Abstract
Background/Aim: Because the skin is exposed to the external environment, it is important that wound healing processes proceed and terminate rapidly to minimize the risk of infection. A previous case report described the promotion of wound healing by transdermal administration of lipopolysaccharide derived from Pantoea agglomerans (LPSp). However, whether the wound healing–promoting effect of LPSp was due to direct activity on skin cells or indirect effects involving macrophages remained unclear. Therefore, this study investigated the wound healing–promoting effect of LPSp, particularly the promotion of keratinocyte migration. Materials and Methods: The migration of HaCaT human keratinocytes over time with and without LPSp was assayed using a cell migration assay kit. Migration was also analyzed using HaCaT cells treated with LPSp and an antibody against Toll-like receptor (TLR) 4, a receptor for LPS. Results: Addition of LPSp significantly enhanced cell migration compared to no LPSp addition. Migration was inhibited by the addition of anti-TLR4 antibody. Conclusion: LPSp acts directly on epidermal cells to promote migration and may be one mechanism by which LPSp promotes wound healing.
Wound healing is a complex process involving multiple cell populations, the extracellular matrix, and soluble mediators, such as growth factors and cytokines (1, 2). This process can be divided into three major phases: inflammation, proliferation, and remodeling. During these phases, the activation, migration, and proliferation of epidermal cells play crucial roles.
Recent studies have highlighted the beneficial interactions between skin cells and skin microbiota in the regulation of innate immune responses during skin wound healing (3). Human skin hosts approximately one million normal flora bacteria per square centimeter, predominantly gram-positive bacteria (4). The gram-positive bacterium Staphylococcus epidermidis contributes to skin homeostasis by secreting glycerol and affecting innate immune activity (5). Gram-negative bacteria are also part of the normal skin flora. The skin microbiota differs significantly between healthy individuals and patients with atopic dermatitis (AD), as AD patients harbor decreased numbers of gram-negative bacteria (6). Studies using a mouse model of AD indicated that Roseomonas mucosa improves barrier function, activates innate immune responses, and inhibits colonization by Staphylococcus aureus on the skin (7, 8). These findings underscore the potential role of gram-negative bacteria in maintaining skin health and promoting wound healing.
Lipopolysaccharide (LPS), an extracellular component of gram-negative bacteria, is a key innate immune activator that exhibits strong activity even at very low levels, functioning as an information molecule between commensal bacteria and the host (9). Understanding the role of LPS in wound healing, particularly LPS produced by normal skin bacteria, is critical for developing new therapeutic approaches.
We have previously reported that oral or transdermal administration of LPS derived from Pantoea agglomerans (LPSp), a gram-negative bacterium isolated from wheat, is an effective and safe method for maintaining skin homeostasis (9, 10). A wound healing–promoting effect was observed when patients with difficult-to-heal wounds were topically treated with a cream containing LPS in combination with other topical medications (11). Furthermore, patients with mild AD treated by topical application of LPS-containing cream experienced symptom relief (12). In a mouse model, topical application of LPS reportedly suppressed contact hypersensitivity reactions (13). These reports suggest that LPSp may promote wound healing of epidermal cells. Another study reported that oral administration of LPS reduced atopic symptoms in a mouse model of AD (14). Oral LPS administration also induced host defense responses in a mouse model of burn wounds by enhancing bactericidal activity and suppressing bacterial translocation (15). A double-blind, placebo-controlled, randomized, parallel-group comparative study found that oral administration of LPS partially improved allergic symptoms (16). Because LPS strongly activates macrophages, the above-mentioned effects of LPS administered orally or transdermally are thought to be primarily mediated indirectly via macrophages (17). During the wound healing process, however, it is unclear whether LPS acts directly on epidermal cells or indirectly via macrophages.
This study investigated the direct effects of LPSp on wound healing using HaCaT cells, a widely used human epidermal cell model. A scratch assay was used to evaluate the migratory response of HaCaT cells to LPS treatment. Additionally, we explored the specific involvement of Toll-like receptor (TLR)4, a known LPS receptor (9), in mediating these effects. Our findings could pave the way for the development of new LPS-based therapeutics for wound healing.
Materials and Methods
Cell culture. Human HaCaT keratinocytes were obtained from CLS Cell Lines Service GmbH (Eppelheim, Germany). HaCaT cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, with high glucose, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 2 mmol/l L-glutamine, 10% fetal bovine serum (FBS, Hyclone™, Grand Island, NY, USA), and 1% antibiotics (10,000 μg/ml streptomycin and 10,000 units/ml penicillin) at 37°C with 5% CO2.
Media, reagents, and chemicals. Purified LPS was obtained from Pantoea agglomerans (Macrophi Inc., Kagawa, Japan). Anti-human TLR4 monoclonal mouse IgG1 (mabg-htlr4), mouse IgG1 isotype control (mabg1-ctrlm), and polyriboinosinic-polyribocytidylic acid (poly I:C) (tlrl-picw) were obtained from InvivoGen (San Diego, CA, USA).
Cell migration assay (Scratch assay). Migration of HaCaT cells was assessed using an Oris cell migration assay kit (Platypus Technologies, Madison, WI, USA) according to the instructions for use. Culture plates (96-well) were stoppered and seeded with 2×104 cells/100 μl/well of HaCaT cells and pre-cultured for 16 h. The stopper was removed, and the cells were washed once in DMEM with high glucose (Fuji Film Wako Pure Chemicals) supplemented with 2 mmol/l L-glutamine, 10 mmol/l N-(2-hydroxyethyl) piperazine-N′-2-ethanesulfonic acid (HEPES), 10% FBS (HycloneTM), and 1% antibiotics (10,000 μg/ml streptomycin and 10,000 units/ml penicillin). The culture medium was replaced with 10% FBS and DMEM containing HEPES (negative control), or 10% FBS and DMEM containing HEPES supplemented with poly I:C (final concentration 100 ng/ml) (positive control), or 10% FBS and HEPES-containing DMEM supplemented with LPSp (test sample) (n=8). LPSp was added at final concentrations of 0.5, 5, 50, or 500 ng/ml. Each well was photographed under a fluorescence microscope (BZ-X810, Keyence Corp., Osaka, Japan) with a microscopic culture system Stage Top incubator® (Tokai Hit., Co, Ltd., Shizuoka, Japan) at 0, 24, 48, and 72 h after medium replacement (0 h). Advanced Analysis Software (BZ-H4A, Keyence Corp.) and Hybrid Cell Count (BZ-H4C, Keyence Corp.) were used to determine the area of cell migration at each elapsed time point. Figure 1A shows images at each time of analysis. The observed areas are marked in red, and blue indicates areas in which no cells migrated.
Promotion of HaCaT cell migration by LPSp addition. (A) Representative photographs showing the remaining HaCaT cell wound area at 0 and 24 h after scratching. Red circle indicates a diameter of 1.9 mm. The blue area is the region in which no cells migrated (remaining wound area), and the area of this region was determined. (B) Migration rate of HaCaT cells over time following the addition of LPSp (n=8). Data are presented as the mean±SEM. *p<0.05 LPSp 50 ng/ml (24 h) vs. control (24 h), poly I:C (24 h), †p<0.05 LPSp 500 ng/ml (24 h) vs. control (24 h), poly I:C (24 h), **p<0.05 LPSp 50 ng/ml (48 h) vs. control (48 h), ‡p<0.05 LPSp 500 ng/ml (48 h) vs. control (48 h), #p<0.05 poly I:C (48 h) vs. control (48 h), ##p<0.05 poly I:C (72 h) vs. control (72 h) according to the Tukey–Kramer test. (C) Cell proliferation rate for each group after 72 h (n=8). Data are presented as the mean±SEM. p<0.05 poly I:C vs. control according to the Dunnett’s test.
Cell proliferation (MTS assay). Cell proliferation was quantified using a CellTiter 96 AQueous one solution cell proliferation assay system (Promega Corporation, Madison, WI, USA) to measure mitochondrial activity, according to the manufacturer’s protocol. In this cell viability/proliferation assay, methoxyphenyl-tetrazolium salt (MTS) is reduced by viable cells to form a colored formazan product, which is quantified colorimetrically at 490 nm using a spectrophotometer.
Inhibition of LPS effect by anti-TLR4 antibody. As in the Cell migration assay (Scratch assay) described above, HaCaT cells were seeded in an Oris cell migration assay kit and pre-cultured. Then, the stopper was removed, and the cells were washed once with culture medium. The culture medium was replaced with 10% FBS and HEPES-containing DMEM (medium-only group, 8 wells), or 10% FBS and HEPES-containing DMEM supplemented with anti-human TLR4 monoclonal mouse IgG1 (final concentration 10 μg/ml) (4 wells), or 10% FBS and HEPES-containing DMEM supplemented with mouse IgG1 (final concentration 10 μg/ml) (4 wells). After 1 h of incubation, LPSp was added to four wells of the medium-only group and four wells of each antibody group to a final concentration of 50 ng/ml. Each well was photographed under a fluorescence contrast microscope at 0, 24, 48, and 72 h. The area of cell migration was determined for each elapsed time point as described above.
Statistical analysis. The statistical analysis was performed using BellCurve for Excel (ver. 4.01, Social Survey Research Information, Tokyo, Japan). Differences in the cell migration activity based on surface area per elapsed time in the Scratch assay were evaluated using ANOVA analysis followed by Tukey–Kramer multiple comparison test. To evaluate differences in the cell proliferation rate, ANOVA analysis followed by Dunnett’s multiple comparison test was used. Differences were considered significant at p<0.05.
Results
Promotion of HaCaT human epidermal cell migration by addition of LPS. After pre-culturing HaCaT cells in 96-well plates of an Oris cell migration assay kit, LPSp was added to final concentrations of 0, 0.5, 5, 50, or 500 ng/ml, and cell migration was observed over time. Figure 1A shows images of cell migration over time. The observed areas are marked in red, and blue indicates areas in which no cells migrated. Figure 1B shows the migration rate of cells in each group at each time point. Poly I:C was used as a positive control in this study, as its effect on migration of HaCaT cells was reported by Takada et al. (18).
Treatment with poly I:C showed statistically significant enhancement of migration at 48 and 72 h compared with the negative control (p=0.042 and p=0.045, respectively), indicating that this evaluation system was valid. LPSp at 50 and 500 ng/ml significantly enhanced cell migration at 24 h compared with the negative control (p<0.001, p=0.002, respectively). Compared with the negative control, significant 1.68- and 1.65-fold increases in migration were observed at 24 h in the LPSp 50 and 500 ng/ml groups, respectively. These groups also showed significantly higher migration compared to cells treated with poly I:C at the same time point (p=0.024, p=0.040, respectively). At 48 h, cells treated with LPSp at 50 and 500 ng/ml showed significantly greater migration than negative control cells (p=0.026 and p=0.046, respectively), but there was no significant difference between poly I:C and LPSp-treated cells (Figure 1B).
Cell proliferation rate with addition of LPSp and Poly I:C. The proliferation rate of HaCaT cells at 72 h after addition of LPSp or poly I:C was determined relative to the negative control group using the MTS assay, which is commonly used to identify viable cells (Figure 1C). The proliferation rate of HaCaT cells in the poly I:C group was 97.7%, which was significantly lower than that of the negative control group (p=0.037). In contrast, there were no significant differences between the LPSp-treated and negative control groups (99.8-101.5%).
Anti-TLR4 antibody inhibits the effect of LPS on cell migration. An antibody against the LPS receptor TLR4 was used to confirm the direct contribution of LPS in promoting HaCaT cell migration. Representative images at various time points are shown in Figure 2A, and the change in the cell migration rate over time in each group is shown in Figure 2B. Migration of LPSp-treated cells was significantly inhibited at 48 and 72 h after the addition of anti-TLR4 antibodies relative to cells treated with LPSp alone (p<0.05). Migration of LPSp-treated cells was also significantly inhibited at 48 and 72 h after anti-TLR4 antibody addition relative to cells treated with LPSp plus isotype control antibody (p<0.05). The LPSp-induced up-regulation of cell migration was significantly inhibited by the addition of anti-TLR4 antibody, reduced to 91.3% at 48 h (p=0.003) and 76.1% at 72 h (p=0.002).
Inhibition of LPSp-induced HaCaT cell migration by anti-TLR4 antibody. (A) Representative photographs showing the remaining HaCaT cell wound area at 0, 24, 48, and 72 h after scratching. The red circle indicates a diameter of 1.9 mm. Blue area is the region in which no cells migrated (remaining wound area), and the area of this region was determined. (B) Cell migration rate per elapsed time with and without addition of anti-TLR4 antibody (n=4). Data are presented as the mean±SEM. *p<0.01 LPSp (48 h) vs. control (48 h), anti-TLR4 Ab + LPSp (48 h), †p<0.001 IgG + LPSp (48 h) vs. control (48 h), anti-TLR4 Ab + LPSp (48 h), **p<0.05 LPSp (72 h) vs. control (72 h), anti-TLR4 Ab + LPSp (72 h), ‡p<0.001 IgG + LPSp (72 h) vs. control (72 h), anti-TLR4 Ab + LPSp (72 h) according to the Tukey–Kramer test.
Discussion
This study found that treatment of human epidermal cells (HaCaT) with LPS promoted cell migration, underscoring the role of the gram-negative bacteria cell wall component LPS in wound healing processes. This effect of LPS on human epidermal cells was direct. LPS is widely present in natural environments, including the soil, trees, foods, and air. LPS activates macrophages via TLR4, thereby contributing to homeostasis maintenance (9, 19). When the skin is injured, the rapid progression of the healing process also contributes to the maintenance of homeostasis.
Previous studies in various cellular and animal models showed that LPS enhances wound healing (20-22), possibly by modulating immune responses and tissue regeneration processes. Low concentrations of Pseudomonas LPS were shown to promote wound healing in human airway epithelial cells (20). In mouse skin wounds, topical administration of LPS also promoted wound healing by enhancing the resolution of inflammation, increasing macrophage infiltration, promoting collagen synthesis, and altering the secretion of many mediators involved in the skin regeneration process, such as cytokines and chemokines (21). Injection of LPS-stimulated mesenchymal stem cells (LPS-primed MSCs) into the wound site of mice resulted in accelerated healing compared to the injection of MSCs without LPS stimulation (22). Furthermore, injection of TLR4-silenced LPS-primed MSCs into the wound site did not promote healing (22). Healing in a patient with a difficult-to-heal wound was reportedly accelerated after application of LPSp cream to the affected area (11).
TLR4, a receptor for LPS, is a representative regulator of innate immune responses, similar to heat shock protein, hyaluronic acid, and fibronectin. A study examining normal human epidermal keratinocytes confirmed that wound stimulation induces the expression of TLR4 mRNA (23). In mouse skin, TLR4 expression was found to be increased in the early stages after wounding, with expression particularly distributed in epidermal cells at the wound edge (23). TLR4 protein expression was also shown to be increased during the wound healing process in mouse corneal epithelium, and TLR4 expression was increased in scratch assays of human corneal epithelial cells (24). Elevated levels of Flightless I, an immunomodulator that negatively regulates TLR4 signaling in macrophages, were shown to decrease the initial expression of TLR4 and delay wound healing in mice (25). These findings suggest that TLR4 plays an important role in wound healing and that LPS, a ligand for TLR4, may also play such a role.
As no studies have reported the effect of LPSp on the migration of HaCaT cells, which are commonly used human epidermal cells, we evaluated the migration-promoting effect of LPSp on HaCaT cells using a scratch assay (26), which is often used to evaluate wound healing. LPSp at 50 and 500 ng/ml significantly enhanced cell migration at 24 h, by 1.68- and 1.65-fold, respectively, compared with the negative control (Figure 1B). In addition, this effect was more pronounced than that observed with poly I:C, the positive control, suggesting that the action of LPSp in promoting cell migration is robust and occurs early after wounding. Interestingly, whereas LPSp enhanced migration, it did not significantly affect HaCaT cell proliferation, as determined by MTS assay (Figure 1C), indicating that the primary role of LPSp in this context is to facilitate cell movement rather than proliferation.
Furthermore, as HaCaT cells express TLR4 (27), we treated HaCaT cells with an antibody against TLR4, the receptor for LPS (anti-TLR4 antibody). After 1 h of culture, LPSp was added, and cell migration was monitored. Treatment with the anti-TLR4 antibody significantly inhibited cell migration at 48 and 72 h compared with no addition of antibody (Figure 2A, B). Thus, the migration-promoting effect of LPSp on HaCaT cells observed in this study is mediated by TLR4.
In airway epithelial cells, LPS binds to TLR4 and then activates the protein kinase C (PKC) isoforms PKCα and PKCβ. In addition, activation of dual oxidase 1 and TNF-α–converting enzyme is followed by the release of soluble transforming growth factor–α, which binds to the epidermal growth factor receptor (EGFR), resulting in phosphorylation of EGFR, which promotes wound healing (20). Pseudomonas LPS is known to activate EGFR (28), and in a study of various epithelial cells, the addition of growth factors activated EGFR and promoted wound healing (20). Stimulation of EGFR induces the expression of interleukin (IL)-8, which induces the migration of neutrophils important in the wound healing process (29). IL-8 also promotes the migration of HaCaT cells, suggesting that IL-8 has a wound healing–promoting effect (18). In a preliminary study, the level of IL-8 in the culture supernatant of HaCaT cells at 72 h after addition of the anti-TLR4 antibody and LPSp was significantly decreased compared to the cells that were treated with LPSp without anti-TLR4 antibody (data not shown). Therefore, IL-8 may play a role in the mechanism of the cell migration–promoting effect of LPS. In this experiment, LPSp promoted the migration of HaCaT cells via TLR4, but details regarding the mechanism, such as whether it involves activation of EGFR and an association with IL-8, have not been fully elucidated, and further analysis is thus needed.
Our previous study examined the effects of LPS with both oral and transdermal administration (9-14, 16, 17). The data suggested that LPS acts indirectly rather than directly. However, keratinocytes stimulated with LPS exhibit increased expression of filaggrin, a barrier function protein (30). Furthermore, keratinocytes produce the antimicrobial compound β-defensin upon LPS stimulation (31). Thus, keratinocytes respond to LPS stimulation by various mechanisms, and LPSp directly promoted keratinocyte migration in the present study. This direct effect of LPSp on keratinocytes suggests one of the mechanisms by which transdermal administration of LPSp promotes wound healing.
Despite continued medical advances, research to identify substances that promote wound healing in the skin is ongoing (32-34), but the development of therapeutic agents without side effects remains challenging. In the broader context of wound healing therapeutics, the role of the skin microbiota, particularly that of gram-negative bacteria and their components, such as LPS, is gaining increasing attention (3). LPS is a component of gram-negative bacteria, which includes normal skin bacteria, and LPSp, in particular, is safe, and used in food products and skin care (12, 17, 19).
Although there have been reports of different results depending on LPS origin, cell types, and experimental conditions (35), our findings suggest that LPSp, derived from a safe and commonly used strain of bacteria, holds potential as a therapeutic agent for enhancing skin wound healing. Future research should explore the varying effects of different LPS types and concentrations on various cell types and under different conditions to fully harness its therapeutic potential.
Conclusion
In this study, we demonstrate that lipopolysaccharide from Pantoea agglomerans (LPSp) directly promotes the migration of human epidermal cells (HaCaT cells) via TLR4. LPSp acts directly on epidermal cells to facilitate their migration to the wound site, suggesting its role in accelerating tissue regeneration. This mechanism provides new insights into the conventional view that the effects of LPSp are exerted indirectly, mainly via macrophages.
Future research should further elucidate the detailed mechanism of LPSp action and develop specific protocols for clinical application. Additionally, the effects of LPSp on other skin cells and immune cells should be investigated to maximize its therapeutic potential by comprehensively understanding its role in wound healing.
Acknowledgements
The Authors thank Macrophi Inc. and Control of Innate Immune Laboratory members for valuable comments on our research.
Footnotes
Authors’ Contributions
H.I., T.N. and G-I.S. conceptualized the study and coordinated the experiments. T.N. performed the experiments, data curation, and formal analysis. H.I., T.N., C.K. and G-I.S. wrote, reviewed and edited the manuscript. All Authors have read and agreed to the published version of the manuscript.
Conflicts of Interest
The Authors declare no conflicts of interest in relation to this study.
Funding
This research received no external funding.
- Received June 13, 2024.
- Revision received July 8, 2024.
- Accepted July 9, 2024.
- Copyright © 2024 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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