Neutrophils and their extracellular traps impair ablative fractional carbon dioxide laser‐induced dermal remolding in mice

Abstract

Objectives: Ablative fractional CO 2 laser (AFL) therapy is an effective intervention to induce dermal remodeling. AFL treatment of the skin triggers the recruitment of immune cells, with neutrophils dominating the early phase. However, the role of recruited neutrophils in AFL-induced microinjuries and their subsequent dermal remodeling capacity remains elusive. Materials and Methods: A mouse model of AFL-induced dermal remodeling was established. RNA sequencing was used to identify the prominent features of AFL-treated tissues. Histological analysis, including H&E and Masson staining, ultrastructure observation by transmission microscopy, immunofluorescence, and quantitative real-time polymerase chain reaction were used for dermal remodeling analysis. Moreover, AFL-treated mice were intraperitoneally injected with antimouse Ly6G antibodies to deplete neutrophils. Neutrophil extracellular traps (NETs) were explored using immunofluorescence, transmission microscopy, and in vitro coculture experiments. Results: Dermal remodeling, characterized by an increased number of CD31-positve vessels and elevated messenger RNA (mRNA) expression of genes encoding transforming growth factor-β (TGF-β), collagen I, and collagen III, was observed at 15 days after AFL treatment. In the AFL-induced inflammation phase, RNA sequencing identified neutrophil chemotaxis, and degranulation genes were significantly enriched. Histology and immunofluorescence staining of human and mouse tissues harvested at Day 1 after AFL treatment revealed significant neutrophil infiltration surrounding thermal-induced microinjuries. Neutrophil depletion decreased the expression of stressrelated genes such as S100A8 and S100A9 in the early phase following AFL treatment. Importantly, neutrophil depletion enhanced dermal remodeling at Day 15, as reflected by enrichment of the extracellular matrix and collagen biosynthesis genes based on RNA sequencing. Moreover, increased collagen I, collagen III, and TGF-β mRNA expression, increased cell proliferation, and vascularity were observed. Interestingly, NETs, which could be induced by AFL-treated fibroblasts in vitro, were identified in both human and mouse tissues on Day 1 after AFL treatment. Conclusions: AFL-treated human and mouse skin recruited a large number of neutrophils. The neutrophil surge impaired dermal remodeling in mice. The microenvironment and fibroblast functional modulation mediated by neutrophil degranulation and NET formation were determined to be the underlying mechanisms. Our results indicate that modification of infiltrated neutrophil activity might be a potential therapeutic target for AFL-induced dermal remodeling.

Ablative fractional CO2 laser treatment promotes wound healing phenotype in skin macrophages

“…We found that AFL treatment induced a pronounced neutrophil infiltration in the skin which may have a negative impact on the healing response, as a previous study found that excessive neutrophil infiltration and inflammation of the skin resulted in an impaired wound healing response in mice. 12 In the current study, histology samples showed a full skin microbeam penetration, and we observed various degrees of ulceration in 8 out of 18 treated zones. In a clinical setting, we would advocate for a considerably lower energy level of the AFL to substantially decrease the risk of ulceration.…”

“…These findings suggest that AFL polarizes the phenotype of the skin macrophage toward a wound healing phenotype which is supported by the observed decrease in the expression of the pro‐inflammatory marker, MHC II. Previous studies have shown that AFL increases TGF‐β mRNA expression 12 . TGF‐β is a potent stimulator of wound healing as it stimulates collagen I and III production in fibroblasts, 27–29 and interestingly, TGF‐β has also been shown to polarize macrophages toward a wound healing phenotype 30 and induce expression of Arg1 in macrophages in vitro 31 …”

“…6,10 Temporal changes in collagen biosynthesis pathways, matrix metalloproteases, and pro-inflammatory and anti-inflammatory cells and cytokines occur after AFL treatment of skin in a strictly coordinated manner that eventually leads to dermal remodeling. 4,[11][12][13][14] Macrophages are key players in coordinating this healing process due to their plethora of functions. Every phase in the healing is dominated by different macrophage phenotypes.…”

“…15,18,20,21 While previous studies have indicated that AFL induces infiltration of myeloid immune cells, e.g., macrophages and neutrophil granulocytes in skin, 12,22,23 no studies have investigated AFL's influence on macrophages' phenotype. 12,22,23 In this study, we hypothesize that AFL treatment polarizes skin macrophages toward a wound healing phenotype characterized by an increased level of anti-inflammatory markers (Arg1 and CD206) and decreased major histocompatibility complex class II molecules (MHC II). 15 Thus, this study aims to investigate AFL's impact on the inflammatory phenotype of macrophages in skin characterized by expression of these inflammatory markers.…”

“…Studies of the underlying biological dynamics after AFL treatment of skin have suggested that AFL reinvigorates tissue homeostasis by inducing a wound healing response involving remodeling the extracellular matrix 6,10 . Temporal changes in collagen biosynthesis pathways, matrix metalloproteases, and pro‐inflammatory and anti‐inflammatory cells and cytokines occur after AFL treatment of skin in a strictly coordinated manner that eventually leads to dermal remodeling 4,11–14 . Macrophages are key players in coordinating this healing process due to their plethora of functions.…”