Downregulated cytotoxic CD8+ T-cell identifies with the NKG2A-soluble HLA-E axis as a predictive biomarker and potential therapeutic target in keloids.

Keloids are an abnormal fibroproliferative wound-healing disease with a poorly understood pathogenesis, making it difficult to predict and prevent this disease in clinical settings. Identifying disease-specific signatures at the molecular and cellular levels in both the blood circulation and primary lesions is urgently needed to develop novel biomarkers for risk assessment and therapeutic targets for recurrence-free treatment. There is mounting evidence of immune cell dysregulation in keloid scarring. In this study, we aimed to profile keloid scar tissues and blood cells and found that downregulation of cytotoxic CD8+ T cells is a keloid signature in the peripheral blood and keloid lesions. Single-cell RNA sequencing revealed that the NKG2A/CD94 complex was specifically upregulated, which might contribute to the significant reduction in CTLs within the scar tissue boundary. In addition, the NKG2A/CD94 complex was associated with high serum levels of soluble human leukocyte antigen-E (sHLA-E). We subsequently measured sHLA-E in our hospital-based study cohort, consisting of 104 keloid patients, 512 healthy donors, and 100 patients with an interfering disease. The sensitivity and specificity of sHLA-E were 83.69% (87/104) and 92.16% (564/612), respectively, and hypertrophic scars and other unrelated diseases exhibited minimal interference with the test results. Furthermore, intralesional therapy with triamcinolone combined with 5-fluorouracil drastically decreased the sHLA-E levels in keloid patients with better prognostic outcomes, while an incomplete reduction in the sHLA-E levels in patient serum was associated with higher recurrence. sHLA-E may effectively serve as a diagnostic marker for assessing the risk of keloid formation and a prognostic marker for the clinical outcomes of intralesional treatment.

Molecular Changes Underlying Hypertrophic Scarring Following Burns Involve Specific Deregulations at All Wound Healing Stages (Inflammation, Proliferation and Maturation).

Excessive connective tissue accumulation, a hallmark of hypertrophic scaring, results in progressive deterioration of the structure and function of organs. It can also be seen during tumor growth and other fibroproliferative disorders. These processes result from a wide spectrum of cross-talks between mesenchymal, epithelial and inflammatory/immune cells that have not yet been fully understood. In the present review, we aimed to describe the molecular features of fibroblasts and their interactions with immune and epithelial cells and extracellular matrix. We also compared different types of fibroblasts and their roles in skin repair and regeneration following burn injury. In summary, here we briefly review molecular changes underlying hypertrophic scarring following burns throughout all basic wound healing stages, i.e. during inflammation, proliferation and maturation.

Scar-reducing effects of gambogenic acid on skin wounds in rabbit ears.

Hypertrophic scar (HS) is a dermal fibroproliferative disease that often occurs following abnormal wound healing. To date, there is no satisfied treatment strategies for improvement of scar formation with few side effects. The effects of gambogenic acid (GNA) on scar hypertrophy has not been studied previously. The present study was undertaken to find out the scar-reducing effects of GNA (0.48, 0.96 or 1.92 mg/ml) on skin wounds in rabbit ears. Scar evaluation index (SEI), collagen I (Col1) and collagen III (Col3), microvascular density (MVD), CD4+T cells and macrophages, vascular endothelial growth factor receptor 2 (VEGFR2), fibroblast growth factor receptor 1 (FGFR1), phospho-VEGFR 2 (p-VEGFR2) and p-FGFR1, interleukin (IL)-1ß, IL-6, IL-10 and tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-ß1 and connective tissue growth factor (CTGF) in scar tissue were detected using various methods, respectively. Our data showed that GNA significantly reduced SEI, and the expression of Col1 and Col3 in scar tissue in a concentration-dependent manner. Also, it decreased MVD, the infiltration of CD4+T cells and macrophages, and the levels of VEGFR2, p-VEGFR2, FGFR1, p-FGFR1, TGF-ß1, CTGF, IL-1ß, IL-6, TNF-α, in addition to upregulated IL-10 in scar tissue. As a result, this study revealed that GNA reduced HS formation, which was associated with the inhibition of neoangiogenesis, local inflammatory response and growth factor expression in scar tissue during wound healing. These findings suggested that GNA may be considered as a preventive and therapeutic candidate for HS.

Human CD206+ Macrophages Show Antifibrotic Effects on Human Fibroblasts through an IL-6-Dependent Mechanism In Vitro.

BACKGROUND: Pathologic scarring including keloid and hypertrophic scar causes aesthetic and physical problems, and there are clinical difficulties (e.g., posttreatment recurrence) in dealing with pathologic scarring. Understanding the mechanisms that underlie scar control in wound healing will help prevent and treat pathologic scarring. The authors focused on CD206+ macrophages in the wound-healing process, and hypothesized that CD206+ macrophages have antifibrotic effects on fibroblasts. METHODS: The authors established a co-culture system for CD206+ macrophages and fibroblasts (cell ratio, 1:1). The authors examined the CD206+ macrophages' antifibrotic effects on fibroblasts after a 72-hour culture, focusing on fibrosis-related genes. To identify key factor(s) in the interaction between CD206+ macrophages and fibroblasts, the authors analyzed cytokines in a conditioned medium of the co-culture system. RESULTS: Under co-culture with CD206+ macrophages, expression of the following in the fibroblasts was significantly down-regulated: type 1 (fold change, 0.38) and type 3 collagen (0.45), alpha smooth muscle actin (0.24), connective tissue growth factor (0.40), and transforming growth factor-beta (0.66); the expression of matrix metalloproteinase 1 was significantly up-regulated (1.92). Conditioned medium in the co-culture showed a high interleukin (IL)-6 concentration (419 ± 88 pg/ml). When IL-6 was added to fibroblasts, antifibrotic changes in gene expression (as observed under the co-culture) occurred in the fibroblasts. CONCLUSIONS: The authors' in vitro results revealed that CD206+ macrophages have antifibrotic effects on fibroblasts by means of a paracrine mechanism involving IL-6. Understanding these effects, especially in vivo, will help elucidate the mechanism of scar control in wound healing and contribute to the development of new scar treatments.

Prognostic tools for hypertrophic scar formation based on fundamental differences in systemic immunity.

Unpredictable hypertrophic scarring (HS) occurs after approximately 35% of all surgical procedures and causes significant physical and psychological complaints. Parallel to the need to understanding the mechanisms underlying HS formation, a prognostic tool is needed. The objective was to determine whether (systemic) immunological differences exist between patients who develop HS and those who develop normotrophic scars (NS) and to assess whether those differences can be used to identify patients prone to developing HS. A prospective cohort study with NS and HS groups in which (a) cytokine release by peripheral blood mononuclear cells (PBMC) and (b) the irritation threshold (IT) after an irritant (sodium lauryl sulphate) patch test was evaluated. Univariate regression analysis of PBMC cytokine secretion showed that low MCP-1, IL-8, IL-18 and IL-23 levels have a strong correlation with HS (P < .010-0.004; AUC = 0.790-0.883). Notably, combinations of two or three cytokines (TNF-a, MCP-1 and IL-23; AUC: 0.942, Nagelkerke R2 : 0.727) showed an improved AUC indicating a better correlation with HS than single cytokine analysis. These combination models produce good prognostic results over a broad probability range (sensitivity: 93.8%, specificity 86.7%, accuracy 90,25% between probability 0.3 and 0.7). Furthermore, the HS group had a lower IT than the NS group and an accuracy of 68%. In conclusion, very fundamental immunological differences exist between individuals who develop HS and those who do not, whereas the cytokine assay forms the basis of a predictive prognostic test for HS formation, the less invasive, easily performed irritant skin patch test is more accessible for daily practice.

The Roles of Inflammation in Keloid and Hypertrophic Scars.

The underlying mechanisms of wound healing are complex but inflammation is one of the determining factors. Besides its traditional role in combating against infection upon injury, the characteristics and magnitude of inflammation have dramatic impacts on the pathogenesis of scar. Keloids and hypertrophic scars are pathological scars that result from aberrant wound healing. They are characterized by continuous local inflammation and excessive collagen deposition. In this review, we aim at discussing how dysregulated inflammation contributes to the pathogenesis of scar formation. Immune cells, soluble inflammatory mediators, and the related intracellular signal transduction pathways are our three subtopics encompassing the events occurring in inflammation associated with scar formation. In the end, we enumerate the current and potential medicines and therapeutics for suppressing inflammation and limiting progression to scar. Understanding the initiation, progression, and resolution of inflammation will provide insights into the mechanisms of scar formation and is useful for developing effective treatments.

Skin tissue regeneration for burn injury.

The skin is the largest organ of the body, which meets the environment most directly. Thus, the skin is vulnerable to various damages, particularly burn injury. Skin wound healing is a serious interaction between cell types, cytokines, mediators, the neurovascular system, and matrix remodeling. Tissue regeneration technology remarkably enhances skin repair via re-epidermalization, epidermal-stromal cell interactions, angiogenesis, and inhabitation of hypertrophic scars and keloids. The success rates of skin healing for burn injuries have significantly increased with the use of various skin substitutes. In this review, we discuss skin replacement with cells, growth factors, scaffolds, or cell-seeded scaffolds for skin tissue reconstruction and also compare the high efficacy and cost-effectiveness of each therapy. We describe the essentials, achievements, and challenges of cell-based therapy in reducing scar formation and improving burn injury treatment.

Regulation of Langerhans cell functions in a hypoxic environment.

UNLABELLED: Langerhans cells (LCs) are a specialized dendritic cell subset that resides in the epidermis and mucosal epithelia and is critical for the orchestration of skin immunity. Recent evidence suggest that LCs are involved in aberrant wound healing and in the development of hypertrophic scars and chronic wounds, which are characterized by a hypoxic environment. Understanding LCs biology under hypoxia may, thus, lead to the identification of novel pathogenetic mechanisms of wound repair disorders and open new therapeutic opportunities to improve wound healing. In this study, we characterize a previously unrecognized role for hypoxia in significantly affecting the phenotype and functional properties of human monocyte-derived LCs, impairing their ability to stimulate naive T cell responses, and identify the triggering receptor expressed on myeloid (TREM)-1, a member of the Ig immunoregulatory receptor family, as a new hypoxia-inducible gene in LCs and an activator of their proinflammatory and Th1-polarizing functions in a hypoxic environment. Furthermore, we provide the first evidence of TREM-1 expression in vivo in LCs infiltrating hypoxic areas of active hypertrophic scars and decubitous ulcers, pointing to a potential pathogenic role of this molecule in wound repair disorders. KEY MESSAGES: Hypoxia modulates surface molecule expression and cytokine profile in Langerhans cells. Hypoxia impairs human Langerhans cell stimulatory activity on naive T cells. Hypoxia selectively induces TREM-1 expression in human Langerhans cells. TREM-1 engagement stimulates Langerhans cell inflammatory and Th1-polarizing activity. TREM-1 is expressed in vivo in Langerhans cells infiltrating hypoxic skin lesions.

Missense Variant in MAPK Inactivator PTPN5 Is Associated with Decreased Severity of Post-Burn Hypertrophic Scarring.

BACKGROUND: Hypertrophic scarring (HTS) is hypothesized to have a genetic mechanism, yet its genetic determinants are largely unknown. The mitogen-activated protein kinase (MAPK) pathways are important mediators of inflammatory signaling, and experimental evidence implicates MAPKs in HTS formation. We hypothesized that single-nucleotide polymorphisms (SNPs) in MAPK-pathway genes would be associated with severity of post-burn HTS. METHODS: We analyzed data from a prospective-cohort genome-wide association study of post-burn HTS. We included subjects with deep-partial-thickness burns admitted to our center who provided blood for genotyping and had at least one Vancouver Scar Scale (VSS) assessment. After adjusting for HTS risk factors and population stratification, we tested MAPK-pathway gene SNPs for association with the four VSS variables in a joint regression model. In addition to individual-SNP analysis, we performed gene-based association testing. RESULTS: Our study population consisted of 538 adults (median age 40 years) who were predominantly White (76%) males (71%) admitted to our center from 2007-2014 with small-to-moderate-sized burns (median burn size 6% total body surface area). Of 2,146 SNPs tested, a rare missense variant in the PTPN5 gene (rs56234898; minor allele frequency 1.5%) was significantly associated with decreased severity of post-burn HTS (P = 1.3×10-6). In gene-based analysis, PTPN5 (P = 1.2×10-5) showed a significant association and BDNF (P = 9.5×10-4) a borderline-significant association with HTS severity. CONCLUSIONS: We report PTPN5 as a novel genetic locus associated with HTS severity. PTPN5 is a MAPK inhibitor expressed in neurons, suggesting a potential role for neurotrophic factors and neuroinflammatory signaling in HTS pathophysiology.

The natural behavior of mononuclear phagocytes in HTS formation.

Hypertrophic scars (HTS) are caused by trauma or burn injuries to the deep dermis and are considered fibrosis in the skin. Monocytes, M1 and M2 macrophages are mononuclear phagocytes. Studies suggest that M2 macrophages are profibrotic and might contribute to HTS formation. Our lab has established a human HTS-like nude mouse model, in which the grafted human skin develops red, raised, and firm scarring, resembling HTS seen in humans. In this study, we observed the natural behavior of mononuclear phagocyte system in this nude mouse model of dermal fibrosis at multiple time points. Thirty athymic nude mice received human skin grafts and an equal number of mice received mouse skin grafts as controls. The grafted skin and blood were harvested at 1, 2, 3, 4, and 8 weeks. Wound area, thickness, collagen morphology and level, the cell number of myofibroblasts, M1- and M2-like macrophages in the grafted skin, as well as monocyte fraction in the blood were investigated at each time points. Xenografted mice developed contracted and thickened scars grossly. The xenografted skin resembled human HTS tissue based on enhanced thickness, fibrotic orientation of collagen bundles, increased collagen level, and infiltration of myofibroblasts. In the blood, monocytes dramatically decreased at 1 week postgrafting and gradually returned to normal in the following 8 weeks. In the xenografted skin, M1-like macrophages were found predominantly at 1-2 weeks postgrafting; whereas, M2-like macrophages were abundant at later time points, 3-4 weeks postgrafting coincident with the development of fibrosis in the human skin tissues. This understanding of the natural behavior of mononuclear phagocytes in vivo in our mouse model provides evidence for the role of M2-like macrophages in fibrosis of human skin and suggests that macrophage depletion in the subacute phases of wound healing might reduce or prevent HTS formation.

Exacerbated and prolonged inflammation impairs wound healing and increases scarring.

Altered inflammation in the early stage has long been assumed to affect subsequent steps of the repair process that could influence proper wound healing and remodeling. However, the lack of explicit experimental data makes the connection between dysregulated wound inflammation and poor wound healing elusive. To bridge this gap, we used the established rabbit ear hypertrophic scar model for studying the causal effect of dysregulated inflammation. We induced an exacerbated and prolonged inflammatory state in these wounds with the combination of trauma-related stimulators of pathogen-associated molecular patterns from heat-killed Pseudomonas aeruginosa and damage-associated molecular patterns from a dermal homogenate. In stimulated wounds, a heightened and lengthened inflammation was observed based on quantitative measurements of IL-6 expression, tissue polymorphonuclear leukocytes infiltration, and tissue myeloperoxidase activity. Along with the high level of inflammation, wound healing parameters (epithelial gap and others) at postoperative day 7 and 16 were significantly altered in stimulated wounds compared to unstimulated controls. By postoperative day 35, scar elevation of stimulated wounds was higher than that of control wounds (scar elevation index: 1.90 vs. 1.39, p < 0.01). Moreover, treatment of these inflamed wounds with Indomethacin (at concentrations of 0.01, 0.1, and 0.4%) reduced scar elevation but with adverse effects of delayed wound closure and increased cartilage hypertrophy. In summary, successful establishment of this inflamed wound model provides a platform to understand these detrimental aspects of unchecked inflammation and to further test agents that can modulate local inflammation to improve wound outcomes.

Inhibition of mechanical stress-induced hypertrophic scar inflammation by emodin.

At least 50% of hypertrophic scarring (HS) is characterized by inflammation, for which there is currently no effective treatment available. Emodin is a major component of the widely used Chinese herb, rhubarb, which has been used to treat inflammation in several types of disease. However, few studies have investigated the efficacy of emodin in the treatment of HS. In the present study, a mouse model with mechanical stress­induced HS was used to investigate the effects of emodin (20, 40, 80, or 120 mg/ml) on HS, and to determine the potential underlying mechanisms. Treatment with emodin significantly attenuated HS inflammation, as determined by histopathological assessment of the scar elevation index, collagen structure and inflammation (P<0.05). Furthermore, treatment with emodin (40 mg/ml) markedly inhibited phosphoinositide 3­kinase (PI3K)/Akt activity (P<0.01) and this attenuation was associated with reduced expression levels of tumor necrosis factor­α, interleukin­6 and monocyte chemoattractant protein­1 (P<0.05) in the HS tissue. The results of the present study indicated that administration of emodin had therapeutic effects on the progression of HS and the underlying mechanism of this may be due to inhibition of the PI3K/Akt signaling pathway.

A novel immune competent murine hypertrophic scar contracture model: a tool to elucidate disease mechanism and develop new therapies.

Hypertrophic scar (HSc) contraction following burn injury causes contractures. Contractures are painful and disfiguring. Current therapies are marginally effective. To study pathogenesis and develop new therapies, a murine model is needed. We have created a validated immune-competent murine HSc model. A third-degree burn was created on dorsum of C57BL/6 mice. Three days postburn, tissue was excised and grafted with ear skin. Graft contraction was analyzed and tissue harvested on different time points. Outcomes were compared with human condition to validate the model. To confirm graft survival, green fluorescent protein (GFP) mice were used, and histologic analysis was performed to differentiate between ear and back skin. Role of panniculus carnosus in contraction was analyzed. Cellularity was assessed with 4',6-diamidino-2-phenylindole. Collagen maturation was assessed with Picro-sirius red. Mast cells were stained with Toluidine blue. Macrophages were detected with F4/80 immune. Vascularity was assessed with CD31 immune. RNA for contractile proteins was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Elastic moduli of skin and scar tissue were analyzed using a microstrain analyzer. Grafts contracted to ∼45% of their original size by day 14 and maintained their size. Grafting of GFP mouse skin onto wild-type mice, and analysis of dermal thickness and hair follicle density, confirmed graft survival. Interestingly, hair follicles disappeared after grafting and regenerated in ear skin configuration by day 30. Radiological analysis revealed that panniculus carnosus doesn't contribute to contraction. Microscopic analyses showed that grafts show increase in cellularity. Granulation tissue formed after day 3. Collagen analysis revealed increases in collagen maturation over time. CD31 stain revealed increased vascularity. Macrophages and mast cells were increased. qRT-PCR showed up-regulation of transforming growth factor beta, alpha smooth muscle actin, and rho-associated protein kinase 2 in HSc. Tensile testing revealed that human skin and scar tissues are tougher than mouse skin and scar tissues.

Effect of lipopolysaccharide on the biological characteristics of human skin fibroblasts and hypertrophic scar tissue formation.

Burn injury-mediated destruction of the skin barrier normally induces microbial invasion, in turn leading to the development of systemic infection and occasional septic shock by the release of endotoxins. The objective of this work was to study the influence of lipopolysaccharide (LPS) on the biological characteristics of normal skin fibroblasts and to elucidate the influence of LPS in the initial stage of skin wound healing. Twenty patients with hypertrophic scar in proliferative stage were selected randomly and primary cultures were established from fibroblasts derived from their hypertrophic scar tissue and normal skin. Normal skin fibroblasts of passage 3 were stimulated with different concentrations of LPS. LPS stimulated the proliferation and collagen synthesis of fibroblasts within a certain extent of concentrations (0.005-0.5 µg/mL) (P < 0.05), whereas at a concentration of 1 µg/mL inhibited the proliferation and collagen synthesis of fibroblasts (P < 0.05). Collagen synthesis by normal skin fibroblasts after LPS stimulation mimicked those derived from hypertrophic scar tissue. LPS of 0.1 µg/mL had significant effect on normal skin fibroblasts-continuous passage of these fibroblasts resulted in ultrastructural pattern similar to fibroblasts derived from hypertrophic scar tissue, and the findings was substantiated by hematoxylin and eosin staining and immunohistochemistry detection of proliferation cell nuclear antigen, type I procollagen and α-smooth muscle actin. Our results suggest that LPS might convert normal skin fibroblasts to hypertrophic scar tissue fibroblasts and participate in the formation of hypertrophic scar; hence, appropriate concentration of LPS may have no effect or be beneficial to skin wound healing, whereas excessive concentration of LPS may delay the time of wound healing.

Tacrolimus fails to regulate collagen expression in dermal fibroblasts.

BACKGROUND: The purpose of this study was to investigate the effects of tacrolimus on human fibroblasts derived from unwounded skin, hypertrophic scars (HTS), and keloids. We hypothesized that tacrolimus, a potent anti-inflammatory and immunosuppressant drug known to attenuate solid organ transplant fibrosis, would block collagen expression in human dermal fibroblasts. METHODS: We performed genomewide microarray analysis on human dermal fibroblasts treated with tacrolimus in vitro. We used principal component analysis and hierarchical clustering to identify targets regulated by tacrolimus. We performed quantitative polymerase chain reaction to validate the effect of tacrolimus on collagen 1 and 3 expression. RESULTS: We identified 62, 136, and 185 gene probes on microarray analysis that were significantly regulated (P < 0.05) by tacrolimus in normal, HTS, and keloid fibroblasts, respectively. Collagen pathways were not blocked after tacrolimus exposure in any of the fibroblast groups; we validated these findings using quantitative polymerase chain reaction for collagen 1 and 3. Microarray gene expression of NME/NM23 nucleoside diphosphate kinase 1 and heterogeneous nuclear ribonucleoprotein H3-2H9 were significantly downregulated (P < 0.05) by tacrolimus in both HTS and keloid fibroblast populations but not normal fibroblasts. CONCLUSIONS: Tacrolimus does not modulate the expression of collagen 1 or 3 in human dermal fibroblasts in vitro. Microarray gene expression of NME/NM23 nucleoside diphosphate kinase 1 and heterogeneous nuclear ribonucleoprotein H3-2H9 are blocked by tacrolimus in pathologic fibroblasts but not normal fibroblasts, and may represent novel genes underlying HTS and keloid pathogenesis. Tacrolimus-based anti-fibrotics might prove more effective if non-fibroblast populations such as inflammatory cells and keratinocytes are targeted.

Transdermal siRNA-TGFß1-337 patch for hypertrophic scar treatment.

Hypertrophic scarring (HSc) is a fibroproliferative disorder of the dermis characterized by erythematous, swollen, and pruritic lesions of healing skin. An increased understanding of the role of TGFß1 in the development of HSc provides the potential for treating HSc by down-regulating TGFß1 expression. siRNAs that effectively interfered with TGFß1 expression were screened. It was concluded that the siRNA-TGFß1-337 was able to effectively down-regulate TGFß1 expression in HSc fibroblasts. The effects of siRNA-TGFß1-337 on cell proliferation, cell cycle, and apoptosis of HSc fibroblasts were investigated. It was shown that it inhibited cell proliferation, arrested cells in the G1 stage of the cell cycle, and induced apoptosis of HSc fibroblasts. The transdermal patch of siRNA-TGFß1-337 was a combination of siRNA-TGFß1-337 and a pressure-sensitive adhesive hydrogel. The treatment effects of the transdermal patch were assessed in an animal model established by transplanting human HSc to nude mice. Decreased expression of TGFß1 was observed with treatment with the transdermal siRNA-TGFß1-337 patch. Consequently, the treatment resulted in type I collagen down-regulation and regularly arranged scar fibroblasts being significantly reduced and undergoing apoptosis; the scar size was decreased significantly. Thus, our findings indicate that a transdermal siRNA-TGFß1-337 patch is a potential treatment for hypertrophic scars.

Evidence of a role for fibrocyte and keratinocyte-like cells in the formation of hypertrophic scars.

Burn injuries affect millions of people every year, and dermal fibrosis is a common complication for the victims. This disfigurement has functional and cosmetic consequences and many research groups have made it the focus of their work to understand the mechanisms that underlie its development. Although significant progress has been made in wound-healing processes, the complexity of events involved makes it very difficult to come up with a single strategy to prevent this devastating fibrotic condition. Inflammation is considered one predisposing factor, although this phase is a necessary aspect of the wound-healing process. Inflammation, driven by infiltrated immune cells, begins minutes after the burn injury and is the prevalent phase of wound healing in the early stages. Accompanying the inflammatory infiltrate, there is evidence that subpopulations of bone marrow-derived cells are also present. These populations include fibrocytes and keratinocyte-like cells, derivatives of CD14 monocytes, a component of the peripheral blood mononuclear cell infiltrate. There is evidence that these cells contribute to regeneration and repair of the wound site, but it is interesting to note that there are also reports that these cells can have adverse effects and may contribute to the development of dermal fibrosis. In this article, the authors present a review of the origin and transdifferentiation of these cells from bone marrow stem cells, the environments that direct this transdifferentiation, and evidence to support their role in fibrosis, as well as potential avenues for therapeutics to control their fibrotic effects.

[Opportunities for effective skin regeneration by topical stimulation of immunity].

A murine model of incisional wound was used to evaluate effect of topical application of purified bacterial lipopolysaccharide on the wound healing process. Thirty five ICR mice were used in the study. It was shown that bacterial lipopolysaccharide is a strong promotor of wound healing. It increases tensile strength, accelerates completion of the inflammatory process, stimulates collagen deposition and early remodeling.

Mechanical force prolongs acute inflammation via T-cell-dependent pathways during scar formation.

Mechanical force significantly modulates both inflammation and fibrosis, yet the fundamental mechanisms that regulate these interactions remain poorly understood. Here we performed microarray analysis to compare gene expression in mechanically loaded wounds vs. unloaded control wounds in an established murine hypertrophic scar (HTS) model. We identified 853 mechanically regulated genes (false discovery rate <2) at d 14 postinjury, a subset of which were enriched for T-cell-regulated pathways. To substantiate the role of T cells in scar mechanotransduction, we applied the HTS model to T-cell-deficient mice and wild-type mice. We found that scar formation in T-cell-deficient mice was reduced by almost 9-fold (P < 0.001) with attenuated epidermal (by 2.6-fold, P < 0.01) and dermal (3.9-fold, P < 0.05) proliferation. Mechanical stimulation was highly associated with sustained T-cell-dependent Th2 cytokine (IL-4 and IL-13) and chemokine (MCP-1) signaling. Further, T-cell-deficient mice failed to recruit systemic inflammatory cells such as macrophages or monocytic fibroblast precursors in response to mechanical loading. These findings indicate that T-cell-regulated fibrogenic pathways are highly mechanoresponsive and suggest that mechanical forces induce a chronic-like inflammatory state through immune-dependent activation of both local and systemic cell populations.

Toll-like receptors expressed by dermal fibroblasts contribute to hypertrophic scarring.

Hypertrophic scar (HTS), a fibroproliferative disorder (FPD), complicates burn wound healing. Although the pathogenesis is not understood, prolonged inflammation is a known contributing factor. Emerging evidence suggests that fibroblasts regulate immune/inflammatory responses through toll-like receptor 4 (TLR4) activated by lipopolysaccharide (LPS) through adaptor molecules, leading to nuclear factor kappa-light-chain-enhancer of activated B cells and mitogen-activated protein kinases activation, cytokine gene transcription and co-stimulatory molecule expression resulting in inflammation. This study explored the possible role of TLR4 in HTS formation. Paired normal and HTS tissue from burn patients was collected and dermal fibroblasts isolated and cultured. Immunohistochemical analysis of tissues demonstrated increased TLR4 staining in HTS tissue. Quantitative RT-PCR of three pairs of fibroblasts demonstrated mRNA levels for TLR4 and its legend myeloid differentiation factor 88 (MyD88) in HTS fibroblasts were increased significantly compared with normal fibroblasts. Flow cytometry showed increased TLR4 expression in HTS fibroblasts compared with normal. ELISA demonstrated protein levels for prostaglandin E2, interleukin (IL)-6, IL-8 and monocyte chemotactic protein-1 (MCP-1) were significantly increased in HTS fibroblasts compared to normal. When paired normal and HTS fibroblasts were stimulated with LPS, significant increases in mRNA and protein levels for MyD88, IL-6, IL-8, and MCP-1 were detected. However, when transfected with MyD88 small interfering RNA (siRNA), then stimulated with LPS, a significant decrease in mRNA and protein levels for these molecules compared to only LPS-stimulated fibroblasts was detected. In comparison, a scramble siRNA transfection did not affect mRNA or protein levels for these molecules. Results demonstrate LPS stimulates proinflammatory cytokine expression in dermal fibroblasts and MyD88 siRNA eliminates the expression. Therefore, controlling inflammation and manipulating TLR signaling in skin cells may result in novel treatment strategies for HTS and other FPD.