Levels of IL-10 mRNA, intracellular IL-10 production and SOCS expression

<p>(<b>A</b>)<b>.</b> Levels of IL-10 mRNA expression in lymph nodes at day 42 in LNT-GFP or LNT-IL-10 mice. (<b>B</b>) The amount of IL-10/cell measured as geometric mean flourescent intensity (MFI) in lymph node CD19⁺MHC II⁺B cells, (<b>C</b>) in lymph node CD19^-MHC II⁺non-B APCs (<b>D</b>) in splenic B cells, (<b>E</b>) in splenic non-B APCs. (<b>F</b>) Typical gating for intracellular cytokine staining showing one sample from an LNT-GFP mouse and an LNT-IL-10 mouse (<b>G</b>) Levels of mRNA SOCS1 and 3 expression in draining lymph nodes at day 42. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049731#pone-0049731-g002" target="_blank">figure 2A–E and G</a> data were analysed by Mann-Whitney U-test. Closed circles represents LNT-GFP and open circles LNT-IL-10 mice.</p

Levels of IL-6 and anti-CII antibodies

<p>(<b>A</b>) Serum protein levels of IL-6 (<b>B</b>) and serum levels of anti-CII IgG were analysed at days 29 and 42 after CII immunisation. Analysed by Mann-Whitney U-test. Closed circles represents LNT-GFP and open circles LNT-IL-10 mice.</p

Disease-Dependent Local IL-10 Production Ameliorates Collagen Induced Arthritis in Mice

<div><p>Rheumatoid arthritis (RA) is a chronic destructive autoimmune disease characterised by periods of flare and remission. Today’s treatment is based on continuous immunosuppression irrespective of the patient’s inflammatory status. When the disease is in remission the therapy is withdrawn but withdrawal attempts often results in inflammatory flares, and re-start of the therapy is commenced when the inflammation again is prominent which leads both to suffering and increased risk of tissue destruction. An attractive alternative treatment would provide a disease-regulated therapy that offers increased anti-inflammatory effect during flares and is inactive during periods of remission. To explore this concept we expressed the immunoregulatory cytokine interleukin (IL)-10 gene under the control of an inflammation dependent promoter in a mouse model of RA - collagen type II (CII) induced arthritis (CIA). Haematopoetic stem cells (HSCs) were transduced with lentiviral particles encoding the IL-10 gene (LNT-IL-10), or a green fluorescence protein (GFP) as control gene (LNT-GFP), driven by the inflammation-dependent IL-1/IL-6 promoter. Twelve weeks after transplantation of transduced HSCs into DBA/1 mice, CIA was induced. We found that LNT-IL-10 mice developed a reduced severity of arthritis compared to controls. The LNT-IL-10 mice exhibited both increased mRNA expression levels of IL-10 as well as increased amount of IL-10 produced by B cells and non-B APCs locally in the lymph nodes compared to controls. These findings were accompanied by increased mRNA expression of the IL-10 induced suppressor of cytokine signalling 1 (SOCS1) in lymph nodes and a decrease in the serum protein levels of IL-6. We also found a decrease in both frequency and number of B cells and serum levels of anti-CII antibodies. Thus, inflammation-dependent IL-10 therapy suppresses experimental autoimmune arthritis and is a promising candidate in the development of novel treatments for RA.</p> </div

Lentiviral gene constructs and clinical development of arthritis.

<p>(<b>A</b>) Lentiviral constructs: LNT-GFP and LNT-IL-10. LTR; long terminal repeat, cPPT; central polypurine tract, pA; polyadenylic acid tail, WPRE; Woodchuck post-transcriptional regulatory element, IL-1E; Interleukin-1 enhancer, IL-6 promoter. (<b>B</b>) Severity of arthritis (mean arthritis score ± SEM). LNT-GFP (day 0–42 n = 18, day 44–49 n = 10) and LNT-IL-10 (day 0–42 n = 25, day 44–49 n = 14)). (<b>C</b>) Histopathological severity of synovitis and cartilage and bone erosivity measured as histological severity score (Y-axis) ranging from 0–3. Data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049731#pone-0049731-g001" target="_blank">figure 1B and C</a> were analysed by Mann-Whitney U-test. Closed circles represents LNT-GFP and open circles LNT-IL-10 mice. Bars in 1C represent the median.</p

Adoptive transfer of T cells, B cells and APCs.

<p><b>(A)</b> Design of experiment. Adoptive transfer of sorted T cells, B cells and APCs from LNT-Ctrl (n = 5) and LNT-CII (n = 6) mice respectively were transferred into naive recipients 2 days before CII immunization. <b>(B)</b> Severity of arthritis after immunization in mice receiving T cells (LNT-Ctrl n = 6, LNT-CII n = 6). <b>(C)</b> B cells (LNT-Ctrl n = 7, LNT-CII n = 7) or <b>(D)</b> APCs (LNT-Ctrl n = 6, LNT-CII n = 4). <b>(E)</b> Histopathological examination of synovitis and bone/cartilage erosivity. The experiments have been performed once. Statistical analysis was performed using linear regression for comparing development of arthritis, Mann-Whitney U-test for comparing histopathological score, mean±SEM.</p

qPCR array and SOCS1 association with LNT-Ctrl vs LNT-CII at day 3 after CII immunization.

<p><b>(A)</b> OPLS-DA scatter dot plot showing the separation of gene expression in tolerized or non-tolerized mice and <b>(B)</b> OPLS-DA column loading plot that depicts the association between LNT-CII and LNT-Ctrl mice with the expression of different genes. X-variables represented with a positive bar are positively associated with LNT-CII mice, whereas variables in the opposite direction are inversely related to this group of mice. The larger the bar and smaller the error bar, the stronger and more certain is the contribution to the model. The final OPLS-DA loading plots are based on parameters with VIP values ≥1.3. R2Y indicates how well the variation of Y is explained, whereas Q2 indicates how well Y can be predicted. Univariate analysis (Student’s t-test) of <b>(C)</b> SOCS1 <b>(D)</b> of IL-10 <b>(E)</b> IFN-γ <b>(F)</b> IL-6, n = 2–4 animals/group, mean ± SEM. <i>Actb</i> and <i>Gadph</i> were selected as housekeeping genes and relative quantification was calculated from the sample of a naïve DBA/1 mouse.</p

Involvement of T cells in tolerance induction.

<p><b>(A)</b> Capacity of Tregs cells to down-regulate effector T cell responses. Sorted Tregs cells from LNT-Ctrl or LNT-CII mice were co-cultured with LNT-Ctrl T effector cells in ratios 1:1, 1:5 and 1:10 for three days and proliferation measured by radioactive thymidine incorporation (counts per minutes). Y-axis—percentage of proliferation compared to the negative control containing sorted T effector cells only (n = 6/group). The experiment has been repeated three times. <b>(B)</b> CII-specific IFN-γ production from stimulated spleen cells obtained 14 days after CII immunization, n = 4–5 and repeated once. Statistical analysis was performed using Two-way ANOVA for evaluation of proliferative responses in (A) and Mann-Whitney U-test in (B), mean±SEM.</p

Additional file 3: of Collagen epitope expression on B cells is sufficient to confer tolerance to collagen-induced arthritis

is Figure S3 showing FACS plots for adoptive transfer of Tregs or B cells during, and after, sort. (EPS 2857 kb