Non-MHC Risk Alleles in Rheumatoid Arthritis and in the Syntenic Chromosome Regions of Corresponding Animal Models

Rheumatoid arthritis (RA) is a polygenic autoimmune disease primarily affecting the synovial joints. Numerous animal models show similarities to RA in humans; some of them not only mimic the clinical phenotypes but also demonstrate the involvement of homologous genomic regions in RA. This paper compares corresponding non-MHC genomic regions identified in rodent and human genome-wide association studies (GWAS). To date, over 30 non-MHC RA-associated loci have been identified in humans, and over 100 arthritis-associated loci have been identified in rodent models of RA. The genomic regions associated with the disease are designated by the name(s) of the gene having the most frequent and consistent RA-associated SNPs or a function suggesting their involvement in inflammatory or autoimmune processes. Animal studies on rats and mice preferentially have used single sequence length polymorphism (SSLP) markers to identify disease-associated qualitative and quantitative trait loci (QTLs) in the genome of F2 hybrids of arthritis-susceptible and arthritis-resistant rodent strains. Mouse GWAS appear to be far ahead of rat studies, and significantly more mouse QTLs correspond to human RA risk alleles

Suppression of Proteoglycan-Induced Autoimmune Arthritis by Myeloid-Derived Suppressor Cells Generated <i>In Vitro</i> from Murine Bone Marrow

<div><p>Background</p><p>Myeloid-derived suppressor cells (MDSCs) are innate immune cells capable of suppressing T-cell responses. We previously reported the presence of MDSCs with a granulocytic phenotype in the synovial fluid (SF) of mice with proteoglycan (PG)-induced arthritis (PGIA), a T cell-dependent autoimmune model of rheumatoid arthritis (RA). However, the limited amount of SF-MDSCs precluded investigations into their therapeutic potential. The goals of this study were to develop an in vitro method for generating MDSCs similar to those found in SF and to reveal the therapeutic effect of such cells in PGIA.</p><p>Methods</p><p>Murine bone marrow (BM) cells were cultured for 3 days in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin-6 (IL-6), and granulocyte colony-stimulating factor (G-CSF). The phenotype of cultured cells was analyzed using flow cytometry, microscopy, and biochemical methods. The suppressor activity of BM-MDSCs was tested upon co-culture with activated T cells. To investigate the therapeutic potential of BM-MDSCs, the cells were injected into SCID mice at the early stage of adoptively transferred PGIA, and their effects on the clinical course of arthritis and PG-specific immune responses were determined.</p><p>Results</p><p>BM cells cultured in the presence of GM-CSF, IL-6, and G-CSF became enriched in MDSC-like cells that showed greater phenotypic heterogeneity than MDSCs present in SF. BM-MDSCs profoundly inhibited both antigen-specific and polyclonal T-cell proliferation primarily via production of nitric oxide. Injection of BM-MDSCs into mice with PGIA ameliorated arthritis and reduced PG-specific T-cell responses and serum antibody levels.</p><p>Conclusions</p><p>Our in vitro enrichment strategy provides a SF-like, but controlled microenvironment for converting BM myeloid precursors into MDSCs that potently suppress both T-cell responses and the progression of arthritis in a mouse model of RA. Our results also suggest that enrichment of BM in MDSCs could improve the therapeutic efficacy of BM transplantation in RA.</p></div

Suppression of antigen (Ag)-specific and non-specific T-cell responses by BM-MDSCs.

<p>(<b>A</b>) T cells, purified from the spleens of mice expressing a PG-specific T cell receptor transgene (PG-TCR-Tg) were cultured for 5 days with dendritic cells (DCs) loaded with recombinant G1 domain of human PG (rhG1) in the absence or presence of the following “suppressors”: BM-MDSCs (red bar), arthritic SF cells (gray bar), or Ly6C^hi (monocytic) cell-depleted BM-MDSCs (black bar). The ability of suppressors to inhibit Ag (rhG1)-specific T-cell proliferation (which is also dependent on Ag presentation by DCs) was assessed on the basis of inhibition of [³H]thymidine incorporation by the T cells. Percent suppression was calculated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111815#s2" target="_blank">Methods</a>. All suppressors exhibited robust inhibition of T-cell proliferation. The results shown are from 5 independent experiments. (<b>B</b>) T cells from PG-TCR-Tg mice were cultured for 2 days with rhG1-loaded DCs and BM-MDSCs as described for panel A. The percent of CD4⁺ T cells containing IFNγ, IL-10, or FoxP3 (CD4⁺CD25⁺FoxP3⁺ T regulatory cells, Tregs) was determined by flow cytometry. The results shown are the individual values (n = 5–6) and the means. On average, the percentages of IFNγ⁺ cells, IL-10⁺ cells, and Tregs were lower in the presence of BM-MDSCs (*p<0.001, 0.001, and 0.05, respectively; Mann-Whitney U test) than in their absence (None). (<b>C</b>) T cells from PG-TCR-Tg mice were cultured in anti-CD3/CD28-coated plates for 4 days in the absence or presence of the listed suppressors. Percent suppression was calculated and results expressed as described for panel A. Non-depleted BM-MDSCs and BM-MDSCs depleted in Ly6C^hi cells were equally potent in suppressing anti-CD3/CD28-induced T-cell proliferation, while arthritic SF cells exhibited much weaker inhibition (*p<0.01, n = 5; Kruskal-Wallis test followed by Dunn’s multiple comparisons test) in this induction system.</p

Effects of BM-MDSCs on arthritis severity and Ag (PG)-specific immune responses in SCID mice with PGIA.

<p>(<b>A</b>) Effect of BM-MDSC transfer on arthritis severity. Arthritis was induced in SCID mice via 2 transfers of spleen cells (black arrows) from wild type mice with PGIA as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111815#s2" target="_blank">Methods</a>. At the early phase of arthritis, one group of the SCID recipients was co-injected with BM-MDSCs (red arrow). Disease severity scores were monitored until day 34. Arthritis progressed rapidly in the control group (black line), but not in the BM-MDSC-treated group (red line) (*p<0.05, n = 10 mice/group; two-way repeated measures analysis of variance). (<b>B</b>) Joint histopathology of control (left panel) and BM-MDSC-treated (right panel) mice on day 34. The ankle joint of the control mouse demonstrated massive leukocyte infiltration (star) in the joint cavity (JC) and synovial tissue (ST) as well as synovial hyperplasia. The articulating surfaces appeared rough due to cartilage damage. In the ankle joint of the BM-MDSC-treated mouse only mild synovial hyperplasia was seen, suggesting the resolution of initial (previous) inflammation. Representative hematoxylin-eosin-stained tissue sections from both groups are shown. (<b>C</b>) Antigen (PG)-specific T-cell responses of control and BM-MDSC-treated mice. T-cell responses were compared between the two groups on day 34 by measuring spleen cell proliferation in the presence or absence of PG in vitro. Results are expressed as stimulation index (SI), a ratio of [³H]thymidine incorporation by PG-stimulated and non-stimulated cultures. The SI of the BM-MDSC-injected group (red bar) was significantly lower than the SI of the control group (black bar) (*p<0.0001, n = 10 mice/group; Student’s t test). (<b>D</b>) Serum levels of anti-PG antibodies in the control and BM-MDSC-treated groups as determined by ELISA. The levels of IgG1 anti-PG antibodies (top) were significantly lower in the sera of BM-MDSC-injected mice than in control mice (*p<0.01, n = 5 samples/group; Mann-Whitney U test), while the levels of IgG2a anti-PG antibodies (bottom) were similar.</p

Osteoarthritis-like pathologic changes in the knee joint induced by environmental disruption of circadian rhythms is potentiated by a high-fat diet.

A variety of environmental factors contribute to progressive development of osteoarthritis (OA). Environmental factors that upset circadian rhythms have been linked to various diseases. Our recent work establishes chronic environmental circadian disruption - analogous to rotating shiftwork-associated disruption of circadian rhythms in humans - as a novel risk factor for the development of OA. Evidence suggests shift workers are prone to obesity and also show altered eating habits (i.e., increased preference for high-fat containing food). In the present study, we investigated the impact of chronic circadian rhythm disruption in combination with a high-fat diet (HFD) on progression of OA in a mouse model. Our study demonstrates that when mice with chronically circadian rhythms were fed a HFD, there was a significant proteoglycan (PG) loss and fibrillation in knee joint as well as increased activation of the expression of the catabolic mediators involved in cartilage homeostasis. Our results, for the first time, provide the evidence that environmental disruption of circadian rhythms plus HFD potentiate OA-like pathological changes in the mouse joints. Thus, our findings may open new perspectives on the interactions of chronic circadian rhythms disruption with diet in the development of OA and may have potential clinical implications

Expression and activity of iNOS in BM-MDSCs.

<p>(<b>A</b>) Comparison of murine iNOS (<i>Nos2</i>) transcript levels in BM-MDSCs and spleen cells revealed that iNOS mRNA was upregulated in BM-MDSCs. The housekeeping gene (<i>Actb</i>, encoding β-actin) was expressed at equal levels. Results of one of 2 replicate experiments (with similar results) are shown. (<b>B</b>) Western blot using an antibody against murine iNOS demonstrated the presence of iNOS protein in BM-MDSCs, but not in spleen cells. The β-actin control blot shows equal sample loading. One of 3 independent Western blots is shown. (<b>C</b>) iNOS activity was assayed on the basis of NO release into the supernatants of cultures containing BM-MDSCs (orange bar) or spleen cells (green bar), and expressed as total nitrate concentration (µM). BM-MDSC-containing cultures produced significantly higher amounts of NO than spleen cells did (*p<0.05, n = 5 cultures/cell type; Mann-Whitney U test). Molecular markers: bp, base pairs; kDa, kilodalton.</p

Phenotype and morphology of myeloid-derived suppressor cell (MDSC)-like cells generated in vitro from murine bone marrow (BM) in comparison with synovial fluid (SF) cells.

<p>(<b>A</b>) Phenotype of MDSCs arising from growth factor-cytokine treated BM cell cultures as determined by flow cytometry. BM cells were cultured in the presence of GM-CSF, IL-6, and G-CSF (10 ng/ml each). On day 3, cells were immunostained for CD11b, Ly6C, and Ly6G. Approximately 80% of the cells expressed the common myeloid marker CD11b (gray bar). Gating on CD11b⁺ cells revealed that the majority of them co-expressed Ly6C (marker of the “monocytic” subset) and Ly6G (marker of the “granulocytic” subset), but cells expressing only one marker were also present (black bars). The results are the means ± SEM of 7 independent BM cultures. (<b>B</b>) In SF, the vast majority of the CD11b⁺ myeloid population (gray bar) was found to be cells co-expressing Ly6G and Ly6C, and lower proportions of cells expressed Ly6C or Ly6G only (black bars) than in the BM-MDSC cell cultures. The results are the means ± SEM of 7 separate pools of SF cells freshly harvested from arthritic mouse joints. (<b>C</b>) Flow cytometry profile of BM-MDSCs (left panels) is shown as an example of subset identification after gating on CD11b⁺ cells. Fluorescence image of EGFP⁺ BM-MDSCs (middle panel) after surface staining with a blue fluorescent antibody to Ly6C and a red fluorescent antibody to Ly6G shows cells expressing one or both markers. BM for culture was obtained from an EGFP-LysM-Tg mouse expressing EGFP (green fluorescence) in myeloid cells. Imaging was performed using two-photon microscopy (TPM). Morphology of BM-MDSCs (right panel) was visualized by Wright-Giemsa staining of a cytospin preparation, which shows both polymorphonuclear granulocyte (neutrophil)-like cells (arrows) and large precursor-like cells (arrowheads). (<b>D</b>) Flow cytometry profile (left) and morphology (right) of SF cells harvested from the arthritic joints of mice with PGIA. While the CD11b⁺ myeloid population is large in both the BM-MDSC culture and arthritic SF, and is dominated by Ly6C/Ly6G double positive cells in both samples (analyzed simultaneously), BM-MDSCs show greater heterogeneity in morphology than SF cells.</p