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. 2019 Nov 1;8(11):527-537.
doi: 10.1089/wound.2019.0981. Epub 2019 Oct 16.

T Lymphocytes Attenuate Dermal Scarring by Regulating Inflammation, Neovascularization, and Extracellular Matrix Remodeling

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Free PMC article

T Lymphocytes Attenuate Dermal Scarring by Regulating Inflammation, Neovascularization, and Extracellular Matrix Remodeling

Xinyi Wang et al. Adv Wound Care (New Rochelle). .
Free PMC article

Abstract

Objective: While tissue injury and repair are known to involve adaptive immunity, the profile of lymphocytes involved and their contribution to dermal scarring remain unclear. We hypothesized that restoration of T cell deficiency attenuates dermal scarring. Approach: We assessed the temporal-spatial distribution of T lymphocytes and their subtypes during the physiological dermal wound repair process in mice. Also, we compared the scarring outcomes between wild-type (WT) and severe combined immunodeficient (SCID) mice, which are lymphocyte deficient. Complementary gain-of-function experiments were performed by adoptively transferring lymphocyte subsets to validate their contribution to tissue repair in wounded SCID mice. Results: CD4+ T lymphocytes were present within dermal wounds of WT mice beginning on day 1 and remained through day 30. Wounds of SCID mice exhibited accelerated closure, increased inflammation, limited neovascularization, and exacerbated scarring compared with WT mice. Conversely, transfer of either mixed B and T lymphocytes or CD4+ lymphocytes alone into SCID mice resulted in moderated healing with less inflammation, collagen deposition, and scarring than control SCID wounds. In contrast, transfer of other lymphocyte subsets, including helper T lymphocytes (CD3+CD4+CD25-), CD8+ T cells and B cells, or regulatory T lymphocytes (CD4+CD25+CD127low), did not reduce scar. Innovation: The finding that lymphocytes delay wound healing but reduce scar is novel and provides new insights into how dermal scarring is regulated. Conclusion: Our data support a suppressive role for CD4+ T cells against inflammation and collagen deposition, with protective effects in early-stage dermal wound healing. These data implicate adaptive immunity in the regulation of scarring phenotypes.

Keywords: adaptive immunity; extracellular matrix; inflammation; lymphocytes; wound healing.

Conflict of interest statement

The article was written by the authors and ghostwriting services were not used. Editorial support provided by Drs. Hector Martinez-Valdez and Monica Fahrenholtz at the Office of Surgical Research Administration at TCH.

Figures

None
Sundeep G. Keswani, MD, FACS, FAAP
<b>Figure 1.</b>
Figure 1.
CD3+ and CD4+ T lymphocytes are present throughout wound repair process up to day 30 post-wounding. (A) Representative images of IF staining of CD3+ cells in unwounded skin (day 0) and in the wounded area at day 1, 3, 7, 14, and 30 post-wounding. Epidermis (E) and dermis (D) are shown for the day 0 image, and dermis only are shown in the remaining time points. White arrows indicate positively stained cells, and white dotted lines indicate the basement membrane zone. Scale bars: 50 μm. (B) Quantification of CD3+ cells per high-power field from images in (A) Green dots/line indicate the average CD3+ T lymphocytes count in the epidermis per high-power field at the specified time points. Columns indicate the average CD3+ T lymphocytes count in dermis of the wound area at specified time points. (C) Representative images of IF staining of CD4+, CD8+, CD25+, and Foxp3+ cells in unwounded skin (day 0) and in the wounded area at day 1, 3, 7, 14, and 30 post-wounding. Epidermis (E) and dermis (D) are shown for the days 0 and 30 images, and dermis only is shown in the remaining time points. Scale bars: 50 μm. IF, immunofluorescence.
<b>Figure 2.</b>
Figure 2.
A deficiency of lymphocytes in vivo is associated with accelerated epithelization and fibrosis. (A) Representative images of H&E staining at day 7 post-wounding in WT and SCID tissue sections. Yellow arrows indicate the original wound edges, and the indigo arrows indicate the neo-epidermis ends (current closed wound edges). The neo-epidermis length is sum of left and right sides epithelial edges. Scale bar: 500 μm. (B) H&E image quantification of (A) demonstrating differences in neo-epidermis length and granulation tissue area (for thickness of the wounds). *p < 0.05. (C) White arrows indicate positively stained cells, and white dotted lines indicate the basement membrane zone. Scale bars: 75 μm. (D) Quantification of fibrosis burden of images in (C) via collagen content (trichrome), collagen bundle maturation (Herovici), and α-SMA as a percentage of total wound area. *p < 0.05. α-SMA, α-smooth muscle actin; H&E, hematoxylin and eosin; IHC, immunohistochemistry; SCID, severe combined immunodeficient; WT, wild type.
<b>Figure 3.</b>
Figure 3.
Excessive inflammation and decreased vessel formation prohibit normal wound healing in SCID mice. (A) Representative images of IHC-labeled CD45+ and F4/80+ cells on day 7 post-wounding in WT and SCID mouse tissue sections. Scale bar: 75 μm. (B) Quantification of CD45+ and F4/80+ cells in WT and SCID mouse wounds. *p < 0.05. (C) Representative images of IHC-labeled CD31+ cells at day 30 post-wounding in WT and SCID mouse tissue sections. Scale bar: 100 μm. (D) Quantification of CD31+ cells in WT and SCID mouse wounds reported as microvascular density per high-power field. *p < 0.05.
<b>Figure 4.</b>
Figure 4.
CD4+ T lymphocytes are associated with improved dermal wound healing in SCID mice. (A) Expansion level of CD3+ lymphocytes after 7 days post-adoptive transfer in each indicated condition through flow cytometry, using WT total lymphocytes as a positive control and untreated SCID lymphocytes as a negative control; n ≥ 3 per condition. (B) Representative images of IHC-labeled CD45+ and F4/80+ cells at 7 days post-wounding in untreated SCID mice and SCID mice with adoptive transfer of one of the following subsets: total lymphocytes, CD4 lymphocytes, CD4+CD25 lymphocytes, or CD4+CD25+ lymphocytes. Scale bars: 75 μm. (C) Quantification of CD45+ and F4/80+ cells in each condition from (B). *p < 0.05 compared with SCID control. (D) Representative images of immunohistochemistry (IHC)-labelled tissue sections of trichrome and Herovici at day 30 post-wounding and alpha-smooth muscle actin (α-SMA) at day 7 post-wounding from WT and SCID tissue. Scale bars: 200 μm. (E) Quantification of collagen content (trichrome) and α-SMA levels from D. *p < 0.05 compared with SCID control. (F) Representative IHC images of CD31 at 30 days post-wounding in control SCID and adoptively transferred SCID with total, CD4, CD4+CD25, or CD4+CD25+ lymphocytes. Scale bars: 200 μm. (G) Quantification of microvascular density from (F). *p < 0.05 compared with SCID control.
<b>Figure 5.</b>
Figure 5.
Schematic illustration of the timeline of T cells involved in wound healing. This schematic illustration reflects what we observed in C57BL/6J WT mouse wound healing at different time points. At all stages of wound healing, we observed T cells in various parts of the skin or wounds, indicating their important role in wound healing. Initially, we observed T cells in only the epidermis of the normal skin. After wounding, a small population of T cells was observed in the deep dermis of the wound edge during the inflammation phase, while macrophages were observed in the upper dermis. More T cells were observed during the proliferation phase, and they were located at the edge and center of the wound dermis. Although we did not observe any T cells in the newly formed epidermis at day 30 post-wounding, they can be seen in the upper dermis or close to the newly formed epidermis.

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