaseIFN therapeutic efficacy, making the expression of SOCS-1and SOCS-3 proteins in liver before treatment an import-ant impact factor in IFN treatment for CHB. SOCS genescan be the promising candidates in gene therapy and drugdevelopment. For example, adenovirus vector expressingthe SOCS proteins can be used to treat rheumatic arthritis[31]. Moreover, RNA interference and antisense nucleicacid could also be used to down-regulate the SOCS expres-sion, and some molecular compound could be used toenhance or block its inhibition of the cytokine signalingtransduction [32]. New drugs for the JAK-STATs pathwaycan be developed if detailed mechanism by which SOCSproteins function under physiological and pathologicalcondition is understood. Anti-cytokine and anti-SOCSproteins therapies might be the alternative opportunitiesfor patients with IFN resistance.

Competing interestsThe contents are solely the responsibility of the authors and do notnecessarily represent the views of the funding source. The authors declarethat they have no competing interests.

Authors’ contributionsTH conceived the study, provided fund support and revised the manuscriptcritically for important intellectual content. DLY, CYL, CEQ, CX and LL madesubstantial contributions to experiment, clinical studies and data analysis.DLY and CYL participated in interpretation of data and manuscriptpreparation. DLY draft the manuscript and revised it according to all author’sopinions. All authors have read and approved the final manuscript.

AcknowledgementsThis study was supported by grants from 973 Project of China (No.2013CB911300) and National Natural Science Foundation of China (No.81071363). We present our special thanks to Prof. Alan McLachlan forplasmid pHBV4.1-DNA. We also thank Ms. Cong Liu and Mr. Dongbo Wu fortheir hard work on scoring the liver tissue sections.

Received: 28 August 2013 Accepted: 6 March 2014Published: 17 March 2014

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doi:10.1186/1743-422X-11-51Cite this article as: Du et al.: Correlation between the suppressor of cytokinesignaling-1 and 3 and hepatitis B virus: possible roles in the resistance tointerferon treatment. Virology Journal 2014 11:51.

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AbstractBackgroundMethodsResultsConclusion

BackgroundMethodsAnimal studiesStudy designTissue sample analysis

Percentage scoreIntensity scoreClinical studiesStudy design

Sample analysisStatistical analysisEthics statement

ResultsAnimal studiesExpression of SOCS-1 in liver tissuesExpression of SOCS-2 in liver tissuesExpression of SOCS-3 in liver tissues

Clinical studiesSubjectsTherapeutic response to PEGINF α-2b in CHB patientsExpression of SOCS-1, 2 and 3 in the liver tissue of normal controls and CHB patients before treatmentFactors affecting the therapeutic efficacy

DiscussionCompeting interestsAuthors’ contributionsAcknowledgementsReferences

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