Interleukin-10: an anti-inflammatory marker to target atherosclerotic lesions via PEGylated liposomes

Gunter Almer, Daniela Frascione, Isabella Pali-Schöll, Caroline Vonach, Anna Lukschal, Caroline Stremnitzer, Susanne C Diesner, Erika Jensen-Jarolim, Ruth Prassl, Harald Mangge, Gunter Almer, Daniela Frascione, Isabella Pali-Schöll, Caroline Vonach, Anna Lukschal, Caroline Stremnitzer, Susanne C Diesner, Erika Jensen-Jarolim, Ruth Prassl, Harald Mangge

Abstract

Atherosclerosis (AS) causes cardiovascular disease, which leads to fatal clinical end points like myocardial infarction or stroke, the most prevalent causes of death in developed countries. An early, noninvasive method of detection and diagnosis of atherosclerotic lesions is necessary to prevent and treat these clinical end points. Working toward this goal, we examined recombinant interleukin-10 (IL-10), stealth liposomes with nanocargo potency for NMRI relevant contrast agents, and IL-10 coupled to stealth liposomes in an ApoE-deficient mouse model using confocal laser-scanning microscopy (CLSM). Through ex vivo incubation and imaging with CLSM, we showed that fluorescently labeled IL-10 is internalized by AS plaques, and a low signal is detected in both the less injured aortic surfaces and the arteries of wild-type mice. In vivo experiments included intravenous injections of (i) fluorescent IL-10, (ii) IL-10 targeted carboxyfluorescin (CF-) labeled stealth liposomes, and (iii) untargeted CF-labeled stealth liposomes. Twenty-four hours after injection the arteries were dissected and imaged ex vivo. Compared to free IL-10, we observed a markedly stronger fluorescence intensity with IL-10 targeted liposomes at AS plaque regions. Moreover, untargeted CF-labeled liposomes showed only weak, unspecific binding. Neither free IL-10 nor IL-10 targeted liposomes showed significant immune reaction when injected into wild-type mice. Thus, the combined use of specific anti-inflammatory proteins, high payloads of contrast agents, and liposome particles should enable current imaging techniques to better recognize and visualize AS plaques for research and prospective therapeutic strategies.

Figures

Figure 1
Figure 1
Coupling reaction of IL-10 to liposomes. Recombinant IL-10 was coupled to PEGylated stealth liposomes via amine-reactive carboxylic acid succinimidyl (NHS) ester binding. The unbound protein was removed by dialysis.
Figure 2
Figure 2
Recombinant IL-10 is internalized by AS plaques. Aortic specimens of ApoE-deficient and wild-type mice were incubated with Atto655 labeled IL-10 (10 μg/mL, shown in green) and stained with Hoechst nucleus dye (blue). (A) Optical sections from aortic preparations projected into one plane. For each image, a series of 20–40 fluorescence images in Z (1 μm consecutive intervals) were projected in a single image. Wild-type mouse aorta (upper panels) and AS plaques of ApoE-deficient mouse aorta (lower panels) are shown. IL-10 slightly adheres to the intact aortic endothelium, but is much more strongly internalized by AS plaques. Small insets show unstained control specimens. Both bars indicate 50 μm. (B) Single vertical and orthogonal Z-plane fluorescence images from plaques stained with recombinant gAd-Atto655 (left panels) and IL-10-Atto655 (right panels) at a depth of 15 μm (marked by the dotted lines). Vertical bar indicates 35 μm. (C) One-plane fluorescence image (upper panel) of an atherosclerotic plaque from an ApoE-deficient mouse intravenously injected with IL-10. The lower panel shows an orthogonal slide of the plaque with additional anti-CD31 endothelial-cell staining (red). The yellow arrow marks comparatively less in vivo accumulation of IL-10. Both bars indicate 20 μm.
Figure 3
Figure 3
Recombinant IL-10 is localized predominantly in the foam cell area of atherosclerotic plaques. For double-staining experiments, aortic specimens of ApoE-deficient mice were coincubated with IL-10-Atto655 (10 μg/mL, shown in green) and AlexaFluor ready-labeled rat anti-mouse antibodies (5 μg/mL). Single Z-plane fluorescence images of AS plaques were taken at a depth of 8 μm (marked by the dotted lines in the orthogonal sections). (A) Double staining of IL-10 (green) with an anti-CD68 monocyte/macrophage marker (shown in red). Partial colocalization is seen in the merged images. Horizontal bar indicates 25 μm; vertical bar indicates 20 μm. (B) Double staining of IL-10 with an anti-CD4 T-cell/macrophage marker (blue). Partial colocalization is seen in the merged images. Horizontal bar indicates 25 μm.
Figure 4
Figure 4
IL-10 receptor is localized on the surface of the aortic endothelium from ApoE-deficient mice. (A) One-plane images of aortic specimens from ApoE- deficient mice coincubated with AF488 labeled anti-IL-10R antibody (20 μg/mL, green) and AF635 ready-labeled anti-CD31 antibody (5 μg/mL, red). IL-10R was detected on uninjured specimens and on atherosclerotic plaques; no direct colocalization between IL-10R and CD31 was found. Bar indicates 50 μm. (B) Single orthogonal Z-plane fluorescence images from the aortic endothelium of ApoE-deficient mice showing the localization of IL-10R on the endothelial surface. Vertical bar indicates 25 μm. (C) One-plane image of an aortic section with AS plaque showing the negative staining with an AF488 ready labeled isotype control antibody. All specimens were stained with Hoechst nucleus dye (blue).
Figure 5
Figure 5
IL-10-targeted PEGylated liposomes are internalized by atherosclerotic plaques after in vivo injection. ApoE-deficient mice were injected with CF-labeled IL-10-targeted stealth liposomes (green). After 24 h, aortic specimens were dissected, costained with AF635 ready-labeled rat anti-mouse antibodies (5 μg/mL, red) and Hoechst nucleus dye (blue), and imaged by CLSM. (A) One-plane and single orthogonal Z-plane fluorescence images from an AS plaque costained with an anti-CD68 monocyte/macrophage marker. Horizontal bar indicates 50 μm, vertical bar 25 μm. (B) The same as shown in panel A, but costained with an anti-CD31 endothelial-cell marker (upper and middle panels). The IL-10-CF-liposomes did not accumulate at the uninjured aortic endothelium (lower panel). (C) Aortic specimens of ApoE-deficient mice dissected 24 h after injection of CF-labeled untargeted liposomes. The CF-liposomes did not accumulate at the uninjured aortic endothelium (small inset) and were just slightly internalized by AS plaques. Bar in inset indicates 50 μm.
Figure 6
Figure 6
Evaluation of antiliposome-antibodies in sera. After immunization of Balb/c mice, IgM- or IgG-antibodies directed against liposomes were detected by ELISA. A significant increase of IgM-antibodies directed against liposomes was induced by immunizations with IL-10-liposomes iv and nontargeted liposomes iv. Importantly, the targeting of liposomes with IL-10 significantly reduced the increase of IgM-antibodies (**p < 0.01, ***p < 0.001). Boxes represent the range of the inner quartiles of the samples divided by the median, and whiskers represent the smallest and the highest value, respectively, of duplicates from two independent experiments.

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