Quorum sensing is a cell density-dependent gene regulation system in bacteria. N-(3-oxododecanoyl) homoserine lactone (3-oxo-C12-HSL) is used in the las quorum-sensing system in Pseudomonas aeruginosa, which is an opportunistic pathogen that causes many human diseases. Although many studies have investigated the sole effects of quorum sensing on several types of mammalian cells, including lung cells, little is known about the effects of quorum sensing on the cells associated with wound healing. To better understand the mechanism of bacterial wound infection, we investigated the effects of 3-oxo-C12-HSL on cells using a rat full-thickness wound-healing model. We found that the wound contraction was significantly increased at 24 h after the administration of 3-oxo-C12-HSL to the surface of granulation tissue. Differentiation of fibroblasts to myofibroblasts was induced in the in vivo wound-healing model and was confirmed in vitro using the rat fibroblastic cell line Rat-1. Cyclooxygenase (Cox)-2 expression was also induced in Rat-1 cells by 3-oxo-C12-HSL. This finding suggested that Cox-2 upregulation may be related to the inflammatory findings in the histological examinations, in which infiltrating polymorphonuclear neutrophils were observed at the wound site. Taken together, these results imply that mammals have a potential defense system against invading pathogens by responding to the presence of 3-oxo-C12-HSL and inducing the differentiation of fibroblasts to myofibroblasts as well as inflammation for accelerating wound healing.
Quorum sensing is a system that regulates gene expression through density-dependent cell-to-cell signaling (Smith & Iglewski, 2003a). The system relies on small diffusible signaling molecules called autoinducers in gram-negative bacteria, including Pseudomonas aeruginosa, which is a common opportunistic pathogen in human diseases (Smith & Iglewski, 2003b). An autoinducer binds to and activates a receptor protein, which is a transcriptional regulator for several virulence genes and an enzyme for the synthesis of autoinducers after the concentration of molecules reaches a threshold level (Pearson et al., 1995). Pseudomonas aeruginosa adopts two quorum-sensing systems: las and rhl. The las and rhl systems use N-(3-oxododecanoyl) homoserine lactone (3-oxo-C12-HSL) and N-butyryl homoserine lactone (C4-HSL) as their autoinducers, respectively, with LasR and RhlR proteins as their respective receptors.
Recent studies have revealed that P. aeruginosa quorum-sensing signals have the potential to alter gene expressions in mammalian cells. Among these studies, the cells in lung tissues, including lung fibroblasts, epithelial cells and innate immune cells, have been investigated widely (Pritchard, 2006). Tateda (2003) previously reported that the P. aeruginosa autoinducer can cause apoptosis of polymorphonuclear neutrophils (PMNs) and macrophages in vitro. They assessed the effects of many types of autoinducers on the induction of apoptosis in neutrophils and macrophages, and revealed that 3-oxo-C12-HSL was able to cause apoptosis in these cells in a dose-dependent manner, which was confirmed by the detection of the apoptosis markers caspase 3, caspase 8 and DNA fragmentation. Although these findings allow increasing insights into the effects of quorum-sensing signals on mammalian cells, there have been few experiments on cells associated with cutaneous wound healing. Wound healing is a potential model for assessing the mechanism of infection through the quorum-sensing system (Nakagami et al., 2008). Wound infection is one of the most difficult complications in the wound management field and effective infection control is the most coveted practice (Healy & Freedman, 2006). One study investigated the effects of 3-oxo-C12-HSL on the cells in mouse skin and found that it induced inflammation in vivo (Smith et al., 2002a). This observation raised the strong possibility that 3-oxo-C12-HSL affects wound healing, but no further information has been published. A cutaneous wound infection is different from other types of infection, including pneumonia and nephritis, in terms of its infectious environment. A cutaneous wound is exposed to the outer environment, including skin-resident flora producing several types of homoserine lactones, which complicates the pathogenesis of cutaneous wound infection. For a detailed understanding of the mechanism of wound infection, investigation of the sole effects of 3-oxo-C12-HSL on wound healing is necessary. Therefore, the objective of the present study was to explore the effects of 3-oxo-C12-HSL on wound-healing properties using a rat full-thickness wound model.
Materials and methods
Male-specific pathogen-free 7-week-old Sprague–Dawley rats were purchased from Japan SLC Inc. (Shizuoka, Japan). Animals were given food and ultrafiltered water ad libitum, and were maintained on a 12-h/12-h light/dark cycle with lights on from 08:00 to 20:00 hours.
The P. aeruginosa las quorum-sensing signal 3-oxo-C12-HSL was purchased from Sigma (St. Louis, MO). A stock solution of 10 mM 3-oxo-C12-HSL was prepared by dissolution in dimethyl sulfoxide (DMSO) and stored in a −20 °C freezer. Just before administration to the animals, the stock solution was diluted to 10 µM with 0.9% sodium chloride. A pure DMSO solution diluted with 0.9% sodium chloride was used in a similar manner as a control. For in vitro experiment for immunocytochemistry analysis, 100 mM 3-oxo-C12-HSL stock solution was used.
Full-thickness cutaneous wound-healing model
Full-thickness wounds were created in both lateroabdominal regions using sterile scissors under sedation with an intraperitoneal injection of Somnopentyl (pentobarbital sodium; Kyoritsu Seiyaku Corporation, Tokyo, Japan) (30 mg kg−1 body weight). The subcutaneous fat layer was completely dissected to expose the fascia. To investigate the effects of 3-oxo-C12-HSL on wound healing, we allowed granulation tissue to develop under moist conditions using a transparent film dressing occlusion, and then challenged the granulation tissue with 3-oxo-C12-HSL on day 5 after wounding. Specifically, 100 µL of 10 µM 3-oxo-C12-HSL solution or control DMSO solution was administered to the surface of the granulation tissue using a micropipette. This concentration was derived from the previous study, which demonstrated that the 10 µM 3-oxo-C12-HSL to the dermis could induce inflammatory cell infiltration and cyclooxygenase (Cox)-2 induction (Smith et al., 2002a). The wound was covered with transparent film dressing after the administration. The wound area was measured every day until 9 days after wounding using image analysis software (imagej version 1.42; NIH, Bethesda, MD) and expressed as relative values to the initial wound area (Pietramaggiori et al., 2008).
The experimental protocol was approved by the Animal Research Committee of The University of Tokyo. All animals were treated according to the Guide for the Care and Use of Laboratory Animals of the NIH.
Wound samples were collected at 24 h after the 3-oxo-C12-HSL challenge. The collected samples were fixed in 4% paraformaldehyde in phosphate buffer, dehydrated with alcohol, cleared with xylene and processed for embedding in paraffin. Sections were prepared at 5-µm interval for hematoxylin and eosin (HE) staining. α-Smooth muscle actin immunostaining was performed as follows: the sections were incubated for 10 min with 3% H2O2 to quench the endogenous peroxidase activity. Between each set of the following steps, the sections were washed three times with phosphate-buffered saline (PBS) for 5 min each. First, the sections were incubated with a mouse monoclonal anti-human actin antibody (Ready To Use; Dako, Tokyo, Japan) as the primary antibody for 1 h at room temperature. Subsequently, the sections were incubated with horseradish peroxidase-conjugated rabbit anti-mouse immunoglobulin (Vector Laboratories Inc., Burlingame, CA) diluted 1 : 1000 for 30 min at room temperature. The bound antibodies were visualized with 3,3′-diaminobenzidine tetrahydrochloride. The numbers of α-smooth muscle actin-positive cells were counted in three high-power (× 400) fields of each section and averaged.
Cell culture and immunocytochemistry
Fibroblastic cell line Rat-1 cells (RIKEN BioResource Center, Ibaraki, Japan) were grown at 37 °C under 5% CO2 in Dulbecco's modified Eagle's medium (Nacalai Tesque, Tokyo, Japan) supplemented with 10% fetal bovine serum (Biowest, Nuaillé, France) and antibiotics (100 U mL−1 penicillin, 100 µg mL−1 streptomycin; Nacalai Tesque). The cells were seeded in 12-well plates at 4 × 104 cells per well. When the cells became subconfluent, a medium containing 1, 10, 50 and 100 µM 3-oxo-C12-HSL or 0.1% DMSO was added. After 24 h of treatment, the cells were fixed with 4% paraformaldehyde in phosphate buffer for 20 min at room temperature, washed three times in PBS containing 0.05% Tween 20 (T-PBS) for 5 min and incubated for 30 min at room temperature with the same anti-α-smooth muscle actin primary antibody as that used for the tissue histological examination. After washing in T-PBS, the cells were incubated with a biotinylated anti-mouse immunoglobulin G secondary antibody (Vector Laboratories Inc.) diluted 1 : 1000 in PBS for 30 min at room temperature. The cells were then washed in T-PBS and incubated with Texas-red-conjugated avidin (Vector Laboratories Inc.) for 30 min at room temperature in the dark. The nuclei were stained with Hoechst 33258. The stained cells were observed using a DMI 4000 B fluorescence microscope (Leica, Wetzlar, Germany). The percentages of α-smooth muscle actin-positive cells relative to the total cell count were calculated to evaluate the effects of 3-oxo-C12-HSL on fibroblast differentiation.
RNA samples were collected from cultured cells treated with 10 µM 3-oxo-C12-HSL using Nucleospin® RNA II (Macherey-Nagel GmbH and Co., Duren, Germany) according to the manufacturer's instructions. RT-PCR amplifications were performed for Cox-2, transforming growth factor (TGF)-β1, and interleukin-6 (IL-6). cDNA was generated using a High Capacity cDNA Reverse Transcription Kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. For quantitative PCR, the amplification of the target-specific region of cDNA was performed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min after preheating at 95 °C for 10 min, and monitored using a real-time PCR system (ABI prism 7700, Applied Biosystems). The relative expression level of the target genes of the AHL-treated cells to the value of the DMSO control was calculated by the Ct method using β-actin gene as an internal control. The sequences of the gene-specific Cox-2, TGF-β1, IL-6 and β-actin primers were as follows: Cox-2 forward, 5′-CCCACTTCAAGGGAGTCTGG-3′ and Cox-2 reverse, 5′-GCAGTCATCAGCCACAGGAG-3′ (GenBank accession no.: NM_017232.2); TGF-β1 forward, 5′-GACCGCAACAACGCAATCTA-3′ and TGF-β1 reverse, 5′-ACGTGTTGCTCCACAGTTGAC-3′ (GenBank accession no.: NM_021578.1); IL-6 forward, 5′-CAGCCACTGCCTTCCCTACT-3′ and IL-6 reverse, 5′-CAGTGCATCATCGCTGTTCAT-3′ (GenBank accession no.: NM_012589.1); β-actin forward, 5′-CCCGCGAGTACAACCTTCTT-3′ and β-actin reverse, 5′-CCACGATGGAGGGGAAGAC-3′ (GenBank accession no.: NM_031144.2).
The significance of differences in the wound area trends between groups was evaluated using repeated-measures anova with group, time and the group and time interaction as fixed effects. Multiple comparisons adjusted by the Bonferroni correction were performed to test the significance of the differences between groups at each time point. The Wilcoxon rank sum test was used to compare the numbers of α-smooth muscle actin-positive cells between two groups. The software SAS ver. 9.1 (SAS Institute Inc., Cary, NC) was used for all statistical analyses. Values are presented as the mean and SD unless otherwise indicated.
3-Oxo-C12-HSL can accelerate wound healing just after administration
Full-thickness wounds were created at lateroabdominal sites on both sides of each animal and kept moist until day 5. After granulation tissue had become established, 3-oxo-C12-HSL or the same concentration of DMSO was administered to the wound surface. Gross observations revealed increased wound contraction after 3-oxo-C12-HSL administration (Fig. 1a). The time course of the changes in the wound area clearly indicated accelerated wound healing at 24 h after the administration of the quorum-sensing signal (Fig. 1b). The interaction of group and time was significant (F=3.03, P=0.002), and multiple comparisons were therefore performed. The relative areas were significantly smaller in the 3-oxo-C12-HSL group than in the vehicle group on days 6, 7, 8 and 9 (P=0.013, P<0.001, P=0.002 and P<0.001, respectively). HE staining of the granulation tissue revealed massive accumulation of fibroblasts in both groups (Fig. 2a). Infiltration of PMNs was also observed on the wound surface in the 3-oxo-C12-HSL group (Fig. 2b). Because wound contraction relies on the differentiation of fibroblasts to myofibroblasts, we further investigated the basis for the accelerated wound contraction by immunostaining of α-smooth muscle actin to assess myofibroblast differentiation (Ishiguro et al., 2009).
3-Oxo-C12-HSL induces faster wound healing. Gross observations (a) and the relative wound areas (b) reveal increased wound contraction induced by 3-oxo-C12-HSL. Control: N=10; 3-oxo-C12-HSL: N=12. Repeated-measures anova for the group and time interaction effect: F=3.03, P=0.02. *P<0.05, **P<0.01, ***P<0.001.
3-Oxo-C12-HSL induces fibroblast accumulation and inflammation. Skin sections were subjected to HE staining at 24 h after the administration of vehicle or 3-oxo-C12-HSL. Insets show × 40 magnified images of the cells associated with the granulation tissue. Magnification in (a) × 4, (b) × 100.
Fibroblasts can differentiate into myofibroblasts under 3-oxo-C12-HSL challenge
The immunostaining revealed that α-smooth muscle actin-positive cells were clearly present across the granulation tissue in the 3-oxo-C12-HSL group, whereas the control DMSO group only contained α-smooth muscle actin-positive cells at the edge of the wound (Fig. 3). The number of α-smooth muscle actin-positive cells per high-power field was significantly higher in the 3-oxo-C12-HSL group than in the control group (P<0.001, Fig. 3). These findings suggested that 3-oxo-C12-HSL had the potential to induce the differentiation of fibroblasts to myofibroblasts, which led to the increased wound contraction.
Myofibroblast differentiation is induced by 3-oxo-C12-HSL in the granulation tissue. Insets show × 40 magnified images of the cells associated with the granulation tissue. P<0.001, Wilcoxon rank sum test. N=6 for each group.
3-Oxo-C12-HSL induces the differentiation of fibroblasts and Cox-2 gene expression in vitro
To confirm the effects of 3-oxo-C12-HSL on cell differentiation, we used the Rat-1 fibroblast cell line. After culture in the presence of various concentrations of 3-oxo-C12-HSL, the number of cells expressing α-smooth muscle actin was increased compared with the control, which was confirmed only from 1 µM through 100 µM (Fig. 4). The representative pictures of 10 µM 3-oxo-C12-HSL-treated fibroblasts are shown. Because the administration of 3-oxo-C12-HSL to subdermal sites was reported to induce inflammation and Cox-2 expression in vivo (Smith et al., 2002a), we measured the expression levels of the Cox-2 gene. The level of Cox-2 expression was increased after the addition of 10 µM of 3-oxo-C12-HSL to the culture medium (Fig. 5). To investigate the differentiation pathway of fibroblasts to myofibroblasts, TGF-β1 and IL-6 gene expressions were examined, but no apparent differences were observed.
3-Oxo-C12-HSL induces Cox-2 gene expression in Rat-1 fibroblast cells.
The effects of the P. aeruginosa quorum-sensing signal 3-oxo-C12-HSL on mammalian cells have been investigated recently in several types of cells. The present study first revealed the effects of 3-oxo-C12-HSL on cutaneous wound healing using an in vivo animal model. The administration of 3-oxo-C12-HSL to the granulation tissue allowed us to evaluate its effects during wound healing. Our results indicated that 3-oxo-C12-HSL accelerated wound healing by inducing fibroblast differentiation to myofibroblasts. Using this wound-healing model, we were able to identify this unique effect of 3-oxo-C12-HSL on host cells.
The wound-healing process is divided into three phases, comprising the inflammation phase, proliferation phase and maturation phase. Fibroblasts play crucial roles in wound healing during the proliferation phase, and therefore, the finding that this P. aeruginosa quorum-sensing molecule can affect their function is of importance. Our in vitro experiments further supported the results of the in vivo experiments. Cox-2 expression was increased in Rat-1 cells, which could lead to the infiltration of neutrophils to induce inflammation (Smith et al., 2002b). Fibroblasts have the possibility of responding to the presence of 3-oxo-C12-HSL by differentiating into myofibroblasts and inducing inflammation. In general, fibroblast migration starts after inflammation is suppressed. However, fibroblasts and PMNs were observed simultaneously in the present study. This can be explained by the expression of Cox-2 by fibroblasts. These findings suggest the possibility that mammals have acquired the potential to accelerate wound healing against pathogen invasion by responding to quorum-sensing molecules. It has already been reported that paraoxonase, which degrades gram-negative quorum-sensing signals, is encoded in mammalian cells (Yang et al., 2005). This observation also indicates a direct defense system against bacterial infection. Taken together, the explanation that wound healing was accelerated by 3-oxo-C12-HSL is quite reasonable as a defense strategy against bacterial infection.
We further explored the mechanism of myofibroblast differentiation by evaluating the expression of TGF-β1 and IL-6, but found little difference between the two groups. A previous report indicated that Cox-2 expression was mediated through the induction of the nuclear factor (NF)-κB. NF-κB could have the potential to interfere with TGF-β signaling, which implies that other pathways are involved in the differentiation mechanism (Werner et al., 2007). One possible pathway involves the IL-6 signaling pathway (Gallucci et al., 2006), since a previous report indicated that increased expression of IL-6 was induced by 3-oxo-C12-HSL in vivo (Smith et al., 2002a); however, our results did not show these possibilities within fibroblasts. Further investigations are needed to elucidate this point.
The phenomena shown in the present study suggest a new strategy for wound management. In general, increased inflammation and wound contraction are unwelcome states for the quality of scar formation after wound healing. Inflammation may induce severe tissue destruction and excessive wound contraction may induce esthetically poor healing under specific conditions. If the quorum-sensing signal can be blocked and/or inflammation and wound contraction may be reduced using anti-inflammatory drugs, the quality of the wound healing will increase. Indeed, foam dressings containing nonsteroidal anti-inflammatory drugs are already commercially available (Cigna et al., 2009). These new strategies will evolve through investigations of the mechanisms of the effects of 3-oxo-C12-HSL on mammalian cells associated with wound healing.
This study was supported by a Grant-in-Aid from the Japan Society for the Promotion of Science (JSPS) (principle investigator: G.N.). There is no conflict of interest to declare.
(2002b) The Pseudomonas autoinducer N-(3-oxododecanoyl) homoserine lactone induces cyclooxygenase-2 and prostaglandin E2 production in human lung fibroblasts: implications for inflammation. J Immunol 169: 2636–2642.