Contribution of Invariant Natural Killer T Cells to Skin Wound Healing
2015; Elsevier BV; Volume: 185; Issue: 12 Linguagem: Inglês
10.1016/j.ajpath.2015.08.012
ISSN1525-2191
AutoresHiromasa Tanno, Kazuyoshi Kawakami, Masae Ritsu, Emi Kanno, Aiko Suzuki, Rina Kamimatsuno, Naoyuki Takagi, Tomomitsu Miyasaka, Keiko Ishii, Y Imai, Ryoko Maruyama, Masahiro Tachi,
Tópico(s)Immune Response and Inflammation
ResumoIn the present study, we determined the contribution of invariant natural killer T (iNKT) cells to the skin wound healing process. In iNKT cell-deficient (Jα18KO) mice lacking iNKT cells, wound closure was significantly delayed compared with wild-type mice. Collagen deposition, expression of α-smooth muscle actin and CD31, and wound breaking strength were significantly attenuated in Jα18KO mice. The adoptive transfer of liver mononuclear cells from wild-type but not from Jα18KO or interferon (IFN)-γ gene-disrupted (IFN-γKO) mice resulted in the reversal of this impaired wound healing in Jα18KO mice. IFN-γ expression was induced in the wounded tissues, which was significantly decreased at 6, 12, and 24 hours, but increased on day 3 after wounding in Jα18KO mice. The main source of the late-phase IFN-γ production in Jα18KO mice were neutrophils rather than NK cells and T cells. Administration of α-galactosylceramide, an activator of iNKT cells, resulted in the acceleration of wound healing on day 3 in wild-type mice. This effect was not observed in IFN-γKO mice. These results indicate that iNKT cells play important roles in wound healing. The iNKT cell-induced IFN-γ production may regulate the wound healing process in the early phase. In the present study, we determined the contribution of invariant natural killer T (iNKT) cells to the skin wound healing process. In iNKT cell-deficient (Jα18KO) mice lacking iNKT cells, wound closure was significantly delayed compared with wild-type mice. Collagen deposition, expression of α-smooth muscle actin and CD31, and wound breaking strength were significantly attenuated in Jα18KO mice. The adoptive transfer of liver mononuclear cells from wild-type but not from Jα18KO or interferon (IFN)-γ gene-disrupted (IFN-γKO) mice resulted in the reversal of this impaired wound healing in Jα18KO mice. IFN-γ expression was induced in the wounded tissues, which was significantly decreased at 6, 12, and 24 hours, but increased on day 3 after wounding in Jα18KO mice. The main source of the late-phase IFN-γ production in Jα18KO mice were neutrophils rather than NK cells and T cells. Administration of α-galactosylceramide, an activator of iNKT cells, resulted in the acceleration of wound healing on day 3 in wild-type mice. This effect was not observed in IFN-γKO mice. These results indicate that iNKT cells play important roles in wound healing. The iNKT cell-induced IFN-γ production may regulate the wound healing process in the early phase. The wound healing process consists of coagulation, inflammation, proliferation, and remodeling phases, which involve a variety of cytokines and growth factors.1Robson M.C. Steed D.L. Franz M.G. Wound healing: biologic features and approaches to maximize healing trajectories.Curr Probl Surg. 2001; 38: 72-140Abstract Full Text Full Text PDF PubMed Google Scholar, 2Rumalla V.K. Borah G.L. Cytokines, growth factors, and plastic surgery.Plast Reconstr Surg. 2001; 108: 719-733Crossref PubMed Scopus (109) Google Scholar, 3Mast B.A. Schultz G.S. Interactions of cytokines, growth factors, and proteases in acute and chronic wounds.Wound Repair Regen. 1996; 4: 411-420Crossref PubMed Scopus (452) Google Scholar, 4Barrientos S. Stojadinovic O. Golinko M.S. Brem H. Tomic-Canic M. Growth factors and cytokines in wound healing.Wound Repair Regen. 2008; 16: 585-601Crossref PubMed Scopus (2392) Google Scholar Both immune and nonimmune cells, including macrophages, neutrophils, fibroblasts, vascular endothelial cells, and keratinocytes, are involved in wound healing. T cells and other immune cells may become involved at later stages such as scar formation.5Efron J.E. Frankel H.L. Lazarou S.A. Wasserkrug H.L. Barbul A. Wound healing and T-lymphocytes.J Surg Res. 1990; 48: 460-463Abstract Full Text PDF PubMed Scopus (75) Google Scholar However, research has found that the epidermal γδT cells promote wound healing by attracting macrophages to the wound bed and modulating the extracellular matrix.6Jameson J.M. Cauvi G. Sharp L.L. Witherden D.A. Havran W.L. Gammadelta T cell-induced hyaluronan production by epithelial cells regulates inflammation.J Exp Med. 2005; 201: 1269-1279Crossref PubMed Scopus (125) Google Scholar In addition, a direct comparison of T cells from acute and chronic wounds found that both αβ and γδ epidermal T cells were activated and produced growth factors only in acute wounds.7Toulon A. Breton L. Taylor K.R. Tenenhaus M. Bhavsar D. Lanigan C. Rudolph R. Jameson J. Havran W.L. A role for human skin-resident T cells in wound healing.J Exp Med. 2009; 206: 743-750Crossref PubMed Scopus (228) Google Scholar Invariant natural killer T (iNKT) cells are a unique lymphocyte subset distinct from T, B, and NK cells and are identified by the co-expression of both αβ T-cell antigen receptor and NK-cell marker. These cells are typically characterized by the expression of a single invariant α chain in the antigen receptor encoded by a rearranged Vα14-Jα18 gene segment coupled with a highly skewed β chain, such as Vβ8.2, Vβ7, or Vβ2.8Godfrey D.I. Hammond K.J. Poulton L.D. Smyth M.J. Baxter A.G. NKT cells: facts, functions and fallacies.Immunol Today. 2000; 21: 573-583Abstract Full Text Full Text PDF PubMed Scopus (763) Google Scholar, 9Kronenberg M. Gapin L. The unconventional lifestyle of NKT cells.Nat Rev Immunol. 2002; 2: 557-568Crossref PubMed Scopus (664) Google Scholar, 10Taniguchi M. Harada M. Kojo S. Nakayama T. Wakao H. The regulatory role of Valpha14 NKT cells in innate and acquired immune response.Annu Rev Immunol. 2003; 21: 483-513Crossref PubMed Scopus (609) Google Scholar α-Galactosylceramide (α-GalCer), a synthetic glycolipid originally isolated from marine sponges, is recognized in a specific manner by iNKT cells in contact with CD1d expressed on antigen-presenting cells, which results in the production of both interferon (IFN)-γ and IL-4.8Godfrey D.I. Hammond K.J. Poulton L.D. Smyth M.J. Baxter A.G. NKT cells: facts, functions and fallacies.Immunol Today. 2000; 21: 573-583Abstract Full Text Full Text PDF PubMed Scopus (763) Google Scholar, 9Kronenberg M. Gapin L. The unconventional lifestyle of NKT cells.Nat Rev Immunol. 2002; 2: 557-568Crossref PubMed Scopus (664) Google Scholar, 10Taniguchi M. Harada M. Kojo S. Nakayama T. Wakao H. The regulatory role of Valpha14 NKT cells in innate and acquired immune response.Annu Rev Immunol. 2003; 21: 483-513Crossref PubMed Scopus (609) Google Scholar iNKT cells play an important role in various aspects of the regulation and effector arms of immune responses, including the regulation of allergic and autoimmune diseases, prevention of tumor metastasis, and protection against bacterial, fungal, and parasitic infections.8Godfrey D.I. Hammond K.J. Poulton L.D. Smyth M.J. Baxter A.G. NKT cells: facts, functions and fallacies.Immunol Today. 2000; 21: 573-583Abstract Full Text Full Text PDF PubMed Scopus (763) Google Scholar, 9Kronenberg M. Gapin L. The unconventional lifestyle of NKT cells.Nat Rev Immunol. 2002; 2: 557-568Crossref PubMed Scopus (664) Google Scholar, 10Taniguchi M. Harada M. Kojo S. Nakayama T. Wakao H. The regulatory role of Valpha14 NKT cells in innate and acquired immune response.Annu Rev Immunol. 2003; 21: 483-513Crossref PubMed Scopus (609) Google Scholar, 11Tupin E. Kinjo Y. Kronenberg M. The unique role of natural killer T cells in the response to microorganisms.Nat Rev Microbiol. 2007; 5: 405-417Crossref PubMed Scopus (365) Google Scholar, 12Kawakami K. Kinjo Y. Uezu K. Yara S. Miyagi K. Koguchi Y. Nakayama T. Taniguchi M. Saito A. Monocyte chemoattractant protein-1-dependent increase of V alpha 14 NKT cells in lungs and their roles in Th1 response and host defense in cryptococcal infection.J Immunol. 2001; 167: 6525-6532Crossref PubMed Scopus (139) Google Scholar, 13Kawakami K. Yamamoto N. Kinjo Y. Miyagi K. Nakasone C. Uezu K. Kinjo T. Nakayama T. Taniguchi M. Saito A. Critical role of Valpha14+ natural killer T cells in the innate phase of host protection against Streptococcus pneumoniae infection.Eur J Immunol. 2003; 33: 3322-3330Crossref PubMed Scopus (161) Google Scholar Schneider et al14Schneider D.F. Palmer J.L. Tulley J.M. Speicher J.T. Kovacs E.J. Gamelli R.L. Faunce D.E. A novel role for NKT cells in cutaneous wound repair.J Surg Res. 2011; 168: 325-333.e1Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 15Schneider D.F. Palmer J.L. Tulley J.M. Kovacs E.J. Gamelli R.L. Faunce D.E. Prevention of NKT cell activation accelerates cutaneous wound closure and alters local inflammatory signals.J Surg Res. 2011; 171: 361-373Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar reported that when NKT cells were absent, initial wound healing was markedly accelerated. However, it is not fully understood how this particular lymphocyte subset is involved in the process of wound healing. Here, to determine whether iNKT cells mediate acute wound healing, we monitored the wound healing process after full-thickness wounding in mice lacking iNKT cells. We found that iNKT cells contribute to the skin wound healing process by inducing the early-phase production of IFN-γ. iNKT cell-deficient (Jα18KO) mice, established by targeted deletion of the Jα18 gene segment,16Cui J. Shin T. Kawano T. Sato H. Kondo E. Toura I. Kaneko Y. Koseki H. Kanno M. Taniguchi M. Requirement for Valpha14 NKT Cells in IL-12-mediated rejection of tumors.Science. 1997; 278: 1623-1626Crossref PubMed Scopus (1154) Google Scholar were kindly provided by Dr. Toshinori Nakayama (Chiba University, Chiba, Japan). IFN-γ gene-disrupted (IFN-γKO) mice were provided by Dr. Yoichiro Iwakura (Tokyo University of Science, Tokyo, Japan).17Tagawa Y. Sekikawa K. Iwakura Y. Suppression of concanavalin A-induced hepatitis in IFN-gamma(-/-) mice, but not in TNF-alpha(-/-) mice: role for IFN-gamma in activating apoptosis of hepatocytes.J Immunol. 1997; 159: 1418-1428PubMed Google Scholar These mice were backcrossed more than eight times with C57BL/6 mice. Wild-type (WT) C57BL/6 mice, purchased from CLEA Japan (Tokyo, Japan), were used as a control. Male or female mice at 6 to 10 weeks old were used in the experiments. All mice were bred in a pathogen-free environment in the Institute for Animal Experimentation, Tohoku University Graduate School of Medicine (Sendai, Japan). All experimental protocols described in the present study were approved by the Ethics Review Committee for Animal Experimentation of Tohoku University. Mice (6 to 10 weeks of age) were anesthetized with an intraperitoneal injection of 40 mg/kg sodium pentobarbital (Nembutal Injection; Dainippon Sumitomo Pharma, Osaka, Japan). The dorsal hairs were shaved to fully expose the skin, which was then rinsed with 70% ethanol. Six full-thickness wounds extending to the panniculus carnosus were created with a 3-mm–diameter biopsy punch (Kai Industries Co., Ltd., Gifu, Japan) under sterile conditions. The six wounds per mouse were at least 5 mm from each other. The injured areas were covered with a polyurethane film (Tegaderm Transparent Dressing; 3M Health Care, St. Paul, MN) and an elastic adhesive bandage (Hilate, Iwatsuki, Tokyo, Japan) for an occlusive dressing. At various time points, mice were sacrificed, and the wound tissues were collected by excising the area with the use of an 8-mm–diameter biopsy punch. The tissues were processed for histologic analysis and measurement of cytokine concentrations. α-GalCer was purchased from Funakoshi (Tokyo, Japan) and dissolved in dimethyl sulfoxide at 5 mg/mL, which was diluted with phosphate-buffered saline (PBS). The final dose for use in vivo was 0.2% dimethyl sulfoxide in PBS. Therefore, PBS that contained 0.2% dimethyl sulfoxide was used as the control vehicle. To activate iNKT cells, mice were injected intraperitoneally with α-GalCer (2 μg per mouse) on day 1 before and on day 3 after wound creation. Morphometric analysis was performed on digital images with the use of a digital camera (CX4; Ricoh, Tokyo, Japan). After the wounds were created, photographs were taken of each wound before dressing. At various time points, the polyurethane films were gently removed from the sacrificed mice, and the wounds were photographed. The wound area was quantified by tracing its margin and calculating the pixel area with the use of AxioVision imaging software Release 4.6 (Carl Zeiss Micro Imaging Japan, Tokyo, Japan). Wound healing was evaluated as percentage of wound closure, which was calculated with the use of the following formula: percentage of wound closure = (1 − wound area at the indicated time point/wound area on day 0) × 100. Mice were sacrificed before or at 6, 12, and 24 hours after wounding. The wound tissues excised with an 8-mm–diameter biopsy punch were incubated in RPMI 1640 medium (Nipro, Osaka, Japan) that was supplemented with 10% fetal calf serum (FCS; BioWest, Nuaillé, France) and 1 mg/mL dispase (Roche Diagnostics, Tokyo, Japan) at 4°C for 16 hours and then incubated with vigorous shaking in 10% FCS RPMI 1640 medium that was supplemented with 10 mmol/L HEPES (Sigma-Aldrich, St. Louis, MO) that contained 1 mg/mL collagenase, 1 mg/mL hyaluronidase (Sigma-Aldrich), and 1 mg/mL DNase I (Roche Diagnostics) at 37°C for 2 hours. After these treatments, the tissues were passed through a 70-μm nylon mesh (BD Falcon, Bedford, MA), then washed three times with 1% FCS RPMI 1640 medium and used as skin leukocyte for flow cytometric analysis. The skin leukocytes were preincubated with anti-FcγRIII monoclonal antibody (mAb), prepared from the culture supernatant fluids of hybridoma cells (clone 2.4G2) with the use of a protein G column kit (Kirkegaard & Perry Laboratories, Gaithersburg, MD), on ice for 15 minutes in PBS that contained 1% FCS and 0.1% sodium azide. The cells were stained with phosphatidylethanolamine-conjugated anti-NK1.1 mAb (clone PK136; e-Bioscience, San Diego, CA), allophycocyanin-conjugated anti-CD3ε mAb (clone 145-2C11; BioLegend, San Diego, CA), and eFluor 450-conjugated anti-CD45 mAb (clone 30-F11; e-Bioscience). In some experiments, the cells were stained with phosphatidylethanolamine-conjugated anti-CD11b (clone M1/70; BioLegend) and fluorescein isothiocyanate-conjugated anti–Gr-1 mAb (clone RB6-8C5; BioLegend). Isotype-matched antibodies were used for control staining. To stain intracellularly expressed IFN-γ, skin leukocytes were incubated in the presence of Cytofix/Cytoperm (BD Biosciences, Franklin Lakes, NJ), washed twice in BD perm/wash solution, and stained with phosphatidylethanolamine-cyanine-7–conjugated anti–IFN-γ mAb (clone XMG1.2; BioLegend) or control rat IgG. The stained cells were analyzed with a BD FACS Canto II flow cytometer (BD Bioscience). The leukocytes were analyzed as CD45+ cells, and NK cells, NKT cells, conventional T cells, and neutrophils were defined as NK1.1+CD3− cells, NK1.1+CD3+ cells, NK1.1−CD3+ cells, and Gr-1bright+CD11bbright+ cells, respectively. Liver mononuclear cells (LMNCs) were prepared as described previously with some modification.18Nakamatsu M. Yamamoto N. Hatta M. Nakasone C. Kinjo T. Miyagi K. Uezu K. Nakamura K. Nakayama T. Taniguchi M. Iwakura Y. Kaku M. Fujita J. Kawakami K. Role of interferon-gamma in Valpha14+ natural killer T cell-mediated host defense against Streptococcus pneumoniae infection in murine lungs.Microbes Infect. 2007; 9: 364-374Crossref PubMed Scopus (77) Google Scholar Briefly, livers were excised from mice anesthetized with ether and teased apart with the use of a stainless-steel mesh in RPMI 1640 medium supplemented with 5 mmol/L HEPES and 2% FCS. After washing, the pellets were suspended in 15 mL of 35% Percoll solution (Pharmacia, Uppsala, Sweden) containing 100 U/mL heparin and centrifuged at 760 × g for 15 minutes at 20°C. The pellet was then resuspended in red blood cell lysis solution (155 mmol/L NH4Cl and 17 mmol/L Tris, pH 7.2) and washed twice with RPMI 1640 that contained 1% FCS. LMNCs were injected intravenously at 1 × 106 per mouse 1 day before wound creation. Total RNA was extracted from wound tissues with the use of ISOGEN (Nippon Gene Co. Ltd., Tokyo, Japan) according to the manufacturer's instructions. The first-strand cDNA was synthesized with a PrimeScript first-strand cDNA synthesis kit (TaKaRa Bio Inc., Shiga, Japan), according to the manufacturer's instructions. Quantitative real-time RT-PCR was performed in a volume of 20 μL with the use of gene-specific primers and FastStart essential DNA green master mix (Roche Applied Science, Branford, CT) in a LightCycler nanosystem (Roche Applied Science). Primers for IFN-γ, IL-4, and hypoxanthine phosphoribosyltransferase were shown in our previous study,19Nakamura Y. Sato K. Yamamoto H. Matsumura K. Matsumoto I. Nomura T. Miyasaka T. Ishii K. Kanno E. Tachi M. Yamasaki S. Saijo S. Iwakura Y. Kawakami K. Dectin-2 deficiency promotes Th2 response and mucin production in the lungs after pulmonary infection with Cryptococcus neoformans.Infect Immun. 2015; 83: 671-681Crossref PubMed Scopus (46) Google Scholar and primers for COL1A1 were as follows: 5′-AAGACAAGGCAGCGGTGGAA-3′ (forward) and 5′-GCAGGGGACAGGAAATAGTT-3′ (reverse). Reaction efficiency with each primer set was determined with standard amplifications. Target gene expression levels and that of hypoxanthine phosphoribosyltransferase as a reference were calculated for each sample with the use of the reaction efficiency. The results were analyzed with a relative quantification procedure and are presented as expression relative to hypoxanthine phosphoribosyltransferase expression. Mice were sacrificed on day 3 after wounding, wounds were removed with an 8-mm–diameter biopsy punch, and total wound hydroxyproline content was assayed. Briefly, all wounds were homogenized and then hydrolyzed in 6N HCl for 21 hours at 120°C. The hydrolysate was neutralized with NaOH. In the next step, 2-mL aliquots were analyzed calorimetrically for hydroxyproline content after addition of 1 mL of 0.05 mol/L chloramine T (Nacalai Tesque, Kyoto, Japan), 1 mL of 3.15 mol/L perchloic acid (Nacalai Tesque), and 1 mL of 20% dimethylaminobenzaldehyde (Nacalai Tesque). The optical density of the samples was determined with a spectrophotometer at 557 nm. The wound tissues that were collected with an 8-mm–diameter biopsy punch were dissected in a caudocranial direction. The tissues were fixed with 4% paraformaldehyde-phosphate buffer solution and embedded in paraffin. Sections of 2-μm thickness were harvested from the central portion of the wound and stained with hematoxylin and eosin, according to the standard method. The extent of re-epithelialization of each wound was measured in these hematoxylin and eosin-stained sections by measuring the distance from the normal wound margin to the edge of the epithelium. The re-epithelialization index was determined on the basis of the percentage of new epithelium present in the total wound. The height of the dermis in the center of the wounds was determined on hematoxylin and eosin-stained sections. For immunohistochemistry, the sections were stained with anti-CD31 antibody (dilution 1:600; Santa Cruz Biotechnology, Santa Cruz, CA) and anti–α-smooth muscle actin (α-SMA) antibody (dilution 1:200; Vector Laboratories, Inc., Burlingame, CA) after endogenous peroxidase and nonspecific binding were blocked. They were then incubated with peroxidase-conjugated secondary antibodies (Histofine Simple Stain MAX-PO; Nichirei Bioscience, Tokyo, Japan). Control sections were treated with nonimmune IgG in place of any of the first antibodies. Five fields (0.2 mm2 in area each) were selected and analyzed under ×400 magnification (Olympus BH-2; Olympus Optical Co., Ltd., Tokyo, Japan). The wounded skin, harvested from WT and iNKT cell-deficient (Jα18KO) mice on days 7 and 10 after wounding, was excised in a strip 5 mm away from the center of the wound with the use of a no. 15 surgical blade (Feather Safety Razor Co., Ltd., Osaka, Japan). Wound breaking strength was measured with IMS-001 (Keisei Medical Industrial Co., Ltd., Tokyo, Japan), as previously described.20Chen L. Mehta N.D. Zhao Y. DiPietro L.A. Absence of CD4 or CD8 lymphocytes changes infiltration of inflammatory cells and profiles of cytokine expression in skin wounds, but does not impair healing.Exp Dermatol. 2014; 23: 189-194Crossref PubMed Scopus (49) Google Scholar Briefly, each side of the strip was pinched by a clip and then pulled at a constant speed of 3 cm/minute until it broke away. The result was expressed as the tensile force necessary to break the repaired wounds. The wound tissues were homogenated with PBS, and concentrations of transforming growth factor (TGF)-β1 and vascular endothelial growth factor (VEGF) in the supernatant fluids were measured by enzyme-linked immunosorbent assay kits (eBioscience for TGF-β1 and R&D Minneapolis, MN, for VEGF). The results were expressed as the values per wound. The detection limit was 8 pg/mL for TGF-β1 and VEGF. Data are expressed as means ± SD. Data analysis was performed with the Welch's t-test. Statistical analysis between more than two groups was performed with an analysis of variance (Dunnett's method). P < 0.05 was considered significant. To examine the possible contribution of iNKT cells to the process of wound healing, the rate of wound closure of Jα18KO mice was compared with that of WT mice. Wound closure was significantly delayed in Jα18KO mice compared with that in WT mice on days 3 and 5, although no significant difference was detected on days 7 and 10 (Figure 1, A and B). In addition, Jα18KO mice showed significantly reduced height of the dermis and impaired re-epithelialization compared with WT mice on days 5 and 7 after wounding (Figure 1, C–E). These results suggest that iNKT cells may contribute substantially to the process of wound healing in skin. We next analyzed the effect of iNKT cells on collagen deposition. The gene expression of COL1A1 was lower in Jα18KO mice than in WT mice (Figure 2A). In addition, the content of collagen-specific amino acid hydroxyproline was significantly reduced in Jα18KO mice compared with that in WT mice (Figure 2B). To further confirm the role of iNKT cells in the late-phase events, we measured the wound breaking strength on days 7 and 10 after wound creation. Consistent with the attenuation of collagen deposition, the wound breaking strength was significantly reduced in Jα18KO mice compared with that in WT mice (Figure 2, B and C). As alternate indicators of wound healing, we analyzed myofibroblast differentiation and angiogenesis. The number of both indicators was significantly decreased in Jα18KO mice (Figure 2, D and E). We next addressed the involvement of iNKT cells on synthesis of TGF-β1 and VEGF, important cytokines for granulation tissue and angiogenesis development, respectively. TGF-β1 production was significantly reduced in Jα18KO mice compared with WT mice on days 1, 2, and 3 (Figure 3A). Furthermore, VEGF production was significantly reduced in Jα18KO mice compared with WT mice on day 1, but not on days 2 and 3 (Figure 3B). In addition, LMNCs from WT mice were transferred into Jα18KO mice, and their effect on the rate of wound closure was tested. The impaired wound closure in Jα18KO mice was almost completely reversed by the transfer of LMNCs obtained from WT mice, whereas this effect was not observed with cells derived from Jα18KO mice (Figure 4A). The decreased number of α-SMA+ cells and CD31+ cells in Jα18KO mice was almost completely reversed by the transfer of LMNCs obtained from WT mice, whereas this effect was not observed with cells derived from Jα18KO mice (Figure 4, B and C). These results indicated that iNKT cells contributed to wound healing. Because iNKT cells are well known to quickly secrete a robust amount of IFN-γ and IL-4 after their activation,8Godfrey D.I. Hammond K.J. Poulton L.D. Smyth M.J. Baxter A.G. NKT cells: facts, functions and fallacies.Immunol Today. 2000; 21: 573-583Abstract Full Text Full Text PDF PubMed Scopus (763) Google Scholar, 9Kronenberg M. Gapin L. The unconventional lifestyle of NKT cells.Nat Rev Immunol. 2002; 2: 557-568Crossref PubMed Scopus (664) Google Scholar, 10Taniguchi M. Harada M. Kojo S. Nakayama T. Wakao H. The regulatory role of Valpha14 NKT cells in innate and acquired immune response.Annu Rev Immunol. 2003; 21: 483-513Crossref PubMed Scopus (609) Google Scholar we examined IFN-γ and IL-4 production in the wounded tissue. The expression of IFN-γ mRNA in the wounded tissues reached peak levels at 12 hours in WT mice, whereas IFN-γ expression in Jα18KO mice remained unchanged. In contrast, Jα18KO mice exhibited a dramatic increase in the expression of IFN-γ mRNA on day 3 (Figure 5A). In further experiments, we examined the cells contributing to the increased expression of IFN-γ on day 3 in Jα18KO mice. NK cells and T cells did not express IFN-γ, but neutrophils showed a considerable expression of this cytokine (Figure 5B). However, IL-4 mRNA expression was not detected at the wound site in two groups (data not shown). To explore if iNKT cell-derived IFN-γ contributed to wound healing, LMNCs from WT or IFN-γKO mice were transferred into Jα18KO mice. The impaired wound closure in Jα18KO mice was almost completely reversed by the transfer of LMNCs obtained from WT mice, whereas this effect was not observed with cells derived from IFN-γKO mice (Figure 5C). The decreased number of α-SMA+ cells and CD31+ cells in Jα18KO mice was almost completely reversed by the transfer of LMNCs obtained from WT mice, whereas this effect was not observed with cells derived from IFN-γKO mice (Figure 5, D and E). These results suggest that iNKT cell-derived IFN-γ contributed to wound healing. To further confirm the possible contribution of iNKT cells, we examined the effect of administration of α-GalCer, which activates iNKT cells in a specific manner, on the process of wound healing. Administration of α-GalCer significantly promoted wound closure compared with treatment with the vehicle on day 3, but not on day 5, in the early phase of wound healing (Figure 6A). Likewise, collagen deposition and the number of CD31+ cells were increased in the α-GalCer–treated group compared with the control group (Figure 6, B and E). However, the production of TGF-β1, number of α-SMA+ cells, and synthesis of VEGF was not significantly different between the two groups (Figure 6, C, D, and F, respectively). Finally, the expression of IFN-γ mRNA was significantly enhanced in the α-GalCer–treated group compared with the control group (Figure 6G), suggesting the contribution of IFN-γ. In agreement with this possibility, α-GalCer–induced promotion of wound healing was canceled in IFN-γKO mice (Figure 6H). The following are the main findings in the present study: i) Jα18KO mice show delayed wound repair compared with WT mice; ii) wound repair-related markers, including hydroxyproline content, COL1A1 expression, α-SMA+ cells, CD31+ cells, and wound breaking strength, are attenuated in Jα18KO mice; iii) TGF-β1 and VEGF production decreases in Jα18KO mice; iv) IFN-γ expression attenuates at 24 hours, but increases on day 3 in Jα18KO mice; v) the impaired wound closure in Jα18KO mice is reversed by the transfer of LMNCs obtained from WT mice, but not from Jα18KO or IFN-γKO mice; and vi) administration of α-GalCer, an activator of iNKT cells, results in accelerated wound repair. These results indicated the critical role of iNKT cells in the process of wound healing. In the present study, we demonstrated, for the first time, that iNKT cells promoted skin wound healing, which was associated with elevated collagen content and increased myofibroblasts at the wound tissues. Consistent with these findings, NKT cells were accumulated in the wound tissues (Supplemental Figure S1). It is well known that TGF-β produced by macrophages promotes the generation of myofibroblasts from some fibroblasts and increases collagen synthesis, which results in the contraction of the extracellular matrix and subsequent wound closure.21Lucas T. Waisman A. Ranjan R. Roes J. Krieg T. Müller W. Roers A. Eming S.A. Differential roles of macrophages in diverse phases of skin repair.J Immunol. 2010; 184: 3964-3977Crossref PubMed Scopus (754) Google Scholar, 22Goldberg M.T. Han Y.-P. Yan C. Shaw M.C. Garner W.L. TNF-alpha suppresses alpha-smooth muscle actin expression in human dermal fibroblasts: an implication for abnormal wound healing.J Invest Dermatol. 2007; 127: 2645-2655Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 23Gabbiani G. The myofibroblast in wound healing and fibrocontractive diseases.J Pathol. 2003; 200: 500-503Crossref PubMed Scopus (1249) Google Scholar In this study, TGF-β1 production was decreased in Jα18KO mice, suggesting that the decreased collagen deposition and number of myofibroblasts may be due to the reduction of TGF-β1 production because of the reduced number of iNKT cells. Similar results were reported, in which the expression of COL1A1, α-SMA, and TGF-β1 mRNA in the liver was reduced in CD1dKO (NKT-deficient) mice, reaching only one-fourth of that in WT mice given the same agents.24Ishikawa S. Ikejima K. Yamagata H. Aoyama T. Kon K. Arai K. Takeda K. Watanabe S. CD1d-restricted natural killer T cells contribute to hepatic inflammation and fibrogenesis in mice.J Hepatol. 2011; 54: 1195-1204Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar However, angiogenesis is also an important event in the skin wound healing process, and VEGF is essential for the development of angiogenesis.25Olsson A.-K. Dimberg A. Kreuger J. Claesson-Welsh L. VEGF receptor signalling - in control of vascular function.Nat Rev Mol Cell Biol. 2006; 7: 359-371Crossref PubMed Scopus (2440) Google Scholar Hijioka et al26Hijioka K. Sonoda K.-H. Tsutsumi-Miyahara C. Fujimoto T. Oshima Y. Taniguchi M. Ishibashi T. Investigation of the role of CD1d-restricted invariant NKT cells in experimental choroidal neovascularization.Biochem Biophys Res Commun. 2008; 374: 38-43Crossref PubMed Scopus (14) Google Scholar reported that neovascularization and VEGF expression in a photocoagulation-induced choroidal neovascularization model were substantially reduced in Jα18KO and CD1dKO mice. In this study, we also found that the number of CD31+ cells and VEGF production were decreased in Jα18KO mice. During the wound healing, macrophages and fibroblasts are the main source of TGF-β and VEGF production.4Barrientos S. Stojadinovic O. Golinko M.S. Brem H. Tomic-Canic M. Growth factors and cytokines in wound healing.Wound Repair Regen. 2008; 16: 585-601Crossref PubMed Scopus (2392) Google Scholar, 21Lucas T. Waisman A. Ranjan R. Roes J. Krieg T. Müller W. Roers A. Eming S.A. Differential roles of macrophages in diverse phases of skin repair.J Immunol. 2010; 184: 3964-3977Crossref PubMed Scopus (754) Google Scholar These results suggest that iNKT cells may promote collagen deposition, myofibroblast differentiation, and angiogenesis by induction of the production of these cytokines by macrophages and fibroblasts. The relation between skin wound healing and IFN-γ was reported, in which the impaired wound healing in diabetic rats was recovered by the injection of monocyte/macrophages stimulated with TNF-α and IFN-γ.27Gu X.Y. Shen S.E. Huang C.F. Liu Y.N. Chen Y.C. Luo L. Zeng Y. Wang A.P. Effect of activated autologous monocytes/macrophages on wound healing in a rodent model of experimental diabetes.Diabetes Res Clin Pract. 2013; 102: 53-59Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar In addition, VEGF production and phagocytic activity were increased in TNF-α plus IFN-γ–stimulated monocytes/macrophages in vitro.27Gu X.Y. Shen S.E. Huang C.F. Liu Y.N. Chen Y.C. Luo L. Zeng Y. Wang A.P. Effect of activated autologous monocytes/macrophages on wound healing in a rodent model of experimental diabetes.Diabetes Res Clin Pract. 2013; 102: 53-59Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar In contrast, Ishida et al28Ishida Y. Kondo T. Takayasu T. Iwakura Y. Mukaida N. The essential involvement of cross-talk between IFN-gamma and TGF-beta in the skin wound-healing process.J Immunol. 2004; 172: 1848-1855Crossref PubMed Scopus (208) Google Scholar showed that IFN-γ disrupted the skin wound healing process via inhibition of TGF-β production. In the present study, IFN-γ expression was substantially reduced in Jα18KO mice compared with WT mice in the early phase, suggesting that iNKT cells contribute to wound healing through inducing the early-phase production of this cytokine. In agreement with this notion, the adoptive transfer of LMNCs from WT mice, but not from Jα18KO or IFN-γKO mice, completely reversed the delayed wound healing and the impaired myofibroblast differentiation and neovascularization in Jα18KO mice, although iNKT cells were not directly transferred. By contrast, the late-phase expression of IFN-γ was substantially increased in Jα18KO mice compared with WT mice. It was also demonstrated that the main source of IFN-γ production was neutrophils rather than NK cells and T cells. It is thus evident that the role of IFN-γ differs between the early and late stage. In the early phase, IFN-γ may activate macrophage phagocytosis and promote VEGF production, but may inhibit TGF-β in the later phase, which may be involved in attenuation of the wound breaking strength. In the present study, we also tested the effect of α-GalCer administration on skin wound healing. Activation of iNKT cells by α-GalCer enhanced the wound closure, collagen deposition, CD31+ cells, and IFN-γ expression. However, the production of TGF-β1 and VEGF and the number of α-SMA+ cells were not increased in α-GalCer–administered mice. Earlier studies reported IFN-γ–induced suppression in the TGF-β production and differentiation of myofibroblasts,29Cornelissen A.M.H. Maltha J.C. Von den Hoff J.W. Kuijpers-Jagtman A.M. Local injection of IFN-gamma reduces the number of myofibroblasts and the collagen content in palatal wounds.J Dent Res. 2000; 79: 1782-1788Crossref PubMed Scopus (37) Google Scholar, 30Hinz B. Formation and function of the myofibroblast during tissue repair.J Invest Dermatol. 2007; 127: 526-537Abstract Full Text Full Text PDF PubMed Scopus (1161) Google Scholar, 31Ulloa L. Doody J. Massagué J. Inhibition of transforming growth factor-beta/SMAD signalling by the interferon-gamma/STAT pathway.Nature. 1999; 397: 710-713Crossref PubMed Scopus (721) Google Scholar which may raise a possibility that excessively produced IFN-γ by α-GalCer–activated iNKT cells obscures the promotion of TGF-β production and the increase in α-SMA+ cells during the process of wound healing. Although the reason for the failure of α-GalCer to promote all wound healing-related events remains unclear, this mechanism might be involved in the inconsistent effects of iNKT cell activation. In earlier studies, Schneider et al14Schneider D.F. Palmer J.L. Tulley J.M. Speicher J.T. Kovacs E.J. Gamelli R.L. Faunce D.E. A novel role for NKT cells in cutaneous wound repair.J Surg Res. 2011; 168: 325-333.e1Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 15Schneider D.F. Palmer J.L. Tulley J.M. Kovacs E.J. Gamelli R.L. Faunce D.E. Prevention of NKT cell activation accelerates cutaneous wound closure and alters local inflammatory signals.J Surg Res. 2011; 171: 361-373Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar reported that wound closure and collagen deposition were accelerated in mice lacking iNKT cells and mice treated with anti-CD1d mAb. In contrast, in the present study, the presence of iNKT cells accelerated wound healing. These contrasting results might be caused by differences in the mouse strains (BALB/c versus C57BL/6) or the treatment of wounded sites: left open versus covered with a polyurethane film. In general, BALB/c mice exhibit a T helper cell type 2-dominant immune response, whereas C57BL/6 mice exhibit a T helper cell type 1-dominant immune response.32Kuroda E. Noguchi J. Doi T. Uematsu S. Akira S. Yamashita U. IL-3 is an important differentiation factor for the development of prostaglandin E2-producing macrophages between C57BL/6 and BALB/c mice.Eur J Immunol. 2007; 37: 2185-2195Crossref PubMed Scopus (16) Google Scholar Different experimental conditions and immune responses may contribute to these opposite results. Further investigation is necessary to determine the exact mechanism. In conclusion, we demonstrated that iNKT cells played crucial roles in the wound healing process and that the agonist affecting their activities enhanced their roles. The present study may therefore shed light on the immunologic mechanisms of wound healing in the skin. This may lead to a novel approach to promote wound healing in chronic wounds.
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