Correlation of the Expression Profile of Peripheral Leukocyte and Liver Tissue Immune Markers With Serum Liver Injury Indices in Children With Biliary Atresia

TL;DR

This study explores how immune markers in blood and liver tissues relate to liver injury in children with biliary atresia.

Contribution

The paper identifies complex immune correlations in biliary atresia children that are not unique to their liver injury profile.

Findings

BA patients show increased frequencies of specific immune cells and altered gene expression in blood and liver.

Strong correlations exist between liver injury markers and immune characteristics like Th17.1 cells and MMPs/TIMPs.

No single immune marker uniquely distinguishes BA from other liver inflammatory diseases.

Abstract

The aim of the study was to find associations between the levels of liver injury serum markers and the selected liver, peripheral leukocytes, and plasma immune characteristics in biliary atresia (BA) children. Twenty-five newly diagnosed BA children aged 4–30 weeks and 12 age-matched controls were included (for leukocytes characteristics) and 19 BA children and 11 controls (for liver studies). The frequencies of T helper 1 (Th1), Th2, Th17, Th17.1 cells as well as numbers of regulatory T (Treg), B cell subsets, and matrix metalloproteinase −2 and −9 (MMP-2 and MMP-9) expressing leukocytes in the whole blood were evaluated by flow cytometry. Plasma concentrations of tissue inhibitors of metalloproteinase (TIMP)−1, −2, MMP-9, interleukin-17A (IL-17A) and IL-6 were assessed by enzyme-linked immunosorbent assay (ELISA). The leukocyte and liver expression of the retinoic acid receptor-related orphan nuclear receptor gamma (RORγT), fork-head winged helix transcription factor P3 (FoxP3), transforming growth factor beta (TGF-β), interleukin-17A (IL-17A), IL-6, IL-1β, IL-21, interleukin 1 receptor antagonist (IL-1Ra), MMP-2, MMP-9, MMP-12 (liver only), TIMP-1, TIMP-2, T-box transcription factor expressed in T cells, also called TBX21 (T-bet), GATA-binding protein 3 (GATA3), and C-type lectin (CD161) mRNA were determined by real time RT-PCR (reverse-transcription polymerase chain reaction). The BA patients were characterized by increased frequencies of peripheral “suppressor” glycoprotein-A repetitions predominant protein (GARP)+latency-associated peptide (LAP)+Treg and activated Treg cells as well as MMP-2 and MMP-9 bearing lymphocytes, elevated plasma TIMP-1 levels, increased leukocyte expression of MMP-9, TIMP-1, TIMP-2, IL-6, and TGF-β, and decreased leukocyte expression of IL-21 and T-bet, increased liver expression of FoxP3, TIMP-1, and decreased liver expression of IL-1β and MMP-2. The following correlations were found between serum markers of liver injury and leukocyte and liver immune characteristics: (a) hemoglobin (Hb) levels correlated negatively with frequency of peripheral “suppressor” GARP+LAP+ Tregs; (b) aspartate aminotransferase (AST) levels correlated positively with frequency of the peripheral Th17.1 subset and expression of leukocyte FoxP3, (c) gamma glutamyltransferase (GGT) levels correlated positively with the peripheral memory B cells frequencies, the leukocyte IL-6 and TIMP-1 gene expression, (d) alanine aminotransferase (ALT) serum levels correlated positively with the naïve B cell frequency and liver TIMP-2 expression, (e) total bilirubin (Bil) levels correlated positively with the leukocyte MMP-9, the plasma IL-6 levels, and the liver TIMP-2 gene expression, (f) direct Bil levels positively correlated with the liver IL-6 and TIMP-2 expression, (g) international normalized ratio of prothrombin time (PT/INR) concentrations correlated positively with the peripheral Th17.1 subset frequency and the leukocyte MMP-9 but negatively with the liver FoxP3 expression. There were numerous strong positive correlations between the BA liver genes known to be involved in upregulation of IL-17 axis and MMPs/TIMPs expression. No prevailing leukocyte or liver single markers were uniquely associated with serum liver injury indices. BA immune profile is very complex with no single characteristics that would distinguish it from other liver inflammatory diseases.