ARTHRITIS & RHEUMATOLOGY Vol. 68, No. 4, April 2016, pp 795–804 DOI 10.1002/art.39514 C 2016, American College of Rheumatology V
Impaired Porphyromonas gingivalis–Induced Tumor Necrosis Factor Production by Dendritic Cells Typifies Patients With Rheumatoid Arthritis Kim C. M. Santegoets,1 Mark H. Wenink,1 Felipe A. Vieira Braga,2 Marta Cossu,3 Femke B. G. Lamers-Karnebeek,2 Piet L. C. M. van Riel,2 Patrick D. J. Sturm,2 Wim B. van den Berg,2 and Timothy R. D. J. Radstake3 Objective. The prevalence of periodontitis is increased in patients with rheumatoid arthritis (RA), and the severity of periodontitis can affect the level of arthritis. Porphyromonas gingivalis is one of the main bacteria involved in periodontitis. Our aim was to determine if there are differences in the innate immune response against P gingivalis between healthy controls and RA patients. Methods. Monocyte-derived dendritic cells (DCs) from healthy controls, RA patients, and patients with psoriatic arthritis (PsA) were stimulated with P gingivalis, a range of other bacteria, and Toll-like receptor agonists. Cytokine production was determined, and blocking studies were performed to determine which receptors were involved in differential recognition of P gingivalis. Effects on T cell cytokines were also determined in cultures of peripheral blood mononuclear cells (PBMCs). Results. Upon stimulation with P gingivalis, RA patient DCs produced less tumor necrosis factor as compared to healthy control DCs, which was not
observed in PsA patients or upon stimulation with other bacteria. In addition, P gingivalis–mediated activation of RA patient PBMCs showed a clear reduction of interferon-g production. Among the various possible underlying mechanisms investigated, only blockade of CR3 abolished the difference between RA patients and healthy controls, suggesting the involvement of CR3 in this process. Conclusion. Immune cells from RA patients display a reduced response to P gingivalis, which has functional consequences for the immune response. This may result in prolonged survival of P gingivalis, possibly driving autoantibody formation and a self-perpetuating loop of chronic inflammation. The possible role of CR3 in this process warrants further investigation. Rheumatoid arthritis (RA) and chronic periodontitis are 2 chronic inflammatory diseases resulting in destruction of the synovial joint or the tissue surrounding the teeth (the periodontium), respectively. Several studies have clearly demonstrated an epidemiologic association between these 2 diseases (1–6). Both diseases share some common susceptibility factors, including genetic factors and environmental factors such as smoking (7,8). This raises the questions of whether periodontitis is a risk factor for the development of RA, whether RA predisposes to periodontal disease, or whether both diseases present together more often because of common risk factors or a similar disease mechanism. Increased prevalence of periodontitis is already seen in patients with early untreated RA, suggesting that periodontitis could be present before RA onset and that the increased prevalence of periodontitis seen in RA patients is not a consequence of RA treatment (4,5). Several small studies indicated that extensive tooth cleaning and oral hygiene instruction to
Supported by the Dutch Arthritis Foundation (Reumafonds) and by The Netherlands Organization for Scientific Research (Vidi grant to Dr. Radstake). 1 Kim C. M. Santegoets, PhD, Mark H. Wenink, MD, PhD: University Medical Center Utrecht, Utrecht, The Netherlands, and Radboud University Medical Center, Nijmegen, The Netherlands; 2 Felipe A. Vieira Braga, MSc, Femke B. G. Lamers-Karnebeek, MD, Piet L. C. M. van Riel, MD, PhD, Patrick D. J. Sturm, MD, PhD, Wim B. van den Berg, PhD: Radboud University Medical Center, Nijmegen, The Netherlands; 3Marta Cossu, MD, Timothy R. D. J. Radstake, MD, PhD: University Medical Center Utrecht, Utrecht, The Netherlands. Address correspondence to Timothy R. D. J. Radstake, MD, PhD, Department of Rheumatology and Clinical Immunology, Laboratory of Translational Immunology, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. E-mail:
[email protected]. Submitted for publication January 26, 2015; accepted in revised form November 12, 2015. 795
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Table 1.
Characteristics of the patients and controls*
Age, mean (range) years Women, % Disease duration, mean (range) years Methotrexate use Sulfasalazine use Hydroxychloroquine use Prednisolone use Leflunomide use Not taking DMARDs or prednisolone ESR, mean (range) mm/hour CRP $5 mg/liter RF positive† ACPA positive‡ Erosions DAS28-ESR, mean (range) TJC, mean (range) SJC, mean (range)
RA patients (n 5 35)
PsA patients (n 5 9)
Healthy controls (n 5 27)
64 (43–88) 69 8.1 (0–26) 23 (66) 10 (29) 7 (20) 3 (9) 1 (3) 4 (11) 12.4 (2–33) 11 (31) 24 (69) 20 (67) 21 (60) 2.8 (1.1–6.0) 3.5 (0–18) 2.5 (0–8)
57 (23–77) 33 8.3 (1–45) 7 (78) 2 (22) 0 (0) 1 (11) 0 (0) 2 (22) 8 (2–18) 5 (56) 1 (17) ND 3 (33) ND 5.2 (0–11) 4.4 (0–11)
37 (22–61) 65 – – – – – – – – – – – – – – –
* Except where indicated otherwise, values are the number (%) of subjects. DMARDs 5 diseasemodifying antirheumatic drugs; ESR 5 erythrocyte sedimentation rate; CRP 5 C-reactive protein; ND 5 not determined; DAS28-ESR 5 Disease Activity Score in 28 joints using the ESR; TJC 5 tender joint count; SJC 5 swollen joint count. † Rheumatoid factor (RF) status was unknown for 3 patients with psoriatic arthritis (PsA). ‡ Anti–citrullinated protein antibody (ACPA) status was unknown for 5 patients with rheumatoid arthritis (RA).
treat periodontitis can also positively affect RA disease activity (9–12). Chronic periodontitis is caused by a complex biofilm containing mainly gram-negative bacteria, often including Porphyromonas gingivalis. This bacterium is specifically interesting in the context of RA because it is the only prokaryotic organism expressing a peptidylarginine deiminase (13), which is able to citrullinate bacterial and human proteins in vitro (14). Anti–citrullinated protein antibodies (ACPAs) are the most sensitive and specific autoantibodies found in RA patients (15,16). These antibodies can often be found several years before the clinical onset of RA and are associated with disease progression (17). This has led to the hypothesis that P gingivalis might be involved in the development of ACPAs and possibly RA in a susceptible host. Associations between antibodies against P gingivalis and ACPA levels in RA patients support this hypothesis (18,19). Prompted by these observations, we set forth to investigate a possible immunologic link between RA and P gingivalis. Hence, we focused on the innate immune response toward P gingivalis in RA patients versus healthy controls, because this is the first line of defense against bacteria like P gingivalis. Dendritic cells (DCs) are important sentinels of the immune system that sample the microenvironment for potential microbial antigens and initiate an adept immune response when necessary. They are found to colocalize with P gin-
givalis proteins in the oral mucosa of patients with periodontitis (20). Furthermore, DNA of P gingivalis has been found in myeloid DCs from the peripheral blood in 72% of orally colonized periodontitis patients (20). These DCs could be involved in the dissemination of bacteria to distal sites such as atherosclerotic plaques or the synovium, where multiple researchers have found DNA from P gingivalis (and other [oral] bacteria) in patients with RA (21–23). We found that the immune response to P gingivalis in RA patients is markedly altered, which might lead to an increased oral bacterial burden, thereby possibly driving the self-perpetuating loop of chronic inflammation. PATIENTS AND METHODS Study population. We included 35 RA patients and 9 patients with psoriatic arthritis (PsA) attending the Department of Rheumatology at Radboud University Medical Center or the Department of Rheumatology and Clinical Immunology at University Medical Center Utrecht. All patients fulfilled the American College of Rheumatology 1987 revised criteria for the classification of RA (24) or the Classification of Psoriatic Arthritis Study Group criteria for PsA (25) at the time of diagnosis and gave their informed consent. In addition, we included 27 healthy controls. Demographic and clinical characteristics of patients and demographic characteristics of controls are shown in Table 1. Patients using biologic agents and high-dose prednisolone were excluded from the study. Experiments were performed in accordance with the Helsinki Declaration and approved by the local Medical Ethics Committees
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of Radboud University Medical Center and University Medical Center Utrecht. Cell isolation and culture of monocyte-derived DCs and macrophages. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized venous blood using density-gradient centrifugation over Ficoll-Paque (Amersham Biosciences). Subsequently, monocytes were obtained using CD14 microbeads and MS columns (Miltenyi Biotec). Isolated monocytes were cultured for 6 days in the presence of granulocyte–macrophage colony-stimulating factor (GM-CSF) (800 units/ml; R&D Systems) and interleukin-4 (IL-4) (500 units/ ml; R&D Systems) to generate monocyte-derived DCs or in the presence of GM-CSF alone (800 units/ml) to generate macrophages. Fresh culture medium with the same growth factors was added on day 3. Cell stimulation. Day 6 monocyte-derived DCs or macrophages were harvested, plated in 96-well culture plates (25,000 cells/well), and stimulated with a range of Toll-like receptor (TLR) agonists or heat-killed bacteria for 18 hours. The following TLR agonists and heat-killed bacteria were used: lipopolysaccharide (LPS) (100 ng/ml, Escherichia coli 0111:B4; Sigma-Aldrich), palmitoyl-3-cysteine-serine-lysine-4 (5 mg/ml; EMC Microcollections), fibroblast-stimulating lipopeptide 1 (1 mg/ml; EMC Microcollections), P gingivalis (ATCC 33277, 1 3 107/ml; InvivoGen), Proteus mirabilis (ATCC 142723, 1 3 107/ml), Salmonella typhimurium (ATCC 13311, 1 3 107/ml), Shigella sonnei (ATCC 11060, 1 3 106/ml), Klebsiella pneumoniae (ATCC 13883, 1 3 107/ml), E coli (1 3 107/ml), Streptococcus mutans (1 3 107/ml), Prevotella intermedia (ATCC 9336, concentration unknown), and purified P gingivalis LPS (1 mg/ml; InvivoGen). E coli LPS was doublepurified to remove any contaminating proteins (26). All bacteria except P gingivalis, S mutans, and E coli were cultured in the Department of Medical Microbiology, Radboud University Medical Center. The bacteria were heat-killed by incubation at 608C for 1 hour. E coli and S mutans were kind gifts from Dr. John Butcher (Institute of Infection and Immunity, Glasgow, UK). CXCR4 and CR3 were blocked by adding antibodies against CXCR4 (clone 12G5, functional grade, 10 mg/ml; eBioscience) or CD18 (clone L19, 5 mg/ml) 30 minutes before stimulation with P gingivalis. An isotype control was used in equal concentrations. PBMCs (0.5 3 106 cells/well) were also directly stimulated with P gingivalis or P intermedia for 7 days to determine effects on T cell cytokines. Flow cytometry. Using standardized flow cytometry protocols as described previously (27), CD18 and CD11b expression was determined on monocyte-derived DCs from RA patients and healthy controls. Clones L19 (anti-CD18) and Bear-1 (anti-CD11b) were kind gifts from Dr. Alessandra Cambi (Department of Tumor Immunology, Radboud University Medical Center). Expression was visualized via a fluorescein isothiocyanate–labeled goat anti-mouse secondary antibody. Cells were analyzed using FlowJo software (Tree Star) for the mean fluorescence intensity relative to cells stained with the appropriate IgG isotypes. Beads labeled with iC3b were prepared by incubating 2 mm Streptavidin Fluoresbrite beads (Polysciences) with biotinconjugated C3b (1 mg/ml) and treating them subsequently with factor H and factor I (3 mg/ml each). These beads were added to monocyte-derived DCs for 60 minutes at a 10:1 ratio, after which binding/uptake was analyzed by flow cytometry.
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RNA isolation and real-time polymerase chain reaction (PCR). Total RNA was extracted in 0.5 ml of TRI Reagent and treated with DNase to remove genomic DNA before being reverse-transcribed into complementary DNA. Messenger RNA for TLRs 1, 2, 4, and 6 and CXCR4 was quantified by quantitative real-time PCR as previously described (28). Quantitative PCR (qPCR) signals were quantified by comparing the Ct value of the gene of interest of each sample with the Ct value of the reference gene GAPDH (DCt) and were calculated as relative expression (22DCt). The following primers were used: for GAPDH, 50 -ATCTTCTTTTGCGTCGCCAG-30 (forward) and 50 -TTCCCCATGGTGTCTGAGC-30 (reverse); for TLR-1, 50 -GCATCTTCCATTTTGCCATT-30 (forward) and 50 -GAACGTGGATGAGACCGTTT-30 (reverse); for TLR-2, 50 -GAATCCTCCATTCAGGCTTCTCT-30 (forward) and 50 -GCCCTGAGGGAATGGAGTTTA-30 (reverse); for TLR-4, 50 -GGCATGCCTGTGCTGAGTT-30 (forward) and 50 -CTGCTACAACAGATACTACAAGCACACT-30 (reverse); for TLR-6, 50 -GGCCGAAACTGGTTTATTGA-30 (forward) and 50 -GGAGTGATGATGGGAGGAGA-30 (reverse); and, for CXCR4, 50 -ATGAAGGAACCCTGTTTCCGT-30 (forward) and 50 -AGATGATGGAGTAGATGGTGGG-30 (reverse). Measurement of cytokines in culture supernatants. Levels of tumor necrosis factor (TNF), IL-6, IL-10, IL-12p70, IL-13, IL-17, and interferon-g (IFNg) were measured in cellfree supernatants using commercially available kits according to the instructions of the manufacturer (Milliplex; Millipore). Milliplex kits were measured on a Luminex 200, and data were analyzed using Bio-Plex Manager software (Bio-Rad). The limit of detection was below 5 pg/ml for all cytokines. Undetectable levels were set at 0.01 pg/ml. Statistical analysis. Differences between groups were analyzed using a Mann-Whitney U test. A Student’s paired t-test was used to compare differences upon stimulation within the same donor. P values less than 0.05 were considered significant.
RESULTS Decreased P gingivalis–induced TNF production by RA patient DCs and macrophages. Monocytederived DCs were cultured from 31 RA patients and 23 healthy controls. Characteristics of patients and controls are shown in Table 1. To determine possible differences between RA patients and healthy controls regarding their response to P gingivalis, monocyte-derived DCs were stimulated with heat-killed P gingivalis. Compared with DCs from healthy controls, DCs from RA patients secreted significantly less TNF upon contact with P gingivalis (median 790 pg/ml versus 285 pg/ml; P 5 0.0001) (Figure 1A). IL-12p70 production also showed a trend toward reduced levels in RA patients (Figure 1B), but this did not reach statistical significance (P 5 0.0751). On the other hand, IL-6 and IL-10 production was similar between patients and controls (Figures 1C and D). To investigate whether our results correlate with RA disease phenotype, RA disease activity, or medication use, we stratified our data accordingly. TNF levels were
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Figure 1. Decreased Porphyromonas gingivalis–induced production of tumor necrosis factor (TNF) in dendritic cells (DCs) from patients with rheumatoid arthritis (RA). Monocyte-derived DCs from healthy controls (HC) and RA patients were stimulated with heat-killed P gingivalis (1 3 107/ml) for 18 hours, and TNF (A), interleukin-12p70 (IL-12p70) (B), IL-6 (C), and IL-10 (D) were measured in the supernatant. Symbols represent individual subjects; horizontal lines indicate the median. *** 5 P , 0.001.
similarly distributed throughout all groups investigated, suggesting that the observation of a decreased DC response in RA patients was a disease-specific phenomenon rather than a phenotype-specific phenomenon and was not caused by medication use (Figure 2A) (see Supplementary Figure 1, available on the Arthritis & Rheumatology web site at http://onlinelibrary.wiley.com/ doi/10.1002/art.39514/abstract). Smoking is a well-known risk factor for RA, and periodontitis and could affect cytokine production. Although information regarding smoking history of our patients and healthy controls was incomplete, we were able to compare the P gingivalis– induced cytokine response of 7 RA patients who were smoking at RA diagnosis with that of 7 RA patients who had never smoked. This analysis did not show any differences, with a median TNF production of 275 pg/ml (interquartile range [IQR] 177–432 pg/ml) in smokers
compared to 232 pg/ml (IQR 116–897 pg/ml) in patients who had never smoked (data not shown), minimizing the chance that smoking is a confounding factor. The specificity of the response for RA disease was further substantiated by the finding that DCs from PsA patients and those from healthy controls secreted similar levels of TNF upon stimulation with P gingivalis (Figure 2B). We next tested whether the altered response to P gingivalis was limited to DCs. To do this, we stimulated RA macrophages with P gingivalis and observed a similar decrease in TNF production (P 5 0.0286), while IL-6 and IL-10 production did not differ between macrophages from RA patients and those from healthy controls (Figure 2C). Reduced IFNg production by RA patient PBMCs upon stimulation with P gingivalis. We stimulated freshly isolated PBMCs from RA patients with
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Figure 2. Decreased response to Porphyromonas gingivalis in immune cells from patients with rheumatoid arthritis (RA) but not those from patients with psoriatic arthritis (PsA). A, P gingivalis–induced levels of tumor necrosis factor (TNF) secreted by monocyte-derived dendritic cells (moDCs) were stratified based on the presence of autoantibodies or erosions. ACPA 5 anti–citrullinated protein antibody; RF 5 rheumatoid factor. B, Monocyte-derived DCs from PsA patients and healthy controls (HC) were stimulated with P gingivalis (1 3 107/ml) for 18 hours, and TNF production was determined. C, Similar experiments were performed with macrophages from RA patients and healthy controls. D, Peripheral blood mononuclear cells (PBMCs) from 10 RA patients and 12 healthy controls were stimulated with P gingivalis for 7 days to determine production of interferon-g (IFNg), interleukin-10 (IL-10), IL-13, and IL-17. Symbols represent individual subjects; horizontal lines indicate the median. * 5 P , 0.05; ** 5 P , 0.01.
P gingivalis for 7 days to determine effects on T cell cytokines. In addition to the clear decrease in TNF production by DCs, RA patient PBMCs produced markedly less IFNg than did PBMCs from controls after stimulation with P gingivalis (median 130.5 pg/ml versus 304 pg/ ml; P 5 0.0085) (Figure 2D). This reduction was specific for IFNg since IL-10 and IL-13 production was similar between patients and controls, and IL-17 production was similar or even slightly increased in RA patients (P 5 0.078). Overall, these data indicate that T cell cytokines are also affected by altered P gingivalis recognition in RA patients, with reduced production of IFNg, a
cytokine that is important for the antimicrobial activity of macrophages/DCs. Species-specific altered cytokine response to P gingivalis in RA. To test the species specificity of the decreased cytokine induction by P gingivalis, we included a multitude of other bacteria that have been implicated in arthritis development—P mirabilis, S typhimurium, S sonnei, K pneumoniae, and E coli and the oral bacteria P intermedia and S mutans (29,30). In contrast to P gingivalis, none of these bacteria induced similar differences in DC TNF production between RA patients and healthy controls (Figure 3A). In addition, healthy con-
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Figure 3. Dendritic cells (DCs) and peripheral blood mononuclear cells (PBMCs) from patients with rheumatoid arthritis (RA) do not respond differentially to a range of other bacteria. A, DCs from RA patients and healthy controls (HC) were stimulated with multiple bacteria, and production of tumor necrosis factor (TNF) was measured in the culture supernatant after 18 hours. DCs from at least 8 healthy controls and 12 RA patients were stimulated with all bacteria shown. B, PBMCs from 9 RA patients and 11 healthy controls were stimulated with Prevotella intermedia for 7 days to determine production of interferon-g (IFNg), interleukin-10 (IL-10), IL-13, and IL-17. Symbols represent individual subjects; horizontal lines indicate the median. ** 5 P , 0.01.
Figure 4. No difference in expression and function of Toll-like receptor 2 (TLR-2) or TLR-4 between patients with rheumatoid arthritis (RA) and healthy controls (HC). A, Expression of TLRs 1, 2, 4, and 6 by monocyte-derived dendritic cells (DCs) from 9 healthy controls and 12 RA patients was determined by quantitative polymerase chain reaction. Values are the mean 6 SEM. B–D, Production of tumor necrosis factor (TNF) was determined in supernatant from unstimulated DCs (medium) or DCs stimulated with the TLR-2/1 ligand palmitoyl-3-cysteine-serine-lysine-4 (5 mg/ml) or with the TLR-2/6 ligand fibroblast-stimulating lipopeptide 1 (1 mg/ml) (B), DCs stimulated with the TLR-4 ligand lipopolysaccharide (LPS) (100 ng/ml) (C), or DCs stimulated with purified Porphyromonas gingivalis LPS (1 mg/ml) (D). Symbols represent individual subjects; horizontal lines indicate the median.
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Figure 5. CR3-dependent difference between patients with rheumatoid arthritis (RA) and healthy controls (HC) upon stimulation with Porphyromonas gingivalis (PG). A, CXCR4 expression was determined in dendritic cells (DCs) from 9 healthy controls and 13 RA patients by quantitative polymerase chain reaction. B, DCs from 5 healthy controls and 5 RA patients were incubated with an anti-CXCR4 antibody (10 mg/ml) or an isotype control (10 mg/ml) for 30 minutes before stimulation with P gingivalis. Production of tumor necrosis factor (TNF) was measured after 18 hours. C, CD18 and CD11b expression on DCs from 8 healthy controls and 8 RA patients was determined by flow cytometry. MFI 5 mean fluorescence intensity. D, DCs from 4 healthy controls and 6 RA patients (3 independent experiments) were incubated with anti-CD18 antibody L19 (5 mg/ml) or an isotype control (5 mg/ml) for 30 minutes before stimulation with P gingivalis. TNF levels were measured in the supernatant after 18 hours. Values are the mean 6 SEM. * 5 P , 0.05.
trol and RA patient PBMCs were also stimulated with the periodontal pathogen P intermedia, and we could not observe the differences in T cell cytokine production that we had observed with P gingivalis (Figures 2D and 3B). These data suggest that the decreased cytokine response toward P gingivalis in RA patients is not only disease specific but also species specific. Altered cytokine response is not caused by altered TLR expression and/or function. The main TLRs thought to be involved in recognition of P gingivalis by immune cells are TLR-2 and TLR-4. TLR-2 functions as a heterodimer with either TLR-1 or TLR-6, and both combinations have been reported to bind P gingivalis (31). In order to increase our understanding of the downstream pathways that may underlie our observed P gingivalis–specific effects, we first determined the ex-
pression of TLRs 1, 2, 4, and 6 on monocyte-derived DCs by qPCR. The expression of these receptors was similar between DCs from RA patients and those from healthy controls (Figure 4A). To exclude functional impairment of these TLRs, DCs were stimulated with ligands for TLR-2/1, TLR-2/6, and TLR-4. Again, no differences were observed between DCs from RA patients and those from controls (Figures 4B and C). A role of endosomal TLRs was excluded by the lack of effect of bafilomycin treatment on P gingivalis–induced TNF production (data not shown). Two important immunogenic parts of P gingivalis are its fimbriae and an LPS structure. This LPS structure is different from that of other bacteria in that it binds mainly to TLR-2 and can either stimulate or inhibit TLR-4 signaling (32). Stimulation with P gingivalis LPS alone induced similar cytokine production in RA patients
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and healthy controls (Figure 4D), indicating that recognition of other P gingivalis parts, such as its fimbriae, is likely responsible for the different cytokine response in RA patients. Attenuation of the difference between RA patients and healthy controls by blocking of CR3. As shown by several studies, binding of P gingivalis fimbriae to either CXCR4 or CR3 can affect TLR-2–induced cytokine production (33–35). Binding of P gingivalis to CXCR4 is reported to suppress TLR-2 signaling (33), and increased CXCR4 expression has been found on RA immune cells (36). Therefore, we determined CXCR4 expression on monocyte-derived DCs, which was found to be similar between RA patients and healthy controls (Figure 5A). In addition, blocking of CXCR4 (which was able to prevent migration toward CXCL12 [data not shown]) did not affect P gingivalis–mediated cytokine production, suggesting that CXCR4 is not the underlying cause of diminished cytokine production in RA (Figure 5B). The other candidate tested was CR3 (also called Mac-1), which has a complex interaction with TLR-2 signaling. Binding of P gingivalis to TLR-2 can activate CR3, and subsequent binding to CR3 by P gingivalis fimbriae contributes to the induction of inflammatory cytokines including TNF (34,35). Both heterodimers of CR3, CD18 and CD11b, were similarly expressed on DCs from RA patients and healthy controls (Figure 5C). Since CR3 function does not always correlate with expression, we stimulated RA patient and healthy control DCs with P gingivalis in the presence of a CD18 blocking antibody. When CR3 was blocked, TNF production upon stimulation with P gingivalis was significantly decreased in healthy controls but not in RA patients, resulting in similar cytokine production in patients and controls, suggesting that the CR3/CD18 pathway could be responsible for the altered activation of RA patient DCs by P gingivalis (Figure 5D). Uptake of iC3b-labeled beads was not affected in monocytederived DCs from RA patients (data not shown), excluding a defect in general CR3-mediated uptake. DISCUSSION We have demonstrated that DCs and macrophages from RA patients produce less TNF upon contact with P gingivalis. This decreased response was both bacterium specific and disease specific. In PBMC cultures, the induction of IFNg, an important activator of the antimicrobial activity of macrophages, was also reduced in RA patients. In this way, the impaired response of RA patient DCs and macrophages to P gingivalis could directly and/or indirectly affect bacterial
clearance. This is supported by a mouse study showing an inverse relationship between proinflammatory potential of P gingivalis subtypes and induction of experimental periodontitis (37). It is tempting to speculate that this decreased cytokine production by RA immune cells could result in impaired clearance and prolonged presence of P gingivalis in RA patients, culminating in intraoral persistence of P gingivalis, which in turn might lead to citrullination of bacterial and human proteins and possibly ACPA development in genetically predisposed individuals (38,39). This hypothesis is supported by a recent article showing that periodontal treatment in patients with RA and periodontitis decreased levels of antibodies to both P gingivalis and citrullinated proteins and decreased RA disease activity (40). The lack of correlation between cytokine production and clinical phenotype in our patients suggests a generally decreased response toward P gingivalis independent of current disease activity or medication use, which might have been present before RA disease onset. Further studies with healthy controls at risk of developing RA might provide more insight into potential underlying mechanisms. Alternatively, a reduced immune response toward P gingivalis might also result in a prolonged low level of systemic inflammation, which could increase the risk of developing further systemic disease including arthritis or atherosclerosis. Increased survival of P gingivalis in DCs could also facilitate extravasation of the bacterium or bacterial fragments to other sites including the joint, a site where P gingivalis DNA has been found (20–22). It remains difficult to predict the exact consequences of these changes in immune mediators. Sufficient immune activation is necessary to eliminate pathogens like P gingivalis and P intermedia in periodontitis, while an overactivation of the immune system is thought to be responsible for eventual tissue damage in both periodontitis and RA. Further studies are needed to enhance our understanding of the potential consequences of our observations. Some possible limitations of our study should be taken into account when interpreting our results. First, information on periodontal status is lacking. This is an unavoidable consequence of the health care setup in The Netherlands, where 2 different specialties, one intramural and the other outside the hospital, see patients with RA or periodontal disease. Second, the age of our healthy donors is lower than that of our RA patients. However, there was no correlation between age and P gingivalis–induced cytokine production by DCs in our healthy donors or RA patients. Finally, medication use in RA and PsA patients was rather similar (Table 1), but P gingivalis–induced cytokine production
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was not influenced by stratification for treatment strategies (see Supplementary Figure 1, available on the Arthritis & Rheumatology web site at http://onlinelibrary. wiley.com/doi/10.1002/art.39514/abstract), thus excluding an effect of medication in our study. In this study, we show that CR3 function could be responsible for reduced TNF production by RA patient DCs upon contact with P gingivalis. Binding of P gingivalis fimbriae to CR3 has been reported to induce bacterial uptake and production of proinflammatory cytokines such as TNF (34,35). Interestingly, early-onset periodontal disease is often seen in patients with a leukocyte adhesion deficiency (41). This is a very rare disease in which patients have a mutation leading to almost absent expression and/or function of CD18, resulting in recurrent infections. Neutrophil dysfunction is described as one of the main causes of disease in these patients. However, impaired recognition and clearance of bacteria such as P gingivalis by macrophages and DCs could also contribute to the development of periodontitis in these patients. Although this falls beyond the scope of this work, our data justify further research into the downstream molecular circuitry underlying possible altered CR3 function in RA. Collectively, our data support an immunologic link between P gingivalis (associated with periodontitis) and RA. RA patient DCs produced less TNF upon contact with P gingivalis, which was accompanied by decreased IFNg production in full PBMC cultures. This could result in impaired clearance and prolonged presence of P gingivalis in RA patients, which in turn might, via citrullination or by inducing a low level of systemic inflammation, contribute to the perpetuation and/or severity of RA. ACKNOWLEDGMENTS We thank Carla Bartels (Department of Medical Microbiology, Radboud University Medical Center) for providing us with most of the heat-killed bacteria used in this study. We thank Suzan Rooijakkers (Department of Medical Microbiology, University Medical Center Utrecht) for her help with the experiments with iC3b-labeled beads. We further thank John Butcher for kindly providing us with heat-killed E coli and S mutans and Alessandra Cambi for the L19 and Bear-1 antibodies. We thank Richard Huijbens (Department of Rheumatology, Radboud University Medical Center) and the Multiplex Core Facility of the Laboratory for Translational Immunology (University Medical Center Utrecht) for help with cytokine measurements. AUTHOR CONTRIBUTIONS All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Radstake had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
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Study conception and design. Santegoets, Wenink, Vieira Braga, Lamers-Karnebeek, van den Berg, Radstake. Acquisition of data. Santegoets, Vieira Braga, Cossu, LamersKarnebeek, van Riel, Sturm. Analysis and interpretation of data. Santegoets, Wenink, Vieira Braga, van den Berg, Radstake.
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