Hydrogen-rich solution alleviates acute radiation pneumonitis by regulating oxidative stress and macrophages polarization.

This study was aimed to investigate the effect of hydrogen-rich solution (HRS) on acute radiation pneumonitis (ARP) in rats. The ARP model was induced by X-ray irradiation. Histopathological changes were assessed using HE and Masson stains. Inflammatory cytokines were detected by ELISA. Immunohistochemistry and flow cytometry were performed to quantify macrophage (CD68) levels and the M2/M1 ratio. Western blot analysis, RT-qPCR, ELISA and flow cytometry were used to evaluate mitochondrial oxidative stress injury indicators. Immunofluorescence double staining was performed to colocalize CD68/LC3B and p-AMPK-α/CD68. The relative expression of proteins associated with autophagy activation and the adenosine 5'-monophosphate-activated protein kinase/mammalian target of rapamycin/Unc-51-like kinase 1 (AMPK/mTOR/ULK1) signaling pathway were detected by western blotting. ARP decreased body weight, increased the lung coefficient, collagen deposition and macrophage infiltration and promoted M1 polarization in rats. After HRS treatment, pathological damage was alleviated, and M1 polarization was inhibited. Furthermore, HRS treatment reversed the ARP-induced high levels of mitochondrial oxidative stress injury and autophagy inhibition. Importantly, the phosphorylation of AMPK-α was inhibited, the phosphorylation of mTOR and ULK1 was activated in ARP rats and this effect was reversed by HRS treatment. HRS inhibited M1 polarization and alleviated oxidative stress to activate autophagy in ARP rats by regulating the AMPK/mTOR/ULK1 signaling pathway.

Metabolic profiling of murine radiation-induced lung injury with Raman spectroscopy and comparative machine learning.

Radiation-induced lung injury (RILI) is a dose-limiting toxicity for cancer patients receiving thoracic radiotherapy. As such, it is important to characterize metabolic associations with the early and late stages of RILI, namely pneumonitis and pulmonary fibrosis. Recently, Raman spectroscopy has shown utility for the differentiation of pneumonitic and fibrotic tissue states in a mouse model; however, the specific metabolite-disease associations remain relatively unexplored from a Raman perspective. This work harnesses Raman spectroscopy and supervised machine learning to investigate metabolic associations with radiation pneumonitis and pulmonary fibrosis in a mouse model. To this end, Raman spectra were collected from lung tissues of irradiated/non-irradiated C3H/HeJ and C57BL/6J mice and labelled as normal, pneumonitis, or fibrosis, based on histological assessment. Spectra were decomposed into metabolic scores via group and basis restricted non-negative matrix factorization, classified with random forest (GBR-NMF-RF), and metabolites predictive of RILI were identified. To provide comparative context, spectra were decomposed and classified via principal component analysis with random forest (PCA-RF), and full spectra were classified with a convolutional neural network (CNN), as well as logistic regression (LR). Through leave-one-mouse-out cross-validation, we observed that GBR-NMF-RF was comparable to other methods by measure of accuracy and log-loss (p > 0.10 by Mann-Whitney U test), and no methodology was dominant across all classification tasks by measure of area under the receiver operating characteristic curve. Moreover, GBR-NMF-RF results were directly interpretable and identified collagen and specific collagen precursors as top fibrosis predictors, while metabolites with immune and inflammatory functions, such as serine and histidine, were top pneumonitis predictors. Further support for GBR-NMF-RF and the identified metabolite associations with RILI was found as CNN interpretation heatmaps revealed spectral regions consistent with these metabolites.

Inhibition of TGFß1 activation prevents radiation-induced lung fibrosis.

BACKGROUND: Radiotherapy is the main treatment modality for thoracic tumours, but it may induce pulmonary fibrosis. Currently, the pathogenesis of radiation-induced pulmonary fibrosis (RIPF) is unclear, and effective treatments are lacking. Transforming growth factor beta 1 (TGFß1) plays a central role in RIPF. We found that activated TGFß1 had better performance for radiation pneumonitis (RP) risk prediction by detecting activated and total TGFß1 levels in patient serum. αv integrin plays key roles in TGFß1 activation, but the role of αv integrin-mediated TGFß1 activation in RIPF is unclear. Here, we investigated the role of αv integrin-mediated TGFß1 activation in RIPF and the application of the integrin antagonist cilengitide to prevent RIPF. METHODS: ItgavloxP/loxP ;Pdgfrb-Cre mice were generated by conditionally knocking out Itgav in myofibroblasts, and wild-type mice were treated with cilengitide or placebo. All mice received 16 Gy of radiation or underwent a sham radiation procedure. Lung fibrosis was measured by a modified Ashcroft score and microcomputed tomography (CT). An enzyme-linked immunosorbent assay (ELISA) was used to measure the serum TGFß1 concentration, and total Smad2/3 and p-Smad2/3 levels were determined via Western blotting. RESULTS: Conditional Itgav knockout significantly attenuated RIPF (p < .01). Hounsfield units (HUs) in the lungs were reduced in the knockout mice compared with the control mice (p < .001). Conditional Itgav knockout decreased active TGFß1 secretion and inhibited fibroblast p-Smad2/3 expression. Exogenous active TGFß1, but not latent TGFß1, reversed these reductions. Furthermore, cilengitide treatment elicited similar results and prevented RIPF. CONCLUSIONS: The present study revealed that conditional Itgav knockout and cilengitide treatment both significantly attenuated RIPF in mice by inhibiting αv integrin-mediated TGFß1 activation. HIGHLIGHTS: Activated TGFß1 has a superior capacity in predicting radiation pneumonitis (RP) risk and plays a vital role in the development of radiation-induced pulmonary fibrosis (RIPF). Conditional knock out Itgav in myofibroblasts prevented mice from developing RIPF. Cilengitide alleviated the development of RIPF by inhibiting αv integrin-mediated TGFß1 activation and may be used in targeted approaches for preventing RIPF.

Cytokine, chemokine alterations and immune cell infiltration in Radiation-induced lung injury: Implications for prevention and management.

Radiation therapy is one of the primary treatments for thoracic malignancies, with radiation-induced lung injury (RILI) emerging as its most prevalent complication. RILI encompasses early-stage radiation pneumonitis (RP) and the subsequent development of radiation pulmonary fibrosis (RPF). During radiation treatment, not only are tumor cells targeted, but normal tissue cells, including alveolar epithelial cells and vascular endothelial cells, also sustain damage. Within the lungs, ionizing radiation boosts the intracellular levels of reactive oxygen species across various cell types. This elevation precipitates the release of cytokines and chemokines, coupled with the infiltration of inflammatory cells, culminating in the onset of RP. This pulmonary inflammatory response can persist, spanning a duration from several months to years, ultimately progressing to RPF. This review aims to explore the alterations in cytokine and chemokine release and the influx of immune cells post-ionizing radiation exposure in the lungs, offering insights for the prevention and management of RILI.

Autophagy-mediated NKG2D internalization impairs NK cell function and exacerbates radiation pneumonitis.

Introduction: Radiation pneumonitis is a critical complication that constrains the use of radiation therapy for thoracic malignancies, leading to substantial morbidity via respiratory distress and lung function impairment. The role of Natural killer (NK) cells in inflammatory diseases is well-documented; however, their involvement in radiation pneumonitis is not fully understood. Methods: To explore the involvement of NK cells in radiation pneumonitis, we analyzed tissue samples for NK cell presence and function. The study utilized immunofluorescence staining, western blotting, and immunoprecipitation to investigate CXCL10 and ROS levels, autophagy activity, and NKG2D receptor dynamics in NK cells derived from patients and animal models subjected to radiation. Result: In this study, we observed an augmented infiltration of NK cells in tissues affected by radiation pneumonitis, although their function was markedly diminished. In animal models, enhancing NK cell activity appeared to decelerate the disease progression. Concomitant with the disease course, there was a notable upsurge in CXCL10 and ROS levels. CXCL10 was found to facilitate NK cell migration through CXCR3 receptor activation. Furthermore, evidence of excessive autophagy in patient NK cells was linked to ROS accumulation, as indicated by immunofluorescence and Western blot analyses. The association between the NKG2D receptor and its adaptor proteins (AP2 subunits AP2A1 and AP2M1), LC3, and lysosomes was intensified after radiation exposure, as demonstrated by immunoprecipitation. This interaction led to NKG2D receptor endocytosis and subsequent lysosomal degradation. Conclusion: Our findings delineate a mechanism by which radiation-induced lung injury may suppress NK cell function through an autophagy-dependent pathway. The dysregulation observed suggests potential therapeutic targets; hence, modulating autophagy and enhancing NK cell activity could represent novel strategies for mitigating radiation pneumonitis.

Amomum subulatum mitigates experimental thoracic radiation-induced lung injury by regulating antioxidant status and inflammatory responses.

Radiation-induced lung injury (RILI) is one of the most prominent complications of thoracic radiotherapy for which effective therapy is still lacking. This study investigates the nutraceutical potential of the culinary spice Amomum subulatum in mitigating thoracic radiation-induced pneumonitis (RP) and pulmonary fibrosis (PF). Mouse models of RP and PF were established by whole thorax irradiation at a dose of 25 gray. C57BL/6 mice were administered with 250 mg per kg body weight of methanolic extract of A. subulatum dry fruits (MEAS) for four consecutive weeks and observed for changes in lung tissue antioxidant activities, oxidative stress parameters, and expression of antioxidant, inflammation, and fibrosis-related genes by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR analysis, and histology analysis. MEAS administration reduced radiation-induced oxidative stress by enhancing the expression of Nrf2 and its target genes. Irradiation increased gene expression of inflammatory mediators and lung histology further confirmed the characteristics of RP, which were reduced by MEAS treatment. Immunohistochemistry analysis revealed the potential of MEAS in reducing the radiation-induced elevation of cyclooxygenase 2 expression in the lungs. The late sequel of RILI was manifested as PF, characterized by the elevated expression of pro-fibrotic genes and increased collagen content. However, MEAS administration markedly reduced radiation-induced fibrotic changes in the lungs. These effects might be attributed to the synergistic effect of bioactive polyphenols in MEAS with antioxidant, anti-inflammatory, and anti-fibrotic efficacies. Taken together, this study demonstrates the potential of MEAS in mitigating RILI, suggesting the possible nutraceutical application of A. subulatum against radiation toxicities.

Involvement of eNAMPT/TLR4 signaling in murine radiation pneumonitis: protection by eNAMPT neutralization.

Therapeutic strategies to prevent or reduce the severity of radiation pneumonitis are a serious unmet need. We evaluated extracellular nicotinamide phosphoribosyltransferase (eNAMPT), a damage-associated molecular pattern protein (DAMP) and Toll-Like Receptor 4 (TLR4) ligand, as a therapeutic target in murine radiation pneumonitis. Radiation-induced murine and human NAMPT expression was assessed in vitro, in tissues (IHC, biochemistry, imaging), and in plasma. Wild type C57Bl6 mice (WT) and Nampt+/- heterozygous mice were exposed to 20Gy whole thoracic lung irradiation (WTLI) with or without weekly IP injection of IgG1 (control) or an eNAMPT-neutralizing polyclonal (pAb) or monoclonal antibody (mAb). BAL protein/cells and H&E staining were used to generate a WTLI severity score. Differentially-expressed genes (DEGs)/pathways were identified by RNA sequencing and bioinformatic analyses. Radiation exposure increases in vitro NAMPT expression in lung epithelium (NAMPT promoter activity) and NAMPT lung tissue expression in WTLI-exposed mice. Nampt+/- mice and eNAMPT pAb/mAb-treated mice exhibited significant histologic attenuation of WTLI-mediated lung injury with reduced levels of BAL protein and cells, and plasma levels of eNAMPT, IL-6,  and IL-1ß. Genomic and biochemical studies from WTLI-exposed lung tissues highlighted dysregulation of NFkB/cytokine and MAP kinase signaling pathways which were rectified by eNAMPT mAb treatment. The eNAMPT/TLR4 pathway is essentially involved in radiation pathobiology with eNAMPT neutralization an effective therapeutic strategy to reduce the severity of radiation pneumonitis.

Potential role of senescent macrophages in radiation-induced pulmonary fibrosis.

Radiation-induced pulmonary fibrosis (RIPF) is a late toxicity of therapeutic radiation in clinic with poor prognosis and limited therapeutic options. Previous results have shown that senescent cells, such as fibroblast and type II airway epithelial cell, are strongly implicated in pathology of RIPF. However, the role of senescent macrophages in the development RIPF is still unknown. In this study, we report that ionizing radiation (IR) increase cellular senescence with higher expression of senescence-associated ß-galactosidase (SA-ß-Gal) and senescence-specific genes (p16, p21, Bcl-2, and Bcl-xl) in irradiated bone marrow-derived monocytes/macrophages (BMMs). Besides, there's a significant increase in the expression of pro-fibrogenic factors (TGF-ß1 and Arg-1), senescence-associated secretory phenotype (SASP) proinflammatory factors (Il-1α, Il-6, and Tnf-α), SASP chemokines (Ccl2, Cxcl10, and Ccl17), and SASP matrix metalloproteinases (Mmp2, Mmp9 and Mmp12) in BMMs exposed to 10 Gy IR. In addition, the percentages of SA-ß-Gal+ senescent macrophages are significantly increased in the macrophages of murine irradiated lung tissue. Moreover, robustly elevated expression of p16, SASP chemokines (Ccl2, Cxcl10, and Ccl17) and SASP matrix metalloproteinases (Mmp2, Mmp9, and Mmp12) is observed in the macrophages of irradiated lung, which might stimulate a fibrotic phenotype in pulmonary fibroblasts. In summary, irradiation can induce macrophage senescence, and increase the secretion of SASP in senescent macrophages. Our findings provide important evidence that senescent macrophages might be the target for prevention and treatment of RIPF.

Effects of diabetes on the development of radiation pneumonitis.

Radiation pneumonia (RP) is a common adverse reaction to radiation therapy in patients with chest tumors. Recent studies have shown that diabetes mellitus (DM), which can cause systemic multisystem damage, specifically targets lungs, and the incidence of RP in patients with a history of diabetes is higher than that in other patients with tumors who have undergone radiotherapy. DM is an important risk factor for RP in tumor patients undergoing RT, and patients with DM should be treated with caution. This article reviews research on the clinical aspects, as well as the mechanism, of the effects of diabetes on RP and suggests future research needed to reduce RP.

PM014 attenuates radiation-induced pulmonary fibrosis via regulating NF-kB and TGF-b1/NOX4 pathways.

Radiation therapy is the mainstay in the treatment of lung cancer, and lung fibrosis is a radiotherapy-related major side effect that can seriously reduce patient's quality of life. Nevertheless, effective strategies for protecting against radiation therapy-induced fibrosis have not been developed. Hence, we investigated the radioprotective effects and the underlying mechanism of the standardized herbal extract PM014 on radiation-induced lung fibrosis. Ablative radiation dose of 75 Gy was focally delivered to the left lung of mice. We evaluated the effects of PM014 on radiation-induced lung fibrosis in vivo and in an in vitro model. Lung volume and functional changes were evaluated using the micro-CT and flexiVent system. Fibrosis-related molecules were evaluated by immunohistochemistry, western blot, and real-time PCR. A orthotopic lung tumour mouse model was established using LLC1 cells. Irradiated mice treated with PM014 showed a significant improvement in collagen deposition, normal lung volume, and functional lung parameters, and these therapeutic effects were better than those of amifostine. PM104 attenuated radiation-induced increases in NF-κB activity and inhibited radiation-induced p65 translocation, ROS production, DNA damage, and epithelial-mesenchymal transition. PM104 effectively alleviated fibrosis in an irradiated orthotopic mouse lung tumour model while not attenuating the efficacy of the radiation therapy by reduction of the tumour. Standardized herbal extract PM014 may be a potential therapeutic agent that is able to increase the efficacy of radiotherapy by alleviating radiation-induced lung fibrosis.

Regulatory T Cells: An Emerging Player in Radiation-Induced Lung Injury.

Regulatory T cells (Tregs), which have long been recognized as essential regulators of both inflammation and autoimmunity, also impede effective antitumor immune response due to their immunosuppressive properties. Combined radiotherapy and immunotherapeutic interventions focusing on the removal of Tregs have recently garnered interest as a promising strategy to reverse immunosuppression. Meanwhile, Tregs are emerging as a key player in the pathogenesis of radiation-induced lung injury (RILI), a frequent and potentially life-threatening complication of thoracic radiotherapy. Recognition of the critical role of Tregs in RILI raises the important question of whether radiotherapy combined with Treg-targeting immunotherapy offers any beneficial effects in the protection of normal lung tissue. This present review focuses on the contributions of Tregs to RILI, with particular emphasis on the suspected differential role of Tregs in the pneumonitic phase and fibrotic phase of RILI. We also introduce recent progress on the potential mechanisms by which Tregs modulate RILI and the crosstalk among Tregs, other infiltrating T cells, fibrocytes, and resident epithelial cells driving disease pathogenesis. Finally, we discuss whether Tregs also hold promise as a potential target for immunotherapeutic interventions for RILI.

Identification of miRNA signatures associated with radiation-induced late lung injury in mice.

Acute radiation exposure of the thorax can lead to late serious, and even life-threatening, pulmonary and cardiac damage. Sporadic in nature, late complications tend to be difficult to predict, which prompted this investigation into identifying non-invasive, tissue-specific biomarkers for the early detection of late radiation injury. Levels of circulating microRNA (miRNA) were measured in C3H and C57Bl/6 mice after whole thorax irradiation at doses yielding approximately 70% mortality in 120 or 180 days, respectively (LD70/120 or 180). Within the first two weeks after exposure, weight gain slowed compared to sham treated mice along with a temporary drop in white blood cell counts. 52% of C3H (33 of 64) and 72% of C57Bl/6 (46 of 64) irradiated mice died due to late radiation injury. Lung and heart damage, as assessed by computed tomography (CT) and histology at 150 (C3H mice) and 180 (C57Bl/6 mice) days, correlated well with the appearance of a local, miRNA signature in the lung and heart tissue of irradiated animals, consistent with inherent differences in the C3H and C57Bl/6 strains in their propensity for developing radiation-induced pneumonitis or fibrosis, respectively. Radiation-induced changes in the circulating miRNA profile were most prominent within the first 30 days after exposure and included miRNA known to regulate inflammation and fibrosis. Importantly, early changes in plasma miRNA expression predicted survival with reasonable accuracy (88-92%). The miRNA signature that predicted survival in C3H mice, including miR-34a-5p, -100-5p, and -150-5p, were associated with pro-inflammatory NF-κB-mediated signaling pathways, whereas the signature identified in C57Bl/6 mice (miR-34b-3p, -96-5p, and -802-5p) was associated with TGF-ß/SMAD signaling. This study supports the hypothesis that plasma miRNA profiles could be used to identify individuals at high risk of organ-specific late radiation damage, with applications for radiation oncology clinical practice or in the context of a radiological incident.

The Intestinal Microbiota Plays as a Protective Regulator Against Radiation Pneumonitis.

Radiation pneumonitis is a common complication of thoracic irradiation for lung cancer patients. The healthy gut microbiota plays an important role in the local mucosal defense process as well as pulmonary immunomodulation of the host. However, the effect of the intestinal microbiota on radiation pneumonitis is not well understood. Here we studied how the intestinal microbiota affected the host response to radiation pneumonitis. C57BL/6 mice were administered antibiotics to induce disequilibrium in the gut microbiota, and subsequently irradiated. We found that the intestinal microbiota served as a protective mediator against radiation pneumonitis, as indicated by decreased body weight and increased mortality in antibiotic-treated mice. In mice with gut microbiota disequilibrium, more serious pathological lung damage was observed at two and four weeks postirradiation. Fecal microbiota transplantation into irradiated mice led to improvement from radiation-induced inflammation two weeks postirradiation. High-throughput sequencing of murine feces displayed conversion of flora diversity, bacterial composition and community structure in the absence of normal intestinal flora. We filtered the potentially important species among the gut microbiota and considered that the tissue-type plasminogen activator might be involved in the inflammatory process. This study reveals that the gut microbiota functions as a protective regulator against radiation pneumonitis. Additionally, fecal microbiota transplantation was shown to alleviate lung injury in the irradiated model. The protective role of the healthy gut microbiota and the utilization of the gut-lung axis show potential for innovative therapeutic strategies in radiation-induced lung injury.

Low-Dose Radiation Therapy Promotes Radiation Pneumonitis by Activating NLRP3 Inflammasome.

PURPOSE: To determine the role of NLRP3 inflammasome activation in low-dose radiation-induced radiation pneumonitis (RP) and to assess whether inhibition or deletion of the NLRP3 inflammasome is critical for conferring protection against RP. METHODS AND MATERIALS: The human monocytic THP-1 cells were treated with increasing doses of radiation to assess the activation of NLRP3 by Western blot and enzyme-linked immunosorbent assay. Reactive oxygen species (ROS) production was measured by flow cytometry, with or without ROS inhibitor treatment. A mouse thoracic radiation model that received different doses of radiation was used, and the lung tissues of thoracic-irradiated nlrp3-/- and wild-type C57BL/6 mice were examined by hematoxylin and eosin and immunofluorescence staining. The concentrations of cytokines in the bronchoalveolar lavage fluid were measured by enzyme-linked immunosorbent assay and Luminex multiplex assays. Lipopolysaccharide (LPS) was administered intranasally 28 days after thoracic irradiation, and NLRP3 inhibitor, MCC950 was administered intraperitoneally after irradiation at 2 different doses. RESULTS: (1) The NLRP3 inflammasome was activated in 2 Gy irradiated THP-1 cells; NLRP3 and cleaved-caspase-1 levels were not associated with dose escalation of irradiation. (2) Activation of the NLRP3 inflammasome was mediated by ROS, and ROS inhibitor treatment decreased the production of IL-1ß and IL-18 in vitro. (3) NLRP3 was activated in mouse lungs by irradiation at 2 Gy, 4 Gy, and 16 Gy, and NLRP3 activation was continuous for 8 weeks. (4) NLRP3 deletion protects against LPS-mediated monocyte infiltration in the mouse lung. (5) The administration of MCC950 decreased the inflammation score of the mice irradiated with 2 Gy or 16 Gy in vivo. CONCLUSIONS: Our results indicate that the NLRP3 inflammasome is activated by low-dose irradiation both in vitro and in vivo. Inhibition or deletion of NLRP3 can specifically alleviate the mouse lung inflammation caused by radiation and LPS treatment. This study reveals the mechanism of low-dose radiation therapy-induced RP and offers a possible treatment strategy.

Ionizing Radiation Promotes Epithelial-to-Mesenchymal Transition in Lung Epithelial Cells by TGF-ß-producing M2 Macrophages.

BACKGROUND/AIM: Ionizing radiation induces pulmonary fibrosis, which is a common dose-limiting complication in patients receiving radiotherapy. Fibrosis occurs through the accumulation of large amounts of ECM components, synthesized by myofibroblasts in damaged lung tissue. Epithelial cells serve as one of the cellular sources of myofibroblasts via the epithelial-to-mesenchymal transition (EMT) process. In this study, we investigated the role of TGF-ß-secreting M2 macrophages in association with ionizing radiation-induced EMT. MATERIALS AND METHODS: The lung epithelial cell line MLE12, was irradiated and the expression of EMT markers and chemokines was examined. Moreover, the mouse lung macrophage MH-S cell line was cultured with conditioned media from irradiated MLE12 cells, to examine the effects of the secreted factors on the migration ability of macrophages. For the murine pulmonary fibrosis model, mice were locally irradiated and the levels of M1 or M2 macrophage-related markers and cytokines were measured in bronchoalvelolar lavage (BAL) fluid and lung tissue. RESULTS: In MLE12 cells, irradiation directly induced expression of EMT-related markers and secretion of various chemokines, which lead to macrophage migration. Interestingly, the sub-population of macrophages recruited in the lung of mice after thoracic irradiation was M2 macrophages that expressed Arg-1 and CD206. M2 macrophages induced the MLE12 to undergo phenotypic conversion to form fibroblast-like cells, which leads to a down-regulation of epithelial markers and an up-regulation of new EMT-related markers. In thoracic irradiated mice, pro-inflammatory cytokines such as IL-1ß, IL-4 and IL-10 were increased at 2 weeks, but returned to normal levels from 16 weeks or 24 weeks after irradiation. However, thoracic irradiation led to a rapid increase of TGF-ß and IGF-1 levels, which lasted up to 24 weeks. It was confirmed that M2 macrophages secreted the high levels of TGF-ß. Moreover, the elimination of TGF-ß from M2 macrophages attenuated mesenchymal transition of MLE12. CONCLUSION: TGF-ß-secreting M2 macrophages play an important regulatory role in mesenchymal transition of epithelial cells in the lung of irradiated mice, thus contributing to radiation-induced pulmonary fibrosis.

Optimization of the adipose-derived mesenchymal stem cell delivery time for radiation-induced lung fibrosis treatment in rats.

The present study attempts to identify the optimal time duration for the administration of Ad-MSCs, in order to maximize its therapeutic benefits, and compare the degree of fibrosis among three different administration time points using the RILF rat model system. Ad-MSCs were delivered to Sprague-Dawley rats through the tail vein at the following different time points after thorax irradiation: two hours, seven days, and two hours + seven days. Post Ad-MSCs transplantation and the histopathological analysis of the lungs were performed along with analysis of inflammatory cytokine levels, including interleukin (IL)-1, IL-2, IL-6, IL-10 and tumor necrosis factor-α (TNF-α). In particular, pro-fibrotic factors (TGF-ß1 and α-SMA) were also evaluated in serum and lung tissues. In addition, it was also determined whether Ad-MSCs had any role in inhibiting the transition of type II alveolar epithelial cells into fibroblasts in the lungs of injured rats. The present results demonstrated that the intravenous delivery of Ad-MSCs twice at the 2-hour and 7-day (R + MSC2h+7d group) was effective in reducing lung fibrosis for long term durations, when compared with single delivery either at the two-hour or 7-day time points. In addition, a marked anti-inflammatory effect was also observed in RILF rats in the R + MSC2h+7d group, as indicated by the reduced serum levels of pro-inflammatory cytokines (TNF-α, IL-1 and IL-6) and increased levels of anti-inflammatory cytokines IL-10 and IL-2. Rats that were delivered twice with Ad-MSCs (R + MSC2h+7d group) exhibited significantly reduced TGF-ß1 and α-SMA levels, in contrast to rats in the R + MSC7d or R + MSC2h groups, after four weeks. Furthermore, it was also noted that after four weeks, Ad-MSCs increased the number of lung epithelial cells (SP-C) and inhibited the lung fibroblastic cells (α-SMA) of rats in the R + MSC2h and R + MSC2h+7d groups. The present study concluded that two injections of Ad-MSCs (R + MSC2h+7d group) appear to be optimal for therapeutic efficacy and safety during RILF.

Regulation of miR-125a expression by rs12976445 single-nucleotide polymorphism is associated with radiotherapy-induced pneumonitis in lung carcinoma patients.

BACKGROUND: The expression of miR-125 is regulated by an single-nucleotide polymorphism (SNP), rs12976445, which may be involved in the risk of pneumonitis among non-small-cell lung carcinoma patients undergoing the radiotherapy. We investigated this hypothesis via clinical data analysis and in vitro experiments. METHODS: An online microRNA (miRNA) database (www.mirdb.org) and luciferase reporter assays were used to confirm the role of transforming growth factor-ß1 (TGFB1) as a target gene of miR-125a. Quantification by real-time PCR and Western blot analysis were used to measure the expression of miRNA-125a and TGFB1 among different groups (carrying CC, CT, and TT genotypes of rs12976445) or cells transfected with a scramble control, miR-125a mimics, TGFB1 siRNA, or miR-125a inhibitors. RESULTS: We evaluated 699 NSCLC patients and found that the patients carrying the TT or CT genotype of rs12976445 had a higher risk of radiotherapy-induced pneumonitis. Computational analysis and luciferase assays validated that TGFB1 is a target gene of miR-125a. The expression level of miR-125a mRNA was significantly downregulated in the CT and TT groups, while the expression levels of TGFB1 and SMAD2 were significantly upregulated in the CC group. The expression of TGFB1 and SMAD2 was regulated by miR-125a in A549 cells. CONCLUSION: The rs12976445 SNP in miR-125a is associated with the risk of pneumonitis after in lung cancer patients undergoing the radiotherapy by regulating the expression of miR-125a and TGFB1.

Recurrent DNA damage is associated with persistent injury in progressive radiation-induced pulmonary fibrosis.

PURPOSE: Radiation-induced lung injuries (RILI), namely radiation pneumonitis and/or fibrosis, are dose-limiting outcomes following treatment for thoracic cancers. As part of a search for mitigation targets, we sought to determine if persistent DNA damage is a characteristic of this progressive injury. METHODS: C57BL/6J female mice were sacrificed at 24 h, 1, 4, 12, 16, 24 and 32 weeks following a single dose of 12.5 Gy thorax only gamma radiation; their lungs were compared to age-matched unirradiated animals. Tissues were examined for DNA double-strand breaks (DSBs) (γ-H2A.X and p53bp1), cellular senescence (senescence-associated beta-galactosidase and p21) and oxidative stress (malondialdehyde). RESULTS: Data revealed consistently higher numbers of DSBs compared to age-matched controls, with increases in γ-H2A.X positivity beyond 24 h post-exposure, particularly during the pathological phases, suggesting periods of recurrent DNA damage. Additional intermittent increases in both cellular senescence and oxidative stress also appeared to coincide with pneumonitis and fibrosis. CONCLUSIONS: These novel, long-term data indicate (a) increased and persistent levels of DSBs, oxidative stress and cellular senescence may serve as bioindicators of RILI, and (b) prevention of genotoxicity, via mitigation of free radical production, continues to be a potential strategy for the prevention of pulmonary radiation injury.

Nebulisation of synthetic lamellar lipids mitigates radiation-induced lung injury in a large animal model.

Methods to protect against radiation-induced lung injury (RILI) will facilitate the development of more effective radio-therapeutic protocols for lung cancer and may provide the means to protect the wider population in the event of a deliberate or accidental nuclear or radiological event. We hypothesised that supplementing lipid membranes through nebulization of synthetic lamellar lipids would mitigate RILI. Following pre-treatment with either nebulised lamellar lipids or saline, anaesthetised sheep were prescribed fractionated radiotherapy (30 Gray (Gy) total dose in five 6 Gy fractions at 3-4 days intervals) to a defined unilateral lung volume. Gross pathology in radio-exposed lung 37 days after the first radiation treatment was consistent between treatment groups and consisted of deep red congestion evident on the pleural surface and firmness on palpation. Consistent histopathological features in radio-exposed lung were subpleural, periarteriolar and peribronchial intra-alveolar oedema, alveolar fibrosis, interstitial pneumonia and type II pneumocyte hyperplasia. The synthetic lamellar lipids abrogated radiation-induced alveolar fibrosis and reduced alpha-smooth muscle actin (ASMA) expression in radio-exposed lung compared to saline treated sheep. Administration of synthetic lamellar lipids was also associated with an increased number of cells expressing dendritic cell-lysosomal associated membrane protein throughout the lung.

A Potential Biomarker for Predicting the Risk of Radiation-Induced Fibrosis in the Lung.

Biomarkers could play an essential role during triage in the aftermath of a radiological event, where exposure to radiation will be heterogeneous and complicated by concurrent trauma. Used alongside biodosimetry, biomarkers can identify victims in need of treatment for acute radiation effects, and might also provide valuable information on later developing consequences that need to be addressed as part of a treatment strategy. Indeed, because the lung is particularly sensitive to radiation and resultant late effects not only affect quality of life, but can also lead to morbidity, the risk of developing downstream pulmonary complications in exposed individuals requires assessment. In this study, analyses of changes in pulmonary and circulating content of club cell secretory protein (CCSP) and surfactant protein D (SP-D), expressed by epithelial club cells and type II pneumocytes in the lung, respectively, were used to evaluate pulmonary epithelial damage in several lung injury models. Using a combined radiation exposure model, fibrosis-susceptible C57BL/6J (C57) and alveolitis-prone C3H/HeJ (C3H) mice received 5 Gy total-body irradiation plus 2.5-10 Gy whole-lung irradiation, and lung and plasma samples were collected throughout the course of the radiation response, at time points ranging from 24 h to 26 weeks postirradiation. Radiation significantly reduced bronchiole CCSP coverage in C57 mice at 26 weeks, a response that varied in extent among animals, but correlated with the severity of fibrosis in each animal. Interestingly, plasma CCSP content was elevated in C57 mice at multiple time points preceding and during the fibrotic period; this response that was not observed in C3H mice. Circulating CCSP/SP-D ratios, calculated as an index of lung integrity, were similarly increased throughout the time course in C57, but not C3H, mice. Furthermore, when the thoracic doses were reduced to subthreshold levels for fibrosis induction (2.5 or 7.5 Gy), although the CCSP/SP-D ratio in lung homogenates demonstrated dose-responsive changes, this was not reflected in the plasma ratios at acute and late time points. Importantly, plasma CCSP/SP-D ratios also were not significantly altered in C57 mice exposed to LPS, and only transiently decreased in influenza-exposed mice, demonstrating a level of specificity for radiation-induced lung injury. These results indicate that the CCSP/SP-D ratio, measured in plasma, is sensitive to individual variation in radiation sensitivity, correlates with fibrosis development, can be detected early after exposure and is specific to radiation-induced injury. This suggests that the CCSP/SP-D ratio may be useful as a biomarker of radiation-induced pulmonary fibrosis.