Rheumatoid arthritis (RA), the most common inflammatory joint disease, exacts a huge toll of disability, deformities, quality-of-life alterations, premature deaths, and economic costs 1. RA is an autoimmune disease characterized by chronic inflammation of the synovial membrane, which is infiltrated by activated immune cells including CD4⁺ T cells, B cells, and antigen-presenting cells such as dendritic cells and macrophages. The factors responsible for RA induction and progression are poorly understood but may involve interactions between innate and adaptive immunity 2. It has been suggested that viruses and bacteria may contribute to initiate or exacerbate RA by binding to Toll-like receptors (TLRs). TLRs are expressed by a variety of immune cells, including B lymphocytes and T lymphocytes, antigen-presenting cells, regulatory T cells and nonimmune cells such as fibroblastic synoviocytes 34567. All of these cell populations are found in the rheumatoid synovium. TLR ligands such as peptidoglycans and double-stranded DNA are also present in the rheumatoid synovium 8, suggesting that innate immunity may be involved in initiating the inflammatory process or in inhibiting regulation mechanisms that normally prevent chronic inflammation.

TLR gene polymorphisms have been tested in several cohorts. A study of Asp299Gly (rs4986790) and Thr399Ile (rs4986791) TLR4 polymorphisms in a cohort of RA patients in Spain found no associations with susceptibility to RA 9. A case–control study of TLR4 Asp299Gly in a cohort in Northern England also found no association, even in the subgroup of patients negative for the shared epitope 10. Interestingly, heterozygous Asp299Gly status was protective in early untreated RA in a case–control study performed in The Netherlands 11. Finally, the Asp299Asp polymorphism was associated with higher remission rates after treatment with disease-modifying antirheumatic drugs, compared with the Asp299Gly polymorphism 12. The role for TLR4 in RA, despite studies in various cohorts, therefore remains unclear.

The TLR2 polymorphisms Arg677Trp (no rs number reported) and Arg753Gln (rs5743708), both implicated in susceptibility to infection, were not associated with arthritis in a cohort in Spain 9. Of note, these two TLR2 gene mutations were associated with reduced induction of IL-10 and IL-12 expression after stimulation 13. In mice injected with TLR2 ligands, regulatory T cells lose their regulatory capacities, suggesting a role for TLR2 in regulatory T cell control 14. Prolonged regulatory T cell stimulation by TRL2 ligands may therefore trigger or exacerbate autoimmune responses. Studies in animal models have established that the TLR2 status influences the outcome of adjuvant-induced arthritis and streptococcal cell wall arthritis. Mice deficient in MyD88, the TLR adaptor molecule, do not develop arthritis. Similarly, TLR2-deficient mice exhibit lower arthritis scores 15.

As TLR1 and TLR6 are TLR2 co-receptors that increase the number of ligands and induce different transduction pathways 16171819, it was of interest to determine whether the TLR1 and TLR6 genes showed polymorphisms that were linked to RA. These polymorphisms have been studied in inflammatory bowel disease 20 but not in joint disease. Studies have shown that TLR1 Arg80Thr (rs5743610), Asn248Ser (rs4833095), and Ser602Ile (rs5743618) SNPs are associated with invasive aspergillosis 21 and with Crohn's disease 20. The TLR6 Ser249Pro SNP (rs5743810) is associated with a reduced risk of asthma and an increased risk of invasive aspergillosis 2122.

Transcription factors that bind to the TLR9 promoter region include GATA-1, GATA-2, c-Ets, and CP2 23. In silico investigations indicate that the -1486 T/C (rs187084) substitution probably creates an SP-1 binding site 24. Nevertheless, neither this SNP nor TLR9 +2848A/G (rs352140) was associated with systemic lupus erythematosus in a cohort in Korea 23. In contrast, the TLR9 -1237C/T (rs5743836) polymorphism was associated with Crohn's disease 25 and with asthma 26.

In the present article, we investigated potential associations between RA and SNPs of TLR1, TLR2, TLR4, TLR6, and TLR9 in a cohort of French Caucasian families with RA. We elected to investigate a range of TLRs believed to interact with viruses, Gram-positive bacteria, or Gram-negative bacteria. We used PCR-RFLP and matrix-assisted laser desorption/ionization–time of flight mass spectrometry to determine the genotypes of 100 family trios, each comprising the index patient and both healthy parents. We analyzed several subgroups of severe RA that might be linked to TLR gene polymorphisms, including the subgroups with rheumatoid factor (RF) or with anti-cyclic citrullinated peptide antibody (anti-CCP), two RA-related autoantibodies, and the subgroup with joint erosions, since these are often associated with autoantibody production.

We did not observe a large effect of the TLR1, TLR2, TLR4, TLR6, or TLR9 genes in a cohort of French Caucasian families with RA. The present study was properly designed since we chose the candidate genes before performing the linkage/association analysis. We used the TDT, which simultaneously evaluates linkage and association, thus avoiding biases due to the inevitable imperfections in matching between cases and controls. Moreover, we had a high homogeneous cohort where all the patients had four European Caucasian grandparents.

To our knowledge, this is the first study of TLR1, TLR6, and TLR9 in a cohort with RA. TLR1 and TLR6 are co-receptors but might display specific polymorphisms, no evidence of which was found in our cohort. TLR9 is involved in autoantibody production, as shown in the model developed by Leadbetter and colleagues 34, and probably but indirectly in inactivated DNAse mice 35. Whether the role for TLR is confined to autoantibody production remains unclear; TLR may exert key effects on interactions between B cells and T cells, as well as on T-cell regulation mechanisms. For this reason, we performed subgroup analysis in patients with RF, anti-CCP antibodies, or joint erosions – and found no effect. Even if this stratification reduces the number of investigated patients, each feature investigated is extremely frequent and so the subgroups maintain a numerosity comparable with the main sample. Moreover, subgroup analysis is justified by the fact that RA is a complex disease that reasonably might have different etiopathogenesis subgroups. If a subgroup matches a certain etiopathogenesis, then the effect size of a genetic variant might be much higher than for the whole RA population on average.

The polymorphisms tested in our study were selected based on frequency and on feasibility of tests; neither their location in exons or introns nor the nature of the polymorphisms was a selection criterion. Sequences located in intron or promoter regions hold appeal for research, because chromosomal interactions occur between genes independently from enhancer sequences known to exist in the regulated gene. Independent genes can therefore exert effects via intra-chromosomal and inter-chromosomal interactions during cell activation 36. As TLR1 and TLR6 act as co-receptors with TLR2 and are promiscuous in the genome, we performed haplotype analysis for the TLR1 and TLR6 genes. We found no associations between the frequent haplotypes and RA susceptibility in the overall group or in subgroups defined by the presence of RF, anti-CCP antibodies, or joint erosions (data not shown).

Because RA patients were eligible for our study only if both their parents were alive, our RA population contains an unusually high proportion of young patients. Conceivably, this bias toward younger patients may have led to unusually high prevalences of criteria for severe RA (RF, anti-CCP antibodies, and rheumatoid nodules), since these allow a definitive diagnosis early in the course of the disease. Our working hypothesis that TLR genes might be associated with greater disease severity by increasing autoantibody production received no support from our findings.

Definitive proof that autoantibodies are involved in the pathophysiology of RA is still lacking, despite accumulating evidence of a role for B cells – including the efficacy of second-line treatments targeting B cells in severe RA 37. B cells express TLR and may exert pathogenic effects in RA after TLR stimulation, independently from autoantibody production, since they are involved in presenting autoantigens to T cells, producing cytokines, and inducing ectopic architecture 38. Furthermore, there is strong evidence of a lack of tolerance in RA, which may be ascribable to regulatory-T-cell impairment at the time of TLR2 stimulation with TLR1 or TLR6 co-engagement by ligands, allowing pathogenic immune cells to escape from normal regulatory mechanisms and to trigger or exacerbate arthritis. Moreover, TLR9 engagement was shown to induce a T-helper type 1 isotypic switch in B lymphocytes, which may be involved in the pathogenesis of RA. Further evidence of the role for TLR9 comes from the efficacy in RA of chloroquine and quinacrine, both of which block TLR9 signaling in antigen-presenting cells 39. Finally, studies on human rheumatoid tissue indicate that TLRs play a potential role in driving inflammation and/or destructive process in RA 40. In our cohort, we found no evidence that the TLR polymorphism influenced the above-described effects.

We found no associations between RA and TLR polymorphisms in more severe subgroups – with RF, or anti-CCP antibodies, or joint erosions – in our cohort of French Caucasians. Similarly, studies conducted in Spain 9 and in England 10 showed no associations between RA and TLR4 or TLR2. A statistically significant decrease in the G allele of TLR4 Asp299Gly (rs4986790) was noted in RA patients in a case–control study in The Netherlands 11. In contrast, in our study there was no significant G-allele enrichment in the RA patients. Our study had a power of 74% to detect a difference at least as great as the one found in the study from The Netherlands 11. Our results therefore rule out a protective role for TLR4 Asp299Gly in our French Caucasian cohort. Nevertheless, although TLR4 may not be involved in initiating RA, it seems to be important in the early treatment response. Remission rates are therefore higher in patients with the A896A genotype than in patients with the uncommon G896G genotype or in heterozygous patients 12.

In conclusion, the role for TLR4 in the pathogenesis of RA remains uncertain. Altogether, published data and our data lead to the prediction that, to improve these data, analyses in larger cohorts with more than 500 patients would be required. Since this sample size is not easy to reach in single-center or even multicenter studies, meta-analytic analysis could probably be the only feasible approach. With that method, our results might help to spread light on the overall contribution of TLR genes in RA.

We tested five out of the 11 members of the TLR family and selected these five members based on their potential role in autoantibody production. Brentano and colleagues showed recently that TLR3 expression was high in RA synovium and increased further after stimulation by TLR3 ligand poly(I–C) or by necrotic RA synovial fluid cells 41. These data suggest that studies of TLR3 polymorphism might be of interest.

Our study rules out a major contribution of the tested TLR polymorphisms to RA in French Caucasians. Our findings need to be confirmed in other cohorts, but already add to the publicity of available data. As we did not observe a large effect it seems that an association between polymorphisms of other TLR genes and RA and/or a functional role for TLR genes in the pathogenesis of RA would be weakly predictable.

anti-CCP: anti-cyclic citrullinated peptide; IL: interleukin; PCR: polymerase chain reaction; RA: rheumatoid arthritis; RF: rheumatoid factor; RFLP: restriction fragment length polymorphism; SNP: single nucleotide polymorphism; TDT: transmission disequilibrium test; TLR: Toll like-receptor.

The authors declare that they have no competing interests.

OJ and HK performed study design, data analysis, manuscript drafting, and data acquisition. PA, EPT, and GF performed study design, data analysis, and manuscript drafting. CP performed data acquisition. LS performed data analysis and manuscript drafting. MCB and FC performed manuscript drafting.