http://informahealthcare.com/jmf ISSN: 1476-7058 (print), 1476-4954 (electronic) J Matern Fetal Neonatal Med, Early Online: 1–5 ! 2015 Informa UK Ltd. DOI: 10.3109/14767058.2015.1028354
ORIGINAL ARTICLE
The role of interleukin-17 in intrahepatic cholestasis of pregnancy Ayse Kirbas1, Ebru Biberoglu1, Ali Ozgur Ersoy1, Asiye Ugras Dikmen2, Cemile Koca3, Seval Erdinc1, Dilek Uygur1, Turhan Caglar1, and Kutay Biberoglu4
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1
Department of Perinatology, Zekai Tahir Burak Women Health Care, Education and Research Hospital, Ankara, Turkey, 2Department of Statistics, Gazi University Medical Faculty, Ankara, Turkey, 3Department of Biochemistry, Ataturk Education and Research Hospital, Ankara, Turkey, and 4 Department of Obstetrics and Gynecology, Gazi University Medical School, Ankara, Turkey Abstract
Keywords
Objective: Intrahepatic cholestasis of pregnancy (ICP) is the most common pregnancy-specific liver disease, is characterized by pruritus, abnormal liver function and elevated serum bile acid levels. The main cause of ICP has not yet been identified. We aimed to provide a new perspective to the pathogenesis of by investigating the possible association of circulating interleukin-17 (IL-17) that is a recently discovered proinflammatory cytokine levels with ICP. Materials and methods: In this controlled cross-sectional study, maternal venous blood samples were obtained from 33 consecutive pregnant women with ICP (15 with mild and 18 with severe forms of the disease) and 25 healthy women with uncomplicated pregnancies (as the control group) and IL-17 levels were compared among the groups. Results: Although serum IL-17 levels were significantly higher in the severe ICP group than in the control group (p ¼ 0.022), there were no significant differences between the mild and severe ICP groups or between the control and mild ICP groups. Conclusion: Explaining the mechanisms of hepatocyte injury might contribute to the existing therapeutic strategies for treating cholestatic diseases. Changes in IL-17 levels may shed light on the pathogenesis of ICP.
Bile acid, cholestasis, inflammation, interleukin 17, pregnancy
Introduction Intrahepatic cholestasis of pregnancy (ICP) is a transient form of cholestasis, which typically occurs in the second half of pregnancy. ICP is characterized by elevated serum total bile acid (BA) levels and/or liver enzymes, as well as itching, localized to the abdomen, legs and palms, that resolves spontaneously within months of delivery [1,2]. ICP is associated with increased risk of fetal complications, such as preterm labor, fetal distress, fetal dysrhythmias and unexplained fetal death, all of which are linked with elevated maternal serum BA levels [1-6]. The etiology of the disease is still unknown; genetic, endocrinologic, nutritional and environmental factors are likely to be important in its pathogenesis [1,2]. The significance of inflammation in cholestatic liver diseases has previously been recognized [7]. Pathologic
Address for correspondence: Dr Ayse Kirbas, Cankaya Mahallesi, Ziaurrahman Caddesi, 4A\2 Postal code: 06680, Cankaya, Ankara, Turkey. Tel: +90 533-646-9213. Fax: +90 312-312-4931. E-mail: [email protected]; [email protected]
History Received 23 January 2015 Revised 27 February 2015 Accepted 9 March 2015 Published online 7 April 2015
concentrations of BAs trigger a strong inflammatory response in hepatocytes, characterized by the induction of diverse classes of cytokines that promote the infiltration of inflammatory cells, including neutrophils, macrophages and lymphocytes. However, the mechanism by which cholestasis initiates an inflammatory response in the liver is not understood. The inflammation might either cause or contribute to cholestasis in liver diseases [7,8]. Interleukin-17 (IL-17), also known IL-17A or CTLassociated antigen 8, is a recently discovered proinflammatory cytokine [9]. There is a growing body of data regarding the implications of T helper (Th17) cells and IL-17 in various liver disorders, including obstructive cholestasis [10,11]. Although several studies have investigated the pathogenesis of ICP, the role of IL-17 has not been explored yet. To our knowledge, this is the first survey designed to do so. Explaining the mechanisms of hepatocyte injury might contribute to the existing therapeutic strategies for treating cholestatic diseases. Although the etiology of ICP is heterogeneous and not fully understood, immunological and inflammatory processes are implicated in its pathogenesis in addition to the known cholestatic effects of pregnancy hormones, and nutritional and environmental factors in genetically predisposed women [7–9].
2
A. Kirbas et al.
In this study, we aimed to provide a new perspective to the pathogenesis of ICP by investigating the possible association of circulating IL-17 levels with ICP.
J Matern Fetal Neonatal Med Downloaded from informahealthcare.com by Nyu Medical Center on 04/11/15 For personal use only.
Materials and methods Thirty-three consecutive pregnant women with ICP (15 with mild and 18 with severe forms of the disease) and 25 healthy women with uncomplicated pregnancies (as the control group), all in the third trimester (33–38 weeks) and matched for maternal and gestational ages, were recruited between February and October, 2014, at the Zekai Tahir Burak Women’s Health Education and Research Hospital, Ankara, Turkey. All of the participants provided written informed consent to donate biological specimens for this study. The Institutional Review Board of the hospital approved the study, and the universal principles of the Helsinki Declaration were applied. None of the patients or controls exhibited signs or symptoms of an infectious disease or a pre-existing chronic systemic disease. Pregnant women with multiple gestations or in active labor were excluded. Patients on any medication at the time of admission to the hospital, including ursodeoxycholic acid (UDCA), were also excluded from the study, due to their well-established ability to reduce the effect of serum BA levels and possible anti-inflammatory effects [12]. The initial evaluation included a thorough physical examination, daily body temperature, basal hematological and biochemical blood sample profile, routine urine culture and testing for hepatitis A, B and C. Further investigations of infection markers are made on clinical ground. Patient with signs and symptoms of active infection are excluded from the study. Intrahepatic cholestasis of pregnancy was diagnosed when a pregnant woman had pruritus without rash and elevated total BA (TBA) (10 lmol/L) and/or aminotransferase levels in her blood sample. The patients were classified as mild or severe ICP based on TBA concentrations of 10–40 or 40 lmol/L, respectively [1]. Gestational age was determined based on the first day of the last menstrual period and/or first trimester ultrasonographic measurements. Preterm birth was defined as delivery before 37 weeks of pregnancy. Venous blood samples for IL-17 were obtained at the time of admission and kept on ice. The samples were centrifuged at 5000 revolutions/minute for 10 min within 5–10 min of blood sampling, and frozen immediately at 80 C until the analyses were carried out. Serum concentration of IL-17 was measured using a commercially available Human Il-17 ELISA Kit (ab100556; ABCAM, Cambridge, MA), according to the protocols provided by the manufacturer. The intra-assay and inter-assay coefficients of variation for serum IL-17 were 8 and 9.5%, respectively. All samples were assessed in duplicate. Following at least 8 h of fasting, about 5 cc of venous blood was drawn from the antecubital vein early in the morning. TBA levels were determined spectrophotometrically using an enzymatic method on a Cobas C501 Analyzer (Roche Diagnostics, Rotkreuz, Switzerland) and reported as micromoles/liter (lmol/L). The rest of the blood analyses
J Matern Fetal Neonatal Med, Early Online: 1–5
were carried out within 2 h of blood sampling, using a hematology analyzer (GEN-S; Beckman-Coulter Inc., Brea, CA) at the central laboratories of Zekai Tahir Burak Women’s Hospital. The clinical information of each participant, including age, maternal weight gain during pregnancy and body mass index (BMI), were recorded, as well as the laboratory results of the hematologic and biochemical parameters and TBA levels. The perinatal outcome parameters, including gestational age at delivery, birth weight and birth weight centile, iatrogenic and spontaneous preterm delivery, stillbirth, meconiumstained amniotic fluid, 5-minute Apgar score 7, and neonatal intensive care unit admission were also assessed. Statistical analysis Statistical analyses were performed using SPSS version 12 (Statistical Package for the Social Sciences, Chicago, IL). Sample size was determined according to the results of the central limit theorem [13]. The data were summarized as mean ± standard deviation and median (minimum–maximum). Comparisons of multiple independent groups were performed using the Kruskal–Wallis test for numerical variables not normally distributed. Proportions were compared with Fisher’s exact test or the chi-square test where appropriate. Correlations between different maternal biochemical parameters were examined using Spearman’s correlation. The Mann–Whitney U-test was used to compare the two independent groups. The level of statistical significance was considered to be p50.05.
Results Following the exclusion of 16 pregnant women with ICP (twin pregnancies in two, active hepatitis in one, active labor in four and current UDCA treatment in nine) from the study group and 21 normal pregnant women (twin pregnancies in four, active labor in 12, symptoms of active infection in five) from the control group, 33 cases with ICP (15 with mild and 18 with severe disease) and 25 normal healthy pregnant women constituted the study and control groups, respectively. Maternal age, gestational week, BMI at assessment, maternal weight gain during pregnancy, maternal hemoglobin levels, and white blood cell counts were comparable between the study and control groups. However, TBA, aspartate aminotransferase and alanine aminotransferase levels were significantly higher in the severe ICP group than in the mild ICP and control groups. The characteristics of the groups are depicted in Table 1. Although serum IL-17 levels were significantly higher in the severe ICP group than in the control group (p ¼ 0.022), there were no significant differences between the mild and severe ICP groups or between the control and mild ICP groups. The mean TBA and IL-17 values in the ICP and control groups are shown in Figure 1. Duration of pregnancy was significantly shorter in the ICP groups compared to the healthy pregnant women. While the mean birth weight was lower in pregnant women with severe ICP compared with the control group, there were no significant differences between the mild and severe ICP groups or between the control and mild ICP groups.
Interleukin-17 in cholestasis of pregnancy
DOI: 10.3109/14767058.2015.1028354
3
Table 1. Comparison of patient parameters in control (group 1) and intrahepatic cholestasis (group 2 mild and group 3 severe disease) cases. p value (groups)
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Maternal age (years) Gestational week at assessment Maternal weight gain (kg) BMI at assessment (kg/m2) Hemoglobin (g/dL) WBC (103/lL) TBA (lmol/L) AST (U/L) ALT (U/L) Maternal IL-17 (pg/mL)
Group 1 (n ¼ 25)
Group 2 (n ¼ 15)
Group 3 (n ¼ 18)
28.44 ± 5.27 37 ± 1.08 11.5 ± 5 28.55 ± 3.88 11.6 ± 0.8 8764 ± 1571 2.48 ± 1.85 17.8 ± 4.03 16.4 ± 3.68 303.38 ± 74.4
28.27 ± 4.89 36.7 ± 1.49 9.9 ± 2.86 28.6 ± 4.51 11.8 ± 0.5 9280 ± 1409 24.8 ± 6.71 59.2 ± 30 76.2 ± 35 348.58 ± 81.5
28.59 ± 4.96 36.2 ± 3.42 10.9 ± 3.8 27.5 ± 5.91 11.4 ± 0.6 8876 ± 1848 63.76 ± 20.8 95.8 ± 31 143.2 ± 74.03 370.52 ± 106.3
1 versus 2
1 versus 3
2 versus 3
50.001 50.001 50.001 NS
NS NS NS NS NS NS 50.001 50.001 50.001 0.022
50.001 50.001 0.005 NS
ALT: alanine aminotransferase; AST: aspartate aminotransferase; BMI: body mass index; ICP: intrahepatic cholestasis of pregnancy; TBA, total bile acids; WBC: white blood cell. Data are expressed as mean ± SD. The mean difference is significant at the 0.05 level. NS: Non-significant.
400
TBA (µmol/L) IL 17 Levels (pg/ml)
350
370.52
348.58
303.38
300 250 200 150 100 50
63.76 2.48
24.8
0 Control Group
Mild ICP Group
Severe ICP Group
Figure 1. The mean values of TBA (lmol/L) and IL-17 levels (pg/mL) in pregnant with ICP and controls. TBA, total bile acid; ICP, intrahepatic cholestasis of pregnancy.
There were a significantly higher number of iatrogenic preterm deliveries in the severe ICP group compared with the mild ICP and control groups. The main indications for admission to the neonatal intensive care unit were preterm delivery and neonate respiratory problems in all groups. There were no cases of intrauterine fetal demise in any of the groups. The perinatal outcomes of the pregnant women with ICP and the control cases are shown in Table 2. Pearson’s correlation analysis revealed a significant negative correlation between TBA level and length of gestation (r¼-0.485, p ¼ 0.049) and between TBA level and mean birth weight (r¼-0.600, p ¼ 0.011) in the severe ICP group. No significant correlations between IL-17 and TBA levels were detected in the severe ICP group (r ¼ 0.424, p ¼ 0.09) (Table 3).
Discussion Th17 cells, a novel subset of T cells that secrete IL-17, are known to be involved in the pathogenesis of autoimmune and inflammatory diseases [9]. It has been suggested that along with regulatory T cells, Th17 cells play a key role in the initiation and maintenance of pregnancy. An imbalance between regulatory and effector cells have been reported to lead to several problems in pregnancy, including recurrent miscarriages [14], preeclampsia [15] and preterm delivery [16].
There is evidence to suggest that ICP is an immunological and inflammatory disease of pregnancy. In a recently published study, the genes associated with immune response were found to be upregulated in women with ICP, suggesting that the immune response in the placentas of patients with ICP is abnormal. The increased infiltration of leucocytes and the presence of both T and B cells in the placentas of women with ICP further support the activated immune response [17]. Along with pregnancy, autoimmune reactions, congenital disorders, drug hepatotoxicities and other forms of liver disease can cause interruptions in the bile flow, resulting in cholestatic liver disease [18]. A common feature of most types of cholestatic liver disease is an elevation in proinflammatory cytokines and hepatic inflammation. It has been suggested that the pathologic concentrations of BAs might cause the release of several proinflammatory mediators and trigger inflammatory response, leading to the accumulation of immune cells in the liver, and finally, to hepatocyte injury [8,11,19,20]. The mechanism by which cholestasis stimulates the production of proinflammatory mediators in the liver is not completely understood. Early growth response factor-1 (Egr-1), which regulates the production of inflammatory mediators, such as cytokines (IL-1b, IL-10), chemokines, adhesion molecules and enzymes in arachidonic acid metabolism, increases after ligation of the bile duct in the murine model [21]. Elevated levels of BAs act as inflammatory agents that stimulate the production of proinflammatory substances by hepatocytes through Egr-1-dependent and -independent pathways. It has been suggested that BAs stimulate the production of cytokines that may promote the accumulation of other immune cells, such as T cells, potentially influencing liver pathology during cholestasis [8]. Interleukin-17 might be the stimulant of the proinflammatory cytokines and neutrophil-attracting chemokines, which activates neutrophils and augments antibody production [21]. The activation and recruitment of neutrophils to the infection area by IL-17 seems crucial in protecting against gramnegative bacteria and fungal infections. On the other hand, once neutrophils are activated, they might also cause damage to normal tissues [22]. Concentrations of Th17 cells and IL-17 have been linked to the pathogenesis of alcohol-induced liver injury [23],
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J Matern Fetal Neonatal Med, Early Online: 1–5
Table 2. The comparison of the perinatal outcomes in the control (group 1) and intrahepatic cholestasis (group 2 mild and group 3 severe disease) pregnant groups cases. p value (groups)
Gestational week at delivery Spontaneous delivery537 weeks Iatrogenic delivery537 weeks Birth weight (g) Birth weight510th percentile Meconium staining of amniotic fluid 5-minute Apgar 7 NICU admission
Group 1 (n ¼ 25)
Group 2 (n ¼ 15)
Group 3 (n ¼ 18)
1 versus 2
1 versus 3
2 versus 3
39.6 ± 2.1 1 (4%) 0 3287 ± 382 0 3 (12%) 1 (4%) 1 (4%)
38.5 ± 0.9 1 (6.7%) 0 3022 ± 467 1 (6.7%) 2 (13.3%) 1 (6.7%) 1 (6.7%)
37.1 ± 1.6 1 (5.8%) 5 (29.4%) 2906 ± 544 1 (5.8%) 4 (23.5%) 1 (5.8%) 1 (5.8%)
50.001 NS NS NS NS NS NS
50.001 NS 50.001 0.002* NS NS NS NS
0.003 NS 50.001 NS NS NS NS NS
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Data are expressed as number (%), mean ± SD; NS: Non-significant; NICU, Neonatal intensive care unit. *The mean difference is significant at the 0.05 level.
Table 3. Correlation analysis within bile acid levels, IL-17, gestational week at delivery and birth weight in ICP and control groups.
Control Group (n ¼ 25) TBA IL-17 Mild ICP group (n ¼ 15) TBA IL-17 Severe ICP group (n ¼ 18) TBA IL-17
TBA r P r P r p r P r p r p
1 0.136 0.516 1 0.154 0.583 1 0.424 0.009
IL-17
Gestational week at delivery
Birth weight
0.136 0.516 1 -
0.130 0.536 0.240 0.288
0.188 0.369 0.67 0.75
0.154 0.583 1 -
0.99 0.726 0.04 0.989
0.76 0.787 0.237 0.396
0.424 0.009 1 -
0.485 0.049 0.308 0.229
0.600 0.011 0.730 0.781
Pearson correlation is significant at the 0.05 level (two-tailed). ICP, Intrahepatic cholestasis of pregnancy ; TBA, total bile acids.
non-alcoholic steatohepatitis [24], chronic hepatitis B [25], hepatitis C virus infection [26] and autoimmune hepatitis [27]. In addition, elevated levels of IL-17-positive cells have been associated with primary biliary cirrhosis [28]. O’Brien et al. [11] explored the effect of IL-17 on the upregulation of Egr-1 by BAs in a bile duct-ligated mice model, and they suggested that IL-17 might interact with other inflammatory substances and might synergistically enhance the BA-induced production of proinflammatory cytokines by hepatocytes in obstructive cholestasis [11]. In the current study, in order to shed light on the pathogenesis of hepatic inflammation during ICP, we aimed to explore the potential link between maternal serum IL-17 and BA levels in a group of women with ICP and compared the results with those of a group of women with healthy pregnancies. We demonstrated that IL-17 serum levels were significantly higher in the severe ICP group than in the control group. We found a significant negative correlation between TBA level and gestational age at delivery and between TBA level and mean birth weight in the severe ICP group; however, there was no significant correlation between IL-17 and TBA level.
The main limitation of this study is that our sample size was rather small - only 1% of pregnancies are affected by ICP, and we had to exclude several patients who were already on a well-known anti-inflammatory drug, UDCA, at the time of admission to our hospital. Nevertheless, our study is the first of its kind to investigate the relationship between a novel Tcell derived cytokine, IL-17 and intrahepatic cholestasis. We believe that explaining the mechanisms of BA-induced hepatocyte injury will contribute to the invention of novel therapeutic strategies for treating human cholestatic diseases. Therefore, further studies that are more comprehensive and include larger sample sizes are needed to determine the role of IL-17-mediated mechanisms in the etiopathogenesis of ICP.
Acknowledgements The authors thank to Dr Ozgur Kirbas for his secretarial help and his valuable advice in the statistical analysis.
Declaration of interest The authors have no conflicts of interest.
DOI: 10.3109/14767058.2015.1028354
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References 1. Williamson C, Geenes V. Intrahepatic cholestasis of pregnancy. Obstet Gynecol 2014;124:120–33. 2. Glantz A, Marschall HU, Mattsson LA. Intrahepatic cholestasis of pregnancy: relationships between bile acid levels and fetal complication rates. Hepatology 2004;40:467–74. 3. Geenes V, Chappell LC, Seed PT, et al. Association of severe intrahepatic cholestasis of pregnancy with adverse pregnancy outcomes: a prospective population-based case-control study. Hepatology 2014; 59:1482–91. 4. Kirbas A, Biberoglu E, Daglar K, et al. Neutrophil-to-lymphocyte ratio as a diagnostic marker of intrahepatic cholestasis of pregnancy. Eur J Obstet Gynecol Reprod Biol 2014;180:12–15. 5. Williamson C, Gorelik J, Eaton BM, et al. The bile acid taurocholate impairs rat cardiomyocyte function: a proposed mechanism for intra-uterine fetal death in obstetric cholestasis. Clin Sci (Lond) 2001;100:363–9. 6. Biberoglu EH, Kirbas A, Kirbas O, et al. Prediction of cardiovascular risk by electrocardiographic changes in women with intrahepatic cholestasis of pregnancy. J Matern Fetal Neonatal Med 2014;30:1–5. 7. Kosters A, Karpen SJ. The role of inflammation in cholestasis: clinical and basic aspects. Semin Liver Dis 2010;30:186–94. 8. Allen K, Jaeschke H, Copple BL. Bile acids induce inflammatory genes in hepatocytes: a novel mechanism of inflammation during obstructive cholestasis. Am J Pathol 2011;178:175–86. 9. Miossec P, Kolls JK. Targeting IL-17 and TH17 cell in chronic inflammation. Nat Rev Drug Discov 2012;11:763–76. 10. Hammerich L, Heymann F, Tacke F. Role of IL-17 and Th17 cells in liver diseases. Clin Dev Immunol 2011;2011:345803. 11. O’Brien KM, Allen KM, Rockwell CE, et al. IL-17A synergistically enhances bile acid-induced inflammation during obstructive cholestasis. Am J Pathol 2013;183:1498–507. 12. Poupon R. Ursodeoxycholic acid and bile-acid mimetics as therapeutic agents for cholestatic liver diseases: an overview of their mechanisms of action. Clin Res Hepatol Gastroenterol 2012; 36:S3–12. 13. Celik Y. Principles of biostatistics. 2nd ed. Diyarbakır: Dicle University Press; 2007. 14. Najafi S, Hadinedoushan H, Eslami G, Aflatoonian A. Association of IL-17A and IL-17 F gene polymorphisms with recurrent pregnancy loss in Iranian women. J Assist Reprod Genet 2014;31:1491–6.
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15. Cornelius DC, Hogg JP, Scott J, et al. Administration of interleukin-17 soluble receptor C suppresses TH17 cells, oxidative stress, and hypertension in response to placental ischemia during pregnancy. Hypertension 2013;62:1068–73. 16. Hee L, Kirkegaard I, Vogel I, Thorsen P, et al. Low serum interleukin-17 is associated with preterm delivery. Acta Obstet Gynecol Scand 2011;90:92–6. 17. Du Q, Pan Y, Zhang Y, et al. Placental gene-expression profiles of intrahepatic cholestasis of pregnancy reveal involvement of multiple molecular pathways in blood vessel formation and inflammation. BMC Med Genomics 2014;7:42. 18. Li MK, Crawford JM. The pathology of cholestasis. Semin Liver Dis 2004;24:21–42. 19. Gujral JS, Farhood A, Bajt ML, Jaeschke H. Neutrophils aggravate acute liver injury during obstructive cholestasis in bile duct-ligated mice. Hepatology 2003;38:355–63. 20. Woolbright BL, Jaeschke H. Novel insight into mechanisms of cholestatic liver injury. World J Gastroenterol 2012;18:4985–93. 21. Kim ND, Moon JO, Slitt AL, Copple BL. Early growth response factor-1 is critical for cholestatic liver injury. Toxicol Sci 2006;90: 586–95. 22. Awasthi A, Kuchroo VK. Th17 cells: from precursors to players in inflammation and infection. Int Immunol 2009;21: 489–98. 23. Lemmers A, Moreno C, Gustot T, et al. The interleukin-17 pathway is involved in human alcoholic liver disease. Hepatology 2009;49: 646–57. 24. Tang Y, Bian Z, Zhao L, et al. Interleukin-17 exacerbates hepatic steatosis and inflammation in non-alcoholic fatty liver disease. Clin Exp Immunol 2011;166:281–90. 25. 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 2010;51:81–91. 26. Rowan AG, Fletcher JM, Ryan EJ, et al. Hepatitis C virus-specific Th17 cells are suppressed by virus-induced TGF-beta. J Immunol 2008;181:4485–94. 27. Harada K, Shimoda S, Sato Y, et al. Periductal interleukin-17 production in association with biliary innate immunity contributes to the pathogenesis of cholangiopathy in primary biliary cirrhosis. Clin Exp Immunol 2009;157:261–70. 28. Lan RY1, Salunga TL, Tsuneyama K, et al. Hepatic IL-17 responses in human and murine primary biliary cirrhosis. J Autoimmun 2009;32:43–51.
ORIGINAL ARTICLE
The role of interleukin-17 in intrahepatic cholestasis of pregnancy Ayse Kirbas1, Ebru Biberoglu1, Ali Ozgur Ersoy1, Asiye Ugras Dikmen2, Cemile Koca3, Seval Erdinc1, Dilek Uygur1, Turhan Caglar1, and Kutay Biberoglu4
J Matern Fetal Neonatal Med Downloaded from informahealthcare.com by Nyu Medical Center on 04/11/15 For personal use only.
1
Department of Perinatology, Zekai Tahir Burak Women Health Care, Education and Research Hospital, Ankara, Turkey, 2Department of Statistics, Gazi University Medical Faculty, Ankara, Turkey, 3Department of Biochemistry, Ataturk Education and Research Hospital, Ankara, Turkey, and 4 Department of Obstetrics and Gynecology, Gazi University Medical School, Ankara, Turkey Abstract
Keywords
Objective: Intrahepatic cholestasis of pregnancy (ICP) is the most common pregnancy-specific liver disease, is characterized by pruritus, abnormal liver function and elevated serum bile acid levels. The main cause of ICP has not yet been identified. We aimed to provide a new perspective to the pathogenesis of by investigating the possible association of circulating interleukin-17 (IL-17) that is a recently discovered proinflammatory cytokine levels with ICP. Materials and methods: In this controlled cross-sectional study, maternal venous blood samples were obtained from 33 consecutive pregnant women with ICP (15 with mild and 18 with severe forms of the disease) and 25 healthy women with uncomplicated pregnancies (as the control group) and IL-17 levels were compared among the groups. Results: Although serum IL-17 levels were significantly higher in the severe ICP group than in the control group (p ¼ 0.022), there were no significant differences between the mild and severe ICP groups or between the control and mild ICP groups. Conclusion: Explaining the mechanisms of hepatocyte injury might contribute to the existing therapeutic strategies for treating cholestatic diseases. Changes in IL-17 levels may shed light on the pathogenesis of ICP.
Bile acid, cholestasis, inflammation, interleukin 17, pregnancy
Introduction Intrahepatic cholestasis of pregnancy (ICP) is a transient form of cholestasis, which typically occurs in the second half of pregnancy. ICP is characterized by elevated serum total bile acid (BA) levels and/or liver enzymes, as well as itching, localized to the abdomen, legs and palms, that resolves spontaneously within months of delivery [1,2]. ICP is associated with increased risk of fetal complications, such as preterm labor, fetal distress, fetal dysrhythmias and unexplained fetal death, all of which are linked with elevated maternal serum BA levels [1-6]. The etiology of the disease is still unknown; genetic, endocrinologic, nutritional and environmental factors are likely to be important in its pathogenesis [1,2]. The significance of inflammation in cholestatic liver diseases has previously been recognized [7]. Pathologic
Address for correspondence: Dr Ayse Kirbas, Cankaya Mahallesi, Ziaurrahman Caddesi, 4A\2 Postal code: 06680, Cankaya, Ankara, Turkey. Tel: +90 533-646-9213. Fax: +90 312-312-4931. E-mail: [email protected]; [email protected]
History Received 23 January 2015 Revised 27 February 2015 Accepted 9 March 2015 Published online 7 April 2015
concentrations of BAs trigger a strong inflammatory response in hepatocytes, characterized by the induction of diverse classes of cytokines that promote the infiltration of inflammatory cells, including neutrophils, macrophages and lymphocytes. However, the mechanism by which cholestasis initiates an inflammatory response in the liver is not understood. The inflammation might either cause or contribute to cholestasis in liver diseases [7,8]. Interleukin-17 (IL-17), also known IL-17A or CTLassociated antigen 8, is a recently discovered proinflammatory cytokine [9]. There is a growing body of data regarding the implications of T helper (Th17) cells and IL-17 in various liver disorders, including obstructive cholestasis [10,11]. Although several studies have investigated the pathogenesis of ICP, the role of IL-17 has not been explored yet. To our knowledge, this is the first survey designed to do so. Explaining the mechanisms of hepatocyte injury might contribute to the existing therapeutic strategies for treating cholestatic diseases. Although the etiology of ICP is heterogeneous and not fully understood, immunological and inflammatory processes are implicated in its pathogenesis in addition to the known cholestatic effects of pregnancy hormones, and nutritional and environmental factors in genetically predisposed women [7–9].
2
A. Kirbas et al.
In this study, we aimed to provide a new perspective to the pathogenesis of ICP by investigating the possible association of circulating IL-17 levels with ICP.
J Matern Fetal Neonatal Med Downloaded from informahealthcare.com by Nyu Medical Center on 04/11/15 For personal use only.
Materials and methods Thirty-three consecutive pregnant women with ICP (15 with mild and 18 with severe forms of the disease) and 25 healthy women with uncomplicated pregnancies (as the control group), all in the third trimester (33–38 weeks) and matched for maternal and gestational ages, were recruited between February and October, 2014, at the Zekai Tahir Burak Women’s Health Education and Research Hospital, Ankara, Turkey. All of the participants provided written informed consent to donate biological specimens for this study. The Institutional Review Board of the hospital approved the study, and the universal principles of the Helsinki Declaration were applied. None of the patients or controls exhibited signs or symptoms of an infectious disease or a pre-existing chronic systemic disease. Pregnant women with multiple gestations or in active labor were excluded. Patients on any medication at the time of admission to the hospital, including ursodeoxycholic acid (UDCA), were also excluded from the study, due to their well-established ability to reduce the effect of serum BA levels and possible anti-inflammatory effects [12]. The initial evaluation included a thorough physical examination, daily body temperature, basal hematological and biochemical blood sample profile, routine urine culture and testing for hepatitis A, B and C. Further investigations of infection markers are made on clinical ground. Patient with signs and symptoms of active infection are excluded from the study. Intrahepatic cholestasis of pregnancy was diagnosed when a pregnant woman had pruritus without rash and elevated total BA (TBA) (10 lmol/L) and/or aminotransferase levels in her blood sample. The patients were classified as mild or severe ICP based on TBA concentrations of 10–40 or 40 lmol/L, respectively [1]. Gestational age was determined based on the first day of the last menstrual period and/or first trimester ultrasonographic measurements. Preterm birth was defined as delivery before 37 weeks of pregnancy. Venous blood samples for IL-17 were obtained at the time of admission and kept on ice. The samples were centrifuged at 5000 revolutions/minute for 10 min within 5–10 min of blood sampling, and frozen immediately at 80 C until the analyses were carried out. Serum concentration of IL-17 was measured using a commercially available Human Il-17 ELISA Kit (ab100556; ABCAM, Cambridge, MA), according to the protocols provided by the manufacturer. The intra-assay and inter-assay coefficients of variation for serum IL-17 were 8 and 9.5%, respectively. All samples were assessed in duplicate. Following at least 8 h of fasting, about 5 cc of venous blood was drawn from the antecubital vein early in the morning. TBA levels were determined spectrophotometrically using an enzymatic method on a Cobas C501 Analyzer (Roche Diagnostics, Rotkreuz, Switzerland) and reported as micromoles/liter (lmol/L). The rest of the blood analyses
J Matern Fetal Neonatal Med, Early Online: 1–5
were carried out within 2 h of blood sampling, using a hematology analyzer (GEN-S; Beckman-Coulter Inc., Brea, CA) at the central laboratories of Zekai Tahir Burak Women’s Hospital. The clinical information of each participant, including age, maternal weight gain during pregnancy and body mass index (BMI), were recorded, as well as the laboratory results of the hematologic and biochemical parameters and TBA levels. The perinatal outcome parameters, including gestational age at delivery, birth weight and birth weight centile, iatrogenic and spontaneous preterm delivery, stillbirth, meconiumstained amniotic fluid, 5-minute Apgar score 7, and neonatal intensive care unit admission were also assessed. Statistical analysis Statistical analyses were performed using SPSS version 12 (Statistical Package for the Social Sciences, Chicago, IL). Sample size was determined according to the results of the central limit theorem [13]. The data were summarized as mean ± standard deviation and median (minimum–maximum). Comparisons of multiple independent groups were performed using the Kruskal–Wallis test for numerical variables not normally distributed. Proportions were compared with Fisher’s exact test or the chi-square test where appropriate. Correlations between different maternal biochemical parameters were examined using Spearman’s correlation. The Mann–Whitney U-test was used to compare the two independent groups. The level of statistical significance was considered to be p50.05.
Results Following the exclusion of 16 pregnant women with ICP (twin pregnancies in two, active hepatitis in one, active labor in four and current UDCA treatment in nine) from the study group and 21 normal pregnant women (twin pregnancies in four, active labor in 12, symptoms of active infection in five) from the control group, 33 cases with ICP (15 with mild and 18 with severe disease) and 25 normal healthy pregnant women constituted the study and control groups, respectively. Maternal age, gestational week, BMI at assessment, maternal weight gain during pregnancy, maternal hemoglobin levels, and white blood cell counts were comparable between the study and control groups. However, TBA, aspartate aminotransferase and alanine aminotransferase levels were significantly higher in the severe ICP group than in the mild ICP and control groups. The characteristics of the groups are depicted in Table 1. Although serum IL-17 levels were significantly higher in the severe ICP group than in the control group (p ¼ 0.022), there were no significant differences between the mild and severe ICP groups or between the control and mild ICP groups. The mean TBA and IL-17 values in the ICP and control groups are shown in Figure 1. Duration of pregnancy was significantly shorter in the ICP groups compared to the healthy pregnant women. While the mean birth weight was lower in pregnant women with severe ICP compared with the control group, there were no significant differences between the mild and severe ICP groups or between the control and mild ICP groups.
Interleukin-17 in cholestasis of pregnancy
DOI: 10.3109/14767058.2015.1028354
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Table 1. Comparison of patient parameters in control (group 1) and intrahepatic cholestasis (group 2 mild and group 3 severe disease) cases. p value (groups)
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Maternal age (years) Gestational week at assessment Maternal weight gain (kg) BMI at assessment (kg/m2) Hemoglobin (g/dL) WBC (103/lL) TBA (lmol/L) AST (U/L) ALT (U/L) Maternal IL-17 (pg/mL)
Group 1 (n ¼ 25)
Group 2 (n ¼ 15)
Group 3 (n ¼ 18)
28.44 ± 5.27 37 ± 1.08 11.5 ± 5 28.55 ± 3.88 11.6 ± 0.8 8764 ± 1571 2.48 ± 1.85 17.8 ± 4.03 16.4 ± 3.68 303.38 ± 74.4
28.27 ± 4.89 36.7 ± 1.49 9.9 ± 2.86 28.6 ± 4.51 11.8 ± 0.5 9280 ± 1409 24.8 ± 6.71 59.2 ± 30 76.2 ± 35 348.58 ± 81.5
28.59 ± 4.96 36.2 ± 3.42 10.9 ± 3.8 27.5 ± 5.91 11.4 ± 0.6 8876 ± 1848 63.76 ± 20.8 95.8 ± 31 143.2 ± 74.03 370.52 ± 106.3
1 versus 2
1 versus 3
2 versus 3
50.001 50.001 50.001 NS
NS NS NS NS NS NS 50.001 50.001 50.001 0.022
50.001 50.001 0.005 NS
ALT: alanine aminotransferase; AST: aspartate aminotransferase; BMI: body mass index; ICP: intrahepatic cholestasis of pregnancy; TBA, total bile acids; WBC: white blood cell. Data are expressed as mean ± SD. The mean difference is significant at the 0.05 level. NS: Non-significant.
400
TBA (µmol/L) IL 17 Levels (pg/ml)
350
370.52
348.58
303.38
300 250 200 150 100 50
63.76 2.48
24.8
0 Control Group
Mild ICP Group
Severe ICP Group
Figure 1. The mean values of TBA (lmol/L) and IL-17 levels (pg/mL) in pregnant with ICP and controls. TBA, total bile acid; ICP, intrahepatic cholestasis of pregnancy.
There were a significantly higher number of iatrogenic preterm deliveries in the severe ICP group compared with the mild ICP and control groups. The main indications for admission to the neonatal intensive care unit were preterm delivery and neonate respiratory problems in all groups. There were no cases of intrauterine fetal demise in any of the groups. The perinatal outcomes of the pregnant women with ICP and the control cases are shown in Table 2. Pearson’s correlation analysis revealed a significant negative correlation between TBA level and length of gestation (r¼-0.485, p ¼ 0.049) and between TBA level and mean birth weight (r¼-0.600, p ¼ 0.011) in the severe ICP group. No significant correlations between IL-17 and TBA levels were detected in the severe ICP group (r ¼ 0.424, p ¼ 0.09) (Table 3).
Discussion Th17 cells, a novel subset of T cells that secrete IL-17, are known to be involved in the pathogenesis of autoimmune and inflammatory diseases [9]. It has been suggested that along with regulatory T cells, Th17 cells play a key role in the initiation and maintenance of pregnancy. An imbalance between regulatory and effector cells have been reported to lead to several problems in pregnancy, including recurrent miscarriages [14], preeclampsia [15] and preterm delivery [16].
There is evidence to suggest that ICP is an immunological and inflammatory disease of pregnancy. In a recently published study, the genes associated with immune response were found to be upregulated in women with ICP, suggesting that the immune response in the placentas of patients with ICP is abnormal. The increased infiltration of leucocytes and the presence of both T and B cells in the placentas of women with ICP further support the activated immune response [17]. Along with pregnancy, autoimmune reactions, congenital disorders, drug hepatotoxicities and other forms of liver disease can cause interruptions in the bile flow, resulting in cholestatic liver disease [18]. A common feature of most types of cholestatic liver disease is an elevation in proinflammatory cytokines and hepatic inflammation. It has been suggested that the pathologic concentrations of BAs might cause the release of several proinflammatory mediators and trigger inflammatory response, leading to the accumulation of immune cells in the liver, and finally, to hepatocyte injury [8,11,19,20]. The mechanism by which cholestasis stimulates the production of proinflammatory mediators in the liver is not completely understood. Early growth response factor-1 (Egr-1), which regulates the production of inflammatory mediators, such as cytokines (IL-1b, IL-10), chemokines, adhesion molecules and enzymes in arachidonic acid metabolism, increases after ligation of the bile duct in the murine model [21]. Elevated levels of BAs act as inflammatory agents that stimulate the production of proinflammatory substances by hepatocytes through Egr-1-dependent and -independent pathways. It has been suggested that BAs stimulate the production of cytokines that may promote the accumulation of other immune cells, such as T cells, potentially influencing liver pathology during cholestasis [8]. Interleukin-17 might be the stimulant of the proinflammatory cytokines and neutrophil-attracting chemokines, which activates neutrophils and augments antibody production [21]. The activation and recruitment of neutrophils to the infection area by IL-17 seems crucial in protecting against gramnegative bacteria and fungal infections. On the other hand, once neutrophils are activated, they might also cause damage to normal tissues [22]. Concentrations of Th17 cells and IL-17 have been linked to the pathogenesis of alcohol-induced liver injury [23],
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Table 2. The comparison of the perinatal outcomes in the control (group 1) and intrahepatic cholestasis (group 2 mild and group 3 severe disease) pregnant groups cases. p value (groups)
Gestational week at delivery Spontaneous delivery537 weeks Iatrogenic delivery537 weeks Birth weight (g) Birth weight510th percentile Meconium staining of amniotic fluid 5-minute Apgar 7 NICU admission
Group 1 (n ¼ 25)
Group 2 (n ¼ 15)
Group 3 (n ¼ 18)
1 versus 2
1 versus 3
2 versus 3
39.6 ± 2.1 1 (4%) 0 3287 ± 382 0 3 (12%) 1 (4%) 1 (4%)
38.5 ± 0.9 1 (6.7%) 0 3022 ± 467 1 (6.7%) 2 (13.3%) 1 (6.7%) 1 (6.7%)
37.1 ± 1.6 1 (5.8%) 5 (29.4%) 2906 ± 544 1 (5.8%) 4 (23.5%) 1 (5.8%) 1 (5.8%)
50.001 NS NS NS NS NS NS
50.001 NS 50.001 0.002* NS NS NS NS
0.003 NS 50.001 NS NS NS NS NS
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Data are expressed as number (%), mean ± SD; NS: Non-significant; NICU, Neonatal intensive care unit. *The mean difference is significant at the 0.05 level.
Table 3. Correlation analysis within bile acid levels, IL-17, gestational week at delivery and birth weight in ICP and control groups.
Control Group (n ¼ 25) TBA IL-17 Mild ICP group (n ¼ 15) TBA IL-17 Severe ICP group (n ¼ 18) TBA IL-17
TBA r P r P r p r P r p r p
1 0.136 0.516 1 0.154 0.583 1 0.424 0.009
IL-17
Gestational week at delivery
Birth weight
0.136 0.516 1 -
0.130 0.536 0.240 0.288
0.188 0.369 0.67 0.75
0.154 0.583 1 -
0.99 0.726 0.04 0.989
0.76 0.787 0.237 0.396
0.424 0.009 1 -
0.485 0.049 0.308 0.229
0.600 0.011 0.730 0.781
Pearson correlation is significant at the 0.05 level (two-tailed). ICP, Intrahepatic cholestasis of pregnancy ; TBA, total bile acids.
non-alcoholic steatohepatitis [24], chronic hepatitis B [25], hepatitis C virus infection [26] and autoimmune hepatitis [27]. In addition, elevated levels of IL-17-positive cells have been associated with primary biliary cirrhosis [28]. O’Brien et al. [11] explored the effect of IL-17 on the upregulation of Egr-1 by BAs in a bile duct-ligated mice model, and they suggested that IL-17 might interact with other inflammatory substances and might synergistically enhance the BA-induced production of proinflammatory cytokines by hepatocytes in obstructive cholestasis [11]. In the current study, in order to shed light on the pathogenesis of hepatic inflammation during ICP, we aimed to explore the potential link between maternal serum IL-17 and BA levels in a group of women with ICP and compared the results with those of a group of women with healthy pregnancies. We demonstrated that IL-17 serum levels were significantly higher in the severe ICP group than in the control group. We found a significant negative correlation between TBA level and gestational age at delivery and between TBA level and mean birth weight in the severe ICP group; however, there was no significant correlation between IL-17 and TBA level.
The main limitation of this study is that our sample size was rather small - only 1% of pregnancies are affected by ICP, and we had to exclude several patients who were already on a well-known anti-inflammatory drug, UDCA, at the time of admission to our hospital. Nevertheless, our study is the first of its kind to investigate the relationship between a novel Tcell derived cytokine, IL-17 and intrahepatic cholestasis. We believe that explaining the mechanisms of BA-induced hepatocyte injury will contribute to the invention of novel therapeutic strategies for treating human cholestatic diseases. Therefore, further studies that are more comprehensive and include larger sample sizes are needed to determine the role of IL-17-mediated mechanisms in the etiopathogenesis of ICP.
Acknowledgements The authors thank to Dr Ozgur Kirbas for his secretarial help and his valuable advice in the statistical analysis.
Declaration of interest The authors have no conflicts of interest.
DOI: 10.3109/14767058.2015.1028354
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