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TLR4, rather than TLR2, regulates wound healing through TGF-β and CCL5 expression
Journal of Dermatological Science 73 (2014) 117–124
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TLR4, rather than TLR2, regulates wound healing through TGF-b and CCL5 expression Hiraku Suga, Makoto Sugaya *, Hideki Fujita, Yoshihide Asano, Yayoi Tada, Takafumi Kadono, Shinichi Sato * Department of Dermatology, Faculty of Medicine, University of Tokyo, Tokyo, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 April 2013 Received in revised form 22 October 2013 Accepted 23 October 2013
Background: Toll-like receptors (TLRs) have a crucial role in early host defense against invading pathogens. Recent studies suggest that TLRs play important roles in non-infections inflammation and tissue repair and regeneration. Objective: To determine the roles of TLR2 and TLR4 in mouse wound healing using TLR2-deficient (TLR2/), TLR4-deficient (TLR4/), and TLR2/TLR4-deficient (TLR2/4/) mice. Methods: Open wounds made in TLR2/, TLR4/, and TLR2/4/ mice were examined clinically and histologically. Cytokine expression in the wounded skin was also investigated. TGF-b production from macrophages stimulated by hyaluronan, a ligand for TLR2 and TLR4, was evaluated by real-time PCR. Results: Wound areas in TLR2/, TLR4/, and TLR2/4/ mice were larger than wild-type mice both at days 3 and 7 after wounding, accompanied by decreased numbers of infiltrating macrophages in the dermis and decreased TGF-b and CCL5 mRNA expression in the wounded skin. Immunohistochemistry showed decreased numbers of macrophages expressing TGF-b and reduced CCL5 expression by keratinocytes in the wounded skin from TLR2/, TLR4/, and TLR2/4/ mice compared to wild-type mice. Moreover, TGF-b production from macrophages induced by hyaluronan stimulation in vitro was significantly decreased in the absence of TLRs, especially TLR4. Interestingly, macrophages and wounded skin from TLR2/ mice showed decreased TLR4 mRNA expression compared to wild-type mice, suggesting that the effect of TLR2 deficiency was at least partially dependent on decrease in TLR4. Topical application of TGF-b and CCL5 significantly improved wound healing in TLR-deficient mice. Conclusion: TLR4, rather than TLR2, regulates wound healing through TGF-b and CCL5 expression. ß 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
Keywords: Wound healing TLR2 TLR4 TGF-b CCL5
1. Introduction Wound healing is a complex but well-orchestrated biological event, with interplay between different tissue structures and a large number of resident and infiltrating cell types [1,2]. The mediators of wound repair have not been fully delineated, but include neutrophils, macrophages, fibroblasts, cytokines, chemokines, and growth factors [1,3]. For example, basic fibroblast growth factor (bFGF) and TGF-b play important roles in wound healing by inducing chemotaxis of various types of cells to the wound site, increasing granulation tissue and collagen formation, and promoting vascular formation [4–7]. Three isoforms of TGF-b (1, 2, and 3) have been identified in mammals, each of which has
* Corresponding authors at: Department of Dermatology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Tel.: +81 3 5800 8661; fax: +81 3 3814 1503. E-mail addresses: [email protected] (M. Sugaya), [email protected] (S. Sato).
distinct roles [8]. Especially, TGF-b1 is reported to enhance wound repair [9,10]. CCL5 is one of the macrophage attractive chemokines, mainly produced by epidermal keratinocytes in wounds [11,12]. This chemokine accelerates wound healing by recruiting macrophages into the wound tissue [13,14]. Toll-like receptors (TLRs) are a family of evolutionarily conserved receptors, which have a crucial role in early host defense against invading pathogens [15,16]. TLR2 recognizes the peptidoglycan and lipopeptide in the cell walls of Gram-positive bacteria, while TLR4 recognizes lipopolysaccharide which is an integral component of the outer membranes of Gram-negative bacteria. TLRs elicit immune responses through their coupling with intracellular adaptor molecules including MyD88. Recent studies suggest that TLRs also play important roles in non-infections inflammation and tissue repair and regeneration [17–19]. In noninfectious inflammation, some endogenous molecules, called endogenous danger signals, activate TLRs. They include extracellular matrix breakdown products (hyaluronan), heat shock proteins, members of the S100 family of proteins, and high
0923-1811/$36.00 ß 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jdermsci.2013.10.009
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mobility group box-1 (HMGB1) [20–22]. Activation of TLRs by these endogenous danger signals initiates sterile inflammatory reactions in a number of physiological circumstances, including wound, shock, and autoimmunity [23]. For example, hyaluronan binds to TLR2 and TLR4, and these interactions provide signals that initiate inflammatory responses, maintain epithelial cell integrity, and promote recovery from acute injury [18]. A quite recent study has shown that TLR4 has an essential role in early wound repair by comparing wound healing in C3H/HeJ and wild-type C3H/HeOuJ mice [24]. C3H/HeJ mice have lost TLR4 gene by spontaneous mutation. They have chromosomal inversion of Chromosome 6, which could lead to unknown abnormalities other than TLR4. A high incidence of hepatomas is reported in C3H mice. Despite the lack of exogenous mouse mammary tumor virus, virgin and breeding females may still develop some mammary tumors later in life. Thus, C3H/HeJ mice have several immunological abnormalities, which may affect the function of TLR4. In order to elucidate the precise role of TLR4 in wound healing, it is more suitable to use the mice whose TLR4 gene has been artificially knocked out. Moreover, we attempted to reveal relative contributions of TLR2 and TLR4 to wound healing. Synergistic effects of these receptors were also examined. For that purpose, we used TLR2-deficient (TLR2/), TLR4-deficient (TLR4/), and TLR2/TLR4-deficient (TLR2/4/) mice, which were all on the C57BL/6 background. Skin wound healing was impaired in TLR2/, TLR4/, and TLR2/4/ mice compared to wild-type mice, with decreased macrophage infiltration and reduced TGF-b and CCL5 mRNA expression. TLR4/ and TLR2/4/ mice tended to show a slower wound healing compared to TLR2/ mice. Moreover, TGF-b production from macrophages induced by hyaluronan stimulation in vitro was significantly decreased in the absence of TLRs, especially TLR4. Taken together, our study suggests that TLR4, rather than TLR2, regulates wound healing through TGF-b and CCL5 expression. 2. Materials and methods 2.1. Mice TLR2/ and TLR4/ mice with C57BL/6 background and C57BL/6 mice were purchased from Jackson Laboratory and TLR2/ 4/ mice with C57BL/6 background were obtained from Oriental BioService (Kyoto, Japan). All mice used were 8–14 weeks old. They were healthy, fertile, and did not display evidence of infection or disease. All animal experiments were approved by the Animal Research Committee of the University of Tokyo.
specific for mouse macrophages (F4/80; Serotec, Oxford, UK) or polyclonal antibodies for TGF-b1 or CCL5 (Santa Cruz Biotechnology, Santa Cruz, CA). Sections were sequentially incubated with biotinylated rabbit anti-rat IgG (Vectastain ABC kit; Vector Laboratories, Burlingame, CA). 2.4. RNA isolation and quantitative reverse transcription-PCR RNA was obtained from the wounded skin with RNeasy Fibrous Tissue Mini Kit (QIAGEN, Valencia, CA). Complementary DNA was synthesized using iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Berkeley, CA). Quantitative RT-PCR was performed as described previously [26]. Primers for mouse TGF-b1, CCL5, CCL2, TLR2, TLR4, inducible nitric oxide synthase (iNOS), Ym1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were as follows: TGF-b1 forward, 50 -TTG CTT CAG CTC CAC AGA GA-30 and reverse, 50 -TGG TTG TAG AGG GCA AGG AC-30 ; CCL5 forward, 50 CAT ATG GCT CGG ACA CCA-30 and reverse, 50 -ACA CAC TTG GCG GTT CCT-30 ; CCL2 forward, 50 -CTG GAT CGG AAC CAA ATG AG-30 and reverse, 50 -CGG GTC AAC TTC ACA TTC AA-30 ; TLR2 forward, 50 TGC AAG TAC GAA CTG GAC TTC T-30 and reverse, 50 -CCA GGT AGG TCT TGG TGT TCA TT-30 ; TLR4 forward, 50 -TAG CCA TTG CTG CCA ACA TCA T-30 and reverse, 50 -AAG ATA CAC CAA CGG CTC TGA A-30 ; iNOS forward, 50 -CGA AAC GCT TCA CTT CCA A-30 and reverse, 50 TGA GCC TAT ATT GCT GTG GCT-30 ; Ym1 forward, 50 -GGG CAT ACC TTT ATC CTG AG-30 and reverse, 50 -CCA CTG AAG TCA TCC ATG TC30 ; GAPDH forward, 50 -ACC CAC TCC TCC ACC TTT GA-30 and reverse, 50 -CAT ACC AGG AAA TGA GCT TGA CAA-30 . Specific primer for bFGF was purchased from Applied Biosystems (Foster City, CA). 2.5. Macrophage isolation and stimulation by hyaluronan Mouse peritoneal macrophages were harvested by washing the peritoneal cavity with ice cold PBS. The cells were allowed to adhere overnight in RPMI 1640 supplemented with 10% FBS and 1% penicillin–streptomycin/1% glutamine before use. All experiments were conducted in serum-free RPMI so as to minimize the effects of serum stimulation. Macrophages (5 105 cells) were cultured in 1 mL of serum-free medium in 6-well flat-bottom plates and stimulated for 12 h with 100 or 200 mg/mL of low molecular weight hyaluronan (R&D systems, Minneapolis, MN). Culture supernatants from unstimulated or stimulated macrophages were analyzed for the production of TGF-b by specific ELISA kit (R&D systems). Keratinocytes from newborn mice were also harvested as described previously [27]. RNA was extracted from peritoneal macrophages and keratinocytes with RNeasy Mini Kit (QIAGEN).
2.2. Wounding and macroscopic examination 2.6. Topical application of TGF-b or CCL5 Mice were anesthetized with diethyl ether and their backs were shaved. Four full-thickness excisional wounds per mouse were made using a disposable sterile 6-mm biopsy punch (Maruho, Osaka, Japan), as described elsewhere [25]. At days 3 and 7 after wounding, areas of open wounds were measured by tracing the wound openings onto a millimeter scale graph paper. All four wounds were analyzed for macroscopic analysis of wound closure. 2.3. Histological assessment of wound healing After mice were sacrificed, wounds were harvested with a 2mm rim of unwounded skin tissue. The wounds were fixed in 10% formalin for 3 days and then paraffin-embedded. Six-mm paraffin sections were stained with hematoxylin and eosin. For immunohistochemistry, frozen tissue sections of skin biopsies were acetone-fixed and then incubated with 10% normal rabbit serum in phosphate-buffered saline (10 min at 37 8C) to block nonspecific staining. Sections were then incubated with monoclonal Ab (mAb)
Recombinant human TGF-b (Peprotech, Rocky Hill, NJ), or bFGF (Kaken Pharmaceutical, Tokyo, Japan), or recombinant murine CCL5 (Peprotech), or recombinant mouse CCL2 (Ray Biotech, Norcross, GA), or hyaluronan (R&D systems) was applied to each wound in 20mL aqueous buffer immediately and 12 h after wounding, and wounds were covered with an occlusive dressing (Tegaderm; 3M, Tokyo, Japan). The amounts of TGF-b, bFGF, CCL5, CCL2, and hyaluronan used in this study were as follows: TGF-b, 20 pg/20 mL or 200 pg/20 mL; bFGF, 1 mg/20 mL; CCL5, 2 ng/20 mL, CCL2, 2 ng/ 20 mL, and hyaluronan, 4 mg/20 mL [11,28–30]. Macroscopic area of open wounds was measured at days 3 and 7 after wounding. 2.7. Statistical analysis Statistical analysis between two groups was performed using the Mann–Whitney U-test. P-values of <0.05 were considered statistically significant.
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3. Results 3.1. Wound healing is delayed in TLR2/, TLR4/, and TLR2/4/ mice At day 3, wound areas in TLR2/, TLR4/, and TLR2/4/ mice were significantly larger than those in wild-type mice (Fig. 1a and b; p < 0.05, p < 0.01, and p < 0.01, respectively). At day 7, wounds in wild-type mice were almost closed. By contrast, TLR2/, TLR4/ , and TLR2/4/ mice still had open wounds, whose areas were significantly larger than those in wild-type mice (p < 0.01, each). TLR4/ and TLR2/4/ mice tended to show wider open wound compared to TLR2/ mice, although the differences were not statistically significant. Re-epithelialization was assessed by microscopically measuring the epithelial gap, the distance between the migrating edges of keratinocytes (Fig. 1a and c). The epithelial gap was significantly longer in mutant mice compared to wild-type mice at both 3 and 7 days after wounding. Thus, loss of TLRs, especially TLR4, inhibited cutaneous wound healing. 3.2. Decreased neutrophil and macrophage infiltration in TLR2/, TLR4/, and TLR2/4/ mice We next evaluated the wounded skin histologically. At days 3 and 7 after wounding, neutrophils and F4/80-positive macrophages infiltrated into the wounded skin (Fig. 2a and b and data not shown). The numbers of neutrophils on day 3 in the wounded skin of TLR2/ mice, TLR4/ mice, and TLR2/4/ mice were significantly lower than those of wild-type mice (Fig. 2c; p 0.05, each), while on day 7, there was no significant difference in the neutrophil numbers among wild-type and TLR-deficient mice (Fig. 2c). Similarly, the numbers of macrophages on days 3
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and 7 in the wounded skin of TLR2/ mice, TLR4/ mice, and TLR2/4/ mice were significantly lower than those of wild-type mice (Fig. 2d). 3.3. TGF-b and CCL5 mRNA expression is decreased in wounded skin tissue from TLR2/, TLR4/, and TLR2/4/ mice We next examined cytokine expression in wounded skin tissue at days 3 and 7 after wounding by real-time reverse transcription (RT)-PCR. We first assessed expression levels of TGF-b and bFGF, both of which are important in wound healing and fibrosis [4–7]. At day 3 after wounding, TGF-b mRNA expression in TLR2/, TLR4/, and TLR2/4/ mice was significantly decreased compared to wild-type mice (Fig. 3a; p < 0.05, p < 0.01, and p < 0.01, respectively). TGF-b mRNA expression in TLR2/4/ mice was significant lower than TLR2/ mice (p < 0.01). On the other hand, there was no significant difference in bFGF mRNA expression among wild-type and TLR-deficient mice. As macrophage infiltration was significantly attenuated in TLR-deficient mice, we also examined expression of CCL5 and CCL2, both of which can attract and activate macrophages [11,13,14,31]. At day 3 after wounding, CCL5 mRNA expression in TLR2/, TLR4/, and TLR2/4/ mice was significantly decreased compared to wild-type mice (Fig. 3a; p < 0.05, p < 0.01, and p < 0.01, respectively). TLR2/4/ mice exhibited significantly lower CCL5 mRNA expression compared to TLR2/ mice (p < 0.05). There was no significant difference in CCL2 mRNA expression among wild-type and TLR-deficient mice. At day 7 after wounding, mRNA expression of TGF-b, CCL5, bFGF, and CCL2 in each mutant mice was similar to that in wild-type mice (data not shown). Moreover, we evaluated the expression status of the four above cytokines in wild-type and mutant mice under normal condition, finding that there was no significant
Fig. 1. Delayed wound healing in TLR2/, TLR4/, and TLR2/4/ mice. (a) Representative pictures of open wounds and histology of wound tissues (epithelial gap; arrows) in wild-type (WT), TLR2/, TLR4/, and TLR2/4/ mice were shown. (b) Wound areas in wild-type (WT), TLR2/, TLR4/, and TLR2/4/ mice were measured 3 and 7 days after wounding. (c) Epithelial gap in wild-type (WT), TLR2/, TLR4/, and TLR2/4/ mice 3 and 7 days after wounding was measured. Each histogram shows the mean + SEM obtained from 8 mice (30 wounds) in each group. Representative results from two independent experiments are shown. *p < 0.05, **p < 0.01.
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Fig. 2. Decreased neutrophil and macrophage infiltration into the wounded skin in TLR2/, TLR4/ and TLR2/4/ mice. (a) Representative pictures of hematoxylin and eosin staining in wild-type (WT) and knockout mice 3 days after wounding were shown (original magnification 400; scale bar = 50 mm). Neutrophils were indicated by arrows. Six mice were used in each condition. (b) Representative pictures of F4/80 staining in wild-type (WT) and knockout mice 3 days after wounding were shown (400; scale bar = 50 mm). Positive cells were indicated by arrows. Six mice were used in each condition. (c) The number of neutrophils per high power filed (HPF, 400) in the wounded skin from wild-type (WT), TLR2/, TLR4/, and TLR2/4/ mice were counted. Each histogram shows the mean SEM obtained from six mice in each group. (d) The number of macrophages per high power filed (HPF, 400) in the wounded skin from wild-type (WT), TLR2/, TLR4/, and TLR2/4/ mice were counted. Each histogram shows the mean + SEM obtained from six mice in each group. *p < 0.05, **p < 0.01.
difference. Thus, decreased CCL5 mRNA expression may account for reduced macrophage infiltration into the wounded skin, while decreased TGF-b mRNA expression may delay cutaneous wound healing in TLR2/, TLR4/, and TLR2/4/ mice. 3.4. Macrophages expressing TGF-b and CCL5 expression in keratinocytes are decreased in wounded skin tissue from TLR2/, TLR4/, and TLR2/4+ mice We next examined the cellular sources of TGF-b and CCL5 in the wounded skin. As previously reported [32–34], immunohistochemical staining for TGF-b using skin samples at day 3 after wounding showed positive staining in the dermal macrophages (Fig. 3b; arrows) in addition to some fibroblasts. Immunohistochemical staining for CCL5 revealed that keratinocytes of the wound margins and the hypertrophic epithelium was the main cells expressing this chemokine within the wound (Fig. 3b), which was consistent with previous reports [11,12]. Decreased CCL5 expression was observed in keratinocytes of the wounded skin in TLR2/, TLR4/, and TLR2/4/ mice compared to those of wildtype mice. Thus, macrophages and keratinocytes were main sources of TGF-b and CCL5, respectively, the expression of which was decreased in TLR-deficient mice.
3.5. TGF-b production in macrophages induced by hyaluronan is decreased in the absence of TLR4 We examined the roles of TLR2 and TLR4 in TGF-b production from macrophages induced by hyaluronan, which is abundant in wounded skin and play important roles in wound repair [18,28]. Stimulation with hyaluronan induced macrophages from wildtype mice to produce TGF-b in a dose-dependent manner (Fig. 4a). Likewise, hyaluronan stimulation (200 mg/mL) increased TGF-b production by macrophages from TLR2/ mice, although it was significantly lower than that of wild-type (p < 0.05). Hyaluronan stimulation only slightly increased TGF-b production by macrophages from TLR4/ and TLR2/4/ mice. With hyaluronan at a concentration of 200 mg/mL, TGF-b production by macrophages from these mutant mice was significantly lower than that of wildtype mice (p < 0.01, each). Moreover, TLR4/ and TLR2/4/ macrophages showed decreased TGF-b production compared to TLR2/ macrophages (p < 0.05, each). These results suggest that TLR4 is more important for hyaluronan-induced TGF-b production than TLR2. We next assessed expression of TLR2 and TLR4 by peritoneal macrophages. There was no significant difference in TLR2 mRNA expression between wild-type and TLR4/ mice, whereas TLR4 mRNA expression from TLR2/ mice was
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Fig. 3. Decreased TGF-b and CCL5 expression in the wounded skin of TLR2/, TLR4/, and TLR2/4/ mice compared to wild-type (WT) mice. (a) Expression of TGF-b, CCL5, basic fibroblast growth factor (bFGF), and CCL2 in the wounded skin at day 3 was examined by real-time RT-PCR. Each histogram shows the mean + SEM obtained from 10 mice (10 wounds) in each group. Representative results from two independent experiments are shown. (b) Immunohistochemisty for TGF-b and CCL5 in the wounded skin. Skin samples were collected 3 days after wounding (original magnification 400). Macrophages positive for TGF-b were indicated by arrows. Representative pictures from six mice in each condition.
significantly lower than wild-type mice (Fig. 4b). Moreover, TLR4 mRNA expression in the wounded skin at day 3 after wounding from TLR2/ mice was significantly lower than that of wild-type mice (data not shown; p < 0.05). Taken together, TGF-b production in macrophages induced by hyaluronan in vitro was significantly decreased in the absence of TLRs, especially TLR4. Decreased TLR4 mRNA expression by macrophages and wounded
skin from TLR2/ mice suggests that the effect of TLR2 deficiency was at least partially dependent on decrease in TLR4. 3.6. Topical application of TGF-b or CCL5 improves wound healing To test the therapeutic potential of TGF-b and CCL5 for wound healing, we applied TGF-b and CCL5 to each wound, and measured
Fig. 4. Decreased TGF-b production in macrophages induced by hyaluronan in vitro in the absence of TLR4. (a) Macrophages were stimulated for 12 h with 100 or 200 mg/mL of hyaluronan. Culture supernatants were analyzed by ELISA. Each point indicates the mean SEM obtained from six mice in each group. (b) TLR2 and TLR4 mRNA expression by peritoneal macrophages from wild-type (WT), TLR2/, TLR4/, and TLR2/4/ mice was examined by real-time PCR. Each histogram shows the mean + SEM obtained from six mice in each group. Representative results from three independent experiments are shown. *p < 0.05, **p < 0.01.
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Fig. 5. Improved wound healing by topical application of TGF-b or CCL5. Wound areas in wild-type (WT), TLR2/, TLR4/, and TLR2/4/ mice with or without treatment with (a) TGF-b (1 ng/mL), or (b) TGF-b (10 ng/mL), or (c) basic fibroblast growth factor (bFGF) (50 mg/mL), or (d) CCL5 (100 ng/mL), or (e) CCL2 (100 ng/mL), or (f) hyaluronan (HA) (200 mg/mL) were measured at days 3 and 7 after wounding. Each histogram shows the mean + SEM obtained from 6 mice (20 wounds) in each group. Representative results from two independent experiments are shown. *p < 0.05, **p < 0.01. #p < 0.05 versus non-treated WT mice. $p < 0.05 versus treated WT mice.
macroscopic areas of open wounds at days 3 and 7 after wounding. We also evaluated the effects of bFGF, CCL2, and hyaluronan on wound healing in TLR-deficient mice. Treatment with TGF-b at a low concentration significantly improved wound healing only at day 7 in TLR2/, TLR4/, and TLR2/4/ mice (Fig. 5a; p 0.05, each). Wound areas of the groups treated with TGF-b at a high concentration were significantly smaller than non-treated groups in TLR2/, TLR4/, and TLR2/4/ mice at days 3 and 7 after wounding (Fig. 5b; p 0.05, each for day 3 and p 0.01, each for day 7). They were comparable to the wound area of non-treated wild-type mice, suggesting that topical application of TGF-b normalized delayed wound healing dose-dependently in TLR-deficient mice. Topical application of bFGF equally improved wound healing in wild-type and mutant mice at days 3 and 7 (Fig. 5c), suggesting that bFGF accelerated wound healing regardless of TLR expression. Next, CCL5 treatment improved wound healing at day 7 in mutant mice (Fig. 5d), which was consistent with the results of real-time PCR. Expectedly, application of CCL2 did not improve wound healing (Fig. 5e). Application of hyaluronan in vivo did not improve wound healing in wildtype or TLR-deficient mice (Fig. 5f), suggesting that there were enough endogenous hyaluronan to stimulate TLRs in wounded skin [35].
4. Discussion In this study, we showed that wound healing was inhibited in TLR2/, TLR4/, and TLR2/4/ mice, accompanied by decreased numbers of macrophages infiltration and decreased TGF-b and CCL5 expression in the wounded skin, compared to wild-type mice. Immunohistochemistry showed decreased numbers of macrophages expressing TGF-b and reduced CCL5 expression by keratinocytes in the wounded skin from TLR2/, TLR4/, and TLR2/4/ mice compared to wild-type mice. Moreover, TGF-b production from macrophages induced by hyaluronan in vitro was significantly decreased in the absence of TLRs, especially TLR4. Interestingly, macrophages and wounded skin from TLR2/ mice showed decreased TLR4 mRNA expression compared to wild-type mice, suggesting that the effect of TLR2 deficiency was at least partially dependent on decrease in TLR4. Application of TGF-b or CCL5 significantly improved wound healing in TLR2/, TLR4/, and TLR2/4/ mice to a level similar to wild-type mice. Taken together, our results suggest that TLR4, rather than TLR2, regulates wound healing through TGF-b and CCL5 expression. TLRs play an essential role in the activation of innate immunity by recognizing specific patterns of microbial components. Recent studies suggest that some endogenous molecules, called endogenous danger signals, activate TLRs. Hyaluronan is one of the
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endogenous ligands which binds to TLR2 and TLR4. HyaluronanTLR2 and hyaluronan-TLR4 interactions stimulate expression of many cytokines such as IL-6, IL-10, TNF-a, and TGF-b [28]. Consistently, peritoneal macrophages stimulated with hyaluronan in this study produced TGF-b in a dose-dependent manner (Fig. 4a). Furthermore, TGF-b production was severely attenuated by TLR4 deficiency. TLR4 mRNA expression in peritoneal macrophages and the wounded skin from TLR2/ mice was significantly lower than that of wild-type mice (Fig. 4b and data not shown), although its mechanism is yet to be elucidated. Our results suggest that TLR4 is more important for hyaluronan-mediated activation of macrophages than TLR2. Consistent with this idea, a previous study shows that peritoneal macrophages from wild-type and TLR2/ mice express high levels of CXCL2 in response to hyaluronan, while those from TLR4/ and TLR2/4/ mice hardly increase CXCL2 expression when stimulated with hyaluronan [18]. Cultured keratinocytes expressed much lower levels of TLR2 and TLR4 than macrophages in this study (data not shown), which made it difficult to investigate the effect of hyaluronan on keratinocytes. Previous studies showing TLR2 and TLR4 expression on keratinocytes [24,36], and increased CCL5 production from renal tubular epithelial cells by TLR ligands, which strictly depends on TLR2 and TLR4 [37], suggest that CCL5 expression by keratinocytes in the wounded skin may be mediated by TLR2 and TLR4. Immunohistochemistry showing very weak CCL5 expression in keratinocytes from TLR2/4/ mice compared to those from TLR2/ or TLR4/ mice (Fig. 3b) suggested that both TLR2 and TLR4 would be important for hyaluronan-mediated activation of keratinocytes after wounding. We also investigated whether the activation markers of macrophages were changed or not. While expression of iNOS, a representative of M1 macrophage marker [38], was not changed, expression of Ym1, which is a M2 macrophage marker [39,40], was significantly decreased in the wounded skin from TLRdeficient mice compared to wild-type mice (data not shown), which was consistent with previous reports that M2 macrophages are involved in wound healing [40,41]. Importance of TLR2 and TLR4 in wound repair and tissue remodeling has been studied using different mouse models. Hyaluronan degradation products required both TLR2 and TLR4 in vitro and in vivo to initiate inflammatory responses in acute lung injury [18,42]. TLR4 played an important role in microvascular leakage and leukocyte adhesion under the inflammatory condition associated with nonseptic thermal injury [43]. Wounds created in diabetic, healing-impaired mice in which TLR2/ mice closed more rapidly than wounds in wild-type mice [44], suggesting that sustained TLR2 activation might be detrimental to diabetic wounds. Similarly, TLR2 inhibition improved healing in mice models of renal dysfunction following ischemia/reperfusion injury and postischemic coronary endothelial dysfunction [45–47]. On the other hand, macrophage-activating lipopeptide-2 was capable of stimulating embryonic fibroblasts to release CCL2 via TLR2 at the wounded site [48,49], leading to increased leukocytes infiltration and accelerated healing in diabetic skin wounds. Thus, the role of TLR2 in wound healing is still controversial. In the most recent report, C3H/HeJ mice, lacking TLR4, showed delayed wound healing accompanied by elevated levels of TLR2 mRNA expression in the wounded skin [24]. We revealed that TLR4 mRNA expression in the wounded skin from TLR2/ mice was significantly decreased compared to wild-type mice. Taken together, TLR expression may affect expression levels of other TLRs, which regulates immune responses differently depending on the disease models. Skin wound repair, whose underlying mechanism still remains unclear, is modulated by numerous cytokines and chemokines. Immunohistochemical staining of the wounded skin showed that macrophages and keratinocytes were main sources of TGF-b and
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CCL5, respectively (Fig. 3b), which was consistent with previous reports [11,12,32–34]. TGF-b induces migration of neutrophils, monocytes, and keratinocytes to the wound site [32–34]. Autocrine expression of TGF-b by leukocytes and fibroblasts, in turn, induces these cells to generate additional cytokines and chemokines [32–34]. The paradoxical actions of TGF-b are best appreciated in inflammation, where dependent upon the state of differentiation of the cell and the context of action, TGF-b acts in a bi-directional manner [50]. Therefore, TGF-b may act as a therapeutic tool in some circumstances, but also a target for therapeutic intervention in others. Interestingly, this character of TGF-b is very similar to that of TLR2 and TLR4 signaling since these TLRs act in a bi-directional manner in wound healing. In a recent report, CCL5-CCR5 interaction was found out to be important for recruiting endothelial progenitor cells and forming new blood vessels in mouse wound healing [11]. It was shown that CCR5 deficiency reduced accumulation of bone marrow-derived vascular endothelial progenitor cells, which could also function as the source of growth factors during tissue repair [11]. Improvement of wound healing by topical application of TGF-b or CCL5 to each wound (Fig. 5a and b) suggests their important roles in tissue repair and remodeling. In summary, we showed TLR4, rather than TLR2, regulates wound healing through TGF-b and CCL5 expression. Although some ligands activate multiple TLRs at the same time, it is important to investigate the role of each TLR separately. Blocking or activating the specific TLR signaling pathway would be a reasonable therapeutic strategy for regulating immune responses. Funding source The Ministry of Education, Culture, Sports, Science and Technology in Japan. Acknowledgments We thank Tamami Kaga and Yoshiko Ito for technical assistance. This study was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology in Japan. References [1] Werner S, Grose R. Regulation of wound healing by growth factors and cytokines. Physiol Rev 2003;83:835–70. [2] Singer AJ, Clark RA. Cutaneous wound healing. N Engl J Med 1999;341:738–46. [3] Gillitzer R, Goebeler M. Chemokines in cutaneous wound healing. J Leukoc Biol 2001;69:513–21. [4] Bryan D, Walker KB, Ferguson M, Thorpe R. Cytokine gene expression in a murine wound healing model. Cytokine 2005;31:429–38. [5] Takamiya M, Saigusa K, Nakayashiki N, Aoki Y. Studies on mRNA expression of basic fibroblast growth factor in wound healing for wound age determination. Int J Legal Med 2003;117:46–50. [6] Badr G, Badr BM, Mahmoud MH, Mohany M, Rabah DM, Garraud O. Treatment of diabetic mice with undenatured whey protein accelerates the wound healing process by enhancing the expression of MIP-1a, MIP-2, KC, CX3CL1 and TGF-b in wounded tissue. BMC Immunol 2012;13:32. [7] Zhang C, Tan CK, McFarlane C, Sharma M, Tan NS, Kambadur R. Myostatin-null mice exhibit delayed skin wound healing through the blockade of transforming growth factor-b signaling by decorin. Am J Physiol Cell Physiol 2012;302:C1213–25. [8] Shah M, Foreman DM, Ferguson MW. Neutralisation of TGF-beta 1 and TGFbeta 2 or exogenous addition of TGF-beta 3 to cutaneous rat wounds reduces scarring. J Cell Sci 1995;108(Pt 3):985–1002. [9] Ishida Y, Gao JL, Murphy PM. Chemokine receptor CX3CR1 mediates skin wound healing by promoting macrophage and fibroblast accumulation and function. J Immunol 2008;180:569–79. [10] Shah M, Revis D, Herrick S, Baillie R, Thorgeirson S, Ferquson M, et al. Role of elevated plasma transforming growth factor-beta1 levels in wound healing. Am J Pathol 1999;154:1115–24. [11] Ishida Y, Kimura A, Kuninaka Y, Inui M, Matsushima K, Mukaida N, et al. Pivotal role of the CCL5/CCR5 interaction for recruitment of endothelial progenitor cells in mouse wound healing. J Clin Invest 2012;122:711–21.
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TLR4, rather than TLR2, regulates wound healing through TGF-b and CCL5 expression Hiraku Suga, Makoto Sugaya *, Hideki Fujita, Yoshihide Asano, Yayoi Tada, Takafumi Kadono, Shinichi Sato * Department of Dermatology, Faculty of Medicine, University of Tokyo, Tokyo, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 April 2013 Received in revised form 22 October 2013 Accepted 23 October 2013
Background: Toll-like receptors (TLRs) have a crucial role in early host defense against invading pathogens. Recent studies suggest that TLRs play important roles in non-infections inflammation and tissue repair and regeneration. Objective: To determine the roles of TLR2 and TLR4 in mouse wound healing using TLR2-deficient (TLR2/), TLR4-deficient (TLR4/), and TLR2/TLR4-deficient (TLR2/4/) mice. Methods: Open wounds made in TLR2/, TLR4/, and TLR2/4/ mice were examined clinically and histologically. Cytokine expression in the wounded skin was also investigated. TGF-b production from macrophages stimulated by hyaluronan, a ligand for TLR2 and TLR4, was evaluated by real-time PCR. Results: Wound areas in TLR2/, TLR4/, and TLR2/4/ mice were larger than wild-type mice both at days 3 and 7 after wounding, accompanied by decreased numbers of infiltrating macrophages in the dermis and decreased TGF-b and CCL5 mRNA expression in the wounded skin. Immunohistochemistry showed decreased numbers of macrophages expressing TGF-b and reduced CCL5 expression by keratinocytes in the wounded skin from TLR2/, TLR4/, and TLR2/4/ mice compared to wild-type mice. Moreover, TGF-b production from macrophages induced by hyaluronan stimulation in vitro was significantly decreased in the absence of TLRs, especially TLR4. Interestingly, macrophages and wounded skin from TLR2/ mice showed decreased TLR4 mRNA expression compared to wild-type mice, suggesting that the effect of TLR2 deficiency was at least partially dependent on decrease in TLR4. Topical application of TGF-b and CCL5 significantly improved wound healing in TLR-deficient mice. Conclusion: TLR4, rather than TLR2, regulates wound healing through TGF-b and CCL5 expression. ß 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
Keywords: Wound healing TLR2 TLR4 TGF-b CCL5
1. Introduction Wound healing is a complex but well-orchestrated biological event, with interplay between different tissue structures and a large number of resident and infiltrating cell types [1,2]. The mediators of wound repair have not been fully delineated, but include neutrophils, macrophages, fibroblasts, cytokines, chemokines, and growth factors [1,3]. For example, basic fibroblast growth factor (bFGF) and TGF-b play important roles in wound healing by inducing chemotaxis of various types of cells to the wound site, increasing granulation tissue and collagen formation, and promoting vascular formation [4–7]. Three isoforms of TGF-b (1, 2, and 3) have been identified in mammals, each of which has
* Corresponding authors at: Department of Dermatology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Tel.: +81 3 5800 8661; fax: +81 3 3814 1503. E-mail addresses: [email protected] (M. Sugaya), [email protected] (S. Sato).
distinct roles [8]. Especially, TGF-b1 is reported to enhance wound repair [9,10]. CCL5 is one of the macrophage attractive chemokines, mainly produced by epidermal keratinocytes in wounds [11,12]. This chemokine accelerates wound healing by recruiting macrophages into the wound tissue [13,14]. Toll-like receptors (TLRs) are a family of evolutionarily conserved receptors, which have a crucial role in early host defense against invading pathogens [15,16]. TLR2 recognizes the peptidoglycan and lipopeptide in the cell walls of Gram-positive bacteria, while TLR4 recognizes lipopolysaccharide which is an integral component of the outer membranes of Gram-negative bacteria. TLRs elicit immune responses through their coupling with intracellular adaptor molecules including MyD88. Recent studies suggest that TLRs also play important roles in non-infections inflammation and tissue repair and regeneration [17–19]. In noninfectious inflammation, some endogenous molecules, called endogenous danger signals, activate TLRs. They include extracellular matrix breakdown products (hyaluronan), heat shock proteins, members of the S100 family of proteins, and high
0923-1811/$36.00 ß 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jdermsci.2013.10.009
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mobility group box-1 (HMGB1) [20–22]. Activation of TLRs by these endogenous danger signals initiates sterile inflammatory reactions in a number of physiological circumstances, including wound, shock, and autoimmunity [23]. For example, hyaluronan binds to TLR2 and TLR4, and these interactions provide signals that initiate inflammatory responses, maintain epithelial cell integrity, and promote recovery from acute injury [18]. A quite recent study has shown that TLR4 has an essential role in early wound repair by comparing wound healing in C3H/HeJ and wild-type C3H/HeOuJ mice [24]. C3H/HeJ mice have lost TLR4 gene by spontaneous mutation. They have chromosomal inversion of Chromosome 6, which could lead to unknown abnormalities other than TLR4. A high incidence of hepatomas is reported in C3H mice. Despite the lack of exogenous mouse mammary tumor virus, virgin and breeding females may still develop some mammary tumors later in life. Thus, C3H/HeJ mice have several immunological abnormalities, which may affect the function of TLR4. In order to elucidate the precise role of TLR4 in wound healing, it is more suitable to use the mice whose TLR4 gene has been artificially knocked out. Moreover, we attempted to reveal relative contributions of TLR2 and TLR4 to wound healing. Synergistic effects of these receptors were also examined. For that purpose, we used TLR2-deficient (TLR2/), TLR4-deficient (TLR4/), and TLR2/TLR4-deficient (TLR2/4/) mice, which were all on the C57BL/6 background. Skin wound healing was impaired in TLR2/, TLR4/, and TLR2/4/ mice compared to wild-type mice, with decreased macrophage infiltration and reduced TGF-b and CCL5 mRNA expression. TLR4/ and TLR2/4/ mice tended to show a slower wound healing compared to TLR2/ mice. Moreover, TGF-b production from macrophages induced by hyaluronan stimulation in vitro was significantly decreased in the absence of TLRs, especially TLR4. Taken together, our study suggests that TLR4, rather than TLR2, regulates wound healing through TGF-b and CCL5 expression. 2. Materials and methods 2.1. Mice TLR2/ and TLR4/ mice with C57BL/6 background and C57BL/6 mice were purchased from Jackson Laboratory and TLR2/ 4/ mice with C57BL/6 background were obtained from Oriental BioService (Kyoto, Japan). All mice used were 8–14 weeks old. They were healthy, fertile, and did not display evidence of infection or disease. All animal experiments were approved by the Animal Research Committee of the University of Tokyo.
specific for mouse macrophages (F4/80; Serotec, Oxford, UK) or polyclonal antibodies for TGF-b1 or CCL5 (Santa Cruz Biotechnology, Santa Cruz, CA). Sections were sequentially incubated with biotinylated rabbit anti-rat IgG (Vectastain ABC kit; Vector Laboratories, Burlingame, CA). 2.4. RNA isolation and quantitative reverse transcription-PCR RNA was obtained from the wounded skin with RNeasy Fibrous Tissue Mini Kit (QIAGEN, Valencia, CA). Complementary DNA was synthesized using iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Berkeley, CA). Quantitative RT-PCR was performed as described previously [26]. Primers for mouse TGF-b1, CCL5, CCL2, TLR2, TLR4, inducible nitric oxide synthase (iNOS), Ym1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were as follows: TGF-b1 forward, 50 -TTG CTT CAG CTC CAC AGA GA-30 and reverse, 50 -TGG TTG TAG AGG GCA AGG AC-30 ; CCL5 forward, 50 CAT ATG GCT CGG ACA CCA-30 and reverse, 50 -ACA CAC TTG GCG GTT CCT-30 ; CCL2 forward, 50 -CTG GAT CGG AAC CAA ATG AG-30 and reverse, 50 -CGG GTC AAC TTC ACA TTC AA-30 ; TLR2 forward, 50 TGC AAG TAC GAA CTG GAC TTC T-30 and reverse, 50 -CCA GGT AGG TCT TGG TGT TCA TT-30 ; TLR4 forward, 50 -TAG CCA TTG CTG CCA ACA TCA T-30 and reverse, 50 -AAG ATA CAC CAA CGG CTC TGA A-30 ; iNOS forward, 50 -CGA AAC GCT TCA CTT CCA A-30 and reverse, 50 TGA GCC TAT ATT GCT GTG GCT-30 ; Ym1 forward, 50 -GGG CAT ACC TTT ATC CTG AG-30 and reverse, 50 -CCA CTG AAG TCA TCC ATG TC30 ; GAPDH forward, 50 -ACC CAC TCC TCC ACC TTT GA-30 and reverse, 50 -CAT ACC AGG AAA TGA GCT TGA CAA-30 . Specific primer for bFGF was purchased from Applied Biosystems (Foster City, CA). 2.5. Macrophage isolation and stimulation by hyaluronan Mouse peritoneal macrophages were harvested by washing the peritoneal cavity with ice cold PBS. The cells were allowed to adhere overnight in RPMI 1640 supplemented with 10% FBS and 1% penicillin–streptomycin/1% glutamine before use. All experiments were conducted in serum-free RPMI so as to minimize the effects of serum stimulation. Macrophages (5 105 cells) were cultured in 1 mL of serum-free medium in 6-well flat-bottom plates and stimulated for 12 h with 100 or 200 mg/mL of low molecular weight hyaluronan (R&D systems, Minneapolis, MN). Culture supernatants from unstimulated or stimulated macrophages were analyzed for the production of TGF-b by specific ELISA kit (R&D systems). Keratinocytes from newborn mice were also harvested as described previously [27]. RNA was extracted from peritoneal macrophages and keratinocytes with RNeasy Mini Kit (QIAGEN).
2.2. Wounding and macroscopic examination 2.6. Topical application of TGF-b or CCL5 Mice were anesthetized with diethyl ether and their backs were shaved. Four full-thickness excisional wounds per mouse were made using a disposable sterile 6-mm biopsy punch (Maruho, Osaka, Japan), as described elsewhere [25]. At days 3 and 7 after wounding, areas of open wounds were measured by tracing the wound openings onto a millimeter scale graph paper. All four wounds were analyzed for macroscopic analysis of wound closure. 2.3. Histological assessment of wound healing After mice were sacrificed, wounds were harvested with a 2mm rim of unwounded skin tissue. The wounds were fixed in 10% formalin for 3 days and then paraffin-embedded. Six-mm paraffin sections were stained with hematoxylin and eosin. For immunohistochemistry, frozen tissue sections of skin biopsies were acetone-fixed and then incubated with 10% normal rabbit serum in phosphate-buffered saline (10 min at 37 8C) to block nonspecific staining. Sections were then incubated with monoclonal Ab (mAb)
Recombinant human TGF-b (Peprotech, Rocky Hill, NJ), or bFGF (Kaken Pharmaceutical, Tokyo, Japan), or recombinant murine CCL5 (Peprotech), or recombinant mouse CCL2 (Ray Biotech, Norcross, GA), or hyaluronan (R&D systems) was applied to each wound in 20mL aqueous buffer immediately and 12 h after wounding, and wounds were covered with an occlusive dressing (Tegaderm; 3M, Tokyo, Japan). The amounts of TGF-b, bFGF, CCL5, CCL2, and hyaluronan used in this study were as follows: TGF-b, 20 pg/20 mL or 200 pg/20 mL; bFGF, 1 mg/20 mL; CCL5, 2 ng/20 mL, CCL2, 2 ng/ 20 mL, and hyaluronan, 4 mg/20 mL [11,28–30]. Macroscopic area of open wounds was measured at days 3 and 7 after wounding. 2.7. Statistical analysis Statistical analysis between two groups was performed using the Mann–Whitney U-test. P-values of <0.05 were considered statistically significant.
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3. Results 3.1. Wound healing is delayed in TLR2/, TLR4/, and TLR2/4/ mice At day 3, wound areas in TLR2/, TLR4/, and TLR2/4/ mice were significantly larger than those in wild-type mice (Fig. 1a and b; p < 0.05, p < 0.01, and p < 0.01, respectively). At day 7, wounds in wild-type mice were almost closed. By contrast, TLR2/, TLR4/ , and TLR2/4/ mice still had open wounds, whose areas were significantly larger than those in wild-type mice (p < 0.01, each). TLR4/ and TLR2/4/ mice tended to show wider open wound compared to TLR2/ mice, although the differences were not statistically significant. Re-epithelialization was assessed by microscopically measuring the epithelial gap, the distance between the migrating edges of keratinocytes (Fig. 1a and c). The epithelial gap was significantly longer in mutant mice compared to wild-type mice at both 3 and 7 days after wounding. Thus, loss of TLRs, especially TLR4, inhibited cutaneous wound healing. 3.2. Decreased neutrophil and macrophage infiltration in TLR2/, TLR4/, and TLR2/4/ mice We next evaluated the wounded skin histologically. At days 3 and 7 after wounding, neutrophils and F4/80-positive macrophages infiltrated into the wounded skin (Fig. 2a and b and data not shown). The numbers of neutrophils on day 3 in the wounded skin of TLR2/ mice, TLR4/ mice, and TLR2/4/ mice were significantly lower than those of wild-type mice (Fig. 2c; p 0.05, each), while on day 7, there was no significant difference in the neutrophil numbers among wild-type and TLR-deficient mice (Fig. 2c). Similarly, the numbers of macrophages on days 3
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and 7 in the wounded skin of TLR2/ mice, TLR4/ mice, and TLR2/4/ mice were significantly lower than those of wild-type mice (Fig. 2d). 3.3. TGF-b and CCL5 mRNA expression is decreased in wounded skin tissue from TLR2/, TLR4/, and TLR2/4/ mice We next examined cytokine expression in wounded skin tissue at days 3 and 7 after wounding by real-time reverse transcription (RT)-PCR. We first assessed expression levels of TGF-b and bFGF, both of which are important in wound healing and fibrosis [4–7]. At day 3 after wounding, TGF-b mRNA expression in TLR2/, TLR4/, and TLR2/4/ mice was significantly decreased compared to wild-type mice (Fig. 3a; p < 0.05, p < 0.01, and p < 0.01, respectively). TGF-b mRNA expression in TLR2/4/ mice was significant lower than TLR2/ mice (p < 0.01). On the other hand, there was no significant difference in bFGF mRNA expression among wild-type and TLR-deficient mice. As macrophage infiltration was significantly attenuated in TLR-deficient mice, we also examined expression of CCL5 and CCL2, both of which can attract and activate macrophages [11,13,14,31]. At day 3 after wounding, CCL5 mRNA expression in TLR2/, TLR4/, and TLR2/4/ mice was significantly decreased compared to wild-type mice (Fig. 3a; p < 0.05, p < 0.01, and p < 0.01, respectively). TLR2/4/ mice exhibited significantly lower CCL5 mRNA expression compared to TLR2/ mice (p < 0.05). There was no significant difference in CCL2 mRNA expression among wild-type and TLR-deficient mice. At day 7 after wounding, mRNA expression of TGF-b, CCL5, bFGF, and CCL2 in each mutant mice was similar to that in wild-type mice (data not shown). Moreover, we evaluated the expression status of the four above cytokines in wild-type and mutant mice under normal condition, finding that there was no significant
Fig. 1. Delayed wound healing in TLR2/, TLR4/, and TLR2/4/ mice. (a) Representative pictures of open wounds and histology of wound tissues (epithelial gap; arrows) in wild-type (WT), TLR2/, TLR4/, and TLR2/4/ mice were shown. (b) Wound areas in wild-type (WT), TLR2/, TLR4/, and TLR2/4/ mice were measured 3 and 7 days after wounding. (c) Epithelial gap in wild-type (WT), TLR2/, TLR4/, and TLR2/4/ mice 3 and 7 days after wounding was measured. Each histogram shows the mean + SEM obtained from 8 mice (30 wounds) in each group. Representative results from two independent experiments are shown. *p < 0.05, **p < 0.01.
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Fig. 2. Decreased neutrophil and macrophage infiltration into the wounded skin in TLR2/, TLR4/ and TLR2/4/ mice. (a) Representative pictures of hematoxylin and eosin staining in wild-type (WT) and knockout mice 3 days after wounding were shown (original magnification 400; scale bar = 50 mm). Neutrophils were indicated by arrows. Six mice were used in each condition. (b) Representative pictures of F4/80 staining in wild-type (WT) and knockout mice 3 days after wounding were shown (400; scale bar = 50 mm). Positive cells were indicated by arrows. Six mice were used in each condition. (c) The number of neutrophils per high power filed (HPF, 400) in the wounded skin from wild-type (WT), TLR2/, TLR4/, and TLR2/4/ mice were counted. Each histogram shows the mean SEM obtained from six mice in each group. (d) The number of macrophages per high power filed (HPF, 400) in the wounded skin from wild-type (WT), TLR2/, TLR4/, and TLR2/4/ mice were counted. Each histogram shows the mean + SEM obtained from six mice in each group. *p < 0.05, **p < 0.01.
difference. Thus, decreased CCL5 mRNA expression may account for reduced macrophage infiltration into the wounded skin, while decreased TGF-b mRNA expression may delay cutaneous wound healing in TLR2/, TLR4/, and TLR2/4/ mice. 3.4. Macrophages expressing TGF-b and CCL5 expression in keratinocytes are decreased in wounded skin tissue from TLR2/, TLR4/, and TLR2/4+ mice We next examined the cellular sources of TGF-b and CCL5 in the wounded skin. As previously reported [32–34], immunohistochemical staining for TGF-b using skin samples at day 3 after wounding showed positive staining in the dermal macrophages (Fig. 3b; arrows) in addition to some fibroblasts. Immunohistochemical staining for CCL5 revealed that keratinocytes of the wound margins and the hypertrophic epithelium was the main cells expressing this chemokine within the wound (Fig. 3b), which was consistent with previous reports [11,12]. Decreased CCL5 expression was observed in keratinocytes of the wounded skin in TLR2/, TLR4/, and TLR2/4/ mice compared to those of wildtype mice. Thus, macrophages and keratinocytes were main sources of TGF-b and CCL5, respectively, the expression of which was decreased in TLR-deficient mice.
3.5. TGF-b production in macrophages induced by hyaluronan is decreased in the absence of TLR4 We examined the roles of TLR2 and TLR4 in TGF-b production from macrophages induced by hyaluronan, which is abundant in wounded skin and play important roles in wound repair [18,28]. Stimulation with hyaluronan induced macrophages from wildtype mice to produce TGF-b in a dose-dependent manner (Fig. 4a). Likewise, hyaluronan stimulation (200 mg/mL) increased TGF-b production by macrophages from TLR2/ mice, although it was significantly lower than that of wild-type (p < 0.05). Hyaluronan stimulation only slightly increased TGF-b production by macrophages from TLR4/ and TLR2/4/ mice. With hyaluronan at a concentration of 200 mg/mL, TGF-b production by macrophages from these mutant mice was significantly lower than that of wildtype mice (p < 0.01, each). Moreover, TLR4/ and TLR2/4/ macrophages showed decreased TGF-b production compared to TLR2/ macrophages (p < 0.05, each). These results suggest that TLR4 is more important for hyaluronan-induced TGF-b production than TLR2. We next assessed expression of TLR2 and TLR4 by peritoneal macrophages. There was no significant difference in TLR2 mRNA expression between wild-type and TLR4/ mice, whereas TLR4 mRNA expression from TLR2/ mice was
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Fig. 3. Decreased TGF-b and CCL5 expression in the wounded skin of TLR2/, TLR4/, and TLR2/4/ mice compared to wild-type (WT) mice. (a) Expression of TGF-b, CCL5, basic fibroblast growth factor (bFGF), and CCL2 in the wounded skin at day 3 was examined by real-time RT-PCR. Each histogram shows the mean + SEM obtained from 10 mice (10 wounds) in each group. Representative results from two independent experiments are shown. (b) Immunohistochemisty for TGF-b and CCL5 in the wounded skin. Skin samples were collected 3 days after wounding (original magnification 400). Macrophages positive for TGF-b were indicated by arrows. Representative pictures from six mice in each condition.
significantly lower than wild-type mice (Fig. 4b). Moreover, TLR4 mRNA expression in the wounded skin at day 3 after wounding from TLR2/ mice was significantly lower than that of wild-type mice (data not shown; p < 0.05). Taken together, TGF-b production in macrophages induced by hyaluronan in vitro was significantly decreased in the absence of TLRs, especially TLR4. Decreased TLR4 mRNA expression by macrophages and wounded
skin from TLR2/ mice suggests that the effect of TLR2 deficiency was at least partially dependent on decrease in TLR4. 3.6. Topical application of TGF-b or CCL5 improves wound healing To test the therapeutic potential of TGF-b and CCL5 for wound healing, we applied TGF-b and CCL5 to each wound, and measured
Fig. 4. Decreased TGF-b production in macrophages induced by hyaluronan in vitro in the absence of TLR4. (a) Macrophages were stimulated for 12 h with 100 or 200 mg/mL of hyaluronan. Culture supernatants were analyzed by ELISA. Each point indicates the mean SEM obtained from six mice in each group. (b) TLR2 and TLR4 mRNA expression by peritoneal macrophages from wild-type (WT), TLR2/, TLR4/, and TLR2/4/ mice was examined by real-time PCR. Each histogram shows the mean + SEM obtained from six mice in each group. Representative results from three independent experiments are shown. *p < 0.05, **p < 0.01.
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Fig. 5. Improved wound healing by topical application of TGF-b or CCL5. Wound areas in wild-type (WT), TLR2/, TLR4/, and TLR2/4/ mice with or without treatment with (a) TGF-b (1 ng/mL), or (b) TGF-b (10 ng/mL), or (c) basic fibroblast growth factor (bFGF) (50 mg/mL), or (d) CCL5 (100 ng/mL), or (e) CCL2 (100 ng/mL), or (f) hyaluronan (HA) (200 mg/mL) were measured at days 3 and 7 after wounding. Each histogram shows the mean + SEM obtained from 6 mice (20 wounds) in each group. Representative results from two independent experiments are shown. *p < 0.05, **p < 0.01. #p < 0.05 versus non-treated WT mice. $p < 0.05 versus treated WT mice.
macroscopic areas of open wounds at days 3 and 7 after wounding. We also evaluated the effects of bFGF, CCL2, and hyaluronan on wound healing in TLR-deficient mice. Treatment with TGF-b at a low concentration significantly improved wound healing only at day 7 in TLR2/, TLR4/, and TLR2/4/ mice (Fig. 5a; p 0.05, each). Wound areas of the groups treated with TGF-b at a high concentration were significantly smaller than non-treated groups in TLR2/, TLR4/, and TLR2/4/ mice at days 3 and 7 after wounding (Fig. 5b; p 0.05, each for day 3 and p 0.01, each for day 7). They were comparable to the wound area of non-treated wild-type mice, suggesting that topical application of TGF-b normalized delayed wound healing dose-dependently in TLR-deficient mice. Topical application of bFGF equally improved wound healing in wild-type and mutant mice at days 3 and 7 (Fig. 5c), suggesting that bFGF accelerated wound healing regardless of TLR expression. Next, CCL5 treatment improved wound healing at day 7 in mutant mice (Fig. 5d), which was consistent with the results of real-time PCR. Expectedly, application of CCL2 did not improve wound healing (Fig. 5e). Application of hyaluronan in vivo did not improve wound healing in wildtype or TLR-deficient mice (Fig. 5f), suggesting that there were enough endogenous hyaluronan to stimulate TLRs in wounded skin [35].
4. Discussion In this study, we showed that wound healing was inhibited in TLR2/, TLR4/, and TLR2/4/ mice, accompanied by decreased numbers of macrophages infiltration and decreased TGF-b and CCL5 expression in the wounded skin, compared to wild-type mice. Immunohistochemistry showed decreased numbers of macrophages expressing TGF-b and reduced CCL5 expression by keratinocytes in the wounded skin from TLR2/, TLR4/, and TLR2/4/ mice compared to wild-type mice. Moreover, TGF-b production from macrophages induced by hyaluronan in vitro was significantly decreased in the absence of TLRs, especially TLR4. Interestingly, macrophages and wounded skin from TLR2/ mice showed decreased TLR4 mRNA expression compared to wild-type mice, suggesting that the effect of TLR2 deficiency was at least partially dependent on decrease in TLR4. Application of TGF-b or CCL5 significantly improved wound healing in TLR2/, TLR4/, and TLR2/4/ mice to a level similar to wild-type mice. Taken together, our results suggest that TLR4, rather than TLR2, regulates wound healing through TGF-b and CCL5 expression. TLRs play an essential role in the activation of innate immunity by recognizing specific patterns of microbial components. Recent studies suggest that some endogenous molecules, called endogenous danger signals, activate TLRs. Hyaluronan is one of the
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endogenous ligands which binds to TLR2 and TLR4. HyaluronanTLR2 and hyaluronan-TLR4 interactions stimulate expression of many cytokines such as IL-6, IL-10, TNF-a, and TGF-b [28]. Consistently, peritoneal macrophages stimulated with hyaluronan in this study produced TGF-b in a dose-dependent manner (Fig. 4a). Furthermore, TGF-b production was severely attenuated by TLR4 deficiency. TLR4 mRNA expression in peritoneal macrophages and the wounded skin from TLR2/ mice was significantly lower than that of wild-type mice (Fig. 4b and data not shown), although its mechanism is yet to be elucidated. Our results suggest that TLR4 is more important for hyaluronan-mediated activation of macrophages than TLR2. Consistent with this idea, a previous study shows that peritoneal macrophages from wild-type and TLR2/ mice express high levels of CXCL2 in response to hyaluronan, while those from TLR4/ and TLR2/4/ mice hardly increase CXCL2 expression when stimulated with hyaluronan [18]. Cultured keratinocytes expressed much lower levels of TLR2 and TLR4 than macrophages in this study (data not shown), which made it difficult to investigate the effect of hyaluronan on keratinocytes. Previous studies showing TLR2 and TLR4 expression on keratinocytes [24,36], and increased CCL5 production from renal tubular epithelial cells by TLR ligands, which strictly depends on TLR2 and TLR4 [37], suggest that CCL5 expression by keratinocytes in the wounded skin may be mediated by TLR2 and TLR4. Immunohistochemistry showing very weak CCL5 expression in keratinocytes from TLR2/4/ mice compared to those from TLR2/ or TLR4/ mice (Fig. 3b) suggested that both TLR2 and TLR4 would be important for hyaluronan-mediated activation of keratinocytes after wounding. We also investigated whether the activation markers of macrophages were changed or not. While expression of iNOS, a representative of M1 macrophage marker [38], was not changed, expression of Ym1, which is a M2 macrophage marker [39,40], was significantly decreased in the wounded skin from TLRdeficient mice compared to wild-type mice (data not shown), which was consistent with previous reports that M2 macrophages are involved in wound healing [40,41]. Importance of TLR2 and TLR4 in wound repair and tissue remodeling has been studied using different mouse models. Hyaluronan degradation products required both TLR2 and TLR4 in vitro and in vivo to initiate inflammatory responses in acute lung injury [18,42]. TLR4 played an important role in microvascular leakage and leukocyte adhesion under the inflammatory condition associated with nonseptic thermal injury [43]. Wounds created in diabetic, healing-impaired mice in which TLR2/ mice closed more rapidly than wounds in wild-type mice [44], suggesting that sustained TLR2 activation might be detrimental to diabetic wounds. Similarly, TLR2 inhibition improved healing in mice models of renal dysfunction following ischemia/reperfusion injury and postischemic coronary endothelial dysfunction [45–47]. On the other hand, macrophage-activating lipopeptide-2 was capable of stimulating embryonic fibroblasts to release CCL2 via TLR2 at the wounded site [48,49], leading to increased leukocytes infiltration and accelerated healing in diabetic skin wounds. Thus, the role of TLR2 in wound healing is still controversial. In the most recent report, C3H/HeJ mice, lacking TLR4, showed delayed wound healing accompanied by elevated levels of TLR2 mRNA expression in the wounded skin [24]. We revealed that TLR4 mRNA expression in the wounded skin from TLR2/ mice was significantly decreased compared to wild-type mice. Taken together, TLR expression may affect expression levels of other TLRs, which regulates immune responses differently depending on the disease models. Skin wound repair, whose underlying mechanism still remains unclear, is modulated by numerous cytokines and chemokines. Immunohistochemical staining of the wounded skin showed that macrophages and keratinocytes were main sources of TGF-b and
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CCL5, respectively (Fig. 3b), which was consistent with previous reports [11,12,32–34]. TGF-b induces migration of neutrophils, monocytes, and keratinocytes to the wound site [32–34]. Autocrine expression of TGF-b by leukocytes and fibroblasts, in turn, induces these cells to generate additional cytokines and chemokines [32–34]. The paradoxical actions of TGF-b are best appreciated in inflammation, where dependent upon the state of differentiation of the cell and the context of action, TGF-b acts in a bi-directional manner [50]. Therefore, TGF-b may act as a therapeutic tool in some circumstances, but also a target for therapeutic intervention in others. Interestingly, this character of TGF-b is very similar to that of TLR2 and TLR4 signaling since these TLRs act in a bi-directional manner in wound healing. In a recent report, CCL5-CCR5 interaction was found out to be important for recruiting endothelial progenitor cells and forming new blood vessels in mouse wound healing [11]. It was shown that CCR5 deficiency reduced accumulation of bone marrow-derived vascular endothelial progenitor cells, which could also function as the source of growth factors during tissue repair [11]. Improvement of wound healing by topical application of TGF-b or CCL5 to each wound (Fig. 5a and b) suggests their important roles in tissue repair and remodeling. In summary, we showed TLR4, rather than TLR2, regulates wound healing through TGF-b and CCL5 expression. Although some ligands activate multiple TLRs at the same time, it is important to investigate the role of each TLR separately. Blocking or activating the specific TLR signaling pathway would be a reasonable therapeutic strategy for regulating immune responses. Funding source The Ministry of Education, Culture, Sports, Science and Technology in Japan. Acknowledgments We thank Tamami Kaga and Yoshiko Ito for technical assistance. This study was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology in Japan. References [1] Werner S, Grose R. Regulation of wound healing by growth factors and cytokines. Physiol Rev 2003;83:835–70. [2] Singer AJ, Clark RA. Cutaneous wound healing. N Engl J Med 1999;341:738–46. [3] Gillitzer R, Goebeler M. Chemokines in cutaneous wound healing. J Leukoc Biol 2001;69:513–21. [4] Bryan D, Walker KB, Ferguson M, Thorpe R. Cytokine gene expression in a murine wound healing model. Cytokine 2005;31:429–38. [5] Takamiya M, Saigusa K, Nakayashiki N, Aoki Y. Studies on mRNA expression of basic fibroblast growth factor in wound healing for wound age determination. Int J Legal Med 2003;117:46–50. [6] Badr G, Badr BM, Mahmoud MH, Mohany M, Rabah DM, Garraud O. Treatment of diabetic mice with undenatured whey protein accelerates the wound healing process by enhancing the expression of MIP-1a, MIP-2, KC, CX3CL1 and TGF-b in wounded tissue. BMC Immunol 2012;13:32. [7] Zhang C, Tan CK, McFarlane C, Sharma M, Tan NS, Kambadur R. Myostatin-null mice exhibit delayed skin wound healing through the blockade of transforming growth factor-b signaling by decorin. Am J Physiol Cell Physiol 2012;302:C1213–25. [8] Shah M, Foreman DM, Ferguson MW. Neutralisation of TGF-beta 1 and TGFbeta 2 or exogenous addition of TGF-beta 3 to cutaneous rat wounds reduces scarring. J Cell Sci 1995;108(Pt 3):985–1002. [9] Ishida Y, Gao JL, Murphy PM. Chemokine receptor CX3CR1 mediates skin wound healing by promoting macrophage and fibroblast accumulation and function. J Immunol 2008;180:569–79. [10] Shah M, Revis D, Herrick S, Baillie R, Thorgeirson S, Ferquson M, et al. Role of elevated plasma transforming growth factor-beta1 levels in wound healing. Am J Pathol 1999;154:1115–24. [11] Ishida Y, Kimura A, Kuninaka Y, Inui M, Matsushima K, Mukaida N, et al. Pivotal role of the CCL5/CCR5 interaction for recruitment of endothelial progenitor cells in mouse wound healing. J Clin Invest 2012;122:711–21.
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