Deficiency of salt-inducible kinase 2 (SIK2) promotes immune injury by inhibiting the maturation of lymphocytes.

Salt-inducible kinase 2 (SIK2) belongs to the serine/threonine protein kinases of the AMPK/SNF1 family, which has important roles in cell cycle, tumor, melanogenesis, neuronal damage repair and apoptosis. Recent studies showed that SIK2 regulates the macrophage polarization to make a balance between inflammation and macrophage. Macrophage is critical to initiate immune regulation, however, whether SIK2 can be involved in immune regulation is not still well understood. Here, we revealed that the protein of SIK2 was highly expressed in thymus, spleen, lung, and brain. And SIK2 protein content increased in RAW264.7 and AHH1 cells with a time and dose-dependent after-ionizing radiation (IR). Inhibition of SIK2 could promote AHH1 cells apoptosis Moreover, we used the Cre-LoxP system to construct the SIK2+/- mice, and the research on function suggested that the deficiency of SIK2 could promote the sensitivity of IR. The deficiency of SIK2 promoted the immune injury via inhibiting the maturation of T cells and B cells. Furthermore, the TCRß rearrangement was inhibited by the deficiency of SIK2. Collectively, this study demonstrated that SIK2 provides an essential function of regulating immune injury, which will provide new ideas for the treatment of immune injury-related diseases.

Discovery of novel exceptionally potent and orally active c-MET PROTACs for the treatment of tumors with MET alterations.

Various c-mesenchymal-to-epithelial transition (c-MET) inhibitors are effective in the treatment of non-small cell lung cancer; however, the inevitable drug resistance remains a challenge, limiting their clinical efficacy. Therefore, novel strategies targeting c-MET are urgently required. Herein, through rational structure optimization, we obtained novel exceptionally potent and orally active c-MET proteolysis targeting chimeras (PROTACs) namely D10 and D15 based on thalidomide and tepotinib. D10 and D15 inhibited cell growth with low nanomolar IC50 values and achieved picomolar DC50 values and >99% of maximum degradation (Dmax) in EBC-1 and Hs746T cells. Mechanistically, D10 and D15 dramatically induced cell apoptosis, G1 cell cycle arrest and inhibited cell migration and invasion. Notably, intraperitoneal administration of D10 and D15 significantly inhibited tumor growth in the EBC-1 xenograft model and oral administration of D15 induced approximately complete tumor suppression in the Hs746T xenograft model with well-tolerated dose-schedules. Furthermore, D10 and D15 exerted significant anti-tumor effect in cells with c-METY1230H and c-METD1228N mutations, which are resistant to tepotinib in clinic. These findings demonstrated that D10 and D15 could serve as candidates for the treatment of tumors with MET alterations.

Tolerogenic dendritic cells in radiation-induced lung injury.

Radiation-induced lung injury is a common complication associated with radiotherapy. It is characterized by early-stage radiation pneumonia and subsequent radiation pulmonary fibrosis. However, there is currently a lack of effective therapeutic strategies for radiation-induced lung injury. Recent studies have shown that tolerogenic dendritic cells interact with regulatory T cells and/or regulatory B cells to stimulate the production of immunosuppressive molecules, control inflammation, and prevent overimmunity. This highlights a potential new therapeutic activity of tolerogenic dendritic cells in managing radiation-induced lung injury. In this review, we aim to provide a comprehensive overview of tolerogenic dendritic cells in the context of radiation-induced lung injury, which will be valuable for researchers in this field.

LncRNA CCTT-mediated RNA-DNA and RNA-protein interactions facilitate the recruitment of CENP-C to centromeric DNA during kinetochore assembly.

Kinetochore assembly on centromeres is central for chromosome segregation, and defects in this process cause mitotic errors and aneuploidy. Besides the well-established protein network, emerging evidence suggests the involvement of regulatory RNA in kinetochore assembly; however, it has remained elusive about the identity of such RNA, let alone its mechanism of action in this critical process. Here, we report CCTT, a previously uncharacterized long non-coding RNA (lncRNA) transcribed from the arm of human chromosome 17, which plays a vital role in kinetochore assembly. We show that CCTT highly localizes to all centromeres via the formation of RNA-DNA triplex and specifically interacts with CENP-C to help engage this blueprint protein in centromeres, and consequently, CCTT loss triggers extensive mitotic errors and aneuploidy. These findings uncover a non-centromere-derived lncRNA that recruits CENP-C to centromeres and shed critical lights on the function of centromeric DNA sequences as anchor points for kinetochore assembly.

Inhibition of cannabinoid receptor type 1 sensitizes triple-negative breast cancer cells to ferroptosis via regulating fatty acid metabolism.

Triple-negative breast cancer (TNBC) is a heterogeneous subtype of breast cancer that displays highly aggressive with poor prognosis. Owing to the limited targets and drugs for TNBC clinical therapy, it is necessary to investigate the factors regulating cancer progression and develop novel therapies for cancer treatment. Ferroptosis, a nonapoptotic form of programmed cell death characterized by accumulation of iron-dependent peroxidation of phospholipids, is regulated by cellular metabolism, redox homeostasis, and various cancer-related signaling pathways. Recently, considerable progress has been made in demonstrating the critical role of lipid metabolism in regulating ferroptosis, indicating potential combinational therapeutic strategies for cancer treatment. In this study, by drug combination screen of lipid metabolism compounds with ferroptosis inducers in decreasing TNBC cell viability, we found potent synergy of the CB1 antagonist rimonabant with erastin/(1 S, 3 R)-RSL3 (RSL3) in inhibiting TNBC cell growth both in vitro and in vivo via promoting the levels of lipid peroxides, malondialdehyde (MDA), 4-hydroxynonenal (4-HNE) and cytosolic reactive oxygen species (ROS) production, enhancing intracellular glutathione (GSH) depletion and inducing G1 cell cycle arrest. We identified that inhibition of CB1 promoted the effect of erastin/RSL3 on inducing ferroptosis and enhanced their inhibitory effect on tumor growth. Using RNA-Seq, fatty acid analyses and functional assays, we found that CB1 regulated stearoyl-CoA desaturase 1 (SCD1)- and fatty acyl desaturase 2 (FADS2)-dependent fatty acid metabolism via phosphatidylinositol 3 kinase (PI3K)-AKT and mitogen-activated protein kinase (MAPK) signaling pathways to modulate ferroptosis sensitivity in TNBC cells. These data demonstrate that dual targeting of CB1 and ferroptosis could be a promising therapeutic strategy for TNBC.

Inhibition of lncRNA RET enhances radio-sensitivity of tumor cells via miR-3179/Slug/PTEN axis.

Radioresistance is one of the key obstacles that may lead to the failure of cancer treatment. The underlying mechanisms of radioresistance remain largely unknown; however, increasing evidence has shown that long noncoding RNAs (lncRNAs) are involved in radiotherapy resistance of several cancers. In the present study, we demonstrated that radiation-elevated transcript (RET), a newly identified lnRNA, was highly expressed in cancer cells. Knockdown of RET significantly inhibited the proliferation and colony formation of cancer cells and markedly inhibited apoptosis. Furthermore, downregulation of RET in cancer cells significantly inhibited cell growth, decreased colony survival fractions, and promoted apoptosis in response to radiation treatment, indicating a role in radiation resistance. Moreover, RET knockdown significantly increased the expression of γ-H2AX, an indicator of DNA double strand damage, and reversed radiation-induced EMT, both of which contributed to its radiation resistance. In addition, a negative correlation was found between the expression of RET and PTEN. Rescue assays confirmed RET knockdown enhanced radiosensitivity of cancer cells by upregulating the expression of PTEN. Mechanistically, RET positively regulated Slug, a repressor of PTEN transcription, by acting as a molecular sponge of miR-3179. Our present study showed that RET conferred radioresistance by regulating miR-3179/Slug/PTEN axis, indicating that RET may be a potential target for the clinical application in cancer patients with radioresistance.

Irradiation Activates MZF1 to Inhibit miR-541-5p Expression and Promote Epithelial-Mesenchymal Transition (EMT) in Radiation-Induced Pulmonary Fibrosis (RIPF) by Upregulating Slug.

Understanding miRNAs regulatory roles in epithelial-mesenchymal transition (EMT) would help establish new avenues for further uncovering the mechanisms underlying radiation-induced pulmonary fibrosis (RIPF) and identifying preventative and therapeutic targets. Here, we demonstrated that miR-541-5p repression by Myeloid Zinc Finger 1 (MZF1) promotes radiation-induced EMT and RIPF. Irradiation could decrease miR-541-5p expression in vitro and in vivo and inversely correlated to RIPF development. Ectopic miR-541-5p expression suppressed radiation-induced-EMT in vitro and in vivo. Knockdown of Slug, the functional target of miR-541-5p, inhibited EMT induction by irradiation. The upregulation of transcription factor MZF1 upon irradiation inhibited the expression of endogenous miR-541-5p and its primary precursor (pri-miR-541-5p), which regulated the effect of the Slug on the EMT process. Our finding showed that ectopic miR-541-5p expression mitigated RIPF in mice by targeting Slug. Thus, irradiation activates MZF1 to downregulate miR-541-5p in alveolar epithelial cells, promoting EMT and contributing to RIPF by targeting Slug. Our observation provides further understanding of the development of RIPF and determines potential preventative and therapeutic targets.

A Biomarker Panel of Radiation-Upregulated miRNA as Signature for Ionizing Radiation Exposure.

Ionizing radiation causes serious injury to the human body and has long-time impacts on health. It is important to find optimal biomarkers for the early quick screening of exposed individuals. A series of miRNAs signatures have been developed as the new biomarkers for diagnosis, survival, and prognostic prediction of cancers. Here, we have identified the ionizing radiation-inducible miRNAs profile through microarray analysis. The biological functions were predicted for the top six upregulated miRNAs by 4 Gy γ-rays: miR-1246, miR-1307-3p, miR-3197, miR-4267, miR-5096 and miR-7641. The miRNA-gene network and target gene-pathway network analyses revealed that DNAH3 is the target gene associated with all the six miRNAs. GOLGB1 is related to 4 miRNAs and other 26 genes targeted by 3 miRNAs. The upregulation of fifteen miRNAs were further verified at 4 h and 24 h after 0 to 10 Gy irradiation in the human lymphoblastoid AHH-1 cells, and some demonstrated a dose-dependent increased. Six miRNAs, including miR-145, miR-663, miR-1273g-3p, miR-6090, miR-6727-5p and miR-7641, were validated to be dose-dependently upregulated at 4 h or 24 h post-irradiation in both AHH-1 and human peripheral blood lymphocytes irradiated ex vivo. This six-miRNA signature displays the superiority as a radiation biomarker for the translational application of screening and assessment of radiation exposed individuals.

Effects of low dose radiation on immune cells subsets and cytokines in mice.

Whole-body exposure to low-dose radiation due to diagnostic imaging procedures, occupational hazards and radiation accidents is a source of concern. In this study, we analyzed the effects of single and long-term low-dose irradiation on the immune system. Male Balb/c mice received a single whole-body dose of irradiation (0.01, 0.05, 0.2, 0.5 or 1 Gy). For long-term irradiation, mice were irradiated 10 times (total dose of 0.2, 0.5 or 1 Gy) over a period of 6 weeks. Two days after single or long-term irradiation, the numbers of splenic macrophages, natural killer cells and dendritic cells were reduced, and the spleen organ coefficient was decreased. At 2 Days after long-term low-dose irradiation, the number of white blood cells in the peripheral blood of the mice decreased. Between 7 and 14 Days after long-term low-dose irradiation, the number of immune cells in the thymus and spleen began to increase and then stabilized. Th1/Th2 cytokines and reactive oxygen species-related proteins first decreased and then increased to a plateau. Our results show a significant difference in the effects of single and long-term low-dose irradiation on the immune system.

MiRNA-21 functions in ionizing radiation-induced epithelium-to-mesenchymal transition (EMT) by downregulating PTEN.

Radiation-induced pulmonary fibrosis (RIPF) results from thoracic radiotherapy and severely limits the use of radiotherapy. Recent studies suggest that epithelium-to-mesenchymal transition (EMT) contributes to pulmonary fibrosis. Although miRNA dysregulation participates in a variety of pathophysiologic processes, their roles in fibrotic lung diseases and EMT are unclear. In this study, we aimed to identify key miRNAs involved in this process using a mouse model of RIPF previously established by irradiation with a single dose (20 Gy) of 60Co γ-rays. At 2-weeks post-irradiation, a set of significantly upregulated miRNAs was identified in lung tissue by miRNA array analysis. This included miR-21, which has been reported to contribute to the pulmonary fibrotic response induced by stereotactic body radiotherapy. Here, we showed that miR-21 expression increased in parallel with EMT progression in the lungs of irradiated mice. Ectopic miR-21 expression promoted EMT progression in lung epithelial cells. Furthermore, downregulation of miR-21 expression by transfection of its inhibitor inhibited ionizing radiation (IR)-induced EMT. Knockdown of PTEN, which is the functional target of miR-21, reversed the attenuation of IR-induced EMT mediated by miR-21 downregulation. Radiation treatment decreased PTEN expression and increased Akt phosphorylation; these effects were abolished by the miR-21 inhibitor. MiR-21 overexpression in lung epithelial cell also downregulated PTEN expression and upregulated Akt phosphorylation. In conclusion, we have demonstrated that miR-21 functions as a key regulator of IR-induced EMT in lung epithelial cells via the PTEN/Akt pathway. Targeting miR-21 is implicated as a novel therapeutic strategy for the prevention of RIPF.

Lung Function Decline after 24 Weeks of Moxa Smoke Exposure in Rats.

OBJECTIVE: Moxibustion is a complementary therapy that has been used for thousands of years. Burning moxa produces smoke and inhalable particulates. Recent research has indicated that smoke inhalation is associated with negative lung effects. This study aimed to evaluate the lung function of rats after moxa smoke exposure at different concentrations. METHODS: Using a randomised block experiment design, 28 male Wistar rats were randomly divided into three moxa smoke groups (opacity) (n=7): low concentration (27.45 mg/m3), medium concentration (168.76 mg/m3), and high concentration (384.67 mg/m3) with a control group. Rats in the moxa smoke groups were exposed in an automatic dynamic exposure device separately with different concentrations for 20 min/d, 6d/week, for 24 weeks. Rats in the control group were exposed in the same space without moxa smoke. Lung function was evaluated by the AniRes 2005 animal pulmonary function analysing system. Statistical Product and Service Solutions 18.0 software was used for data analysis. RESULTS: In the study, no deaths were found in any group. There was no difference of forced expiratory volume in one second/forced vital capacity percentage (FEV1/FVC%), inspiratory resistance (Ri), and expiratory resistance (Re) among each group after 24 weeks of moxa smoke exposure (P>0.05). Compared with the control group (0.33 ml/cmH20), dynamic compliance (Cdyn) was reduced in the medium (0.29 ml/cmH20) and high (0.25 ml/cmH20) concentration groups (P<0.05); however, Cdyn in the low concentration group (0.29 ml/cmH20) was not significantly affected. CONCLUSION: Moxa smoke exposure at low concentrations did not affect the rat's lung function. Moxa smoke of medium and high concentrations destroyed the lung function represented by decreased Cdyn. However, moxa smoke of low concentrations (27.45 mg/m3) is much higher than the concentration in a regular moxibustion clinic (3.54 mg/m3). Moxa smoke at higher concentrations might destroy the lung function. The safety evaluation of moxa smoke requires further research.

Autophagy induced by low concentrations of crotonaldehyde promotes apoptosis and inhibits necrosis in human bronchial epithelial cells.

Crotonaldehyde is a common environmental contaminant. Autophagy, apoptosis, and necrosis, were all respectively reported to be induced by crotonaldehyde. However, the relationships between programmed cell deaths, especially between autophagy and apoptosis, have not been elucidated. In the present study, alterations of autophagy, apoptosis and necrosis were investigated in human bronchial epithelial cells (BEAS-2B) exposed to crotonaldehyde, and effects of autophagy on apoptosis and necrosis were detected. We found that a high concentration (160 µmol/L, µM) of crotonaldehyde did not induce apoptosis, while a low concentration (80 µM) of crotonaldehyde induced autophagy, apoptosis and necrosis. In 80 µM crotonaldehyde-exposed BEAS-2B cells, autophagy and apoptosis exhibited a trend of increasing prior to decreasing with the increase of time, while the time point inducing the highest level of autophagy was 2 h, and that of apoptosis was 4 h. With the pretreatment of bafilomycin A1, the apoptosis was inhibited and the necrosis was enhanced significantly in cells exposed to 80 µM crotonaldehyde. Autophagy mediated the induction of apoptosis via the intrinsic apoptotic pathway. The results indicate that autophagy mediates the initiation of apoptosis and plays a role in protecting from necrosis in low concentrations of crotonaldehyde-exposed BEAS-2B cells.

Acute exposure to crotonaldehyde induces dysfunction of immune system in male Wistar rats.

Crotonaldehyde is a ubiquitous air pollutant in the environment. It is reported to be harmful to the biosystems in vivo and in vitro. The exposure to crotonaldehyde irritates the mucous membranes and induces edema, hyperemia, cell necrosis, inflammation, and acute respiratory distress syndrome in the lungs. However, the effects of crotonaldehyde on the immune system have not been reported. In the present study, 6-8 weeks old male Wistar rats were exposed to crotonaldehyde by intratracheal instillation at doses of 4, 8, and 16 µL/kg body weight (b.w.). The general damage in the animals was investigated; the cell counting and the biochemical analysis in the peripheral blood were tested. Furthermore, we investigated the functions of alveolar macrophages (AMs), the alterations of the T-lymphocyte subsets, and the cell composition in the bronchoalveolar lavage fluid (BALF). We found that the activities of the animals were changed after exposure to crotonaldehyde, the cellular ratios and the biochemical components in the peripheral blood were altered, the ratio of mononuclear phagocytes decreased, and the ratios of lymphocytes and granulocytes elevated significantly in BALF. Meanwhile, crotonaldehyde altered the ratio of the T-lymphocyte subsets, and the phagocytic rates and indices of AMs increased obviously. In conclusion, crotonaldehyde induces dysfunction of immune system in male Wistar rats.

Repeated exposure to moxa-burning smoke: its acute and chronic toxicities in rats.

OBJECTIVE: To assess toxicities of the air in Chinese medicine clinics polluted by moxa-burning smoke due to moxibustion-derived burning products (MBP). METHODS: Both acute and chronic toxicity studies were conducted. For the acute toxicity study, five groups of Wistar rats (n = 16/group, male: female = 1∶1) were exposed to five different concentrations (95%, 90%, 85%, 80% and 75%, respectively) of MBP for 2 h. For the chronic toxicity study, another three groups of male rats (n = 21/group) were exposed to MBP in three concentrations (10%, 40% and 70%, respectively) and one control group exposed to clean air 20 min/d for 144 d. Routine examinations were performed and analyzed by analysis of variance and dose-response relationship. RESULTS: In the acute toxicity study, the number of dead rats in the 95%, 90%, 85%, 80% and 75% groups were 16, 13, 7, 6 and 3, respectively, with LD50 of 86.274% after or during the 2 h exposure. In the chronic toxicity study, MBP exposure induced a decline in activity of the rats. Rats in the 10% group showed no signs of toxicity, while those in the 40% MBP group showed toxicity effects on the body weights (P < 0.05) and lung. Rats in the 70% MBP group also presented with reversible damage in the blood coagulation system (P < 0.05). CONCLUSION: Exposure to 10% MBP, which is equivalent to 27.45 mg/m3, was under the critical threshold for male rats' safety. Exposure to MBP above that limit induced lung damage. MBP in clinics need to be reduced to a safe level with enhanced ventilation.

Immunomodulatory effects of cigarette smoke condensate in mouse macrophage cell line.

Increasing evidence has demonstrated that the secretion of cytokines may be associated with cigarette smoke-induced immunomodulatory effects, but a comprehensive analysis of the cytokine profile for cigarette smoke condensate (CSC) exposure is lacking. The aims of this study were to (1) examine the release of 20 cytokines induced by CSC from 12 brands of cigarettes in macrophages cells (Ana-1) and (2) to investigate the general characteristics of the immunomodulatory effects of CSC. Luminex technology was used to simultaneously determine the levels of 20 cytokines (interleukin (IL)-1α, IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-17, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), keratinocyte-derived Chemokine (KC), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1α (MIP-1α), induced protein 10 (IP-10), tumor necrosis factor α (TNF-α), vascular endothelial growth factor (VEGF), monkine inducible by γ interferon (MIG), and fibroblast growth factor (FGF)-basic) in the supernatants from Ana-1 cells treated with the CSC. The results showed that the release of eight cytokines was altered (IL-5, IL-6, IL-12, TNF-α, VEGF, IP-10, MCP-1, and MIP-1α) compared with the control. These cytokines fall into two major subtypes: proinflammatory cytokines, including IL-5, IL-6, IL-12, TNF-α, and VEGF, and chemokines, including IP-10, MCP-1, and MIP-1α. Compared with control, the remaining 12 cytokines were not significantly affected by CSC from the 12 brands of cigarettes. As a general characteristic, CSC exerts potently suppressive immunomodulatory effects on cytokine production of Ana-1 cells. Proinflammatory cytokines and chemokines may account for or contribute to the immunosuppressive properties of CSC.

Crotonaldehyde induces autophagy-mediated cytotoxicity in human bronchial epithelial cells via PI3K, AMPK and MAPK pathways.

Crotonaldehyde is an ubiquitous hazardous pollutant in the environment which can be produced naturally, artificially and endogenously. Acute exposure of crotonaldehyde was reported to induce severe lung injury in humans and experimental animals. However, the exact toxicity mechanisms of crotonaldehyde in organisms have not been fully explored. In the present study, we explored the role autophagy played in the cytotoxicity induced by crotonaldehyde in human bronchial epithelial cells (BEAS-2B), and the pathways that mediated autophagy, including the phosphatidylinositol 3-kinase (PI3K) pathway, the AMP-activated protein kinase (AMPK) pathway and the mitogen-activated protein kinase (MAPK) pathways, were examined and validated. We found that crotonaldehyde induced cytotoxicity and autophagy simultaneously in BEAS-2B cells, and blockage of autophagic flux significantly elevated the viability of BEAS-2B exposed to high concentrations of crotonaldehyde. Crotonaldehyde down-regulated the activity of PI3K pathway, and elevated the activities of AMPK and MAPK pathways. Pretreatment of specific agonist or antagonist of these pathways could inhibit autophagy and partly improve the viability. These results suggested that acute exposure of crotonaldehyde induced cell death mediated by autophagy, which might be helpful to elucidate the toxicity mechanisms of crotonaldehyde and contribute to environmental and human health risk assessment.

[Effects of moxa smoke with different concentrations on expression of SOD and MDA in lung and serum of rats].

OBJECTIVE: To observe the effects of intervention of moxa smoke with different concentrations on superoxide dismutase (SOD) and malondialdehyde (MDA) in serum and lung of male rats, so as to explore the safety concentration of moxa smoke. METHODS: A total of 32 Wistar male rats were randomly divided into a control group, a low-concentration group, a moderate-concentration group and a high-concentration group, 8 rats in each one. All the rats were exposed in the full-automatic toxicant exposure cabinet, and the overshadow of moxa smoke was set at 0%, 10%, 40% and 70%, respectively. Each rat was exposed for 20 min per day. After 26 weeks, the activities of SOD and content of MDA in serum, lung organ and bronchoalveolar lavage fluid were tested. RESULTS: Compared with the control group, the activities of serum SOD in the high-concentration group were reduced (P< 0. 05), but those in the low-concentration group and moderate-concentration group were not significantly different (both P>0. 05). Compared with the control group, the content of serum MDA in the low-concentration group, moderate-concentration group and high-concentration group was increased insignificantly (all P>0. 05). There were no significant differences regarding activities of SOD and content of MDA in lung organ and bronchoalveolar lavage fluid among each moxa smoke group (all P>0. 05). CONCLUSION: There is no obvious toxic reaction in the low-concentration group and moderate-concentration group; in the high-concentration group the antioxidant ability is damaged due to long-term exposure.

Regulatory T Cells Promote ß-Catenin--Mediated Epithelium-to-Mesenchyme Transition During Radiation-Induced Pulmonary Fibrosis.

PURPOSE: Radiation-induced pulmonary fibrosis results from thoracic radiation therapy and severely limits radiation therapy approaches. CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) as well as epithelium-to-mesenchyme transition (EMT) cells are involved in pulmonary fibrosis induced by multiple factors. However, the mechanisms of Tregs and EMT cells in irradiation-induced pulmonary fibrosis remain unclear. In the present study, we investigated the influence of Tregs on EMT in radiation-induced pulmonary fibrosis. METHODS AND MATERIALS: Mice thoraxes were irradiated (20 Gy), and Tregs were depleted by intraperitoneal injection of a monoclonal anti-CD25 antibody 2 hours after irradiation and every 7 days thereafter. Mice were treated on days 3, 7, and 14 and 1, 3, and 6 months post irradiation. The effectiveness of Treg depletion was assayed via flow cytometry. EMT and ß-catenin in lung tissues were detected by immunohistochemistry. Tregs isolated from murine spleens were cultured with mouse lung epithelial (MLE) 12 cells, and short interfering RNA (siRNA) knockdown of ß-catenin in MLE 12 cells was used to explore the effects of Tregs on EMT and ß-catenin via flow cytometry and Western blotting. RESULTS: Anti-CD25 antibody treatment depleted Tregs efficiently, attenuated the process of radiation-induced pulmonary fibrosis, hindered EMT, and reduced ß-catenin accumulation in lung epithelial cells in vivo. The coculture of Tregs with irradiated MLE 12 cells showed that Tregs could promote EMT in MLE 12 cells and that the effect of Tregs on EMT was partially abrogated by ß-catenin knockdown in vitro. CONCLUSIONS: Tregs can promote EMT in accelerating radiation-induced pulmonary fibrosis. This process is partially mediated through ß-catenin. Our study suggests a new mechanism for EMT, promoted by Tregs, that accelerates radiation-induced pulmonary fibrosis.

Treg depletion attenuates irradiation-induced pulmonary fibrosis by reducing fibrocyte accumulation, inducing Th17 response, and shifting IFN-γ, IL-12/IL-4, IL-5 balance.

Irradiation-induced pulmonary fibrosis results from thoracic radiotherapy and severely limits radiotherapy approaches. CD4(+) CD25(+) FoxP3(+) regulatory T cells (Tregs) are involved in experimentally induced murine lung fibrosis. However, the precise contribution of Tregs to irradiation-induced pulmonary fibrosis still remains unclear. We have previously established the mouse model of irradiation-induced pulmonary fibrosis and observed an increased frequency of Tregs during the process. This study aimed to investigate the effects of Treg depletion on irradiation-induced pulmonary fibrosis and on fibrocyte, Th17 cell response and production of multiple cytokines in mice. Treg-depleted mice were generated by intraperitoneal injection with anti-CD25 mAb 2h after 20 Gy (60)CO γ-ray thoracic irradiation and every 7 days thereafter. Pulmonary fibrosis was semi-quantitatively assessed using Masson's trichrome staining. The proportions of Tregs, fibrocyte and Th17 cells were detected by flow cytometry. Th1/Th2 cytokines were assessed by Luminex assays. We found that Treg depletion decelerated the process of irradiation-induced pulmonary fibrosis and hindered fibrocyte recruitment to the lung. In response to Treg depletion, the number of CD4(+) T lymphocytes and Th17 cells increased. Moreover, Th1/Th2 cytokine balance was disturbed into Th1 dominance upon Treg depletion. Our study demonstrates that Tregs are involved in irradiation-induced pulmonary fibrosis by promoting fibrocyte accumulation, attenuating Th17 response and regulating Th1/Th2 cytokine balance in the lung tissues, which suggests that Tregs may be therapeutically manipulated to decelerate the progression of irradiation-induced pulmonary fibrosis.