International Immunopharmacology 67 (2019) 372–377 Contents lists available at ScienceDirect International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp Association of CCL11, CCL24 and CCL26 with primary biliary cholangitis T Feng Lina,1, Hong Shib,1, Donghong Liuc,1, Zhencheng Zhangc, Wanwan Luoc, Panying Maoc, ⁎ ⁎ Renqian Zhongb, Yan Liangb, , Zaixing Yangc, a Department of General Surgery, Huangyan Hospital of Wenzhou Medical University, Taizhou First People's Hospital, Taizhou, Zhejiang, China b Department of Laboratory Diagnostics, Changzheng Hospital, Second Military Medical University, Shanghai, China c Department of Laboratory Medicine, Huangyan Hospital of Wenzhou Medical University, Taizhou First People's Hospital, Taizhou, Zhejiang, China ARTICLE INFO ABSTRACT Keywords: Background: CCL11, CCL24 and CCL26 are potent chemokines for eosinophils. Since there has been no study CCL11 reporting the association serum CCL11, CCL24 and CCL26 with fibrotic progression of PBC, the aim of this study CCL24 is to explore the association. CCL26 Methods: One hundred and eight PBC patients, 52 patients with chronic hepatitis B (CHB) and 50 healthy Primary biliary cholangitis controls (HC) were recruited. The sera were detected for CCL11, CCL24 and CCL26 using multiplex im- munoassay. Other laboratory indicators were routinely measured. PBC was divided into four stages according to Scheuer's classification. Results: Serum CCL11, CCL24 and CCL26 levels were significantly higher in PBC patients than those with CHB and HC (P < 0.05). The ROC analyses showed that all of the three CCLs performed well for identification of PBC (all P < or =0.001). The multiple linear regression analysis showed an independent relationship of CCL26 with APRI and FIB-4 in PBC patients, but no relationship of CCL11 and CCL24 with fibrotic indicators. Additionally, serum CCL11 and CCL26 were negatively correlated with histological stage of PBC, while serum CCL24 showed no statistical correlation. Conclusion: Serum CCL11, CCL24 and CCL26 are upregulated in PBC. CCL11 and CCL26 are associated with fibrotic progression of PBC, but CCL24 is not. 1. Introduction eosinophil activation/degranulation in PBC patients, further supporting the speculation about the association of eosinophil with PBC [4–6]. Primary biliary cholangitis (PBC) is a chronic autoimmune liver However, the mechanism by which eosinophil was recruited into portal disease of unknown cause, characterized by the inflammatory de- tract in PBC has remained challenging. struction of intrahepatic bile ducts, affecting predominantly middle- Eotaxins, including C-C motif chemokine ligand 11 (CCL11) (eo- aged women. If untreated, the disease may progress toward cirrhosis, taxin), CCL24 (eotaxin-2) and CCL26 (eotaxin-3), belong to the family liver failure and even liver cancer. Although anti-mitochondrial anti- of CC chemokines. These three eotaxins, as potent chemoattractants for bodies (AMAs) are key diagnostic markers of PBC, with a prevalence of eosinophils, have been reported to be increased in some allergic dis- 90% or so for PBC, they have no ability to identify the severity and eases, such as allergic asthma, allergic rhinitis, and atopic dermatitis progression of PBC [1]. Therefore, up to date, the search for new serum [7–9]. Additionally, the increased CCL26 is also correlated with bullous markers associated with disease severity and progression has been a hot pemphigoid [10], eosinophilic granulomatosis with polyangiitis [11] spot in PBC study field. and eosinophilic esophagitis [12]. A recent study found the decreased Although the pathogenesis of PBC has remained elusive, several serum CCL11 levels and increased CCL26 in PBC patients [13], but the studies showed a large number of eosinophils infiltrating in portal tract association of these two eotaxins with staging and fibrosis progression of liver in PBC patients, suggesting that eosinophils may play an im- of PBC has remained unclear. portant role in PBC [2–4]. Furthermore, some studies indicated that In the present study, we determined serum CCL11, CCL24 and treatment of ursodeoxycholic Acid (UDCA), one of the most important CCL26 levels and explored whether or not they were associated with drugs for PBC treatment, can ameliorate eosinophilia and inhibit the fibrotic progression of PBC which is divided into four histological ⁎ Corresponding authors. E-mail addresses: [email protected] (Y. Liang), [email protected] (Z. Yang). 1 Feng Lin, Hong Shi and Donghong Liu contributed equally to this work. https://doi.org/10.1016/j.intimp.2018.12.026 Received 30 May 2018; Received in revised form 15 November 2018; Accepted 11 December 2018 Available online 22 December 2018 1567-5769/ © 2018 Published by Elsevier B.V. F. Lin et al. International Immunopharmacology 67 (2019) 372–377 Table 1 immunoassay according to manufacturer's instructions (MSD SCALE Clinical, laboratory, and histological characteristics of PBC patients. DISCOVERY, Rockville, MD). Other laboratory indicators were routi- fl Characteristics PBC patients CHB patients HC nely measured. Brie y, Antinuclear antibodies (ANA) and AMA were detected by indirect immunofluorescence (IIF) (EUROPIN, Germany), n Results n Results n Results and anti-centromere, gp210 and sp100 antibodies by immunoblot assay (EUROPIN, Germany). Blood routine testing was performed in Sysmex Age (years) 108 59 ± 11 52 52 ± 9 50 55 ± 7 Female, n (%) 108 96 (89) 52 41 (79) 50 44 (88) XE-2100 analyzer (Sysmex Corporation, Kobe, Japan). Alanine amino- WBC count (109/L) 106 3.95 (2.50–5.30) / / / / transferase (ALT), Aspartate aminotransferase (AST), alkaline phos- Neutrophil (109/L) 106 2.01 (1.38–3.02) / / / / phatase (ALP), gamma-glutamyltranspeptidase (GGT) and total bilir- 9 Lymphocyte (10 /L) 106 1.21 (0.80–1.74) / / / / ubin were measured using the Cobas P800 analyzer (Roche diagnostic 9 – Eosinophil (10 /L) 106 0.08 (0.05 0.14) system). Platelet (109/L) 106 121 (65–171) / / / / NLR 106 1.71 (1.23–2.36) / / / / PLR 106 105.06 //// 2.3. Calculation of indirect laboratory indicators (71.55–133.33) – Bilirubin (mg/dl) 108 19 (13 45) / / / / The indirect laboratory indicators included Neutrophil- (NLR) and ALT (IU/L) 108 45 (24–88) / / / / AST (IU/L) 108 59 (36–103) / / / / platelet-lymphocyte (PLR), AST to ALT ratio, AST to platelet ratio index ALP (IU/L) 108 179 (122–298) / / / / (APRI) calculated by Wai's formula: (AST/upper limit of normal) / GGT (IU/L) 108 127 (64–283) / / / / platelet count (109/L) × 100 [15], and FIB-4 index calculated by AST/ALT ratio 108 1.32 (0.91–1.83) / / / / Sterling's formula as: age (years) × AST (IU/L) / platelet count (×109/ – APRI 106 1.45 (0.70 2.55) / / / / L) × √ALT (IU/L) [16]. FIB-4 index 106 4.16 (2.68–9.99) / / / / AMA positive, n (%) 108 97 (90) / / / / ANA positive, n (%) 108 78 (72) / / / / 2.4. Histological staging Anti-centromere 108 30 (28) / / / / positive, n (%) PBC is divided into four histological stages in accordance with Anti-gp210 positive, 108 35 (32) / / / / fi fl n (%) Scheuer's classi cation [17]. In Stage 1, in ammatory damage is loca- Anti-sp100 positive, 108 15 (14) / / / / lized to the portal triads. In stage 2 shows the damage extending be- n (%) yond the portal triads into the surrounding parenchyma and the loss of Scheuer's stage 78 18:15:25:20 / / / / some bile ducts. Stage 3 shows the damage characterized by fibrous (I:II:III:IV) septa linking adjacent portal triads. In stage 4, cirrhosis is clear. Age is expressed as mean ± SD, while other continuous variables as median and interquartile range. Categorical variables were expressed as total number 2.5. Statistical analysis (%). WBC, white blood cell; NLR, neutrophil to lymphocyte ratio; PLR, platelet to Statistical analysis was performed by SPSS 17.0 software. lymphocyte ratio; ALT, alanine aminotransferase; AST, aspartate amino- Mean ± standard deviation (SD) and median (interquartile range transferase; ALP, alkaline phosphatase; GGT, gamma-glutamyltransferase; (IQR)) were used to describe normal-distributed and skewed variables, APRI, AST to platelet ratio index; FIB-4, fibrosis index based on the 4 factors; respectively. All the continuous variables were compared by Student's t- AMA, anti-mitochondrial antibody; ANA, antinuclear antibody; PBC, primary test or Mann–Whitney U test when appropriate. Receiver operating biliary cholangitis. characteristic (ROC) curves were plotted and the areas under the curves (AUC) were calculated for assessing the diagnostic value of CCL11, stages in accordance with internationally recognized Scheuer's classi- CCL24 or CCL26 for PBC. Spearman's correlation coefficient was used to fi cation. evaluate the correlation between CCL11, CCL24 or CCL26 and other continuous variables. The multiple linear regression analysis was used 2. Patients and methods to assess the independent relationships of CCL11 and CCL26 with other variables. Results were considered significant if the two-tailed P-value 2.1. Subjects was < 0.05. We recruited 108 treat-naïve patients from Changzheng Hospital 3. Results between January 2009 and October 2018. All of them fulfilled the criteria from the American Association for the Study of Liver Diseases 3.1. The comparisons of serum CCL11, CCL24 and CCL26 levels between for PBC [14]. Of those, 78 patients received liver biopsy. The exclusion different populations criteria included primary sclerosing cholangitis, autoimmune hepatitis, other focal intrahepatic or extrahepatic lesions or obstruction, any The median levels of serum CCL11, CCL24 and