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. 2019 Oct 4;9(1):14293.
doi: 10.1038/s41598-019-49435-z.

A Modified Collagen Dressing Induces Transition of Inflammatory to Reparative Phenotype of Wound Macrophages

Amitava Das  1   2 , Motaz Abas  2 , Nirupam Biswas  1   2 , Pradipta Banerjee  1   2 , Nandini Ghosh  1   2 , Atul Rawat  1 , Savita Khanna  1   2 , Sashwati Roy  1   2 , Chandan K Sen  3   4
Affiliations

A Modified Collagen Dressing Induces Transition of Inflammatory to Reparative Phenotype of Wound Macrophages

Amitava Das et al. Sci Rep. .

Abstract

Collagen containing wound-care dressings are extensively used. However, the mechanism of action of these dressings remain unclear. Earlier studies utilizing a modified collagen gel (MCG) dressing demonstrated improved vascularization of ischemic wounds and better healing outcomes. Wound macrophages are pivotal in facilitating wound angiogenesis and timely healing. The current study was designed to investigate the effect of MCG on wound macrophage phenotype and function. MCG augmented recruitment of macrophage at the wound-site, attenuated pro-inflammatory and promoted anti-inflammatory macrophage polarization. Additionally, MCG increased anti-inflammatory IL-10, IL-4 and pro-angiogenic VEGF production, indicating a direct role of MCG in resolving wound inflammation and improving angiogenesis. At the wound-site, impairment in clearance of apoptotic cell bioburden enables chronic inflammation. Engulfment of apoptotic cells by macrophages (efferocytosis) resolves inflammation via a miR-21-PDCD4-IL-10 pathway. MCG-treated wound macrophages exhibited a significantly bolstered efferocytosis index. Such favorable outcome significantly induced miR-21 expression. MCG-mediated IL-10 production was dampened under conditions of miR-21 knockdown pointing towards miR-21 as a causative factor. Pharmacological inhibition of JNK attenuated IL-10 production by MCG, implicating miR-21-JNK pathway in MCG-mediated IL-10 production by macrophages. This work provides direct evidence demonstrating that a collagen-based wound-care dressing may influence wound macrophage function and therefore modify wound inflammation outcomes.

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Conflict of interest statement

The dressing and unrestricted research development funding was provided to The Ohio State University by Southwest Technologies. CKS serves as a scientific consultant to Southwest Technologies.

Figures

Figure 1
Figure 1
MCG increased macrophage infiltration at wound-site. Wound inflammatory cells were harvested from MCG treated PVA sponges on day 3 (A,B) and day 7 (C,D) post-implantation from C57BL/6 mice. (A,C) The cells were immunostained with F4/80 and subjected to flow cytometry analysis. (B,D) F4/80+ cells were quantified from the gated cell populations at both time points. Data are mean ± SEM (n = 3); *p < 0.05 compared to cells harvested from untreated PVA sponges.
Figure 2
Figure 2
MCG attenuated mϕinf and promoted mϕheal polarization of wound macrophage in the inflammatory phase. d3 wound macrophages (CD11b+) were harvested from MCG treated PVA sponges subcutaneously implanted in C57BL/6 mice. The cells were immune-stained using PE conjugated mϕinf/mϕheal markers and co-immunostained with FITC conjugated F4/80 and subjected to flow cytometry analysis. (A) Gating strategy in which the mϕinf/mϕheal markers were determined in double positive cells (quadrant Q2). (B–G) Quantitative analysis of the expression (mean fluorescence intensity, MFI) is expressed as bar graphs for individual mϕinf/mϕheal markers. Data are mean ± SEM (n = 6); *p < 0.05 compared to cells harvested from untreated PVA sponges.
Figure 3
Figure 3
MCG induced IL-10 & VEGF release by murine wound cells. Wound inflammatory cells on d3 were harvested from MCG treated PVA sponges subcutaneously implanted in C57BL/6 mice. The wound inflammatory cells were harvested from the sponges, and subjected to ELISA for (A) IL-10 protein expression analysis. (B) d3 wound macrophages (CD11b+) were harvested from MCG treated PVA sponges subcutaneously implanted in C57BL/6 mice and subjected to ELISA for IL-10 protein expression. (C,D) d7 wound macrophages (CD11b+) were harvested from MCG treated PVA sponges subcutaneously implanted in C57BL/6 mice and subjected to ELISA for (C) IL-10 and (D) VEGF protein expression. Data are mean ± SEM (n = 5–6); *p < 0.05 compared to wound cells/macrophages harvested from untreated PVA sponges.
Figure 4
Figure 4
Direct MCG treatment to human cultured macrophages induces IL-10 & VEGF production. THP-1 cells were differentiated to macrophage with PMA (20 ng/ml, 48 h). The differentiated cells were then treated with MCG (100 mg/ml; 72 h) (A) IL-10 (B) IL-4 and (C) VEGF protein released from THP-1 differentiated human macrophages measured by ELISA. Data are mean ± SEM (n = 4–5); *p < 0.05 compared to cells harvested from untreated THP-1 cells.
Figure 5
Figure 5
MCG promotes macrophage anti-inflammatory phenotype via promoting efferocytosis-JNK-miR-21 pathway. (A) PVA sponges were treated with MCG (2.5 g/ml), implanted subcutaneously in C57BL/6 mice. Day 3 wound cells were harvested from the sponges and subjected to efferocytosis assay. Representative images showing harvested MCG-treated macrophages (green, F4/80) cultured with apoptotic thymocytes (red, CMTMR cell tracker). (B) Efferocytosis index of apoptotic thymocytes engulfed by macrophages, calculated as total number of apoptotic cells engulfed by macrophages in a field of view divided by total number of macrophage present in the same field of view. Data are mean ± SEM (n = 4); *p < 0.05 compared to control. (C) miR-21 expression in mouse inflammatory cells collected from MCG-treated sponges at day 3 post-implantation. Data are mean ± SEM (n = 4); *p < 0.05 compared to control. (D) IL-10 production in miR-21-zip cells after treatment with MCG(100 mg/ml). Data are mean ± SEM (n = 3–5); *p < 0.05 compared with MCG untreated miR-000-zip (control) cells; †p < 0.05 compared with MCG treated miR-000–zip cells. (E) IL-10 production in differentiated THP-1 cells after treatment with pharmacological JNK inhibitor (420119 JNK Inhibitor II, 20 µM) and MCG (100 mg/ml). Data are mean ± SEM (n = 4); *p < 0.05 compared with MCG untreated (control) cells; †p < 0.05 compared with MCG-treated and JNK inhibitor untreated cells. (F) IL-10 production in THP-1 cells transfected with mimic miR-21 and si-cJun followed by treatment with MCG. Data are mean ± SEM (n = 5); *p < 0.05 compared with mimic control + si control transfected cells; †p < 0.05 compared with mimic miR-21 + si control transfected cells.
Figure 6
Figure 6
Proposed mechanism of action of Modified Collagen Gel-induced resolution of inflammation.
See this image and copyright information in PMC

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