The handle
http://hdl.handle.net/1887/136520
holds various files of this Leiden University
dissertation.
Author: Hafkenscheid, L.
Title: Anti Citrullinated Protein Antibodies-IgG variable domain glycosylation in
rheumatoid arthritis
Rheumatoid arthritis:
Rheumatoid arthritis (RA) is an inflammatory autoimmune disease. RA is the most common form of arthritis, affecting around 0.5% till 1% of the population (1). RA is found three times more frequent in females than in men, although the ratio can be variable among different population groups and ages (2, 3). This disease primarily affects the small joints of the hands and feet (1), but can also lead to inflammation and destruction in all other synovial joints. In RA, the joints can be inflamed, swollen and painful which, when left untreated, leads to joint destruction and deformities. RA is a systemic autoimmune disease and with other manifestations as some other autoimmune diseases where only one specific tissue is affected. RA is a complex disease where patients can also develop several systemic complications such as rheumatoid nodules, interstitial lung disease and vasculitis. The most prominent inflammation however takes place in the synovium; a thin layer of cells lining the inside of the joint(4). Synovial tissue surrounds, as a fibrous capsel, the joint and creates a membrane that is attached to the bone-cartilage interface. The synovium consists of two layers, the first layer is the synovial intima and is composed of synovial lining cells (SLC) and it has a thickness of about three cell layers. The second layer, the sub lining, contains blood vessels, lymphatic vessels and nerves. The synovium in a RA patient undergoes major changes. First the synovial tissue is thickened to 10 to 20 cell layers as a consequence of hyperplasia due to the chronic inflammatory process. Secondly, the lining layer becomes infiltrated by a variety of immune cells such as T lymphocytes, dendritic cells, macrophages and B cells that are likely contributing to inflammation and chronicity (5). Finally, a proliferation of blood vessels is observed probably by the presence of angiogenetic factors in the inflamed, hypoxic environment. This histological change of the synovium is leading to pannus tissue and characteristic for RA (6).
Classification and diagnosis of Rheumatoid Arthritis:
Auto-antibodies:
RA is hallmarked by the presence of autoantibodies and, as mentioned, their presence is part of the 2010 ACR/EULAR classification criteria for RA. Rheumatoid factor is the most commonly used test and one of the first tests used to aid diagnosing RA (2). Rheumatoid factors are auto-antibodies targeting the Fc-region of IgG antibodies, and can found in different immunoglobulin isotypes such as IgG-RF and IgA-RF. The most frequently detected isotype however is IgM-RF. Although RF is used as a standard disease marker in RA, it is only detected in 60-70% of the RA patients. In addition, it is also detected in other inflammatory diseases like systemic lupus (SLE) and sometimes even healthy individuals. Nevertheless, RF is a good predictor of clinical disease activity and joint damage (8-10). In 1964, it was first shown that RA patients also harbor other antibodies that were named anti-perinuclear factor or anti-keratin antibodies (11, 12). In the late 1990s, these were found to be directed against citrullinated proteins (11-13). Citrullination is a post translation modification were a positively charged arginine is converted to a neutrally charged citrulline by the enzyme Peptidyl Arginine Deiminase (PAD) (14). The antibodies against citrullinated proteins are now commonly known as anti-citrullinated protein autoantibodies, shortly, ACPA (15, 16). These autoantibodies have been shown to be highly specific for the disease and are now used in clinical routine as well. The ACPA test has a higher specificity than the IgM-RF and a sensitivity of around 50% till 80% depending on the cohort analyzed. The presence of ACPA can also predict different clinical outcomes such as the progression of joint destruction in RA (17, 18).
Figure 1: 2010 ACR/EULAR Classification criteria for RA (7).
Pathogenicity of ACPA in RA:
Whether an anti-citrulline response can be induced in mouse is debated (19-22). However, it was reported that infusion of ACPA resulted in long-lasting pain-like behavior in mouse without signs of inflammation. It was suggested that this was coupled to the activation of osteoclast that releases the chemokine CXCL1 (analogue to the human IL-8) that was causing pain sensation in mice (23), although recent retractions and notes of caution indicate that these notions are probably incorrect (24, 25). In humans, ACPA have been implicated in the facilitation of pro-inflammatory effects as well; one of the first observations of the pro-inflammatory behavior of ACPA was observed on macrophages that were stimulated with ACPA. The macrophages showed a skewed phenotype toward M1 macrophages able to secrete pro-inflammatory cytokines once exposed to ACPA (26). ACPA were also found to be able to activate the complement system and platelets (27, 28). Furthermore, purified ACPA-IgG and not non-ACPA-IgG are described to induce a variety of other cells as wells, including mast cells and neutrophils (29, 30). Recently, it was also reported that ACPA-IgG, but not control IgG, could induce NET formation by neutrophils (31). These data, together with the notion that the presence of ACPA is associated with more severe disease is suggesting that ACPA is more than “just” a biomarker but conceivably also directly involved in mediating inflammation.
Risk factors for RA:
Both environmental risks as well as genetics risk factors are described to contribute to RA development. The best-known environmental risk is smoking and is estimated to account for 20-30% of the environmental risk for RA development. Interestingly, this risk is predominantly associated with auto-antibody-positive disease (32). Some studies have suggested that exposure to smoke can lead to increased citrullination in the exposed tissues (33-35). More specifically, smoking has been suggested to contribute to ACPA-positive RA by increasing citrullinated in exposed tissues such as lung through the induction of low-grade inflammation, leading to the activation of neutrophils and the release of PADs followed by the formation of extra-cellular citrullinated proteins (36). However, as it is now clear that smoking does not predispose specifically to the formation of ACPA, the precise biological underpinnings explaining the connection between smoking and RA remains an unresolved issue. In addition, to smoking also microbes such as
Porphyromonas Gingivalis and Aggregatibacter Actinomycetemcomitans are proposed to
shown to express a toxin called leukotoxin A. This toxin is able to induce citrullination in neutrophils, thereby creating potential autoantigens relevant for ACPA-positive RA (37). Nevertheless, no direct relation between the presence of anti-LtxA antibodies as proxy for the presence of Aggregatibacter Actinomycetemcomitans and RA was observed (42). Next to the environmental risks also genetics can play a role in the development of RA. For example, the genetic risk was investigated by analyzing the genetic variants in large populations of RA-patients and controls by “Genome Wide Association Studies” (GWAS). By now, 101 risk alleles have been identified that are thought to be involved in different molecular pathways. The molecular pathway enrichments analysis revealed pathways that involve the activation of T cells, B cells and cytokines signaling pathways (43). However, the strongest genetic risk factor that is associated with ACPA positive disease is located within the HLA class II gene locus. It is estimated that this locus account for 20%-30% of the genetic variation in RA. Intriguingly, this locus and many other genetic risk factors for RA mainly predispose to ACPA-positive disease and not, or far less, to seronegative RA. (44, 45). The strongest association reported is found with the HLA-DRB1 alleles that share a common amino acid sequence (46), the shared epitope, and hence are called the shared epitopes (SE) alleles (47-50). It is hypothesized that the SE alleles, contain a positively charged pocket favoring the binding of citrulline over an arginine-residue (51), although this does not seem to apply for all HLA-SE-alleles (52) .
The two-hit model for ACPA development:
As mentioned above, the HLA SE alleles are associated with ACPA-positive disease. Intriguingly, there is the HLA SE alleles do not associate with the presence of ACPA in health (53, 54). Therefore, it is believed that the development of ACPA-positive disease is a multistep process, figure 2 (55, 56). The “first hit” may drive the initial break of tolerance by environmental and/or stochastic events which leads to the production of ACPA. This process is supposedly independent from the presence of HLA-SE-alleles. The ACPA production that precedes the onset of RA can be present years before disease onset without signs of clinical symptoms (57). Upon a certain trigger, the so-called “second hit” the citrullinated protein-directed B cells will receive T cell help which leads to the maturation of the ACPA response. Current immunogenic evidence indicates that the HLA-SE-alleles primarily associate with the “second hit”. The HLA association strengthens the idea that T cell help is involved in this process (54). Indeed, before the disease onset, epitope spreading of the ACPA-response is observed, as well as isotype switching and increase in ACPA-titer, all indicators for the presences of T cell help (58).
Figure 2: The Two Hit hypothesis of ACPA-positive disease development. IgG structure and Fc-glycosylation
Figure 3: Schematic representation of IgG (on the left) and N-linked glycan structure
(right side).
ACPA-IgG glycosylation:
As indicated, the Fc-glycosylation can change or is altered during inflammatory conditions. By now, it is firmly established that lower galactosylation-levels in the IgG-Fc is associated with disease activity in RA (65). More specifically, we and others have shown that ACPA-IgG harbour lower levels of galactosylation and sialylation but higher fucosylation-levels compared to conventional IgG (66, 67). Furthermore, the changes in Fc glycosylation that associate with inflammation are more pronounced for ACPA-IgG isolated from the synovial fluid, the site of inflammation, as compared to ACPA isolated from serum. In addition, lower galactosylation and sialylation of the ACPA-Fc-IgG can already be detected a few months before disease onset (68). However, next to the Fc-glycosylation, about 15-20% of the antibodies carry V-domain glycans (69). Intriguingly, we observed recently that ACPA-IgG is extensively glycosylated in the Fab region (70).
Thesis outline:
The aim of this thesis is to provide a better understanding and characterization of ACPA-IgG V-region glycosylation in the context of RA. To do so, an overview is provided in
chapter 2 of what is known about V-domain glycosylation. More specifically, the chapter
describes which type of glycans have been identified on V-domains of antibodies in different situations. Likewise, an overview is given how the V-domain glycans could be involved in health and disease and the possible different effector functions (69). With this background, in chapter 3 studies are described aiming to characterize the type of glycans present in the Fab region of ACPA-IgG as well as the extent to which they differ from the glycans present on the V-domain on conventional IgG. The studies presented in this chapter show that ACPA-IgG is over 90% V-domain glycosylated compared to only 17% found on conventional IgG. In addition, the glycans found on the Fab-region of ACPA-IgG are “hypersialylated” compared the V-domain glycan of conventional IgG (71). The studies described in chapter 4 focus on the sequencing of ACPA-B cell receptors
(BCRs). In this study, it was determined whether the glycosylation-sites present in ACPA are introduced upon somatic hypermutations (SHM) that are typically occurring during a germinal center reaction or are rather derived from expansion of BCRs that harbor a germ-line encoded N-linked glycosylation-site. Furthermore, it is described that the ACPA-IgG had undergone more extensive SHM compared to anti-tetanus IgG. However, as the level of glycosylation-site introduction did not coincidence with the high mutation rate, the results presented in this chapter indicate that there is a selecting pressure for introducing a glycosylation site in ACPA-expressing B-cells. Additionally, in the context of this study, also the glycosylation-sites were modelled to study the location of the glycan sites in the V-region of ACPA-IgG. This revealed that the glycosylation sites of ACPA are more at the exterior of the antibody (72). In a preparation for the study described in
chapter 6, we developed a bioinformatics tool named “HappyTools”. This tool allowed us
to analyze quickly a considerable number of chromatograms produced by the Ultra High-Performance Liquid Chromatography (UHPLC) in a high throughput manner. The study is presented in chapter 5. Chapter 6 describes a high throughput method to analyze ACPA-IgG glycosylation using a microbeads assay. In this study, we analyzed glycans released from ACPA-IgG of healthy ACPA positive individuals that transitioned to RA or not by UHPLC chromatograms. We provide a new view on “the second hit” hypothesis by studying the V-domain glycosylation of ACPA-IgG. In addition, we found that the ACPA-IgG V-domain glycosylation could function as a predictive marker in ACPA-positive individuals at risk. The study presented in chapter 7 is addressing the possible functions of the V-domain glycans in relation to antigen binding. ACPA-IgG is of low avidity and we hypothesize that the glycans can hinder antigen binding. For this study, monoclonal ACPA with- or without V-domain glycans were used to address the role of these glycans in antigen binding. Besides the question if the glycans can influence antigen binding, it was also examined whether there was a role for sialic acid in this context. Finally, in
chapter 8 a summary and discussion of the implications of the findings are presented
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