Liposomal targeting of PLP to the inflamed synovial lining strongly suppresses joint inflammation during AIA.

<p>A: Knee joint swelling as measured by ^99MTc-uptake is strongly suppressed after a single injection of Lip-PLP. B: Photomicrographs of frontal knee joint sections of mice with AIA at day 5 after treatment and naïve mice. Note that the inflammatory infiltrate is reduced in mice treated with Lip-PLP. Original magnification ×100, Asterisks points to synovial infiltrate, hash sign points to inflammatory exudates. C: Histological scoring of synovial infiltration at day 1 and day 5 after systemic treatment with Lip-PLP or saline. D: Silver staining of frontal knee joint sections of mice with AIA, treated by intravenous injection with gold-containing liposomes. Note that the silver staining of the gold particles is mostly observed within the synovial lining cells (arrows). Mice were treated at day 3 after induction of AIA. Values are the mean of 8 mice per group. Original magnification ×100; insert ×400. F = femur, JS = joint space. Statistical significance was determined by Student's t-test. * = P<0.05 compared to saline treatment.</p

Effect of Lip-PLP on M1 macrophages <i>in vitro</i>.

<p>Cells and supernatant of bone-marrow macrophages (BMM) and M1 macrophages (stimulated with LPS and IFN-γ for 24 hours) were obtained 24 hours after treatment with Lip-PLP or saline. A: Uptake of fluorescent liposomes by BMM and M1 macrophages as measured by flow cytometry. Note that uptake of liposomes is dependent on the amount of liposomes but not on PLP content. B: Protein levels of M1 cytokines TNF-α, IL-6 and IL-12 within the supernatant and surface expression of M1 marker CD86 as determined by flow cytometry. C: Gene expression of M2 markers. RE = Relative Expression compared to values of GAPDH. Data are expressed as mean +/− S.D. UD = undetectable. Three independent experiments were performed. Statistical significance was determined by Student's t-test. * = P<0.05, ** = P<0.01 compared to saline treatment.</p

Effect of Lip-PLP on M1 and M2 marker expression within the synovium during ICA.

<p>A: Photomicrographs of frontal knee joint sections of mice with ICA at day 1 after treatment. B: Histological scoring of synovial infiltration mice with ICA at day 1 after treatment with saline or Lip-PLP. C+D: Expression of M1 (C) and M2 (D) markers in the synovium during ICA. RE = Relative Expression compared to values of GAPDH. Mice were treated at day 1 after induction of ICA and biopsies were obtained at day 1 after treatment. Data are expressed as mean +/− SD of eight animals. UD = undetectable. Statistical significance was determined by Student's t-test. * = P<0.05, ** = P<0.01 compared to saline treatment.</p

Immune complexes can inhibit TLR2, 3, 4 and 7/8 induced cytokine production in gmMφ.

<p>gmMφ and mMφ were stimulated with ICs (50 µg/ml) (<b>A</b>), LPS (100 ng/ml) or LPS+ICs (<b>B</b>) and TNFα and IL-10 were measured in culture supernatants after 20 hours. (<b>C</b>) gmMφ were stimulated with Pam3CSK4 (5 µg/ml), Poly(I:C) (25 µg/ml) or R848 (2 µg/ml) in the presence or absence of ICs. After 20 hours, supernatants were collected and analyzed for TNFα levels. Figure shows data of at least 7 donors for all stimulations. *P<0.05 difference with and without IC.</p

TLR and Tck inhibition by IC is mediated via FcγRIIb and PI3K.

<p>gmMφ were pre-incubated for 30 minutes with the FcγRIIb specific blocking antibody 2B6 (10 µg/ml) or an isotype control before stimulation with IC (50 µg/ml) and LPS (100 ng/ml) (<b>A</b>) or Tck (<b>B</b>). gmMφ were pre-incubated with Wortmannin (0.1 µM) or a Cox2 inhibitor (20 µM) for 1 hr before stimulation with IC and LPS (<b>C</b>) or Tck (<b>D</b>). 20 hour supernatants were collected to analyze TNFα levels. In the graphs showing TLR stimulation (A and C) the percentage of TNFα production is plotted with the LPS only stimulation set at 100%. In the Tck graphs (B and D) absolute values are shown. All graphs show the mean and SEM of at least 3 experiments. *P<0.05.</p

gmMφ express high FcγRIIb levels, while mMφ express more FcγRIIa and FcγRIII.

<p>Monocytes were cultured for 6 days with GM-CSF or M-CSF into gmMφ and mMφ respectively. Expression of CD14, CD163 and MHC-II (<b>A</b>) and all FcγRs (<b>B</b>) was determined by flow cytometry. (<b>B</b>) For FcγR expression, representative FACS plots are shown together with bar graphs showing mean (and SEM of) MFI and percentage of positive cells from 11 donors. Histograms show isotype control (thin line) and FcγR specific antibody (solid grey). (<b>C</b>) mRNA expression of FcγRIIb1 and FcγRIIb2 were determined by qPCR and plotted as relative expression compared to GAPDH. Bars are mean and SEM of 7 donors. (<b>D</b>) gmMφ and mMφ were incubated with FITC-labeled ICs (50 µg/ml) for 30 min at 4°C for binding and 37°C for uptake. IC uptake was determined in the presence of trypan blue. Bars are mean and SEM from 4 donors. *P<0.05, **P<0.01 and ***P<0.001 compared to mφ-1.</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

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

Localization splenic GFP expression and identification of transduced cell types by cell surface marker analysis.

<p>A) Increasing doses of lentivirus encoding GFP were injected via the tail vein and GFP expression was analyzed by immunohistochemistry ten days after virus injection. Representative histological images of experiment (magnification 200×), dotted line outlines white pulpa. W  =  white pulpa, R  =  red pulpa. Arrow heads indicate GFP positive cells. B) Flow cytometric analysis of GFP expressing cells 4 and 7 days after virus injection. Data represented as average (±SEM) GFP positive cells in gate indicated on x-axis (n = 4). C) Representative plots of cell marker and GFP FACS analysis.</p