IL‐10 alleviates lipopolysaccharide‐induced skin scarring via IL‐10R/STAT3 axis regulating TLR4/NF‐κB pathway in dermal fibroblasts

Abstract Hypertrophic scar (HS) is a severe fibrotic skin disease. It has always been a major problem in clinical treatment, mainly because its pathogenesis has not been well understood. The roles of bacterial contamination and prolonged wound inflammation were considered significant. IL‐10 is a potent anti‐inflammatory cytokine and plays a pivotal role in wound healing and scar formation. Here, we investigate whether IL‐10 alleviates lipopolysaccharide (LPS)‐induced inflammatory response and skin scarring and explore the possible mechanism of scar formation. Our results showed that the expression of TLR4 and pp65 was higher in HS and HS‐derived fibroblasts (HSFs) than their counterpart normal skin (NS) and NS‐derived fibroblasts (NSFs). LPS could up‐regulate the expression of TLR4, pp65, Col I, Col III and α‐SMA in NSFs, but IL‐10 could down‐regulate their expression in both HSFs and LPS‐induced NSFs. Blocking IL‐10 receptor (IL‐10R) or the phosphorylation of STAT3, their expression was up‐regulated. In addition, in vitro and in vivo models results showed that IL‐10 could alleviate LPS‐induced fibroblast‐populated collagen lattice (FPCL) contraction and scar formation. Therefore, IL‐10 alleviates LPS‐induced skin scarring via IL‐10R/STAT3 axis regulating TLR4/NF‐κB pathway in dermal fibroblasts by reducing ECM proteins deposition and the conversion of fibroblasts to myofibroblasts. Our results indicate that IL‐10 can alleviate the LPS‐induced harmful effect on wound healing, reduce scar contracture, scar formation and skin fibrosis. Therefore, the down‐regulation of inflammation may lead to a suitable scar outcome and be a better option for improving scar quality.


| INTRODUC TI ON
Skin injury activates physiological responses, which can be identified as inflammation, proliferation and remodelling phases. [1][2][3] Although these phases are recognized as distinct entities, they usually overlap throughout the wound healing process. Inflammation may play a pivotal role in determining wound healing and scar outcome. [4][5][6] The most desirable scar is thin, flat and almost invisible.
Severe trauma (eg deep burns) and sometimes even standard surgery can result in adverse scar formation, known as a hypertrophic scar (HS). 2,7,8 HS is a firm, raised, red, itchy, sometimes painful fibrotic skin disease that usually develops within 4-8 weeks post-injury and may recede over time. 2,[6][7][8] Usually, the life quality of patients is declining because of loss of joint mobility and disfigurement. 6,9 There is currently no satisfactory clinical treatment for HS. The present studies suggest that HS is characterized by excessive abnormal deposition and metabolism disorders of collagen-based extracellular matrix (ECM) proteins, mainly including type I collagen (Col I), type III collagen (Col III) and the transformation of fibroblasts to myofibroblasts during scar formation. [9][10][11][12][13][14] To date, the pathogenesis of HS has not been fully understood, but the role of bacterial contamination and prolonged wound inflammation is considered important. 2,[15][16][17] IL-10 is a potent anti-inflammatory cytokine 18,19 that prevents fibrosis in several models, including dermal wounds, 20-23 myocardial infarction, 24 lung 25 and kidney injury. 26 Emerging reports have shown that IL-10 plays a key role in wound healing, 21,23,27 and it has been identified as a promising therapeutic agent that can reduce scar formation. 12,14,23,[28][29][30] However, the molecular mechanism of IL-10-mediated scar-improvement is still unclear. IL-10 can inhibit pro-inflammatory mediators including TNF-α, IL-1β and IL-8 and is thought to act through STAT3-mediated signalling pathways. 21,27 Specifically, dimerized IL-10s bind to an IL-10 receptor (IL-10R) complex. STAT3 is phosphorylated, inducing its dimerization and translocation into the nucleus to activate target gene expression. 21,31,32 So far, whether the IL-10R-STAT3 pathway plays a major role in mediating the function of IL-10 is still controversial.
Fibroblasts are one of the most important effector cell types responsible for scar formation. 12,29,30 For a long time, HS fibroblasts (HSFs) have been considered to be responsible only for the overproduction of ECM components and the transformation of fibroblasts into myofibroblasts, 9,10,[12][13][14]30 which is a major difference in comparison with normal skin fibroblasts (NSFs). Emerging evidence also shows that fibroblasts can also participate in immunological responses in direct response to pro-inflammatory signals, as well as regulation of normal barrier function of the epithelium, 33 infected tissue remodelling 34,35 and the infiltration behaviour of leucocytes to inflammatory sites. 36,37 Lipopolysaccharide (LPS) can activate toll-like receptor 4 (TLR4) in dermal fibroblasts through NF-κB, leading to the production of pro-inflammatory cytokines, which in turn causes inflammation. 16,38 Based on the known anti-inflammatory properties of IL-10 and the lack of inflammation and inflammatory mediators in scarless wound repair, we suggested that modulation of the inflammatory response by IL-10 would convert the wound healing phenotype from scar formation to scarless healing. To test this hypothesis, we evaluated the effect of IL-10 on the production of inflammatory mediators and the presence of an inflammatory response in LPS-stimulated NSFs, in an attempt to further clarify the role of IL-10 during scar formation. Therefore, the aim of this study was to investigate whether IL-10 can improve LPS-induced inflammatory responses in dermal fibroblasts and skin scarring and to explore the possible mechanism of regulating TLR4/NF-κB pathway during scar formation.  Figure S3).

| Immunostaining and ultrastructure
Immunohistochemistry was performed as previously reported. 23,39,40 In brief, the skin tissue fixed in 10% formalin buffer was embedded in paraffin blocks and cut into 4 μm-thick tissue sections. The pro-

| RT-qPCR
RT-qPCR was performed as previously reported. 23,39,40 In brief, the total RNA was extracted from cultured cells using an RNA isolation kit (Takara, Japan). The purity of RNA was calculated as follows: A260/A280 (1.9-2.0). Table 2 lists the human primer pairs used to amplify genes from cDNA templates. The mRNA levels of genes were normalized to the housekeeping gene encoding GAPDH.

| Western blot
Cultured fibroblasts, with a 70%-80% confluence after incubation for 12-16 hours in serum-free medium, were stimulated with LPS

| Animal model and treatment
A rabbit ear scar model was according to previous description. 39 and IL-10+ LPS treatment groups (6 scars for each group). IL-10 was injected into the scars for 24 hours before LPS injection, and they were applied to scars two times in a week.

| Statistical analysis
All results were obtained from at least three independent experiments and analysed using SPSS 20.0 software as the mean ± standard error (SEM). Quantitative data between two groups were analysed using Student's t test, and the comparisons among multiple groups were conducted using analysis of variance followed by Turkey's post hoc test. A value of P < .05 was considered statistically significant.

| HS is a serious fibrotic skin disease characterized by excessive ECM proteins deposition
HE staining showed an abundance of fibroblasts in HS ( Figure   were remarkably up-regulated to 85% ( Figure 1A-C, **P ˂ .01) and 87% ( Figure 1E-G, ***P ˂ .001) in HS than NS, respectively. RT-qPCR result showed the mRNA levels of TLR4 and p65 in HS/HSFs were much higher compared to NS/NSFs ( Figure 1D,H, *P ˂ .05, **P ˂ .01).
Immunofluorescence results showed that TLR4, pp65 and p65 were also expressed in cultured HSFs ( Figure S4). These findings confirm that TLR4 and pp65 are existed in HS/HSFs and higher expressed in HS/HSFs than their counterparts NS/NSFs.

| LPS induces NSFs to HSFs and participates in HS formation
To investigate whether the observed effects after LPS treatment were owing to its impact on ECM proteins expression in cultured

| LPS induces the expression of key molecules in TLR4/NF-κB pathway and the downstream inflammatory cytokines in NSFs
The next to investigate was whether the observed effects after LPS treatment were owing to its impact on the key molecules in TLR4/ NF-κB pathway and the downstream inflammatory cytokines. After treating NSFs with 1.0 μg/mL LPS, Western blot results showed that TLR4 and pp65 progressively increased from 3 to 72 hours compared to control group ( Figure 3A-C, **P ˂ .01, ***P ˂ .001), and the peak expression of TLR4 was from 12 to 48 hours ( Figure 3A,B), and pp65 was from 3 to 72 hours ( Figure 3A

| IL-10 suppresses the expression of inflammatory molecules and ECM proteins in HSFs and LPS-stimulated NSFs
IL-10 is a potent anti-inflammatory cytokine 18,19 and plays a pivotal role in wound healing. 21,23,27 Recently, it has been identified as a promising new therapeutic agent that can reduce scar After IL-10 treatment, the expression of TLR4 and pp65 was downregulated in some extend ( Figure 5K-M, *P ˂ .05). Therefore, the anti-inflammatory function of IL-10 is through regulating TLR4 and pp65 in TLR4/NF-κB pathway.

| IL-10 inhibits LPS-induced FPCL contracture
To showed that the architecture in PBS group takes the shape of uniform lattice arrangement ( Figure 7D). But this architecture was not appeared in LPS group ( Figure 7D). In contrast, LPS treatment led to the deposition of collagen in FPCL gel ( Figure 7D,E, ***P ˂ .001).
Interestingly, the architecture of FPCL was significantly improved in IL-10 treatment group compared to LPS group ( Figure 7D
Gram-negative bacteria are common microorganisms in burn wound infections and release endotoxins to the wound surface. 49,50 LPS is the main component of endotoxin, and its biological activity, cytotoxicity and immunological activity determine the process to a large extent and scar formation. It has been suggested that fibroblasts regulate immune/inflammatory response through TLR4 activated by LPS, leading to NF-κB activation, cytokine gene transcription and co-stimulatory molecule expression resulting in inflammation during HS formation. 16,[51][52][53][54] In fact, TLR4 has been shown to be overexpressed at both mRNA and protein levels in HSFs in comparison with NSFs. 16 The pro-inflammatory cytokines recruit polymorphonuclear cells, monocytes and macrophages to the wound, thereby producing more pro-inflammatory cytokines, amplifying the inflammatory response and stimulating scar formation. [55][56][57][58] Our results confirmed that TLR4 was expressed in dermal fibroblasts ( Figures 1,3-5 and Figure S4). LPS stimulated the expression of TLR4 and pp65 (Figures 3 and 4), increased pro-inflammatory cytokines (including IFN-γ, IL-1β, IL-6, IL-8 and MCP-1, Figure 3) secretion in NSFs, and the ultrastructure of LPS-stimulated NSFs mimicked their counterparts HSFs (Figure 2). These pro-inflammatory cytokines then stimulate collagen synthesis and ECM deposition and are also related to other pathological fibrotic disease models. 21,59,60 Therefore, LPS induces the inversion of NSFs to HSFs and leads to scar formation and skin fibrosis.
IL-10, an anti-inflammatory cytokine, has been involved in the attenuating inflammation phase of healing and is known to be elevated in foetal tissues and amniotic fluid. 21,55,61 Van den Broek 2 found a decrease in IL-10 production in HS compared with normal scars. In a study analysing IL-10 embryonic knockout mice, injury to these mice produced scars not observed in wild-type counterparts. 29,56 When IL-10 was administered to adult mice, a scarless  5) in HSFs. Convincingly, after IL-10RA and Cry treatment of IL-10-stimulated HSFs, the expression of TLR4 and pp65 was up-regulated ( Figure 5). And the expression change of inflammatory molecules was consistent with those of fibrotic proteins ( Figure 6).
Combining these findings, these data support the notion that the anti-inflammatory and anti-fibrotic function of IL-10 is promoting the phosphorylation of STAT3 (pSTAT3) through IL-10R, and regulating TLR4/NF-κB pathway by the IL-10R/STAT3 axis in dermal fibroblasts.
After tissue injury, persistent activation of fibroblasts and increased deposition of ECM proteins were usually observed in pathological scars. [63][64][65] As collagen is one of the key components in ECM, the continuous expression of collagen is an important histological feature to distinguish HS from NS (Figures S1-S3). 6,66,67 In addition, activated fibroblast overexpresses α-SMA, which is a well-known marker for myofibroblasts and promotes scar contracture. 30,63,68 Therefore, the regulation of fibroblast excess ECM protein deposition and the transformation to myofibroblasts are the main focus to study the prevention and treatment of HS.
In HS, molecules that regulate intracellular signalling cascades leading to the production of inflammatory mediators are being stud- NSFs. Therefore, LPS-stimulated fibroblasts will express pro-inflammatory cytokines (IFN-γ, IL-1β, IL-6, IL-8 and MCP-1), which cause persistent inflammation in injured tissue, thus promoting HS development. Therefore, controlling bacterial contamination and manipulating TLR4 signalling in injured skin fibroblasts may lead to novel strategies for the treatment of HS.
In summary, we have elucidated a novel mechanism for IL-10 to alleviate LPS-induced skin scarring. As illustrated schematically in Figure 7I, LPS up-regulates the expression of inflammatory cytokines, ECM proteins, and the transformation of fibroblasts to myofibroblasts through the TLR4/NF-κB pathway. Applying of IL-10RA or Cyr, the expression of inflammatory cytokine is up-regulated, leading to excess ECM proteins deposition and fibroblasts-to-myofibroblasts transformation. We suggest that excess LPS is detrimental to wound healing and ultimately lead to scar tissue formation. Therefore, it is of great significance to understand how natural endotoxin released at the wound microenvironment dictates scar formation. And a down-regulation of inflammation may be a better option for a suitable scar outcome and improved scar quality.