J Mol Med DOI 10.1007/s00109-016-1389-0

ORIGINAL ARTICLE

Far-infrared promotes burn wound healing by suppressing NLRP3 inflammasome caused by enhanced autophagy Hui-Wen Chiu 1,2 & Cheng-Hsien Chen 1,3 & Jen-Ning Chang 1 & Chien-Hsiung Chen 4 & Yung-Ho Hsu 1

Received: 6 August 2015 / Revised: 10 January 2016 / Accepted: 28 January 2016 # Springer-Verlag Berlin Heidelberg 2016

Abstract Understanding the underlying molecular mechanisms in burn wound progression is crucial to providing appropriate diagnoses and designing therapeutic regimens for burn patients. When inflammation becomes unregulated, recurrent, or excessive, it interferes with burn wound healing. Autophagy, which is a homeostatic and catabolic degradation process, was found to protect against ischemic injury, inflammatory diseases, and apoptosis in some cases. In the present study, we investigated whether far-infrared (FIR) could ameliorate burn wound progression and promote wound healing both in vitro and in a rat model of deep second-degree burn. We found that FIR induced autophagy in differentiated THP-1 cells (human monocytic cells differentiated to macrophages). Furthermore, FIR inhibited both the NLRP3 inflammasome and the production of IL-1β in lipopolysaccharide-activated THP-1 macrophages. In addition, FIR induced the ubiquitination of ASC, which is the adaptor protein of the inflammasome, by

increasing tumor necrosis factor receptor-associated factor 6 (TRAF6), which is a ubiquitin E3 ligase. Furthermore, the exposure to FIR then promoted the delivery of inflammasome to autophagosomes for degradation. In a rat burn model, FIR ameliorated burn-induced epidermal thickening, inflammatory cell infiltration, and loss of distinct collagen fibers. Moreover, FIR enhanced autophagy and suppressed the activity of the NLRP3 inflammasome in the rat skin tissue of the burn model. Based on these results, we suggest that FIRregulated autophagy and inflammasomes will be important for the discovery of novel therapeutics to promote the healing of burn wounds.

Electronic supplementary material The online version of this article (doi:10.1007/s00109-016-1389-0) contains supplementary material, which is available to authorized users.

Key messages & Far-infrared (FIR) induced autophagy in THP-1 macrophages. & FIR suppressed the NLRP3 inflammasome through the activation of autophagy. & FIR induced the ubiquitination of ASC by increasing TRAF6. & FIR ameliorated burn wound progression and promoted wound healing in a rat burn model.

* Yung-Ho Hsu [email protected]

Keywords Far-infrared . Burn wound healing . Autophagy . NLRP3 inflammasome

1

Division of Nephrology, Department of Internal Medicine, Shuang Ho Hospital, Taipei Medical University, No. 291, Zhongzeng Rd., Zhonghe District, New Taipei City 23561, Taiwan

2

Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan

3

Department of Internal Medicine, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan

4

Department of Industrial and Commercial Design, National Taiwan University of Science and Technology, Taipei, Taiwan

Introduction Burn wound progression makes treatment more difficult, prolongs hospital stays, increases costs, and increases the likelihood of scarring. Burn injuries are highly variable in terms of the tissue affected, the severity, and the resultant complications [1]. A number of mechanisms are involved in the process of burn wound progression, including local tissue

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hypoperfusion, edema, prolonged inflammation, hypercoagulability, free radical damage, and the accumulation of cytotoxic cytokines [2, 3]. Importantly, inflammation is a natural reaction to injury and is essential for the induction of tissue repair. However, when it becomes dysregulated, recurrent, or excessive, therapeutic inhibition of the inflammation is required. The standard treatments including steroids and nonsteroidal anti-inflammatory drugs have severe side effects, such as gastrointestinal ulcers and bleeding, thromboembolic events, kidney and liver toxicity, Cushing’s syndrome, and infections [4]. Therefore, there is a need for novel therapeutic approaches to prevent burn wound progression. Autophagy is a cellular response to starvation that can deliver damaged organelles and long-lived proteins from the cytoplasm to lysosomes for degradation [5]. The accumulated evidence indicates that defective autophagy is linked to many human diseases and to the function of cells of the immune response [6]. Previous studies have demonstrated that blocking autophagy through the genetic deletion of Atg16L1 potentiates the NLRP3 inflammasome activity induced by TLR4 signaling [7]. Furthermore, autophagy inhibits the inflammasome activity by degrading the inflammasome via ASC ubiquitination and the recruitment of p62 and LC3 [6]. Therefore, ubiquitination is an important step in the regulation of inflammasomes. Previous research has shown that inflammasomes are multiprotein complexes that are activated after cellular infection or stress. Their activation triggers caspase 1 activation and the maturation of interleukin 1β (IL-1β) and IL-18 to engage the innate immune defenses [8]. To date, four inflammasomes have been identified, including NLRP1, NLRP3, NLRC4, and AIM2 [9]. The most extensively studied inflammasome, NLRP3, responds to numerous physically and chemically diverse stimuli [10]. Recent evidence has shown that the process of cellular trauma during an acute injury activates the inflammasomes due to the endogenous alarm signals that are released from damaged cells [11]. Autophagy could ameliorate burn wound progression and promote wound healing [12]. However, the relationship between autophagy and inflammasome activity during wound healing is unclear. Infrared radiation is an invisible electromagnetic wave with a longer wavelength than that of visible light. Infrared radiation is subdivided into three categories: near-infrared radiation (IR-A, 0.7 to 1.4 μm), middle-infrared radiation (IR-B, 1.4 to 3 μm), and far-infrared (FIR) radiation (IR-C, 3 to 1000 μm), according to the International Commission on Illumination. There are only a few reports on the biological activities of FIR, and most of these address the hyperthermic effect of FIR. However, data accumulated by us and others have revealed that FIR has both a hyperthermic effect and biological effect of FIR [13–15]. Our previous study found that FIR is a potential therapeutic modality to maintain vascular endothelial health and function, which acts by inducing the nuclear

translocation of promyelocytic leukemia zinc finger protein (PLZF). Furthermore, this molecular mechanism was independent of any thermal effect [15]. In addition, FIR therapy exerts a nitric oxide-mediated biological effect to increase skin microcirculation in rats [14]. Previous research has shown that FIR promotes skin wound healing, as indicated by histological evidence of greater collagen regeneration and the infiltration of fibroblasts into the wounds [13]. Nevertheless, the biological effects of FIR on wound healing are still poorly understood. The present study aimed to test the hypothesis that FIR is effective in promoting burn wound healing. We analyzed whether FIR could enhance autophagy and inhibit the NLRP3 inflammasome while promoting wound healing both in vitro and in a rat model of deep second-degree burn wounds.

Materials and methods Cell culture The monocytic leukemia cell line THP-1 (ATCC TIB-202) was obtained from the American Type Culture Collection (ATCC). The cells were cultured in RPMI 1640 medium (Gibco BRL, Grand Island, NY) supplemented with 100 nM penicillin/streptomycin (Gibco BRL, Grand Island, NY), 0.05 mM 2-mercaptoethanol, 10 mM HEPES, 1 mM sodium pyruvate, and 10 % fetal bovine serum (HyClone, South Logan, UT, USA). The THP-1 monocytic cells were differentiated into macrophages by culturing for 48 h in dishes or wells containing the RPMI 1640 medium supplemented with 5 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma Chemical Co.). The murine macrophage cell line J774A.1 (ATCC TIB-67) was obtained from the ATCC. The cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco BRL, Grand Island, NY) supplemented with 100 nM penicillin/streptomycin (Gibco BRL, Grand Island, NY) and 10 % fetal bovine serum (HyClone, South Logan, UT, USA). The two cell lines were incubated in a humidified atmosphere containing 5 % CO2 at 37 °C.

FIR exposure A ceramic FIR generator, a WS TY301 FIR emitter (WS Far Infrared Medical Technology, Taipei, Taiwan), was used to provide the FIR exposure. This FIR emitter generates electromagnetic waves with wavelengths in the range of 3~25 μm. During the FIR exposure, an experimental group and a negative control covered with aluminum foil were set up in a culture chamber of a LiveCell™ system (Pathology Devices, Westminster, MD, USA) at 37 °C with a 5 % CO2 atmosphere. The details are described in our previous study [15].

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Cell viability assay Cellular viability was determined using the MTS assay, which measures the reduction of (3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium) (MTS) to formazan in viable cells. Briefly, the cells were plated into 96 multiwell plates (Costar, Corning, NY). After the FIR treatment, MTS was added to the cells, and the incubation at 37 °C was continued for 1 h. The formazan absorbance was then measured at 490 nm. The mean absorbance of the nonexposed cells was the reference value for calculating 100 % cellular viability. Detection of autophagy Cell staining with acridine orange (AO) (Sigma Chemical Co., USA) was performed according to published procedures [16].

Diego, CA, USA); anti-ubiquitin antibody was obtained from Proteintech Group (Chicago, IL, USA); and anti-p62/ SQSTM1 antibody was obtained from MBL (Nagoya, Japan). RNA interference We used OMNIfect transfection reagent (transOMIC technologies Inc., AL, USA) to transfect cells according to the manufacturer’s protocol. RNAi reagents were obtained from the National RNAi Core Facility located at the Institute of Molecular Biology/Genomic Research Center, Academia Sinica, supported by the National Core Facility Program for Biotechnology Grants of NSC (NSC 100-2319-B-001-002). The human library is referred to as TRC-Hs 1.0. Individual clones are identified as small hairpin RNA (shRNA) TRCN0000072184 and shRNA TRCN0000356147.

Immunofluorescence microscopy

Immunoprecipitation

The cells were cultured on coverslips. After the FIR treatment, the cells were fixed in 4 % paraformaldehyde and blocked with 1 % BSA for 30 min. This was followed by incubation with a specific antibody against LC3 (MBL, Japan) for 1 h. After washing, the cells were labeled with a DyLight™ 488conjugated affinipure goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, PA, USA) for 1 h and stained with DAPI. Finally, the cells were washed in PBS, coverslipped, and examined with a fluorescence microscope or confocal microscope (Carl Zeiss LSM780, Instrument Development Center, NCKU).

The cells were lysed with protein extraction buffer as above. The soluble fraction was incubated overnight at 4 °C with an antibody to anti-ASC Adipogen (San Diego, CA, USA); subsequently, A/G agarose beads (Merck Millipore, MA, USA) were added to the solution, and the incubation was continued for an additional hour at 4 °C. The beads were washed three times with the protein extraction buffer, resuspended in SDS sample buffer, and boiled for 5 min. The supernatant was subjected to western blot analysis using an ASC antibody performed as described above. Rat model of deep second-degree burn

Transmission electron microscopy The cells were trypsinized and harvested followed by 1 h fixation in a solution containing 2.5 % glutaraldehyde and 2 % paraformaldehyde in 0.1 M cacodylate buffer, pH 7.3. After fixation, the samples were postfixed with buffer containing 1 % OsO4 for 30 min. Ultrathin sections were subsequently examined using a transmission electron microscope (Hitachi HT-7700). Western blot analysis Total cellular protein lysates were prepared by harvesting the cells in a protein extraction buffer for 1 h at 4 °C, as described previously [17]. GAPDH expression was used as the protein loading control. Anti-GAPDH, anti-Beclin-1, anti-tumor necrosis factor receptor-associated factor 6 (TRAF6), anti-procaspase 1 and anti-cleaved-caspase 1 antibodies were obtained from Abcam (Cambridge, MA, USA); anti-LC3 antibody was obtained from Abgent (San Diego, CA, USA); anti-NLRP3 and anti-ASC antibodies were obtained from Adipogen (San

Male Wistar rats weighing 200 to 220 g were obtained from BioLasco Taiwan (Taipei, Taiwan). The rats were housed for at least 7 days prior to the experiments in a ventilated and temperature-controlled room and had access to water ad libitum. The rats were anesthetized via inhalation with Attane Isoflurane (Minrad Inc, NY, USA). The hair on the dorsal skin of the rats was removed using electric clippers. A 1.0-cm-diameter brass rod was heated to 100 °C and applied to the skin for 6 s to produce a deep second-degree burn [18]. Three burns were created on each half of the dorsal skin at 1 cm apart, for a total of six burns on each rat. The rats were randomized into three treatment groups (five rats per group): (1) normal, (2) burned (rats were burned and then covered with aluminum foil during the exposure to FIR for 30 min per day), and (3) burned+FIR (rats were burned and exposed to FIR for 30 min per day). The rats were sacrificed using CO2 exposure. After sacrifice, some skin tissues were snap frozen in liquid nitrogen and stored at −80 °C, and the others were formalin-fixed and paraffin-embedded for immunohistochemistry.

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Histological analysis The tissues were fixed in 10 % formalin (in normal saline). After 3 days, the tissues were sectioned using a microtome and stained with hematoxylin and eosin (H & E) for histological analyses. The slides were examined microscopically and the images were recorded. Immunohistochemical staining analysis The paraffin-embedded tissue sections were dried, deparaffinized, and rehydrated. Following a microwave pretreatment in citrate buffer (pH 6.0; for antigen retrieval), the slides were immersed in 3 % hydrogen peroxide for 20 min to block the activity of endogenous peroxidase activity. After extensive washing with PBS, the slides were incubated overnight at 4 °C with the anti-LC3 (MBL, Japan), anti-caspase 1 (Abcam, MA, USA), or anti-TRAF6 (Abcam, MA, USA) antibody. The sections were then incubated with the secondary antibody for 1 h at room temperature, and the slides were developed using the UltraVision Quanto HRP Detection kit (Thermo Scientific, IL, USA). Finally, the slides were counterstained using hematoxylin. Each slide was imaged. Detection of IL-1β by ELISA The THP-1 cells or plasma of rats were collected to measure IL-1β using ELISA (eBioscience, San Diego, CA, USA; R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. The optical density of the peroxidase product was read using an ELISA reader (Emax, Molecular Devices, Sunnyvale, CA, USA) at 450 nm. Based on the standard curve, the concentrations of IL-1β in each sample were determined. Statistical analysis The data are expressed as the means ± SD. Statistical significance was determined using Student’s t test to compare between the means or one-way analysis of variance with post hoc Dunnett’s test [19]. The differences were considered to be significant when p < 0.05.

Results FIR induces autophagy but does not cause cell death in differentiated THP-1 cells We investigated whether FIR induced autophagy in the differentiated THP-1 cells (THP-1 monocytic cells that had been differentiated into macrophages). Autophagy is characterized by the formation of numerous acidic vesicles that are called

acidic vesicular organelles (AVOs) [20]. AVOs were evaluated as green and red fluorescence in the AO-stained cells by using flow cytometry (Fig. 1a, b). The quantitative results showed that a significant increase in AO-positive cells was found for the cells that received the FIR treatment compared with the control-differentiated THP-1 cells. Furthermore, we performed transmission electron microscopy (TEM), which is a powerful technique for evaluating subcellular structural changes. To determine whether the changes in the cytoplasm could be associated with FIR, we collected images of the differentiated THP-1 cells treated with FIR (Fig. 1c). The cells in the FIR-treated samples showed nuclei similar to the untreated control and no significant chromatin condensation or nuclear pyknosis, both of which are characteristic of apoptosis. However, the FIR-treated cells displayed an increased formation of autophagic vesicles in the cytoplasm. Previous studies have demonstrated that LC3 is the most reliable marker to detect and monitor autophagy [21]. Therefore, we applied fluorescence microscopy to identify cells with punctate LC3 staining (Fig. 2a). The results showed that FIR increased the LC3 puncta in the differentiated THP-1 cells. We also detected the expression of autophagy-related proteins by western blotting (Fig. 2b). The expression levels of LC3-II, Beclin1, and p62 proteins increased with FIR treatment. Furthermore, FIR treatment increased LC3-II and Beclin-1 in J774A.1 cells (a murine macrophage cell line) (Fig. S1A). However, it is important to note that the role of autophagy in regulating cell death or survival remains controversial. Figures 2c and S2A showed that FIR did not reduce the viability of cells and affect the percentage of apoptosis in a timedependent manner. In addition, FIR did not increase the expression level of cleaved-caspase 3 (Fig. S2B). These results indicated that FIR treatment induced autophagy but did not affect the viability of the macrophages. FIR inhibits NLRP3 inflammasome in lipopolysaccharide-activated THP-1 macrophages Lipopolysaccharide (LPS) is an inducer of inflammatory responses and the NLRP3 inflammasome in macrophages [22]. We examined whether FIR could affect activation of the NLRP3 inflammasome. As shown in Fig. 3a, FIR significantly inhibited the LPS-induced expression of NLRP3, ASC, and cleaved-caspase 1. The activation of the NLRP3 inflammasome has been reported to cause the production of IL-1β [8]. Next, to investigate whether cytokine production was influenced by FIR, the differentiated THP-1 cells were treated with FIR in the presence of LPS. The cytokine secretion into the medium of the cells was then measured using western blot analysis and ELISA. Our results showed that FIR inhibited the LPS-increased IL-1β secretion (Fig. 3a, b). A similar effect was obtained when FIR was used to prevent NLRP3 inflammasome complex formation and IL-1β

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Fig. 1 Measurement of AVOs and TEM microphotographs in differentiated THP-1 cells treated with FIR. a Development of AVOs in differentiated THP-1 cells. Detection of green and red fluorescence in AO-stained cells was performed using flow cytometry. The cells were treated with FIR for 10, 20, or 30 min and cultured for 1 h. b Quantification of AVOs in differentiated THP-1 cells treated with FIR

using flow cytometry. The data presented as the means ± standard deviation from three independent experiments. *p < 0.05, control versus FIR. c The ultrastructure of differentiated THP-1 cells treated with FIR for 30 min and cultured for 1 h was analyzed by TEM. N indicates nucleus. Black arrows indicate autophagic vesicles

secretion in J774A.1 cells (Fig. S1B). We also evaluate if FIR plays a role in NLRP3 inflammasome activation by other NLRP3 inflammasome activator (monosodium urate (MSU) crystals) (Fig. S3B). FIR suppressed NLRP3 inflammasome activation and IL-1β secretion by MSU. These results showed that FIR treatment reduced IL-1β secretion and the NLRP3 inflammasome activity. In addition, we examined the effect of FIR on another inflammasome (AIM2 inflammasome) (Fig. S3A). However, FIR did not change the poly(dA:dT)-induced expression of AIM2 and IL-1β secretion.

methyladenine (3-MA) or enhanced autophagy by treatment with rapamycin. As shown in Fig. 4a, pretreatment of cells with 3-MA showed a significant decrease in the expression of LC3-II and rapamycin showed a significant increase compared with the combined treatment with LPS and FIR. Of note, LPS can cause activation of autophagy. Our observations are quite parallel with those of other investigators [23, 24]. Furthermore, we found that pretreatment with 3-MA resulted in enhanced IL-1β secretion and in the expression of NLRP3, ASC, and cleaved-caspase 1 in the differentiated THP-1 cells. In contrast, pretreatment of the cells with rapamycin, an autophagy inducer, resulted in a marked decrease in IL-1β secretion and in the expression of the NLRP3 inflammasome. Our results showed that autophagy interacted with the NLRP3 inflammasome in FIR-treated macrophages. More recently, the accumulated evidence has revealed that autophagy can limit the inflammasome activity via ubiquitination of ASC [6]. We found that FIR induced ASC ubiquitination but inhibited the LPS-induced ASC expression (Fig. 4b). Furthermore, we investigated which ubiquitin E3 ligase ubiquitinates ASC. p62 can trigger ubiquitination of

FIR suppresses the NLRP3 inflammasome through the activation of autophagy and the ubiquitination of ASC There is an intimate interaction between the inflammasomes and autophagy [6]. We speculated that FIR-induced autophagy would suppress the NLRP3 inflammasome. To study this possibility, we examined the NLRP3 inflammasome activity under conditions in which we blocked autophagy using 3-

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Fig. 2 Measurement of autophagy and cell viability in differentiated THP-1 cells treated with FIR. a The differentiated THP-1 cells that had been treated with FIR were stained for immunofluorescence detection of LC3. b The expression of the autophagic-associated proteins was measured by western blot analysis following treatment with FIR. The

cells were treated with FIR for 10, 20, or 30 min and cultured for 1 h. c Cytotoxicity was measured in differentiated THP-1 cells treated with FIR for 10, 20, or 30 min and cultured for 24 h. The data are presented as the means ± standard deviation of three independent experiments

TRAF6, which is an E3 ligase. Furthermore, TRAF6mediated ubiquitination of Beclin-1 facilitates the induction of autophagy [25, 26]. Therefore, we analyzed whether FIR

affected TRAF6. We also observed an increase in the expression of TRAF6 following the FIR treatment (Fig. 5a). We also used TRAF6 shRNA to inhibit TRAF6. As shown in Fig. 5b,

Fig. 3 FIR attenuates the LPS-induced NLRP3 inflammasome. a The expression of NLRP3 inflammasome-associated proteins was measured by western blot analysis following treatment with FIR in the supernatants (Sup) or cell lysates (Lys). b The levels of IL-1β in the culture medium were measured by ELISA. The differentiated THP-1 cells were incubated

for 12 h with or without 1 μg/ml of LPS and then treated with FIR for 10, 20, or 30 min and cultured for 3 h longer. The data are presented as the mean ± standard deviation of three independent experiments. *p < 0.05, LPS versus LPS+FIR

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Fig. 4 FIR-induced autophagy affects NLRP3 inflammasomes. a Effects of 3-MA or rapamycin on the expression of NLRP3 inflammasomeassociated proteins and the levels of IL-1β. The differentiated THP-1 cells were incubated for 12 h with 1 μg/ml of LPS and then treated with 3-MA or rapamycin for 1 h. The cells were then treated with FIR for 30 min and cultured for 3 h longer. *p < 0.05, LPS versus LPS+FIR. #p < 0.05, LPS+FIR versus LPS+FIR+3-MA or rapamycin. b Immunoprecipitation (IP) assay for ASC ubiquitination. The

Fig. 5 FIR affects the E3 ligase TRAF6. a The expression levels of TRAF6 in differentiated THP1 cells were analyzed by western blot analysis following FIR treatment. The cells were treated with FIR for 10, 20, or 30 min and cultured for 1 h. b TRAF6 protein expression in differentiated THP1 cells transfected with the control or TRAF6 shRNA for 24 h. c IP assay of ubiquitination of ASC. The differentiated THP-1 cells were transfected with the control or TRAF6 shRNA for 24 h and then incubated for 12 h with or without 1 μg/ml of LPS. The cells were then treated with FIR for 30 min and cultured for 3 h longer

differentiated THP-1 cells were incubated for 12 h with or without 1 μg/ml of LPS and then treated with FIR for 20 or 30 min and cultured for 3 h longer. The total cellular protein lysates prepared from the differentiated THP-1 cells were immunoprecipitated with anti-ASC antibody. The immune complexes and the input were analyzed using western blot analysis with an antibody specific to ubiquitin. Asterisk (*) shows the IgG heavy chains

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the expression of the TRAF6 proteins was markedly decreased in the cells treated with the TRAF6 shRNA compared with the control shRNA. Transfection with TRAF6 shRNA significantly inhibited the ASC ubiquitination induced by FIR treatment (Fig. 5c). Thus, TRAF6 could regulate the ubiquitination of ASC in FIR-treated macrophages.

FIR promotes wound healing by enhancing autophagy and inhibiting the NLRP3 inflammasome in a rat model of deep second-degree burn wounds To evaluate the roles of FIR in burn wound progression, we used a rat model of deep second-degree burn wounds. At 10 days postburn, the epidermal changes in tissues were characterized by thickening (Fig. 6b, e). Furthermore, the boundary between the epidermis and dermis showed edema, as indicated by the black arrows (Fig. 6b, e), and inflammatory cell infiltration (Fig. 6b). Unburned rat dermis showed distinct individual collagen fibers and bundles. In contrast, the dermis of rats after burn treatment showed a gray background fluid with the collagen, and distinct collagen fibers, glands, and hair follicle were absent (Fig. 6b, h). In addition, quantification of epidermal thickness showed a significant decrease in the FIR treatment group than in the burned group (Fig. S4A). Our results indicated that FIR ameliorated the burn-induced epidermal thickening, subepidermal vesicle formation, inflammatory cell infiltration, and loss of distinct collagen fibers (Fig. 6c, f, i). Next, LC3, caspase 1, and TRAF6 expression levels were examined in the

Fig. 6 H & E staining of normal skin and burn wounds. Six burns were created on each rat. The rats were randomized into three treatment groups: normal, burned (rats were burned and then covered with aluminum foil during the exposure to FIR for 30 min per day), and burned+FIR (rats were burned and exposed to FIR for 30 min per day). a–c Low magnification skin tissue section showing the representative dermal change in the burn, ×100. The boundary between the epidermis and dermis showed edema as indicated by the black arrows. d–f High magnification skin tissue section showing the representative epidermis of a burn, ×400. g–i High magnification skin tissue section showing the representative collagen structure in the burn, ×400

skin tissue using immunohistochemical (IHC) staining (Figs. 7a and S4B). We found that LC3 expression was increased in the burned group. These observations are quite consistent with those of other investigators [12]. Significant increases in the cells that expressed LC3 and TRAF6 were observed in the FIR treatment group compared with the burned group. However, FIR decreased the caspase 1 expression as compared with the burned group rats. In addition to IHC staining, proteins extracted from the skin tissue were assayed by western blotting, and the results are shown in Fig. 7b. LC3-II and TRAF6 protein levels were increased, and the expression of NLRP3 was decreased in the FIR treatment group compared with the burned treatment group. In addition, an ELISA specific for rat IL-1β was used to detect and quantify the IL-1β in plasma of the rats (Fig. 7c). The expression of IL-1β in the burned group was higher than that in the FIR groups, which demonstrated that FIR indeed decreased the inflammatory effect.

Discussion Autophagy is a cell death process and has been referred to as type II programmed cell death. However, autophagy is also involved in cellular metabolic turnover and physiological homeostasis [27, 28]. Therefore, the role of autophagy in the regulation of cell death or survival remains controversial. There are few published articles describing the relationship between autophagy and burn wound progression. Xiao et al.

J Mol Med Fig. 7 The protein expression and the levels of IL-1β in a rat model of deep second-degree burn wounds. a IHC staining of skin tissue. IHC was used to determine the expression levels of LC3, caspase 1, and TRAF6. The percentages of LC3-, caspase 1-, and TRAF6-positive cells were determined using HistoQuest software (TissueGnostics). b Western blot analysis of protein expression in skin tissue. N indicates the normal group. B indicates the burned group. F indicates the burned+FIR group. c The levels of IL-1β in the rat plasma (n = 5 rats per group) were measured by ELISA. *p < 0.05, burned versus burned+FIR

indicated that enhanced autophagy may be a prosurvival mechanism that protects against ischemia and inflammation after burn injury [1]. Another recent study concluded that rapamycin, an autophagy inducer, ameliorates burn wound progression and promotes wound healing by enhancing autophagy [12]. In the present study, we found that FIR induced autophagy but did not reduce cell viability in THP-1

macrophages (Figs. 1 and 2). In the rat model of deep second-degree burn wounds, the level of autophagy in the burn wounds was increased when compared with normal tissues (Fig. 7a, b). Our observations are quite consistent with those of other investigators [1, 29]. Moreover, autophagy in the FIR treatment group was significantly increased compared with the burned group (Fig. 7a, b).

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Burn injury is a complex traumatic event that has various local and systemic effects and affects several organ systems. A major burn injury is followed by an enormous inflammatory response [30]. Immediately after the injury, components of the coagulation cascade, inflammatory pathways, and the immune system are activated. Evidence has been presented that indicates that inflammasomes play an important role in wound healing [11]. It has been demonstrated that autophagy inhibited inflammasome activity [6]. However, the relationship between autophagy and inflammasomes in wound healing has yet to be fully elucidated. In this study, we provide evidence that FIR can significantly inhibit the LPS-induced NLRP3 inflammasome and IL-1β secretion (Fig. 3). In addition, we find that FIR suppressed the NLRP3 inflammasome by activating autophagy and inducting of ASC ubiquitination (Fig. 4a, b). In the in vivo study, the NLRP3 inflammasomes were activated in the skin tissue of the burned rats, as indicated by the results of the IHC staining and western blotting (Fig. 7b, c). Importantly, p62 interacts with TRAF6, which is a lysine 63 (K63) E3 ubiquitin ligase, and it then promotes TRAF6 oligomerization, thus increasing its E3 ligase activity [31]. Our results found that FIR increased the expression of p62. Thus, TRAF6 could be a candidate enzyme for the ubiquitination of ASC. In the current study, we observed the increased expression of TRAF6 following FIR treatment (Fig. 5a). Furthermore, TRAF6 shRNA significantly inhibited the ASC ubiquitination induced by FIR treatment (Fig. 5c). However, whether TRAF6 directly or indirectly regulated the ubiquitination of ASC in FIR-treated macrophages requires further investigations. Nowadays, FIR has many different uses in medical applications. However, the exact mechanisms of the biological activities of FIR are still poorly understood [32]. Leung et al. found that ceramic-emitted FIR (cFIR) significantly inhibited intracellular peroxide levels and LPS-induced peroxide production by macrophages. Furthermore, cFIR blocked ROSmediated cytotoxicity [33]. Previous studies have demonstrated that FIR upregulated the expression of arterial endothelial nitric oxide synthase (eNOS) and increases angiogenesis in mice with hindlimb ischemia [34]. In addition, data accumulated by us and others have revealed that FIR had promoting effects on skin wound healing [13–15]. Previous research has shown that FIR can penetrate through the skin and transfer energy into deep tissue gradually through a resonanceabsorption mechanism of organic and water molecules [35]. Another recent study indicated that FIR exerts a nitric oxidemediated biological effect to increase skin microcirculation in rats [14]. It is worth mentioning that FIR caused collagen regeneration and infiltration of fibroblasts that expressed transforming growth factor-β1 (TGF-β1) in wounds [13]. In our study, we found that FIR could ameliorate burn wound progression using an animal model (Figs. 6 and 7). Mechanistically, FIR inhibited the NLRP3 inflammasome by

the inducting autophagy and the ubiquitination of ASC. Moreover, FIR improved burn-induced epidermal thickening, edema formation, inflammatory cell infiltration, and loss of distinct collagen fibers (Fig. 6). Additionally, the traditional treatments for burn injuries including steroids and nonsteroidal anti-inflammatory drugs have severe side effects [4]. FIR therapy uses low-energy light and there were no obvious adverse events in our in vivo study. To the best of our knowledge, this study is the first to demonstrate that FIR promotes the healing of burn wounds. Our study provides evidence that FIR might promote burn wound healing by suppressing the NLRP3 inflammasome through an enhancement of autophagy. Furthermore, FIR limited inflammasome activity via ASC ubiquitination. TRAF6, which is an E3 ubiquitin ligase, may regulate the ubiquitination of ASC in FIRtreated macrophages. In a rat model of burn wounds, FIR improved burn wound progression. Therefore, FIR may be an effective therapeutic strategy for burn wound treatment. Acknowledgments This study was supported by the Taipei Medical University-National Taiwan University of Science and Technology Joint Research Program (TMU-NTUST-102-13) and the Taipei Medical University (TMU103-AE1-B28). Compliance with ethical standards Conflict of interest The authors declare that they have no competing interests.

References 1.

Xiao M, Li L, Li C, Zhang P, Hu Q, Ma L, Zhang H (2014) Role of autophagy and apoptosis in wound tissue of deep second-degree burn in rats. Acad Emerg Med 21:383–391 2. Shupp JW, Nasabzadeh TJ, Rosenthal DS, Jordan MH, Fidler P, Jeng JC (2010) A review of the local pathophysiologic bases of burn wound progression. J Burn Care Res 31:849–873 3. Singh V, Devgan L, Bhat S, Milner SM (2007) The pathogenesis of burn wound conversion. Ann Plast Surg 59:109–115 4. Stein C, Kuchler S (2012) Non-analgesic effects of opioids: peripheral opioid effects on inflammation and wound healing. Curr Pharm Des 18:6053–6069 5. Behrends C, Sowa ME, Gygi SP, Harper JW (2010) Network organization of the human autophagy system. Nature 466:68–76 6. Shi CS, Shenderov K, Huang NN, Kabat J, Abu-Asab M, Fitzgerald KA, Sher A, Kehrl JH (2012) Activation of autophagy by inflammatory signals limits IL-1beta production by targeting ubiquitinated inflammasomes for destruction. Nat Immunol 13: 255–263 7. Saitoh T, Fujita N, Jang MH, Uematsu S, Yang BG, Satoh T, Omori H, Noda T, Yamamoto N, Komatsu M et al (2008) Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1beta production. Nature 456:264–268

J Mol Med 8. 9. 10. 11. 12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

Schroder K, Tschopp J (2010) The inflammasomes. Cell 140:821– 832 Gross O, Thomas CJ, Guarda G, Tschopp J (2011) The inflammasome: an integrated view. Immunol Rev 243:136–151 Chen S, Sun B (2013) Negative regulation of NLRP3 inflammasome signaling. Protein Cell 4:251–258 Artlett CM (2013) Inflammasomes in wound healing and fibrosis. J Pathol 229:157–167 Xiao M, Li L, Hu Q, Ma L, Liu L, Chu W, Zhang H (2013) Rapamycin reduces burn wound progression by enhancing autophagy in deep second-degree burn in rats. Wound Repair Regen 21: 852–859 Toyokawa H, Matsui Y, Uhara J, Tsuchiya H, Teshima S, Nakanishi H, Kwon AH, Azuma Y, Nagaoka T, Ogawa T et al (2003) Promotive effects of far-infrared ray on full-thickness skin wound healing in rats. Exp Biol Med (Maywood) 228:724–729 Yu SY, Chiu JH, Yang SD, Hsu YC, Lui WY, Wu CW (2006) Biological effect of far-infrared therapy on increasing skin microcirculation in rats. Photodermatol Photoimmunol Photomed 22:78– 86 Hsu YH, Chen YC, Chen TH, Sue YM, Cheng TH, Chen JR, Chen CH (2012) Far-infrared therapy induces the nuclear translocation of PLZF which inhibits VEGF-induced proliferation in human umbilical vein endothelial cells. PLoS One 7:e30674 Chiu HW, Lin JH, Chen YA, Ho SY, Wang YJ (2010) Combination treatment with arsenic trioxide and irradiation enhances cell-killing effects in human fibrosarcoma cells in vitro and in vivo through induction of both autophagy and apoptosis. Autophagy 6:353–365 Chiu HW, Ho SY, Guo HR, Wang YJ (2009) Combination treatment with arsenic trioxide and irradiation enhances autophagic effects in U118-MG cells through increased mitotic arrest and regulation of PI3K/Akt and ERK1/2 signaling pathways. Autophagy 5: 472–483 Yuhua S, Ligen L, Jiake C, Tongzhu S (2012) Effect of Poloxamer 188 on deepening of deep second-degree burn wounds in the early stage. Burns 38:95–101 Peng PL, Kuo WH, Tseng HC, Chou FP (2008) Synergistic tumorkilling effect of radiation and berberine combined treatment in lung cancer: the contribution of autophagic cell death. Int J Radiat Oncol Biol Phys 70:529–542 Paglin S, Hollister T, Delohery T, Hackett N, McMahill M, Sphicas E, Domingo D, Yahalom J (2001) A novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles. Cancer Res 61:439–444 Hansen TE, Johansen T (2011) Following autophagy step by step. BMC Biol 9:39

22.

Ding Z, Liu S, Wang X, Dai Y, Khaidakov M, Deng X, Fan Y, Xiang D, Mehta JL (2014) LOX-1, mtDNA damage, and NLRP3 inflammasome activation in macrophages: implications in atherogenesis. Cardiovasc Res 103:619–628 23. Li S, Zhou Y, Fan J, Cao S, Cao T, Huang F, Zhuang S, Wang Y, Yu X, Mao H (2011) Heat shock protein 72 enhances autophagy as a protective mechanism in lipopolysaccharide-induced peritonitis in rats. Am J Pathol 179:2822–2834 24. Wang F, Xiao J, Shen Y, Yao F, Chen Y (2014) Estrogen protects cardiomyocytes against lipopolysaccharide by inhibiting autophagy. Mol Med Rep 10:1509–1512 25. Wooten MW, Geetha T, Seibenhener ML, Babu JR, Diaz-Meco MT, Moscat J (2005) The p62 scaffold regulates nerve growth factor-induced NF-kappaB activation by influencing TRAF6 polyubiquitination. J Biol Chem 280:35625–35629 26. Shi CS, Kehrl JH (2010) TRAF6 and A20 regulate lysine 63-linked ubiquitination of Beclin-1 to control TLR4-induced autophagy. Sci Signal 3:ra42 27. Chen Y, Klionsky DJ (2011) The regulation of autophagy—unanswered questions. J Cell Sci 124:161–170 28. Noguchi M, Hirata N, Suizu F (2014) The links between AKT and two intracellular proteolytic cascades: ubiquitination and autophagy. Biochim Biophys Acta 1846:342–352 29. Tan JQ, Zhang HH, Lei ZJ, Ren P, Deng C, Li XY, Chen SZ (2013) The roles of autophagy and apoptosis in burn wound progression in rats. Burns 39:1551–1556 30. Evers LH, Bhavsar D, Mailander P (2010) The biology of burn injury. Exp Dermatol 19:777–783 31. Moscat J, Diaz-Meco MT, Wooten MW (2007) Signal integration and diversification through the p62 scaffold protein. Trends Biochem Sci 32:95–100 32. Vatansever F, Hamblin MR (2012) Far infrared radiation (FIR): its biological effects and medical applications. Photon Lasers Med 4: 255–266 33. Leung T, Lin Y, Lee C, Chen Y, Shang H, Hsiao S, Chang H, Chao J (2011) Direct and indirect effects of ceramic far infrared radiation on the hydrogen peroxide-scavenging capacity and on murine macrophages under oxidative stress. J Med Biol Eng 31:345–351 34. Akasaki Y, Miyata M, Eto H, Shirasawa T, Hamada N, Ikeda Y, Biro S, Otsuji Y, Tei C (2006) Repeated thermal therapy upregulates endothelial nitric oxide synthase and augments angiogenesis in a mouse model of hindlimb ischemia. Circ J 70:463–470 35. Inoue S, Kabaya M (1989) Biological activities caused by farinfrared radiation. Int J Biometeorol 33:145–150