Autoimmune correlation of rheumatoid arthritis and periodontitis.

Rheumatoid arthritis and periodontitis, both, chronic inflammatory diseases share certain common diagnostic, pathological, immunogenetic and therapeutic features. A recently discovered enzymatic mimicry between human and bacterial species is novel and it opens up a new terrain for therapeutic blockade in the management of rheumatoid arthritis.

INTRODUCTION

Periodontitis, a chronic inflammatory disease of infectious origin, is one of the most prevalent oral diseases. The severity of the disease depends upon the various parameters like the virulence of microorganisms, plaque levels, genetic and environmental risk factors, but most of all is the host immune response elicited towards the disease. Although infectious in nature, the pathogenesis is similar to other inflammatory diseases in its progression. Over the past few decades, the oral-systemic link has become stronger. Periodontitis has been shown to have a causative or modulating role in diabetes, heart disease, respiratory disorders and adverse pregnancy outcomes, and recently, a number of studies have shown a potential relationship between periodontitis and rheumatoid arthritis (RA). RA, an autoimmune disease is characterized by chronic inflammation of joints, bone destruction, and damage to adjacent soft tissues. The prevalence of rheumatoid arthritis in the adult Indian population is 0.75%,[1] which projected to the whole population, would give a total of about seven million patients in India. Both seem to share similar immunopathogenic profile, with activation of immune cells, production of inflammatory mediators like interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), as well as matrix metalloproteinases (MMP's), resulting in tissue damage. This cross susceptibility and remarkable similarities between the two provide useful insights in to these diseases.

Causal or casual association between rheumatoid arthritis and periodontitis?

The etiological association between the two has been shown by many epidieomological studies. It has been reported that patients with longstanding active RA have a significantly increased incidence of periodontitis when compared with healthy subjects[2] and that patients with periodontitis have a higher prevalence of RA than patients without Periodontitis.[3] A recent study evaluated patients with RA for the prevalence and severity of periodontitis and their relationship to RA disease activity and severity with patients with osteoarthritis (OA) serving as controls. Periodontitis was more common and severe in patients with RA when compared with patients with OA.[4]

GENETIC LINK

The relationship between human leukocyte antigens (HLA) determinants and periodontitis is well studied. Examination of the DRB1 * 04 alleles that code HLA-DR4 in patients with rapidly progressive periodontitis (RPP), showed a significantly higher frequency (42%) of one of the DRB1 subtypes than the control group (7%).[5] These DRB1 subtypes are part of the so-called shared epitope genotypes, which also play a role in other inflammatory diseases like RA. Hitchon and colleagues[6] reported an association between immune responses to the oral pathogen Porphyromonas gingivalis (P. gingivalis) and the presence of anti-citrullinated protein antibodies (ACPA) in a population with a high background prevalence of RA predisposing HLA-DRB1 alleles. This gene environment interaction may result in breaking self-tolerance to citrullinated antigens or amplification of these autoimmune responses or both and may ultimately lead to the development of RA.

SUPERANTIGENS

Superantigens are proteins that bind simultaneously to V beta (VβV) chain of the T-cell receptors (TCRs) and α chain of the major histocompatibility complex II molecules. They induce T-cell activation and proliferation by binding the TCR and MHC II simultaneously. TCRs of the Vβ gene (Vβ 6, 8, 14, and 17) are more frequent in patients with RA than in the control groups. These superantigens of RA can be influenced by oral bacteria - Prevotella intermedia (P. intermedia) stimulates the expression of Vβ 8 and Vβ 17 genes in CD4 (+) T cells, and both bacteria P. gingivalis and P. intermedia can increase the expression of Vβ 6 and Vβ 8.[7]

Heat shock proteins

Heat shock proteins form an ancient, primary system for “intracellular self-defense”. Heat shock proteins (HSPs) are present in cells under normal conditions, but are expressed at high levels when exposed to a sudden temperature jump or other stress. Heat shock proteins stabilize proteins and are involved in the folding of denatured proteins. The presence of 70 kDa Prevotella melaninogenica (P. melaninogenica) HSP and P. intermedia HSP is established in periodontal disease. However, HSP 70 antibodies are also found in the synovium of patients with RA and occur in the synovialis if the HSP 70 expression is triggered by specific stress factors and pro-inflammatory cytokines.[8]

Cytokine link

The role of the T-helper 17 (Th 17) cells in the host defense is not completely known. It is shown that IL-17 stimulates the generation and mobilization of neutrophils and is instrumental in the defense of extracellular bacteria. Th 17 cells and IL-17 play an important role in the pathogenesis of RA. Also, Th 17 cells are also present in chronic periodontal disease,[9] contributing to the etiopa-thogenesis of periodontal disease.

IL-1 cytokines are key mediators of immune responses, inflammation and tissue destruction in both RA and periodontitis. IL-1β levels are elevated in synovial fluids from RA patients[10] and IL-1β is produced by synovial tissue macrophages, activated T cells, fibroblasts and chondrocytes. IL-1β is also prominent in periodontal tissue and gingival crevicular fluid of patients with periodontitis and is stimulated in a variety of resident and immune cells by components of oral bacteria like lipopolysaccharide (LPS).[11] Excessive IL-1β in both disorders accounts for increased local blood flow, neutrophil infiltration and activation of connective tissue turnover via stimulation of MMP secretion from osteoclasts, fibroblasts and neutrophils.

IL-18 is present in the synovial membranes of patients with RA and is thought to amplify the inflammatory response by promoting the release of other cytokines, in particular TNF-α, granulocyte-macrophage colony stimulating factor (GM-CSF) and interferon g (IFN-g). IL-18 has also been shown to promote angiogenesis, prevent endothelial cell and fibroblast apoptosis and modulate various cell lineages, including keratinocytes, osteoblasts, osteoclasts and chondrocytes, in RA.[12] Measurements of IL-18 in periodontal tissue and in the circulation indicate that IL-18 is associated with active periodontitis[13] although there are, as yet, no direct functional data linking this cytokine with destructive processes in periodontitis.

IL-33, in its intracellular form, is highly expressed within endothelial cells in the RA synovium, suggesting a pathogenic role.[14] Increased microvasculature is a prominent histological finding in periodontitis, so it would be interesting to investigate expression of IL-33 associated with endothelial cells in periodontal tissue.

Periodontal microbiota and rheumatoid arthritis

Several research groups have reported an increased variety and number of oral bacterial DNA and antibodies targeting these bacteria in serum and synovial fluid of patients with RA. Antibody levels to P. gingivalis have been shown to correlate with anti-CCP (anti-cyclic citrullinated peptide) antibody levels,[15] making this periodontopathic oral bacterium an attractive candidate environmental trigger in the development of RA. Bacterial antibody (IgG) levels of P. gingivalis, P. intermedia, P. melaninogenica, and Bacteroides forsythus (B. forsythus) were found to be significantly higher in RA patients when compared with those of the controls, which could be important to the etiopathogenesis of RA.[16]

P. gingivalis is currently the only known bacterium with expression of peptidyl arginine deiminase (PAD), which represents an important pathogenic factor of RA. The PAD expressed by P. gingivalis is not entirely homolog to human PAD but leads to an irreversible, post-translational conversion of arginine to citrulline. It has been proposed that oral citrullination of human and bacterial proteins by P.gingivalis peptidyl arginine deiminase (PPAD) in an infectious context prior to the onset of RA could break tolerance and trigger a latent antibody response against citrullinated protein.[17] Once tolerance is breached, citrullination of host proteins by human PAD's perpetuates the immune response through epitope spreading and cross-reactivity, resulting in chronic inflammatory disease.

CITRULLINATION

Arginine is a positively charged, hydrophilic amino acid that is found on protein surface and participates in ionic interactions with other amino acid side chains. It is a key amino acid in structural organization of proteins.

The term citrullination refers to the deimination of arginine side chains (peptidyl arginine) to peptidyl citrulline or simply conversion of peptidyl arginine to peptidyl citrulline. This plays an important role in normal physiological process, involved in formation of rigid structures like hair follicle, epidermal differentiation, in myelin sheaths. Aberrant citrullination is noticed in skin disorders like psoriasis, nervous system disorders like Alzheimer disease, multiple sclerosis and inflammatory arthritis like rheumatoid arthritis, and more recently cancer. This aberrant citrullination can alter the three-dimensional structure and function and expose the previously hidden epitopes to the immune system, giving new antigenic properties to the peptide.

In RA, anti-citrullinated protein (anti-CCP) autoantibodies are seen to citrullinated fibrinogen, filagrine, keratine, vimentin. Anti-CCP is one of the earliest autoantibodies of RA, and are found in 80% of sera of patients with RA, with 99% specificity.[18]

Peptidyl arginine deiminase enzymes

Citrullination is catalyzed by a family of calcium dependent PAD enzymes. This enzyme is a common enzyme of human cells, and consists of five isoforms, with isoform 4 (PADI 4) being the most important for autoimmunity. It is expressed by many cells - B and T lymphocytes, neutrophils, eosinophils, monocytes and natural killer (NK) cells. Normally, this enzyme which is inactive becomes active, during oxidative stress or apoptosis, when calcium enters the cell and causes citrullination of vimentin, fibrin, collagen and α-enolase, making them more antigenic and are recognized by anti-CCP antibodies.[19]

Bacterial peptidyl arginine deiminase

P. gingivalis, the primary pathogen in chronic periodontitis, is the only anaerobic prokaryote till date, to express a functional endogenous peptidyl arginine deiminase (PPAD). This PPAD though not in complete homology with all PAD enzymes, can cause citrullination, thus generating autoantibodies in RA patients. The ability of P. gingivalis to express PAD enzyme, indicates or suggests that periodontal infection can induce or accelerate RA. The three-dimensional structure of PPAD was predicted to have aminoterminal catalytic α/β propeller domain, followed by immunoglobulin like β sandwich which is comparable to the structure of human PADI4 which is composed of two aminoterminal immunoglobulin like β sandwich domains followed by catalytic α/β propeller domain. Unlike mammalian enzyme, PPAD can deiminate both free arginine and peptidyl arginine. Moreover, this citrullination is not calcium dependent like the human PAD.

The physiological role of PPAD is not clear. The production of ammonia during deimination promotes the survival of P. gingivalis within the periodontal pocket.[20] The ammonia produced neutralizes the acidic environment, making gingipains more active, inactivates hemagglutins and has a negative effect on neutrophil function. This bacterial citrullination depends on another virulence factor of P. gingivalis - gingipains. The preference for carboxy terminal arginine residues could be mediated through arginine-gingipains which generate the arginine residues that are subsequently citrullinated by PPAD.[21] It is also speculated that PPAD acts as a virulent factor by producing citrullinated products that help the bacterium to circumvent humoral immune response. Therefore, PPAD may be more important in initiation of autoimmunity, distant from the joint, like the gingiva and human PAD's may be more relevant in generating autoantigens in RA, once tolerance is breached. A study by Mikuls et al.,[15] has shown that increased titers of anti-P.gingivalis antibodies in RA subjects correlated with anti-CCP IgM and IgG2 subtypes for RA. Another study has shown anti-CCP IgG2 antibodies are predominantly elevated in P. gingivalis infection. Thus, the citrullination of bacterial and host proteins by PPAD, along with the infectious context, paves way for breach of self-tolerance, leading to an autoimmune response in the development of RA [Figure 1].

Figure 1

Oral citrullination and RA

In addition, PPAD citrullinates α-enolase enzyme also. α-enolase has a multifunctional role in glucose metabolism and also as a plasminogen-binding protein on surface of various cell types. A sequence of nine amino acids (Asp-Ser-Arg-Gly-Asn-Pro-Thr-Val-Glu) spanning the immunodominant epitope on the citrullinated enolase peptide-1 (CEP-1) is 100% identical to the corresponding region in P.gingivalis enolase, and affinity-purified antibodies to CEP-1 react with recombinant enolase citrullinated in vitro from both humans and P. gingivalis,[22] indicating a role for bacterial infection, particularly with P. gingivalis, in priming autoimmunity in a subset of patients with RA.

PPAD - Possible therapeutic target?

(PPAD, though not in homology with human PPAD, is unique and provides an attractive target in the treatment of RA. The native form of PPAD, in its full length is not stable and has shown less activity. McGraw et al.,[20] tested various compounds and reported that the serine- and cysteine protease inhibitor leupeptin is able to completely inhibit PPAD at millimolar levels, with other inhibitors such as thiourea, thio-l-citrulline, and the serine and cysteine protease inhibitor TLCK (N-alpha-p-tosyl-l-lysine chloromethyl ketone) being inhibitory at higher concentrations. These compounds have low inhibitory capacity, and are either toxic or unselective.

CONCLUSION

Rheumatoid arthritis and periodontitis, both, chronic inflammatory diseases, are characterized by bone and soft tissue loss having similar immune response as well as some common genetic and therapeutic features. Evidence linking the two is many and the contribution of periodontal bacteria is clear as their DNA and antibodies against these bacteria have been detected in the synovium of RA patients. Strengthening the microbial link is the recent discovery of the enzymatic mimicry between human and P. gingivalis PAD. Citrullination by human and bacterial PAD enzymes offer a platform for inhibiting the host immune response at an early stage in the development of rheumatoid arthritis. More research in identifying selective and potent inhibitors of PAD with further studies elucidating the role of PPAD in RA disease etiology/pathology will be useful in treating RA, at an early stage in the progression of the disease.