The appropriate inflammatory response is essential for normal wound repair, and skin commensal Staphylococcus epidermidis has been shown to regulate TLR3-mediated inflammatory response to maintain skin homeostasis after injury. However, the underlying mechanism by which S. epidermidis regulates wound-induced inflammation remains largely unexplored. In this study we identified a previously unknown lipopeptide 78 (LP78) from S. epidermidis and showed that LP78 inhibited TLR3-mediated skin inflammation to promote wound healing. Skin injury activated TLR3/NF-κB to promote the interaction of p65 and PPARγ in nuclei and then initiated the inflammatory response in keratinocytes. LP78 activated TLR2-SRC to induce β-catenin phosphorylation at Tyr654. The phospho–β-catenin translocated into nuclei to bind to PPARγ, thus disrupting the interaction between p65 and PPARγ. The disassociation between p65 and PPARγ reduced the expression of TLR3-induced inflammatory cytokines in skin wounds of normal and diabetic mice, which correlated with accelerated wound healing. Our data demonstrate that S. epidermidis–derived LP78 inhibits skin inflammation to promote wound healing and suggest that LP78 might be a potential compound for the treatment of delayed or unhealed wounds.

Staphylococcus epidermidis, the most abundant bacterium that resides on skin, is regarded as an opportunistic pathogen that induces inflammation when present below the dermis but is tolerated on the epidermal surface without initiating inflammation (1). Accumulating evidence shows that S. epidermidis plays important roles in host defense via regulating host immune responses or producing antimicrobial peptides against pathogen colonization or invasion (25). However, the role of S. epidermidis in wound healing remains largely unexplored.

Wound healing is a fundamental physiological process restoring the integrity of the skin barrier and consists of four sequential but overlapping phases: hemostasis, inflammation, proliferation, and tissue remodeling (6). Inflammation in wound healing is a double-edged sword. It is essential but must be tightly regulated both temporally and spatially. Any pathological process that interferes with this self-limited physiological process can result in non-healed wounds. For example, a persistent high level of proinflammatory cytokines (TNF-α, IL-1, and IL-6) in diabetic skin wounds exerts a negative effect on wound cells such as keratinocytes, fibroblasts, pericytes, and smooth muscle cells (79), which results in delayed or unhealed wounds in diabetes (10). Therefore, homeostatic control of inflammatory responses in wounds might be an effective way to improve wound repair.

TLR3 is a key element in the initiation of inflammatory responses after skin injury (2, 11), and increased production of proinflammatory cytokines is also dependent on the activation of TLR3 in skin wounds of diabetes (12). To limit TLR3-mediated inflammation in skin wounds, the antimicrobial protein REG3A induces a negative regulator Src homology region 2 domain–containing protein tyrosine phosphatase 1 (SHP-1) to inactivate TLR3-induced JNK2 phosphorylation, thus inhibiting wound-induced inflammation to promote wound healing in diabetes (12). Moreover, lipoteichoic acid (LTA) from the <10-kDa product of S. epidermidis culture medium (SECM) has been shown to induce TNFR-associated factor 1 (TRAF1) to bind to TRIF, an adaptor of TLR3, thus suppressing TLR3-mediated inflammation in skin wounds (2). Although LTA has been identified as a key molecule from SECM to inhibit wound-induced inflammation, the inhibitory effect of LTA was not as potent as SECM (2). We therefore hypothesized there might be another molecule from SECM that could modulate inflammatory responses in skin wounds. In this study, we successfully identified one previously unknown lipopeptide 78 (LP78) from SECM, verified the inhibitory effect of LP78 on the inflammation in skin wounds of both normal and diabetic mice, and further delineated the mechanism by which LP78 induced β-catenin to inhibit TLR3-mediated inflammation. Our findings reveal the potential use of commensal bacterium-derived lipopeptides in treatment of delayed or unhealed wounds.

S. epidermidis 12228 and 1457 were stored in our laboratory and cultured at 37°C for 16 h in Tryptic Soy Broth (TSB) medium (Sigma-Aldrich, St Louis, MO).

Tlr2−/− (Stock No. 004650) and Tlr3−/− (Stock No. 009675) breeding pairs were purchased from The Jackson Laboratory. Mice were housed in the animal facilities in East China Normal University. All animal experiments were approved by East China Normal University Animal Care and Use Committee.

S. epidermidis 1457 was grown in 3% TSB medium at 37°C overnight. The overnight bacterial culture was diluted 1:100 by fresh TSB and then continued to culture for another 16 h. Bacterial culture medium was centrifuged at 10,000 × g for 30 min to remove the bacteria. The supernatant was filtered by 0.22-μm Stericup (Millipore, Shanghai). The sterile supernatant was adjusted to pH 2, and then set at 4°C for overnight precipitation. The next day, the precipitate was collected by centrifuge (10,000 × g) at 4°C for 30 min, and then extracted by methanol overnight. After TLC separation, the band containing LP78 was collected to load on HPLC (Agilent Technologies 1200 Series) with a C18 column for further purification. The amino acid sequence of LP78 was analyzed by quadrupole time-of-flight tandem mass spectrometry de novo sequencing (acquity ultra-performance liquid chromatography coupled with a quadrupole time-of-flight premier mass spectrometer; Waters). The lipid structure of LP78 was analyzed by gas chromatography/mass spectrometry (GC/MS-QP2010; Shimadzu).

To identify whether S. epidermidis 12228 also produced LP78, 20 μl of synthetic LP78 (1 mg/ml; APeptide) was loaded as the standard on a C18 column (Eclipse XDB C18 column, 5 μm, 4.6 mm × 250 mm). The column was eluted at a flow rate of 1.5 ml/min by the following procedure: 10−60% solvent B (methanol) and 90–40% solvent A (0.5% CF3COOH in water) for 5 min, followed by 60−90% solvent B and 40–10% solvent A for 10 min, 90–95% solvent B and 10–5% solvent A for 5 min, and 95–100% solvent B and 5–0% solvent A for 5 min. After that, 20 μl of the culture medium from S. epidermidis 12228 was loaded on the same C18 column and eluted by the same procedure as the above.

Age-matched wild-type (WT) C57BL/6 mice, Tlr2−/− C57BL/6, Tlr3−/− C57BL/6 mice (7–8 wk) were used to induce diabetes. Fifty milligrams per kilogram body weight of streptozotocin (STZ) was injected i.p. into the mice for 5 d. After 10 d, blood glucose levels were measured by using glucose meters (Jinque, China). The mice were considered diabetic if the nonfasted glycaemia was higher than 20 mM.

Dorsal hair of mice was shaved and removed by using chemical depilation (Veet). One hundred micrograms of LP78 was intradermally injected into mouse dorsal skin 24 and 2 h before wounding. Wounds were made by 6-mm biopsy punches and photographed every day. The healing area was calculated by Image J. Three days later, 2 mm of skin around wound edges was collected either for quantitative RT-PCR or stored in formalin for H&E staining.

Neonatal human epidermal keratinocytes (NHEKs; Cascade Biologics) were cultured in EpiLife medium supplemented with EpiLife Defined Growth Supplement and 0.06 mM CaCl2 (Cascade Biologics). Murine primary keratinocytes were isolated from newborn mice by using dispase II (Sigma-Aldrich) and cultured in 154CF medium supplemented with HKGS and 0.05 mM CaCl2 (Invitrogen; Shanghai, China). Subconfluent cells were seeded in plates to grow to 70% confluence. To test the role of LP78, various concentrations of LP78 were used to treat NHEKs 10 min before stimulation with polyriboinosinic polyribocytidylic acid [poly(I:C)]. Cells were harvested and the expression of genes was analyzed by using real-time RT-PCR.

Oligonucleotides encoding human β-catenin short hairpin RNA (shRNA) (Supplemental Table I) were designed. Blast search was performed by using the National Center for Biotechnology Information database to ensure that the shRNA constructs were targeting only human β-catenin. The oligonucleotides were annealed and cloned into the pLL3.7 vector as the manufacturer described. 4 μg of pLL3.7 vector containing β-catenin shRNAs, 4 μg of packaging plasmid psPAX2 (Addgene), and 2 μg of envelope plasmid pMD2.G (Addgene) were used to transfect HEK293 T cells by calcium phosphate precipitation. Forty-eight hours later, lentiviruses containing β-catenin shRNAs were collected and used to transfect NHEK cells.

The biopsies of mouse skin were homogenized using mini-beadbeater. Total RNA of skin biopsies or cells was extracted using TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions. Real-time RT-PCR was conducted on Mx3005P (Stratagene) using SYBR Premix Ex Taq (Takara). The quantification of gene expression was determined by the comparative 2ΔΔ threshold cycle method. The primers used in this manuscript are shown in Supplemental Table I. The relative expression levels were determined by normalizing expression to 18s rRNA or GAPDH. All samples were performed in triplicate and repeated at least twice.

NHEKs were grown to 70–80% confluence in a 10-cm dish and then treated with poly(I:C) and/or LP78. The cells were lysed in cell lysis buffer (10 mM HEPES, 10 mM NaCl, 1 mM KH2PO4, 5 mM NaHCO3, 5 mM EDTA, 1 mM CaCl2, 0.5 mM MgCl2, 0.1 mM PMSF, 10 mM NaF, and 1 mM NaVO3, pH 7.4). After pipetting, sucrose was added (0.27 M) and centrifuged (6300 × g) at 4°C for 15 min. The precipitate was washed by TSE buffer (10 mM Tris, 300 mM sucrose, 1 mM EDTA, and 0.1% Nonidet P-40, pH 7.4) and centrifuged (4000 × g) at 4°C for 5 min. The precipitated protein was dissolved and loaded for gel electrophoresis.

Cells or 2 mm of mouse skin around wound edges were lysed with RIPA buffer (pH 7.4) containing protease inhibitor mixture (Roche). Sixty micrograms of total protein was used for immunoprecipitation. Cell lysates were incubated with 1 μg of anti-PPARγ (Cell Signaling Technology) at 4°C overnight, followed by incubation with 30 μl of prewashed protein A/G plus–agarose at 4°C for 1 h. The precipitated complexes were separated on SDS–polyacrylamide gels and immunoblotted with anti-p65 (Abcam, Cambridge, MA) or anti–β-catenin (Cell Signaling Technology) Ab.

For immunofluorescent staining, NHEKs were grown on glass slides and treated with LP78 or poly(I:C). After fixation with 4% paraformaldehyde, the cells were blocked with 5% BSA for 30 min at room temperature, then stained with anti-p65 or anti–β-catenin or control IgG at 4°C overnight. The next day, the cells were reprobed with anti-rabbit FITC-conjugated Ab and mounted in ProLong Gold Antifade reagent with DAPI and visualized by the microscope (Leica).

All data are present as mean ± SEM. Two-tailed t test was used to determine significances between two groups. The significances among multiple groups were determined by one-way or two-way ANOVA with Bonferroni posttest of GraphPad Prism Version 6 (San Diego, CA). For all statistical tests, we considered p values <0.05 to be statistically significant.

To discover new molecules from S. epidermidis that could regulate skin inflammation, we used acid precipitation and methanol extraction to collect lipopeptides from SECM. TLC suggested that band 2 contained the most abundant lipopeptide in SECM, because only band 2 was observed on TLC plate after ninhydrin reaction, even though three bands were shown on TLC plate by H2O (Supplemental Fig. 1A). We then further purified the lipopeptide in band 2 by HPLC and used it for the structure identification. de novo peptide sequencing and gas chromatography/mass spectrometry revealed that the lipopeptide, named as LP78, had one heneicosanoic acid C20H40COOH bound to aspartic acid (D1) at N terminus of its peptide chain (Fig. 1A). In addition to S. epidermidis 1457, LP78 was detected in S. epidermidis 12228 culture medium (Fig. 1B). These data demonstrate that LP78 is a previously unknown lipopeptide regularly produced by S. epidermidis.

FIGURE 1.

LP78 is a previously unknown lipopeptide from S. epidermidis. (A) The molecular structure of LP78. (B) HPLC analysis of LP78 produced by S. epidermidis 12228.

FIGURE 1.

LP78 is a previously unknown lipopeptide from S. epidermidis. (A) The molecular structure of LP78. (B) HPLC analysis of LP78 produced by S. epidermidis 12228.

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To evaluate whether LP78 could regulate inflammatory response in skin, we first used LP78 to treat NHEKs in the presence or absence of TLR3 ligand poly(I:C). LP78 significantly inhibited poly(I:C)-induced TNF-α and IL-6 in a dose-dependent manner (Fig. 2A, 2B). Notably, the inhibitory effect of LP78 on poly(I:C)-induced inflammatory cytokines was similar to LTA, but less potent than SECM, whereas the combination of LP78 and LTA was almost as potent as SECM to inhibit poly(I:C)-induced inflammatory cytokines in NHEKs (Fig. 2B). Consistent with the inhibitory effect of LP78 in keratinocytes, the intradermal injection of LP78 markedly inhibited poly(I:C)-induced cutaneous inflammation in mouse ears (Supplemental Fig. 1B–D). The decrease in inflammation corresponded with a decrease in the expression of TNF-α and IL-6 (Fig. 2C). These data suggest that LP78 regulates TLR3-mediated inflammatory responses in keratinocytes and skin.

FIGURE 2.

LP78 inhibits TLR3-mediated inflammation to promote wound healing. (A) Quantification of TNF-α and IL-6 mRNA expression in NHEKs treated with different doses of LP78 (2–8 μg/ml) in the presence or absence of 3 μg/ml of poly(I:C) for 24 h. (B) Quantification of TNF-α and IL-6 mRNA expression in NHEKs treated with SECM, LTA, and LP78 in the presence or absence of 3 μg/ml of poly(I:C) for 24 h. (C) The expression of TNF-α and IL-6 in mouse ears intradermally injected with poly(I:C) (25 μg/ear) and/or LP78 (25 μg/ear) (n = 5). (D) Quantification of TNF-α and IL-6 expression in skin wounds of C56BL/6 normal mice (n = 6) or STZ-induced diabetic mice (n = 6) treated with PBS or LP78 (50 μg/mouse). (E) Photographs of wounds after injury in normal mice and STZ-induced diabetic mice treated as in (D). Wound closure was quantified and analyzed by Image J. (F) Quantification of TNF-α and IL-6 protein in skin wounds of WT and Tlr3-deficient mice under normal and diabetic condition (n = 3–6) treated with PBS or LP78 (50 μg/mouse). Data are means ± SEM and representative of two to three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. The p values were determined by one-way ANOVA (C) or two-way ANOVA (A, B, and D–F). n.s., no significance.

FIGURE 2.

LP78 inhibits TLR3-mediated inflammation to promote wound healing. (A) Quantification of TNF-α and IL-6 mRNA expression in NHEKs treated with different doses of LP78 (2–8 μg/ml) in the presence or absence of 3 μg/ml of poly(I:C) for 24 h. (B) Quantification of TNF-α and IL-6 mRNA expression in NHEKs treated with SECM, LTA, and LP78 in the presence or absence of 3 μg/ml of poly(I:C) for 24 h. (C) The expression of TNF-α and IL-6 in mouse ears intradermally injected with poly(I:C) (25 μg/ear) and/or LP78 (25 μg/ear) (n = 5). (D) Quantification of TNF-α and IL-6 expression in skin wounds of C56BL/6 normal mice (n = 6) or STZ-induced diabetic mice (n = 6) treated with PBS or LP78 (50 μg/mouse). (E) Photographs of wounds after injury in normal mice and STZ-induced diabetic mice treated as in (D). Wound closure was quantified and analyzed by Image J. (F) Quantification of TNF-α and IL-6 protein in skin wounds of WT and Tlr3-deficient mice under normal and diabetic condition (n = 3–6) treated with PBS or LP78 (50 μg/mouse). Data are means ± SEM and representative of two to three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. The p values were determined by one-way ANOVA (C) or two-way ANOVA (A, B, and D–F). n.s., no significance.

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Because the inflammatory responses in skin wounds of normal and diabetic mice is dependent on the activation of TLR3 (2, 12), and LP78 inhibited poly(I:C)-induced inflammation in skin, we next examined whether LP78 would inhibit inflammation in skin wounds. As expected, the application of LP78 on mouse skin significantly inhibited the expression of wound-induced TNF-α and IL-6 in both WT normal and diabetic mice (Fig. 2D), which was accompanied by improved wound healing (Fig. 2E). Moreover, LP78 had no marked effect on the injury response in Tlr3−/− normal and diabetic mice (Fig. 2F). These data reveal that LP78 inhibits TLR3-mediated inflammation to promote wound healing in both normal and diabetic mice.

To explore the mechanism by which LP78 inhibits TLR3-mediated inflammatory responses, we first examined the possible downstream signaling pathways involved in TLR3 signaling. It has been reported that activation of TLR3 leads to NF-κB nuclear translocation to induce the transcription of proinflammatory genes (13). In this study, we analyzed the phosphorylation and nuclear translocation of p65 protein, a subunit of NF-κB, which reflects the activation of NF-κB signal (14). As expected, treatment of NHEKs with poly(I:C) induced p65 translocation from the cytoplasm into the nuclei of NHEKs by immunofluorescent staining (Fig. 3A). NF-κB inhibitor, Bay 11, decreased poly(I:C)-induced TNF-α and IL-6 mRNA expression in NHEKs (Fig. 3B). We next evaluated whether LP78 inhibited TLR3-induced inflammation via blocking TLR3-activated NF-κB. To our surprise, LP78 had no effects on poly(I:C)-induced p65 phosphorylation (Fig. 3C) and also failed to block p65 translocation into nuclei of NHEKs, as p65 remained predominantly in the nuclei of keratinocytes by nuclear fractionation and immunofluorescent staining (Fig. 3D, 3E). All these data demonstrate that the inhibitory effect of LP78 on TLR3-induced inflammation is not via blocking TLR3-activated NF-κB phosphorylation and translocation.

FIGURE 3.

LP78 does not block TLR3-activated NF-κB translocation into nuclei of NHEKs. (A) Immunofluorescent staining of p65 in NHEK treated with 3 μg/ml poly(I:C). Scale bar, 25 μm. (B) Quantification of TNF-α and IL-6 mRNA expression in NHEKs treated with 3 μg/ml poly(I:C) in the presence or absence of NF-κB inhibitor Bay 11-7082(5 μM). (C) Immunoblotting of phosphorylated p65 and total p65 in NHEKs treated with poly(I:C) and/or LP78. (D) Immunoblotting of p65 in the nuclear fraction of NHEKs treated with poly(I:C) and/or LP78. Histone was used as an endogenous control of nuclear proteins. (E) Immunofluorescent staining of p65 in NHEK treated with 3 μg/ml poly (I:C) and/or LP78. Scale bar, 50 μm. Data are means ± SEM and representative of three independent experiments. ***p < 0.001. The p values were determined by one-way ANOVA.

FIGURE 3.

LP78 does not block TLR3-activated NF-κB translocation into nuclei of NHEKs. (A) Immunofluorescent staining of p65 in NHEK treated with 3 μg/ml poly(I:C). Scale bar, 25 μm. (B) Quantification of TNF-α and IL-6 mRNA expression in NHEKs treated with 3 μg/ml poly(I:C) in the presence or absence of NF-κB inhibitor Bay 11-7082(5 μM). (C) Immunoblotting of phosphorylated p65 and total p65 in NHEKs treated with poly(I:C) and/or LP78. (D) Immunoblotting of p65 in the nuclear fraction of NHEKs treated with poly(I:C) and/or LP78. Histone was used as an endogenous control of nuclear proteins. (E) Immunofluorescent staining of p65 in NHEK treated with 3 μg/ml poly (I:C) and/or LP78. Scale bar, 50 μm. Data are means ± SEM and representative of three independent experiments. ***p < 0.001. The p values were determined by one-way ANOVA.

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The above data suggest that there should be another mechanism by which LP78 inhibited TLR3-mediated inflammatory response. β-Catenin is an essential component of canonical Wnt pathway and widely expressed in numerous cells, including keratinocytes (15). Specifically, activation of β-catenin has been show to limit the inflammatory response in inflammatory bowel disease (16). Thus, we next evaluated whether β-catenin would be involved in LP78 inhibition of TLR3-mediated inflammatory response in skin wounds. Immunoblotting analysis showed that treatment NHEKs with LP78 induced the phosphorylation of β-catenin at Tyr654 in a time-dependent manner (Fig. 4A). Because the phosphorylation of Tyr654 is essential for β-catenin translocation into nuclei (17), we then analyzed whether LP78 would induce β-catenin translocation. Both immunofluorescent staining and nuclear fractionation results showed that LP78 increased β-catenin accumulation in the nuclei of NHEKs (Fig. 4B, 4C). Lithium chloride (LiCl), an inhibitor of glycogen synthetase kinase-3β (GSK-3β), increased the nuclear accumulation of β-catenin and also significantly inhibited poly(I:C)-induced inflammatory response (Supplemental Fig. 2A, 2B). Moreover, silencing β-catenin by β-catenin shRNA abrogated the inhibitory effect of LP78 on poly(I:C)-induced TNF-α and IL-6 expression in keratinocytes (Fig. 4D). The administration of β-catenin inhibitor FH535 completely blocked the capacity of LP78 to inhibit wound-induced TNF-α and IL-6 in skin wounds of diabetes (Fig. 4E). In line with this, LP78 markedly induced β-catenin phosphorylation at Tyr654 in skin wounds of diabetes, and this induction was inhibited by β-catenin inhibitor FH535 (Fig. 4F). Taken together, these results demonstrate that LP78 activates β-catenin to inhibit TLR3-mediated inflammation in skin wounds.

FIGURE 4.

LP78 activates β-catenin to inhibit TLR3-mediated inflammation in skin wounds. (A) Immunoblotting of phospho-Tyr654 of β-catenin induced by LP78 in NHEKs. (B) LP78 increased β-catenin translocation into nuclei in a time-dependent manner. Histone was loaded as endogenous control. (C) Immunofluorescent staining of β-catenin of NHEK treated with 8 μg/ml LP78 for 4 h or PBS. Scale bar, 50 μm. (D) Quantification of TNF-α and IL-6 expression in NHEK treated with 3 μg/ml poly (I:C) before and after β-catenin was silenced by shRNA. (E) Quantification of TNF-α and IL-6 expression in skin wounds of diabetic mice (n = 7) treated with β-catenin inhibitor FH535 (3 μg/mouse). (F) β-Catenin inhibitor FH535 decreased β-catenin phosphorylation induced by LP78 in skin wounds of diabetic mice. Data are means ± SEM and representative of two to three independent experiments. *p < 0.05. The p values were determined by two-way ANOVA. n.s., no significance.

FIGURE 4.

LP78 activates β-catenin to inhibit TLR3-mediated inflammation in skin wounds. (A) Immunoblotting of phospho-Tyr654 of β-catenin induced by LP78 in NHEKs. (B) LP78 increased β-catenin translocation into nuclei in a time-dependent manner. Histone was loaded as endogenous control. (C) Immunofluorescent staining of β-catenin of NHEK treated with 8 μg/ml LP78 for 4 h or PBS. Scale bar, 50 μm. (D) Quantification of TNF-α and IL-6 expression in NHEK treated with 3 μg/ml poly (I:C) before and after β-catenin was silenced by shRNA. (E) Quantification of TNF-α and IL-6 expression in skin wounds of diabetic mice (n = 7) treated with β-catenin inhibitor FH535 (3 μg/mouse). (F) β-Catenin inhibitor FH535 decreased β-catenin phosphorylation induced by LP78 in skin wounds of diabetic mice. Data are means ± SEM and representative of two to three independent experiments. *p < 0.05. The p values were determined by two-way ANOVA. n.s., no significance.

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Because TLR2 is a well-known receptor for lipopeptides (18), and the activation of TLR2 has been shown to regulate multiple downstream molecules including β-catenin (19, 20), we hypothesized that the activation of β-catenin by LP78 would be dependent on TLR2. Treatment NHEKs with TLR2 inhibitor OxPAPC markedly reduced the accumulation of nuclear β-catenin induced by LP78 (Fig. 5A and Supplemental Fig. 2C). Consistent with this, OxPAPC completely abrogated the inhibitory effect of LP78 on poly(I:C)-induced TNF-α and IL-6 in NHEKs (Fig. 5B). Moreover, LP78 failed to inhibit poly(I:C)-induced TNF-α and IL-6 in Tlr2-deficient mouse ears and keratinocytes (Fig. 5C, 5D) as well as wound-induced TNF-α and IL-6 in dorsal skin of Tlr2-deficient mice (Fig. 5E). All these data confirm that LP78 activates TLR2 to induce β-catenin for suppressing TLR3-mediated inflammation in skin wounds.

FIGURE 5.

LP78 activates TLR2-SRC to induce β-catenin activation. (A) TLR2 inhibitor, OxPAPC, inhibited LP78-induced β-catenin translocation into nuclei of NHEKs. (B) TLR2 inhibitor OxPAPC blocked the inhibitory effect of LP78 on poly(I:C)-induced TNF-α and IL-6 expression in NHEKs. (C) Quantification of TNF-α and IL-6 mRNA expression in WT (n = 4) or Tlr2-deficient (n = 4) mouse ears treated with poly(I:C) (25 μg/ear) and/or LP78 (25 μg/ear). (D) Quantification of TNF-α and IL-6 mRNA expression in primary murine keratinocytes treated with 5 μg/ml poly(I:C) and/or 6 μg/ml LP78. (E) Quantification of TNF-α and IL-6 expression in skin wounds of WT (n = 5) and Tlr2-deficient (n = 5) mice injected with LP78 (50 μg/mice) or PBS. (F) SRC inhibitor PP2 (5 μM) inhibited LP78-induced β-catenin phosphorylation. (G) The effects of TLR2 inhibitor OxPAPc and β-catenin inhibitor Cardamonin on SRC activation. Data are representative of two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. The p values were determined by two-way ANOVA. n.s., no significance.

FIGURE 5.

LP78 activates TLR2-SRC to induce β-catenin activation. (A) TLR2 inhibitor, OxPAPC, inhibited LP78-induced β-catenin translocation into nuclei of NHEKs. (B) TLR2 inhibitor OxPAPC blocked the inhibitory effect of LP78 on poly(I:C)-induced TNF-α and IL-6 expression in NHEKs. (C) Quantification of TNF-α and IL-6 mRNA expression in WT (n = 4) or Tlr2-deficient (n = 4) mouse ears treated with poly(I:C) (25 μg/ear) and/or LP78 (25 μg/ear). (D) Quantification of TNF-α and IL-6 mRNA expression in primary murine keratinocytes treated with 5 μg/ml poly(I:C) and/or 6 μg/ml LP78. (E) Quantification of TNF-α and IL-6 expression in skin wounds of WT (n = 5) and Tlr2-deficient (n = 5) mice injected with LP78 (50 μg/mice) or PBS. (F) SRC inhibitor PP2 (5 μM) inhibited LP78-induced β-catenin phosphorylation. (G) The effects of TLR2 inhibitor OxPAPc and β-catenin inhibitor Cardamonin on SRC activation. Data are representative of two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. The p values were determined by two-way ANOVA. n.s., no significance.

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To further dissect the downstream signaling pathway involved in TLR2-activated β-catenin, keratinocytes were treated with inhibitors of different signaling pathways, including PP2 for nonreceptor tyrosine kinase family member SRC, Bay-11 for NF-κB, FH535 for β-catenin, and SB431542 for TGF-β receptor (Supplemental Fig. 2D). Among these inhibitors, SRC inhibitor PP2 and β-catenin inhibitor FH535 blocked LP78-induced β-catenin phosphorylation (Fig. 5F, Supplemental Fig. 2D). Moreover, the induction of SRC phosphorylation by LP78 was significantly impaired by TLR2 inhibitor OxPAPC but not by Cadamonin, another β-catenin inhibitor (Fig. 5G). All these data suggest that LP78 activates TLR2-SRC to induce β-catenin activation.

Having established the involvement of β-catenin in LP78 inhibition of TLR3-mediated inflammation, we next sought to explore the mechanism by which LP78-induced β-catenin inhibits TLR3 signaling. Besides NF-κB, we observed that PPARγ was required for poly(I:C) to induce inflammatory cytokine expression in NHEKs, as the GW9662, an inhibitor of PPARγ, significantly inhibited poly(I:C)-induced TNF-α and IL-6 (Fig. 6A). It has been reported that PPARγ binds to NF-κB subunit p65 to initiate inflammatory responses after NF-κB translocation into nuclei (21). Moreover, β-catenin has been implicated to inhibit inflammatory response via interacting with PPARγ to disrupt the association between p65 and PPARγ, thus inhibiting NF-κB–mediated inflammation (22). We thereby speculated that LP78 might inhibit TLR3-mediated inflammation via inducing β-catenin to block the interaction between p65 and PPARγ. To test this, we did immunoprecipitation assays and confirmed that poly(I:C)-activated p65 and LP78-activated β-catenin bound to PPARγ in nuclei, respectively (Fig. 6B, 6C). When keratinocytes were treated with both LP78 and/or poly(I:C), poly(I:C)-induced p65 translocated into the nuclei but reduced p65 in the cytoplasm of NHEKs, whereas LP78 increased the accumulation of β-catenin in the nuclei but deceased β-catenin in the cytoplasm of NHEKs (Fig. 6D, 6E). Consistently, the decreased association between poly(I:C)-activated p65 and PPARγ, but the increased interaction between LP78-activated β-catenin and PPARγ in nuclei of NHEKs was observed (Fig. 6F, 6G). Moreover, immunofluorescent staining showed that LP78 stimulation decreased the colocalization of PPARγ and poly(I:C)-activated p65 but increased the colocalization of PPARγ and LP78-activated β-catenin in the nuclei of NHEKs (Fig. 6H). All these data confirm that LP78 induces β-catenin translocation into nuclei to disrupt the interaction between TLR3-activated p65 and PPARγ, thus inhibiting TLR3-mediated inflammation in skin wounds.

FIGURE 6.

β-Catenin disrupts the interaction between p65 and PPARγ. (A) Quantification of TNF-α and IL-6 mRNA expression in NHEKs treated with 3 μg/ml poly(I:C) in the presence or absence of PPARγ inhibitor GW9662 (5 μM). (B) Immunoblotting of PPARγ after immunoprecipitation with anti-p65 in poly(I:C)-stimulated NHEKs. (C) Immunoblotting of PPARγ after immunoprecipitation with anti–β-catenin in LP78-stimulated NHEKs. (D) Immunoblotting of β-catenin, p65, and PPARγ in the nuclei and cytoplasm of NHEKs that were treated with poly(I:C) and/or LP78, respectively. (E) The densitometry of the bands of β-catenin, p65, and PPARγ corresponding to (D). (F)Immunoblotting of p65 or β-catenin after immunoprecipitation with anti-PPARγ in NHEKs that were treated with poly(I:C) and/or LP78. (G) The densitometry of the bands of β-catenin and p65 corresponding to (F). (H) Immunofluorescence staining of p65, PPARγ, and β-catenin in NHEKs treated with LP78, poly(I:C) or LP78 + poly(I:C). Scale bars, 100 μm. Data are representative of two independent experiments. **p < 0.01, ***p < 0.001. The p values were determined by one-way ANOVA (A) or two-way ANOVA (E and G).

FIGURE 6.

β-Catenin disrupts the interaction between p65 and PPARγ. (A) Quantification of TNF-α and IL-6 mRNA expression in NHEKs treated with 3 μg/ml poly(I:C) in the presence or absence of PPARγ inhibitor GW9662 (5 μM). (B) Immunoblotting of PPARγ after immunoprecipitation with anti-p65 in poly(I:C)-stimulated NHEKs. (C) Immunoblotting of PPARγ after immunoprecipitation with anti–β-catenin in LP78-stimulated NHEKs. (D) Immunoblotting of β-catenin, p65, and PPARγ in the nuclei and cytoplasm of NHEKs that were treated with poly(I:C) and/or LP78, respectively. (E) The densitometry of the bands of β-catenin, p65, and PPARγ corresponding to (D). (F)Immunoblotting of p65 or β-catenin after immunoprecipitation with anti-PPARγ in NHEKs that were treated with poly(I:C) and/or LP78. (G) The densitometry of the bands of β-catenin and p65 corresponding to (F). (H) Immunofluorescence staining of p65, PPARγ, and β-catenin in NHEKs treated with LP78, poly(I:C) or LP78 + poly(I:C). Scale bars, 100 μm. Data are representative of two independent experiments. **p < 0.01, ***p < 0.001. The p values were determined by one-way ANOVA (A) or two-way ANOVA (E and G).

Close modal

Increasing evidence shows that S. epidermidis regulates immune responses of keratinocytes that dictates susceptibility to autoimmunity and infection (2, 4, 23), but how S. epidermidis regulates inflammatory responses in skin wounds remains largely unknown. In this study, we identify a previously unknown lipopeptide LP78 from S. epidermidis, and reveal that LP78 inhibits TLR3-mediated inflammation to promote wound healing. The inhibitory effect is mediated by LP78-actviated β-catenin via TLR2-SRC signaling. The mechanism for LP78/β-catenin–mediated suppression of TLR3 signaling is accomplished by β-catenin binding to PPARγ and then disrupting the interaction between TLR3-activated p65 and PPARγ, an event we show has a major role in inhibiting TLR3-mediated inflammation in skin wounds. More importantly, the inhibition of inflammation by LP78 promotes wound healing in diabetes. Thus, this study suggests that LP78 from skin resident bacterium S. epidermidis is able to limit the extent of inflammation in skin wounds via the activation of β-catenin. These findings also provide potential strategies for the treatment of delayed or unhealed wounds.

Multiple lipopeptides from different bacterial species exert important biological functions. For example, Daptomycin from Streptomyces roseosporus (24), Surfactin (25) and Fengycin (26) from Bacillus subtilis show direct antimicrobial activities. The cyclic lipopeptide (CLP) from B. natto T-2 induces apoptosis to inhibit the growth of human leukemic K562 cells (27). Moreover, a cyclic Surfactin from Bacillus sp. BML752-121F2 (28) and the diacylated lipoprotein from S. aureus (29) exhibit immunomodulatory effects. However, whether skin commensal S .epidermidis would secret lipopeptides to regulate immune responses in skin was largely unclear. Previously, we have identified the lipopeptide LP01 from S. epidermidis and show that LP01 increased the expression of antimicrobial peptides hBD2 and hBD3 against S. aureus skin infection (30). In this study we have further identified another lipopeptide LP78 from S. epidermidis and demonstrated that LP78 significantly inhibited TLR3-mediated inflammatory response to promote wound healing in both normal and diabetic mice. Interestingly, LP78 is an isomer of LP01, and the difference between these two lipopeptides is that heneicosanoic acid binds to different amino acids of the peptide chain. Specifically, heneicosanoic acid of LP01 binds to Lys11 whereas heneicosanoic acid of LP78 binds to N-terminal Asp1. LP78 was not able to induce the expression of antimicrobial peptides, and LP01 failed to inhibit TLR3-induced cytokines (Supplemental Fig. 3A, 3B). These data suggest that lipopeptides with different structures have their own unique function.

Excessive and persistent inflammation results in the failure of wound healing (6). Therefore, restraining inflammation to the proper level is critical for ensuring a normal wound healing. We have shown that LTA from S. epidermidis either induces miR-143 to inhibit TLR2-mediated inflammation (31) or induces a negative regulator TRAF1 for suppressing TLR3 signaling (2), thus limiting the extent of cutaneous inflammation in normal mice. However, we did not observe that LP78 induced the expression of miR-143 or TRAF1 in keratinocytes (Supplemental Fig. 3C, 3D), suggesting that LP78 exerts anti-inflammatory function by a completely different mechanism with LTA. It has been reported that β-catenin exerts anti-inflammatory effects by inducing anti-inflammatory mediators such as IL-10 or TGF-β (16) or reducing NF-κB–mediated proinflammatory activity (22, 32), and the activation of β-catenin in dendritic cells protects mice from Th17/Th1-mediated autoimmune neuroinflammation (19). We thereby assumed that LP78 might induce β-catenin to inhibit TLR3-mediated inflammation. Our results clarify the role of LP78 in the inhibition of TLR3-mediated inflammation via the activation of β-catenin at Tyr654 and further delineate that activated β-catenin is translocated into nuclei of keratinocytes to disrupt the interaction between TLR3-activated p65 and PPARγ, thus blocking TLR3-induced inflammation. However, β-catenin has multiple phosphorylation sites (33) and the activation of β-catenin signaling can also trigger inflammatory responses (34), further investigation is needed to dissect whether LP78 would induce β-catenin phosphorylation at other residues rather than Tyr654 to exert an inflammatory or anti-inflammatory function. Moreover, U1 RNA from UVB-damaged keratinocytes is another endogenous ligand of TLR3 and has been shown to activate TLR3 to induce inflammatory responses after solar injury (35), and whether LP78 could suppress U1 RNA–mediated inflammatory responses also needs further investigation.

In addition to LP78, we have observed that LTAs from both S. epidermidis and S. aureus induce TRAF1 to inhibit TLR3-mediated inflammation after a sterile skin injury (2). This inhibitory effect of LTAs on wound inflammation might also contribute to skin infections by staphylococci when they breach skin barrier in the context of diseases such as diabetes. Moreover, the relative absence of commensal bacterial strain S. epidermidis is most evident on individuals colonized by S. aureus (36), and increased population of S. aureus, not S. epidermidis, has been observed in skin wounds of diabetes compared with controls (37, 38). Therefore, LP78, as a unique lipopeptide from S. epidermidis, would not be manipulated by S. aureus to evade skin to exacerbate inflammatory responses, and might be a potential therapeutic target to control inflammation in skin wounds of diabetes.

In conclusion, these findings support our discovery that the lipopeptide LP78 from normal skin commensal S. epidermidis plays a crucial role in regulating inflammatory response in skin wounds. Activation of β-catenin at Tyr654 by LP78 is critical for the inhibition of wound-induced inflammation and might be a previously unknown key element in the control of skin inflammation. The identification of the unique lipopeptide from skin commensals advances our understanding of the important mutualistic relationship that exists between humans and skin microbiome. These findings also suggest that LP78 might be a potential therapeutic strategy for management of skin inflammation in diabetes.

This work was supported by the National Natural Science Foundation of China (31670925 and 31470878), the National Key Research and Development Program of China (2016YFC0906200), the Henry Fok Educational Foundation (141017), the National Program for Support of Top-Notch Young Professionals (to Y.L.), and the Fundamental Research Funds for the Central Universities.

The online version of this article contains supplemental material.

Abbreviations used in this article:

LP78

lipopeptide 78

LTA

lipoteichoic acid

NHEK

neonatal human epidermal keratinocyte

poly(I:C)

polyriboinosinic polyribocytidylic acid

SECM

S. epidermidis culture medium

shRNA

short hairpin RNA

STZ

streptozotocin

TRAF1

TNFR-associated factor 1

TSB

Tryptic Soy Broth

WT

wild-type.

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D.L. and Y.L. are named inventors on a patent filed regarding LP78 in the treatment of skin inflammation, titled “Anti-inflammatory lipopeptide and preparing method and application thereof.” The other authors have no financial conflicts of interest.

Supplementary data