The Effects Of Radiation-Induced Bystander Responses On Skin Wound Healing And The Underlying Mechanisms

Objective:As the biggest organ of human body,which provides physical and immunological barrier against the surrounding environment,the skin is vulnerable to ionizing radiation-induced injury.The symptoms of acute radiation skin injury include erythema,epilation,dry desquamation and hyperpigmentation,which are resulted from immediate radiation-induced damage to basal keratinocytes,hair follicle stem cells,melanocytes,etc.In addition to dose,dose rate and skin thickness,radiation type is another important factor that affects the severity of radiation skin injury.In another word,the same absorbed dose of different types of radiation causes different level of skin damage.Due to its short range,alpha particles emitted non-homogeneously from alpha particle sources on the skin surface will irradiate only a proportion of keratinocytes in the out layers of the skin at low doses.However,because of the occurrence of radiation-induced bystander effects(RIBEs),skin fibroblasts in the unexposed dermis,which are one kind of key cells in wound healing process,could be damaged via bystander signaling thus affecting efficiency of skin repair.Therefore,we investigated whether ?-particles at relatively low dose would cause biological effects on the skin and its molecular mechanism.Methods:The aim of the first part of this study was to investigate whether exposure to low dose of alpha particles would affect the morphology and function of the skin e.g.wound healing capability.The wounds of BALB/c mice were either created in directly irradiated area or covered with irradiated skin,the healing process was monitored.The changes in skin morphology and the thickness of the epidermis were evaluated using HE staining of the skin paraffin sections,the size of wound was measured by using vernier calipers.In the second part of this study we set a cell model with HaCaT cells as irradiatedsignaling cells and WS1 cells as unirradiated bystander cells,and they shared the medium by co-culture.We used the WST-1 kit to evaluate the proliferation of cells,and the scratch assay to measure the migration of bystander cells.To explore the molecular mechanisms of the change of migration of WS1 cells,we mainly focused on three aspects,including oxidative stress,which was evaluated via the detection of the intracellular ROS levels of WS1 cells,the roles of miRNAs,which was measured using RT-PCR,western blot,luciferase report gene experiment,gene knockdown by virus infection,etc.and the transportation of bystander factors from HaCaT cells to WS1 cells through exosomes,which were studied using electron microscopy,particle size measurement and the fluorescent probe.In the third part of this study,the exosomes secreted by irradiated HaCaT cells and the recombinant protein MMP2 were subcutaneously injected around the wound of mice,respectively.Then the wound healing process was monitored to verify whether the exosomes and MMP2 affected the animal wound healing.In the fourth part of this research,we used WS1 fibroblasts and HaCaT keratinocytes to construct a multicellular experimental 3-D model,simply to simulate the structure of normal human skin tissue.We also investigated the effects of fractionated low dose alpha irradiation on mono-layer HaCaT cells and three-dimensional skin tissue by performing HE staining of paraffin sections,E-cadherin and Ki67 immunohistochemistry.Results:1.Balb/c mice were exposed to 0.56 Gy of ?-particles in the lower back.Symptoms such as erythema and epilation were not observed in irradiated skin area within 10 days post irradiation.However,when a wound(1 cm×1.5 cm)was created in the irradiated area 30 min after ?-irradiation,we found that the healing of the wounds in irradiated skins was delayed when compared with the wounds in unirradiated skins.In addition to that,after the wounds had healed,we found that the epidermis growing back in irradiated areas were markedly thicker than those in unirradiated areas.Moreover,the wound covered with ?-irradiated skin healed more slowly than the wound covered with sham-irradiated skin.In addition,the epidermis growing back in the wound covered with ?-irradiated skin was thicker compared with that in the wound covered with sham-irradiated skin,although the difference was not statistically significant.All these data indicated that the wound healing may be affected by low dose of ?-particles,andbystander factors from the irradiated skin may contribute to wound healing process.2.At cellular level,first we found that after co-culture with HaCaT keratinocytes irradiated with ?-particles,unirradiated bystander WS1 fibroblasts did not show any obvious changes in proliferation,while co-culturing with X-irradiated HaCaT cells even accelerated the proliferation in bystander WS1 cells.However,compared with the corresponding controls,the wound closure of unirradiated WS1 cells was significantly slower after co-culture with HaCaT cells irradiated with either ?-particles or X-rays.Since the proliferation of bystander WS1 cells was not inhibited after co-culture with irradiated HaCaT cells,these wound scratch assay data suggested that irradiated keratinocytes did slow fibroblast migration via bystander signaling in vitro.In addition,we found that after co-culture with HaCaT keratinocytes irradiated with ?-particles,unirradiated bystander HaCaT cells did show an acceleration both in the proliferation and migration.We observed an increase in the levels of miR-27 a in irradiated HaCaT cells and bystander WS1 cells after co-culture with HaCaT cells irradiated with both ?-particles and X-rays,which showed slowed migration.Additionally,we observed an increase in intracellular ROS levels in bystander WS1 cells after co-culture with HaCaT cells irradiated with either ?-particles or X-rays.However,WS1 cells overexpressing SOD2 did not display slowed migration after co-culture with irradiated HaCaT cells.Moreover,the intracellular ROS levels in WS1 cells transfected with miR-27 a mimics were significantly elevated compared with cells transfected with negative control(NC).When WS1 cells overexpressing SOD2 were transfected with miR-27 a mimics,no slowed migration was observed,which was in contrast to WS1 cells transfected with NC.On the other hand,co-culturing WS1 cells with irradiated HaCaT cells transfected with miR-27 a inhibitors did not cause an increase in intracellular ROS levels and a reduction of cell migration rate in WS1 cells.Thus we hypothesized that mi R-27 a might be secreted by irradiated HaCaT cells as a RIBE signal.MiR-27 a levels in exosomes from irradiated HaCaT cells collected 3 and 6 h post radiation increased obviously compared with the relevant exosomes from unirradiated cells.However,no increase in miR-27 a levels was observed when exosomes were extracted 12 h post radiation.All these data suggested that irradiated HaCaT cells secreted exosome-encapsulated miR-27 a.More importantly,when WS1 cells were cultured with exosomes collected from irradiated HaCaT cells 3 or 6 h post radiation for 1 h,the intracellular ROS levelsof WS1 cells were significantly elevated compared with WS1 cells cultured with exosomes from unirradiated HaCaT cells.But the increase was not observed when WS1 cells were cultured with exosomes extracted from irradiated HaCaT cells 12 h post radiation.And culturing WS1 cells with exosomes from irradiated HaCaT cells 3 and 6but not 12 h post radiation also induced significant reduction in WS1 migration rate.In addition,no increase in miR-27 a levels was detected in the exosomes isolated from irradiated HaCaT cells pre-transfected with miR-27 a inhibitors.Most importantly,the elevation of intracelluar ROS levels of the recipient WS1 cells and the slowed cell migration were significantly inhibited after uptake of these exosomes.Compared with WS1 cells cultured with exosomes from unirradiated HaCaT cells,culturing WS1 cells with exosomes from irradiated HaCaT cells 3 h post radiation for 1 and 12 h induced an increase in miR-27 a expression levels of WS1 cells.On the contrary,only slight increase was observed in the recipient WS1 cells after 1 h of culture with the exosomes from irradiated HaCaT cells pre-transfected with miR-27 a inhibitors,but no increase was detected after 12 h of culture.Therefore,it was very likely that exosomes from irradiated HaCaT cells induced bystander effects in unexposed WS1 cells via up-regulating their miR-27 a expression levels,and ROS played an important role in bystander effects such as slowed migration in WS1 cells after co-culture with irradiated HaCaT cells.The fluorescent reporter assay was performed to verify that MMP2 is a target of miR-27 a.Compared with the negative control,transfection of miR-27 a mimic significantly reduced the luciferase activity of the wild type MMP2 5'-UTR,but had no effect on the luciferase activity of mutated MMP2 5'-UTR.Furthermore,MMP2 expression levels of unexposed WS1 cells were found to reduce obviously after co-culture with irradiated HaCaT cells for 12 h.And this reduction was also abolished in bystander WS1 cells when they were over-expressed with SOD2 and when they were co-cultured with HaCaT cells transfected with miR-27 a inhibitors prior to irradiation.In addition,the exosomes from X-irradiated HaCaT cells 3 or 6 h but not 12 h post radiation induced a decrease in MMP2 expression in the recipient WS1 cells.We also used shRNA to knock down the MMP2 of WS1 cells.Not unexpectedly,miR-27a-containing exosomes from irradiated HaCaT cells failed to induce a significant reduction in the cell migration rate of the recipient WS1 cells deficient in MMP2.All these data indicated that miR-27 a directly targeted MMP2,leading to the reduction of MMP2 expression and the delayed cell migration.3.Injection of exosomes from irradiated HaCaT cells 3 h post radiation delayed wound closure of mice compared with injection of exosomes from unirradiated HaCaT cells.Moreover,the epidermis was found to be thicker 10 days after injection with the exosomes from irradiated keratinocytes compared with the exosomes from unirradiated keratinocytes.All of these results indicated that the exosomes derived from irradiated keratinocytes could inhibit wound healing in vivo.In addition,when subcutaneously injecting 1 Gy-exosomes and human recombinant MMP2 protein at the wound on one side of the back in mice,and injecting 1 Gy-exosomes in PBS on the other side,no statistical difference in the wound healing process was observed between both sides.4.After fractionated alpha particle irradiation,E-cadherin expression were significantly decreased in both two-dimensional monolayer HaCaT cells and HaCaT cells in the three dimensional skin constructs.In addition,with the increase of the irradiation dose,the number of WS1 fibroblasts in three dimensional skin and the expression of Ki67 obviously increased,suggesting that its proliferation capability was markedly enhanced.Conclusions:1.Low dose ?-irradiation did not cause perceptible skin damage symptoms but delayed wound healing,and radiation-induced bystander effects contributed to the delayed skin wound healing.2.The miR-27 a level of irradiated HaCaT cells had a significant increase,and then the high level of miR-27a-containing exosomes secreted by irradiated HaCaT cells were taken in by WS1 cells through the culture medium.Next,the miR-27 a and ROS level of WS1 cells were increased,while the MMP2 expression of WS1 cells was decreased.Finally,the migration of WS1 cells slowed down.In addition,there was a mutual regulatory effect between miR-27 a and ROS level,and the redox status of WS1 cells had important effects on the occurrence of radiation-induced bystander effect.3.Exosomes secreted by irradiated HaCaT cells inhibited wound healing in vivo,and the role of MMP2 in the phenomenon was not clear so far.4.Fractionated low dose of alpha irradiation decreased E-cadherin expression in HaCaT cells in both two-dimensional monolayer and three-dimensional construct,suggesting a possibility of occurrence of EMT;The proliferation enhancement of WS1 cells in three-dimensional skin tissue might be induced by continuous low dose of alpha particle irradiation.