Department of Health Technology, Technical University of Denmark, Kongens Lyngby, Denmark.
Department of Dermatology, Copenhagen University Hospital-Bispebjerg, Copenhagen, Denmark.
Lasers Surg Med. 2024 Mar;56(3):270-278. doi: 10.1002/lsm.23772. Epub 2024 Feb 26.
Ablative fractional laser (AFL) treatment is a well-established method for reducing signs of skin photoaging. However, the biological mechanisms underlying AFL-induced healing responses and skin rejuvenation remain largely unknown. It is known that macrophages play an important role in orchestrating healing, normalization, and remodeling processes in skin. Macrophage phenotypes are characterized by inflammatory markers, including arginase-1 (Arg1), major histocompatibility class II molecules (MHC II), and CD206. This study aims to explore AFL's effect on macrophage phenotype by evaluating changes in inflammatory markers and the potential concurrent accumulation of Arg1 in the skin.
Mice (n = 9) received a single AFL treatment on the left side of the back skin (100 mJ/microbeam, 5% density) while the right side of the back remained untreated as control. Treated and untreated skin from each mouse were collected Day 5 posttreatment for flow cytometry and histology analysis. Flow cytometry evaluated the immune infiltration of macrophages and the expression of macrophage inflammatory markers (Arg1, MHC II, and CD206). In addition, Arg1 presence in the skin was evaluated through antibody staining of histology samples and quantification was performed using QuPath image analysis software.
Following AFL, the number of macrophages increased 11-fold (p = 0.0053). Phenotype analysis of AFL-treated skin revealed an increase in the percentage of macrophages positive for Arg1 (p < 0.0001) and a decrease in the percentage of macrophages positive for MHC II (p < 0.0001) compared to untreated skin. No significant differences were observed in percentage of CD206-positive macrophages (p = 0.8952). Visualization of AFL-treated skin demonstrated a distinct pattern of Arg1 accumulation that correlated with the microscopic treatment zones (MTZ). Quantification of the percentage of Arg1-positive area in epidermis and dermis showed a significant increase from 3.5% ± 1.2% to 5.2% ± 1.7 (p = 0.0232) and an increase from 2.2% ± 1.2% to 9.6% ± 3.3 (p < 0.0001) in whole skin samples.
AFL treatment polarizes macrophages toward a wound healing phenotype and induces Arg1 accumulation in the MTZ. We propose that the polarized wound healing macrophages are a major source for the increased Arg1 levels observed in the skin following treatment.
烧蚀性分数激光（AFL）治疗是一种成熟的方法，可减少皮肤光老化的迹象。然而，AFL 诱导的愈合反应和皮肤年轻化的生物学机制在很大程度上仍不清楚。众所周知，巨噬细胞在协调皮肤愈合、正常化和重塑过程中起着重要作用。巨噬细胞表型的特征是存在炎症标志物，包括精氨酸酶-1（Arg1）、主要组织相容性复合体 II 分子（MHC II）和 CD206。本研究旨在通过评估炎症标志物的变化和 Arg1 在皮肤中的潜在累积，探讨 AFL 对巨噬细胞表型的影响。
将 9 只小鼠（n=9）的背部皮肤一侧（100mJ/microbeam，5%密度）接受单次 AFL 治疗，另一侧背部皮肤作为对照未治疗。在治疗后第 5 天，每组小鼠的治疗和未治疗皮肤均用于流式细胞术和组织学分析。流式细胞术评估了巨噬细胞的免疫浸润和巨噬细胞炎症标志物（Arg1、MHC II 和 CD206）的表达。此外，还通过组织学样本的抗体染色评估了皮肤中 Arg1 的存在，并使用 QuPath 图像分析软件进行定量。
AFL 后，巨噬细胞数量增加了 11 倍（p=0.0053）。与未治疗皮肤相比，AFL 治疗皮肤中 Arg1 阳性巨噬细胞的百分比增加（p<0.0001），而 MHC II 阳性巨噬细胞的百分比减少（p<0.0001）。CD206 阳性巨噬细胞的百分比无显著差异（p=0.8952）。AFL 治疗皮肤的可视化显示出 Arg1 积累的明显模式，与微观治疗区（MTZ）相关。表皮和真皮中 Arg1 阳性区域百分比的定量显示，全皮肤样本中从 3.5%±1.2%增加到 5.2%±1.7%（p=0.0232），从 2.2%±1.2%增加到 9.6%±3.3%（p<0.0001）。
AFL 治疗使巨噬细胞向伤口愈合表型极化，并在 MTZ 中诱导 Arg1 积累。我们提出，极化的伤口愈合巨噬细胞是治疗后皮肤中 Arg1 水平升高的主要来源。
