Elsevier

Journal of Autoimmunity

Volume 36, Issue 1, February 2011, Pages 76-86
Journal of Autoimmunity

Cell-specific expression of TLR9 isoforms in inflammation

https://doi.org/10.1016/j.jaut.2010.11.001Get rights and content

Abstract

Toll-like receptors (TLRs) are key pattern recognition receptors during an immune response. With five isoforms of human TLR9 described, we hypothesised that differential expression of TLR9 isoforms in different cell types would result in variable contributions to the overall input from TLR9 during inflammation. We assessed the molecular expression of the TLR9 isoforms, TLR9-A, -C and -D. In normal peripheral blood mononuclear cells, B-lymphocytes express ∼100-fold more TLR9-A transcript than monocytes or T-lymphocytes, which predominantly express the TLR9-C transcript. Switches in isoform predominance accompany B-lymphocyte development. TLR9 protein expression in rheumatoid inflammatory lesions reflected the TLR9 isoform expression by immune cells. Herein we suggest that B-lymphocytes and plasmacytoid dendritic cells contribute the ∼3-fold higher TLR9-A transcript levels observed in inflamed synovium when compared to subcutaneous rheumatoid nodules. In contrast, macrophages and T-lymphocytes contribute the ∼4-fold higher TLR9-C transcript levels seen in nodules, compared to synovia. From protein sequence, predictions of subcellular localisation suggest TLR9-B may locate to the mitochondria, whereas TLR9-D adopts an opposing orientation in the endoplasmic reticulum. Consistent with this, structure models raise the possibility of alternative ligands for the TLR9-B and TLR9-D variants. Our results highlight differences in the expression of human TLR9 isoforms in normal and inflamed tissues, with differing contributions to inflammation.

Introduction

Toll-like receptors (TLRs) are key innate immune receptors, which mediate defense against pathogens. To date, 10 members of the TLR family (TLR1–TLR10) have been reported in humans. TLRs recognise a range of pathogen-associated peptides or nucleic acids. Amongst these, TLR9 recognises hypomethylated CpG motifs in pathogen DNA. Synthetic CpG oligodeoxynucleotides (CpG-ODNs) that mimic these pathogen-derived CpG motifs stimulate innate and/or adaptive immune responses with outcomes depending on the cell type involved and features of the CpG-ODN.

Human B-lymphocytes and plasmacytoid dendritic cells (pDCs) are the primary target cells for CpG-ODNs. Stimulation is dependent on the internalisation of CpG-ODNs and translocation of TLR9 from the endoplasmic reticulum (ER) to endolysosomes [1]. Different CpG-ODN types have been described, based on the response elicited by these two cell types [2]. CpG-A/D stimulates strong production of type I interferons (IFN) by pDC, while weakly stimulating B-lymphocytes. In contrast, CpG-B/K induces weak pDC IFN production, but stimulates B-lymphocyte activation and proliferation. The effects of CpG-C are highly stimulatory for both B-lymphocytes and pDCs. Other cell types including monocytes, T-lymphocytes and natural killer cells also respond to CpG-ODN stimulation with pro-inflammatory cytokine production, and cytotoxic T-lymphocyte differentiation [3], [4], [5].

Synergy between B-cell receptor (BCR) and TLR9 signalling in B-lymphocytes contributes to the initiation and persistence of autoimmune responses in a number of disease settings. Activation of autoreactive B-lymphocytes by CpG-ODNs enables these cells to avoid negative selection [6]. One consequence is the production of (pathogenic) auto-antibodies that is amplified by the synergy between BCR and TLR9 signalling [7]. In patients with RA, autoreactive B-lymphocytes make a substantial contribution to the inflammation [8]. Depletion of these B-lymphocytes using anti-CD20 therapy results in clinical improvement in RA [9] and has similar outcomes in other disorders where B-lymphocytes and TLR9 make a contribution, including systemic lupus erythematosus, Sjogren’s syndrome, graft vs host disease, and multiple sclerosis (reviewed by [10]). Furthermore the combination of anti-CD20 therapy with CpG-ODNs as adjuvant leads to a greater depletion of B-lymphocytes than anti-CD20 alone [11]. Taken together these data provide strong evidence that TLR9 activation of B-lymphocytes is important to the pathogenesis of a number of autoimmune diseases.

The human TLR9 gene has five isoforms generated by alternative splicing of transcript. The first report of human TLR9-A and TLR9-B isoforms came from cloning of the TLR9 gene from monocytic THP-1 cells in 2000 [12]. At that time TLR9, TLR9.0, TLR9.1 and TLR9.2 were also cloned from human placenta, and later renamed TLR9-A, TLR9-C, TLR9-D and TLR9-E respectively [13]. In the intervening period, there has been no extensive investigation into the expression of human TLR9 isoforms. While multiple TLR9 isoforms have not been reported in rodents, they are present in other species [14], [15] where they show differential patterns of expression among tissues and developmental stages, as well as following infection.

In the assessment of human TLR9 gene expression, few studies consider the different TLR9 isoforms. Most assess TLR9 gene expression based on the detection of exon 2 that is common to all known isoforms (total TLR9 expression). A large percentage of human genes undergo alternative splicing at the transcript level [16], [17] with functional consequences for the encoded protein variants. Amongst TLRs, there is evidence that one of the alternatively spliced TLR4 variants adopts an inhibitory role [18]. Consequently alternative splicing of TLR9 has the capacity to impact on function through differential expression of isoforms by different cell types in different inflammatory settings.

The aim of this study was to examine the expression of human TLR9 isoforms (TLR9-A, -C and -D) and establish any differences associated with inflammation. Initial investigation considered normal peripheral blood mononuclear cells (PBMCs) and immune cell-rich tissues. Consequently the expression of TLR9 isoforms was assessed in different rheumatoid inflammatory lesions where the cellular composition of the infiltrate also differs [19], [20], [21]. These tissues provide a model in which to investigate differential expression of the various TLR9 isoforms and to assess their contribution to different forms of inflammation, in this case inflammation of the joint lining and rheumatoid subcutaneous granulomas.

Section snippets

Patients and tissues

Fifteen joint synovia and 15 rheumatoid subcutaneous nodules (granulomas) were obtained from 24 different RA patients who fulfilled the American College of Rheumatology diagnostic criteria for RA [22]. Synovial tissue was obtained during joint replacement surgery and nodules following elective surgery. Details of all patients are shown in Table 1. Ethics approval was obtained from the Multi-region Ethics Committee, New Zealand. Human tonsil was used as the positive control in all experiments.

Genomic origin of human TLR9-C and TLR9-D isoforms

Five variants of human TLR9 protein have been described [12], [13]. These are illustrated in Fig. 1A. TLR9-A provides the canonical protein sequence. In addition, there are two alternatively spliced variants, TLR9-C and TLR9-D, that include an additional 23 or 15 amino acids (aa) respectively at their N-terminal, and two truncated variants, TLR9-B and TLR9-E, lacking 42 and 15 aa respectively. A 975 aa region is common to all TLR9 protein variants. Consequently, at a molecular level only

Discussion

A key molecule in the B-lymphocyte armory is TLR9, a dsDNA receptor that plays a fundamental role in innate immunity and appears of increasing importance in a number of autoimmune diseases. The principal objective of this study was to determine if differential expression of TLR9 isoforms occurs according to different cell types and between different tissues that might mediate differences in inflammatory responses. We were particularly interested in variable expression of TLR9 isoforms between

Role of funding source

This work was supported in part by Arthritis New Zealand and by the Health Research Council of New Zealand; KMcK was the recipient of a Top Achiever Doctoral Scholarship from the Tertiary Education Commission, New Zealand.

Disclosure statement

The authors have no actual or potential conflict of interest to disclose.

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