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. 2015 Jan:11:37-47.
doi: 10.1016/j.actbio.2014.09.022. Epub 2014 Sep 19.

Loss of monocyte chemoattractant protein-1 alters macrophage polarization and reduces NFκB activation in the foreign body response

Laura Beth Moore  1 , Andrew J Sawyer  2 , Antonios Charokopos  3 , Eleni A Skokos  2 , Themis R Kyriakides  4
Affiliations

Loss of monocyte chemoattractant protein-1 alters macrophage polarization and reduces NFκB activation in the foreign body response

Laura Beth Moore et al. Acta Biomater. 2015 Jan.

Abstract

Implantation of biomaterials elicits a foreign body response characterized by fusion of macrophages to form foreign body giant cells and fibrotic encapsulation. Studies of the macrophage polarization involved in this response have suggested that alternative (M2) activation is associated with more favorable outcomes. Here we investigated this process in vivo by implanting mixed cellulose ester filters or polydimethylsiloxane disks in the peritoneal cavity of wild-type (WT) and monocyte chemoattractant protein-1 (MCP-1) knockout mice. We analyzed classical (M1) and alternative (M2) gene expression via quantitative polymerase chain reaction, immunohistochemistry and enzyme-linked immunosorbent assay in both non-adherent cells isolated by lavage and implant-adherent cells. Our results show that macrophages undergo unique activation that displays features of both M1 and M2 polarization including induction of tumor necrosis factor α (TNF), which induces the expression and nuclear translocation of p50 and RelA determined by immunofluorescence and Western blot. Both processes were compromised in fusion-deficient MCP-1 KO macrophages in vitro and in vivo. Furthermore, inclusion of BAY 11-7028, an inhibitor of NFκB activation, reduced nuclear translocation of RelA and fusion in WT macrophages. Our studies suggest that peritoneal implants elicit a unique macrophage polarization phenotype leading to induction of TNF and activation of the NFκB pathway.

Keywords: Foreign body giant cell; Foreign body response; Inflammation; Macrophage.

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Figures

Figure 1
Figure 1
mRNA expression of M1 and M2 polarization markers in non-adherent (lavage) and implant-adherent cells at 2, 4, and 7 days after implantation. At each time point, five mice per genotype were used. Analysis was performed in triplicate. Fold-change was determined in comparison to WT samples at 24 h. Results are given as mean (SEM) fold change and are representative of two independent experiments that were normalized relative to the housekeeping gene GAPDH (n = 5). * indicates p ≤ 0.05 when comparing lavage and adherent cells and ¶ indicates p ≤ 0.05 when comparing WT and MCP-1 KO samples, respectively.
Figure 2
Figure 2
(A) Enzyme-linked immunosorbent assay analysis of d 2 lavage fluid revealed similar levels of IL-6, IL-10 and IL-1β in WT and MCP1-KO mice. (B – C) Representative images of d 2 peritoneal implants from WT (B) and MCP-1 KO (C) mice stained with anti-IL-1β Ab. Immunoreactivity was detected using the peroxidase reaction (brown), and nuclei were counterstained with methyl green. (D) Image analysis of samples from 2,4, and 7 d revealed similar IL-1β expression in WT and MCP-1 KO mice. (n = 5). Scale bar = 50 μm.
Figure 3
Figure 3
Representative images of d 2 peritoneal implants from WT (A) and MCP-1 KO (B) mice stained with anti-TNF-α Ab. Immunoreactivity was detected using the peroxidase reaction (brown), and nuclei were counterstained with methyl green. (C) Image analysis of samples from 2, 4, and 7 d implants revealed increased expression of TNF in a temporal fashion in WT mice that was reduced in MCP-1 KO mice at all time points. (D) Enzyme-linked immunosorbent assay analysis of confirmed reduced levels of TNF in d 2 lavage fluid from MCP-1 KO. ¶ and * indicate P ≤ 0.05 when comparing levels within a group and between WT and MCP-1 KO samples, respectively. (n = 5) Scale bar = 50 μm.
Figure 4
Figure 4
IL-4-induced activation of NFkB. Representative images of untreated bone marrow derived macrophages (A, C, F) or treated with IL-4 (B, D, G) for 3 d and stained with anti p50 (A–B) or anti-RelA (C–D,F–G) are shown. Images A–D are from conventional fluorescence and F–G from confocal microscopy. Immunoreactivity was detected with FITC-conjugated secondary Ab. Cytoskeleton and nuclei were stained with rhodamine-phalloidin and DAPI, respectively. Arrows in B, C, D, G indicate nuclear translocation and cytoplasmic localization in F. Bottom images show enlarged areas defined by rectangles in F and G. (E) Western blot analysis of macrophages at d 3 following IL-4 treatment. Cells plated on either petri dish (PD) or tissue culture plastic (TC) were analyzed for the expression of phosphor-RelA. Scale bar = 50 μm (A–D, F, G).
Figure 5
Figure 5
Induction and nuclear localization of NFκB in vivo. Representative images of PDMS disks implanted in the peritoneum of WT (A, B, E, F) and MCP-1 KO (C, D, G, H) mice for 2 or 4 d and stained with anti-p50 Ab (A–D) or anti-RelA Ab (E–H). Images on the right show enlarged areas defined by rectangles in B, D, F, and H. Immunoreactivity was detected with FITC-conjugated secondary Ab. Nuclei were stained with DAPI. (A–D) Arrows indicate p50 induction (in B,D) and nuclear translocation (in B). (E–H) Arrows indicate RelA induction (in E–H) and nuclear translocation (in F). Scale bar = 50 μm.
Figure 6
Figure 6
Inhibition of NFκB reduces IL-4-induced fusion. Representative images of WT macrophages treated with IL-4 for 3 d (A, B) or 5 d (C, D) in the presence of BAY 11 (B, D) and stained with anti-RelA (A, B) or Giemsa and May Grunwald (C, D) are shown. RelA immunoreactivity was detected with FITC-conjugated secondary Ab. Cytoskeleton and nuclei were stained with rhodamine-phalloidin and DAPI, respectively. Scale bar = 50 μm. (E) Quantification of fusion was performed from randomly selected (10 per well) in triplicate wells from three independent experiments. Values represent mean + SEM, n = 3. ** * indicates p ≤ 0.0001.
Figure 7
Figure 7
Representative images of d 2 peritoneal implants from WT (A) and MCP-1 KO (B) mice stained with anti-TGF-β Ab. Immunoreactivity was detected using the peroxidase reaction (brown), and nuclei were counterstained with methyl green. (C) Image analysis of samples from 2, 4, and 7 d implants revealed increased expression of TGF-β at d 7 in WT mice, which was not evident in MCP-1 KO mice. (D) Enzyme-linked immunosorbent assay analysis of confirmed reduced levels of TGF-β in d d7 protein extracts in MCP-1 KO. ¶ and * indicate P ≤ 0.05 when comparing levels within a group and between WT and MCP-1 KO samples, respectively. (n = 5) Scale bar = 50 μm.
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