Increased serum levels of TNF-like cytokine 1A (TL1A) and its decoy receptor 3 (DcR3) predict absence of fibrosis in chronic hepatitis B virus infection A.-K. Gantzarou1*, V. Kotoula2, S. Kim3, G. Koustiani1, M. Pape4, G. Germanidis5, K. Mandraveli4, P. Hytiroglou2, E. Akriviadis1. 14th Unit of Internal Medicine, Hippokration General Hospital of Thessaloniki, 2Department of Pathology, Aristotle University of Thessaloniki, Thessaloniki, Greece, 3BioPower Tech, Tuscaloosa, AL, USA, 4Laboratory of infectious diseases, AHEPA University Hospital of Thessaloniki, Thessaloniki, Greece, 51st Internal Medicine Unit, AHEPA University Hospital of Thessaloniki, Thessaloniki, Greece. *alexgan@freemail.gr

Background and aims: TL1A is a ligand of the TNF superfamily (TNFSF15), with apoptotic, immunostimulant and angiostatic properties, expressed by endothelial cells, tissue macrophages, lymphocytes and dendritic cells. It can bind to both its functional receptor DR3 and to its inhibitory decoy receptor DcR3. DcR3, apart from TL1A, can also bind to FasL and LIGHT and inhibit their actions. Via its inhibitory nature it expresses immunosupressive and antiapoptotic properties. CK18 is an intermediate filament epithelial cell cytoskeleton protein, highly expressed in parenchymal liver cells. Serum detection of caspase-cleaved apoptotic CK18 fragments has been used as a hepatocellular apoptosis quantification method. The aim of this study was to explore the possible involvement of the system of TL1A and DcR3 molecules in Chronic Hepatitis B Virus infection, and their potential correlation to hepatocellular apoptosis, and Liver Histology.

Methods: Sera and liver biopsies were collected from 46 chronic hepatitis B patients. HBeAg/anti-HBe status was determined and serum HBVDNA was quantitatively measured. Grade of histological activity was evaluated from 0-18 according to modified HAI system (Ishak et al, 1995) and semiquantitatively determined as minimal (HAI=0-4), mild (HAI=5-8), moderate (HAI=9-12) and severe (HAI=13-18). Stage of fibrosis was evaluated in a 5 degrees scale (0-4). DcR3 serum levels were measured by ELISA from BioPowerTech, Tuscaloosa, AL, USA (capture and detection antibody MD3E2 and MD3B1, respectively). TL1A levels were measured by ELISA from BioPowerTech, Tuscaloosa, AL, USA (capture and detection antibody MTLD2 and MTLG6 , respectively). CK18 levels were measured by M30-Apoptosense ELISA (Peviva, Sweden). Comparison to normal controls (n=30) was performed by 2-tailed t-test. Correlations among the 3 molecules, as well as variations according to the histological grade and stage of disease, were analyzed with Pearson’s correlation, and ANOVA, followed by multiple Bonferroni comparisons, respectively.

Results(1): DcR3, TL1A and CK18 levels were elevated in chronic hepatitis B patients, compared to normal controls (p=0.037, 0.043 and 0.01, respectively). (Fig. 1-3)

Fig.1 : Serum DcR3 levels in CHB patients and Normal Controls *p<0,05. Columns depict mean values and bars on top of them represent SE.

Fig. 2: Serum TL1A levels in CHB patients and Normal Controls *p<0,05. Columns depict mean values and bars on top of them represent SE.

Fig.3 : Serum CK18 levels in CHB patients and Normal Controls **p=0,01. Columns depict mean values and bars on top of them represent SE.

Results (2) DcR3 and TL1A levels are elevated in both HBeAg positive and anti-HBe positive CHB. There is a trend for DcR3 levels to be elevated in anti-HBe positive CHB patients with low viral load.(Fig. 4) CK18 levels are elevated in anti-HBe positive CHB only. DcR3, TL1A and CK18 levels according to histological grade, stage and steatosis are shown in Tables 1-3 and figures 5-8 Correlations between serum levels of the three molecules are shown in table 4.

Fig. 4: Serum DcR3 levels according to viral load in anti-HBe positive patients p=0,064 for the difference between low (HBVDNA<2000 IU/ml) and high (HBVDNA≥2000 IU/ml) viral load

Histological activity grade 45 0,337 Minimal Mild Moderate & Severe 11 Table 1. DcR3 and CHB Ν Mean (ng/ml) SD SE 95% CI p ANOVA Histological activity grade 45 0,337 Minimal Mild Moderate & Severe 11 18 16 5,63 2,12 2,45 10,31 5,70 3,33 3,11 1,34 0,83 -1,29-12,56 -0,71-4,96 0,68-4,23 Histological fibrosis stage <0,0001 1 2 3 4 5 8 20,03 1,51 1,98 2,38 1,57 16,22 0,93 3,79 4,51 1,91 9,36 0,42 0,89 1,59 0,58 -20,26-60,31 0,35-2,66 0,09-3,86 -1,39-6,15 0,29-2,85 Histological steatosis grade 44 0,850 No Moderate Severe 9 28 4,46 2,92 3,24 0,02 7,69 6,76 5,65 0,00 2,56 1,28 2,53 -1,45-10,37 0,30-5,54 -3,77-10,26 0,02-0,02

Histological activity grade 35 0,476 Minimal Mild Moderate & Severe 10 Table 2. TL1A and CHB Ν Mean (ng/ml) SD SE 95% CI p ANOVA Histological activity grade 35 0,476 Minimal Mild Moderate & Severe 10 14 11 0,242 0,120 0,103 0,459 0,213 0,104 0,145 0,057 0,031 -0,086-0,570 -0,003-0,243 0,032-0,172 Histological fibrosis stage <0,0001 1 2 3 4 15 6 7 0,813 0,076 0,080 0,107 0,090 0,734 0,040 0,073 0,119 0,082 0,424 0,020 0,019 0,048 -1,011-2,636 0,013-0,139 0,040-0,121 -0,018-0,231 0,015-0,166 Histological steatosis grade 0,894 No Moderate Severe 8 21 0,176 0,162 0,079 0,042 0,274 0,326 0,043 0,012 0,097 0,071 0,022 0,009 -0,053-0,406 0,014-0,311 0,010-0,148 -0,067-0,150

Histological activity grade 35 0,013 Minimal Mild Moderate & Severe 10 Table 3. CK18 and CHB Ν Mean (ng/ml) SD SE 95% CI p ANOVA Histological activity grade 35 0,013 Minimal Mild Moderate & Severe 10 14 11 217,9 303,2 821,4 100,0 194,2 830,6 31,6 51,9 250,4 146,4-289,4 191,1-415,3 263,4-1379,4 Histological fibrosis stage 0,712 1 2 3 4 15 6 7 181,9 275,8 405,6 634,9 559,7 48,8 95,9 381,0 1145,3 305,0 28,2 47,9 98,4 467,6 115,3 60,7-303,2 123,2-428,4 194,6-616,6 -567,0-1836,7 277,6-841,8 Histological steatosis grade 0,704 No Moderate Severe 8 21 290,4 518,1 466,5 195,1 203,2 663,7 318,8 33,7 71,8 144,8 159,4 23,9 120,5-460,3 216,0-820,2 -40,8-973,8 -108,1-498,3

Correlation between serum DcR3 and TL1A levels Table 4. Correlations between serum levels of the three molecules Correlation between serum DcR3 and TL1A levels Correlation between serum DcR3 and CK18 levels Correlation between serum TL1A and CK18 levels N=36 r=0,922 p<0,0001 N=36 r=-0,006 p=0,973 N=36 r=0,026 p=0,878

Fig.5: Serum CK18 levels according to histological activity grade *p<0.05 for both differences between patients with moderate/severe and patients with mild as well as minimal histological activity

Fig.6: Correlation between serum CK18 levels and Histological Activity Index (HAI)

Fig.7: Serum DcR3 levels according to histological fibrosis stage ****p<0,0001 for all differences in DcR3 levels between stage 0 and all other stages. p=NS for all other differences

Fig.8: Serum TL1A levels according to histological fibrosis stage ***p<0.001 for all differences in TL1A levels between stage 0 and all other stages, p=NS for all other differences

Discussion We found circulating DcR3 levels significantly elevated in CHB, with a strong positive correlation with TL1A levels, but not with hepatocellular apoptosis. Hepatocellular apoptosis marker CK18 levels were also significantly elevated in CHB patients, showing significant variation according to histological grade, since patients with moderate/severe inflammation had significantly higher CK18 levels than patients with no or mild inflammation. On the contrary, CK18 levels showed no variation according to stage, whereas DcR3 and TL1A levels were significantly elevated in patients with absence of fibrosis, in comparison to all other stages. Thus, combined CK18 and DcR3/TL1A measurements might be of possible future use in evaluating noninvasively both grade and stage of disease. Given the inverse relation of high serum DcR3/TL1A levels with the absence of fibrosis, low serum CK18 levels combined with high DcR3/TL1A levels might be used for the identification of true inactive HBV carriers , in which both histological activity and fibrosis are absent. The tendency for higher DcR3 levels in patients with low (<2000IU/ml) viral load, among which inactive carriers are included, reinforces the above scenario. Given a)that DcR3 is an inhibitory molecule, which suppresses the transfer of costimulatory signals between T lymphocytes thus exhibiting immunocompromising actions, whereas TL1A has the opposite immunostimulatory effect and b)that we observed that both DcR3 and TL1A levels are especially elevated in a milieu of minimal immunological activity, as in the inactive carrier stage, one can suppose that DcR3 immunocompromising actions predominate over TL1A immunostimulant actions in CHB. It is possible that the target of DcR3 elevation in this group of patients is protection of certain cells against the action of the other two DcR3 ligands, namely FasL and LIGHT, and that TL1A elevation is a secondary phenomenon, in the effort of the system to keep a balance in TL1A actions. Further studies are needed to precisely identify the biological role of all these interconnected ligands and receptors in CHB.

Conclusions Circulating DcR3 levels are significantly elevated in chronic hepatitis B, with a strong positive correlation with TL1A levels, but not with hepatocellular apoptosis. CK18 levels are related to the grade of disease, whereas DcR3 and TL1A levels are inversely related to the stage of disease, specifically to the absence of fibrosis. Therefore, these 3 markers could potentially be used in a noninvasive panel for the clinical assessment and follow-up of patients with chronic HBV infection.