Periodontal disease,Porphyromonas gingivalis,and rheumatoid arthritis: what triggers autoimmunity and clinical disease?

Rheumatoid arthritis, currently regarded as a complex multifactorial disease, was initially characterized as such at the turn of the 19th century. Ever since, multiple lines of investigation have attempted to elucidate the etiological factor(s) involved in disease incidence. Genes – including those risk alleles within HLA-DR4 – have been implicated but are insufficient to explain the vast majority of cases. Several environmental factors, therefore, are being studied. Among them, the role of periodontal disease and Porphyromonas gingivalis in the pathogenesis of rheumatoid arthritis has attracted both clinical and bench interest given supportive epidemiologic and mechanistic data.The notion that rheumatoid arthritis (RA) is a polygenic autoimmune disorder that requires environmental factors in order to become clinically apparent is not novel. Since the very beginning, infectious agents have been implicated. This includes early theories such as the ‘oral sepsis’ hypothesis, which supported the notion that periodontal infections were the true etiologic factors behind many chronic diseases. Soon after, dental extraction became a central part of the RA therapeutic armamentarium.Over the last decade, an ever-increasing body of literature has been devoted to study the association between periodontal disease, Porphyromonas gingivalis, and RA. In this issue of Arthritis Research & Therapy, Arvikar and colleagues [1] demonstrate a positive correlation between P. ginigivalis antibody responses and presence/levels of anti-cyclic citrullinated peptide antibodies in a subset of patients with early RA. Moreover, subjects with serological reactivity toward P. ginvgivalis also tended to have higher RA disease activity as measured by Disease Activity Score in 28 Joints and Clinical Disease Activity Index. This occurred both at baseline – that is, in recently diagnosed patients who have not previously been treated with disease-modifying anti-rheumatic drugs (DMARDs) – and 12 months after initiation of therapy.The study had several strengths. First, the authors enrolled early RA patients who were DMARD-naïve at the time of antibody measurements and clinical assessments. Second, patients were followed for a period of 1 year to address biologic and phenotypic alterations after initiation of therapy. This is of utmost importance since very few studies have addressed clinical and immunologic changes at the very onset of disease [2,3]. Too often, however, the natural history of RA is studied without considering the confounding effects of long-standing systemic inflammation or immunosuppressive therapies or both. It is almost certain that the use of DMARDs and biologics alters the quantification and behavior of multiple immune cells and proteins (including auto- and alloantibody responses), thus distorting the true understanding of disease pathogenesis. Efforts to elucidate the earliest changes in pre-clinical and clinical RA are underway [2,4].An accumulating body of evidence suggests a role for clinical periodontal diseases in RA pathogenesis. Periodontitis was more common and severe in patients with RA compared with osteoarthritis [5], and subjects with RA had an increased likelihood of periodontitis compared with controls [6]. Multiple recent studies have specifically implicated P. gingivalis, a periodontopathic bacterium, as a possible triggering factor. This microorganism has gained scientific attention given its ability to citrullinate peptides via unique enzymatic properties conferred by peptydil arginine deiminase (PAD), which reportedly promotes the generation of neoantigens and the subsequent production of antibodies to citrullinated protein antigens (ACPAs). Experimentally, P. gingivalis-PAD is capable of citrullinating human peptides [7] and ACPAs have proven pathogenic in murine models of arthritis [8].A decade ago, it was postulated that a specific humoral immune response to P. gingivalis was the actual stimulus for the development of RA [9]. Since then, multiple reports used serological methods [10,11] to correlate the generation of antibodies to P. gingivalis with autoimmunity (that is, ACPA antibodies) and clinical RA. Several lessons can be learned from these types of approaches. First, as in the case of reports by Arvikar and colleagues [1] and others [2,10,11], the methodology and antigens used to quantify anti-P. gingivalis antibodies have been heterogeneous. Prior studies used antibodies against whole-cell, bacterial lipopolysaccharide, or P. gingivalis-specific chaperone protein. The sensitivity and specificity of each one of these antibodies (and their measurements through different phases of disease) add to the complexity of correlating P. gingivalis serologic responses to RA pathogenesis. A concerted effort toward standardization is warranted in the interest of scientific validation and replication. Second, very few studies have reported the direct presence of P. gingivalis (or other periodontopathic bacteria) in subgingival biofilms of patients with RA. This can now be achieved without the need for laborious, classic microbiologic culture techniques. The advent of high-throughput, bacterial DNA sequencing has allowed taxonomic classification of multiple bacterial species within hundreds of samples (that is, microbiome analysis) in a matter of days.Finally, and perhaps more importantly, virtually all studies consistently reported only a small fraction of RA patients as being exposed to P. gingivalis (serologically, microbiologically, or both). This can have several (and possibly complementary) explanations. It is conceivable that the overabundance of other, non-measured, periodontopathic bacteria (or the lack of protective flora or both) contributes to disease initiation. Moreover, exposure to bacterial antigenic burden at other body sites, such as the lung or the gut, may represent triggering factors for RA. The intestinal microbiome, for example, is vast and diverse. It contains 100 times more protein-coding genes than the human genome and harbors 100 trillion cells (10-fold the amount of total host human cells). Studies in animal models support the notion that the oral, lung, or intestinal microbiome (or a combination thereof) is required to develop inflammatory arthritis. This is based on the fact that rodents do not develop joint inflammation under germ-free conditions or when treated with antibiotics. It is plausible, therefore, that an alteration in the bacterial taxa of several mucosal sites (including oral, lung, and intestinal microbiomes) is required for the transition from a pre-clinical, autoimmune phase of RA into clinically classifiable disease.Novel and comprehensive approaches for the study of the microbiome and the initiation of RA are now possible. Immunologic and microbiome analyses in prospective cohorts of subjects with periodontal disease and other risk factors for the development of RA (for example, first-degree relatives, discordant twins, or asymptomatic individuals with circulating autoantibodies or a combination thereof) may help elucidate some of these questions and ultimately target these organisms (or their components) as a diagnostic or even preventive strategy for RA.     

RANK ligand, RANK, and OPG expression in type II collagen-induced arthritis mouse

Rheumatoid arthritis (RA) is a systemic disorder characterized by synovial inflammation and subsequent destruction and deformity of synovial joints. The articular lesions start with synovitis, focal erosion of unmineralized cartilage, and then culminate in the destruction of subarticular bone by pannus tissue. Periarticular osteopenia and systemic osteoporosis follow as late complications of RA. Osteoclasts, specialized cells that resorb bone, play a central role in developing these osteolytic lesions. To elucidate the mechanism of osteoclastogenesis and bone destruction in autoimmune arthritis, we investigated the expression of RANK ligand (RANKL), RANK, and osteoprotegerin (OPG) mRNA in a mouse type II collagen-induced arthritis (CIA) model by in situ hybridization. The results indicated that most of the TRAP-positive mono- and multinucleated cells in the inflamed and proliferating synovium and in the pannus were RANK-positive authentic osteoclasts and their precursors. In the inflamed synovium and pannus of the mouse CIA model, synovial fibroblastic cells around these RANK-positive cells were strongly positive for RANKL. Moreover, RANKL-positive osteoblasts on the endosteal bone surface, at a distance from the affected synovial joints, increased significantly in the mouse CIA model prior to periarticular osteopenia and systemic osteoporosis. These data indicated that the RANKL-RANK system plays an important role for osteoclastogenesis in both local and systemic osteolytic lesions in autoimmune arthritis, and can therefore be a good target for therapeutic intervention.     

Sustained biologic-free and drug-free remission in rheumatoid arthritis,where are we now?

The advent of new medications and new treatment strategies for rheumatoid arthritis has made it possible to achieve remission in more patients than before. Furthermore, recent clinical trials and register studies suggest that some patients who initially required aggressive therapy may achieve biologic-free remission or even the ultimate goal of therapy, drug-free remission, resembling recovery. Here, we present a discursive review of the most important studies addressing these issues. Based on the overall results, it remains unclear if achieving biologic-free and drug-free remissions are primarily due to the natural course of the disease or to the early therapeutic intervention according to the ‘window of opportunity’ hypothesis. Although medication-free remission is only achievable in a small subset of patients, characterizing this patient cohort may provide important information about beneficial prognostic factors and the underlying mechanisms. In summary, in a subset of patients biologic-free and even drug-free remission can be achieved; pursuing these possibilities in practice may decrease the risk for long-term side effects and attenuate the economic burden of the disease.     

Tumor necrosis factor α-induced adipose-related protein expression in experimental arthritis and in rheumatoid arthritis

Introduction  

Tumor necrosis factor-alpha (TNFα) plays a pivotal role in rheumatoid arthritis (RA); however, the mechanism of action of TNFα antagonists in RA is poorly defined. Immunization of DBA/1 mice with glucose-6-phosphate isomerase (GPI) induces severe acute arthritis. This arthritis can be controlled by TNFα antagonists, suggesting similar etiology to RA. In this study, we explored TNFα-related mechanisms of arthritis.     

MMP-2, TNF-α and NLRP1 polymorphisms in Chinese patients with ankylosing spondylitis and rheumatoid arthritis

Rheumatoid arthritis (RA) and ankylosing spondylitis (AS) are autoimmune, inflammatory diseases with substantial genetic contributions. Matrix metalloproteinase (MMP)-2, tumor necrosis factor (TNF)-α and NLR family pyrin domain-containing 1 (NLRP1) play important roles in the immune response. We studied the MMP-2 rs243865 C/T, TNF-α rs1800629 A/G, NLRP1 rs878329 C/G and NLRP1 rs6502867 C/T polymorphisms in a Chinese cohort of 520 patients with RA, 100 with AS and 520 controls. Genotyping was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Using the MMP-2 rs243865 CC homozygote genotype as the reference group, the CT and TT/CT genotypes were associated with significantly reduced risks of AS. However, logistic regression analyses revealed that the MMP-2 rs243865 C/T polymorphism was not associated with risk of RA. TNF-α rs1800629 A/G, NLRP1 rs878329 C/G and NLRP1 rs6502867 C/T polymorphisms were not associated with risk of RA or AS. These findings suggest that the MMP-2 rs243865 C/T polymorphism is associated with AS development.     

Tumor necrosis factor α-induced adipose-related protein expression in experimental arthritis and in rheumatoid arthritis

Introduction

Broad-range rDNA PCR provides an alternative, cultivation-independent approach for identifying bacterial DNA in reactive and other form of arthritis. The aim of this study was to use broad-range rDNA PCR targeting the 16S rRNA gene in patients with reactive and other forms of arthritis and to screen for the presence of DNA from any given bacterial species in synovial fluid (SF) samples.

Methods

We examined the SF samples from a total of 27 patients consisting of patients with reactive arthritis (ReA) (n = 5), undifferentiated arthritis (UA) (n = 9), rheumatoid arthritis (n = 7), and osteoarthritis (n = 6) of which the latter two were used as controls. Using broad-range bacterial PCR amplifying a 1400 bp fragment from the 16S rRNA gene, we identified and sequenced at least 24 clones from each SF sample. To identify the corresponding bacteria, DNA sequences were compared to the EMBL (European Molecular Biology Laboratory) database.

Results

Bacterial DNA was identified in 20 of the 27 SF samples (74, 10%). Analysis of a large number of sequences revealed the presence of DNA from more than one single bacterial species in the SF of all patients studied. The nearly complete sequences of the 1400 bp were obtained for most of the detected species. DNA of bacterial species including Shigella species, Escherichia species, and other coli-form bacteria as well as opportunistic pathogens such as Stenotrophomonas maltophilia and Achromobacter xylosoxidans were shared in all arthritis patients. Among pathogens described to trigger ReA, DNA from Shigella sonnei was found in ReA and UA patients. We also detected DNA from rarely occurring human pathogens such as Aranicola species and Pantoea ananatis. We also found DNA from bacteria so far not described in human infections such as Bacillus niacini, Paenibacillus humicus, Diaphorobacter species and uncultured bacterium genera incertae sedis OP10.

Conclusions

Broad-range PCR followed by cloning and sequencing the entire 16S rDNA, allowed the identification of the bacterial DNA environment in the SF samples of arthritic patients. We found a wide spectrum of bacteria including those known to be involved in ReA and others not previously associated with arthritis.     

Lack of association or interactions between the IL-4, IL-4Rα and IL-13 genes, and rheumatoid arthritis

Introduction  

A feature of rheumatoid arthritis (RA) is an imbalance between proinflammatory and anti-inflammatory cytokines. Several recent studies have implicated polymorphism in the IL-4 signalling pathway in the development of erosive RA. The aim of the present study was to investigate the role of polymorphism in the IL-4, IL-4Rα and IL-13 genes in RA, including an examination of epistasis.     

Smoking–gender interaction and risk for rheumatoid arthritis

The present case–control study was conducted to investigate the relationship between smoking and rheumatoid arthritis, and to investigate formally the interaction between sex, smoking, and risk for developing rheumatoid arthritis. The study was performed in the Central District of Finland. Cases were patients with rheumatoid arthritis and the control group was a random sample of the general population. Logistic regression models were used to evaluate the effect of smoking on risk for rheumatoid arthritis, after adjusting for the effects of age, education, body mass index, and indices of general health and pain. Overall, 1095 patients with rheumatoid arthritis and 1530 control individuals were included. Patients were older, less well educated, more disabled, and had poorer levels of general health as compared with control individuals (all P < 0.01). Preliminary analyses revealed the presence of substantial statistical interaction between smoking and sex (P < 0.001). In separate multivariable analyses, past history of smoking was associated with increased risk for rheumatoid arthritis overall in men (odds ratio 2.0, 95% confidence interval 1.2–3.2) but not in women. Among men, this effect was seen only for rheumatoid factor-positive rheumatoid arthritis. There were significant interactions between smoking and age among women but not among men. We conclude that sex is a biologic effect modifier in the association between smoking and rheumatoid arthritis. The role of menopause in the etiology of rheumatoid arthritis merits further research.     

Aspects of early arthritis. What determines the evolution of early undifferentiated arthritis and rheumatoid arthritis? An update from the Norfolk Arthritis Register

Over 3500 patients with recent onset inflammatory polyarthritis (IP) have been recruited by the Norfolk Arthritis Register (NOAR) since 1990. Longitudinal data from this cohort have been used to examine the prevalence and predictors of remission, functional disability, radiological outcome, cardiovascular mortality and co-morbidity and the development of non-Hodgkin's lymphoma. Rheumatoid factor titre, high baseline C-reactive protein and high baseline HAQ score are all predictors of a poor outcome. There is a strong association between possession of the shared epitope and the development of erosions. Patients who satisfy the American College of Rheumatology criteria for rheumatoid arthritis (RA) have a worse prognosis than those who do not. However, it appears that these patients are a poorly defined subset of all those with IP rather than having an entirely separate disease entity. New statistical techniques offer exciting possibilities for using longitudinal datasets such as NOAR to explore the long-term effects of treatment in IP and RA.     

Treatment with recombinant interferon-β reduces inflammation and slows cartilage destruction in the collagen-induced arthritis model of rheumatoid arthritis

We investigated the therapeutic potential and mechanism of action of IFN-β protein for the treatment of rheumatoid arthritis (RA). Collagen-induced arthritis was induced in DBA/1 mice. At the first clinical sign of disease, mice were given daily injections of recombinant mouse IFN-β or saline for 7 days. Disease progression was monitored by visual clinical scoring and measurement of paw swelling. Inflammation and joint destruction were assessed histologically 8 days after the onset of arthritis. Proteoglycan depletion was determined by safranin O staining. Expression of cytokines, receptor activator of NF-κB ligand, and c-Fos was evaluated immunohistochemically. The IL-1-induced expression of IL-6, IL-8, and granulocyte/macrophage-colony-stimulating factor (GM-CSF) was studied by ELISA in supernatant of RA and osteoarthritis fibroblast-like synoviocytes incubated with IFN-β. We also examined the effect of IFN-β on NF-κB activity. IFN-β, at 0.25 μg/injection and higher, significantly reduced disease severity in two experiments, each using 8–10 mice per treatment group. IFN-β-treated animals displayed significantly less cartilage and bone destruction than controls, paralleled by a decreased number of positive cells of two gene products required for osteoclastogenesis, receptor activator of NF-κB ligand and c-Fos. Tumor necrosis factor α and IL-6 expression were significantly reduced, while IL-10 production was increased after IFN-β treatment. IFN-β reduced expression of IL-6, IL-8, and GM-CSF in RA and osteoarthritis fibroblast-like synoviocytes, correlating with reduced NF-κB activity. The data support the view that IFN-β is a potential therapy for RA that might help to diminish both joint inflammation and destruction by cytokine modulation.     

Distinct synovial immunopathology in Behçet disease and psoriatic arthritis

Introduction

The aim of the study was to investigate synovial immunopathology differences between early Behçet disease (BD) and psoriatic arthritis (PsA).

Methods

Needle arthroscopy of an inflamed knee joint was performed in patients with early untreated BD (n = 8) and PsA (n = 9). Synovial fluid (SF) was collected for cytokines, perforin, and granzyme analysis. Eight synovial biopsies per patient were obtained for immunohistochemical analysis of the cellular infiltrate (T cells, natural killer cells, macrophages, B cells, plasma cells, mast cells, and neutrophils), blood vessels as well as expression of perforin and granzyme. The stained slides were evaluated by digital image analysis.

Results

The global degree of synovial inflammation was similar in the two types of arthritis. In the analysis of the innate immune cell infiltration, there was a striking neutrophilic inflammation in BD synovitis whereas PsA displayed significantly higher numbers of cells positive for c-kit, a marker of mast cells. As for lymphocytes, CD3⁺ T cells, but neither CD20⁺ B cells nor CD138⁺ plasma cells, were significantly increased in BD versus PsA. Further analysis of the T-lymphocyte population showed no clear shift in CD4/CD8 ratio or Th1/Th2/Th17 profile. The SF levels of perforin, an effector molecule of cytotoxic cells, displayed a significant four- to fivefold increase in BD.

Conclusions

This systematic comparative analysis of early untreated synovitis identifies neutrophils and T lymphocytes as important infiltrating cell populations in BD. Increased levels of perforin in BD suggest the relevance of cytotoxicity in this disease.     

Genes and environment in arthritis: can RA be prevented?

Understanding of how interactions between genes and environment contribute to the development of arthritis is a central issue in understanding the etiology of rheumatoid arthritis (RA), as well as for eventual subsequent efforts to prevent the disease. In this paper, we review current published data on genes and environment in RA as well as in certain induced animal models of disease, mainly those in which adjuvants only or adjuvants plus organ-specific autoantigens are used to induce arthritis. We refer to some new data on environmental and genetic factors of importance for RA generated from a large case-control study in Sweden (1200 patients, 1200 matched controls). We found an increased risk of seropositive but not of seronegative RA in smokers, and there are indications that this effect may be due to a gene-environment interaction involving MHC class II genes. We also found an increased risk of RA in individuals heavily exposed to mineral oils. This was of particular interest because mineral oils are strong inducers of arthritis in certain rodent strains and because polymorphisms in human genetic regions syntenic with genes predisposing for oil-induced arthritis in rats have now been shown to associate with RA in humans. Taken together, our data support the notion that concepts and data on gene-environment interactions in arthritis can now be taken from induced animal models of arthritis to generate new etiological hypotheses for RA.     

Epstein-Barr virus and rheumatoid arthritis: is there a link?

Rheumatoid arthritis is a systemic autoimmune disease characterized by chronic, destructive, debilitating arthritis. Its etiology is unknown; it is presumed that environmental factors trigger development in the genetically predisposed. Epstein-Barr virus, a nearly ubiquitous virus in the human population, has generated great interest as a potential trigger. This virus stimulates polyclonal lymphocyte expansion and persists within B lymphocytes for the host's life, inhibited from reactivating by the immune response. In latent and replicating forms, it has immunomodulating actions that could play a role in the development of this autoimmune disease. The evidence linking Epstein-Barr virus and rheumatoid arthritis is reviewed.     

Increased levels of circulating microparticles in primary Sj?gren's syndrome,systemic lupus erythematosus and rheumatoid arthritis and relation with disease activity

Introduction

Cell stimulation leads to the shedding of phosphatidylserine (PS)-rich microparticles (MPs). Because autoimmune diseases (AIDs) are characterized by cell activation, we investigated level of circulating MPs as a possible biomarker in primary Sjögren''s syndrome (pSS), systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA).

Methods

We measured plasma levels of total, platelet and leukocyte MPs by prothrombinase capture assay and flow cytometry in 43 patients with pSS, 20 with SLE and 24 with RA and in 44 healthy controls (HCs). Secretory phospholipase A2 (sPLA2) activity was assessed by fluorometry. Soluble CD40 ligand (sCD40L) and soluble P-selectin (sCD62P), reflecting platelet activation, were measured by ELISA.

Results

Patients with pSS showed increased plasma level of total MPs (mean ± SEM 8.49 ± 1.14 nM PS equivalent (Eq), P < 0.0001), as did patients with RA (7.23 ± 1.05 n PS Eq, P = 0.004) and SLE (7.3 ± 1.25 nM PS Eq, P = 0.0004), as compared with HCs (4.13 ± 0.2 nM PS Eq). Patients with AIDs all showed increased level of platelet MPs (P < 0.0001), but only those with pSS showed increased level of leukocyte MPs (P < 0.0001). Results by capture assay and flow cytometry were correlated. In patients with high disease activity according to extra-glandular complications (pSS), DAS28 (RA) or SLEDAI (SLE) compared with low-activity patients, the MP level was only slightly increased in comparison with those having a low disease activity. Platelet MP level was inversely correlated with anti-DNA antibody level in SLE (r = -0.65; P = 0.003) and serum β2 microglobulin level in pSS (r = -0.37; P < 0.03). The levels of total and platelet MPs were inversely correlated with sPLA2 activity (r = -0.37, P = 0.0007; r = -0.36, P = 0.002, respectively). sCD40L and sCD62P concentrations were significantly higher in pSS than in HC (P ≤ 0.006).

Conclusions

Plasma MP level is elevated in pSS, as well as in SLE and RA, and could be used as a biomarker reflecting systemic cell activation. Level of leukocyte-derived MPs is increased in pSS only. The MP level is low in case of more severe AID, probably because of high secretory phospholipase A2 (sPLA2) activity, which leads to consumption of MPs. Increase of platelet-derived MPs, sCD40L and sCD62P, highlights platelet activation in pSS.