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• W2004651892 abstract "Toll-like receptors (TLRs) are pattern-recognition receptors and have a critical role in both innate and adaptive responses to tissue injury. Our previous study showed that wound healing was impaired in TLR3-deficient mice. In this study, we investigated the capacity of the TLR3 agonist polyriboinosinic-polyribocytidylic acid (poly(I:C)) to promote the healing of skin wounds in humans and mice. We found that topical application with poly(I:C) accelerated the closure of wounds in patients with laser plastic surgery. In a mouse model, topical application of poly(I:C) markedly enhanced re-epithelialization, granulation, and neovascularization required for wound closure. Further studies revealed that poly(I:C) treatment resulted in enhanced recruitment of neutrophils and macrophages in association with upregulation of a chemokine, macrophage inflammatory protein-2 (MIP-2/CXCL2), in the wounds. The effect of poly(I:C) was abolished in TLR3-deficient mice or by treatment with MIP-2/CXCL2-neutralizing antibodies. These results suggest a potential therapeutic value of the TLR3 activator poly(I:C) for wound healing. Toll-like receptors (TLRs) are pattern-recognition receptors and have a critical role in both innate and adaptive responses to tissue injury. Our previous study showed that wound healing was impaired in TLR3-deficient mice. In this study, we investigated the capacity of the TLR3 agonist polyriboinosinic-polyribocytidylic acid (poly(I:C)) to promote the healing of skin wounds in humans and mice. We found that topical application with poly(I:C) accelerated the closure of wounds in patients with laser plastic surgery. In a mouse model, topical application of poly(I:C) markedly enhanced re-epithelialization, granulation, and neovascularization required for wound closure. Further studies revealed that poly(I:C) treatment resulted in enhanced recruitment of neutrophils and macrophages in association with upregulation of a chemokine, macrophage inflammatory protein-2 (MIP-2/CXCL2), in the wounds. The effect of poly(I:C) was abolished in TLR3-deficient mice or by treatment with MIP-2/CXCL2-neutralizing antibodies. These results suggest a potential therapeutic value of the TLR3 activator poly(I:C) for wound healing. antibodies macrophage inflammatory protein-2 polyriboinosinic-polyribocytidylic acid Toll-like receptor Toll/IL-1R domain-containing adaptor-inducing IFN-β Wound healing of the skin represents a dynamic process involving re-epithelialization, fibroplasias, and angiogenesis (37Singer A.J. Clark R.A. Cutaneous wound healing.N Engl J Med. 1999; 341: 738-746Crossref PubMed Scopus (4660) Google Scholar). These processes are anteceded by recruitment of leukocytes spatiotemporally and differentially regulated by chemokines. The expression of chemokine receptors by resident cells such as keratinocytes and endothelial cells suggests that chemokines may also contribute to the regulation of epithelialization, tissue remodeling, and angiogenesis (27McKay I.A. Leigh I.M. Epidermal cytokines and their roles in cutaneous wound healing.Br J Dermatol. 1991; 124: 513-518Crossref PubMed Scopus (189) Google Scholar). Thus, chemokines are in an exclusive position to integrate inflammatory events and reparative processes (7Gillitzer R. Goebeler M. Chemokines in cutaneous wound healing.J Leukoc Biol. 2001; 69: 513-521PubMed Google Scholar). In addition to chemokines, the repair process is executed and promoted by complicated signaling network that involves numerous growth factors and cytokines to regulate the growth, differentiation, and metabolism of a target cell via paracrine, autocrine, juxtacrine, or endocrine mechanisms. The usage of transgenic and gene knockout mice provides important results that reveal the in vivo function of mediators in wound repair (41Werner S. Grose R. Regulation of wound healing by growth factors and cytokines.Physiol Rev. 2003; 83: 835-870Crossref PubMed Scopus (2614) Google Scholar). The importance of cognate receptors responding to these mediators in cutaneous wound repair, such as TCRγ (10Jameson J. Ugarte K. Chen N. et al.A role for skin gammadelta T cells in wound repair.Science. 2002; 296: 747-749Crossref PubMed Scopus (517) Google Scholar), TNFR p55 (28Mori R. Kondo T. Ohshima T. et al.Accelerated wound healing in tumor necrosis factor receptor p55-deficient mice with reduced leukocyte infiltration.FASEB J. 2002; 16: 963-974Crossref PubMed Scopus (221) Google Scholar), chemokine receptor CXCR2 (5Devalaraja R.M. Nanney L.B. Du J. et al.Delayed wound healing in CXCR2 knockout mice.J Invest Dermatol. 2000; 115: 234-244Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar), and CX3CR1 (9Ishida Y. Gao J.L. Murphy P.M. Chemokine receptor CX3CR1 mediates skin wound healing by promoting macrophage and fibroblast accumulation and function.J Immunol. 2008; 180: 569-579Crossref PubMed Scopus (223) Google Scholar), as well as Toll-like receptors (TLRs), have been demonstrated. As members of the pattern-recognition receptors that build the first defense line of the body, mammalian TLRs are linkers of innate and adaptive responses. TLRs recognize microbial, as well as endogenous, ligands from necrotic cells to induce inflammatory responses (17Lin Q. Li M. Fang D. et al.The essential roles of Toll-like receptor signaling pathways in sterile inflammatory diseases.Int Immunopharmacol. 2011; 11: 1422-1432Crossref PubMed Scopus (79) Google Scholar). For example, TLR3 recognizes double-stranded RNAs derived from viruses (26Matsumoto M. Seya T. TLR3: interferon induction by double-stranded RNA including poly(I:C).Adv Drug Deliv Rev. 2008; 60: 805-812Crossref PubMed Scopus (488) Google Scholar), and RNA released from damaged tissue. Ligands contained within endocytosed cells also activate TLR3 from within the cells (12Kariko K. Ni H. Capodici J. et al.mRNA is an endogenous ligand for Toll-like receptor 3.J Biol Chem. 2004; 279: 12542-12550Crossref PubMed Scopus (827) Google Scholar). Studies revealed that TLRs are crucial in the response to tissue injuries. A markedly slower healing of wounds has been observed in MyD88-deficient mice (22Macedo L. Pinhal-Enfield G. Alshits V. et al.Wound healing is impaired in MyD88-deficient mice: a role for MyD88 in the regulation of wound healing by adenosine A2A receptors.Am J Pathol. 2007; 171: 1774-1788Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), whereas the absence of TLR2 decreases inflammation and improves wound healing (4Dasu M.R. Thangappan R.K. Bourgette A. et al.TLR2 expression and signaling-dependent inflammation impair wound healing in diabetic mice.Lab Invest. 2010; 90: 1628-1636Crossref PubMed Scopus (56) Google Scholar). A recent study reported that TLR9 activation by its ligand CpG ODN accelerated wound healing in mice (32Sato T. Yamamoto M. Shimosato T. et al.Accelerated wound healing mediated by activation of Toll-like receptor 9.Wound Repair Regen. 2010; 18: 586-593Crossref PubMed Scopus (57) Google Scholar). In addition to MyD88-dependent signaling, Toll/IL-1R domain-containing adaptor-inducing IFN-β (TRIF) activated by TLR3 was also involved in wound healing based on our previous findings that skin healing was impaired in TLR3-deficient mice with defective expression of chemokines and recruitment of myeloid cells (16Lin Q. Fang D. Fang J. et al.Impaired wound healing with defective expression of chemokines and recruitment of myeloid cells in TLR3-deficient mice.J Immunol. 2011; 186: 3710-3717Crossref PubMed Scopus (81) Google Scholar). These results raised the possibility that activation of TLR3 in vivo with its exogenous agonists may promote wound closure. To test this hypothesis, we investigated the effect of topical application with TLR3 agonist polyriboinosinic-polyribocytidylic acid (poly(I:C)), a common injection solution for the treatment of viral diseases such as chronic viral hepatitis in clinic, on skin restoration, as well as the underlying mechanisms. Our results showed that topical application with poly(I:C) accelerated wound closure in patients with post laser surgery injury. We also found that in a mouse model activation of the TLR3 pathway by poly(I:C) promoted the healing of excisional skin wounds by enhancement of the production of a chemokine macrophage inflammatory protein-2 (MIP-2/CXCL2), which recruited myeloid cells to the wounds for repair. These results indicate a therapeutic potential for poly(I:C) to promote wound healing in humans. To study the clinical effect of poly(I:C) on wound healing, 18 patients with laser surgery skin wounds were enrolled into the study. Eighteen patients with skin wounds caused by laser plastic surgery were enrolled into the study. Ethics approval for the study was granted by the local research ethics committee. The patients with laser wounds were randomly divided into saline-treated and poly(I:C)-treated groups. The solution of poly(I:C; Guangdong Bangmin Pharmaceutical) or saline were topically applied to wounds once a day, and no patient showed systemic or local side effects during or after treatment. Among 18 patients with laser-caused wounds, 12 patients underwent laser surgery to remove nevus, 4 patients were operated to remove nevus of Ota, 1 patient had freckles, and 1 had a tattoo (Table 1). Poly(I:C) application markedly enhanced skin wound healing as compared with treatment with saline solution (Table 1 and Figure 1). In the group of 12 patients of nevus surgery (Table 1), 6 patients who received poly(I:C) treatment had wound closure time of 5.35±1.03 days, whereas the other 6 patients treated with saline needed 7.35±1.51 days to achieve wound closure. The difference in 2 days was statistically significant with a P-value of 0.023.Table 1Effects of topical poly(I:C) application on patient skin woundsPoly(I:C)SalinePatient numberAgeWound ofComplete healing (day)Patient numberAgeWound ofComplete healing (day)143Nevus515Nevus7211Nevus of Ota5218Nevus of Ota10314Nevus6314Tattoo7417Nevus of Ota5419Nevus of Ota759Nevus552Nevus5622Nevus5635Nevus7720Nevus4731Nevus9848Nevus7825Nevus9938Freckle6917Nevus7Average: 5.3 (days)Average: 7.6 (days)Abbreviation: Poly(I:C), polyriboinosinic-polyribocytidylic acid.Wounds in patients receiving laser surgery were treated with poly(I:C) or saline daily until healing. Open table in a new tab Abbreviation: Poly(I:C), polyriboinosinic-polyribocytidylic acid. Wounds in patients receiving laser surgery were treated with poly(I:C) or saline daily until healing. To study the basis of the mechanism for the capacity of poly(I:C) to promote skin wound healing, we used a mouse model. Four excision wounds with 4mm diameter were performed symmetrically on the back skin of mice. Two right side wounds were treated with 1mgml−1 poly(I:C) (Sigma) and two left side wounds were treated with delivery vehicle (Figure 2a). Wound areas were analyzed throughout the healing process. The results showed that treatment with poly(I:C) decreased wound area beginning on day 3 and became significant on day 5 after injury. Healing was completed on day 10 following surgery in poly(I:C)-treated wounds, which was 2 days earlier than in vehicle-treated wounds (Figure 2b and c). Microscopic examination on day 3 revealed that although the control skin wound displayed similar granulation tissue and wound gap area as compared with the poly(I:C)-treated wound (Figure 2d and e), poly(I:C) markedly enhanced re-epithelialization (Figure 2h and i), organized granulation tissue formation (Figure 2f and g), and neovascularization (Figure 2j and k), with a more promoted effect on day 5. The enhancing effect of poly(I:C) was not observed after denaturation (Supplementary Figure S1 online), confirming the specificity of the poly(I:C)-promoted wound healing. However, poly(I:C) failed to enhance wound healing in TLR3−/- mice throughout the 14-day period (Supplementary Figure S2a and b online) and failed to enhance re-epithelialization, granulation tissue formation, and neovascularization (Supplementary Figure S2c online). We also examined the effect of poly(I:C) on the expression of TLR3 and its adaptor protein TRIF in wounded skin. Quantitative PCR showed that TLR3 and TRIF mRNA was significantly upregulated in wounded skins of wild-type mice (Figure 2l and Supplementary Figure S3 online). Wounds with poly(I:C) treatment showed an increase in the expression of TLR3 and TRIF as compared with samples tissues without poly(I:C) treatment (Figure 2l). These findings indicate that poly(I:C) accelerates skin wound healing in a TLR3-dependent manner. Download .pdf (.78 MB) Help with pdf files Supplementary Information We next examined the effect of poly(I:C) on leukocyte infiltration at the excisional wound sites. The influx of neutrophils reached the peak on day 1 and gradually reduced until day 6 in all wounds without poly(I:C) treatment. Neutrophil infiltration was significantly enhanced by poly(I:C) treatment (Figure 3a and d). The difference was pronounced between days 1 and 3 post surgery, with a 41% increase on day 1 as compared with the wounds treated with phosphate-buffered saline (PBS; Figure 3a and d). Macrophages began to accumulate on day 1 after injury and reached maximal levels on day 6 (Figure 3b and e). The number of macrophages was notably increased in poly(I:C)-treated wounds on days 3 and 6 (Figure 3b and e). In contrast, CD3+ T cells in the wound site were in small numbers after injury. The kinetics and extent of CD3+ T-cell recruitment were similar in poly(I:C)-treated and control wounds (Figure 3c and f). Thus, poly(I:C) specifically promoted the recruitment of neutrophils and macrophages, but not T cells, into the wounds. Macrophages in wounds can be divided into alternatively activated macrophages and “classically” activated macrophages (38Stein M. Keshav S. Harris N. et al.Interleukin 4 potently enhances murine macrophage mannose receptor activity: a marker of alternative immunologic macrophage activation.J Exp Med. 1992; 176: 287-292Crossref PubMed Scopus (1378) Google Scholar). The classically activated macrophages tend to elicit tissue injury, whereas alternatively activated macrophages exhibit a high endocytic and phagocytotic capacity (8Goerdt S. Orfanos C.E. Other functions, other genes: alternative activation of antigen-presenting cells.Immunity. 1999; 10: 137-142Abstract Full Text Full Text PDF PubMed Scopus (607) Google Scholar), promote angiogenesis (13Kodelja V. Muller C. Tenorio S. et al.Differences in angiogenic potential of classically versus alternatively activated macrophages.Immunobiology. 1997; 197: 478-493Crossref PubMed Scopus (160) Google Scholar), and contribute to wound healing (33Schebesch C. Kodelja V. Muller C. et al.Alternatively activated macrophages actively inhibit proliferation of peripheral blood lymphocytes and CD4+ T cells in vitro.Immunology. 1997; 92: 478-486Crossref PubMed Scopus (110) Google Scholar). A number of markers for the alternatively activated macrophages including FIZZ1/ RELMα and YM1 have been identified (30Raes G. De Baetselier P. Noel W. et al.Differential expression of FIZZ1 and Ym1 in alternatively versus classically activated macrophages.J Leukoc Biol. 2002; 71: 597-602PubMed Google Scholar). In our study, there were numerous F4/80 and Ym1 or F4/80 and FIZZ1 double-positive macrophages in both control and poly(I:C)-treated wound tissue 6 days after injury (Figure 3g and h). The number of Ym1- or FIZZ1-positive macrophages in poly(I:C)-treated wounds was higher than that in control wounds (Figure 3g and h). However, poly(I:C) treatment failed to enhance leukocyte accumulation in the wounds of TLR3−/- animals (Supplementary Figure S4 online). These data indicate that activation of TLR3 by poly(I:C) enhanced the recruitment of wound healing/alternatively activated/M2 macrophages into the wounds. Markedly enhanced infiltration of neutrophils and macrophages implied that chemokine expression might be upregulated in poly(I:C)-treated mouse skin wounds. Quantitative PCR arrays revealed that poly(I:C) treatment markedly increased the expression of the genes for MIP-2/CXCL2, MIP-1α/CCL3, MCP-1/CCL2, and RANTES/CCL5, with an elevated expression of MIP-2/CXCL2 up to 10.5-fold on day 1 (Figure 4a). The protein level of MIP-2/CXCL2 was also determined. In intact skin, MIP-2/CXCL2 protein was below detectable level (data not shown). The level of MIP-2/CXCL2 protein was significantly increased, reaching the peak on day 1, declining on day 3, and was still detectable until day 6 after injury (Figure 4b). At every checked point, poly(I:C) significantly enhanced MIP-2/CXCL2 production. The most notable increase was seen on day 1, with up to a 2.5-fold increase above the control level (Figure 4b). MIP-2/CXCL2 is a ligand for the receptor CXCR2 (11Jerva L.F. Sullivan G. Lolis E. Functional and receptor binding characterization of recombinant murine macrophage inflammatory protein 2: sequence analysis and mutagenesis identify receptor binding epitopes.Protein Sci. 1997; 6: 1643-1652Crossref PubMed Scopus (27) Google Scholar). CXCR2 expression was therefore examined in wounded skin by immunohistochemical staining. CXCR2 protein was detectable in normal skin, with increased expression in both the dermis and subcutaneous tissue of wounds particularly after poly(I:C) treatment (Figure 4c). Similar to protein expression, the CXCR2 mRNA was highly expressed in wounds on day 1 after injury, which was further enhanced by poly(I:C) treatment (Figure 4d). However, poly(I:C) treatment failed to upregulate MIP-2/CXCL2 and CXCR2 expression in TLR3−/- mouse wounds (Supplementary Figure S5 online). These data suggest that MIP-2/CXCL2 induced by poly(I:C) has an important role in the recruitment of myeloid cells in TLR3-mediated wound healing. To further confirm the role of MIP-2/CXCL2 in wound healing, we treated mouse wounds with anti-MIP-2/CXCL2 antibodies (Abs) before poly(I:C) application. As shown in Figure 5a, the effect of poly(I:C) on wound closure was abrogated by neutralizing MIP-2/CXCL2 Ab treatment. Notably, in poly(I:C)-treated wounds, MIP-2/CXCL2 neutralization also reduced the mRNA expression of monocyte-specific chemokine MCP-1/CCL2, MIP-1α/CCL3, and RANTES/CCL5 (Figure 5b), as well as epithelialization, granulation tissue formation (Figure 5c), angiogenesis, and macrophage accumulation in the wounds (Figure 5c and d). These results confirm that MIP-2/CXCL2 is a critical mediator for poly(I:C)-promoted wound healing. The primary function of the skin is to serve as a protective barrier against environment insults. Loss of the integrity of large portions of the skin as a result of injury or illness may lead to severe disability or even death. The primary goals of the treatment of wounds are promoting rapid wound closure and the formation of a functional and aesthetically satisfactory scar (37Singer A.J. Clark R.A. Cutaneous wound healing.N Engl J Med. 1999; 341: 738-746Crossref PubMed Scopus (4660) Google Scholar). The present study demonstrates that in the clinic topical treatment with TLR3 agonist poly(I:C) accelerated wound closure in patients. The mouse model study further showed that topical application of poly(I:C) promoted wound healing by enhancing re-epithelialization, granulation tissue formation, and angiogenesis during the proliferative phase. Moreover, poly(I:C) treatment enhanced inflammatory cell infiltration associated with MIP-2/CXCL2 upregulation in wild-type mice. Poly(I:C) was an effective adjuvant for Th1/17 cell responses (21Longhi M.P. Trumpfheller C. Idoyaga J. et al.Dendritic cells require a systemic type I interferon response to mature and induce CD4+ Th1 immunity with poly IC as adjuvant.J Exp Med. 2009; 206: 1589-1602Crossref PubMed Scopus (488) Google Scholar; 31Ren X. Zhou H. Li B. et al.Toll-like receptor 3 ligand polyinosinic:polycytidylic acid enhances autoimmune disease in a retinal autoimmunity model.Int Immunopharmacol. 2011; 11: 769-773Crossref PubMed Scopus (13) Google Scholar). Poly(I:C) also has important regulatory activity on the production of proinflammatory cytokines and chemokines (2Cavassani K.A. Ishii M. Wen H. et al.TLR3 is an endogenous sensor of tissue necrosis during acute inflammatory events.J Exp Med. 2008; 205: 2609-2621Crossref PubMed Scopus (374) Google Scholar; 20Liu Y. Kimura K. Yanai R. et al.Cytokine, chemokine, and adhesion molecule expression mediated by MAPKs in human corneal fibroblasts exposed to poly(I:C).Invest Ophthalmol Vis Sci. 2008; 49: 3336-3344Crossref PubMed Scopus (62) Google Scholar). In human skin, poly(I:C) induced MIP-1α or IL-8 production by keratinocytes via TLR3 (39Tohyama M. Dai X. Sayama K. et al.dsRNA-mediated innate immunity of epidermal keratinocytes.Biochem Biophys Res Commun. 2005; 335: 505-511Crossref PubMed Scopus (39) Google Scholar; 25Matsukura S. Kokubu F. Kurokawa M. et al.Synthetic double-stranded RNA induces multiple genes related to inflammation through Toll-like receptor 3 depending on NF-kappaB and/or IRF-3 in airway epithelial cells.Clin Exp Allergy. 2006; 36: 1049-1062Crossref PubMed Scopus (94) Google Scholar). In an in vivo study on skin wounds, it was found that poly(I:C) was a potent inducer of proinflammatory cytokines IL-6 and tumor necrosis factor-α (14Lai Y. Di Nardo A. Nakatsuji T. et al.Commensal bacteria regulate Toll-like receptor 3-dependent inflammation after skin injury.Nat Med. 2009; 15: 1377-1382Crossref PubMed Scopus (519) Google Scholar). Recent reports showed that poly(I:C) binds to cytosolic receptors including PKR, retinoic acid–inducible gene-I (RIG-I), and melanoma differentiation–associated antigen 5 (MDA5) in addition to TLR3. In this study, we found that the stimulatory effect of poly(I:C) was absent in TLR3−/- mice, indicating that TLR3 signaling is critical in poly(I:C)-enhanced wound healing. Our findings are consistent with the study by Maheshwari's group showing that poly(I:C) enhanced wound healing in rats and mice by upregulating adhesion molecules, extracellular matrix proteins, and TGF-β1 (1Bhartiya D. Sklarsh J.W. Maheshwari R.K. Enhanced wound healing in animal models by interferon and an interferon inducer.J Cell Physiol. 1992; 150: 312-319Crossref PubMed Scopus (33) Google Scholar; 36Sidhu G.S. Thaloor D. Singh A.K. et al.Enhanced biosynthesis of extracellular matrix proteins and TGF-beta 1 by polyinosinic-polycytidylic acid during cutaneous wound healing in vivo.J Cell Physiol. 1996; 169: 108-114Crossref PubMed Scopus (10) Google Scholar). Our data also demonstrate that poly(I:C) promotes wound healing by activating TLR3 and its signal adaptor TRIF to upregulate the expression of type I INF (data not shown), as well as the chemokine MIP-2/CXCL2 to enhance macrophage accumulation in the wounds. To our knowledge, these observations delineate a previously unreported mechanism by which poly(I:C) accelerates wound healing. Leukocyte infiltration is a hallmark of the inflammatory phase of skin wound healing (19Lingen M.W. Role of leukocytes and endothelial cells in the development of angiogenesis in inflammation and wound healing.Arch Pathol Lab Med. 2001; 125: 67-71PubMed Google Scholar). The impaired cutaneous wound healing in mice deficient in CXCR2 (5Devalaraja R.M. Nanney L.B. Du J. et al.Delayed wound healing in CXCR2 knockout mice.J Invest Dermatol. 2000; 115: 234-244Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar) or mice with morphine administration were linked to the defective neutrophil recruitment (23Martin J.L. Koodie L. Krishnan A.G. et al.Chronic morphine administration delays wound healing by inhibiting immune cell recruitment to the wound site.Am J Pathol. 2010; 176: 786-799Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Consistently, our data show that the stimulatory effect of poly(I:C) on early immune cell recruitment, which might have resulted from elevated MIP-2/CXCL2 expression, leads to a net acceleration in wound healing. We speculate that as neutrophils migrate into the site by 24hours post injury more MIP-1α/CCL3, MCP-1/CCL2, and RANTES/CCL5 were produced, resulting in a chemoattractant gradient that is essential for the subsequent recruitment of macrophages. It is generally agreed that wound macrophages have a pivotal role in the transition between inflammation and repair during cutaneous healing, by virtue of their function as a rich source of mediators, which are necessary for the initiation and propagation of new tissue formation in wounds (37Singer A.J. Clark R.A. Cutaneous wound healing.N Engl J Med. 1999; 341: 738-746Crossref PubMed Scopus (4660) Google Scholar; 41Werner S. Grose R. Regulation of wound healing by growth factors and cytokines.Physiol Rev. 2003; 83: 835-870Crossref PubMed Scopus (2614) Google Scholar; 6Eming S.A. Krieg T. Davidson J.M. Inflammation in wound repair: molecular and cellular mechanisms.J Invest Dermatol. 2007; 127: 514-525Abstract Full Text Full Text PDF PubMed Scopus (1444) Google Scholar; 34Schurmann C. Seitz O. Sader R. et al.Role of wound macrophages in skin flap loss or survival in an experimental diabetes model.Br J Surg. 2010; 97: 1437-1451Crossref PubMed Scopus (18) Google Scholar). The notion was supported by in vivo studies in which depletion of macrophages caused impaired collagen deposition (40van Amerongen M.J. Harmsen M.C. van Rooijen N. et al.Macrophage depletion impairs wound healing and increases left ventricular remodeling after myocardial injury in mice.Am J Pathol. 2007; 170: 818-829Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar) and defective wound repair (15Leibovich S.J. Ross R. The role of the macrophage in wound repair. A study with hydrocortisone and antimacrophage serum.Am J Pathol. 1975; 78: 71-100PubMed Google Scholar). Other studies demonstrated that injection of macrophages into healing cutaneous wounds augments the repair process (3Danon D. Kowatch M.A. Roth G.S. Promotion of wound repair in old mice by local injection of macrophages.Proc Natl Acad Sci USA. 1989; 86: 2018-2020Crossref PubMed Scopus (199) Google Scholar). Our study also showed that macrophages could sustain the angiogenesis cascade under inflammatory conditions in a positive feedback loop through the activation of the HMGB1-TLR4 signaling pathway (18Lin Q. Yang X.P. Fang D. et al.High-mobility group box-1 mediates Toll-like receptor 4-dependent angiogenesis.Arterioscler Thromb Vasc Biol. 2011; 31: 1024-1032Crossref PubMed Scopus (75) Google Scholar). It is known that alternatively activated macrophages are a main subpopulation with beneficial effect on wound healing (24Martinez F.O. Helming L. Gordon S. Alternative activation of macrophages: an immunologic functional perspective.Annu Rev Immunol. 2009; 27: 451-483Crossref PubMed Scopus (2055) Google Scholar). Our study showed an increase of Fizz1 and Ym1 double-positive wound-healing/alternatively activated/M2 macrophages (30Raes G. De Baetselier P. Noel W. et al.Differential expression of FIZZ1 and Ym1 in alternatively versus classically activated macrophages.J Leukoc Biol. 2002; 71: 597-602PubMed Google Scholar; 29Nair M.G. Cochrane D.W. Allen J.E. Macrophages in chronic type 2 inflammation have a novel phenotype characterized by the abundant expression of Ym1 and Fizz1 that can be partly replicated in vitro.Immunol Lett. 2003; 85: 173-180Crossref PubMed Scopus (187) Google Scholar) in poly(I:C)-treated wounds, suggesting that the enhanced macrophage recruitment may be a key step in the wound healing promoted by poly(I:C). Furthermore, we found that neutralization of MIP-2/CXCL2 abolished the effect of poly(I:C) on wound healing, confirming that poly(I:C)-enhanced macrophage accumulation was dependent on elevated MIP-2/CXCL2. Chronic skin wounds affect 6.5 million patients in the United States (35Sen C.K. Gordillo G.M. Roy S. et al.Human skin wounds: a major and snowballing threat to public health and the economy.Wound Repair Regen. 2009; 17: 763-771Crossref PubMed Scopus (1953) Google Scholar). The immense economic and social impact of wounds calls for a better understanding of the biological mechanisms underlying cutaneous wound complications. In the clinic, the poly(I:C) injection is currently used as an IFN-α/β inducer for the treatment of viral diseases such as chronic hepatitis, flu, and herpes zoster. In our study, topical administration of poly(I:C) solution in patients accelerated skin wound healing as compared with the saline treatment. These observations indicate the therapeutic effect of poly(I:C) topical administration in human skin wound and suggest a potential therapeutic value for other abnormal wound healing. Poly(I:C), goat anti-rabbit and anti-rat IgG-peroxidase Abs, and rabbit anti-goat IgG-peroxidase Ab were purchased from Sigma. TRIzol reagent and Hoechst 33342 were from Invitrogen (Carlsbad, CA). The ExScript RT reagent kit was from TaKaRa (TaKaRa Biotechnology, DaLian, China). Brilliant SYBR Green QPCR Master Mix was from Stratagene (La Jolla, CA). FITC-conjugated anti-mouse F4/80 and FITC-conjugated anti-mouse CD3 were from eBioscience (San Diego, CA). Rabbit anti-mouse CXCR2, goat anti-mouse PECAM-1 (CD31), goat anti-mouse myeloperoxidase, FITC-conjugated donkey anti-goat, and PE-conjugated donkey anti-rabbit IgG polyclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-mouse Ym1 polyclonal antibody was from StemCell Technologies (Vancouver, British Columbia, Canada). Rabbit anti-mouse Fizz1 was from PeproTech (London, UK). Anti-mouse MIP-2/CXCL2 mAb and the mouse MIP-2/CXCL2 ELISA kit were from R&D Systems (Minneapolis, MN). For the clinical trial, the poly(I:C) injection solution (1mgml−1) was purchased from Guangdong Bangmin Pharmaceutical (JiangMen, China). Eighteen patients with skin wounds caused by laser plastic surgery were enrolled into the study. Ethics approval for the study was granted by the local research ethics committee. The study was conducted according to the Declaration of Helsinki Principles and in compliance with the International Conference on Harmonization Harmonized Tripartite Guideline for Good Clinical Practice. All patients provided written informed consent before participation. The radiant flux of the laser is 11mJs−1, which could create a skin lesion reaching the dermis. It was a double-blind, within-patient, saline-controlled, randomized study to investigate the topical efficacy of poly(I:C) on wound healing. The wounds were digitally photographed to determine the size of the areas. The laser wounds of nine patients were treated daily with topical application of poly(I:C) (2mg per 10 × 10cm2, 1mgml−1, in sodium chloride injection solution). As controls, the other nine patients were daily treated with saline solution. The wounds were digitally photographed at the indicated time intervals. TLR3−/- mice were purchased from Taconic Farms (Rockville, MD). These mice were backcrossed ten or more generations onto the C57BL/6 background, and were then intercrossed to obtain the knockout genotypes and wild-type mice. Littermates of both sexes aged between 8 and 12 weeks old were used in all experiments. Animals were housed individually in cages under specific pathogen-free conditions and given water and standard laboratory chow ad libitum during the experiments. Genotyping of animals was performed by PCR of DNA obtained from tail biopsies. Primers were created according to the genotyping protocol (Stock number: 005217) from the Jackson Laboratory (Bar Harbor, ME). Animal care and all experimental procedures were approved by the Institutional Laboratory Animal Care and Use Committee. Full-thickness wounds were created and analyzed as described previously (16Lin Q. Fang D. Fang J. et al.Impaired wound healing with defective expression of chemokines and recruitment of myeloid cells in TLR3-deficient mice.J Immunol. 2011; 186: 3710-3717Crossref PubMed Scopus (81) Google Scholar). After wound surgery, 20μl 1mgml−1 poly(I:C) in PBS were locally applied to the two wounds on the right side daily, whereas the other two wounds on the left side were treated with an equal volume of PBS alone as control. These conducts were paralleled with another two negative control groups in which the two skin wounds on the left were treated with 20μl PBS or 1mgml−1 of denatured poly(I:C), which was heated at 95°C for 5min and cooled immediately on ice, whereas the wounds on the right were not treated throughout the entire observation period. In another series of experiments, ahead of being treated with poly(I:C), the two wounds on the right side were topically applied with 20μl of anti-MIP-2/CXCL2-neutralizing polyclonal Ab (endotoxin level: <0.1EU per 1μg of the antibody) or control IgG at a concentration of 100μgml−1 into wild-type mice for 30min. Wounds were redressed daily, and fresh control or test solutions were applied. The procedures were described previously (16Lin Q. Fang D. Fang J. et al.Impaired wound healing with defective expression of chemokines and recruitment of myeloid cells in TLR3-deficient mice.J Immunol. 2011; 186: 3710-3717Crossref PubMed Scopus (81) Google Scholar). Wound specimens were fixed and the sections were stained routinely with hematoxylin and eosin for histological analysis. Immunohistochemical analyses were performed using anti-CXCR2 (or -MIP-2/CXCL2) Abs. Frozen cryostat sections of skin wound tissues were immunostained with anti-myeloperoxidase, -F4/80, and -CD3 Ab for neutrophilic granulocytes, macrophages, and T cells, respectively, and double-color immunofluorescence analysis was performed to identify the types of Ym1- or Fizz1-positive macrophages. Wound samples were excised and homogenized in 0.4ml lysis buffer (10mM PBS, 0.1% SDS, 1% Nonidet P-40, and 5mM EDTA) containing Protease Inhibitors. The homogenates were centrifuged at 12,000r.p.m. for 15min. Supernatants were used to determine MIP-2/CXCL2 levels with commercial ELISA kits according to the manufacturer’s instructions. Total protein in the supernatants was measured following the Bradford method. The data were expressed as the target molecule (ng) per total protein (mg) for each sample. Total RNA isolation and quantitative real-time PCR were performed as described previously (16Lin Q. Fang D. Fang J. et al.Impaired wound healing with defective expression of chemokines and recruitment of myeloid cells in TLR3-deficient mice.J Immunol. 2011; 186: 3710-3717Crossref PubMed Scopus (81) Google Scholar). Oligonucleotide primers (Supplementary Table S1 online) were synthesized by Invitrogen Biotechnology (Shanghai, China). The values for the initial concentrations of unknown samples were calculated using a software (version 1.7) provided with the ABI 7700 system. The tested mRNA expression in each sample was finally determined after correction with GAPDH expression. Each measurement of a sample was recorded in duplicate. Laboratory experiments were repeated at least twice. All figures show pooled data from replicate experiments or representative experiments as indicated. Data are expressed as mean±SEM for the indicated number of independently performed duplicated experiments. Statistical significance between means was analyzed by one-way ANOVA or two-tailed Student's t test using the SPSS 13.0 version (IBM, Armonk, NY). A value of P<0.05 was considered statistically significant. We are grateful to Dr Ji Ming Wang of the National Cancer Institute, NIH, for helpful critique of the manuscript. This project was supported by the National Natural Science Foundation of China (81072483, 31170861, and 30872281). Supplementary material is linked to the online version of the paper at http://www.nature.com/jid" @default.
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• W2004651892 title "Toll-Like Receptor 3 Ligand Polyinosinic:Polycytidylic Acid Promotes Wound Healing in Human and Murine Skin" @default.
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