Prevention of NKT cell activation accelerates cutaneous wound closure and alters local inflammatory signals

Abstract

We previously reported that in the absence of NKT cells, wound closure was accelerated in a murine excisional punch wound model. Here, we explored whether purposefully inhibiting NKT cell activation had similar effects on wound closure and the dermal inflammatory response to injury. We found that prevention of NKT cell activation accelerated wound closure in a dose-responsive manner. If anti-CD1d was administered before wounding, NKT cell infiltration into cutaneous wounds was diminished without quantitative changes in cellular infiltrates. Furthermore, prevention of NKT cell activation transiently enhanced the local production of a subset of chemokines, including MIP-2, MCP-1, MIP-1α, and MIP-1β, and altered the relative expression of CD69 and CXCR2 on the surface of both circulating and wound NKT cells. Taken together, these findings suggest that wounding activates NKT cells via CD1d presentation of glycolipid antigen and help further define a role for NKT cells in the regulation of wound inflammation and closure. Many soluble factors have been targeted as potential wound healing therapies, but their clinical success has been limited. Given our findings, the NKT cell may be an attractive target for wound healing therapies.

FIG. 1

Comparison of wound closure in anti-CD1d versus IgG treated mice. Mice were each given six 3mmdorsal excisional punch wounds followed immediately by either anti-CD1d mAb or control IgG (i.v.). At d 1, 5, 7, and 10 post-injury, pelts were removed and wounds were photographed from a fixed distance. Day 0 wounds are from animals euthanized immediately after wounding. Ruled lines are 1 mm apart. (Color version of figure is available online.)

FIG. 2

Measurement of wound closure in anti-CD1d versus IgG treated mice. The percent of original open wound area was compared between groups; n = 4 per group. *P < 0.05. Similar results were obtained from three separate experiments.

FIG. 3

Comparison of wound closure in various dosages of anti-CD1d versus IgG treated mice. Mice were each given six 3 mm dorsal punch excisional wounds followed immediately by either anti-CD1d mAb (at doses indicated in μg) or control IgG (i.v.). At 5 d post-injury, pelts were removed and wounds were photographed. The percent of original open wound area was compared between groups; n = 4 per group. *P < 0.05 compared with the IgG treated group. Similar results were obtained from two separate experiments.

FIG. 4

Wound chemokine content in IgG versus anti-CD1d treated mice. Mice were each given six 3 mm dorsal punch excisional wounds followed immediately by either anti-CD1d mAb or control IgG (i.v.). At 1, 3, and 7 d pos-injury, wounds were excised and homogenized. Chemokine content was determined by ELISA. Skin biopsies from uninjured animals were used as time (d) zero controls. Data are represented as mean pg chemokine per mL of homogenate (1 mL = 1 wound); n = 4 mice per group. *P < 0.05. Similar results were obtained from two separate experiments.

FIG. 5

Systemic immune consequences in IgG versus anti-CD1d treated mice. Uninjured mice were dosed with either IgG or anti-CD1d mAb; 24 h later, the animals were euthanized and splenocytes cell suspensions stained and analyzed by flow cytometry. Results are displayed as the percentage of live cells positive for the cell surface markers indicated (A). In separate experiments, uninjured mice were dosed with either IgG or anti-CD1d mAb; 24 h later, the animals were euthanized and whole blood collected via cardiac puncture. Serum was analyzed for the chemokines indicated by ELISA (B); n = 4 per group.

FIG. 6

Comparison of pre and post-wounding treatment with IgG or anti-CD1d. Mice were each given six 3 mm dorsal punch excisional wounds. At the times indicated, parallel groups of mice received either anti-CD1d mAb (100 mcg) or control IgG (i.v.). Time 0 groups received antibodies immediately following punch wounding. Three days post-injury, pelts were removed and wounds were photographed. The percent of original open wound area was compared between groups; n = 4 per group. *P < 0.05. Similar results were obtained from two separate experiments.

FIG. 7

Wound NKT cell content with varied administration times of IgG or anti-CD1d. Mice were each given six 3 mm dorsal punch excisional wounds. At the times indicated before or after wounding, parallel groups of mice received either anti-CD1d mAb or control IgG (i.v.). Three days post-injury, wounds were excised, and wound cell suspensions were stained and analyzed by flow cytometry. Results are expressed as the percentage of wound lymphocytes that are NKT cells (A), as determined by the number of Dimer+ CD3+ cells (B). The dot plot is representative of the 6 h pretreatment groups (B). n = 4 mice per group. *P < 0.05. Similar results were obtained from three separate experiments. (Color version of figure is available online.)

FIG. 8

Day 1 wound and circulating NKT cell activation status in IgG versus anti-CD1d treated mice. Parallel groups of mice received either anti-CD1d mAb or control IgG (i.v.). Six hours later, all mice were given six 3 mm dorsal punch excisional wounds. One day post injury, wounds were excised, and wound cell suspensions were stained and analyzed by flow cytometry (A), (B). Blood was also collected from each animal. After red cell lysis, cells were stained and analyzed by flow cytometry (C), (D). Data is represented as the percentage of NKT cell population that is CD69+ (A), (C) and the mean fluorescence intensity (MFI) of CD69 (B), (D). The dashed lines (C), (D) represent the percentage of CD69+ circulating NKT cells (C), and the MFI of CD69 on circulating NKT cells (D) in uninjured animals; n = 4 mice per group. Similar results were obtained from two separate experiments. *P < 0.05.

FIG. 9

Wound and circulating NKT cell CXCR2 expression. Parallel groups of mice received either anti-CD1d mAb or control IgG (i.v.). Six hours later, all mice were given six 3mmdorsal punch excisional wounds. One day post injury, wounds were excised, and cell suspensions were stained and analyzed by flow cytometry (A), (B). Blood was also collected from each animal. After red cell lysis, cells were stained and analyzed by flow cytometry (C), (D). Data is represented as the percentage of NKT cell population that is CXCR2+ (A), (C) and the mean fluorescence intensity (MFI) of CDXCR2 (B), (D). The dashed lines (C), (D) represent the percentage of CXCR2+ circulating NKT cells (C), and the MFI of CXCR2 on circulating NKT cells (D) in uninjured animals; n = 4 mice per group. Similar results were obtained from two separate experiments. *P < 0.05.