N-3 polyunsaturated fatty acids restore Th17 and Treg balance in collagen antibody-induced arthritis

<div><p>N-3 polyunsaturated fatty acids (PUFA) have anti-inflammatory effects and were considered useful for the treatment of rheumatoid arthritis (RA). Recently, several studies suggested that n-3 PUFAs attenuated arthritis in animal model and human, however the mechanism is still unclear. Interleukin 17 (IL-17) is a pro-inflammatory cytokine mainly produced by T helper 17 (Th17) cells which cause tissue inflammation and bone erosion leading to joint destruction. In contrast, regulatory T (Treg) cells down-regulate various immune responses by suppression of naïve T cells. The imbalance between Th17 cells and Tregs cell is important for the pathogenesis of RA. Here, we investigated whether n-3 PUFAs attenuate arthritis in collagen antibody-induced arthritis (CAIA) model. We used fat-1 transgenic mice expressing the Caenorhabditis elegans fat-1 gene encoding an n-3 fatty acid desaturase that converts n-6 to n-3 fatty acids, leading to abundant n-3 fatty acids without the need of a dietary n-3 supply. Clinical arthritis score was significantly attenuated in fat-1 mice compared to wild type (WT) mice on day 7 (1.6±1.8, p = 0.012) and day 9 (1.5±1.6, p = 0.003). Ankle thickness also decreased significantly in fat-1 mice compared to WT mice (1.82±0.11, p = 0.008). The pathologic finding showed that inflammatory cell infiltration and bone destruction were reduced in fat-1 mice compared to WT. The expression levels of IL-17 and related cytokines including IL-6 and IL-23 decreased in the spleen and ankle joint tissue of fat-1 mice compared to WT mice. Furthermore, Treg cells were expanded in the spleen of fat-1 mice and Treg cell differentiation was significantly higher in fat-1 mice than in wild type (p = 0.038). These data suggest that n-3 PUFAs could attenuate arthritis through increasing the expression of FoxP3 and the differentiation of Treg, while reducing IL-17 production. Therefore, dietary supplementation of n-3 PUFAs could have a therapeutic potential for the treatment of RA.</p></div

Clinical disease scores and ankle thickness in the WT and fat-1 mice in which arthritis was induced by anti-collagen antibodies.

<p>Arthritis was induced with 4mg of collagen antibodies in the WT and fat-1 mice. Clinical arthritis scores (A) and ankle thickness (B) were monitored every other day until day 9. Values are the mean±SD (n = 5 mice/group). (*p<0.05, **p<0.01) WT: wild type. Statistical significance test was done by Mann-Whitney U-test.</p

Hypothetical mechanism by which n-3 PUFAs reduce the severity of arthritis in RA model.

<p>Imbalance of Th17/Treg cells in the CAIA model could be restored by n-3 PUFA.</p

Sequences and accession numbers for primers used in RT-PCR.

<p>Sequences and accession numbers for primers used in RT-PCR.</p

N-3 PUFAs inhibits ankle joint inflammation and bone and cartilage damage in CAIA mice.

<p>Ankle joints were collected on day 9, sectioned and stained with H&E in WT (A) and fat-1 mice (B). Photomicrographs (x40) are representative of at least three independent experiments. Further magnification of black-bordered box (x100) showed the typical inflammatory injuries in WT (C) and fat-1 mice (D). (E) Pathologic scores in the joint tissues of both groups were determined as described in the Materials and Methods. Values are the mean±SD (n = 5 mice/group).</p

Pro-inflammatory cytokine production in the joint, spleen and serum of fat-1 and WT mice.

<p>Histological features of joint tissue were evaluated in the WT and fat-1 mice. Immunohistochemical staining (A) of cytokines was conducted in joint sections from WT and fat-1 mice. Ankle joints were collected on day 9, sectioned and stained with anti-IL-6, IL-23 and IL-17 antibodies. Western blots were conducted in the joint lysates from the WT and fat-1 mice (B). The joint lysates were collected on day 9 and blotted with anti-IL-6, IL-23 and IL-17 antibodies. The spleens were collected from WT and fat-1 mice on day 0 and 9. The expression of various pro-inflammatory cytokine in the spleen were measured by real-time Q-PCR (C). The serum were collected from WT and fat-1 mice on day 9. The expression of various pro-inflammatory cytokine were measured by ELISA (D). The results of three independent experiments with 5 mice/group are expressed as mean±SD. (*p<0.05, **p<0.01) Statistical significance test was done by Mann-Whitney U-test. WT: wild type, IL-7: interleukin 17, IL-6: interleukin 6, IL-23: interleukin 23.</p

Additional file 1: Table S1. of An HLA-C amino-acid variant in addition to HLA-B*27 confers risk for ankylosing spondylitis in the Korean population

Effect estimates for each residue at HLA-B amino-acid positions 70, 97, and 114. Table S2. Association of HLA-B amino-acid position 97 with ankylosing spondylitis in Korean and European populations. (PDF 84 kb

Bone Morphogenetic Protein 6 Polymorphisms Are Associated with Radiographic Progression in Ankylosing Spondylitis

<div><p>Background and Object</p><p>Nearly 25 genetic loci associated with susceptibility to ankylosing spondylitis (AS) have been identified by several large studies. However, there have been limited studies to identify the genes associated with radiographic severity of the disease. Thus we investigated which genes involved in bone formation pathways might be associated with radiographic severity in AS.</p><p>Methods</p><p>A total of 417 Korean AS patients were classified into two groups based on the radiographic severity as defined by the modified Stoke’ Ankylosing Spondylitis Spinal Score (mSASSS) system. Severe AS was defined by the presence of syndesmophytes and/or fusion in the lumbar or cervical spine (n = 195). Mild AS was defined by the absence of any syndesmophyte or fusion (n = 170). A total of 251 single nucleotide polymorphisms (SNPs) within 52 genes related to bone formation were selected and genotyped. Odds ratios (OR) and 95% confidence interval (95% CI) were analysed by multivariate logistic regression controlling for age at onset of symptoms, sex, disease duration, and smoking status as covariates.</p><p>Results</p><p>We identified new loci of bone morphogenetic protein 6 (<i>BMP6</i>) associated with radiographic severity in patients with AS that passed false discovery rate threshold. Two SNPs in <i>BMP6</i> were significantly associated with radiologic severity [r<i>s270378</i> (OR 1.97, p = 6.74×10⁻⁴) and <i>rs1235192</i> [OR 1.92, p = 1.17×10⁻³]) adjusted by covariates.</p><p>Conclusion</p><p>This is the first study to demonstrate that <i>BMP6</i> is associated with radiographic severity in AS, supporting the role wingless-type like/BMP pathway on radiographic progression in AS.</p></div

Association of genotype of BMP6 with total mSASSS.

<p>Data represent mean (95% CI). p = 0.046 by the Kruskal-Wallis test. *Mann Whitney P.</p

Clinical characteristics of study cohort.

<p>Data were shown to mean ± SD or n (%).</p><p>*Continuous NSAID intake was defined as 70 or more the score. AS; ankylosing spondylitis, HLA-B27: human leukocyte antigen-B27; ESR: erythrocyte sedimentation rate; CRP: C-reactive protein; Msasss: modified stokes AS spine score; NSAID: non-steroidal anti-inflammatory drugs.</p