A Novel Role of Matrix Metalloproteinase-8 in Macrophage Differentiation and Polarization - PubMed (original) (raw)

A Novel Role of Matrix Metalloproteinase-8 in Macrophage Differentiation and Polarization

Guanmei Wen et al. J Biol Chem. 2015.

Abstract

Matrix metalloproteinase-8 (MMP8) has been shown to influence various cellular functions. As monocytes and macrophages (Mφ) express MMP8, we investigated if MMP8 played a role in macrophage differentiation and polarization. MMP8 expression was significantly increased during monocyte differentiation into Mφ. Monocyte-derived Mφ from MMP8-deficient mice expressed higher levels of M1-Mφ markers but lower levels of M2-Mφ markers than monocyte-derived Mφ from wild-type mice. Although Mφ from either MMP8-deficient or wild-type mice were inducible by interferon-γ into M1-Mφ, only wild-type Mφ but not MMP8-deficient Mφ could be induced into M2-Mφ by interleukin-4. However, MMP8-deficient Mφ exposed to conditioned culture media of wild-type Mφ developed a M2-Mφ phenotype. Compared with conditioned culture media of wild-type Mφ, conditioned culture media of MMP8-deficient Mφ contained a lower concentration of active transforming growth factor-β (TGF-β), an M2-Mφ inducer. Moreover, evidence also showed that the degradation of the TGF-β sequester, fibromodulin, was modulated by MMP8. The data indicate a previously unknown role of MMP8 in M2-Mφ polarization by cleaving fibromodulin and therefore increasing the bioavailability of the M2-Mφ inducer TGF-β.

Keywords: extracellular matrix protein; inflammation; macrophage; macrophage polarization; matrix metalloproteinase (MMP); matrix metalloproteinase-8; transforming growth factor β (TGF-β).

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Figures

FIGURE 1.

MMP8 is up-regulated during Mφ differentiation. Bone marrow monocytes were induced to differentiate into Mφ by M-CSF. Total RNAs, conditioned culture medium (CM), and cell lysate were harvested at the indicated times, and subjected to RT-qPCR (A), ELISA (B), and Western blot (C) analyses, respectively. M1 (MCP-1 and Arg II) or M2 (CD206, Arg I and CD163)-Mφ genes/proteins were examined along with MMP8. D, immunofluorescence staining analyses of MMP8 expression in the 14 days of differentiated Mφ. The data presented here are an average or representative of three independent experiments. *, p < 0.05 (versus day 0).

FIGURE 2.

MMP8 is required for M2-Mφ differentiation. A, RT-qPCR analysis of expression levels of M1- and M2-Mφ genes in the day 14 of differentiated bone marrow Mφ (BMMφ). B, Western blot analyses show that the protein expression levels of Arg I, Arg II, and MMP8 in WT and MMP8KO BMMφ. C, ELISA analysis of the inflammatory cytokine levels in the culture medium conditioned by BMMφ. D, immunofluorescence staining shows that the percentage of M2-Mφ in MMP8KO (ApoE−/−/MMP8−/−) differentiated BMMφ is much lower than that of WT (ApoE−/−/MMP8+/+) BMMφ. Arrows indicate M1-Mφ (cells are positive for Arg II) or M2-Mφ (cells are positive for Arg I). E, immunofluorescence staining of MMP8 in WT and MMP8KO BMMφ. F, RT-qPCR analysis of M1- and M2-Mφ gene expression levels in the naive peritoneal Mφ (pMφ) isolated from the peritoneal cavity of WT and MMP8KO mice. G, ELISA analysis of the inflammatory cytokine levels in the culture medium conditioned by WT and MMP8-deficient pMφ. H, immunofluorescence staining shows that MMP8 deficiency results in a M1-Mφ phenotype in pMφ. The data presented here are representative or an average of three to six independent experiments. *, p < 0.05 (versus WT). Shown in panels D and H are representative images each from three independent experiments, and column charts of the percentage of M1- and M2-Mφ. *, p < 0.05 (versus WT).

FIGURE 3.

MMP8 is required for M2-Mφ polarization. A and B, IFN-γ significantly up-regulated M1-Mφ gene (A) and protein (B) expression levels in both WT and MMP8-deficient BMMφ. C and D, IL-4 significantly up-regulated M2-Mφ gene (C) and IL-10 protein (D) expression levels in WT, but not in MMP8-deficient BMMφ. E and F, IL-4 significantly increased M2-Mφ gene (E) and IL-10 protein (F) expression levels in WT, but not in MMP8 KO pMφ. G and H, IFN-γ up-regulated M1-Mφ gene (G) and protein (H) expression levels in both WT and MMP8KO pMφ. The data presented here are an average of three to four independent experiments. *, p < 0.05 (MMP8KO versus WT); #, p < 0.05 (Mφ inducers versus control).

FIGURE 4.

MMP8 plays a similar role in M2-Mφ polarization of Raw264.7 cells and BMMφ differentiated from C57BL/6 bone marrow monocytes. A, RT-qPCR analyses. RAW264.7 cells were infected with non-target or Mmp8 shRNA lentivirus and cultured in the presence of 5 ng/ml of M-CSF for 3 days. Gene expression levels for Mmp8, M1 (Arg II, Mcp-1, and _Tnf_-α), and M2-Mφ (Arg I, Cd163, and Cd206) genes were analyzed. B, IL-4 significant up-regulated M2-Mφ genes in control Raw264.7 cells, but not in Mmp8 knockdown Raw264.7 cells. C, IFN-γ significant up-regulated M1-Mφ genes. D, RT-qPCR analyses. BMMφ differentiated from C57BL/6 bone marrow monocytes were infected with non-target or Mmp8 shRNA lentivirus and cultured for another 2 days. Gene expression levels for Mmp8, M1, and M2-Mφ genes were analyzed. E, IL-4 significant up-regulated M2-Mφ genes in control BMMφ, but not in Mmp8 knockdown BMMφ. F, IFN-γ significant up-regulated M1-Mφ genes in both control and Mmp8 knockdown BMMφ. The data presented here are an average of three to four independent experiments. *, p < 0.05 (Mmp8 shRNA versus non-target shRNA); #, p < 0.05 (Mφ inducers versus control).

FIGURE 5.

Conditioned medium from WT Mφ stimulated with IL-4 rescues M2-Mφ gene expression in MMP8KO Mφ. A and B, monocytes isolated from WT and MMP8KO bone marrow were cultured in the complete medium containing 5 ng/ml of M-CSF for 7 days, followed by IL-4 polarization for 24 h, then subjected to culture medium swapping (WT/MMP8KO-CM indicates that the culture medium for WT Mφ was replaced with the conditioned medium harvested from MMP8KO Mφ, whereas KO/WT-CM indicates that the culture medium for MMP8KO Mφ was replaced with the conditioned medium harvested from WT Mφ; WT/WT-CM and KO/KO-CM indicate that there were no culture medium swapping) and cultured for a further 24 h. Total RNA and culture medium were harvested at the end of medium swapping experiments and subjected to RT-qPCR (A) and ELISA (B) analyses, respectively. C and D, peritoneal macrophages isolated from WT and MMP8KO mice were cultured overnight in the complete medium containing 5 ng/ml of M-CSF, followed by IL-4 polarization for 24 h, then subjected to culture medium swapping as described above and cultured for a further 24 h. Total RNA and culture medium were harvested and subjected to RT-qPCR (C) and ELISA (D) analyses, respectively. The data presented here are an average of five independent experiments. *, p < 0.05 (MMP8KO versus WT); #, p < 0.05 (after versus before medium swapping).

FIGURE 6.

Bioavailability of TGF-β was mediated by MMP8 during macrophage differentiation and polarization. Cells were cultured and treated as described previously. Conditioned culture medium (CM) were harvested and subjected to ELISA analyses. A, both total and active TGF-β levels were significantly increased during bone marrow macrophage differentiation from monocytes. B and C, active, not total TGF-β levels in MMP8-deficient BMMφ (B) and pMφ (C) were lower than that of WT macrophages. D, active TGF-β levels in the peritoneal cavity fluid of MMP8-deficient mice were lower than that of WT mice. E and F, active TGF-β levels in CM of WT and MMP8-deficient BMMφ (E) and pMφ (F) in response to IL-4 polarization. The data presented here are an average of three to four independent experiments. *, p < 0.05 (day 14 versus 7 or MMP8KO versus WT); #, p < 0.05 (Mφ inducers versus control). G-J, MMP8-deficiency results in higher amount of LAP on BMMφ (G and H) and pMφ (I and J). Cells were fixed and subjected to immunofluorescence staining analyses with antibody against LAP (N terminus of TGF-β1). Shown in the figure are representative images each from three independent experiments, and column charts of mean fluorescence intensity (mean ± S.E., n = 20). *, p < 0.05 (versus controls). K and L, Western analyses of the expression levels of TGF-β and pSMAD3. Proteins were harvested and subjected to Western blot analyses with the antibodies against the C terminus of TGF-β and pSMAD3 (phospho-Ser423/Ser425). Note: the molecular mass for the bands of TGF-β1 (precursor) and TGF-β1 (mature) in panel K are ∼37.5 and ∼12.5 kDa, respectively. GAPDH and total SMAD3 were included as internal controls. Shown in the figure are representative images each from three independent experiments, and column charts of relative protein levels (mean ± S.E., n = 3). *, p < 0.05 (versus WT/vehicle).

FIGURE 7.

TGF-β activity is responsible for MMP8-mediated M2 macrophage polarization. A and B, the protein levels of active TGF-β in CM of WT and MMP8-deficient BMMφ (A) and pMφ (B) after medium swapping as described in the legend to Fig. 5. C and D, exogenous active TGF-β significantly up-regulated M2-Mφ gene expression levels in both WT and MMP8-deficient BMMφ (C) and pMφ (D). The data presented here are an average of three to five independent experiments. *, p < 0.05 (MMP8KO versus WT); #, p < 0.05 (after versus before medium swapping, or Mφ inducers versus control).

FIGURE 8.

MMP8 increases TGF-β1 bioavailability and regulates M2-Mφ polarization by increasing fibromodulin cleavage. A, WT and MMP8KO BMMφ were incubated with IL-4 for 48 h. Proteins were harvested and subjected to Western blot analyses with antibodies against the C terminus of fibromodulin. Shown are representative images each from three independent experiments, and column charts of relative protein levels (mean ± S.E., n = 3). *, p < 0.05 (versus controls). B-D, MMP8 increases fibromodulin cleavage, TGF-β bioavailability, and rescues the M2-Mφ phenotype of MMP8-deficient Mφ. WT and MMP8KO BMMφ were incubated with vehicle or 50 ng/ml of MMP8 for 48 h. Cell lysates, conditioned culture medium, and total RNAs were harvested and subjected to Western blot (B), ELISA (C), and RT-qPCR (D) analyses, respectively. The data presented here are an average of three independent experiments. *, p < 0.05 (treatments versus WT/vehicle); #, p < 0.05 (MMP8 versus vehicle in MMP8KO Mφ).

FIGURE 9.

Exogenous MMP8 restores M2-Mφ polarization (functional properties) of MMP8-deficient Mφ. WT and MMP8 knock-out (MMP8KO) bone marrow (BM) Mφ were incubated with vehicle or 50 ng/ml of MMP8 for 48 h. Cells were fixed and subjected to immunofluorescence staining with antibodies against Arg I (A) and CD206 (B), respectively. Shown in the figure are representative images each from three independent experiments, and column charts of mean fluorescence intensity (MFI; mean ± S.E., n = 20) of Arg I (A) or CD206 (B) on Mφ. *, p < 0.05 (versus controls).

FIGURE 10.

Fibromodulin and TGF-β signaling play a role in IL-4-mediated M2-Mφ polarization. A, knockdown of fibromodulin-promoted M2-Mφ polarization. BMMφ were transfected as control or _Fibromodulin_-specific siRNAs, followed by IL-4 priming. Total RNAs were harvested and subjected to RT-qPCR analyses. B and C, TGF-β activation is required for IL-4-induced M2-Mφ gene expression. BMMφ were preincubated with 10 μ

SB-431542 for 3 h, followed by IL-4 priming. Cell lysate and total RNAs were harvested and subjected to Western blot (B) and RT-qPCR (C) analyses, respectively. The data presented here are an average or representative of three independent experiments. *, p < 0.05 (versus control siRNA/vehicle or DMSO/vehicle); #, p < 0.05 (versus DMSO/IL-4).

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A Novel Role of Matrix Metalloproteinase-8 in Macrophage Differentiation and Polarization - PubMed (original) (raw)