Analysis of gene expression in ZFL cell culture (A) and trout macrophage primary cell culture (B) after 16 h exposure to liposomes.

<p>NL_2,n = liposomes without immunostimulants (750 µg/ml), NL_c Dose 1 = liposomes (750 µg/ml) containing 25 µg/ml poly (I:C) and 12.5 µg/ml LPS, NL_c Dose 2 = liposomes (375 µg/ml) containing 12.5 µg/ml poly (I:C) and 6.25 µg/ml LPS, and LPS+poly (I:C) = stimulation control (25 µg/ml poly (I:C), 12.5 µg/ml LPS). Elongation factor (EF1) was used as reference gene for ZFL cells and 18S for trout macrophages. IFN (φ for ZFL and α for macrophages), GIG2, CCL4, IL-6 and TNFα abundance was analyzed by Q-PCR (left panel) and conventional PCR (right panel). Data represent means ± SD of 3 independent experiments. Values with asterisk are statistically significant relative to the control (*, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001) and values with letters (_a,_b) are statistically significant relative to NL_c Dose 1 (_a, <i>p</i><0.001, _b, <i>p</i><0.05). Differences were analyzed using One-way ANOVA and Tukey's post test. (<b>C</b>) TNFα secretion from trout macrophages stimulated with liposomes for 16 h was assessed by Western blot. NL_c Dose 2 = 375 µg/ml liposomes, 12.5 µg/ml poly (I:C), 6.25 µg/ml LPS, NL_2,n = empty liposomes (375 µg/ml) and LPS = stimulation control (6.25 µg/ml). A representative Western Blot is shown.</p

Endocytosis of NL_c formulation by trout macrophages.

<p>(<b>A</b>) Flow cytometry time-course comparison of the membrane-bound (dark grey bar) versus the endocyted liposomes (light grey bar) after incubation with 750 µg/ml liposome-encapsulated 25 µg/ml poly (I:C) and 12.5 µg/ml LPS at the indicated times. Data represent means ± SD of three independent experiments. (<b>B</b>) Effect of chemical inhibitors on the endocytosis of NL_c (750 µg/ml liposome-encapsulated 25 µg/ml poly (I:C) and 12.5 µg/ml LPS) macrophages uptake. Inhibitors were used at the following concentrations: MβCD at 5 mM, EIPA at 50 µM, sucrose at 150 mM and W at 100 nM. The uptake of cells not treated with inhibitors (NL_c bar) was used as 100% uptake control and non-treated cells were used as control (control bar). Data represent means ± SD of 3 independent experiments. Differences were analyzed using One-way ANOVA followed by Newman-Keuls post-test. *, <i>p</i><0.05; **, <i>p</i><0.01. (<b>C</b>) Confocal microscopy images of fluorescent liposomes (NL_c) endocyted by macrophages. Cells incubated 30 min, 1 h and 16 h with NL_c containing DHPE-Fluorescein (green) at a 0.05 molar ratio. Cell membranes were stained with CellMask (red) and nucleus with Hoechst (blue).</p

Characterization of liposomal formulations.

<p>(<b>A</b>) Representative Cryo-TEM image of DLPC/Chol/Cholesteryl/PEG₆₀₀-Chol (5∶3.5∶1∶0.5) liposomes extruded through a 200 nm pore size membrane. (<b>B</b>) Confocal fluorescence image of a single liposome tagged on its lipid bilayer with Marina Blue-DHPE (blue) and its corresponding fluorescence intensity profile. (<b>C</b>) Confocal fluorescence image of a single Marina Blue-labeled liposome containing AlexaFluor594-labeled LPS (red) and their corresponding fluorescence intensity profiles. (<b>D</b>) Confocal fluorescence image of a single Marina Blue-labeled liposome containing fluorescein-labeled poly (I:C) and their corresponding fluorescence intensity profiles. (<b>E</b>) Schematic representation of the liposomal IS-cocktail (NL_c) showing the presence of both encapsulated LPS (red) and poly (I:C) (green) in the lipidic bilayer of liposomes. (<b>F</b>) Confocal fluorescence image of a single liposome containing both fluorescein-labeled poly (I:C) (green) and AlexaFluor594-labeled LPS (red) and their corresponding fluorescence intensity profiles.</p

Endocytosis of NL_c formulation by ZFL cells.

<p>(<b>A</b>) Flow cytometry time-course comparison of the membrane-bound (dark grey bar) versus the endocyted liposomes (light grey bar) after incubation with NL_c (750 µg/ml liposome, 25 µg/ml poly (I:C) and 12.5 µg/ml LPS) at the indicated times. Data represent means ± SD of three independent experiments. (<b>B</b>) Effect of chemical inhibitors on the endocytosis of the NL_c (750 µg/ml liposome, 25 µg/ml poly (I:C) and 12.5 µg/ml LPS). Inhibitors were used at the following concentrations: MβCD at 5 mM, EIPA at 50 µM, sucrose at 300 mM and W at 100 nM. The uptake of cells without inhibitors (NL_c bar) was used as 100% uptake control and non-treated cells were used as control (control bar). Data represent means ± SD of three independent experiments. Differences were analyzed using One-way ANOVA followed by Tukey's post test. *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001. (<b>C</b>) Confocal microscopy images of fluorescent liposomes (NL_c) endocyted by ZFL cells. Cells were incubated for 30 min, 1.5 h and 16 h with NL_c containing DHPE-Fluorescein (green) at a 0.05 molar ratio. Cell membranes were stained with CellMask (red) and the nucleus was stained with Hoechst (blue).</p

Composition and characterization of non-loaded liposomal formulations.

<p>Composition and characterization of non-loaded liposomal formulations.</p

Cytotoxicity of NL_{2, LPS}, NL_{2, poly (I:C)}, and NL_c formulations in ZFL cells by MTT-based assay.

<p>(<b>A</b>) Viability of ZFL after 24 h incubation with liposome-encapsulated LPS (NL_{2, LPS}, green bars) at Dose 1 = 1 mg/ml liposome with 50 µg/ml LPS, Dose 2 = 0.5 mg/ml liposome with 25 µg/ml LPS and Dose 3 = 0.20 mg/ml liposome with 10 µg/ml LPS. The white bar is the empty liposome control (NL_2,n, 1 mg/ml liposome) and the blue bar is the free LPS control (50 µg/ml). (<b>B</b>) Viability of ZFL after 24 h incubation the liposome-encapsulated poly (I:C) (NL_{2, poly (I:C)}, green bars) at Dose 1 = 1.5 mg/ml liposome with 50 µg/ml poly (I:C), Dose 2 = 0.75 mg/ml liposome with 25 µg/ml poly (I:C) and Dose 3 = 0.375 mg/ml liposome with 10 µg/ml poly (I:C). The white bar is the empty liposome control treatment (NL_2,n, 1.5 mg/ml liposome) and the red bar is the non-encapsulated poly (I:C) control (50 µg/ml). (<b>C</b>) Viability of ZFL cells after 24 h incubation with liposomal LPS-poly (I:C) cocktail (NL_c, green bars) at Dose 1 = 1.5 mg/ml liposome with 50 µg/ml poly (I:C) and 25 µg/ml LPS, Dose 2 = 0.75 mg/ml liposome with 25 µg/ml poly (I:C) and 12.5 µg/ml LPS and Dose 3 = 0.375 mg/ml liposome with 12.5 µg/ml poly (I:C) and 6.25 µg/ml LPS. The white bar is the empty liposome control treatment (NL_2,n, 1.5 mg/ml liposome), the blue bar indicates the free LPS (25 µg/ml) and the red bar is the free (I:C) control (50 µg/ml). Non-treated cells were used as 100% viability control (dotted line). Data represent means ± SD of three independent experiments. Differences were analyzed using One-way ANOVA followed by Tukey's post test. **, <i>p</i><0.01; ***, <i>p</i><0.001.</p

Effects of the capture of PS-liposomes in DCs phenotype.

<p>A) DCs viability assessed by annexin V and 7aad staining. White symbols represent iDCs, before (triangles) and after the capture of PS-liposomes (squares) or PSAB-liposomes (circles), 24 hours after culture. Black symbols represent viability of mature DCs (mDCs) before (triangles) and after the capture of PS-liposomes (squares) or PSAB-liposomes (circles) after proinflammatory stimulus (LPS). Lines show the mean of at least eight independent experiments. B) Median of fluorescence intensity (MFI) for CD86, CD40, MHC Class I and MHC Class II membrane expression on DCs before and after liposome capture (white symbols) and after exposure to LPS (black symbols). Lines show the mean of at least four independent experiments. Comparisons within each group and between paired maturation conditions showed significant differences (*p<0.05, Wilcoxon test). C) Quantification of the PGE₂ production by immature DCs (iDCs) in culture medium (med, white triangles), loaded with PS-liposomes (PS-lipo, white squares) or PSAB-liposomes (PSAB-lipo, white circles), after 24 hours of culture. Data are represented as pg/10⁶ cells. Lines show the mean of a minimum of seven independent experiments. Comparisons between groups showed significant differences (**p<0.01 and *p<0.05, Wilcoxon test).</p

Liposome features.

<p>A) Cryogenic transmission electron microscopy images of PSAB-liposomes. Bar = 0.2 μm. B) Time course analysis of the capture of 100 μM OG488 labeled PS-liposomes (OG488 PS-liposome) by DCs at 37°C (white squares) and at 4°C (black squares). Results are expressed as mean±SD of three independent experiments (***p<0.001, **p<0.01, Two-way ANOVA). C) Flow cytometry contour plots of the uptake of PS-liposomes (OG488+) by DCs (CD11c+). From left to right, control DCs, DCs cocultured with OG488 PS-liposome at 4°C and at 37°C. One representative experiment of three is shown. Percentage of liposome capture (thick line) is referred to CD11c+ cell subset (thin line).</p

Use of Autoantigen-Loaded Phosphatidylserine-Liposomes to Arrest Autoimmunity in Type 1 Diabetes

<div><p>Introduction</p><p>The development of new therapies to induce self-tolerance has been an important medical health challenge in type 1 diabetes. An ideal immunotherapy should inhibit the autoimmune attack, avoid systemic side effects and allow β-cell regeneration. Based on the immunomodulatory effects of apoptosis, we hypothesized that apoptotic mimicry can help to restore tolerance lost in autoimmune diabetes.</p><p>Objective</p><p>To generate a synthetic antigen-specific immunotherapy based on apoptosis features to specifically reestablish tolerance to β-cells in type 1 diabetes.</p><p>Methods</p><p>A central event on the surface of apoptotic cells is the exposure of phosphatidylserine, which provides the main signal for efferocytosis. Therefore, phosphatidylserine-liposomes loaded with insulin peptides were generated to simulate apoptotic cells recognition by antigen presenting cells. The effect of antigen-specific phosphatidylserine-liposomes in the reestablishment of peripheral tolerance was assessed in NOD mice, the spontaneous model of autoimmune diabetes. MHC class II-peptide tetramers were used to analyze the T cell specific response after treatment with phosphatidylserine-liposomes loaded with peptides.</p><p>Results</p><p>We have shown that phosphatidylserine-liposomes loaded with insulin peptides induce tolerogenic dendritic cells and impair autoreactive T cell proliferation. When administered to NOD mice, liposome signal was detected in the pancreas and draining lymph nodes. This immunotherapy arrests the autoimmune aggression, reduces the severity of insulitis and prevents type 1 diabetes by apoptotic mimicry. MHC class II tetramer analysis showed that peptide-loaded phosphatidylserine-liposomes expand antigen-specific CD4⁺ T cells <i>in vivo</i>. The administration of phosphatidylserine-free liposomes emphasizes the importance of phosphatidylserine in the modulation of antigen-specific CD4⁺ T cell expansion.</p><p>Conclusions</p><p>We conclude that this innovative immunotherapy based on the use of liposomes constitutes a promising strategy for autoimmune diseases.</p></div

Immunotherapy using PS-liposomes filled with insulin peptides decreases T1D incidence.

<p>Cumulative incidence (percentage) of T1D in NOD mice treated with PSAB-liposomes (PSAB-lipo, circles, n = 12), PS-liposomes (PS-lipo, squares, n = 18), and sham group (triangles, n = 26). Significant differences were found when compared group treated with PSAB-liposomes <i>versus</i> sham group (*p≤0.05, Kaplan-Meier log-rank analysis).</p