Genetic polymorphisms of toll-like receptor 9 influence the immune response to CpG and contribute to hyper-IgM in primary biliary cirrhosis☆

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Abstract

The serum hallmark of primary biliary cirrhosis (PBC) is the presence of anti-mitochondrial antibodies (AMA), found in 95% of patients. However, nearly every patient with PBC, including those who are AMA-negative, has an elevation in serum IgM. This hyper-IgM is neither representative of other Ig isoforms, nor is due to the levels of AMA. In fact, we have recently reported that the hyper-IgM is an innate immune response and can be induced with CpG-B with concurrent up-regulation of toll-like receptor 9 (TLR9). Based on these observations, we performed a two-tier study. First, we quantitated TLR9 genotypes in patients with PBC and controls and correlated these data with the B cell response to CpG-B. Second, based on these data, we performed an extensive TLR9 genotyping in a large cohort of patients and controls. We report herein that the 2848 AA TLR9 genotype is associated with enhanced gene expression and higher frequency of intracellular IgM+ B cells following CpG stimulation. Interestingly, however, despite the functional association, there is no difference in the distribution of TLR9 genotypes between patients and controls. Our data emphasize the importance of dissecting the innate immune response in PBC.

Introduction

Primary biliary cirrhosis (PBC) is an autoimmune liver disease of unknown etiology characterized by chronic non-suppurative destructive cholangitis that eventually leads to cirrhosis and liver failure [1]. High-titer serum anti-mitochondrial antibodies (AMA) and elevated levels of IgM (hyper-IgM) are common in PBC [2], [3], with AMA detected in 90% of affected individuals [4]. The causes of hyper-IgM in PBC are still poorly understood. Similar to other autoimmune diseases, molecular mimicry by microorganisms, in particular bacteria, has been proposed as the mechanism leading to the breakdown of tolerance in PBC [5]. We have recently reported that hyper-IgM could be the result of a chronic polyclonal innate immune response to bacterial stimuli represented by unmethylated CpG motifs that share immunostimulatory effects in humans [6]. Following stimulation with synthetic oligodeoxynucleotides containing such motifs, cultured peripheral blood mononuclear cells (PBMC) from patients with PBC produced higher amount of IgM compared to controls. Moreover, we also demonstrated that the expression level of toll-like receptor 9 (TLR9), specifically binding CpG motifs [7], was increased after CpG stimulation and directly correlated with IgM production [6]. These observations prompted us to investigate whether TLR9 polymorphisms could determine the levels of gene expression following CpG stimulation, similar to that reported for TLR4 in lipopolysaccharide (LPS) responsiveness [8]. Although several TLR9 haplotypes have been described, the genotyping of −1237 T/C and 2848 G/A single nucleotide polymorphisms (SNPs) represents all variations [9]. We herein report the correlation of TLR9 polymorphisms with gene expression levels and frequency of IgM-producing B cells following CpG stimulation. We also describe the allelic frequencies of the TLR9 2848 G/A SNP in a large series of patients with PBC and controls.

Section snippets

Subjects

First, peripheral blood samples were obtained from 20 patients with PBC (median age 59 years, range 37–71) and 15 healthy controls (62 years, 34–71). All sera from patients with PBC tested positive for AMA using recombinant mitochondrial antigens [10]. Eight (40%) patients with PBC had signs of fibrosis or cirrhosis at histology (stages III and IV according to Ludwig [11]). Total serum IgM levels were measured using the Immuno-Tek human IgM EIA Kit (ZeptoMetrix Corporation, Buffalo, NY). Hence,

Intracellular-IgM positive B cells and TLR9 intensity in B cells following CpG

PBMC from 20 PBC patients and 15 healthy controls were co-stained for both intracellular IgM and TLR9 after culturing for 4 days in presence or absence of CpG. Intracellular IgM+ B cells were virtually non-detectable in cultures without CpG. In the presence of CpG, the percentage of intracellular IgM+ B cells in PBC patients was significantly higher than controls (14.9% vs. 9.4%, P = 0.002) (Fig. 1). The TLR9 staining intensity in B cells was lower in PBC compared to controls when the cells were

Discussion

Unmethylated CpG motifs are prevalent in bacterial DNA and have been shown to activate host defense mechanisms leading to both innate and acquired immune responses [12]. We have previously used CpG as a tool to establish a model of innate immune response to bacterial molecules, and our data suggested that hyper-IgM in PBC patients could be caused by chronic bacterial exposure [6]. Such previous data, however, did not account for potential genetic mechanisms underlying the response to the CpG

Acknowledgements

The authors are grateful to Mrs. Marcy Crees for helping in the collection of samples and Dr. Yasunori Ichiki for the helpful contribution and fruitful discussion.

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    ☆

    This work was supported in part by NIH grants DK39588 and DK92310.

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