Recently, we used this approach to search for T cell epitopes of proteins derived from microbes implicated in RA. We report here the identification of an HLA–DR–presented peptide (T cell epitope) derived from a P copri 27‐kd protein ( Pc ‐p27), which stimulated Th1 responses in 42% of RA patients. We then found that P copri induced differential antibody responses to this protein or the whole organism in a substantial portion of RA patients. These observations provide evidence of the immune relevance of P copri in the pathogenesis of RA.

Whereas the previous studies used unbiased, discovery‐based approaches to assess dysbiosis of microorganisms in the oral or gut microbiome, we developed an unbiased, discovery‐based approach to identify novel, immunogenic T cell epitopes in patients with chronic inflammatory arthritis. With this approach, in vivo HLA–DR–presented peptides are identified directly from patients' synovial tissue, synovial fluid mononuclear cells (SFMCs), or peripheral blood mononuclear cells (PBMCs) by liquid chromatography tandem mass spectrometry (LC‐MS/MS) 13 , 14 , followed by testing the antigenicity of identified peptides and their source proteins using patients' samples 15 - 19 .

A second metagenome‐wide analysis of fecal samples from RA patients showed dysbiosis in the gut as well as in the mouth and salivary glands 11 . Moreover, a recent study in mice showed that dysbiosis contributes to arthritis development via activation of autoreactive T cells in the intestine 12 . However, it is unclear whether overexpansion of P copri in the human gut has the potential to affect immune cell functions at both mucosal and systemic sites, thereby contributing to RA disease pathogenesis.

Using high‐throughput sequencing, Scher et al 8 showed that Prevotella copri in the gut microbiota was overexpanded in stool samples from patients with new‐onset RA compared with patients with chronic RA, patients with psoriatic arthritis, or healthy individuals. In new‐onset RA patients, Prevotella abundance in the gut was at the expense of Bacteroides fragilis , an organism that is important for Treg function 9 , 10 .

Rheumatoid arthritis (RA) results from a complex interplay between genetic and environmental factors 1 , 2 . Great progress has been made in the identification of genetic factors and inflammatory pathways that influence the disease 1 , 3 , but environmental factors are only now being determined 4 . A key hypothesis is that specific organisms in the mouth or microbiota in the gut, the composition of which is strongly influenced by environmental cues, may shape mucosal and systemic immune responses that affect joints in RA patients 4 - 7 .

Categorical data were analyzed by Fisher's exact test, and quantitative data were analyzed using unpaired t ‐test with Welch's correction. Correlations were sought using Pearson's correlation test. All analyses were performed using GraphPad Prism 6 software. All P values were two‐tailed. P values less than or equal to 0.05 were considered statistically significant.

DNA was isolated from 200 μl of serum or SF using a QIAamp DNA Mini kit (Qiagen). Nested PCR primers were designed to detect DNA for P copri or B fragilis 16S rDNA using Primer3 software (data not shown). DNA was amplified for outer PCR using species‐specific forward and reverse primers; 1 μl of the amplified DNA (diluted 1:10 in sterile distilled water) was then used for the nested PCR reaction. All reactions were carried out using 2.5 units of HotStarTaq DNA polymerase (Qiagen). The amplification program included 40 cycles with denaturation at 94°C for 30 seconds, annealing at 59°C for 30 seconds, extension at 72°C for 50 seconds, and a final extension for 10 minutes. For both outer and nested PCR reactions, a positive control ( P copri or B fragilis DNA) and a negative control (sterile distilled water) were included. When sufficient DNA was available, samples were tested in duplicate. Amplified products (10 μl) were visualized by electrophoresis in a 2% agarose gel. The identity of the PCR products was validated by direct DNA sequencing, which was carried out at the Center for Computational and Integrative Biology DNA Core facility at MGH. The sequenced product was aligned with all human and known microbial genomes using Genomic Blast Sequence (NCBI).

The levels of 14 cytokines and chemokines associated with innate immune responses (IFNα, tumor necrosis factor, macrophage inflammatory protein 1α [MIP‐1α], and MIP‐1β) and with Th1 (IFNγ, IL‐12, CXCL9, and CXCL10) or Th17 (IL‐1β, IL‐17A, IL‐17E, IL‐17F, IL‐22, and IL‐23) adaptive immune responses were determined in serum or SF samples from RA patients. Samples were diluted 1:5 in PBS and incubated with HeteroBlock at a concentration of 150 μg/ml to limit the possible confounding effects of RF. Protein levels of all 14 mediators of inflammation in serum or SF were assessed in 1 complete experiment using bead‐based Luminex assays (EMD‐Millipore) coupled with a Luminex‐200 System Analyzer. Data were assessed using xPonent 3.1 software.

IgG and IgA antibody responses to whole‐cell P copri, B fragilis , or E coli were determined by ELISA. The plates were coated overnight at 4˚C with suspensions of inactivated bacterial cells (10 6 cells/ml). Afterwards, plates were incubated for 1 hour with blocking buffer. Patients' serum samples (diluted 1:100) were added in duplicate wells for 1.5 hours, followed by HRP‐conjugated goat anti‐human IgG or HRP‐conjugated goat anti‐human IgA and then TMB substrate. For interplate standardization, 2 control samples were included on each plate. The ELISA for P gingivalis was performed as previously described 21 .

The P copri type strain (DSM 18205) was obtained from the Leibniz Institute DSMZ, German Collection of Microorganisms and Cell Cultures. Bacteroides fragilis (ATCC 25285), Escherichia coli (ATCC 25922), and Porphyromonas gingivalis (ATCC 33277) isolates were obtained from the American Type Culture Collection. The bacterial cultures were inactivated in 1% formalin for 24 hours, washed twice in PBS, and diluted in PBS at a final concentration of 10 6 cells/ml.

ELISA plates were coated overnight at 4°C with 0.25 μg/ml of the P copri protein Pc ‐p27 (GenScript). Afterwards, plates were incubated for 1 hour with blocking buffer (5% nonfat dry milk in phosphate buffered saline [PBS]–Tween). Each patient's serum sample (diluted 200‐fold) was then added in duplicate wells for 1.5 hours, followed by horseradish peroxidase (HRP)–conjugated goat anti‐human IgG (item sc‐2453; Santa Cruz Biotechnology) or HRP‐conjugated goat anti‐human IgA (Bio‐Rad) and then tetramethylbenzidine (TMB) substrate (BD). For interplate standardization, 2 control samples were included on each plate. In addition, using HeteroBlock (Omega Biologicals), we tested serum samples from 15 patients who had a range of optical density values on ELISA and confirmed that RF did not alter the ELISA results. Therefore, HeteroBlock was not used in subsequent antibody determinations.

The detailed methods for isolation and identification of HLA–DR–presented peptides are described in our previous publication 13 . In the present study, 1 microbial peptide ( 2 KRIILILTVLLAMLGQVAY 20 ) derived from a 27‐kd P copri protein (WP_022121928.1) was identified in the PBMCs from 1 RA patient. This P copri peptide and 2 additional peptides from the same protein, which were predicted to be promiscuous T cell epitopes ( 52 DYRGYWTMRYQFDSATVS 69 and 118 EKINSLPTSSTGI 130 ), were synthesized and purified by high‐performance liquid chromatography in the Core Proteomics Laboratory at MGH. PBMCs from RA patients were then stimulated with the peptides (1 μ M ) in duplicate along with positive (phytohemagglutinin) and negative (no antigen) controls, and incubated for 5 days in culture at 37°C in a 5% CO 2 incubator. Cells were then transferred to Dual‐Color ELISpot plates coated with interferon‐γ (IFNγ)/interleukin‐17 (IL‐17) antibodies (Cellular Technology Limited) and incubated overnight at 37°C. Images of wells were captured using an ImmunoSpot series 3B analyzer, and spots were counted using ImmunoSpot software. A positive T cell response was defined as 3 SD above the mean value in healthy subjects.

We obtained synovial tissue, SFMCs, or PBMCs from 5 patients with RA to use for isolation of HLA–DR–presented peptides. To test implicated peptides and their source proteins for immunoreactivity in larger numbers of patients, we used our cohort of patients with new‐onset RA from whom systematic clinical information, PBMCs, serum samples, and in some cases, SF samples were available. For comparison, PBMCs and serum samples were available from patients with Lyme arthritis. In addition, we used our cohort of patients with chronic RA from whom serum and sometimes SF samples were collected. Serum samples were obtained from patients with other types of arthritis or connective tissue diseases (CTDs) as well as from healthy control subjects. HLA–DR typing was performed on blood samples from RA patients at the American Red Cross Laboratory in Dedham, MA. Anti–citrullinated protein antibodies (ACPAs) and rheumatoid factor (RF) determinations were made in the clinical laboratories at MGH.

The study was approved by the Human Investigations Committee at Massachusetts General Hospital (MGH); all subjects gave written informed consent. All RA patients met the American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) criteria for the disease 20 . All study patients with RA or other rheumatic diseases were recruited from the Rheumatology Clinic at MGH or from suburban MGH clinics.

RESULTS

Identification of naturally presented, microbial HLA–DR–presented peptides Using LC‐MS/MS, we identified HLA–DR–presented peptides in synovial tissue (n = 4), SFMCs (n = 3), or PBMCs (n = 2) from 5 patients with new‐onset RA or chronic RA 14. From the 17 HLA–DR–presented peptides identified in the PBMCs from 1 patient with chronic RA (patient RA1), 1 P copri sequence was found (Figure 1A). In contrast, no sequences from P gingivalis or from Borrelia burgdorferi, the agent of Lyme disease, were identified in any sample. Figure 1 Open in figure viewer PowerPoint Identification of a broadly immunogenic Prevotella copri T cell epitope. A, Liquid chromatography tandem mass spectrometry (LC‐MS/MS) spectrum of the P copri 27‐kd protein, Pc‐p27 2–20 peptide. Consensus peptide identification as 2KRIILILTVLLAMLGQ(deamidated)VAY20 was achieved by OMSSA and X!Tandem analysis. Inset, Findings of the interferon‐γ (IFNγ) enzyme‐linked immunospot (ELISpot) assay using matching peripheral blood mononuclear cells (PBMCs) from a patient with chronic rheumatoid arthritis (patient RA1) stimulated with the peptide (1, 2, and 4 μM). Reactivity of >3 times background (no antigen [Ag]) was considered positive. SFU = spot‐forming units. B, Findings of the IFNγ ELISpot assay using PBMCs from patients with RA, patients with Lyme arthritis (LA), and healthy control (HC) subjects. Cells were incubated with the HLA–DR–presented peptide identified from the PBMCs of patient RA1 (peptide 1; 1 μM). C, IFNγ secretion of PBMCs from the same groups of patients and controls, incubated with 2 predicted promiscuous HLA–DR–binding peptides from Pc‐p27 (1 μM each). A positive response was defined as a value >3 SD above the mean in healthy controls (area above the shaded region). In B and C, each symbol represents a single subject; horizontal lines show the mean. Star represents patient RA1. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/journal/doi/10.1002/art.40003/abstract. At disease onset, patient RA1, who had 2 copies of the RA shared epitope (HLA–DRB1*0401 and 0101), had severe symmetric polyarthritis of the large and small joints. During the course of the disease, tests for ACPAs, but not for RF, became positive. Despite treatment with disease‐modifying antirheumatic drugs (DMARDs), she had recurrent episodes of knee swelling, with evidence of destructive changes in cartilage and bone. The HLA–DR–presented peptide derived from P copri was identified from PBMCs obtained during 1 such episode 7 years after disease onset. The peptide sequence of 19 amino acids had 100% sequence homology with part of the signal sequence of a 27‐kd protein of P copri (Pc‐p27, WP_022121928.1) (Figure 1A). The peptide had minimal sequence homology with any human peptide, suggesting that it was not a human protein erroneously assigned with a microbial database. Using signalP 4.0 software 22, this HLA–DR–presented P copri peptide was predicted to be part of the Sec secretion signal peptide sequence (D score = 0.869), strongly suggesting that the peptide would be cleaved from the source protein. This signal peptide was not predicted to be lipidated (LipoP 1.0 Server). In addition, the algorithm TEPITOPE predicted that the peptide was highly promiscuous, as is typical of signal peptides, and would bind all 25 HLA–DR molecules modeled in the program 23, including the patient's DRB1*0101 and 0401 molecules. When her PBMCs were stimulated with this P copri peptide in an IFNγ ELISpot assay, her T cells secreted levels of IFNγ that were >3 times the background levels (insert in Figure 1A), attesting to the immunogenicity of the peptide.

T cell reactivity with P copri peptides in patients with new‐onset RA To determine the immunogenicity of HLA–DR–presented peptides of Pc‐p27 more broadly, we used PBMCs obtained from our cohort of patients with new‐onset RA who were seen prior to DMARD therapy, the time when immune responses would be expected to be most robust. All patients met the ACR/EULAR criteria for the disease 20. When PBMCs from 39 new‐onset RA patients and from case patient RA1 (a patient with chronic RA) were stimulated with Pc‐p27 peptide 1, a total of 17 of these 40 patients (42.5%) secreted levels of IFNγ that were >3 SD above the mean value in healthy control subjects (P = 0.0002), as determined with an IFNγ/IL‐17 double‐color ELISpot assay (Figure 1B). In comparison, patients with Lyme arthritis lacked reactivity with this peptide (P < 0.0001). Stimulation of cells with phytohemagglutinin (used as a positive control) verified the viability of cells in all patients. The predominant response to stimulation with Pc‐p27 in the RA patients was a Th1‐type response, whereas PBMCs from only 1 RA patient secreted small amounts of IL‐17 (data not shown). To determine whether patients had reactivity with other epitopes of the Pc‐p27 protein, TEPITOPE was used to predict 2 additional promiscuous peptides derived from the same protein (Pc‐p27 peptides 2 and 3). The 2 peptides together were predicted to be presented by all 25 HLA–DR molecules in the program, and therefore, these peptides were pooled for testing. PBMCs from 14 of the 40 patients (35%) secreted levels of IFNγ to peptides 2 and 3 that were >3 SD above the mean value in healthy controls (P = 0.0007) or in patients with Lyme arthritis (P = 0.006) (Figure 1C). Altogether, PBMCs from 24 of the 40 patients (60%) had reactivity with 1 or more of the 3 P copri peptide sequences, showing that Th1 immune responses to this protein were common in new‐onset RA patients. Because of the importance of ACPAs in the diagnosis and pathogenesis of RA 1, 24, peptide 1 was resynthesized with a citrulline in place of the only arginine in the peptide, which was predicted to be in the –P1‐flanking position of the HLA–DR–binding pocket. However, the results suggested that the Pc‐p27 signal peptide sequence was probably not citrullinated in vivo (data not shown). This does not preclude citrullination of other parts of the protein, including B cell epitopes.

IgG and IgA antibody responses to Pc‐p27 and whole P copri We next determined antibody responses to Pc‐p27 in serum samples from 303 individuals. These included samples from 127 patients with new‐onset or chronic RA, 28 patients with CTDs (14 with systemic lupus erythematosus, 4 with mixed CTD, 4 with scleroderma, and 6 with Sjögren's syndrome), 28 patients with spondyloarthritides (SpA) (15 with psoriatic arthritis, 10 with ankylosing spondylitis, and 3 with reactive arthritis), 70 patients with Lyme arthritis, and 50 healthy subjects. Of the 78 new‐onset RA patients, 10 (13%) had IgG antibody responses to Pc‐p27 that were >3 SD above those in healthy controls (P < 0.0001) (Figure 2A). Moreover, 10 of 49 patients with chronic RA (20%) had IgG antibody responses to the protein (P < 0.0001), including patient RA1, in whom 4 serial samples obtained 4–9 years after disease onset yielded positive results. In contrast, only 1 patient with SpA and 1 healthy subject had borderline positive IgG antibody responses to the protein. Figure 2 Open in figure viewer PowerPoint IgG and IgA responses to Prevotella copri in rheumatoid arthritis (RA) patients and control subjects. Serum samples from 303 individuals (healthy control [HC] subjects and patients with connective tissue diseases [CTDs], spondyloarthritides [SpA], Lyme arthritis [LA], new‐onset RA [NORA], or chronic RA [CRA]) were tested for P copri antibodies. Enzyme‐linked immunosorbent assays were performed to measure levels of IgG (A) and IgA (B) against the P copri 27‐kd protein (Pc‐p27) as well as levels of IgG (C) and IgA (D) against 1% formalin–inactivated P copri (whole organism). A positive response was defined as a value >3 SD above the mean in healthy controls (area above the shaded region). Each symbol represents a single subject; horizontal lines show the mean. Star represents patient RA1. Only significant P values relative to healthy controls are shown. Because the first interactions between P copri and immune cells would presumably occur in the gut mucosa, we also determined IgA antibody responses to the organism. About 10% of the patients in both the new‐onset RA and chronic RA groups had IgA antibody responses to Pc‐p27 (P ≤ 0.0002 and P ≤ 0.02, respectively), and the responses tended to be more robust in those with new‐onset RA (Figure 2B). In contrast, only 1 patient with Lyme arthritis and 1 healthy subject had IgA antibody reactivity with the protein. Except for 2 RA patients who had both IgG and IgA responses to Pc‐p27, the other Pc‐p27–positive patients had either an IgG or an IgA response, but not both. Altogether, 24% of the 127 RA patients had IgG or IgA antibody responses to Pc‐p27. When both T and B cell responses were considered together, 3 of the 24 patients who had T cell reactivity with Pc‐p27 peptides also had IgG Pc‐p27 antibody responses, but none had IgA responses to the protein. In comparison, among 16 patients lacking T cell reactivity with Pc‐p27 peptides, only 1 had an IgG antibody response to the protein, but 5 had IgA responses (P = 0.05). The frequencies of shared epitope alleles in patients with P copri T cell or B cell responses was not significantly different from those in patients who lacked these responses (data not shown). In an effort to confirm these findings, we determined IgG and IgA antibody responses to whole P copri using the same set of 303 serum samples. Using PCR, we confirmed that this strain expressed Pc‐p27 (data not shown). Six of the 78 new‐onset RA patients (8%) and 5 of the 49 chronic RA patients (10%) had IgG antibody responses to P copri (Figure 2C). Similarly, 6 of 78 new‐onset RA patients (8%) had IgA antibody responses to P copri (P ≤ 0.004), and 7 of 49 patients with chronic RA (14%) had elevated IgA antibody levels to the organism (P ≤ 0.002) (Figure 2D). Among the 19 patients who had positive IgG or IgA responses to P copri, only 5 (26%) had both responses. No patient with CTD, SpA, or Lyme arthritis had IgG or IgA antibodies to the organism. Altogether, 15% of 127 RA patients had P copri IgG or IgA antibody responses. When the antibody responses to whole P copri or recombinant Pc‐p27 were combined, 41 of the 127 RA patients (32%) had IgG or IgA antibody reactivity with the organism. Thus, antibody responses to P copri were common in RA patients, both early and late in the disease, yet they were rarely found in patients with other types of arthritis, implying specificity in RA.

Antibody responses to other oral or commensal bowel flora To examine the specificity of antibody responses to P copri in RA patients, the same serum samples were also tested for reactivity with whole P gingivalis, an oral periodontal pathogen implicated in RA 25, and with 2 common gut commensal organisms, Bacteroides fragilis and Escherichia coli. Similar to previous studies 26, 27, IgG antibody responses to P gingivalis were found in ∼25% of our new‐onset RA and chronic RA patients, and these responses tended to be higher in RA patients than in the other comparison groups (Figure 3A). However, in contrast with P copri antibody responses, IgG antibodies to P gingivalis were also present in small percentages of patients with other rheumatic diseases or in healthy control subjects, and IgA antibody reactivity with P gingivalis was not increased in RA patients compared with the other groups (Figure 3B). Moreover, in contrast with the dichotomy between IgG and IgA antibody responses to P copri, all RA patients with IgA antibodies to P gingivalis also had IgG responses to this microbe. Importantly, 66 of the 127 RA patients (52%) had antibody responses to either P copri or P gingivalis, but only 8 of the 66 patients (12%) had antibody responses to both microbes. Thus, minimal overlap was observed in the antibody responses to these 2 organisms, indicating that these responses are largely independent, and only the response to P copri was specific for RA. Figure 3 Open in figure viewer PowerPoint IgG and IgA responses to other organisms in rheumatoid arthritis (RA) patients and control subjects. Serum samples from the same 303 individuals as in Figure 2 (healthy control [HC] subjects and patients with connective tissue diseases [CTDs], spondyloarthritides [SpA], Lyme arthritis [LA], new‐onset RA [NORA], or chronic RA [CRA]) were tested for antibody responses to Porphyromonas gingivalis, Bacteroides fragilis, and Escherichia coli. Enzyme‐linked immunosorbent assays (ELISAs) were performed to measure levels of IgG (A) and IgA (B) against P gingivalis, levels of IgG (C) and IgA (D) against B fragilis, as well as levels of IgG (E) and IgA (F) against E coli. All ELISAs used 1% formalin–inactivated bacteria. A positive response was defined as a value >2 SD (P gingivalis, as previously reported [26]) or >3 SD (B fragilis and E coli) above the mean in healthy controls (area above the shaded region). Each symbol represents a single subject; horizontal lines show the mean. Only significant P values are shown. Very few RA patients or those with other rheumatic diseases had IgG or IgA antibody responses to B fragilis or E coli that were >3 SD above the mean values in healthy control subjects (Figures 3C–F). However, IgG absorbance values for B fragilis were significantly lower in new‐onset RA patients than in patients in the other groups (P ≤ 0.03) (Figure 3C), consistent with the decrease in the abundance of B fragilis noted previously in new‐onset RA patients 8. Conversely, IgG and IgA absorbance values for B fragilis in the CTD group were significantly higher than those in the other groups. Thus, in contrast with P copri antibody responses, antibody levels to B fragilis and E coli were similar or lower in RA patients than in patients with other types of arthritis or in healthy subjects.

Clinical features of patients with P copri or P gingivalis antibody responses Because P copri and P gingivalis have both been implicated in RA, we compared the clinical findings in our cohorts of new‐onset and chronic RA patients who did or did not have IgG or IgA antibody responses to these microbes. Because the findings were similar in the 2 groups of RA patients, the groups were combined for presentation here. Several significant differences between these groups were found (Table 1). First, 45 of the 50 patients (90%) with P copri antibody responses were female compared with 60 of the 86 patients (70%) who lacked such responses (P = 0.006). In contrast, the percentages of female and male patients were not significantly different in those with P gingivalis antibody responses. Second, only 37% of the RA patients with IgG P copri antibody responses had ACPAs, compared with 74% of those who had IgA P copri antibodies (P = 0.01) and 71% who lacked P copri antibodies (P = 0.003). There was a similar trend with RF (P = 0.08). In contrast, patients with P gingivalis IgG and IgA antibody responses had higher frequencies of ACPA and RF than did those who lacked these responses. Finally, at study entry, there was a trend toward higher disease activity scores (DAS28) in new‐onset RA patients with either P copri or P gingivalis IgG antibody responses. There were no significant differences among the groups in age, body mass index, or smoking history. Thus, P copri antibodies were found primarily in women, and ACPAs were less common in patients with IgG P copri antibodies, whereas neither of these factors correlated with P gingivalis antibody responses, again indicating that these microbes induce distinct responses. Table 1. Demographic and clinical findings in patients with rheumatoid arthritis, according to the presence of antibodies to Prevotella copri and Prevotella gingivalis P copri antibody response P gingivalis antibody response IgG (n = 27) IgA (n = 23) None (n = 86) IgG (n = 33) IgA (n = 12) None (n = 94) Age, median (range) years 47 (19–85) 53 (19–71) 57 (23–84) 58 (21–84) 62 (41–74) 54 (19–85) Sex, no. female/male 24/3 21/2 60/26b 23/10 7/5 73/21 Body mass index, median 27 28 27 29 34 27 Smoking history, no. (%) Current or former 9 (33) 9 (39) 38 (44) 13 (39) 6 (50) 39 (41) Never 18 (67) 14 (61) 48 (56) 20 (61) 6 (50) 55 (59) Autoantibodies, no. (%) RF positive 9 (33) 12 (52) 46 (53) 20 (61) 8 (67) 43 (46) ACPA positive 10 (37)c 17 (74) 61 (71) 26 (79) 11 (92)d 58 (62) Markers of inflammation, median (range) ESR, mm/hour 12.9 (1–84) 17 (4–34) 16 (1–111) 19.2 (1–67) 36 (10–67)d 15.4 (1–111) CRP, mg/liter 8.5 (0.2–70) 4.5 (0.1–15) 8.2 (0.1–87) 9.2 (0.1–68) 7.3 (0.1–66) 6.8 (0.1–87) Disease activity, median DAS28‐ESR 5.2 4.4 4.2 5.2 5 4.5 DAS28‐CRP 4.6 3.8 3.9 4.9 5 4

Correlation of P copri antibodies with serum cytokine and chemokine levels In an effort to link P copri with inflammatory responses and autoantibody production, IgG and IgA P copri antibody values were correlated with serum cytokine levels in 120 of the 127 RA patients in whom sufficient serum samples were still available. The 14 cytokines and chemokines measured were representative of innate, Th1, and Th17 immune responses. The assays were performed with HeteroBlock to limit possible interference by RF. Because the results were similar in new‐onset RA and chronic RA patients, they were combined for presentation here. When the magnitude of P copri IgG or IgA antibodies in the 37 antibody‐positive patients were correlated with the cytokine levels, strong positive correlations were found between the IgA antibody values and the levels of 3 innate cytokines (IFNα, MIP‐1α, and MIP‐1β), 2 Th1‐associated cytokines (IFNγ and IL‐12), and 3 Th17‐associated cytokines (IL‐17F, IL‐22, and IL‐1β) (Figures 4A–C). In contrast, IgG P copri antibody values correlated only with levels of the Th1 chemoattractant CXCL10 (Figure 4B). Figure 4 Open in figure viewer PowerPoint Correlation between cytokine levels and Prevotella copri antibody responses in antibody‐positive rheumatoid arthritis patients. Pearson's correlation test was used to determine correlations between P copri–specific IgG or IgA responses and cytokines associated with innate immunity (A) (interferon‐α [IFNα], macrophage inflammatory protein 1α [MIP‐1α], and MIP‐1β), Th1 immunity (B) (CXCL10, IFNγ, and interleukin‐12 [IL‐12]), or Th17 immunity (C) (IL‐17F, IL‐22, and IL‐1β). When P copri IgA absorbance values in all 120 RA patients, including those with positive and negative results, were correlated with the cytokine levels, even stronger associations were found with innate (MIP‐1α and MIP‐1β), Th1 (IFNγ and IL‐12), and Th17 (IL‐23, IL‐22, IL‐17A, IL‐17E, and IL‐17F) cytokines (data not shown). In contrast, IgG absorbance values did not correlate with any cytokine or chemokine levels. Similarly, P gingivalis IgG and IgA antibodies did not correlate with any cytokine or chemokine level (data available upon request from the corresponding author), further indicating that these microbes induce distinct responses at different mucosal sites.