Evidence of the Immune Relevance of Prevotella copri, a Gut Microbe, in Patients With Rheumatoid Arthritis

Abstract

Objective: Prevotella copri, an intestinal microbe, may overexpand in stool samples from patients with new-onset rheumatoid arthritis (RA), but it is not yet clear whether the organism has immune relevance in RA pathogenesis.

Methods: HLA-DR-presented peptides (T cell epitopes) from P copri were sought directly in the patients' synovial tissue or peripheral blood mononuclear cell (PBMC) samples using tandem mass spectrometry. The antigenicity of peptides or their source proteins was examined in samples from the RA patients or comparison groups. T cell reactivity was determined by enzyme-linked immunospot assay; antibody responses were measured by enzyme-linked immunosorbent assay, and cytokine/chemokine determinations were made by bead-based assays. Serum and synovial fluid samples were examined for 16S ribosomal DNA for P copri using nested polymerase chain reaction analysis.

Results: In PBMCs, we identified an HLA-DR-presented peptide from a 27-kd protein of P copri (Pc-p27), which stimulated Th1 responses in 42% of patients with new-onset RA. In both new-onset RA patients and chronic RA patients, 1 subgroup had IgA antibody responses to either Pc-p27 or the whole organism, which correlated with Th17 cytokine responses and frequent anti-citrullinated protein antibodies (ACPAs). The other subgroup had IgG P copri antibodies, which were associated with Prevotella DNA in synovial fluid, P copri-specific Th1 responses, and less frequent ACPAs. In contrast, P copri antibody responses were rarely found in patients with other rheumatic diseases or in healthy controls.

Conclusion: Subgroups of RA patients have differential IgG or IgA immune reactivity with P copri, which appears to be specific for this disease. These observations provide evidence that P copri is immune-relevant in RA pathogenesis.

Figure 1. Identification of a Broadly Immunogenic P. copri T Cell Epitope

(A) LC/MS/MS spectrum of the Pc-p27_2-20 peptide. Consensus peptide identification was achieved as KRIILILTVLLAMLGQ(deamidated)VAY by OMSSA and X!Tandem. The insert panel shows the IFN-γ ELISpot assay using matching patient's PBMC stimulated with the peptide (1, 2 and 4 μM). Reactivity of >3 times background (no antigen) was considered positive. (B) IFN-γ ELISpot assay using PBMC from patients with rheumatoid arthritis (RA), Lyme arthritis (LA), and healthy controls (HC) incubated with the HLA-DR-presented peptide identified from the PBMC of patient RA1 (Peptide1, 1 μM). (C) IFN-γ secretion of PBMC from patients and controls incubated with 2 predicted promiscuous HLA-DR binding peptides from Pc-p27 (1 μM each). A positive response was defined as >3 SD above the mean value of the HC (area above the shaded region). The value for patient RA1 is indicated with a star. Horizontal lines represent the mean values of each group.

Figure 2. IgG and IgA Responses to P. copri in RA Patients and Control Subjects

Serum samples from 303 individuals with NORA, CRA, other rheumatic diseases or from healthy controls were tested for P. copri antibodies. IgG (A) and IgA (B) ELISAs against the bacterial protein Pc-p27; IgG (C) and IgA (D) ELISA against 1% formalin-inactivated P. copri. Positivity was defined as >3 SD above the mean value of healthy controls (area above the shaded region). Symbols represent values in individual patients and horizontal lines show mean values. The value for patient RA1 is indicated with a star. Only significant P values relative to HC are shown. HC, healthy control; CTD, connective tissue diseases; SpA, spondyloarthropathies; LA, Lyme arthritis; NORA, new onset rheumatoid arthritis; CRA, chronic rheumatoid arthritis.

Figure 3. IgG and IgA Responses to Other Organisms in RA Patients and Controls

Serum samples from the same 303 individuals, shown in figure 2, were tested for antibody responses to other bacteria. IgG (A) and IgA (B) ELISAs against 1% formalin-inactivated P. gingivalis. Positivity was defined as >2SD above the mean value of normal control negative for periodontitis as determined in our previous publication (26). IgG (C) and IgA (D) ELISAs against 1% formalin-inactivated B. fragilis; IgG (E) and IgA (F) ELISAs against 1% formalin-inactivated E. coli. For these analyses, positivity was defined as >3 SD above the mean value of healthy controls (area above the shaded region). Symbols represent values in individual patients and horizontal lines show mean values. Only significant P values are shown. HC, healthy control; CTD, connective tissue diseases; SpA, spondyloarthropathies; LA, Lyme arthritis; NORA, new onset rheumatoid arthritis; CRA, chronic rheumatoid arthritis.

Figure 4. Correlation between Cytokine Levels and P. copri Antibody Responses in Antibody-Positive Patients

Correlation between P. copri specific IgG or IgA responses and cytokines associated with innate immunity (A), Th1 immunity (B), or Th17 immunity (C). Correlations were performed using the Pearson's correlation test.

Figure 5. PCR Testing for Prevotella copri 16S rDNA in Concomitant Serum and SF Samples of RA Patients

(A) Nested PCR of P. copri 16S rDNA amplicons (254 bp) analyzed on 1.5% agarose gels stained with ethidium bromide. Results from serum and SF samples from the 3 positive patients are shown. Patient RA1 had 4 paired serum and SF samples; patient RA2 had 1 serum and 2 SF samples, and patient RA5 had 1 serum and 1 SF samples. In patients RA2 and RA5, enough material was available for testing in duplicate. M, 100 bp DNA ladder; +, positive control (P. copri DSM 18205); H, water control. (B) Sequence alignment of the 16S gene amplicons obtained from patient RA1, RA2 and RA5 using CLC Genomic Workbench software. The sequence of P. copri (DSM 18205) 16S gene is shown as the reference and the conservation of all sequence positions is shown below the alignment.