Blockade of Mast Cell Activation Reduces Cutaneous Scar Formation

Damage to the skin initiates a cascade of well-orchestrated events that ultimately leads to repair of the wound. The inflammatory response is key to wound healing both through preventing infection and stimulating proliferation and remodeling of the skin. Mast cells within the tissue are one of the first immune cells to respond to trauma, and upon activation they release pro-inflammatory molecules to initiate recruitment of leukocytes and promote a vascular response in the tissue. Additionally, mast cells stimulate collagen synthesis by dermal fibroblasts, suggesting they may also influence scar formation. To examine the contribution of mast cells in tissue repair, we determined the effects the mast cell inhibitor, disodium cromoglycate (DSCG), on several parameters of dermal repair including, inflammation, re-epithelialization, collagen fiber organization, collagen ultrastructure, scar width and wound breaking strength. Mice treated with DSCG had significantly reduced levels of the inflammatory cytokines IL-1a, IL-1b, and CXCL1. Although DSCG treatment reduced the production of inflammatory mediators, the rate of re-epithelialization was not affected. Compared to control, inhibition of mast cell activity caused a significant decrease in scar width along with accelerated collagen re-organization. Despite the reduced scar width, DSCG treatment did not affect the breaking strength of the healed tissue. Tryptase b1 exclusively produced by mast cells was found to increase significantly in the course of wound healing. However, DSCG treatment did not change its level in the wounds. These results indicate that blockade of mast cell activation reduces scar formation and inflammation without further weakening the healed wound

Blockade of mast cell activation reduces cutaneous scar formation.

Damage to the skin initiates a cascade of well-orchestrated events that ultimately leads to repair of the wound. The inflammatory response is key to wound healing both through preventing infection and stimulating proliferation and remodeling of the skin. Mast cells within the tissue are one of the first immune cells to respond to trauma, and upon activation they release pro-inflammatory molecules to initiate recruitment of leukocytes and promote a vascular response in the tissue. Additionally, mast cells stimulate collagen synthesis by dermal fibroblasts, suggesting they may also influence scar formation. To examine the contribution of mast cells in tissue repair, we determined the effects the mast cell inhibitor, disodium cromoglycate (DSCG), on several parameters of dermal repair including, inflammation, re-epithelialization, collagen fiber organization, collagen ultrastructure, scar width and wound breaking strength. Mice treated with DSCG had significantly reduced levels of the inflammatory cytokines IL-1α, IL-1β, and CXCL1. Although DSCG treatment reduced the production of inflammatory mediators, the rate of re-epithelialization was not affected. Compared to control, inhibition of mast cell activity caused a significant decrease in scar width along with accelerated collagen re-organization. Despite the reduced scar width, DSCG treatment did not affect the breaking strength of the healed tissue. Tryptase β1 exclusively produced by mast cells was found to increase significantly in the course of wound healing. However, DSCG treatment did not change its level in the wounds. These results indicate that blockade of mast cell activation reduces scar formation and inflammation without further weakening the healed wound

Toll-like receptor 4 plays an essential role in early skin wound healing

TLR4 plays a key role in the initiation of innate immunity and in the regulation of adaptive immune responses. Using microarray analysis and PCR, TLR4 expression was observed to increase in murine skin wounds at the early stages. The cellular location of TLR4 was primarily in keratinocytes at the wound edges. Closure of excisional wounds was significantly delayed in TLR4 deficient (C3H/HeJ) as compared to wild type mice, and both IL-1β and IL-6 production were significantly lower in the wounds of TLR4 deficient mice. EGF also markedly decreased in the wound edge of epidermis in TLR4 deficient mice. In vitro studies confirmed that a wound stimulus induces TLR4 mRNA expression in primary normal human epidermal keratinocytes (NHEK). In vitro injury also induced the phosphorylation of p38 and JNK MAPK and the expression of IL-1β and TNF-α by NHEK. Blockade of TLR4 delayed NHEK migration and abolished the phosphorylation of p38 and JNK MAPK, and blockade of TLR4 and/or p38/JNK abolished IL-1β production. The results suggest that inflammatory cytokine production by injured NHEK is stimulated via the TLR4-p38 and JNK MAPK signaling pathway. Together, the results provide evidence for a role of TLR4 at sites of injury, and suggest that TLR4 is an important regulator of wound inflammation

Tryptase β1 expression in skin wounds.

<p>(a). Tryptase β1 transcript levels during the course of wound healing were determined by microarray analysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085226#pone.0085226-Chen1" target="_blank">[31]</a>. Tryptase β1 gene expression significantly increased started at 6 hours after wounding, and remained increased through day 10 (p<0.05 by a one –way ANOVA test, n = 3 at each time points). (b). Western blot analysis of the protein levels of tryptase β1 in skin wounds after DSCG or PBS treatment. α-tubulin was used as a protein loading control. (c). Relative intensities of the bands shown in (b) after being normalized to α-tubulin. N: normal skin.</p

Tryptase β1 induces α-SMA and collagen I expression in dermal fibroblasts.

<p>(a). Tryptase β1 induces differentiation of dermal fibroblasts into myofibroblasts. Dermal fibroblasts were treated with tryptase β1 for 72 hours and α-SMA expression was detected using direct immunofluorescence. Green stained cells are α-SMA positive myofibroblasts. DAPI was used to counterstain nuclei (blue). (b). Percent of α-SMA positive myofibroblasts. ND: not detectable. (c). Relative mRNA expression of collagen I 24 hours after tryptase β1 treatment determined by real time PCR. Results were the averages of triplicate wells.</p

Re-epithelialization of wounds in DSCG treated mice.

<p>The rate of re-epithelialization was measured by histomorphometric analysis of tissue sections from the central portion of the wound.</p

Ultrastructural analysis of collagen fibril content.

<p>(a) Transmission Electron Microscopy of normal skin and the tongue. n = 5 for all groups. TEM was performed to examine collagen fibril structure in the dermis of control mice (upper panels) and mice treated with DSCG (lower panels). (b). The diameter of individual collagen fibrils was determined for between 1100 and 2100 fibrils per section. Two separate wound sections per mouse were examined and four micrographs per mouse were analyzed. The mean fibril diameter per mouse was calculated. The average collagen diameter (n = 5) at each time point is shown. (c). Measurement of fibrillar density after DSCG treatment. The mean area density was determined by image analysis of TEM sections of wounds from either DSCG or PBS treated mice. The average area density was calculated from 10 micrographs per mouse. Data are expressed as mean ± SEM (n = 5). *p<0.01 by t-test.</p

Microscopic assessment of scar tissue by Picrosirius Red staining.

<p>Sections of scar tissue from either DSCG treated mice or PBS treated mice at day 21 post-wounding were stained with picrosirius red and viewed under polarized light to detect collagen fibers. Mature collagen fibers appear red-orange and immature collagen fibers appear yellow and green. Results are representative to three independent experiments. Dot line framed areas are wounds.</p

Effect of mast cell inhibition with DSCG on scar width and wound breaking strength.

<p>(a) Average scar width in mice treated with DSCG compared to PBS treated mice. H & E staining was used to measure scar with at 7, 14 and 21 days post-wounding in DSCG treated mice (black bars) and control mice (white bars). N = 5. *p<0.05. (b) Effect of DSCG treatment on wound breaking strength. Incisional wounds were prepared on the back of mice treated with DSCG (black bar) or PBS (white bar). N = 10 for both groups. Wounds were excised at day 14, and two skin strips per mouse were used from the upper and lower back of the mouse. Both strips were subjected to tensiometric analysis. The bars indicate the means of wound breaking strength in grams.</p