Imiquimod regulating Th1 and Th2 cell‐related chemokines to inhibit scar hyperplasia

Abstract

Immunological factors play important roles in the occurrence of hypertrophic scars. Imiquimod can be used as an immunosuppressive agent to regulate the function of T‐helper (Th) cell subsets Th1 and Th2. In this article, we explored the impact of imiquimod on scar hyperplasia through Th cells. A rabbit ear hypertrophic scar model was built. Four round wounds were cut in each rabbit's ears ventrally with a diameter of 1 cm and bilateral symmetry. All the right ear wounds were treated with 5% imiquimod cream. The blank control group contained all the left ear wounds, which were treated with Vaseline ointment at the same time. Haematoxylin and eosin and Masson staining showed that imiquimod collagen deposition was significantly reduced compared with the control group, scar index (SEI) showed that the proliferative degree reached its peak on the 28th day after operation in blank group, and the degree of hyperplasia was significantly higher than that of the imiquimod group (P < .05). Real‐time Polymerase chain reaction results showed that the imiquimod induced the expression of Th2 cell‐related chemokines CCL2, CCL3, CCL5, CCL7, and CCL13 at each time point, which were significantly lower than that of the blank control group, and the expressions of Th1 cell‐associated chemokines CXCL10 and CXCL12 at each time point was significantly higher than the blank control group (P < .05). Imiquimod can be used to regulate the expression of Th1 and Th2 cell‐associated chemokines to control scar hyperplasia.

1. INTRODUCTION

A hypertrophic scar is a common, frequently occurring disease that is featured by a large amount of collagen deposition, and the pathogenesis is unclear. Research has confirmed that immune factors are critical in the formation and development of scars.1 In between, helper T lymph (Th) cell population is an important group in the body's immune system. It is the main immune cell group during scar hyperplasia.2 The major subsets of Th1 and Th2 cells are chemotactic and are recruited from the peripheral blood to local scar tissues, which in turn produce a variety of cytokines involved in the formation of scars.3, 4, 5, 6 Imiquimod is a new synthetic immunomodulator. Studies have shown3 that imiquimod can inhibit rabbit ear scar hyperplasia, but how to regulate Th1 and Th2 cytokine expression to inhibit scar hyperplasia is still unknown. Therefore, this experiment was designed to study local imiquimod use for scars in their proliferation phase by regulating Th1 and Th2 cell chemotactic factors CXCL10, CXCL12, CCL2, CCL3, CCL5, CCL7, and CCL13 expression so as to provide a more theoretical basis.

2. MATERIALS AND METHODS

2.1. Experimental animals

Sixteen New Zealand healthy white rabbits, weighing 2.3 to 3.0 kg, both males and females, were used for this study. The rabbits were provided and reared by the Experimental Animal Center of Fourth Military Medical University with single cage. Experimental materials included as follows:

Five percentage imiquimod ointment was purchased from Sichuan Mingxin Pharmaceutical Co., Ltd.; Masson trichrome staining reagent was purchased from Nanjing Senbei Jia Biotechnology Co., Ltd.; trizol reagent was provided by Shanghai Shengongji; the primer was synthesised by Biotech (Beijing); and the real‐time polymerase chain reaction (PCR) kits required were purchased from Dalian Bao Biological Engineering Co., Ltd. (reagent code: RR047A, RR025A, RR082A).

2.2. Experimental method

A rabbit ear hypertrophic scar model was established according to the literature.7, 8 First, rabbits were slowly injected with 3% sodium pentobarbital through the rabbit ear veins and then anaesthetised (3.0 m L/kg). Under aseptic conditions, four clear circular wounds with a diameter of 1 cm were made on the ventral side of each rabbit to avoid obvious small veins. The full‐thickness skin tissue and perichondrium were removed. The distance between them was about 1.5 cm, and they were symmetrical on both sides. Finally, electrocoagulation haemostasis was carried out. The wound was exposed. For the first 3 days after surgery, iodophor disinfection was used for the wound once or twice a day so as to assess the rabbits' own healing. A total of 14 rabbits was modelled. Rabbits with wounds on the left ear were one group, and rabbits with wounds on the right ear were the other group, and there were 56 wounds in each group. The right ear group was set as the imiquimod‐treated group. Imiquimod was applied to the wounds on the 14th day after operation; 0.1% ointment with 5% imiquimod was applied to each wound. The left ear group was a blank control group that was administered the same dose of petrolatum ointment. It was administered once daily for 1 month.

2.3. Specimens collected

The specimens were collected and euthanised by air embolism. Two healthy non‐trauma adult rabbits were used, and the rabbit ear skin tissues were collected as a part of the experimental normal tissue control group. Fourteen rabbits were randomly sacrificed on the 21st day, 28th day, 35th day, 42th day, 49th day, 56th day, and 63th day after operation. Peripheral scarring was extended for 0.5 cm and cut with a ring. The line passing the most protruding point was made the scar diameter, and the scar was divided into halves along the line. Normal rabbit ear skin tissue and all scar specimens were immediately placed in 4% paraformaldehyde solution to be fixed and were then paraffin embedded, and the other half was immediately stored in a refrigerator at −80°C to extract real‐time RNA that was used for fluorescence quantitative PCR detection. After that, haematoxylin and eosin (HE) staining and Masson trichrome staining were carried out.

2.4. Scar index

According to the HE staining and sectioned microscope findings, the ratio of the vertical distance between the most protruding point of the scar to the surface of the ear cartilage and the vertical distance between the normal skin outside the ear and the cartilage surface is scar index (SEI) (as shown in Figure 1). The higher the degree of scar hyperplasia, the greater the index value. Under normal circumstances, an SEI value>1.6 can be considered a hypertrophic scar. Measuring and calculating SEI can be performed using IPP software.

Figure 1.

Scar index calculation

2.5. Real‐time PCR detection

Primer design and Tm values were prepared. The specimens were cut into about 150 mg tissues in sequence at different time points. After freezing in liquid nitrogen and pulverised with a mortar, 1 mL of trizol was added to extract the total RNA. The purity and concentration of the sample were detected using an ultraviolet spectrophotometer. After DNA was removed, cDNA was reverse‐transcribed. Real‐time PCR was used to detect the expression of Th1 and Th2 cell chemokines CXCL10, CXCL12, CCL2, CCL3, CCL5, CCL7, and CCL13.

2.6. Statistical analysis

The data obtained were expressed as mean ± SD (x ± s), and SPSS16.0 statistical software was used for data processing. The t‐test was used to compare the data. The difference was statistically significant with * P < .05 and ** P < .01.

3. RESULTS

3.1. General observation of scar model

The period of epithelialisation was about 13 to 15 days with no significant secretion of infection, perforation, and so on. A total of 35 days after surgery, the scars of the blank control group and the imiquimod group changed with time and presented varying degrees of proliferation: the control blank group showed scar hypertrophy with red scar colour, the hard touching scar, and the thickest part of the wound in the centre; the imiquimod group showed scars with lighter colour, slightly higher than the surrounding normal skin surface. The surface was flat, the scar was soft, and wound surface hyperplasia was significantly reduced (Figure 2). The blank control group reached its peak at 28 to 35 days after operation.

Figure 2.

General observation the 28th and 42nd days. Less scar hyperplasia can be found in imiquimod group (B) compared with the blank group (A)

3.2. HE staining results

Microscopically, on the 35th day postoperative, the collagen fibres were coarse and dense and disordered, and a large deposition of extracellular matrix was presented in the blank control group. The collagen fibres in the imiquimod group were significantly reduced and mostly arranged in parallel with less extracellular matrix deposition (Figure 3).

Figure 3.

Haematoxylin and eosin staining on the 28th and 42nd days (×100) in imiquimod group (B) and blank group (A)

3.3. Masson staining results

Microscopic examination 35 days after operation showed dense fibres in the blank control group and swirling structural disorder. In the imiquimod group, collagen fibres decreased and were arranged smoothly (Figure 4).

Figure 4.

Masson staining on the 28th and 42nd days (×100) in imiquimod group (B) and blank group (A)

3.4. Scar hyperplasia index

The scar index at the 21st, the 28th, the 45th, the 42th, the 49th, the 56th and the 63rd day after the operation in imiquimod group were 1.68 ± 0.32, 1.81 ± 0.35, 1.75 ± 0.27, 1.46 ± 0.29, 1.43 ± 0.28, 1.37 ± 0.31, 1.25 ± 0.21, while the blank control group were 1.86 ± 0.34, 2.88 ± 0.32, 2.49 ± 0.29, 1.84 ± 0.28, 1.67 ± 0.32, 1.58 ± 0.21, 1.49 ± 0.21, as shown in Table.1.

Table 1.

Scar index results at each time point

Group	21 days	28 days	35 days	42 days	49 days	56d	63d
Imiquimod	1.78 ± 0.13	2.01 ± 0.12	1.77 ± 0.11	1.56 ± 0.15	1.43 ± 0.12	1.37 ± 0.14	1.25 ± 0.16
Blank	1.84 ± 0.12	2.62 ± 0.14	2.51 ± 0.13	1.91 ± 0.13	1.71 ± 0.14	1.65 ± 0.15	1.43 ± 0.17

After statistical analysis, the difference was statistically significant (P < .05) between imiquimod group and the blank control group. The SEI at each time point was compared as shown in Table.1. The index of scar hyperplasia in blank control group was always higher than that of imiquimod group at each time point, and reached its peak at 28 days.

Therefore, imiquimod can inhibit rabbit ear scar hyperplasia, and the results and the general conclusion of morphological findings are consistent.

3.5. Real‐time PCR test results

Compared with the m RNA level of normal rabbit ear skin tissue (N), it can be seen in the proliferative phase of rabbit ventral scars that most of the Th2 cell‐associated chemokines CCL2, CCL3, CCL5, CCL7, and CCL13 in the blank control group at each time point are highly expressed. There was almost no expression of Th1 cell‐associated chemokines CXCL10 and CXCL12. The expressions of CCL2, CCL3, CCL5, CCL7, and CCL13 in the imiquimod group were significantly decreased, and the expression of CXCL10 and CXCL12 in scar tissue was significantly increased.* P < .05, ** P < .01; the difference was statistically significant (Figures 5 to 14.

Figure 5.

The expression of 10 chemokines in imiquimod group and blank group

4. DISCUSSION

Hypertrophic scar is the most common disease, often formed after various traumas, burns, or surgeries. However, the exact pathogenesis is not clear, and there is a lack of safe, effective, and targeted treatment. Studies have shown that the occurrence and development of scars are closely related to the involvement of the immune system.1 The Th cell population is an important group in the body's immune system and is a major immune cell group in the process of scar hyperplasia2 and plays an important role in the development of pathological scars. The major subsets of Th cells are Th1 cells and Th2 cells. Studies have shown that Th1 and Th2 cells play an important role in the formation of fibrosis.9, 10, 11, 12 Th1 cells predominantly produce cytokines IFN‐γ and IL‐12. IFN‐γ can inhibit collagen synthesis and increase collagenase expression and activity, thereby promoting collagen remodelling.9 Therefore, in Th1‐based immune responses to reduce the degree of fibrosis, extracellular matrix degradation phenomenon was observed.10 Th2 cells produce a variety of cytokines including IL‐4, IL‐5, IL‐6, IL‐10, IL‐13, etc. Among them, IL‐4 is a potent fibrotic factor that acts on fibroblasts to stimulate the production of collagen and fibronectin.11 Studies have shown that Th2 cell response is predominant in proliferative scarring.12

Imiquimod is a novel immunomodulatory agent that can be recognised by antigen‐presenting cells and mediate early immune recognition and modulation by binding to these cell surface receptors, such as toll receptor 7 (TLR 7), to stimulate immunity factor Interferons (IFN)‐α. tumour necrosis factor (TNF)‐α and interleukin (IL)‐12 and other syntheses activate the Th1 cellular immune pathway and then increase the production of Th1 cytokines IFN‐γ and IL‐12. At the same time, this production can also be inhibited by inhibiting the production of Th2 cytokine IL‐4, IL‐5, etc and then inhibiting Th2 cellular immune response, making it tend to form a Th1 cellular immune response‐based reaction.13, 14, 15, 16 Some studies also confirmed the use of imiquimod ointment on rabbit ear scars to decrease IL‐4 expression and increase IFN‐γ expression.3 So, is the primary mechanism of action of imiquimod for the inhibition of scar hyperplasia modulating the level of cytokines such as increased IFN‐γ or decreased IL‐4 expression? Researchers have treated scar tissue with topical injection of IFN‐γ to prevent recurrence but did not achieve results. That alone can not change the level of cytokine treatment and prevention of scar hyperplasia.4, 5, 6

Th1 and Th2 cells are taken from the peripheral blood to the local tissue under the action of the corresponding chemokines, thereby generating a variety of cytokines involved in scar formation. Chemokine CXCL10, which interacts with Th1 cell surface receptor CXCR3, a potent Th1 cell promoter, is involved in Thl‐mediated cellular immune responses.17, 18, 19 CXCL12 also favours the selective chemotaxis of Th1 cells in chronic inflammatory reactions.20 The literature shows that CCL2, CCL3, CCL5, CCL7, and CCL13 are Th2 cell‐related chemokines.21, 22, 23 The difference of expression type and quantity of chemotactic factor in local tissue can significantly affect the local expression level of Th1 and Th2 cells and further affect the formation of scars. Compared with the m RNA level of normal rabbit ear skin tissue (N), it can be seen in the proliferative phase of rabbit ventral scars that most of the Th2 cell‐associated chemokines CCL2, CCL3, CCL5, CCL7, and CCL13 in the blank control group at the time point was overexpressed. Th1 cell‐associated chemokines CXCL10 and CXCL12 showed no expression. The expressions of CCL2, CCL3, CCL5, CCL7, and CCL13 in the imiquimod group were significantly decreased, and the expression of CXCL10 and CXCL12 in scar tissue was significantly increased. In the experimental results, compared with normal rabbit ear skin tissue (N) of mRNA, no expression of Th1 Cell‐associated chemokines CXCL10 and CXCL12 was observed at all times in the blank group in the process of hyperplasia in the rabbit ventral scars, and Th2 cell‐related chemokines CCL2, CCL3, CCL5, CCL7, and CCL13 were highly expressed at most of the time points, indicating that Th2 cell responses predominate during this proliferative process. In the imiquimod group (treatment group), Th2 cell‐associated chemokines CL2, CCL3, CCL5, CCL7, and CCL13 expression significantly decreased, and Th1 cell chemotactic factors CXCL10 and CXCL12 significantly increased in scar tissue expression so that chemotaxis to local scar tissue increased; Th1 cells and Th2 cells decreased, Th1 cell response increased, Th2 cell response was inhibited, and their secretion of the cytokine IFN‐γ increased; IL‐4, IL‐5 levels decreased, thereby reducing collagen deposition and the degree of fibrosis.

Pathological scarring is not only a localised problem. The simple treatment of local fibroblasts or cytokines levels is often difficult to achieve the desired effect. Only by blocking the interaction of circulating blood‐derived immune cells and local cells of the lesion can we hopefully achieve more effective results. Approaches that can be used include small molecule chemokine receptor antagonists, neutralising antibodies, and immunosuppressants. We chose to use the target to study more accurate immunosuppressive agents of imiquimod to observe its effect on scar hyperplasia, and at the same time, we used immunosuppressive agents to detect the upstream interaction between immunocompromised immune cells and lesion local cells—the role of chemokine expression to further clarify its mechanism of action.

The results of this study show that imiquimod inhibits scarring and can regulate the interaction between immune cells and local fibroblasts by regulating the expression of important Th1‐ and Th2‐related cytokines in the immune pathway so as to inhibit rabbit ear scar hyperplasia. This experiment helps to clarify the role of T‐cell‐derived signals in scar hyperplasia, provide new ideas for the prevention and treatment of hypertrophic scars, and provide a more accurate target for treatment. Currently, small‐molecule chemokine receptor antagonists, neutralising antibodies, etc are still in the research and development stage and are not widely used in clinical practice. This experiment provides an experimental idea for the application of more accurate drug targets in the field of scar treatment, and in‐depth studies are necessary. With regard to the expression of chemokines in pathological scar, most of the existing studies focus on a single factor or several factors or a factor cross‐sectional study. However, in this experiment, the process of hyperplasia of hypertrophic scar with cell‐related chemokines for long‐term expression studies has been described with a more accurate expression curve of chemokines. In terms of the scar prevention and control methods, it provides a wealth of data and research background in the intervention of T lymphocyte chemotaxis.

Chen B, Li H, Xia W. Imiquimod regulating Th1 and Th2 cell‐related chemokines to inhibit scar hyperplasia. Int Wound J. 2019;16:1281–1288. 10.1111/iwj.13183