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Additional file 1: Table S1. Clinical characteristics of patients. Table S2. The information of primers, products, and annealing temperatures. Table S3. The information of mice genotyping primers, products, and hSlc38a6-v2 primers. Figure S1. The agarose gel electrophoresis for genotyping. (A) Slc38a6 knock-out mice genotyping. (B) Slc38a6 conditional knock-out mice genotyping. (C) Lyz-CRE mice genotyping. (D) Tlr4 knock-out mice genotyping. Figure S2. (A) Il-6 relative expression level in PLF among control group (n = 4), 10 mg/kg LPS i.p. group (n = 4) and 20 mg/kg LPS group (n = 4). (B) TNF-α concentration in serum between LyzCRESlc38a6fl/fl and Slc38a6fl/fl group at 24 h post injection by ELISA (n = 7 or 12). (C) Relative expression of Il-1β and Slc38a6 after LPS stimulation in RAW264.7. (D) Relative expression of Tnf-α and Slc38a6 after LPS stimulation in RAW264.7. Data are presented as mean ± SEM of independent experiments. *p < 0.05. Figure S3. (A) Flow cytometry strategy for peripheral blood. (B) Staining of unstained samples with different antibodies. Figure S4. (A) Flow cytometry strategy for spleen cells. (B) Staining of unstained samples with different antibodies. Figure S5. (A) Flow cytometry strategy for PLF. (B) Staining of unstained samples with different antibodies. Figure S6. (A) Flow cytometry strategy for BAL fluid. (B) Staining of unstained samples with different antibodies. Figure S7. Macrophage percentage in BALF between Slc38a6+/− and Slc38a6−/− mice (A) and splenocytes (B) at 72 h post injection (n = 5). Monocyte/macrophage percentage in splenocytes (C) and in BALF (D) at 72 h post injection (n = 4–6 or 8).

Additional file 1: Supplementary Table S1. Cyclic human COL2 peptide sequences (ACC10 and their corresponding arginine peptides). Amino acids differing from mouse are indicated in italic. Supplementary Table S2. Detailed information of antibody response to ACC10 in different sera subset. Supplementary Table S3. Information of other antigen peptides. Supplementary Table S4. Triple-helical COL2 peptide sequences. Supplementary Table S5. Peptides to which private sera have specific responses. Fig. S1. Some characteristics from patients with promiscuous or private ACPA. Supplementary Figure S2. Monoclonal ACPA and COL2 reactive antibody in vitro binding to newly developed arthritic joint tissue (arthritic score=13).

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Additional file of Cystatin C influences the autoimmune but not inflammatory response to cartilage type II collagen leading to chronic arthritis development

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Additional file 3: Supplementary Table 3. Baseline characteristics in CCP2-positive and -negative RA. The table show baseline characteristics for the EIRA patients included in the study, based on anti-CCP2 IgG status; p-values indicate differences between subsets with respect to: age, female-to-male ratio, number of smokers, HLA-DRB1 SE-positivity, PTPN22-positivity, DAS28 and CRP.

Additional file 1: Table S1. Primers for expression analyses.

Additional file 7: Supplementary Table 6. Associations between different ACPA fine-specificities and HLA-DRB1 SE, in anti-CCP2-positive and anti-CCP2-negative RA. Odds ratios with 95% confidence intervals are shown for associations between HLA-DRB1 SE and presence/absence of 19 different ACPA fine-specificities, in anti-CCP2-positive and -negative RA: p-values indicate differences in ORs between ACPA fine-specificity-positive and -negative subsets in the anti-CCP2-positive subset.

Additional file 1 : Table S1. Showing information about peptides sequences. The cyclic peptide \P6-R247 (239CHADSVLERDGSRSSVC255) shares the same core sequence with P6-R-R but contains additional Cys residues at both N and C terminus to facilitate the formation of an intra-chain disulfide bond and thus a cyclic form of the peptide in neutral buffer solution. c=citrulline.

Additional file 6: Supplementary Table 5. Associations between different autoantibodies and PTPN22 polymorphism, in anti-CCP2-negative RA. Odds ratios with 95% confidence intervals are shown for associations between PTPN22 polymorphism and presence/absence of ACPA, RF, other autoantibodies, or "any autoantibody" in anti-CCP2-negative RA.

Additional file 6: Supplementary Table 5. Associations between different autoantibodies and PTPN22 polymorphism, in anti-CCP2-negative RA. Odds ratios with 95% confidence intervals are shown for associations between PTPN22 polymorphism and presence/absence of ACPA, RF, other autoantibodies, or "any autoantibody" in anti-CCP2-negative RA.

Additional file 7: Supplementary Table 6. Associations between different ACPA fine-specificities and HLA-DRB1 SE, in anti-CCP2-positive and anti-CCP2-negative RA. Odds ratios with 95% confidence intervals are shown for associations between HLA-DRB1 SE and presence/absence of 19 different ACPA fine-specificities, in anti-CCP2-positive and -negative RA: p-values indicate differences in ORs between ACPA fine-specificity-positive and -negative subsets in the anti-CCP2-positive subset.

Additional file 4: Supplementary Figure 1. ACPA fine-specificity levels in anti-CCP2-negative RA and controls. ACPA levels for 19 individual ACPA fine-specificities are shown in separate graphs with AU values on the Y-axes. Only ACPA levels above cutoff for positivity are shown.

Additional file 4: Supplementary Figure 1. ACPA fine-specificity levels in anti-CCP2-negative RA and controls. ACPA levels for 19 individual ACPA fine-specificities are shown in separate graphs with AU values on the Y-axes. Only ACPA levels above cutoff for positivity are shown.

Additional file 8: Supplementary Table 7. Associations between RF isotypes and smoking, in anti-CCP2-positive and anti-CCP2-negative RA. Odds ratios with 95% confidence intervals are shown for associations between smoking and presence/absence of IgM, IgG or IgA RF, in anti-CCP2-positive and -negative RA; p-values indicate differences in ORs between RF isotype-positive and -negative subsets.

Additional file 3 : Table S3. Showing disease incidence and maximum arthritis score with isotype control antibodies. Isotype controls: G11 + L243 + Hy2.15; Mice: 12-weeks-old males; 5-6 mice/group. B-LPS: before lipopolysaccharide injection; A-LPS: after lipopolysaccharide injection.

Additional file 8: Supplementary Table 7. Associations between RF isotypes and smoking, in anti-CCP2-positive and anti-CCP2-negative RA. Odds ratios with 95% confidence intervals are shown for associations between smoking and presence/absence of IgM, IgG or IgA RF, in anti-CCP2-positive and -negative RA; p-values indicate differences in ORs between RF isotype-positive and -negative subsets.

Additional file 2 : Table S2. Showing disease incidence and maximum arthritis score in BQ.Ncf1* mice. Comparisons were done between 2mg/4mg Cab3 and 2mg/4mg anti-CII in regard of max arthritis score. Significant difference was observed between 4mg Cab3 and 4mg anti-CII after LPS injection.

By their interaction with IgG immune complexes, FcγR and complement link innate and adaptive immunity, showing functional redundancy. In complement-deficient mice, IgG downstream effector functions are often impaired, as well as adaptive immunity. Based on a variety of model systems using FcγR-knockout mice, it has been concluded that FcγRs are also key regulators of innate and adaptive immunity; however, several of the model systems underpinning these conclusions suffer from flawed experimental design. To address this issue, we generated a novel mouse model deficient for all FcγRs (FcγRI/II/III/IV-/- mice). These mice displayed normal development and lymphoid and myeloid ontogeny. Although IgG effector pathways were impaired, adaptive immune responses to a variety of challenges, including bacterial infection and IgG immune complexes, were not. Like FcγRIIb-deficient mice, FcγRI/II/III/IV-/- mice developed higher Ab titers but no autoantibodies. These observations indicate a redundant role for activating FcγRs in the modulation of the adaptive immune response in vivo. We conclude that FcγRs are downstream IgG effector molecules with a restricted role in the ontogeny and maintenance of the immune system, as well as the regulation of adaptive immunity.

Rheumatoid arthritis is a chronic inflammatory joint disorder characterised by erosive inflammation of the articular cartilage and by destruction of the synovial joints. It is regulated by both genetic and environmental factors, and, currently, there is no preventative treatment or cure for this disease. Genome-wide association studies have identified ∼100 new loci associated with rheumatoid arthritis, in addition to the already known locus within the major histocompatibility complex II region. However, together, these loci account for only a modest fraction of the genetic variance associated with this disease and very little is known about the pathogenic roles of most of the risk loci identified. Here, we discuss how rat models of rheumatoid arthritis are being used to detect quantitative trait loci that regulate different arthritic traits by genetic linkage analysis and to positionally clone the underlying causative genes using congenic strains. By isolating specific loci on a fixed genetic background, congenic strains overcome the challenges of genetic heterogeneity and environmental interactions associated with human studies. Most importantly, congenic strains allow functional experimental studies be performed to investigate the pathological consequences of natural genetic polymorphisms, as illustrated by the discovery of several major disease genes that contribute to arthritis in rats. We discuss how these advances have provided new biological insights into arthritis in humans., Summary: Using experimental models of rheumatoid arthritis, natural genetic polymorphisms associated with arthritis are identified and characterised in rats, bringing in new biological insights into arthritis in humans.

Background An infection-immune association of periodontal disease with rheumatoid arthritis has been suggested. This study aimed to investigate the effect of pre-existing periodontitis on the development and the immune/inflammatory response of pristane-induced arthritis. Methods We investigated the effect of periodontitis induced by ligature placement and Porphyromonas gingivalis (P. gingivalis) infection, in combination with Fusobacterium nucleatum to promote its colonization, on the development of pristane-induced arthritis (PIA) in rats (Dark Agouti). Disease progression and severity of periodontitis and arthritis was monitored using clinical assessment, micro-computed tomography (micro-CT)/intraoral radiographs, antibody response, the inflammatory markers such as α-1-acid glycoprotein (α-1-AGP) and c-reactive protein (CRP) as well as cytokine multiplex profiling at different time intervals after induction. Results Experimentally induced periodontitis manifested clinically (P

Illustration of a conditional inference tree for developing RA in pre-symptomatic individuals compared with control subjects. CCP2 anti-CCP2 antibodies, Filaggrin anti-filaggrin antibodies, CEP-1 = anti-α-enolase antibodies, IgM-RF immunoglobulin M rheumatoid factor, HLA-SE human leukocyte antigen shared epitope, n number of individuals, Pr probability of detecting a pre-symptomatic individual, + = positive, − = negative. (DOCX 29 kb)

Prevalence, sensitivity, specificity and OR of numbers of RF isotypes, alone and/or in combination with ten different ACPA specificities, anti-CCP2 antibodies and anti-carbamylated protein antibodies in pre-symptomatic individuals and population controls. (DOCX 17kb)

Illustration of a conditional inference tree for developing RA in pre-symptomatic individuals compared with control subjects. CCP2 anti-CCP2 antibodies, Filaggrin anti-filaggrin antibodies, CEP-1 = anti-α-enolase antibodies, IgM-RF immunoglobulin M rheumatoid factor, HLA-SE human leukocyte antigen shared epitope, n number of individuals, Pr probability of detecting a pre-symptomatic individual, + = positive, − = negative. (DOCX 29 kb)

Additional file 1: Supplementary Table 1. Citrullinated peptide antigens on the multiplex microarray. The table lists citrullinated antigens used on the multiplex microarray, including name, protein of origin, and amino acid sequence, as well as references.

Peptides recognized by anti-CCP-2-negative EAC patients. Peptides recognized by anti-CCP-2-negative EAC patients who are positive in the multiplex assay for a single epitope (nâ =â 143). CCP cyclic citrullinated peptide, EAC Leiden Early Arthritis Clinic, RA rheumatoid arthritis. (PDF 24 kb)

Peptides recognized by anti-CCP-2-negative EAC patients. Peptides recognized by anti-CCP-2-negative EAC patients who are positive in the multiplex assay for a single epitope (nâ =â 143). CCP cyclic citrullinated peptide, EAC Leiden Early Arthritis Clinic, RA rheumatoid arthritis. (PDF 24 kb)

Additional file 1: Figure S1. Detection of bacterial DNA. a Total load of non-species-specific bacterial gDNA and b specific P. gingivalis DNA (relative quantification) from ligatures of rats with pre-existing periodontitis, collected at endpoint. DNA from P. gingivalis CCUG 14449 strain; Positive Ctrl positive control, NTC non-templated control (PCR-graded water).

Diagnosis of anti-CCP-2-negative non-RA patients. Diagnoses of anti-CCP-2-negative non-RA patients (n = 135) as assessed by an experienced rheumatologist after 1 year of follow-up stratified for multiplex positivity. CCP cyclic citrullinated peptide, RA rheumatoid arthritis. (PDF 14 kb)

Additional file 5: Supplementary Table 4. Other autoantibodies in anti-CCP2-positive and -negative RA and controls. Frequencies of 17 other autoantibodies in anti-CCP2-positive RA, anti-CCP2-negative RA and controls are shown, as well as median antibody levels in anti-CCP2-positive and -negative RA (p-values indicate differences between anti-CCP2-negative and anti-CCP2-positive RA subsets).

Diagnosis of anti-CCP-2-negative non-RA patients. Diagnoses of anti-CCP-2-negative non-RA patients (n = 135) as assessed by an experienced rheumatologist after 1 year of follow-up stratified for multiplex positivity. CCP cyclic citrullinated peptide, RA rheumatoid arthritis. (PDF 14 kb)

Additional file 9: Supplementary Figure 2. Antibody levels in EIRA RA cases based on smoking status. IgM, IgG and IgA RF levels, as well as anti-CCP2 IgG levels, are shown for never, current and former smokers in all EIRA RA patients where smoking data was available.

Additional file 1: Supplementary Table 1. Citrullinated peptide antigens on the multiplex microarray. The table lists citrullinated antigens used on the multiplex microarray, including name, protein of origin, and amino acid sequence, as well as references.

Additional file 5: Supplementary Table 4. Other autoantibodies in anti-CCP2-positive and -negative RA and controls. Frequencies of 17 other autoantibodies in anti-CCP2-positive RA, anti-CCP2-negative RA and controls are shown, as well as median antibody levels in anti-CCP2-positive and -negative RA (p-values indicate differences between anti-CCP2-negative and anti-CCP2-positive RA subsets).

Additional file 2: Supplementary Table 2. Other (non-citrullinated) autoantigens on the multiplex microarray. The table lists the other antigens used on the multiplex microarray, including name, protein of origin, and major associated disease(s).

Additional file 9: Supplementary Figure 2. Antibody levels in EIRA RA cases based on smoking status. IgM, IgG and IgA RF levels, as well as anti-CCP2 IgG levels, are shown for never, current and former smokers in all EIRA RA patients where smoking data was available.

Baseline characteristics of anti-CCP-2-negative RA patients. Baseline characteristics of anti-CCP-2-negative RA patients (nâ =â 279) that are multiplex-negative or -positive. CCP cyclic citrullinated peptide, RA rheumatoid arthritis. (PDF 40 kb)

Baseline characteristics of anti-CCP-2-negative RA patients. Baseline characteristics of anti-CCP-2-negative RA patients (nâ =â 279) that are multiplex-negative or -positive. CCP cyclic citrullinated peptide, RA rheumatoid arthritis. (PDF 40 kb)

Citrulline reactivities to Fibα36-50 and Fibβ563-583. a Citrulline reactivities toward Fibβ563-583 in anti-CCP-2-positive (n = 285) and anti-CCP-2-negative (n = 279) RA patients and non-RA patients (n = 149) in the EAC. b Citrulline reactivities in arbitrary units (AUs) toward Fibβ563-583 in anti-CCP-2-positive (n = 174) and anti-CCP-2-negative (n = 92) RA patients in the IMPROVED cohort. c Citrulline reactivities toward Fibα36-50 in anti-CCP-2-positive (n = 285) and anti-CCP-2-negative (n = 279) RA patients and non-RA patients (n = 149) in the EAC. d Citrulline reactivities toward Fibα36-50 in anti-CCP-2-positive (n = 174) and anti-CCP-2-negative (n = 92) RA patients in the IMPROVED cohort. The dotted line indicates the cutoff. Reactivities are plotted in AUs. Each dot indicates a unique individual. CCP cyclic citrullinated peptide, EAC Leiden Early Arthritis Clinic, IMPROVED, RA rheumatoid arthritis. (PDF 397 kb)

Additional file 1: Figure S1. Detection of bacterial DNA. a Total load of non-species-specific bacterial gDNA and b specific P. gingivalis DNA (relative quantification) from ligatures of rats with pre-existing periodontitis, collected at endpoint. DNA from P. gingivalis CCUG 14449 strain; Positive Ctrl positive control, NTC non-templated control (PCR-graded water).

Citrulline reactivities to Fibα36-50 and Fibβ563-583. a Citrulline reactivities toward Fibβ563-583 in anti-CCP-2-positive (n = 285) and anti-CCP-2-negative (n = 279) RA patients and non-RA patients (n = 149) in the EAC. b Citrulline reactivities in arbitrary units (AUs) toward Fibβ563-583 in anti-CCP-2-positive (n = 174) and anti-CCP-2-negative (n = 92) RA patients in the IMPROVED cohort. c Citrulline reactivities toward Fibα36-50 in anti-CCP-2-positive (n = 285) and anti-CCP-2-negative (n = 279) RA patients and non-RA patients (n = 149) in the EAC. d Citrulline reactivities toward Fibα36-50 in anti-CCP-2-positive (n = 174) and anti-CCP-2-negative (n = 92) RA patients in the IMPROVED cohort. The dotted line indicates the cutoff. Reactivities are plotted in AUs. Each dot indicates a unique individual. CCP cyclic citrullinated peptide, EAC Leiden Early Arthritis Clinic, IMPROVED, RA rheumatoid arthritis. (PDF 397 kb)