Radiation Dermatitis: Radiation-Induced Effects on the Structural and Immunological Barrier Function of the Epidermis.

An important hallmark of radiation dermatitis is the impairment of the mitotic ability of the stem/progenitor cells in the basal cell layers due to radiation-induced DNA damage, leading to suppressed cell renewal in the epidermis. However, this mechanism alone does not adequately explain the complex pathogenesis of radiation-induced skin injury. In this review, we summarize the latest findings on the complex pathogenesis of radiation dermatitis and correlate these with the clinical features of radiation-induced skin reactions. The current studies show that skin exposure to ionizing radiation induces cellular senescence in the epidermal keratinocytes. As part of their epithelial stress response, these senescent keratinocytes secrete pro-inflammatory mediators, thereby triggering skin inflammation. Keratinocyte-derived cytokines and chemokines modulate intercellular communication with the immune cells, activating skin-resident and recruiting skin-infiltrating immune cells within the epidermis and dermis, thereby orchestrating the inflammatory response to radiation-induced tissue damage. The increased expression of specific chemoattractant chemokines leads to increased recruitment of neutrophils into the irradiated skin, where they release cytotoxic granules that are responsible for the exacerbation of an inflammatory state. Moreover, the importance of IL-17-expressing γδ-T cells to the radiation-induced hyperproliferation of keratinocytes was demonstrated, leading to reactive hyperplasia of the epidermis. Radiation-induced, reactive hyperproliferation of the keratinocytes disturbs the fine-tuned keratinization and cornification processes, leading to structural dysfunction of the epidermal barrier. In summary, in response to ionizing radiation, epidermal keratinocytes have important structural and immunoregulatory barrier functions in the skin, coordinating interacting immune responses to eliminate radiation-induced damage and to initiate the healing process.

Cultured Human Foreskin as a Model System for Evaluating Ionizing Radiation-Induced Skin Injury.

PURPOSE: Precise molecular and cellular mechanisms of radiation-induced dermatitis are incompletely understood. Histone variant H2A.J is associated with cellular senescence and modulates senescence-associated secretory phenotype (SASP) after DNA-damaging insults, such as ionizing radiation (IR). Using ex vivo irradiated cultured foreskin, H2A.J was analyzed as a biomarker of radiation-induced senescence, potentially initiating the inflammatory cascade of radiation-induced skin injury. METHODS: Human foreskin explants were collected from young donors, irradiated ex vivo with 10 Gy, and cultured in air-liquid interphase for up to 72 h. At different time-points after ex vivo IR exposure, the foreskin epidermis was analyzed for proliferation and senescence markers by immunofluorescence and immunohistochemical staining of sectioned tissue. Secretion of cytokines was measured in supernatants by ELISA. Using our mouse model with fractionated in vivo irradiation, H2A.J expression was analyzed in epidermal stem/progenitor cell populations localized in different regions of murine hair follicles (HF). RESULTS: Non-vascularized foreskin explants preserved their tissue homeostasis up to 72 h (even after IR exposure), but already non-irradiated foreskin epithelium expressed high levels of H2A.J in all epidermal layers and secreted high amounts of cytokines. Unexpectedly, no further increase in H2A.J expression and no obvious upregulation of cytokine secretion was observed in the foreskin epidermis after ex vivo IR. Undifferentiated keratinocytes in murine HF regions, by contrast, revealed low H2A.J expression in non-irradiated skin and significant radiation-induced H2A.J upregulations at different time-points after IR exposure. Based on its staining characteristics, we presume that H2A.J may have previously underestimated the importance of the epigenetic regulation of keratinocyte maturation. CONCLUSIONS: Cultured foreskin characterized by highly keratinized epithelium and specific immunological features is not an appropriate model for studying H2A.J-associated tissue reactions during radiation-induced dermatitis.

Focused Ion Microbeam Irradiation Induces Clustering of DNA Double-Strand Breaks in Heterochromatin Visualized by Nanoscale-Resolution Electron Microscopy.

BACKGROUND: Charged-particle radiotherapy is an emerging treatment modality for radioresistant tumors. The enhanced effectiveness of high-energy particles (such as heavy ions) has been related to the spatial clustering of DNA lesions due to highly localized energy deposition. Here, DNA damage patterns induced by single and multiple carbon ions were analyzed in the nuclear chromatin environment by different high-resolution microscopy approaches. MATERIAL AND METHODS: Using the heavy-ion microbeam SNAKE, fibroblast monolayers were irradiated with defined numbers of carbon ions (1/10/100 ions per pulse, ipp) focused to micrometer-sized stripes or spots. Radiation-induced lesions were visualized as DNA damage foci (γH2AX, 53BP1) by conventional fluorescence and stimulated emission depletion (STED) microscopy. At micro- and nanoscale level, DNA double-strand breaks (DSBs) were visualized within their chromatin context by labeling the Ku heterodimer. Single and clustered pKu70-labeled DSBs were quantified in euchromatic and heterochromatic regions at 0.1 h, 5 h and 24 h post-IR by transmission electron microscopy (TEM). RESULTS: Increasing numbers of carbon ions per beam spot enhanced spatial clustering of DNA lesions and increased damage complexity with two or more DSBs in close proximity. This effect was detectable in euchromatin, but was much more pronounced in heterochromatin. Analyzing the dynamics of damage processing, our findings indicate that euchromatic DSBs were processed efficiently and repaired in a timely manner. In heterochromatin, by contrast, the number of clustered DSBs continuously increased further over the first hours following IR exposure, indicating the challenging task for the cell to process highly clustered DSBs appropriately. CONCLUSION: Increasing numbers of carbon ions applied to sub-nuclear chromatin regions enhanced the spatial clustering of DSBs and increased damage complexity, this being more pronounced in heterochromatic regions. Inefficient processing of clustered DSBs may explain the enhanced therapeutic efficacy of particle-based radiotherapy in cancer treatment.

Assessment of DNA damage by 53PB1 and pKu70 detection in peripheral blood lymphocytes by immunofluorescence and high-resolution transmission electron microscopy.

PURPOSE: 53BP1 foci detection in peripheral blood lymphocytes (PBLs) by immunofluorescence microscopy (IFM) is a sensitive and quantifiable DNA double-strand break (DSB) marker. In addition, high-resolution transmission electron microscopy (TEM) with immunogold labeling of 53BP1 and DSB-bound phosphorylated Ku70 (pKu70) can be used to determine the progression of the DNA repair process. To establish this TEM method in the PBLs of patients with cancer, we analyzed and characterized whether different modes of irradiation influence the formation of DSBs, and whether accompanying chemotherapy influences DSB formation. METHODS: We obtained 86 blood samples before and 0.1, 0.5, and 24 h after irradiation from patients (n = 9) with head and neck or rectal cancers receiving radiotherapy (RT; n = 4) or radiochemotherapy (RCT; n = 5). 53BP1 foci were quantified by IFM. In addition, TEM was used to quantify gold-labelled pKu70 dimers and 53BP1 clusters within euchromatin and heterochromatin of PBLs. RESULTS: IFM analyses showed that during radiation therapy, persistent 53BP1 foci in PBLs accumulated with increasing numbers of administered RT fractions. This 53BP1 foci accumulation was not influenced by the irradiation technique applied (3D conformal radiotherapy versus intensity-modulated radiotherapy), dose intensity per fraction, number of irradiation fields, or isodose volume. However, more 53BP1 foci were detected in PBLs of patients treated with accompanying chemotherapy. TEM analyses showed that DSBs, indicated by pKu70, were present for longer periods in PBLs of RCT patients than in PBLs of RT only patients. Moreover, not every residual 53BP1 focus was equivalent to a remaining DSB, since pKu70 was not present at every damage site. Persistent 53BP1 clusters, visualized by TEM, without colocalizing pKu70 likely indicate chromatin alterations after repair completion or, possibly, defective repair. CONCLUSION: IFM 53BP1 foci analyses alone are not adequate to determine individual repair capacity after irradiation of PBLs, as a DSB may be indicated by a 53BP1 focus but not every 53BP1 focus represents a DSB.

Histone Variant H2A.J Marks Persistent DNA Damage and Triggers the Secretory Phenotype in Radiation-Induced Senescence.

Irreparable double-strand breaks (DSBs) in response to ionizing radiation (IR) trigger prolonged DNA damage response (DDR) and induce premature senescence. Profound chromatin reorganization with formation of senescence-associated heterochromatin foci (SAHF) is an essential epigenetic mechanism for controlling the senescence-associated secretory phenotype (SASP). To decipher molecular mechanisms provoking continuous DDR leading to premature senescence, radiation-induced DSBs (53BP1-foci) and dynamics of histone variant H2A.J incorporation were analyzed together with chromatin re-modeling in human fibroblasts after IR exposure. High-resolution imaging by transmission electron microscopy revealed that persisting 53BP1-foci developed into DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS), consistently located at the periphery of SAHFs. Quantitative immunogold-analysis by electron microscopy revealed that H2A.J, steadily co-localizing with 53BP1, is increasingly incorporated into DNA-SCARS during senescence progression. Strikingly, shRNA-mediated H2A.J depletion in fibroblasts modified senescence-associated chromatin re-structuring and abolished SASP, thereby shutting down the production of inflammatory mediators. These findings provide mechanistic insights into biological phenomena of SASP and suggest that H2A.J inhibition could ablate SASP, without affecting the senescence-associated growth arrest.

Hair Follicle Stem Cell Faith Is Dependent on Chromatin Remodeling Capacity Following Low-Dose Radiation.

The main function of the skin, to protect against the environment, is supported by the activity of different stem cell populations. The main focus of this study was elucidating the coping mechanisms of stem cells against the stimulation of constant exposure to genotoxic stresses, both endogenous and exogenous, to ensure long-term function. Investigation of various mouse strains, differing in their DNA repair capacity, enables us to clarify fractionated low-dose irradiation (LDR)-induced consequences for different stem cell populations of the murine hair follicle (HF) in their physiological stem cell niche. Using microscopic techniques combined with flow cytometry, we could show that LDR induces accumulation of persisting; pKu70-independent 53BP1-foci ("chromatin-alterations") in heterochromatic regions of the HF stem cells (HFSCs). These remaining chromatin-alterations result in varying stem cell consequences. CD34-positive HFSCs react by ataxia telangiectasia mutated-dependent, premature senescence, which correlates with global chromatin compaction, whereby apoptosis is prevented by the activity of DNA-dependent protein kinase catalytic subunit. However, distinctively highly damaged HFSCs seem to be sorted out of the niche by differentiation, transferring their chromatin-alterations to more proliferative G protein-coupled receptor 5-positive stem cells. Consequentially, the loss of basal HFSCs is compensated by increased proliferation within the stem cell pool. Despite the initial success of these mechanisms in stem cell population maintenance, the combined effect of the chromatin-alterations and the modification in stem cell pool composition may lead to downstream long-term functional loss of tissue or organs. Stem Cells 2018;36:574-588.

TLR2 is a primary receptor for Alzheimer's amyloid ß peptide to trigger neuroinflammatory activation.

Microglia activated by extracellularly deposited amyloid ß peptide (Aß) act as a two-edged sword in Alzheimer's disease pathogenesis: on the one hand, they damage neurons by releasing neurotoxic proinflammatory mediators (M1 activation); on the other hand, they protect neurons by triggering anti-inflammatory/neurotrophic M2 activation and by clearing Aß via phagocytosis. TLRs are associated with Aß-induced microglial inflammatory activation and Aß internalization, but the mechanisms remain unclear. In this study, we used real-time surface plasmon resonance spectroscopy and conventional biochemical pull-down assays to demonstrate a direct interaction between TLR2 and the aggregated 42-aa form of human Aß (Aß42). TLR2 deficiency reduced Aß42-triggered inflammatory activation but enhanced Aß phagocytosis in cultured microglia and macrophages. By expressing TLR2 in HEK293 cells that do not endogenously express TLR2, we observed that TLR2 expression enabled HEK293 cells to respond to Aß42. Through site-directed mutagenesis of tlr2 gene, we identified the amino acids EKKA (741-744) as a critical cytoplasmic domain for transduction of inflammatory signals. By coexpressing TLR1 or TLR6 in TLR2-transgenic HEK293 cells or silencing tlrs genes in RAW264.7 macrophages, we observed that TLR2-mediated Aß42-triggered inflammatory activation was enhanced by TLR1 and suppressed by TLR6. Using bone marrow chimeric Alzheimer's amyloid precursor transgenic mice, we observed that TLR2 deficiency in microglia shifts M1- to M2-inflammatory activation in vivo, which was associated with improved neuronal function. Our study demonstrated that TLR2 is a primary receptor for Aß to trigger neuroinflammatory activation and suggested that inhibition of TLR2 in microglia could be beneficial in Alzheimer's disease pathogenesis.

Immunomodulatory Effects of Histone Variant H2A.J in Ionizing Radiation Dermatitis.

PURPOSE: Histone variant H2A.J is associated with premature senescence after ionizing radiation (IR) and modulates senescence-associated secretory phenotype (SASP). Using constitutive H2A.J knock-out mice, the role of H2A.J was investigated in radiation dermatitis. METHODS AND MATERIALS: H2A.J wild-type (WT) and knock-out (KO) mice were exposed to moderate or high IR doses (≤20 Gy, skinfold IR). Radiation-induced skin reactions were investigated up to 2 weeks post-IR at macroscopic and microscopic levels. H2A.J and other senescence markers, as well as DNA damage and proliferation markers, were studied by immunohistochemistry, immunofluorescence, and electron microscopy. After high-dose IR, protein-coding transcriptomes were analyzed by RNA sequencing, immune cell infiltration by flow cytometry, and gene expression by reverse transcription polymerase chain reaction in (non-) irradiated WT versus KO skin. RESULTS: In WT skin, epidermal keratinocytes showed time- and dose-dependent H2A.J accumulation after IR exposure. Unexpectedly, stronger inflammatory reactions with increased epidermal thickness and progressive hair follicle loss were observed in irradiated KO versus WT skin. Clearly more radiation-induced senescence was observed in keratinocyte populations of KO skin after moderate and high doses, with hair follicle stem cells being particularly badly damaged, leading to follicle atrophy. After high-dose IR, transcriptomic analysis revealed enhanced senescence-associated signatures in irradiated KO skin, with intensified release of SASP factors. Flow cytometric analysis indicated increased immune cell infiltration in both WT and KO skin; however, specific chemokine-mediated signaling in irradiated KO skin led to more neutrophil recruitment, thereby aggravating radiation toxicities. Increased skin damage in irradiated KO skin led to hyperproliferation, abnormal differentiation, and cornification of keratinocytes, accompanied by increased upregulation of transcription-factor JunB. CONCLUSIONS: Lack of radiation-induced H2A.J expression in keratinocytes is associated with increased senescence induction, modulation of SASP expression, and exacerbated inflammatory skin reactions. Hence, epigenetic H2A.J-mediated gene expression in response to IR regulates keratinocyte immune functions and plays an essential role in balancing the inflammatory response during radiation dermatitis.

Inducible response required for repair of low-dose radiation damage in human fibroblasts.

Ionizing radiation (IR) induces a variety of DNA lesions among which DNA double-strand breaks (DSBs) are the biologically most significant. It is currently unclear if DSB repair is equally efficient after low and high doses. Here, we use gamma-H2AX, phospho-ATM (pATM), and 53BP1 foci analysis to monitor DSB repair. We show, consistent with a previous study, that the kinetics of gamma-H2AX and pATM foci loss in confluent primary human fibroblasts are substantially compromised after doses of 10 mGy and lower. Following 2.5 mGy, cells fail to show any foci loss. Strikingly, cells pretreated with 10 microM H(2)O(2) efficiently remove all gamma-H2AX foci induced by 10 mGy. At the concentration used, H(2)O(2) produces single-strand breaks and base damages via the generation of oxygen radicals but no DSBs. Moreover, 10 microM H(2)O(2) up-regulates a set of genes that is also up-regulated after high (200 mGy) but not after low (10 mGy) radiation doses. This suggests that low radical levels induce a response that is required for the repair of radiation-induced DSBs when the radiation damage is too low to cause the induction itself. To address the in vivo significance of this finding, we established gamma-H2AX and 53BP1 foci analysis in various mouse tissues. Although mice irradiated with 100 mGy or 1 Gy show efficient gamma-H2AX and 53BP1 foci removal during 24 h post-IR, barely any foci loss was observed after 10 mGy. Our data suggest that the cellular response to DSBs is substantially different for low vs. high radiation doses.

Radiation-Induced Brain Injury: Age Dependency of Neurocognitive Dysfunction Following Radiotherapy.

Cranial radiotherapy is a known risk factor for neurocognitive impairment in cancer survivors. Although radiation-induced cognitive dysfunction is observed in patients of all ages, children seem to be more vulnerable than adults to suffering age-related deficits in neurocognitive skills. So far, the underlying mechanisms by which IR negatively influences brain functions as well as the reasons for the profound age dependency are still insufficiently known. We performed a comprehensive Pubmed-based literature search to identify original research articles that reported on age dependency of neurocognitive dysfunction following cranial IR exposure. Numerous clinical trials in childhood cancer survivors indicate that the severity of radiation-induced cognitive dysfunction is clearly dependent on age at IR exposure. These clinical findings were related to the current state of experimental research providing important insights into the age dependency of radiation-induced brain injury and the development of neurocognitive impairment. Research in pre-clinical rodent models demonstrates age-dependent effects of IR exposure on hippocampal neurogenesis, radiation-induced neurovascular damage and neuroinflammation.

Measuring out-of-field dose to the hippocampus in common radiotherapy indications.

BACKGROUND: The high susceptibility of the hippocampus region to radiation injury is likely the causal factor of neurocognitive dysfunctions after exposure to ionizing radiation. Repetitive exposures with even low doses have been shown to impact adult neurogenesis and induce neuroinflammation. We address the question whether the out-of-field doses during radiotherapy of common tumour entities may pose a risk for the neuronal stem cell compartment in the hippocampus. METHODS: The dose to the hippocampus was determined for a single fraction according to different treatment plans for the selected tumor entities: Point dose measurements were performed in an anthropomorphic Alderson phantom and the out-of-field dose to the hippocampus was measured using thermoluminescence dosimeters. RESULTS: For carcinomas in the head and neck region the dose exposure to the hippocampal region for a single fraction ranged from to 37.4 to 154.8 mGy. The hippocampal dose was clearly different for naso-, oro- and hypopharynx, with maximal values for nasopharynx carcinoma. In contrast, hippocampal dose levels for breast and prostate cancer ranged between 2.7 and 4.1 mGy, and therefore significantly exceeded the background irradiation level. CONCLUSION: The mean dose to hippocampus for treatment of carcinomas in the head and neck region is high enough to reduce neurocognitive functions. In addition, care must be taken regarding the out of field doses. The mean dose is mainly related to scattering effects, as is confirmed by the data from breast or prostate treatments, with a very different geometrical set-up but similar dosimetric results.

Automated Image Analysis of Transmission Electron Micrographs: Nanoscale Evaluation of Radiation-Induced DNA Damage in the Context of Chromatin.

BACKGROUND: Heavy ion irradiation (IR) with high-linear energy transfer (LET) is characterized by a unique depth dose distribution and increased biological effectiveness. Following high-LET IR, localized energy deposition along the particle trajectories induces clustered DNA lesions, leading to low electron density domains (LEDDs). To investigate the spatiotemporal dynamics of DNA repair and chromatin remodeling, we established the automated image analysis of transmission electron micrographs. METHODS: Human fibroblasts were irradiated with high-LET carbon ions or low-LET photons. At 0.1 h, 0.5 h, 5 h, and 24 h post-IR, nanoparticle-labeled repair factors (53BP1, pKu70, pKu80, DNA-PKcs) were visualized using transmission electron microscopy in interphase nuclei to monitor the formation and repair of DNA damage in the chromatin ultrastructure. Using AI-based software tools, advanced image analysis techniques were established to assess the DNA damage pattern following low-LET versus high-LET IR. RESULTS: Low-LET IR induced single DNA lesions throughout the nucleus, and most DNA double-strand breaks (DSBs) were efficiently rejoined with no visible chromatin decondensation. High-LET IR induced clustered DNA damage concentrated along the particle trajectories, resulting in circumscribed LEDDs. Automated image analysis was used to determine the exact number of differently sized nanoparticles, their distance from one another, and their precise location within the micrographs (based on size, shape, and density). Chromatin densities were determined from grayscale features, and nanoparticles were automatically assigned to euchromatin or heterochromatin. High-LET IR-induced LEDDs were delineated using automated segmentation, and the spatial distribution of nanoparticles in relation to segmented LEDDs was determined. CONCLUSIONS: The results of our image analysis suggest that high-LET IR induces chromatin relaxation along particle trajectories, enabling the critical repair of successive DNA damage. Following exposure to different radiation qualities, automated image analysis of nanoparticle-labeled DNA repair proteins in the chromatin ultrastructure enables precise characterization of specific DNA damage patterns.

Role of Histone Variant H2A.J in Fine-Tuning Chromatin Organization for the Establishment of Ionizing Radiation-Induced Senescence.

PURPOSE: Radiation-induced senescence is characterized by profound changes in chromatin organization with the formation of Senescence-Associated-Heterochromatin-Foci (SAHF) and DNA-Segments-with-Chromatin-Alterations-Reinforcing-Senescence (DNA-SCARS). Importantly, senescent cells also secrete complex combinations of pro-inflammatory factors, referred as Senescence-Associated-Secretory-Phenotype (SASP). Here, we analyzed the epigenetic mechanism of histone variant H2A.J in establishing radiation-induced senescence. EXPERIMENTAL DESIGN: Primary and genetically-modified lung fibroblasts with down- or up-regulated H2A.J expression were exposed to ionizing radiation and were analyzed for the formation of SAHF and DNA-SCARS by immunofluorescence microscopy. Dynamic changes in chromatin organization and accessibility, transcription factor recruitment, and transcriptome signatures were mapped by ATAC-seq and RNA-seq analysis. The secretion of SASP factors and potential bystander effects were analyzed by ELISA and RT-PCR. Lung tissue of mice exposed to different doses were analyzed by the digital image analysis of H2A.J-immunohistochemistry. RESULTS: Differential incorporation of H2A.J has profound effects on higher-order chromatin organization and on establishing the epigenetic state of senescence. Integrative analyses of ATAC-seq and RNA-seq datasets indicate that H2A.J-associated changes in chromatin accessibility of regulatory regions decisively modulates transcription factor recruitment and inflammatory gene expression, resulting in an altered SASP secretome. In lung parenchyma, pneumocytes show dose-dependent H2A.J expression in response to radiation-induced DNA damage, therefore contributing to pro-inflammatory tissue reactions. CONCLUSIONS: The fine-tuned incorporation of H2A.J defines the epigenetic landscape for driving the senescence programme in response to radiation-induced DNA damage. Deregulated H2A.J deposition affects chromatin remodeling, transcription factor recruitment, and the pro-inflammatory secretome. Our findings provide new mechanistic insights into DNA-damage triggered epigenetic mechanisms governing the biological processes of radiation-induced injury.

Imaging doses for different CBCT protocols on the Halcyon 3.0 linear accelerator - TLD measurements in an anthropomorphic phantom.

INTRODUCTION: Image guided radiotherapy allows for particularly conformal tumour irradiation through precise patient positioning. Becoming the standard for radiotherapy, this increases imaging doses to the patient. The Halcyon 3.0 linear accelerator (Varian Medical Systems, Palo Alto, CA) requires daily imaging due to its geometry. For this reason, the accelerator is equipped with on-line kV and MV imaging. However, daily CBCT images required for irradiation apply additional radiation, which increases the dose to normal tissue and therefore can affect the patient's secondary cancer risk. In this study, actual organ doses were measured for the kV system, and a comparison of normal tissue doses for all available kV CBCT protocols was presented to demonstrate differences in imaging doses across entities and protocols. In addition, effective dose and secondary cancer risk from imaging are evaluated. MATERIAL AND METHODS: Measurements were performed with thermoluminescent dosimeters in an anthropomorphic phantom positioned according to each entity (brain, head and neck, breast, lung, pelvis). CBCT images were obtained, using all available pre-set protocols without further adjustment of the parameters. Measured doses for each position and each protocol were then compared and secondary cancer risk of relevant and specifically radiosensitive organs was calculated. RESULTS: It was found that imaging doses for protocols such as Pelvis and Head could be reduced by up to half using the corresponding Fast and Low Dose modes, respectively. On the other hand, larger field sizes or the Large mode yielded higher doses than their initial protocols. Image Gently was found to spare normal tissue best, however it is not suitable for certain entities due to low image quality or insufficient projection data. DISCUSSION: By using appropriate kV-CBCT protocols, it is possible to reduce imaging doses to a significant extent and therefore spare healthy tissue. Combined with studies of image quality, the results of this study could lead to adjustments in workflow regarding the choice of protocols used in daily routine. This could prevent unnecessary radiation exposure and reduce secondary cancer risk.

Myeloid differentiation factor 88-deficient bone marrow cells improve Alzheimer's disease-related symptoms and pathology.

Alzheimer's disease is characterized by extracellular deposits of amyloid ß peptide in the brain. Increasing evidence suggests that amyloid ß peptide injures neurons both directly and indirectly by triggering neurotoxic innate immune responses. Myeloid differentiation factor 88 is the key signalling molecule downstream to most innate immune receptors crucial in inflammatory activation. For this reason, we investigated the effects of myeloid differentiation factor 88-deficient bone marrow cells on Alzheimer's disease-related symptoms and pathology by establishing bone marrow chimeric amyloid ß peptide precursor transgenic mice, in which bone marrow cells differentiate into microglia and are recruited to amyloid ß peptide deposits. We observed that myeloid differentiation factor 88-deficient bone marrow reconstruction reduced both inflammatory activation and amyloid ß peptide burden in the brain. In addition, synaptophysin, a marker of neuronal integrity, was preserved and the expression of neuronal plasticity-related genes, ARC and NMDA-R1, was increased. Thus, myeloid differentiation factor 88-deficient microglia significantly improved the cognitive function of amyloid ß peptide precursor protein transgenic mice. Myeloid differentiation factor 88-deficiency enhanced amyloid ß peptide phagocytosis by microglia/macrophages and blunted toxic inflammatory activation. Both the expression of amyloid ß peptide precursor protein and amyloid ß peptide degrading enzymes and also the efflux of amyloid ß peptide from brain parenchyma were unaffected by myeloid differentiation factor 88-deficient microglia. By contrast, the activity of ß-secretase was increased. ß-Secretase is expressed primarily in neurons, with relatively little expression in astrocytes and microglia. Therefore, microglial replenishment with myeloid differentiation factor 88-deficient bone marrow cells might improve cognitive functions in Alzheimer's disease mouse models by enhancing amyloid ß peptide phagocytosis and reducing inflammatory activation. These results could offer a new therapeutic option that might delay the progression of Alzheimer's disease.

Region-Specific Effects of Fractionated Low-Dose Versus Single-Dose Radiation on Hippocampal Neurogenesis and Neuroinflammation.

BACKGROUND: Despite technical advances in hippocampus-sparing radiotherapy, radiation-induced injury to neural stem cell compartments may affect neurocognitive functions. In pre-clinical mouse models with fractionated low-dose radiation (FLDR) and single-dose radiation (SDR), the accurate response to radiation-induced injury was analyzed in different hippocampal subregions. METHODS: Adult and juvenile C57BL/6NCrl mice were exposed to FLDR (20 × 0.1 Gy, daily exposure from Monday to Friday for 4 weeks) or SDR (1 × 2 Gy). In addition, 72 h after the last exposure, neuroglia (astrocytes and microglia) and neuroprogenitor cells were characterized and quantified in the hippocampal cornu ammonis (CA) and dentate gyrus (DG) by immunofluorescence studies. RESULTS: After analyzing different hippocampal subregions, it was observed that radiation responses varied between non-neurogenic CA, with no detectable inflammatory alterations, and neurogenic DG, characterized by impaired neurogenesis and subsequent neuroinflammation. Age-dependent differences in radiosensitivity appeared to depend on the varying proliferative potential of neural stem cell niches. Using the same overall dose for FLDR and SDR (2 Gy), both the cumulative dose over time and also the single dose fraction have decisive impacts on hippocampal damage. CONCLUSION: Region-specific effects of radiation-induced hippocampal injury relies primarily on cell deaths of proliferating neuroprogenitors. Dose per fraction defines the extent of neuronal injury, and subsequently activated microglia and reactive astrocytes modulate dynamic processes of neuroinflammation. Thus, limiting both cumulative doses and dose fractions to hippocampal DG is an important issue of clinical radiotherapy to preserve neurocognitive functions.

Nuclear Fragility in Radiation-Induced Senescence: Blebs and Tubes Visualized by 3D Electron Microscopy.

Irreparable DNA damage following ionizing radiation (IR) triggers prolonged DNA damage response and induces premature senescence. Cellular senescence is a permanent state of cell-cycle arrest characterized by chromatin restructuring, altered nuclear morphology and acquisition of secretory phenotype, which contributes to senescence-related inflammation. However, the mechanistic connections for radiation-induced DNA damage that trigger these senescence-associated hallmarks are poorly understood. In our in vitro model of radiation-induced senescence, mass spectrometry-based proteomics was combined with high-resolution imaging techniques to investigate the interrelations between altered chromatin compaction, nuclear envelope destabilization and nucleo-cytoplasmic chromatin blebbing. Our findings confirm the general pathophysiology of the senescence-response, with disruption of nuclear lamin organization leading to extensive chromatin restructuring and destabilization of the nuclear membrane with release of chromatin fragments into the cytosol, thereby activating cGAS-STING-dependent interferon signaling. By serial block-face scanning electron microscopy (SBF-SEM) whole-cell datasets were acquired to investigate the morphological organization of senescent fibroblasts. High-resolution 3-dimensional (3D) reconstruction of the complex nuclear shape allows us to precisely visualize the segregation of nuclear blebs from the main nucleus and their fusion with lysosomes. By multi-view 3D electron microscopy, we identified nanotubular channels formed in lamin-perturbed nuclei of senescent fibroblasts; the potential role of these nucleo-cytoplasmic nanotubes for expulsion of damaged chromatin has to be examined.

Adipose tissue-derived microvascular fragments promote lymphangiogenesis in a murine lymphedema model.

Chronic lymphedema after cancer treatment is common and there is still no cure for this disease. We herein investigated the lymphangiogenic capacity of adipose tissue-derived microvascular fragments (MVF), which contain stem cells and lymphatic vessel fragments. Secondary lymphedema was induced in the hindlimbs of C57BL/6J mice. Green fluorescence protein (GFP)+ MVF were isolated from transgenic C57BL/6Tg (CAG-EGFP)1Osb/J mice, suspended in collagen hydrogel, and injected in the lymphadenectomy defect of wild-type animals. This crossover model allowed the detection of MVF-derived blood and lymphatic vessels after transplantation. The MVF group was compared with animals receiving collagen hydrogel only or a sham intervention. Lymphangiogenic effects were analyzed using volumetry, magnetic resonance (MR) lymphography, histology, and immunohistochemistry. MVF injection resulted in reduced hindlimb volumes when compared to non-treated controls. MR lymphography revealed lymphatic regeneration with reduced dermal backflow after MVF treatment. Finally, MVF transplantation promoted popliteal angiogenesis and lymphangiogenesis associated with a significantly increased microvessel and lymphatic vessel density. These findings indicate that MVF transplantation represents a promising approach to induce therapeutic lymphangiogenesis.

Fractionated Low-Dose Radiation Induces Long-Lasting Inflammatory Responses in the Hippocampal Stem Cell Niche.

PURPOSE: Despite major technical advances in hippocampus-sparing radiation therapy, radiation-induced injury to the neural stem cell compartment may affect neurocognitive functions. In the brain, glial cells modulate neuronal functions and are major mediators of neuroinflammation. In a preclinical mouse model with fractionated low-dose radiation (LDR), the complex response to radiation-induced injury was analyzed in the hippocampal stem cell compartment over a period of 6 months. METHODS AND MATERIALS: Adult and juvenile C57BL/6NCrl mice were exposed to low doses of ionizing radiation (IR; 20 fractions of 0.1 Gy, for up to 4 weeks) daily. At 72 hours and 1, 3, and 6 months after fractionated LDR, magnetic resonance imaging (9.4 T) was conducted to detect structural and functional abnormalities in the hippocampal region. Using immunofluorescence and histologic studies, neuroglia cells (astrocytes, microglia, oligodendrocytes) were quantified and neuroinflammatory responses were characterized in the hippocampal dentate gyrus. Using in vivo bromodeoxyuridine labeling, the cell fate of newly generated progenitor cells was tracked in the subgranular zone of the dentate gyrus during fractionated LDR. RESULTS: Low doses of IR induced long-lasting inflammatory responses with local increases of activated microglia and reactive astrocytes, which were most pronounced in the juvenile hippocampus within the first months after LDR. Glial activation with the consequent release of proinflammatory mediators increased local blood flow and vascular permeability in the hippocampal region. Cell fate mapping of progenitors located in the subgranular zone revealed a transient shift from neurogenesis to gliogenesis. CONCLUSIONS: Glial cell activation and transient neuroinflammation may reflect radiation-induced neuronal damage in the hippocampal stem cell niche. The increased proliferative capacity of the developing brain may explain the enhanced hippocampal radiosensitivity, with stronger inflammatory reactions in the juvenile hippocampus. Thus, limiting the radiation dose to the hippocampal region is an important issue of clinical radiation therapy at all ages to preserve neurocognitive functions.

Human skin aging is associated with increased expression of the histone variant H2A.J in the epidermis.

Cellular senescence is an irreversible growth arrest that occurs as a result of damaging stimuli, including DNA damage and/or telomere shortening. Here, we investigate histone variant H2A.J as a new biomarker to detect senescent cells during human skin aging. Skin biopsies from healthy volunteers of different ages (18-90 years) were analyzed for H2A.J expression and other parameters involved in triggering and/or maintaining cellular senescence. In the epidermis, the proportions of H2A.J-expressing keratinocytes increased from ≈20% in young to ≈60% in aged skin. Inverse correlations between Ki67- and H2A.J staining in germinative layers may reflect that H2A.J-expressing cells having lost their capacity to divide. As cellular senescence is triggered by DNA-damage signals, persistent 53BP1-foci, telomere lengths, and telomere-associated damage foci were analyzed in epidermal keratinocytes. Only slight age-related telomere attrition and few persistent nuclear 53BP1-foci, occasionally colocalizing with telomeres, suggest that unprotected telomeres are not a significant cause of senescence during skin aging. Quantification of integrin-α6+ basal cells suggests that the number and function of stem/progenitor cells decreased during aging and their altered proliferation capacities resulted in diminished tissue renewal with epidermal thinning. Collectively, our findings suggest that H2A.J is a sensitive marker of epidermal aging in human skin.