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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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Expression of M1 and M2 markers within the inflamed synovium during AIA as determined by micro-array analysis.Gene expression was determined at day 1, 3,5 and 7 after induction of AIA. Fold increase of gene expression was compared to synovium of naïve mice. A: Expression of M1 markers (IL-1β, IL-6, FcγRI and CD86). B: Expression of M2 markers (IL-1RII, CD163, CD206, Arg1, FIZZ1 and Ym1). Note that expression of M1 markers is highly upregulated compared to M2 markers, with the exception of Arg1 and Ym1. Values are presented as the fold change in mean gene expression levels (relative to GAPDH) from mean gene expression levels of inflamed synovium (n = 8) compared to synovium drived from naïve mice (n = 3).
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pone-0054016-g001: Expression of M1 and M2 markers within the inflamed synovium during AIA as determined by micro-array analysis.Gene expression was determined at day 1, 3,5 and 7 after induction of AIA. Fold increase of gene expression was compared to synovium of naïve mice. A: Expression of M1 markers (IL-1β, IL-6, FcγRI and CD86). B: Expression of M2 markers (IL-1RII, CD163, CD206, Arg1, FIZZ1 and Ym1). Note that expression of M1 markers is highly upregulated compared to M2 markers, with the exception of Arg1 and Ym1. Values are presented as the fold change in mean gene expression levels (relative to GAPDH) from mean gene expression levels of inflamed synovium (n = 8) compared to synovium drived from naïve mice (n = 3).

Mentions: To characterize the expression of M1 and M2 markers in the inflamed synovium during experimental arthritis, we isolated messenger RNA from synovial biopsies in a standard manner [21] at various time points (days 1, 3, 5 and 7) after induction of antigen-induced arthritis (AIA). Gene expression in inflamed synovium was determined by microarray as described earlier [10] and compared with control synovium obtained from normal mouse knee joints. Microarray analysis showed that various M1 markers (IL-1β, IL-6, FcγRI and CD86) were strongly upregulated at day 1 after induction of arthritis up to day 7 (Fig. 1A). The majority of M2 markers (IL-1RII, CD163, CD206, Arg1 and Ym1) were also somewhat upregulated during the course of arthritis, although in lesser extent than the M1 markers, with the exception of Arg1 and Ym1 which were especially high at day 1 after induction of AIA (Fig. 1B). Altogether though, this suggests a shift towards a M1 signature in the inflamed synovium during AIA.


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)

Expression of M1 and M2 markers within the inflamed synovium during AIA as determined by micro-array analysis.Gene expression was determined at day 1, 3,5 and 7 after induction of AIA. Fold increase of gene expression was compared to synovium of naïve mice. A: Expression of M1 markers (IL-1β, IL-6, FcγRI and CD86). B: Expression of M2 markers (IL-1RII, CD163, CD206, Arg1, FIZZ1 and Ym1). Note that expression of M1 markers is highly upregulated compared to M2 markers, with the exception of Arg1 and Ym1. Values are presented as the fold change in mean gene expression levels (relative to GAPDH) from mean gene expression levels of inflamed synovium (n = 8) compared to synovium drived from naïve mice (n = 3).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585322&req=5

pone-0054016-g001: Expression of M1 and M2 markers within the inflamed synovium during AIA as determined by micro-array analysis.Gene expression was determined at day 1, 3,5 and 7 after induction of AIA. Fold increase of gene expression was compared to synovium of naïve mice. A: Expression of M1 markers (IL-1β, IL-6, FcγRI and CD86). B: Expression of M2 markers (IL-1RII, CD163, CD206, Arg1, FIZZ1 and Ym1). Note that expression of M1 markers is highly upregulated compared to M2 markers, with the exception of Arg1 and Ym1. Values are presented as the fold change in mean gene expression levels (relative to GAPDH) from mean gene expression levels of inflamed synovium (n = 8) compared to synovium drived from naïve mice (n = 3).
Mentions: To characterize the expression of M1 and M2 markers in the inflamed synovium during experimental arthritis, we isolated messenger RNA from synovial biopsies in a standard manner [21] at various time points (days 1, 3, 5 and 7) after induction of antigen-induced arthritis (AIA). Gene expression in inflamed synovium was determined by microarray as described earlier [10] and compared with control synovium obtained from normal mouse knee joints. Microarray analysis showed that various M1 markers (IL-1β, IL-6, FcγRI and CD86) were strongly upregulated at day 1 after induction of arthritis up to day 7 (Fig. 1A). The majority of M2 markers (IL-1RII, CD163, CD206, Arg1 and Ym1) were also somewhat upregulated during the course of arthritis, although in lesser extent than the M1 markers, with the exception of Arg1 and Ym1 which were especially high at day 1 after induction of AIA (Fig. 1B). Altogether though, this suggests a shift towards a M1 signature in the inflamed synovium during AIA.

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