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 When the disease is in remission the therapy is withdrawn but withdrawal attempts often results in inflammatory flares, and re-start of the therapy is commenced when the inflammation again is prominent which leads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

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

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

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

Highly reduced Th17 differentiation in the absence of both IL-6 and IL-21 signaling.

<p>WT and IL-21R^-/- naïve T cells were stimulated for four days with a differentiation cocktail either with or without IL-6 as described in <i>Methods</i>. Representative flow cytometry dot plots reflecting the IL-17+ fraction of the CD4+ population. Gates were set at a maximum of 0.3% IL-17-positive cells in the ‘fluorescence minus one’ control per condition, without addition of IL-17A antibodies (A). Summary of the relative proportion of IL-17+ cells within the CD4+ population (B). Culture supernatant levels of IL-17 (C), IL-21 (D), and IL-22 (E). 6–10 mice/group; *p<0.05, **p<0.01, ***p<0.001 versus WT + IL-6; ^###p<0.001 versus WT—IL-6; ^p<0.05, ^^^p<0.001 versus IL-21R^-/- + IL-6; A—Kruskal-Wallis, B-D—One-way ANOVA.</p

IL-6 and IL-21 play an important role during in vivo Th17 differentiation and antibody production.

<p>AIA was induced in WT, IL-6^-/-, IL-21R^-/-, and IL-6^-/- x IL-21R^-/- mice. Draining lymph node CD4+IL-17+ cell fraction two days after arthritis induction as measured using flow cytometry (A; n = 10/group). Serum IgG1, IgG2b, and total IgG levels as measured by ELISA (B; n = 5/group). *p<0.05, **p<0.01, ***p<0.001 versus WT; ^##p<0.01 versus IL-6^-/-; ^^^p<0.001 versus IL-21R^-/-; A—One-way ANOVA, B—Kruskal-Wallis.</p

Effect of anti-IL-6 and/or anti-IL-21 treatment on Th17, Th1, and antibody development in CIA mice.

<p>Th17 (A) and Th1 (B) levels in draining lymph nodes as measured using flow cytometry, and anti-CII antibody level in serum (C) as measured by ELISA of mice with CIA receiving anti-IL-6 and/or anti-IL-21 treatment. n = 5/group; **p<0.01 versus Rat IgG1; A+C—Kruskal-Wallis, B—One-way ANOVA.</p

Antigen-induced arthritis severity is potently reduced by combinatorial blockade of IL-6 and IL-21 signaling pathways.

<p>Joint swelling of WT, IL-6^-/-, IL-21R^-/-, and IL-6^-/- x IL-21R^-/- mice at day 1 (A), day 2 (B), and day 4 (C) after arthritis induction, depicted as ratio between right and left knee joint, measured by ^99mTechnetium pertechnetate uptake in the joint (n = 6/group). Histologically scored inflammation, bone erosion (both H&E staining, scale bar 500 μM), and cartilage proteoglycan depletion (SafO staining, scale bar 200 μM) (D; n = 10/group). *p<0.05, **p<0.01, ***p<0.001 versus WT; ^#p<0.05, ^###p<0.001 versus IL-6^-/-; ^p<0.05 versus IL-21R^-/-; A+B One-way ANOVA, C+D Kruskal-Wallis.</p

Arthritis incidence and severity of mice receiving anti-IL-6 and/or anti-IL-21 treatment late in disease development.

<p>Collagen induced arthritis was initiated in DBA-1 mice, subsequently treated with anti-IL-6R antibodies and/or sIL-21R.Fc from the day of booster injection (d = 21; n = 5/group). Arthritis incidence (A) and severity (B) based on macroscopic scoring. Bone damage as measured using the Faxitron depicted as radiological damage (C). Histologically scored inflammation, bone erosion (both H&E staining, scale bar 500 μM), and cartilage proteoglycan depletion (SafO staining, scale bar 200 μM) (D). *p<0.05 versus Rat IgG1; One-way ANOVA.</p