Cytokines as Predictors for Moderate Inflammation in Patients With ALT Less Than 2 × ULN
For patients with ALT < 2 × ULN, clinical parameters such as age, gender, BMI, HBV DNA/HBeAg status, ALP, and TBil were not correlated with liver inflammation. PLT was significantly lower, and AST and GGT were higher in patients with at least moderate inflammation. Within cytokines and chemokines, only serum CXCL-11 was significantly increased with increased HAI scores (P < 0.05). Multivariate analysis indicated that CXCL-11, CXCL-10 (P = 0.053), AST, GGT, and HBeAg/HBV DNA were independently associated with moderate or severe inflammation. However, P values for CXCL-10, GGT, and AST were not significant (P > 0.05) (Table 4).
Based on the variables above, we developed an inflammation-index by binary logistic regression.
Inflammation-index = exp(g× 1)/[1 + exp(g× 1)]
Cytokines as Predictors for Significant Fibrosis in Patients With ALT Less Than 2 × ULN
For patients with ALT < 2 × ULN, clinical parameters such as age, gender, BMI, HBV DNA/HBeAg status, ALP, and TBil were not correlated with significant fibrosis stages. PLT was significantly lower, and AST and GGT were higher in patients with significant fibrosis. Serum levels of IL-8 (44.80 ± 49.87 pg/mL, P = 0.043), TNF-α (13.48 ± 16.41 pg/mL, P = 0.060), CXCL-10 (60.49 ± 41.40 pg/mL, P = 0.007), IL-2R (633.55 ± 257.71 pg/mL, P = 0.004), and TGF-α (11.90 ± 44.12 pg/mL, P = 0.041) were higher in patients with significant fibrosis than in those without. Multivariate analysis showed TGF-α, IL-2R, platelets, and HBeAg/HBV DNA were independent predictors for significant fibrosis in patients with ALT < 2 × ULN (Table 5).
Fib-index was developed to predict significant fibrosis by binary logistic regression, using TGF-α, L-2R, platelet, and HBeAg/HBV DNA as variables.
Fib-index = exp(g × 2)/[1 + exp(g × 2)]
Predictive Performance of Cytokine-Based Scores for Moderate or Severe Inflammation and Fibrosis in Patients With ALT < 2 × ULN
The inflammation-index had an areas under the receiver operating characteristics curve (AUROC) of 0.75 (95% CI 0.66–0.84) for diagnosing moderate or greater inflammation in patients with ALT < 2 × ULN. Using a cutoff of ≥0.46, moderate or greater inflammation could be diagnosed with a sensitivity of 48.9%, specificity of 91.2%, NPV of 80.2%, PPV of 71%, and accuracy of 78.2%. Fib-index, APRI, and FIB-4 showed AUCs of 0.82 (95% CI 0.75, 0.90), 0.74 (95% CI 0.65, 0.83), and 0.67 (95% CI 0.57, 0.76), respectively, for diagnosing significant fibrosis in patients with ALT < 2 × ULN. Applying a cutoff of ≥0.44, significant fibrosis could be diagnosed by fib-index with sensitivity of 63.6%, specificity of 90.6%, NPV of 85.7%, PPV of 73.7%, and accuracy of 82.7% (Fig. 3).
To validate these noninvasive models for predicting moderate or greater inflammation and significant fibrosis, LOOCV was performed. LOOCV showed that 74.5% cross-validation grouped cases were correctly classified by inflammation-index and 81.3%, 74.3%, and 64.2% classified by fib-index, APRI, and FIB-4, respectively, in patients with ALT < 2 × ULN (Supplemental Table S2, http://links.lww.com/MD/A510).
In the total population, the inflammation-index had an AUC of 0.76 for moderate inflammation, while fib-index, APRI, and FIB-4 showed AUCs of 0.78, 0.66, and 0.63 for significant fibrosis, respectively (Supplemental Table S2, http://links.lww.com/MD/A510).
This study showed that selected circulating cytokines were associated with liver inflammation and fibrosis. This implies that there are likely various mechanisms of liver inflammation and fibrosis during the progression of chronic HBV infection. Previous studies have shown that cytokines initiate downstream signaling pathways by binding to specific receptors expressed on the target cells, and which subsequently secrete various cytokines.26 However, the exact mechanisms by which cytokines mediate liver inflammation and fibrogenesis are not fully understood. INF-γ is well known to be the main mediator for control of HBV infection.8 In the progression to HBV-induced inflammation, INF-γ may be overproduced to clear HBV infection, but may be exhausted in the late stages of chronic inflammation. The inability to control the viral infection leads to the recruitment of inflammatory infiltrates into the liver parenchyma by IFN-γ-inducible CXCL-10 and -11 chemokines.27 Some of the results in the current study could be explained by the fact that INF-γ showed a declining trend while CXCL-10 and CXCL-11 levels increased in patients with moderate and severe inflammation compared to those without.
The current study also showed that circulating IL-8, TGF-α, and IL-2R levels were increased in patients with significant fibrosis. IL-8 has been proven to play a role in recruitment and activation of hepatic macrophages by CXCR1 in human liver cirrhosis.28 TGF-α is a ligand for the epidermal growth factor receptor, which is active in many biological processes, and is a central mediator in the initiation and maintenance of fibrosis in many diseases.29 In in-vitro studies, TGF-α has been found to attenuate hepatic fibrosis through upregulation of MMP-1 and inhibition of type I procollagen peptide synthesis.30,31 To the best of our knowledge, this is the first study to demonstrate that circulating TGF-α levels were independently associated with liver fibrosis. IL-2R is primarily secreted by activated T-helper lymphocytes and is widely expressed on activated T lymphocytes, regulatory T cells, B cells, and monocytes.32 Some studies have examined IL-2R in chronic liver disease and found increased IL-2R in hepatic cirrhosis.33–36 However, the performance of IL-2R in diagnosing liver inflammation and fibrosis had not been determined. In the present study, circulating IL-2R levels increased in both moderate inflammation and significant fibrosis in CHB patients.
Serum ALT is the most widely used parameter to screen for and to monitor patients with liver disease. However, patients with HBV infection, a disease in which apoptosis is likely the dominant mechanism of cell death, can have normal or only minimally elevated serum ALT levels.37 A recent meta-analysis concluded that 20.7% of 830 CHB patients with ALT levels ≤40 IU/L had significant fibrosis.38 The current study showed that 31.7% and 29.1% of 151 patients with ALT levels <2 × ULN had moderate inflammation and significant fibrosis, respectively. It should be noted that patients with significant fibrosis or moderate/severe necro-inflammation should be treated immediately according to current guidelines.6,7 Therefore, the use of serum ALT levels underestimates the proportion of patients who should urgently receive antiviral therapy. There are no reliable noninvasive markers as surrogate for ALT to assess histological liver damage in patients with ALT levels <2 × ULN. In this study, we assessed the relationship between circulating cytokines and liver inflammation and fibrosis in patients with ALT levels <2 × ULN. Our results indicated CXCL-11 as a biomarker independent of other biochemical parameters and known risk factors for moderate greater inflammation. We also found IL-2R and TGF-α to be independent indicators for significant fibrosis.
The APRI and FIB-4 scores derived from patients with HCV infection are the 2 most widely studied noninvasive tools for assessing liver fibrosis in CHB patients.39,40 Studies on APRI and FIB-4 have been validated in patients with normal and mildly elevated ALT, showing AUROCs of 0.71 and 0.72, respectively.41 We employed the 2 scores in our study to evaluate significant fibrosis, obtaining AUROCs of 0.74 and 0.67. Compared to the existing scores, the fib-index performed better in diagnosing significant fibrosis for patients with ALT < 2 × ULN.
The current study has limitations such as a lack of a validation group. A proper internal validation is necessary for the development of a reliable and reproducible prognostic model for external validation.42 Although a validation set was not designed in this study, LOOCV was utilized to detect the reliability of inflammation-index and fib-index. The LOOCV approach has the advantage of producing model estimates easier and with less bias in smaller samples.43 Another limitation of our study is lack of longitudinal data; this will require future work. Finally, not all the cytokines which could be detected were measured.
In conclusion, the present study supports the concept that cytokines contribute significantly to the development of liver inflammation and fibrosis in CHB. Various cytokines have been found to be associated with liver inflammation and fibrosis, which implies that there are likely various mechanisms by which liver inflammation and fibrosis occur and immune cells become activated. Cytokines CXCL-11, INF-γ, and IL-17A are associated with liver inflammation, whereas CXCL-10, IL-2R, TGF-α, and IL-8 are correlated with liver fibrosis in chronic HBV-infected patients. In chronic HBV-infected patients with ALT < 2 × ULN, CXCL-11 was a marker of moderate or severe inflammation independent of other biochemical parameters and known risk factors. IL-2R and TGF-α were independent predictors for significant fibrosis. An IL-2R and TGF-α-based score fib-index was superior to the existing scores APRI and FIB-4 for predicting significant fibrosis in chronic HBV-infected patients. This represents a promising tool for non-invasive diagnosis of fibrosis in patients with normal and mildly elevated ALT levels.
The members of China HepB Related Fibrosis Assessment Research Group: An-Lin Ma, MD, from departments of Infectious Disease, China-Japan Friendship Hospital, Beijing; Shi-Bin Xie, MD, from departments of Infectious Disease, The Third Affiliated Hospital Sun Yat-Sen University, Guangzhou, Guangdong; Xu-Qing Zhang, MD, from department of Infectious Diseases, South West Hospital affiliated to Third Military Medical University, Chongqing; Da-Zhi Zhang, MD, from department of Infectious Diseases, Second Affiliated Hospital Of Chongqing Medical University, Chongqing; Qing Xie, MD, from department of Infectious Diseases, Rui Jin Hospital Shanghai Jiao Tong University School of Medicine, Shanghai; Guo Zhang, MM, from department of Infectious Diseases, The People's Hospital Of Guang Xi Zhuang Autonomous Region, Nanning, Guangxi; Jia Shang, MD, from department of Infectious Diseases, The People's Hospital Of He Nan province, Zheng Zhou, Henan; Jun Chen, MD, from department of Infectious Diseases, Di Tan Hospital affiliated to Capital Medical University, Beijing; Wei-Feng Zhao, MM, from department of Infectious Diseases, Xinxiang Medical University Third Hospital, Xinxiang, Henan; Zhi-Qiang Zhou, MM, from department of Infectious Diseases, Yantai City Hospital for Infectious Disease, Yantai, Shandong; Ying-Xia Liu, MM, from department of Infectious Diseases, Shenzhen Third People's hospital of, Shenzhen; Zhan-Qin Zhang, MD, from department of Infectious Diseases, Public Health Clinical Center Affiliated to Fudan University, Shanghai; Xun Peng, MM, Xie-Wen Sun, MM, from department of Infectious Diseases, Third Hospital Of Qin Huang Dao, Qin Huang Dao, Hebei; Ming-Xiang Zhang, MM, from department of Infectious Diseases, Shenyang Sixth People's Hospital, Shenyang, Liaoning; Fu-Dong Lv, MM, from department of pathology, You An Hospital affiliated to Capital Medical University, Beijing; Jun Li, MD, from department of Infectious Diseases, Jiang Su Province Hospital, Nanjing, Jiangsu; Wan-Li Sun, MM, from department of Infectious Diseases, Dailian Sixth People's Hospital, Dalian, Shandong; Lang Bai, MD, from department of Infectious Diseases, West China School of Medicine/West China Hospital Sichuan University, Chengdu, Sichuan; Li-Ming Chen, MD from department of Infectious Diseases; Yong-Qiong Deng, MD, Hong Zhao, MD, Ji-Yuan Zhou, MD, Yu-Qing Fang, MD, Wei Jia, MD, Dan Liu, MM, Hong-Li Xi, MM, Yi-Hang Zhou, MM, Min Yu, MM, Gui-Qiang Wang, MD, from department of Infectious Disease, Center for Liver Disease, Peking University First Hospital, Beijing. All of the members has participated in acquisition of data, revision of the manuscript for important intellectual content. The authors also thank China HepB Related Fibrosis Assessment Research Group: Professor Xin Xu (Nanfang Medical University, China) for his guidance with the statistics.
1. Lavanchy D. Hepatitis B virus epidemiology, disease burden, treatment, and current and emerging prevention and control measures. J Viral Hepat
2. Alessandro R. Zanettia, Pierre Van Dammeb, Daniel Shouvalc. The global impact of vaccination against hepatitis B: A historical overview. Vaccine
3. Chan HL, Jia J. Chronic hepatitis B in Asia—new insights from the past decade. J Gastroenterol Hepatol
4. Liang X, Bi S, Yang W, et al. Reprint of: epidemiological serosurvey of hepatitis B in China-declining HBV prevalence due to hepatitis B vaccination. Vaccine
2013; 31 (Suppl 9):21–28.
5. Kim WR, Flamm SL, Di Bisceglie AM, et al. Serum activity of alanine aminotransferase (ALT) as an indicator of health and disease. Hepatology
6. Lok AS, McMahon BJ. Chronic hepatitis B: update. Hepatology
7. Liaw YF, Kao JH, Piratvisuth T, et al. Asian-pacific consensus statement on the management of chronic hepatitis B: a 2012 update. Hepatol Int
8. Li C, Liu X, Tian L, et al. Cytokine-mediated immunopathogenesis of hepatic B virus infection. Clin Rev Allergy Immunol
2014; [Epub ahead of print].
9. Steinke JW, Borish L. 3. Cytokines and chemokines. J Allergy Clin Immunol
10. Seki E, Schwabe RF. Hepatic inflammation and fibrosis: functional links and key pathways. Hepatology
11. Liang SC, Tan XY, Luxenberg DP, et al. Interleukin (IL)-22 and IL-17 are coexpressed by Th17 cells and cooperatively enhance expression of antimicrobial peptides. J Exp Med
12. Zhao J, Zhang Z, Luan Y, et al. Pathological functions of interleukin-22 in chronic liver inflammation and fibrosis with HBV infection by promoting Th17 cell recruitment. Hepatology
13. Meng F, Wang K, Aoyama T, et al. Interleukin-17 signaling in inflammatory, Kupffer cells, and hepatic stellate cells exacerbates liver fibrosis in mice. Gastroenterology
14. Zhang JY, Zhang Z, Lin F, et al. Interleukin-17-producing CD4(+) T cells increase with severity of liver damage in patients with chronic hepatitis B. Hepatology
15. Poovorawan K, Tangkijvanich P, Chirathaworn C, et al. Circulating cytokines and histological liver damage in chronic hepatitis B infection. Hepat Res Treat
16. Akcam FZ, Tigli A, Kaya O, et al. Cytokine levels and histopathology in chronic hepatitis B and chronic hepatitis C. J Interferon Cytokine Res
17. Akpolat N, Yahsi S, Godekmerdan A, et al. Relationship between serum cytokine levels and histopathological changes of liver in patients with hepatitis B. World J Gastroenterol
18. Lian JQ, Yang XF, Zhao RR, et al. Expression profiles of circulating cytokines, chemokines and immune cells in patients with hepatitis B virus infection. Hepat Mon
19. Zeremski M1, Dimova R, Astemborski J, et al. CXCL9 and CXCL10 chemokines as predictors of liver fibrosis in a cohort of primarily African-American injection drug users with chronic hepatitis C. J Infect Dis
20. Wai CT, Greenson JK, Fontana RJ, et al. A simple noninvasive index can predict both significant fibrosis and cirrhosis in patients with chronic hepatitis C. Hepatology
21. Vallet-Pichard A1, Mallet V, Nalpas B, et al. FIB-4: an inexpensive and accurate marker of fibrosis in HCV infection. comparison with liver biopsy and fibrotest. Hepatology
22. Jia W, Song LW, Fang YQ, et al. Antibody to hepatitis B core antigen levels in the natural history of chronic hepatitis B. Medicine (Baltimore)
23. Ishak K, Baptista A, Bianchi L, et al. Histological grading and staging of chronic hepatitis. J Hepatol
24. Alex YH, Henry Lik YC, Vincent W, et al. Identification of chronic hepatitis B patients without significant liver fibrosis by a simple noninvasive predictive model. Am J Gastroenterol
25. Wai CT, Joel KG, Robert JF, et al. A simple noninvasive index can predict both significant fibrosis and cirrhosis in patients with chronic hepatitis C. Hepatology
26. Li X, Liu X, Tian L, et al. Cytokine-mediated immunopathogenesis of hepatitis B virus infections. Clin Rev Allergy Immunol
2014; (in press).
27. Zeremski M, Petrovic LM, Talal AH. The role of chemokines as in-flammatory mediators in chronic hepatitis C virus infection. J Viral Hepat
28. Zimmermann HW, Seidler S, Gassler N, et al. Interleukin-8 is activated in patients with chronic liver diseases and associated with hepatic macrophage accumulation in human liver fibrosis. PLoS One
29. Madala SK, Korfhagen TR, Schmidt S, et al. Inhibition of the αvβ6 integrin leads to limited alteration of TGF-α-induced pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol
30. Ohyama T, Yamazaki Y, Sato K, et al. Transforming growth factor-α attenuates hepatic fibrosis: possible involvement of matrix metalloproteinase-1. Liver Int
31. Kato J, Sato Y, Inui N, et al. Ethanol induces transforming growth factor-alpha expression in hepatocytes, leading to stimulation of collagen synthesis by hepatic stellate cells. Alcohol Clin Exp Res
2003; 27 (8 Suppl):S58–S63.
32. Nelson DL, Rubin LA, Kurman CC, et al. An analysis of the cellular requirements for the production of soluble interleukin-2 receptors in vitro. J Clin Immunol
33. Seidler S, Zimmermann HW, Weiskirchen R, et al. Elevated circulating soluble interleukin-2 receptor in patients with chronic liver diseases is associated with non-classical monocytes. BMC Gastroenterol
34. El-Shanawani FM, Abdel-Hadi AA, Abu Zikri NB, et al. Clinical significance of aflatoxin, mutant P53 gene and sIL-2 receptor in liver cirrhosis and hepatocellular carcinoma. J Egypt Soc Parasitol
35. Garcia Ruiz P, Canora Lebrato J, Diez Ruiz A, et al. Soluble interleukin-2 and tumor necrosis factor receptor in liver cirrhosis. Relationship with clinical severity and prognosis. Med Clin (Barc)
36. Barak V, Selmi C, Schlesinger M, et al. Serum inflammatory cytokines, complement components, and soluble interleukin 2 receptor in primary biliary cirrhosis. J Autoimmun
37. Luedde T, Kaplowitz N, Schwabe RF. Cell death and cell death responses in liver disease: mechanisms and clinical relevance. Gastroenterology
38. Chao DT, Lim JK, Ayoub WS, et al. Systematic review with meta-analysis:the proportion of chronic hepatitis B patients with normal alaninetransaminase ≤40 IU/L and significant hepatic fibrosis. Aliment Pharmacol Ther
39. Teshale E, Lu M, Rupp LB, et al. APRI and FIB-4 are good predictors of the stage of liver fibrosis in chronic hepatitis B: the Chronic Hepatitis Cohort Study (CHeCS). J Viral Hepat
40. Xiao G, Yang J, Yan L. Comparison of diagnostic accuracy of aspartate aminotransferase to platelet ratio index and fibrosis-4 index for detecting liver fibrosis in adult patients with chronic hepatitis B virus infection: a systemic review and meta-analysis. Hepatology
41. Wang H, Xue L, Yan R, et al. Comparison of FIB-4 and APRI in Chinese HBV-infected patients with persistently normal ALT and mildly elevatedALT. J Viral Hepatol
42. Rushing C, Bulusu A, Hurwitz HI, et al. A leave-one-out cross-validation SAS macro for the identification of markers associated with survival. Comput Biol Med
43. Molinaro AM, Simon R, Pfeiffer RM. Prediction error estimation: a comparison of resampling methods. Bioinformatics
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