Two rheumatoid arthritis-specific autoantigens correlate microbial immunity with autoimmune responses in joints

Abstract

In rheumatoid arthritis (RA), immunological triggers at mucosal sites, such as the gut microbiota, may promote autoimmunity that affects joints. Here, we used discovery-based proteomics to detect HLA-DR-presented peptides in synovia or peripheral blood mononuclear cells and identified 2 autoantigens, N-acetylglucosamine-6-sulfatase (GNS) and filamin A (FLNA), as targets of T and B cell responses in 52% and 56% of RA patients, respectively. Both GNS and FLNA were highly expressed in synovia. GNS appeared to be citrullinated, and GNS antibody values correlated with anti-citrullinated protein antibody (ACPA) levels. FLNA did not show the same results. The HLA-DR-presented GNS peptide has marked sequence homology with epitopes from sulfatase proteins of the Prevotella sp. and Parabacteroides sp., whereas the HLA-DR-presented FLNA peptide has homology with epitopes from proteins of the Prevotella sp. and Butyricimonas sp., another gut commensal. Patients with T cell reactivity with each self-peptide also had responses to the corresponding microbial peptides, and the levels were directly correlated. Furthermore, HLA-DR molecules encoded by shared-epitope (SE) alleles were predicted to bind these self- and microbial peptides strongly, and these responses were more common in RA patients with SE alleles. Thus, sequence homology between T cell epitopes of 2 self-proteins and a related order of gut microbes may provide a link between mucosal and joint immunity in patients with RA.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. T cell reactivity to GNS and FLNA peptides in RA patients and comparison group subjects.

In initial experiments, (A) PBMCs from patients with RA or LA or from HC subjects were stimulated with a pool of 4 peptides, including the single GNS HLA-DR–presented peptide isolated from the synovial tissue of patient RA1, and 3 predicted promiscuous HLA-DR–binding peptides from GNS (1 μM each). (B) PBMCs from patients and HCs were incubated with a pool of 4 peptides, including the single FLNA HLA-DR–presented peptide identified from the synovial tissue and PBMCs from patient RA1, and 3 predicted promiscuous HLA-DR–binding peptides from FLNA (1 μM each). In each assay, a positive control (phytohemagglutinin) and a negative control (no peptide) were included. The amount of IFN-γ secretion, as determined by an ELISpot assay, is shown. A positive response was defined as greater than 3 SD above the mean value for HCs (area above the shaded region). The values for patient RA1 are indicated with a star. Horizontal lines represent the mean values for each group. P values were determined by unpaired, 2-tailed t test with Welch’s correction. SFU, spot-forming units per million PBMCs.

Figure 2. IgG and IgA responses to GNS and FLNA in RA patients and comparison group subjects.

Serum samples from 259 patients with RA, patients with other forms of chronic inflammatory arthritis, and HCs were tested by ELISA for autoantibodies. (A and C) Plates were coated with the GNS protein and incubated with serum from patients or HCs. All serum samples were tested in duplicate for anti-GNS IgG (A) or IgA (C) antibody responses. (B and D) Plates were coated with the FLNA protein and incubated with serum from patients or control subjects. All serum samples were tested in duplicate for anti-FLNA IgG (B) or IgA (D) antibody responses. For all analyses, positivity was defined as greater than 3 SD above the mean value for HCs (area above the shaded region). Symbols represent values in individual patients, and horizontal lines show the mean values. Values for patient RA1 are indicated with a star. Only significant P values, determined by unpaired, 2-tailed t test with Welch’s correction, are shown.

Figure 3. Autoantibody correlations with P. copri and P. gingivalis antibodies.

Correlations between anti-GNS or anti-FLNA antibodies (IgG or IgA) and antibodies against P. copri (A) or P. gingivalis (B) in 101 RA patients. The r and P values for the corresponding statistical comparisons were determined by Spearman’s correlation test.

Figure 4. Autoantibody responses to citrullinated GNS and FLNA, and correlations with ACPAs.

Serum samples from 46 patients with RA and 15 healthy individuals were tested for IgG antibody responses against citrullinated versus uncitrullinated GNS or FLNA. Plates were coated with GNS (A) or FLNA (C), with or without citrullination, incubated with serum from patients or HC subjects, and tested in duplicate. Symbols represent values for individual patients, and horizontal lines indicate the mean values. In A and C, only significant P values, calculated by an unpaired, 2-tailed t test with Welch’s correction, are shown. (B) Correlation between IgG antibody responses against citrullinated GNS or citrullinated FLNA (D) and ACPA levels in the 46 patients with RA. The r and P values shown in B and D were determined by Spearman’s correlations. citGNS, citrullinated GNS; citFLNA, citrullinated FLNA.

Figure 5. GNS and FLNA protein levels in RA patients and comparison group subjects.

GNS and FLNA protein concentrations were measured in serum and SF samples from patients with RA, serum samples from patients with CTD, SpA, or LA, and serum samples from HCs. (A) GNS protein concentrations and (B) FLNA protein concentrations are shown, as measured by ELISA assay. For both analyses, positivity was defined as greater than 3 SD above the mean value for HC subjects (area above the shaded region). Symbols represent values for individual patients, and horizontal lines indicate the mean values. The values for patient RA1 are indicated with a star. Only significant P values, determined by unpaired, 2-tailed t test with Welch’s correction, are shown. SLE, systemic lupus erythematosus.

Figure 6. Immunohistochemical staining of synovial tissue for GNS and FLNA.

Representative synovial tissue images from 1 patient with RA, 1 with LA, and 1 with OA are shown for expression of GNS or FLNA protein. Brown color indicates specific staining of GNS or FLNA self-proteins, and purple indicates hematoxylin staining. Images were taken at ×20 magnification, and ×40 magnification was used for the RA patient to highlight the staining around blood vessels.

Figure 7. Sequence homology between self- and microbial peptides.

(A) Sequence alignment of the self- and corresponding microbial peptides is shown (Clustal Omega), and the predicted binding frames of the self-peptides are given for the HLA-DRB1*0101 and *0401 molecules. Red residues indicate the P1 position (TEPITOPE predicted 3 binding registers for GNS [both HLA-DRB1*0101 and *0401], 2 for FLNA [HLA-DRB1*0401], and 1 for FLNA [HLA-DR*0101]), and blue residues indicate positions P2 through P9. The line through the amino acid residues indicates that the peptide-binding register contains an amino acid with an R-group that may not interact favorably with one of the MHC-binding pockets. (B) PBMCs from 24 RA patients and 10 HCs were incubated with 1 of the 2 self-peptides (GNS or FLNA) or each of the 2 corresponding microbial peptides (1 μM each). In each assay, a positive control (phytohemagglutinin) and a negative control (no peptide) were included. The amount of IFN-γ secretion is shown, as determined by ELISpot assay. A positive response was defined as greater than 3 SD above the mean value for the HCs (area above the shaded region). Horizontal lines represent the mean values for each group. *P < 0.05 and **P < 0.005, by unpaired, 2-tailed t test with Welch’s correction. (C) Correlations between the T cell reactivity to the GNS peptide and the 2 corresponding microbial peptides, 1 derived from the Prevotella arylsulfatase protein and the other from the Parabacteroides GNS protein. (D) Correlations between the T cell reactivity to the FLNA peptide and the 2 corresponding microbial peptides derived from 2 hypothetical proteins, 1 from the Prevotella sp. and the other from the Butyricimonas sp. P and r values shown in C and D were calculated using Spearman’s correlation test.

Figure 8. HLA-DR–binding prediction for self- and microbial peptides.

Prediction analyses were performed using the IEDB Analysis Resource consensus tool. A low percentile rank indicates good peptide binders. (A) MHC class II–binding prediction of the GNS peptide and the corresponding microbial peptides. (B) MHC class II–binding prediction of the FLNA peptide and the corresponding microbial peptides. SE alleles: *0101, *0102, *0401, *0404, and *1001. Non-SE alleles: *0103, *0301, *0403, *0803, *1101, *1201, *1302, *1501, and *1601. Data represent median values with interquartile ranges. **P < 0.005, by Mann-Whitney U test.