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Liposomal targeting of prednisolone phosphate to synovial lining macrophages during experimental arthritis inhibits M1 activation but does not favor M2 differentiation.

Hofkens W, Schelbergen R, Storm G, van den Berg WB, van Lent PL - PLoS ONE (2013)

Bottom Line: In vitro studies showed that Lip-PLP phagocytosed by M1 macrophages resulted in a suppression of the M1 phenotype and induction of M2 markers (IL-10, TGF-β, IL-1RII, CD163, CD206 and Ym1).This study clearly shows that systemic treatment with PLP-liposomes selectively targets synovial lining macrophages and inhibits M1 activation.In contrast to in vitro findings, PLP-liposomes do not cause a shift of synovial lining macrophages towards M2.

View Article: PubMed Central - PubMed

Affiliation: Rheumatology Research and Advanced Therapeutics, Department of Rheumatology, Radboud University Medical Centre, Nijmegen, The Netherlands.

ABSTRACT

Background: To determine the effects of liposomal targeting of prednisolone phosphate (Lip-PLP) to synovial lining macrophages on M1 and M2 polarization in vitro and during experimental arthritis.

Material and methods: Experimental arthritis (antigen and immune complex induced) was elicited in mice and prednisolone containing liposomes were given systemically. Synovium was investigated using microarray analysis, RT-PCR and histology. Bone-marrow macrophages were stimulated towards M1 using LPS and IFNγ before treatment by PLP-liposomes. M1 and M2 markers were determined using RT-PCR.

Results: Microarray analysis of biopsies of inflamed synovium during antigen induced arthritis (AIA) showed an increased M1 signature characterized by upregulation of IL-1β, IL-6 and FcγRI starting from day 1 and lasting up until day 7 after arthritis induction. The M2 signature remained low throughout the 7 day course of arthritis. Treatment of AIA with intravenously delivered Lip-PLP strongly suppressed joint swelling and synovial infiltration whereas colloidal gold containing liposomes exclusively targeted the macrophages within the inflamed synovial intima layer. In vitro studies showed that Lip-PLP phagocytosed by M1 macrophages resulted in a suppression of the M1 phenotype and induction of M2 markers (IL-10, TGF-β, IL-1RII, CD163, CD206 and Ym1). In vivo, Lip-PLP treatment strongly suppressed M1 markers (TNF-α, IL-1β, IL-6, IL-12p40, iNOS, FcγRI, Ciita and CD86) after local M1 activation of lining macrophages with LPS and IFN-γ and during experimental AIA and immune complex arthritis (ICA). In contrast, M2 markers were not significantly upregulated in antigen-induced arthritis and down regulated in immune complex arthritis.

Conclusion: This study clearly shows that systemic treatment with PLP-liposomes selectively targets synovial lining macrophages and inhibits M1 activation. In contrast to in vitro findings, PLP-liposomes do not cause a shift of synovial lining macrophages towards M2.

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Effect of Lip-PLP on M1 macrophages in vitro.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.
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pone-0054016-g003: Effect of Lip-PLP on M1 macrophages in vitro.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.

Mentions: To study the direct effect of Lip-PLP on activated macrophages, we first investigated whether liposomal PLP may alter M1 macrophages into an M2 phenotype in vitro. Bone marrow-derived macrophages (BMMs) were stimulated towards an M1 type using IFN-γ (10 ng/ml) and LPS (100 ng/ml) for 24 hours and subsequently treated with Lip-PLP for another 24 hours. Liposomes were directly engulfed by non-stimulated macrophages and M1 macrophages as measured by flow cytometry of fluorescently labeled empty and PLP-liposomes (10, 100 and 500 µg/ml, Fig. 3A). PLP-liposomes did not cause cell death as measured by trypan blue uptake and by counting living cells and flow cytometry of apoptotic cells with 7-AAD staining (data not shown).


Liposomal targeting of prednisolone phosphate to synovial lining macrophages during experimental arthritis inhibits M1 activation but does not favor M2 differentiation.

Hofkens W, Schelbergen R, Storm G, van den Berg WB, van Lent PL - PLoS ONE (2013)

Effect of Lip-PLP on M1 macrophages in vitro.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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585322&req=5

pone-0054016-g003: Effect of Lip-PLP on M1 macrophages in vitro.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.
Mentions: To study the direct effect of Lip-PLP on activated macrophages, we first investigated whether liposomal PLP may alter M1 macrophages into an M2 phenotype in vitro. Bone marrow-derived macrophages (BMMs) were stimulated towards an M1 type using IFN-γ (10 ng/ml) and LPS (100 ng/ml) for 24 hours and subsequently treated with Lip-PLP for another 24 hours. Liposomes were directly engulfed by non-stimulated macrophages and M1 macrophages as measured by flow cytometry of fluorescently labeled empty and PLP-liposomes (10, 100 and 500 µg/ml, Fig. 3A). PLP-liposomes did not cause cell death as measured by trypan blue uptake and by counting living cells and flow cytometry of apoptotic cells with 7-AAD staining (data not shown).

Bottom Line: In vitro studies showed that Lip-PLP phagocytosed by M1 macrophages resulted in a suppression of the M1 phenotype and induction of M2 markers (IL-10, TGF-β, IL-1RII, CD163, CD206 and Ym1).This study clearly shows that systemic treatment with PLP-liposomes selectively targets synovial lining macrophages and inhibits M1 activation.In contrast to in vitro findings, PLP-liposomes do not cause a shift of synovial lining macrophages towards M2.

View Article: PubMed Central - PubMed

Affiliation: Rheumatology Research and Advanced Therapeutics, Department of Rheumatology, Radboud University Medical Centre, Nijmegen, The Netherlands.

ABSTRACT

Background: To determine the effects of liposomal targeting of prednisolone phosphate (Lip-PLP) to synovial lining macrophages on M1 and M2 polarization in vitro and during experimental arthritis.

Material and methods: Experimental arthritis (antigen and immune complex induced) was elicited in mice and prednisolone containing liposomes were given systemically. Synovium was investigated using microarray analysis, RT-PCR and histology. Bone-marrow macrophages were stimulated towards M1 using LPS and IFNγ before treatment by PLP-liposomes. M1 and M2 markers were determined using RT-PCR.

Results: Microarray analysis of biopsies of inflamed synovium during antigen induced arthritis (AIA) showed an increased M1 signature characterized by upregulation of IL-1β, IL-6 and FcγRI starting from day 1 and lasting up until day 7 after arthritis induction. The M2 signature remained low throughout the 7 day course of arthritis. Treatment of AIA with intravenously delivered Lip-PLP strongly suppressed joint swelling and synovial infiltration whereas colloidal gold containing liposomes exclusively targeted the macrophages within the inflamed synovial intima layer. In vitro studies showed that Lip-PLP phagocytosed by M1 macrophages resulted in a suppression of the M1 phenotype and induction of M2 markers (IL-10, TGF-β, IL-1RII, CD163, CD206 and Ym1). In vivo, Lip-PLP treatment strongly suppressed M1 markers (TNF-α, IL-1β, IL-6, IL-12p40, iNOS, FcγRI, Ciita and CD86) after local M1 activation of lining macrophages with LPS and IFN-γ and during experimental AIA and immune complex arthritis (ICA). In contrast, M2 markers were not significantly upregulated in antigen-induced arthritis and down regulated in immune complex arthritis.

Conclusion: This study clearly shows that systemic treatment with PLP-liposomes selectively targets synovial lining macrophages and inhibits M1 activation. In contrast to in vitro findings, PLP-liposomes do not cause a shift of synovial lining macrophages towards M2.

Show MeSH
Related in: MedlinePlus