Ionizing radiation induces cutaneous lipid remolding and skin adipocytes confer protection against radiation-induced skin injury

Journal Pre-proof Ionizing radiation induces cutaneous lipid remolding and skin adipocytes confer protection against radiation-induced skin injury Yuji Xiao, Wei Mo, Huimin Jia, Daojiang Yu, Yuyou Qiu, Yang Jiao, Wei Zhu, Hiroshi Koide, Jianping Cao, Shuyu Zhang

PII:

S0923-1811(20)30039-6

DOI:

https://doi.org/10.1016/j.jdermsci.2020.01.009

Reference:

DESC 3563

To appear in:

Journal of Dermatological Science

Received Date:

22 May 2019

Revised Date:

17 January 2020

Accepted Date:

21 January 2020

Please cite this article as: Xiao Y, Mo W, Jia H, Yu D, Qiu Y, Jiao Y, Zhu W, Koide H, Cao J, Zhang S, Ionizing radiation induces cutaneous lipid remolding and skin adipocytes confer protection against radiation-induced skin injury, Journal of Dermatological Science (2020), doi: https://doi.org/10.1016/j.jdermsci.2020.01.009

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

Title page

Ionizing radiation induces cutaneous lipid remolding and skin adipocytes confer protection against radiation-induced skin injury

Running title: Role of skin fat in radiation-induced skin injury

Yuji Xiaoa,b,#, Wei Moa,b,#, Huimin Jiaa,b, Daojiang Yuc, Yuyou Qiud, Yang Jiaoa,b, Wei

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Zhua,b, Hiroshi Koidee, Jianping Caoa,b,*, Shuyu Zhangf,g, *

School of Radiation Medicine and Protection, Medical College of Soochow University,

Suzhou 215123, China

Key Laboratory of Radiation Medicine and Protection and Collaborative Innovation

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b State

University, Suzhou 215123, China

Department of Plastic Surgery, the Second Affiliated Hospital of Soochow University,

Suzhou 215004, China d Department

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c

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Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow

of Radiology, Shanghai Tenth People’s Hospital, Tongji University School of

e Laboratory

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Medicine, Shanghai, 200072, China

of Molecular and Biochemical Research, Research Support Center, Juntendo

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University Graduate School of Medicine, Tokyo 113-8421, Japan. The Second Affiliated Hospital of Chengdu Medical College, China National Nuclear

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Corporation 416 Hospital, Chengdu 610051, China. g

West China Second University Hospital, Sichuan University, Sichuan University,

Chengdu 610041, China.

#The

first two authors contributed equally to this work.

*Corresponding authors: Zhang S, The Second Affiliated Hospital of Chengdu Medical College, China National

Nuclear Corporation 416 Hospital, Chengdu 610051, Tel./Fax:+86-28-82991366; E-mail: [email protected]. Cao J, No. 199 Ren’ai Rd, Medical College of Soochow University, Suzhou 215123,

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Tel./Fax:+86-512-65880037; E-mail: [email protected].

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Highlights

Radiation modulates the expression of lipid metabolism-related genes and

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decreases skin fat mass with altered lipid metabolite profiles. Increased skin adipose mass induced by a high-fat diet confers resistance against radiogenic skin injury.

Mature adipocytes from skin could promote the migration of co-cultured skin

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

keratinocytes and fibroblasts. Fatty acid-binding protein 4 (FABP4) could be incorporated into skin cells and

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

promote DNA damage repair in irradiated skin fibroblasts.

Abstracts Background: Radiation-induced skin injury is a serious concern during radiotherapy and radiation accidents. Skin fat represents the dominant architectural component of the human skin. However, the interplay between skin fat and the progression of radiation-induced skin injury remains largely unexplored. Objective: This study aims to elucidate the interplay between skin fat and the progression of radiation-induced skin injury. Methods: SD rats were irradiated with an electron beam. mRNA profiles were determined

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by RNA-Seq. The skin lipid mass was monitored by magnetic resonance imaging (MRI) and lipid profiles were measured by liquid chromatography-mass spectrometry (LC-MS).

Human mature adipocytes isolated from dermal and subcutaneous white adipose tissues (WATs) were co-cultured with human keratinocytes (HaCaT) and skin fibroblasts (WS1) in

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the transwell culture system. Cell migration ability was measured by migration assay.

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Results: Radiation modulated cutaneous lipid metabolism by downregulating multiple pathways. Moreover, radiation decreased skin fat mass with altered lipid metabolite

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profiles. The rats fed with a high-fat diet showed resistance to radiogenic skin injury compared with that with a control diet, indicating that skin lipid plays a radioprotective role. Mature adipocytes promoted the migration but not the proliferation of co-cultured skin

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keratinocytes and fibroblasts. Palmitic acid, the most abundant fatty acid in skin tissues, facilitated the migration of WS1 cells. Moreover, fatty acid-binding protein 4 (FABP4)

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could be incorporated into skin cells and promote DNA damage repair in irradiated skin fibroblasts.

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Conclusion: Radiation induces cutaneous lipid remolding, and skin adipocytes confer a protective role against radiation-induced skin injury.

Key words: radiation; skin injury; lipid metabolism; fatty acid; fatty acid binding protein 4 (FABP4)

Introduction

Radioactive materials have been widely used in industry, medicine, science, the military and nuclear facilities, which has significantly increased the potential of large-scale, uncontrolled exposure to radiation [1,2]. The skin represents the largest organ of the body; the integumentary system is one of the radiosensitive organ systems because the skin is a continuously renewing organ containing rapidly proliferating and maturing cells [3,4] Radiation-induced skin injury is a deterministic effect of accidental exposure of skin to radiation. For example, in the Chernobyl nuclear accident, many people's skin and clothing surfaces were covered with radioactive materials, which resulted in most patients

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complicated or even die [5]. Moreover, ~95% of cancer patients receiving radiation therapy will develop some form of radiodermatitis, including erythema, dry desquamation, and moist desquamation [4,6]. Radiation-induced skin injury can negatively affect the process of radiotherapy and the quality of life of patients [7]. Despite substantial

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improvements in radiation technology, radiation-induced skin toxicity remains a serious concern [8]. Radiation generates a large amount of reactive oxygen species (ROS), which

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irreversibly attack biomacromolecules, including nuclear DNA, mtDNA, proteins and lipids

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[9]. Numerous cytokines and chemokines, such as TGF-β, TNFα, IL-1α, IL-1β, IL-6, and IFNγ, are produced in response to radiation exposure [4,10,11]. However, the exact pathogenic molecular mechanism of radiation-induced skin injury is still unclear.

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The skin is comprised several distinct layers, including the epidermis, dermis, dermal white adipose tissue (WAT), and subcutaneous WAT [12,13]. Mature adipocytes in the

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skin have been implicated in multiple physiological and pathological processes, including lipid storage and release, adipokine secretion, glucose and lipid metabolism, and hair

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follicle and fibroblast regeneration after injury [12,13]. In vertebrates, adipocytes store energy in the form of fatty acids that can be released under tight metabolic control [14]. Free fatty acids, which are relatively insoluble and potentially toxic, can be transported by noncatalytic binding proteins to other cells [15]. Fatty acid-binding proteins (FABPs) are an intracellular protein family that demonstrate high affinity for noncovalent binding to long-chain fatty acids [16]. Among the 12 members in the human FABP family, FABP4 is the main FABP present in adipose tissues or mature adipocytes with secretory potential

[17,18]. Although our understanding of the importance of WATs is expanding, including energy storage, hormone secretion, and infection defense [13], the role of skin fat and adipocytes on the progression of radiation-induced skin injury remains largely unexplored. High-throughput RNA sequencing (RNA-Seq) is a powerful tool for obtaining comprehensive transcriptome information for the progression of diseases [19]. Using RNA-Seq, we found herein that radiation modulated lipid metabolism pathways in a rat model, which was accompanied by decreased adipose mass and altered lipid profiles. We further observed that skin adipocytes could modulate the migration and repair of skin cells. results suggest that

skin

adipocytes have

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protective

role

against

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radiation-induced skin injury.

Materials and methods

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Reagents and materials

Dimethylsulfoxide (DMSO) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium

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bromide (MTT) were purchased from Solarbio (Beijing, China). Palmitic acid (PA) and

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BMS309403 were obtained from Sigma-Aldrich (St. Louis, MO). Full-length cDNA of an adenovirus-carrying human FABP4 was constructed by ViGene Biotech (Jinan, China). 4’-6-diamidino-2-phenylindole (DAPI) was purchased from Beyotime Biotech (Nantong,

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China). BODIPY fluorophore 493/503 for lipid droplets was obtained from Molecular Probes (Eugene, OR).

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Animal studies

The protocols for experiments involving animals were approved by the Animal

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Experimentation Ethics Committee at Soochow University (Suzhou, China). Male Sprague Dawley (SD) rats (4 weeks of age) were purchased from the Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China) These animals were housed in a pathogen-free environment at the facilities of Medical College of Soochow University. We randomly selected rats and classified them into the control group; they were fed with a control diet (5% fat). The other rats were fed with a high-fat diet (42% fat; both diets were obtained from Shanghai SLAC Laboratory Animal Co., Ltd.) to increase skin adipose mass. Both

diet and water were supplied ad libitum. After three months of dietary intervention, both groups of rats were used for the experiments. For irradiation, the rats were anesthetized with an intraperitoneal injection of ketamine (75 mg/kg) and xylazine (10 mg/kg) (Sigma-Aldrich, St. Louise, MO), and the hair on the gluteal region of the rats was shaved using a razor. The animals were immobilized using adhesive tape on a plastic plate to minimize motion during radiation exposure. A 3-cm-thick piece of lead was used to shield the animals and localize the radiation field (3 × 4 cm). Irradiation was performed on the treatment area at a dose rate of 750 cGy/min

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using a 6-MeV electron beam accelerator (Clinac 2100EX, Varian Medical Systems, Palo Alto, CA) as reported previously [20]. Hematoxylin and eosin (H&E) staining

The skin tissues were fixed in 10% neutral-buffered formalin and embedded in paraffin.

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Three-micrometer paraffin sections were deparaffinized and heat-treated using citrate

using H&E (ZSGB-Bio, Beijing, China).

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buffer (pH, 6.0) for 7 min following an epitope retrieval protocol. The sections were stained

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Liquid chromatography-mass spectrometry (LC-MS)-based lipidomic analysis The rats were irradiated using a 45-Gy electron beam as described above [20], and skin and subcutaneous tissues (both irradiated and non-irradiated areas, n = 12) were

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collected 10 days post-radiation. The LC-MS-based lipidomic analysis was conducted at Suzhou BioNovoGene Co., Ltd. The detailed methods are described in the

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Supplementary materials and methods. Isolation of mature adipocytes from human skin tissues

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Human dermal and subcutaneous WATs were obtained from patients who experienced trauma. All patients provided written informed consent for their tissues to be used for scientific research. Ethical approval of the study was obtained from the Second Affiliated Hospital of Soochow University (Suzhou, China). Adipocytes were isolated from the adipose tissues as described elsewhere [21]. White adipose deposits were digested for 1 h at 37°C using Dulbecco’s Modified Eagle’s Medium (DMEM) containing 1 mg/mL

collagenase type I and 0.1% BSA. Mature adipocytes were collected from the upper phase after centrifugation at 100 × g for 3 min. Cell culture and irradiation Human keratinocyte HaCaT and human skin fibroblast WS1 cells were maintained in DMEM. All culture media were supplemented with 10% FBS (Gibco, Grand Island, NY). The cells were grown at 37°C in 5% CO2 incubators. They were then exposed to different doses of ionizing radiation using an X-ray linear accelerator (Rad Source, Suwanee, GA) at a fixed dose rate of 1.15 Gy/min.

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Adipocyte and skin cell co-culture We used the transwell culture system (3-μm pore size; Corning, NY) for the co-culture study. Adipocytes (1 × 105) were placed in the upper chamber, and WS1 or HaCaT cells

(1 × 105 cells) were seeded in the bottom. WS1 or HaCaT cells grown in mono-cultures (in

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the absence of adipocytes) in the culture medium were considered the controls. Immunofluorescence

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The cells were fixed in 4% paraformaldehyde, washed with PBS, and permeabilized with

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1% Triton X-100 in PBS. They were then blocked with blocking buffer (PBS, 1% Triton X-100, and 5% BSA) and incubated at 4°C with a γ-H2AX antibody (Abcam; #ab 81299) overnight. Next, a rhodamine-conjugated goat anti-rabbit antibody (1:100) was added for

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30 min at room temperature. The nuclei were counterstained with DAPI. Migration assay

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The cells were seeded onto 6-well plates and allowed to form a confluent monolayer for 24 h. After treatment, the monolayer was scratched with the tip of a 200-μL pipette and

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then washed twice with PBS to remove the floating and detached cells. Thereafter, a fresh serum-free medium was added, and images were taken at 0-24 h to assess cell migration using a microscope (Olympus, Tokyo, Japan). Statistical analysis Data were expressed as mean ± SEM of at least three independent experiments. The results were evaluated using one-way ANOVA to determine statistical significance. The

statistical analyses were performed using the SPSS software. The differences were considered significant at P < 0.05.

The other materials and methods are available in the Supplementary Materials and Methods.

Results Profiling of mRNAs in response to radiation reveals aberrant expression of lipid

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metabolism-related genes in rat skin To characterize mRNA changes in response to radiation, RNA-Seq was used to measure the profiles of rat skin with or without 45-Gy electron beam irradiation (n = 3). The RNA-Seq data are accessible at GEO database (accession number GSE86252). A total of

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639 genes (287 upregulated and 352 downregulated genes) were identified with a

significant expression difference of 2-fold or greater between the two groups (Figures

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1A-1C). The top 20 up- and downregulated mRNAs are shown in Figure 1D. For example,

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Igfn1, Rnase2, Svop, and Clrn1 mRNA were increased, whereas Krt27, Krt81, Krt85, and Radi3 transcripts were downregulated in the irradiated skin tissues (Figure 1D). The KEGG-based pathway analysis revealed that radiation exposure affected multiple

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pathways. The upregulated pathways included the immune system process, inflammatory response, and chemotaxis (Supplementary Figure 1). Interestingly, the data revealed

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profound perturbations of lipid metabolism because the downregulated pathways were mainly associated with lipid metabolism (Figures 1E and 1F), such as fatty acid

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metabolism (P = 2.17 × 10-11), steroid biosynthesis (P = 2.59 × 10-10), fatty acid biosynthesis (P = 4.59 × 10-8), PPAR signaling pathway (P = 2.04 × 10-5), and fatty acid elongation (P = 4.83 × 10-5). Several key genes in lipid metabolism, such as Elovl3, Elovl5, FABP5, and FASN, were confirmed to be significantly reduced in the irradiated skin tissues of the rats on real-time PCR (Figure 1G).

Local radiation modulates skin fat mass Because adipocytes are actively involved in lipid metabolism [22], the abovementioned results from RNA-Seq indicate the possible involvement of dermal and subcutaneous adipocytes in radiation response by modulating lipid metabolism. Therefore, we investigated whether radiation-induced lipid dysregulation is associated with changed skin fat disposition. The SD rats were irradiated using 45-Gy electron beam irradiation as reported previously, and skin lipid mass was monitored using MRI. Compared with the non-irradiated field, the irradiated skin region showed decreased adipose tissue mass 3

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days after local radiation, which recovered 10 days after radiation without affecting the volume of abdominal skin adipose tissue and visceral adipose tissue both in the SD and Wistar rats (Figure 2A). Radiation-induced decrease in fat mass was confirmed via oil red

O staining (Figures 2B and 2C). Moreover, H&E staining showed that radiation induced

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atrophy of adipocytes in subcutaneous WAT of rats 7 days after radiation

fat mass.

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Radiation modifies skin lipid profiles

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(Supplementary Figure 2). These results indicate that radiation modulates subcutaneous

The abovementioned results suggest potential lipid abnormalities in irradiated skin and subcutaneous tissues. Therefore, we explored the lipid profiles between the

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non-irradiated and irradiated skin tissues of the rats using the high-throughput LC-MS-based lipidomic method [24]. The typical positive and negative total ion

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chromatograms of the samples are shown in Supplementary Figure 3. The entire lipid species were loaded into a principal component analysis (PCA) using SIMCA-P (version

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11). As shown in Figure 2D, the metabolites of the radiation-treated group could be clearly distinguished from those of the non-irradiated control group according to the PCA score plots. These variables were further loaded into a supervised PLS-DA model. As shown in Figure 2E, the differences between the two groups were also depicted by the PLS-DA score. The abovementioned score plots suggest that the metabolic pattern of lipid species in cutaneous and subcutaneous tissues is altered by radiation.

The relative concentrations of the lipid species were calculated, and 518 individual lipid metabolites were identified and classified into 8 categories. Among them, 178 lipid metabolites (66 upregulated and 112 downregulated) were found to be significantly dysregulated between the non-irradiated and irradiated skin tissues of the rats (Supplementary Table 1). The fold-change in these metabolites was also illustrated by a heatmap as shown in Supplementary Figure 4. The dysregulated lipid metabolites included 25 ceramides (CERs), 1 cardiolipin (CL), 1 diglyceride (DG), 1 phosphatidic acid(PA), 40 phosphatidylcholines (PCs), 20 phosphatidylethanolamines (PEs), 5

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phosphatidylglycerols (PGs), 2 phosphatidylinositols(PIs), 6 phosphatidylserines (PSs), 8 sphingomyelins (SMs), 2 sphingosines (Sos)and 67 triglycerides (TGs)(Supplementary Table 1 and Figure 2F). These results indicate that radiation induced a lipid remolding of skin tissues, and dysregulated lipid metabolites may act as potentially promising

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biomarkers in monitoring and/or diagnosing radiation-induced skin injury. High-fat diet confers resistance to radiogenic skin injury

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The fact that radiation reduced skin adipose mass prompted us to postulate that

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increasing adipose mass may modulate radiation-induced cutaneous reaction. To confirm this hypothesis, the SD rats were divided into two groups. One group of rats was fed with a high-fat diet; the other group was fed with a comparable control diet for 3 months. The

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skin adipose tissue mass of the two groups as measured on MRI is shown in Figure 3A. Thereafter, the rats were irradiated using the 45-Gy electron beam as reported previously

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[20]. After 20 days, radiogenic dermatitis was present in the SD rats fed with a control diet; no obvious skin reaction was observed in the rats fed with a high-fat diet (Figure 3B). H&E

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staining indicated epidermis loss and inflammatory cell infiltration in the skin tissues of the control diet rats after 45-Gy irradiation; these effects were not observed in the skin tissues of the rats fed with a high-fat diet (Figure 3C). Similar results were obtained in the Wistar rats, showing radioresistance of rat skin with high-fat diet (Figure 3D). These data suggest the notion that increased skin adipose mass induced by a high-fat diet may confer resistance against radiogenic skin injury.

Adipocytes facilitate the repair of irradiated skin cells by PA and FABP4 Because keratinocytes and skin fibroblasts grow in the anatomical vicinity of dWAT and subcutaneous WAT, we then examined if these adipocytes affect the biological behavior of neighboring keratinocytes and skin fibroblasts. We dissected the dermal and subcutaneous adipose tissues from human patients and isolated the mature adipocytes from the adipose tissues (Figure 4A and 4B). We then co-cultured these isolated adipocytes with human keratinocytes (HaCaT) and skin fibroblasts (WS1) in the transwell culture system as described under “Materials and methods” (Figure 4C). We first

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examined if lipids in adipocytes could be transferred to co-cultured WS1 cells. As shown in Figure 4D, the co-cultured human keratinocytes and fibroblasts had an obvious

increase in cytoplasmic lipid droplet accumulation compared with the mono-cultured

control cells. Moreover, skin adipocytes could facilitate the migration (Figure 4E and 4F)

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but not the proliferation of co-cultured keratinocytes and skin fibroblasts (Supplementary

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Figure 5). And the migration of irradiated WS1 and HaCaT cells were also facilitated after co-cultured with adipocytes (Figure 4E and 4F). We examed other factors involved in

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radiation-induced skin damage. Cell cycle analysis shows that adipocytes could increase the number of dividing cells but after radiation this trend disappears (Figure 4G). Apoptosis rates of skin cells co-cultured with adipocytes did not show significant

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differences with or without radiation (Supplementary Figure 6). Evidence has shown that mature adipocytes are an exogenous source of fatty acids,

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including PA [23,24]. We first examined the concentration of free fatty acids with or without radiation in skin tissues of a rat model via HPLC. After radiation, several fatty

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acids with a relatively high content were significantly reduced in the skin tissues (Figure 5A). Since PA was the most abundant fatty acid in the skin tissues (Figure 5A), we evaluated the effect of PA on cell migration. Less than 100-μM PA did not show obvious toxicity to WS1 cells (Supplementary Figure 7). Migration assay shows that 25-100 μM PA can promote cell migration to varying degrees (Figure 5B). PA could promote the migration of irradiated cells as well (Figure 5C).

FABP4 has also been proven to be an adipose-derived cytokine that could be released into

circulation

[25].

Therefore,

we

investigated

whether

FABP4

from

the

microenvironment could be incorporated by skin cells. Human FABP4 was fused with EGFP at the C-terminal to obtain an FABP4-EGFP fusion protein. As presented in Supplementary Figure 8, this fusion protein was expressed at a very high level. The fusion protein was purified via affinity chromatography and subsequently confirmed via Western blotting (Supplementary Figure 9 and Figure 5D). The HaCaT cells were incubated with a fresh medium containing EGFP or EGFP-tagged FABP4 protein (FABP4-EGFP). After 4

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h, it was observed that FABP4-EGFP, but not EGFP, could be incorporated to the HaCaT cells (Figure 5E). Moreover, the results revealed that FABP4-EGFP entered the skin

fibroblasts in a dose-dependent manner (Figure 5F). Exogenous addition of FABP4 reduced radiation-induced DNA damage, suggesting a role in promoting DNA repair

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(Supplementary Figure 10 and Figure 5G). This result was further confirmed by

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adenovirus-mediated FABP4 overexpression. However, the DNA repair promoting the role of FABP4 was abrogated by the FABP4 inhibitor BMS309403 (Supplementary Figure

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10), indicating that FABP4 catalytic activity was required for DNA repair. However, FABP4 did not affect the migration of WS1 cells (Supplementary Figure 11). Taken together, these results indicate that adipocytes facilitate the migration and repair of irradiated skin

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Discussion

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cells at least by fatty acids and FABP4.

Radiation-induced skin injury remains a serious concern after ionizing radiation exposure,

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including nuclear accidents, terrorist attacks and radiotherapy. however, there is currently no effective treatment to prevent or mitigate radiogenic skin injury [3,4]. In the present study, we showed that radiation modulates dozens of lipid metabolism-associated genes and multiple pathways in the skin tissues of rats using RNA-Seq. Further, MRI and oil red O staining confirmed that radiation decreased skin fat mass. Lipid profiling showed that radiation modulates the content of 178 lipid metabolites. To our knowledge, this is the first report illustrating the influence of ionizing radiation on skin lipid mass and profiles.

Consistently, Mitani et al reported that UVA irradiation causes the disappearance of dermal adipocytes and triggers cutaneous fibrosis [26]. Several studies confirmed that dermal WATs in mice are reduced by either UVA [24] or UBV radiation [27,28]. Another study showed that near-infrared irradiation non-thermally affects subcutaneous adipocytes [29]. How radiation or light modifies lipid warrants further investigation. Most of the early work concerning radiation-induced skin injury focuses on the role of fibroblasts, endothelial cells, and T cells in response to radiation in the skin [30–32].Although dermal and subcutaneous adipocytes represent the dominant

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architectural component of the human skin, conventional wisdom confines their biological key functions to energy storage, physical buffer, thermoregulation, and thermo-insulation [33]. In this study, we found that the rats fed with a high-fat diet with increased adipose accumulation showed resistance to radiation-induced skin injury, indicating that skin

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adipose plays a radio-protective role. It has been reported that dermal adipocytes promote fibroblast recruitment during skin wound healing [34]. As a natural extension, we found

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that PA and FABP4, which can be released from mature adipocytes [23,25], contributed to

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the repair of irradiated skin cells. During wound healing, the skin usually requires more energy from body energy stores for the building of new cells [35]. Moreover, the skin needs fat for fast keratinization and the barrier function of the stratum corneum, which is

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embedded in a lipid matrix existing in the form of lipid bilayers [36,37]. Several skin diseases, such as psoriasis and atopic dermatitis, are associated with depletion of skin

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lipids [38]. Therefore, increased PA and FABP4 by adipocytes in the microenvironment may also contribute to repair of the irradiated skin.

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In conclusion, we found that radiation modulated lipid mass and profiles during the progression of radiation-induced skin injury. We further observed that skin adipocytes contribute to the repair of radiation-induced skin injury at least by PA and FABP4.

Conflict of interest The authors state no conflict of interest.

Acknowledgements This work is supported by the National Natural Science Foundation of China (31770911, 81522039, U1967220, 81703157 and 81773226), the Social Development Program of Jiangsu Province (BE2017652, BK20161152 and KYCX18_2520) and the Fundamental

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Research Funds for the Central Universities.

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Figure 1. Profiling of mRNAs in response to radiation reveals aberrant expression of lipid metabolism-related genes in rat skin. (A)Number of the identified and dysregulated mRNAs between the non-irradiated and irradiated skin tissues of the rats. (B) Volcano plot of the differentially expressed mRNAs between the two groups. (C) Heatmap of the dysregulated mRNAs between the two groups (n = 3). Expression levels are reflected by color change. Red denotes higher expression, whereas green denotes lower expression. (D) Top 20 upregulated and downregulated mRNAs. (E) KEGG analysis of the downregulated mRNAs between the

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normal and irradiated skin tissues. (F) GOTree analysis of the downregulated and expressed mRNAs between the normal and irradiated skin tissues. (G) Real-time PCR analysis of the mRNAs related to lipid metabolism from the RNA sequencing between the

non-irradiated and irradiated skin tissues of the rats (n = 5). Relative mRNA expression

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ratios between the normalized values were calculated. *, P < 0.05; **, P < 0.01.

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Figure 2. Radiation induces lipid remolding in rat skin tissues.

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The gluteal skin of the SD and Wistar rats was irradiated with a single dose of 45-Gy electron beam. (A) MRI showing the skin fat of an SD rat before and after radiation (on

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day 3 and day 10). MRI showing the skin fat of a Wistar rat before and after radiation (on day 3 and day 10). The volume of back skin adipose tissue at different times after

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radiation was calculated. The volume of abdominal skin adipose tissue and visceral adipose tissue was accounted as well. Data are presented as mean ± SEM and

normalized to the control rats. * P < 0.05; ** P < 0.01. (B) Oil red O staining of the skin tissues of the SD rats with and without 45-Gy electron beam radiation. (C) Oil red O staining of the skin tissues of the Wistar rats with and without 45-Gy electron beam radiation. (D) PCA and (E) PLS-DA score plot based on the profiling of the non-irradiated 21

and irradiated skin tissues of the rats (n = 12). The score plot indicated a clear separation between the two groups. (F) Number of the dysregulated lipid metabolites of each lipid category. SD, Sprague Dawley; MRI, magnetic resonance imaging; PCA, principal

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component analysis; LC-MS, liquid chromatography-mass spectrometry.

Figure 3. High-fat diet confers resistance from radiogenic skin injury. The SD rats were divided into two groups: One group of rats was fed with a high-fat diet, and the other group was fed with a comparable control diet for 3 months. (A) MRI showing 22

the skin adipose mass of the rats fed with a control diet or high-fat diet. (B) The SD rat gluteal skin was irradiated with a single dose of 45-Gy electron beam; representative skin images 20 days after radiation. (C) H&E staining of the skin tissues in each group of SD rats. (D) H&E staining of the skin tissues in each group of Wistar rats. SD, Sprague

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Dawley; MRI, magnetic resonance imaging; H&E, hematoxylin and eosin.

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Figure 4. Adipocytes facilitate the migration of irradiated skin cells.

(A) Representative image of an isolated mature adipocyte. Mature adipocytes were isolated from the subcutaneous tissues of human patients. (B) Adipocytes were stained with DAPI and BODIPY. White arrow indicates the nucleus. (C) Co-culture diagram

of

mature

adipocytes

with

skin

fibroblasts.

(D)

Representative

photomicrographs of BODIPY fluorophore 493/503 staining for lipid droplets. The cells were observed using a confocal microscope (Olympus, Tokyo, Japan). (E) Migration

assay of WS1 cells and HaCaT with or without co-culture of adipocytes. Migration was

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observed 18 h after the treatment, and the open wound area was normalized to the

area at the initial time that the wound was made. Data are presented as mean ± SEM and normalized to the control cells. (F) Migration assay of WS1 cells and HaCaT with

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or without co-culture of adipocytes after 5 Gy or 20 Gy X-ray radiation. (G) Cell cycle

assay of WS1 cells and HaCaT cells with or without co-culture of adipocytes were

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examed at 12h after 5 Gy or 20 Gy X-ray radiation using a flow cytometry (BD, USA). Data are presented as mean ± SEM and normalized to the control cells. * P < 0.05; **

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P < 0.01.

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Figure 5. Palmitic acid promotes the migration of and FABP4 contributes to DNA repair of irradiated skin fibroblasts. (A) The SD rat gluteal skin was irradiated with a single dose of 45-Gy electron beam; quantification of fatty acids with or without radiation by HPLC. (B) Effect of palmitic acid on the migration of WS1 cells. Migration was observed 24h after

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the treatment, and the open wound area was normalized to the area at the initial time that the wound was made. Data are presented as mean ± SEM and

normalized to the control cells. * P < 0.05; ** P < 0.01. (C) Effect of 50 μM

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palmitic acid on the migration of WS1 cells after 5 Gy X-ray radiation.

Migration was observed 0, 12, and 24 h after the treatment, and the open

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wound area was normalized to the area at the initial time that the wound was made. (D) Diagram of the FABP4-EGFP fusion protein and Western blotting

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analysis of the purified FABP4-EGFP fusion protein. (E) Uptake of FABP4-EGFP by the HaCaT cells. The HaCaT cells were treated with 10-μM FABP4-EGFP

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fusion protein for 4 h, and fluorescence was observed using a confocal microscope. The nuclei were counter-stained with DAPI. (F) Uptake of FABP4-EGFP by WS1 cells. The WS1 cells were treated with indicated

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concentrations of the FABP4-EGFP fusion protein for 2 or 4 h, and fluorescence was

observed

using

a

confocal

microscope.

(G)

FABP4

reduced

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radiation-induced DNA damage. The HaCaT and WS1 cells were treated with or without recombinant FABP4 protein (8 μM) for 4 h. One hour after radiation, immunofluorescence assay was performed using an antibody against γ-H2AX. Data are presented as mean ± SEM and normalized to the control cells. * P < 0.05,

** P < 0.01. SD, Sprague Dawley; FABP, fatty acid-binding protein 26

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