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Low Dose Radiation Effects on the Homeostasis and Activation of Immune Cells

A special issue of Cells (ISSN 2073-4409). This special issue belongs to the section "Cellular Immunology".

Deadline for manuscript submissions: closed (10 November 2021) | Viewed by 24697

Special Issue Editors


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Guest Editor
French Alternative Energies and Atomic Energy Commission (CEA), 38000 Grenoble, France
Interests: radiation biology; immunology; low dose; bystander effects; normal tissues; inflammation; lymphocyte development; V(D)J recombination

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Guest Editor

Special Issue Information

Dear Colleagues,

The interactions between the immune system and the response to radiation are complex. On the one hand, irradiation of any part of the body directly affects the immune system, as circulating immune cells are distributed throughout the organism via the blood. Furthermore, immune cells can be concentrated in normal and pathological structures such as lymph nodes and solid tumors, which may be the target of radiation, for example during cancer treatment. On the other hand, as part of the organism’s defenses, the immune system will try to counteract the detrimental effects of radiation exposure and participate in the restoration of tissue homeostasis and functions.

A lot of knowledge have accumulated over time on the effects of high dose radiation on the immune system, and especially on the various forms of radiation-induced cell death and the consequences for immune system activation. In contrast, much less data are available on the effects of low dose radiation exposure, despite the surge in interest for this topic during the last years. Indeed, ionizing radiation exposure in the low to intermediate range induces much less genotoxic damage and cell death, and the effects of such exposures will be dominated by the consequences of cell stress and damage to the various cellular compartments, including an imbalance of the redox status. These effects may manifest as cell autonomous events, or be more diffuse, transmitted to neighboring cells, irradiated or not, by inter-cellular communication, via either cell-cell contact or soluble factors, including inflammatory mediators, and extracellular vesicles. Thus, these effects can result from direct exposure of immune cells, or from their response to irradiated cells, or a combination of both.

In this special issue, we welcome articles from all fields of radiation biology (environmental, occupational and accidental exposure, in vitro and in vivo pre-clinical models, medical applications of ionizing radiation) addressing the specific effects of low dose radiation (<100 mGy) on the homeostasis and direct or indirect activation of the immune system, when compared to intermediate and higher dose exposures. These articles can be original research articles or reviews aimed at summarizing a dedicated aspect of the effects of low dose exposure on immune cells.

Dr. Serge Candéias
Dr. Katalin Lumniczky
Guest Editors

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Keywords

  • low dose ionizing radiation
  • immune system
  • quality of radiation
  • homeostasis
  • activation
  • development
  • inflammation
  • bystander effects
  • exosomes

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Published Papers (7 papers)

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14 pages, 1441 KiB  
Article
Genotoxicity Associated with 131I and 99mTc Exposure in Nuclear Medicine Staff: A Physical and Biological Monitoring Study
by Justyna Miszczyk, Aleksander Gałaś, Agnieszka Panek, Aldona Kowalska, Magdalena Kostkiewicz, Eliza Borkowska and Kamil Brudecki
Cells 2022, 11(10), 1655; https://doi.org/10.3390/cells11101655 - 16 May 2022
Cited by 4 | Viewed by 2867
Abstract
Nuclear medicine staff are constantly exposed to low doses of ionizing radiation. This study investigated the level of genotoxic effects in hospital employees exposed to routinely used 131I and 99mTc in comparison with a control group. The study compared the results [...] Read more.
Nuclear medicine staff are constantly exposed to low doses of ionizing radiation. This study investigated the level of genotoxic effects in hospital employees exposed to routinely used 131I and 99mTc in comparison with a control group. The study compared the results of physical and biological monitoring in peripheral blood lymphocytes. The effects of confounding factors, such as smoking status and physical activity, were also considered. Physical dosimetry monitoring revealed differences in the individual annual effective dose as measured by finger ring dosimeter and whole-body dosimeter between the 131I- and 99mTc-exposed groups. The DNA damage studies revealed differences between the groups in terms of excess premature chromosome condensation (PCC) fragments and tail DNA. Physical activity and smoking status differentiated the investigated groups. When assessed by the level of physical activity, the highest mean values of tail DNA were observed for the 99mTc group. When assessed by work-related physical effort, excess PCC fragments were significantly higher in the 131I group than in the control group. In the investigated groups, the tail DNA values were significantly different between non-smokers and past or current smokers, but excess PCC fragments did not significantly differ by smoking status. It is important to measure exposure to low doses of ionizing radiation and assess the potential risk from this exposure. Such investigations support the need to continue epidemiological and experimental studies to improve our understanding of the mechanisms of the health effects of radionuclides and to develop predictive models of the behavior of these complex systems in response to low-dose radiation. Full article
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Figure 1

Figure 1
<p>Mean values of biomarkers (NDI, MN, PCC, tail DNA) for human peripheral blood lymphocytes (HPBLs) of the <sup>131</sup>I, <sup>99m</sup>Tc, and control groups. The error bars represent the standard deviation. Tail DNA is DNA damage detected by a single-cell gel electrophoresis assay.</p>
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<p>Mean values of tail DNA for normal (NA) and high (HA) leisure time physical activity (<b>a</b>), and low (LA) and high (HA) work-related physical effort (<b>b</b>) in the three studied groups. The error bars represent the standard deviation. * <span class="html-italic">p</span> ≤ 0.01; ** <span class="html-italic">p</span> ≤ 0.05; *** <span class="html-italic">p</span> ≤ 0.001.</p>
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<p>Frequencies of excess PCC fragments in NA and HA LTPA (<b>a</b>) and LA and HA WRPE (<b>b</b>) in peripheral lymphocytes of the three groups. * <span class="html-italic">p</span> ≤ 0.05.</p>
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<p>Tail DNA (<b>a</b>) and excess PCC fragments (<b>b</b>) stratified by smoking status (non-smokers (NS) and past or current smokers (PCS)) in the examined groups. ** <span class="html-italic">p</span> ≤ 0.05.</p>
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23 pages, 5231 KiB  
Article
Radon Improves Clinical Response in an Animal Model of Rheumatoid Arthritis Accompanied by Increased Numbers of Peripheral Blood B Cells and Interleukin-5 Concentration
by Lisa Deloch, Stephanie Hehlgans, Michael Rückert, Andreas Maier, Annika Hinrichs, Ann-Sophie Flohr, Denise Eckert, Thomas Weissmann, Michaela Seeling, Falk Nimmerjahn, Rainer Fietkau, Franz Rödel, Claudia Fournier, Benjamin Frey and Udo S. Gaipl
Cells 2022, 11(4), 689; https://doi.org/10.3390/cells11040689 - 16 Feb 2022
Cited by 4 | Viewed by 3943
Abstract
Radon treatment is used as an established therapy option in chronic painful inflammatory diseases. While analgesic effects are well described, little is known about the underlying molecular effects. Among the suspected mechanisms are modulations of the anti-oxidative and the immune system. Therefore, we [...] Read more.
Radon treatment is used as an established therapy option in chronic painful inflammatory diseases. While analgesic effects are well described, little is known about the underlying molecular effects. Among the suspected mechanisms are modulations of the anti-oxidative and the immune system. Therefore, we aimed for the first time to examine the beneficial effects of radon exposure on clinical outcome as well as the underlying mechanisms by utilizing a holistic approach in a controlled environment of a radon chamber with an animal model: K/BxN serum-induced arthritic mice as well as isolated cells were exposed to sham or radon irradiation. The effects on the anti-oxidative and the immune system were analyzed by flow-cytometry, qPCR or ELISA. We found a significantly improved clinical disease progression score in the mice, alongside significant increase of peripheral blood B cells and IL-5. No significant alterations were visible in the anti-oxidative system or regarding cell death. We conclude that neither cell death nor anti-oxidative systems are responsible for the beneficial effects of radon exposure in our preclinical model. Rather, radon slightly affects the immune system. However, more research is still needed in order to fully understand radon-mediated effects and to carry out reasonable risk-benefit considerations. Full article
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Graphical abstract

Graphical abstract
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<p>Exposure to radon gas for one hour results in an improved clinical score in K/BxN serum-induced C57Bl76 mice in comparison to mock-treated controls. Female C57Bl/6 mice were injected i.p. with 200 µL or 300 µL, depending on the respective serum batch, of pooled K/BxN serum. Within a few days, mice develop a visible swelling of the paws (<b>A</b>,<b>B</b>). On d3 after the injection, mice were scored and randomly distributed into two groups (<b>E</b>) and placed into the radon chamber (<b>C</b>) for treatment. While one group received mock treatment, the other group was exposed to radon gas. Both groups were scored again on d7 and d10. On d10 mice were sacrificed and samples (whole blood, serum, bone marrow and hind feet) were collected for further analysis ((<b>D</b>), timeline). Clinical score of serum-induced mice was determined according to swelling (0—no swelling, 5—complete swelling of paws and toes), whereas the score of both hindlegs/mouse was added up for final evaluation. 6 out of 9 animals in the radon group (<b>F</b>) showed an improvement in clinical score whereas only one in 8 control animals (<b>G</b>) showed improvement during the time of observation. These effects are also visualized in (<b>H</b>), where the Δ clinical score [d7–d10] of clinical progression is significantly better in radon treated animals when compared to mock controls. Figure shows data of two independent experiments with in total 10 mice/group and is shown as Median + IQR. Animals that did not show swelling on d3 were removed from the experiment; n<sub>control</sub> = 7; n<sub>Radon</sub> = 9. Statistics were calculated using Mann-Whitney-U test; (* <span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Exposure to radon gas for one hour results in a slightly increased expression of anti-oxidative enzymes Glutathione Peroxidase (GPx1) and Catalase in the peripheral blood of K/BxN serum-induced C57Bl76 mice in comparison to mock-treated controls. Serum-induced mice were either exposed to radon or were mock-treated (w/o) on day 3. After 7 days (day 10), mice were sacrificed and peripheral blood was collected and subjected to RNA isolation and quantitative real-time PCR to measure the expression of <span class="html-italic">Superoxide Dismutase 1</span> (<span class="html-italic">SOD1</span>) (<b>A</b>), <span class="html-italic">Glutathione Peroxidase 1</span> (<span class="html-italic">GPx1</span>) (<b>B</b>), <span class="html-italic">Catalase</span> (<b>C</b>) and <span class="html-italic">Nuclear factor-erythroid-2-related factor 2</span> (<span class="html-italic">Nrf2</span>) (<b>D</b>). Data are presented as Median + IQR and were derived from 2 independent experiments n<sub>control</sub> = 7; n<sub>Radon</sub> = 9. Statistics were calculated using Mann-Whitney-U test.</p>
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<p>Inhalation of radon gas results only in minor immune cell alterations of the bone marrow in exposed mice. Bone marrow of the long bones of K/BxN serum-induced age and sex matched C57Bl/6 mice was collected, followed by lysis of erythrocytes and multicolor flow cytometry analysis for immune cell subtypes was carried out (<b>A</b>–<b>I</b>). Depicted are various immune cell subsets of radon and mock treated mice. Data shows two independent experiments with in total n<sub>control</sub> = 7; n<sub>Radon</sub> = 9 mice/group and is presented as Median + IQR. Statistics were calculated using Mann-Whitney-U test.</p>
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<p>Inhalation of radon gas results in immune cell alterations in the peripheral blood of radon treated mice. Peripheral blood of K/BxN serum-induced age and sex matched C57Bl/6 mice was collected, followed by lysis of erythrocytes and subsequent multicolor flow cytometry analysis for immune cell subtypes was carried out (<b>A</b>–<b>I</b>). Depicted are various immune cell subsets of radon and mock treated mice. Data shows one independent experiment with in total n<sub>control</sub> = 4; n<sub>Radon</sub> = 5 mice/group and is presented as Median + IQR. Statistics were calculated using Mann-Whitney-U test (* <span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Exposure to radon gas for one hour results in altered cytokine expression levels in the serum of treated animals in comparison to mock treated animals. Serum of K/BxN serum-induced age and sex matched C57Bl/6 mice was collected and analyzed via MSD Multiplex Assay (<b>A</b>–<b>J</b>). Data shows cytokine levels of two independent experiments with in total n<sub>control</sub> = 7; n<sub>Radon</sub> = 9 mice/group and is presented as Median + IQR. Statistics were calculated using Mann-Whitney-U test (* <span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Ex vivo treatment of bone marrow cells as well as monocytes and macrophages with radon does not enhance cell death. Isolated bone marrow cells (168 kBq/m<sup>3</sup>) from C57Bl/6 mice as well as bone marrow-derived monocytes and macrophages (177 kBq/m<sup>3</sup>) were exposed to radon or mock treatment for one hour under standard cell culture conditions. 24 h after treatment, cells were collected, stained for AxV/PI and subsequently analyzed (<b>A</b>–<b>C</b>). For bone marrow cells (<b>D</b>–<b>F</b>), 4 cell pools, each derived from 10 C57Bl/6 mice, were exposed in two independently performed experiments. For monocytes (<b>G</b>–<b>I</b>) and macrophages (<b>J</b>–<b>L</b>), 3 and 4 cell pools were exposed in 3 independently carried out experiments. Data is presented as Median + IQR. Statistics were calculated using Mann-Whitney-U test.</p>
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<p>Ex vivo radon treatment of bone-marrow derived monocytes and macrophages has no effects on the anti-oxidative system while stimulation with TNF-α induces significant effects in macrophages. C57Bl/6 bone marrow-derived monocytes and macrophages were exposed to radon (177 kBq/m<sup>3</sup>) or mock treatment for one hour under standard cell culture conditions. 24 h after radon treatment, cells were collected, and either treated with DCF solution (<b>C</b>,<b>I</b>), stained with antibodies for multicolor flow cytometry analysis (<b>A</b>,<b>B</b>,<b>H</b>), or were collected for subsequent RNA analysis (<b>D</b>–<b>G</b>,<b>J</b>–<b>M</b>). Monocytes (<b>A</b>–<b>G</b>) and macrophages (<b>H</b>–<b>M</b>), 3 and 4 cell pools, respectively, were exposed in 3 independently carried out experiments. Data is presented as Median + IQR. Statistics were calculated using Mann-Whitney-U test (* <span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Ex vivo radon treatment of bone-marrow derived from C57Bl/6 mice has no effects on the anti-oxidative system. C57Bl/6-derived bone marrow was exposed to radon for 1 h at standard cell culture conditions. The expression of <span class="html-italic">SOD1</span> (<b>A</b>), <span class="html-italic">GPx1</span> (<b>B</b>), <span class="html-italic">Catalase</span> (<b>C</b>) and <span class="html-italic">Nrf2</span> (<b>D</b>) was measured by qPCR 24 h after radon exposure of bone marrow. 4 cell pools were exposed in 2 independent experiments. Data is presented as Median + IQR. Statistics were calculated using Mann-Whitney-U test.</p>
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<p>Ex vivo treatment of bone marrow cells with radon significantly increases B cell numbers and CD34+ hematopoietic precursor cells. Isolated bone marrow cells derived from C57Bl/6 mice were exposed to radon (168 kBq/m<sup>3</sup>) or mock treatment for one hour under standard cell culture conditions. 24 h after treatment cells were collected, stained for multicolor flow-cytometry analysis and subsequently analyzed (<b>A</b>–<b>K</b>). Four cell pools, each derived from 10 C57Bl/6 mice, were exposed in two independently performed experiments. Data is presented as Median + IQR. Statistics were calculated using Mann-Whitney-U test (* <span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Exemplary gating strategy for 2′,7′-Dichlordihydrofluorescein-diacetat (DCF) measurements. For each condition, two wells were prepared, treated and washed. One well was subsequently treated with 2 µM DCF (+DCF), while the other one received 0 µM DCF (−DCF), followed by a 90 min incubation period at standard culture conditions in the dark. Afterward, cells were detached and DCF activity was measured. For analysis, ∆MFI was calculated from MFI(+DCF)–MFI(−DCF).</p>
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<p>Exemplary gating strategy for ex vivo differentiated monocytes and macrophages. In both cases, singlets were pre-chosen, followed by gating of viable cells and a live/dead control using Zombie NIR. Afterward, cells were gated for F4/80+ or F4/80−. In case of monocytes, F4/80− cells were further classified into “classical” (F4/80-CD11b+Ly6C+) or “non-classical” (F4/80-CD11b+Ly6C−) monocytes. In case of ex vivo differentiated macrophages, F4/80+CD11b+ cells were considered to be macrophages.</p>
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25 pages, 5004 KiB  
Article
Extracellular Vesicles Derived from Bone Marrow in an Early Stage of Ionizing Radiation Damage Are Able to Induce Bystander Responses in the Bone Marrow
by Dávid Kis, Ilona Barbara Csordás, Eszter Persa, Bálint Jezsó, Rita Hargitai, Tünde Szatmári, Nikolett Sándor, Enikő Kis, Katalin Balázs, Géza Sáfrány and Katalin Lumniczky
Cells 2022, 11(1), 155; https://doi.org/10.3390/cells11010155 - 4 Jan 2022
Cited by 7 | Viewed by 3067
Abstract
Ionizing radiation (IR)-induced bystander effects contribute to biological responses to radiation, and extracellular vesicles (EVs) play important roles in mediating these effects. In this study we investigated the role of bone marrow (BM)-derived EVs in the bystander transfer of radiation damage. Mice were [...] Read more.
Ionizing radiation (IR)-induced bystander effects contribute to biological responses to radiation, and extracellular vesicles (EVs) play important roles in mediating these effects. In this study we investigated the role of bone marrow (BM)-derived EVs in the bystander transfer of radiation damage. Mice were irradiated with 0.1Gy, 0.25Gy and 2Gy, EVs were extracted from the BM supernatant 24 h or 3 months after irradiation and injected into bystander mice. Acute effects on directly irradiated or EV-treated mice were investigated after 4 and 24 h, while late effects were investigated 3 months after treatment. The acute effects of EVs on the hematopoietic stem and progenitor cell pools were similar to direct irradiation effects and persisted for up to 3 months, with the hematopoietic stem cells showing the strongest bystander responses. EVs isolated 3 months after irradiation elicited no bystander responses. The level of seven microRNAs (miR-33a-3p, miR-140-3p, miR-152-3p, miR-199a-5p, miR-200c-5p, miR-375-3p and miR-669o-5p) was altered in the EVs isolated 24 hour but not 3 months after irradiation. They regulated pathways highly relevant for the cellular response to IR, indicating their role in EV-mediated bystander responses. In conclusion, we showed that only EVs from an early stage of radiation damage could transmit IR-induced bystander effects. Full article
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Graphical abstract

Graphical abstract
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<p>Gating Strategy for the Identification of Different Bone Marrow Cell Populations by Flow Cytometry. (<b>A</b>) Lymphoid progenitor cells were identified as CD45 and CD90.2 double positive cells in bone marrow cells. (<b>B</b>) Granulocytes/monocytes were characterized as Gr1 and CD11b double positive cells in bone marrow cells. (<b>C</b>) Hematopoietic stem and progenitor cells were identified as Sca1 and cKit double positive cells in the Lineage negative bone marrow cells. (<b>D</b>) Hematopoietic stem cell subpopulation characterization was done by using the CD34 and CD135 markers in the hematopoietic stem and progenitor cell pool. Long-term hematopoietic stem cells were identified as CD34 and CD135 double negative cells, short-term hematopoietic stem cells were CD34+CD135- cells, multipotent progenitors were characterized as CD34 and CD135 double positive cells. (<b>E</b>) Mesenchymal stem and stromal cells were identified as Sca1 and CD44 double positive cells in the lineage-negative bone marrow cells.</p>
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<p>Characterization of Bone Marrow-Derived Extracellular Vesicles. (<b>A</b>) Representative transmission electron microscopy images of extracellular vesicles isolated from the bone marrow of mice irradiated with the indicated doses of ionizing radiation. (<b>B</b>) Size and concentration of extracellular vesicle suspensions were examined by tunable resistance pulse sensing. Mean values of extracellular vesicle size (<b>B/1</b>) and mean extracellular vesicle particle numbers released by 10<sup>6</sup> bone marrow cells (<b>B/2</b>) are shown with bars representing standard deviations (SD). <span class="html-italic">n</span> = 3, significance tested by Student’s <span class="html-italic">t</span>-test, <span class="html-italic">p</span> * &lt; 0.05. (<b>C</b>) Representative Western blot analysis of whole cell lysates and extracellular vesicles isolated from the bone marrow of mice irradiated with the indicated doses of ionizing radiation. Lane 1: protein ladder, lane 2: whole cell lysate, lane 3: extracellular vesicle sample from unirradiated mice, lane 4-5: extracellular vesicle samples from mice irradiated with 0.1Gy and 2Gy.</p>
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<p>Schematic Presentation of the Workflow of the Study. C57Bl/6 mice were total-body irradiated with different doses (0Gy, 0.1Gy, 0.25Gy and 2Gy) of ionizing radiation. Mice were humanely killed 4 h, 24 h or 3 months later and the bone marrow and spleen were collected. Bone marrow-derived extracellular vesicles were isolated from the bone marrow supernatant of age-matched control and irradiated mice. Bystander effects were monitored in non-irradiated, healthy mice after injecting them with bone marrow-derived extracellular vesicles. Bystander mice were humanely killed 4 h, 24 h or 3 months after extracellular vesicle injection and the same organs were harvested as from the directly irradiated animals. Apoptosis in the bone marrow stem and progenitor cells was measured by TUNEL assay. Bone marrow hematopoietic stem, progenitor and stromal cell subpopulations were characterized phenotypically by flow cytometry. MiRNA expression of BM-EVs was investigated by qRT-PCR.</p>
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<p>Bone Marrow-Derived Extracellular Vesicles from Irradiated Mice Induce Long-Term Hematopoietic Stem Cell Depletion in the Bone Marrow of Bystander Mice. Irradiation, extracellular vesicle treatment and bone marrow cell phenotyping were carried out as described in the Materials and Methods section. Lin-Sca1+cKit+ cells were considered hematopoietic stem cells. (<b>A</b>) Relative changes in hematopoietic stem cell numbers were evaluated 24 h after treatment. Grey bars represent total-body irradiated mice; red bars represent mice treated with bone marrow-derived extracellular vesicles isolated from mice 24 h after irradiation; yellow bars represent mice treated with bone marrow-derived extracellular vesicles isolated from mice 3 months after irradiation. (<b>B</b>) Relative changes in hematopoietic stem cell numbers were evaluated 3 months after treatment. Grey bars represent total-body irradiated mice; blue bars represent mice treated with bone marrow-derived extracellular vesicles isolated from mice 24 h after irradiation. Bars represent mean values of relative hematopoietic stem cell numbers, dots show individual values, error bars represent SD. <span class="html-italic">n</span> = 6–11, significance tested by Student’s <span class="html-italic">t</span>-test, <span class="html-italic">p</span> ** &lt; 0.01, <span class="html-italic">p</span> *** &lt; 0.001.</p>
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<p>Both Direct Irradiation and Extracellular Vesicles-Mediated Bystander Effects Induce Persistent Redistribution between Multipotent Progenitors and Long-Term Hematopoietic Stem Cells in the Bone Marrow. Irradiation, extracellular vesicle treatment and bone marrow cell phenotyping was carried out as described in the Materials and Methods section. Cellular subpopulations were identified as Lin-Sca1+cKit+CD34-CD135- long-term hematopoietic stem cells (LT-HSC), Lin-Sca1+cKit+CD34+CD135- short-term hematopoietic stem cells (ST-HSC) and Lin-Sca1+cKit+CD34+CD135+ multipotent progenitor cells (MPP) [<a href="#B36-cells-11-00155" class="html-bibr">36</a>,<a href="#B37-cells-11-00155" class="html-bibr">37</a>] and were measured by flow cytometry. (<b>A</b>) multipotent progenitors; (<b>C</b>) short-term hematopoietic stem cells; (<b>E</b>) long-term hematopoietic stem cells. Relative changes in hematopoietic stem cell subpopulations were evaluated 24 h after treatment. Grey bars represent total-body irradiated mice; red bars represent mice treated with bone marrow-derived extracellular vesicles isolated from mice 24 h after irradiation; yellow bars represent mice treated with bone marrow-derived extracellular vesicles isolated from mice 3 months after irradiation. (<b>B</b>) multipotent progenitors; (<b>D</b>) short-term hematopoietic stem cells; (<b>F</b>) long-term hematopoietic stem cells. Relative changes in hematopoietic stem cell numbers were evaluated 3 months after treatment. Grey bars represent total-body irradiated mice; blue bars represent mice treated with bone marrow-derived extracellular vesicles isolated from mice 24 h after irradiation. (<b>G</b>) Distribution of individual subpopulations within the hematopoietic stem and progenitor cells was evaluated 24 h after treatment. Left columns represent total-body irradiated mice; middle columns represent mice treated with bone marrow-derived extracellular vesicles isolated from mice 24 h after irradiation; right columns represent mice treated with bone marrow-derived extracellular vesicles isolated from mice 3 months after irradiation. (<b>H</b>) Distribution of individual subpopulations within the hematopoietic stem and progenitor cells was evaluated 3 months after treatment. Left columns represent total-body irradiated mice; right columns represent mice treated with bone marrow-derived extracellular vesicles, which were isolated from mice 24 h after irradiation. Bars represent mean fraction of subpopulations, dots show individual values, error bars represent SD. <span class="html-italic">n</span> = 6–8, significance tested for individual subtypes of hematopoietic stem cells by Student’s <span class="html-italic">t</span>-test, <span class="html-italic">p</span> * &lt; 0.05, <span class="html-italic">p</span> ** &lt; 0.01, <span class="html-italic">p</span> *** &lt; 0.001.</p>
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<p>High Dose Irradiation Induces Persistent Depression of Lymphoid Progenitor Numbers, Extracellular Vesicle-Mediated Mild Bystander Effects Develop Late after Treatment. Irradiation, extracellular vesicle treatment and bone marrow cell phenotyping was carried out as described in the Materials and methods section. CD45+CD90.2+ cells were considered lymphoid progenitors. (<b>A</b>) Relative changes in lymphoid progenitor numbers were evaluated 24 h after treatment. Grey bars represent total-body irradiated mice; red bars represent mice treated with bone marrow-derived extracellular vesicles isolated from mice 24 h after irradiation; yellow bars represent mice treated with bone marrow-derived extracellular vesicles isolated from mice 3 months after irradiation. (<b>B</b>) Relative changes in lymphoid progenitor numbers were evaluated 3 months after treatment. Grey bars represent total-body irradiated mice; blue bars represent mice treated with bone marrow-derived extracellular vesicles isolated from mice 24 h after irradiation Bars represent mean values of relative lymphoid progenitor numbers, dots show individual values, error bars represent SD. <span class="html-italic">n</span> = 5–11, significance tested by Student’s <span class="html-italic">t</span>-test, <span class="html-italic">p</span> * &lt; 0.05, <span class="html-italic">p</span> ** &lt; 0.01, <span class="html-italic">p</span> *** &lt; 0.001.</p>
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<p>Long-Term Direct Radiation and Extracellular Vesicles-Mediated Bystander Effects on the Granulocytes/Monocytes Pool Are More Severe than Acute Effects. Irradiation, extracellular vesicles treatment and bone marrow cell phenotyping was carried out as described in the Materials and methods section. Gr1+CD11b+ cells were considered granulocytes/monocytes. (<b>A</b>) Relative changes in the numbers of granulocytes/monocytes were evaluated 24 h after treatment. Grey bars represent total-body irradiated mice; red bars represent mice treated with bone marrow-derived extracellular vesicles isolated from mice 24 h after irradiation; yellow bars represent mice treated with bone marrow-derived extracellular vesicles isolated from mice 3 months after irradiation. (<b>B</b>) Relative changes in the numbers of granulocytes/monocytes were evaluated 3 months after treatment. Grey bars represent total-body irradiated mice; blue bars represent mice treated with bone marrow-derived extracellular vesicles isolated from mice 24 h after irradiation. Bars represent mean values of relative granulocytes/monocytes progenitor numbers, dots show individual values, error bars represent SD. <span class="html-italic">n</span> = 5–11, significance tested by Student’s <span class="html-italic">t</span>-test, <span class="html-italic">p</span> * &lt; 0.05, <span class="html-italic">p</span> ** &lt; 0.01, <span class="html-italic">p</span> *** &lt; 0.001.</p>
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<p>Short-Term Direct Irradiation and Extracellular Vesicle-Mediated Bystander Effects Affecting the Mesenchymal Stromal Cell Pool Are More Severe than Long-Term Effects. Irradiation, extracellular vesicle treatment and bone marrow cell phenotyping was done as described in the Materials and Methods section. Lin-Sca1+CD44+ cells were considered mesenchymal stromal cells. (<b>A</b>) Relative changes in mesenchymal stromal cell numbers were evaluated 24 h after treatment. Grey bars represent total-body irradiated mice; red bars represent mice treated with bone marrow-derived extracellular vesicles isolated from mice 24 h after irradiation; yellow bars represent mice treated with bone marrow-derived extracellular vesicles isolated from mice 3 months after irradiation. (<b>B</b>) Relative changes in mesenchymal stromal cell numbers were evaluated 3 months after treatment. Grey bars represent total-body irradiated mice; blue bars represent mice treated with bone marrow-derived extracellular vesicles isolated from mice 24 h after irradiation. Bars represent mean values of relative mesenchymal stromal cell numbers, dots show individual values, error bars represent SD. <span class="html-italic">n</span> = 5–11, significance tested by Student’s <span class="html-italic">t</span>-test, <span class="html-italic">p</span> * &lt; 0.05, <span class="html-italic">p</span> ** &lt; 0.01, <span class="html-italic">p</span> *** &lt; 0.001.</p>
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<p>Transfer of Bone Marrow-Derived Extracellular Vesicles from Irradiated Mice Is Able to Induce Apoptosis in a Bystander Manner in Hematopoietic Stem Cells and Lymphoid Progenitors but Not Mesenchymal Stromal Cells. Bone-marrow single-cell suspension was prepared from directly irradiated and extracellular vesicle-treated mice 4 h after treatment and apoptosis was measured by the Tunnel assay as described in the Materials and Methods section. The relative change in apoptosis frequency compared to control mice (either non-irradiated or treated with extracellular vesicles originating from the bone marrow of non-irradiated mice) is shown for hematopoietic stem and progenitor cells (<b>A</b>), lymphoid progenitors (<b>B</b>) and mesenchymal stromal cells (<b>C</b>). Mean, minimum and maximum values are shown, error bars represent SD. <span class="html-italic">n</span> = 4, significance tested by Student’s <span class="html-italic">t</span>-test, <span class="html-italic">p</span> * &lt; 0.05; <span class="html-italic">p</span> ** &lt; 0.01.</p>
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<p>Bone Marrow-Derived Extracellular Vesicles from Low-Dose Irradiated Mice Induce Migration of Hematopoietic Stem and Progenitor Cells into the Spleen in Bystander Mice. Lin-Sca1+cKit+ hematopoietic stem and progenitor cells (<b>A</b>) and Lin-Sca1+CD44+ mesenchymal stromal cells (<b>B</b>) in the spleen were measured by flow cytometry 24 h after irradiation or injection of extracellular vesicles. Isolation of spleens and splenocyte phenotyping was performed as described in the Materials and Methods section. Black bars represent positive control mice treated with AMD3100. Mean, minimum and maximum values are shown, error bars represent SD. <span class="html-italic">N</span> = 6, significance tested by Student’s <span class="html-italic">t</span>-test, <span class="html-italic">p</span> * &lt; 0.05, <span class="html-italic">p</span> ** &lt; 0.01, <span class="html-italic">p</span> *** &lt; 0.001.</p>
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<p>Pathways Related to Cellular Response to Ionizing Radiation Prevail in miRNAs Differentially Expressed in the Bone Marrow-Derived Extracellular Vesicles Isolated from Mice 24 Hours after Irradiation. (<b>A</b>) Extracellular vesicles were isolated from the bone marrow of mice, miRNAs purified from extracellular vesicles and the relative concentration of miRNAs was measured by qRT-PCR as described in the Materials and Methods section. <span class="html-italic">n</span> = 3; * indicate significant changes (<span class="html-italic">p</span> ˂ 0.05) compared to control (0Gy extracellular vesicles samples). Arrows show increased (red arrows) or decreased (green arrows) expression in miRNAs from extracellular vesicles isolated 24 h or 3 months after irradiation. (<b>B</b>) KEGG analysis of differentially expressed 7 miRNAs in murine bone marrow-derived extracellular vesicles isolated 24 h but not 3 months after irradiation. A pathway was considered significant if the <span class="html-italic">p</span>-value was ˂0.05 (−log10 (0.05) indicated by the dashed line.</p>
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<p>The Influence of Differentially Expressed miRNAs on the Wnt Pathway. The Wnt signal transduction pathway is an evolutionary conserved pathway regulating basic developmental processes such as progenitor cell proliferation and cell-fate specification [<a href="#B63-cells-11-00155" class="html-bibr">63</a>], and it can be downregulated upon exposure to ionizing radiation [<a href="#B64-cells-11-00155" class="html-bibr">64</a>,<a href="#B65-cells-11-00155" class="html-bibr">65</a>]. The transcription of Wnt-related genes can be regulated by the cytoplasmic concentration of the β-catenin intracellular signal transducer. Without Wnt ligand binding to its receptor Frizzeld [<a href="#B66-cells-11-00155" class="html-bibr">66</a>] and co-receptor low-density lipoprotein receptor related 5/6 (LRP5/6) [<a href="#B67-cells-11-00155" class="html-bibr">67</a>,<a href="#B68-cells-11-00155" class="html-bibr">68</a>,<a href="#B69-cells-11-00155" class="html-bibr">69</a>], β-catenin is degraded by a destruction complex, which contains axin, adenomatosus polyposis coli (APC), protein phosphatase 2A (PP2A), and two kinases, glycogen synthase kinase 3 (GSK3) and casein kinase 1α (CK1α). In the β-catenin destruction complex, GSK3 and CK1α phosphorylate β-catenin, leading to its proteosomal degradation [<a href="#B70-cells-11-00155" class="html-bibr">70</a>]. Upon Wnt ligand binding, the destruction complex is disrupted, and β-catenin is enriched in the cytoplasm, which leads to its nuclear transport. In the nucleus β-catenin binds to lymphoid enhancer-binding factor/T-cell factor (LEF/TCF) proteins, transforming it into a transcriptional activator, leading to the transcription of Wnt target genes [<a href="#B71-cells-11-00155" class="html-bibr">71</a>,<a href="#B72-cells-11-00155" class="html-bibr">72</a>,<a href="#B73-cells-11-00155" class="html-bibr">73</a>,<a href="#B74-cells-11-00155" class="html-bibr">74</a>]. In the absence of β-catenin, LEF/TCF block the transcription of Wnt target genes [<a href="#B75-cells-11-00155" class="html-bibr">75</a>,<a href="#B76-cells-11-00155" class="html-bibr">76</a>]. The Wnt pathway was targeted by multiple differentially expressed miRNAs in the extracellular vesicles. Targets were associated with miRNAs by Diana mirPath v.3. We present our hypothesis of how differentially expressed miRNAs in 2Gy extracellular vesicles isolated from the bone marrow of mice 24 h after irradiation lead to the repression of the Wnt pathway. MiRNAs mostly repress the expression of their targets, so Wnt components targeted by upregulated miRNAs (illustrated in red in the Figure) are supposed to be downregulated, and components targeted by downregulated miRNAs (illustrated in green in the Figure) are supposed to be upregulated, compared to 0Gy samples. Arrows indicate the possible changes in the expression of the proteins targeted by the differentially expressed miRNAs: a red down arrow indicates decreased expression upon miRNA interaction, and a green up arrow indicates increased target expression. Mmu-miR-33-3p, a down-regulated miRNA targets a secreted frizzled-related protein 2 (Sfrp2), which is a Wnt antagonist [<a href="#B77-cells-11-00155" class="html-bibr">77</a>,<a href="#B78-cells-11-00155" class="html-bibr">78</a>], and nemo-like kinase (NLK), which is an inactivator of β-catenin TCF/LEF transcription complex formation [<a href="#B79-cells-11-00155" class="html-bibr">79</a>]. The other downregulated miRNA mmu-miR-669o-5p interacts with the BMP and Activin Membrane Bound Inhibitor (Bambi) which can both up- and downregulate the β-catenin signaling [<a href="#B80-cells-11-00155" class="html-bibr">80</a>,<a href="#B81-cells-11-00155" class="html-bibr">81</a>,<a href="#B82-cells-11-00155" class="html-bibr">82</a>]. The upregulated mmu-miR-152-3p targets Wnt10b, which promotes the β-catenin-dependent Wnt signaling pathway [<a href="#B83-cells-11-00155" class="html-bibr">83</a>]. Wnt receptors are also targeted by upregulated miRNAs: LRP5 by mm-miR-375-3p and FZD5 by mmu-miR-199a-5p. Two Wnt inhibitors are targeted by two upregulated miRNAs: GSK-3β by mmu-miR-199a-5p, and Ctbp2 by mmu-miR-375-3p. Ctbp2 is originally an inhibitor, but some studies indicate that it may also act as an activator of TCF [<a href="#B84-cells-11-00155" class="html-bibr">84</a>,<a href="#B85-cells-11-00155" class="html-bibr">85</a>]. Thus, it can be seen that downregulated miRNAs target inhibitors, while upregulated miRNAs mostly target Wnt pathway initiators, which indicate an overall downregulation of the Wnt signaling pathway.</p>
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20 pages, 3316 KiB  
Article
ROS- and Radiation Source-Dependent Modulation of Leukocyte Adhesion to Primary Microvascular Endothelial Cells
by Denise Eckert, Felicitas Rapp, Ayele T. Tsedeke, Jessica Molendowska, Robert Lehn, Markus Langhans, Claudia Fournier, Franz Rödel and Stephanie Hehlgans
Cells 2022, 11(1), 72; https://doi.org/10.3390/cells11010072 - 27 Dec 2021
Cited by 15 | Viewed by 3221
Abstract
Anti-inflammatory effects of low-dose irradiation often follow a non-linear dose–effect relationship. These characteristics were also described for the modulation of leukocyte adhesion to endothelial cells. Previous results further revealed a contribution of reactive oxygen species (ROS) and anti-oxidative factors to a reduced leukocyte [...] Read more.
Anti-inflammatory effects of low-dose irradiation often follow a non-linear dose–effect relationship. These characteristics were also described for the modulation of leukocyte adhesion to endothelial cells. Previous results further revealed a contribution of reactive oxygen species (ROS) and anti-oxidative factors to a reduced leukocyte adhesion. Here, we evaluated the expression of anti-oxidative enzymes and the transcription factor Nrf2 (Nuclear factor-erythroid-2-related factor 2), intracellular ROS content, and leukocyte adhesion in primary human microvascular endothelial cells (HMVEC) upon low-dose irradiation under physiological laminar shear stress or static conditions after irradiation with X-ray or Carbon (C)-ions (0–2 Gy). Laminar conditions contributed to increased mRNA expression of anti-oxidative factors and reduced ROS in HMVEC following a 0.1 Gy X-ray and 0.5 Gy C-ion exposure, corresponding to reduced leukocyte adhesion and expression of adhesion molecules. By contrast, mRNA expression of anti-oxidative markers and adhesion molecules, ROS, and leukocyte adhesion were not altered by irradiation under static conditions. In conclusion, irradiation of endothelial cells with low doses under physiological laminar conditions modulates the mRNA expression of key factors of the anti-oxidative system, the intracellular ROS contents of which contribute at least in part to leucocyte adhesion, dependent on the radiation source. Full article
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Figure 1
<p>Low-dose X-irradiation modulates mRNA expression of anti-oxidative factors, reduces reactive oxygen species (ROS) and peripheral blood lymphocyte (PBL) adhesion to human microvascular endothelial cells (HMVEC) under laminar conditions. (<b>A</b>) Experimental setup: For analysis of the mRNA expression of anti-oxidative factors, ROS and adhesion molecules under shear stress, laminar flow conditions were applied 24 h after plating of HMVEC. (<b>B</b>) After irradiation and stimulation with tumor necrosis factor alpha (TNF-α), RNA was isolated to measure the mRNA expression of anti-oxidative factors (Nrf2, Nuclear factor-erythroid-2-related factor 2; SOD1, Superoxide Dismutase 1; GPx1, Glutathione Peroxidase 1; Catalase) by quantitative PCR (qPCR) (mean + SEM; <span class="html-italic">n</span> = 6) or (<b>C</b>) cells were subjected to analysis of ROS by flow cytometry (mean + SEM; <span class="html-italic">n</span> = 8–12). (<b>D</b>) For adhesion assays, PBL were added 23.5 h after irradiation and adhesion was allowed for 0.5 h under constant laminar flow. (<b>E</b>) Measurement of adhesion molecules vascular cellular adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1, and E-selectin expression was performed by qPCR (mean + SEM; <span class="html-italic">n</span> = 6). (<b>F</b>) For adhesion assays, HMVEC were plated on glass cover slips, PBL were stained with CellTracker Green CMFDA (5-chloromethylfluorescein diacetate) and added 23.5 h after irradiation for 30 min to the medium reservoir under permanent laminar flow. HMVEC cells and adhered PBL were fixed and stained with 4′,6-diamidino-2-dhenylindol (DAPI) and phalloidin-tetramethylrhodamine B isothiocyanate (phalloidin-TRITC). PBL adhesion was evaluated by microscopic counting of PBL relative to the number of endothelial cells (mean + SEM; <span class="html-italic">n</span> = 5–9). * <span class="html-italic">p</span> &lt; 0.05, Kruskal-Wallis test vs. 0 Gy.</p>
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<p>X-irradiation under static conditions does not modulate the mRNA expression of anti-oxidative enzymes in HMVEC cells nor PBL adhesion. (<b>A</b>) HMVEC were plated and incubated for 24 h. Medium was changed 24 h before irradiation and TNF-α treatment and ROS measurement and RNA isolation were performed at 24 h thereafter. (<b>B</b>) mRNA expression of anti-oxidative factors was evaluated by qPCR (mean + SEM; <span class="html-italic">n</span> = 5–6 * <span class="html-italic">p</span> &lt; 0.05; Kruskal-Wallis test vs. 0 Gy). (<b>C</b>) Intracellular ROS were measured by flow cytometry (mean + SEM; <span class="html-italic">n</span> = 12). (<b>D</b>) Schematic setup of adhesion experiments. (<b>E</b>) mRNA expression of indicated adhesion molecules was measured by qPCR (mean + SEM; <span class="html-italic">n</span> = 6). (<b>F</b>) Microscopic evaluation of PBL adhesion was performed after fixation and DAPI and phalloidin-TRITC staining and values, relative to non-irradiated samples are shown (mean + SEM; <span class="html-italic">n</span> = 9).</p>
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<p>X-irradiation under laminar conditions reduces adhesion molecule surface expression. (<b>A</b>) For analysis of adhesion molecule protein expression under laminar conditions, HMVEC were exposed to laminar shear stress for 24 h, followed by irradiation and TNF-α stimulation. At 24 h after irradiation, cells were collected for flow cytometric analysis of ICAM-1, VCAM-1 and E-Selectin surface expression. Mean fluorescence intensities (MFI) + SEM are shown relative to non-irradiated cells. <span class="html-italic">n</span> = 4; * <span class="html-italic">p</span> &lt; 0.05 (Mann-Whitney U test vs. +TNF-α). (<b>B</b>) For analysis of adhesion molecule protein expression under static conditions, HMVEC were plated, medium was changed and irradiation and TNF-α was applied. Adhesion molecule protein expression on the cell surface was measured by flow cytometry, 24 h after irradiation, and displayed as MFI relative to non-irradiated cells + SEM (<span class="html-italic">n</span> = 4).</p>
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<p>Carbon ion irradiation modulates the anti-oxidative defense of HMVEC cells and leukocyte adhesion. (<b>A</b>) Experimental settings: At 24 h after plating, HMVEC cells were exposed to laminar shear stress conditions. Irradiation with Carbon (C)-ions and TNF-α stimulation was applied. After 24 h, cells were collected, followed by ROS analysis and RNA isolation. (<b>B</b>) mRNA expression of anti-oxidative factors was measured by qPCR and values were displayed relative to non-irradiated cells (mean + SEM; <span class="html-italic">n</span> = 4; * <span class="html-italic">p</span> &lt; 0.05, Kruskal-Wallis test vs. 0 Gy). (<b>C</b>) ROS analysis was performed by flow cytometry (mean + SEM; <span class="html-italic">n</span> = 4). (<b>D</b>) For adhesion experiments, PBL were added 23.5 h after irradiation to the flow chamber and adhesion was subsequently measured by microscopic evaluation. (<b>E</b>) mRNA expression of indicated adhesion molecules relative to non-irradiated cells is shown (mean + SEM; <span class="html-italic">n</span> = 3–4). (<b>F</b>) Adhesion of PBL was calculated relative to non-irradiated samples (mean + SEM; <span class="html-italic">n</span> = 6; * <span class="html-italic">p</span> &lt; 0.05, one-way ANOVA vs. 0 Gy).</p>
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<p>C-ion irradiation moderately changes the mRNA expression of anti-oxidative factors, ROS and adhesion of HMVEC cells under static conditions. (<b>A</b>) Experimental time course of analysis of C-ion irradiation-induced changes of the anti-oxidative defense of HMVEC, cultured under normal static conditions. (<b>B</b>) Relative mRNA expression of Nrf2, SOD1, GPx1 and Catalase at 24 h after C-ion irradiation (mean + SEM; <span class="html-italic">n</span> = 8; * <span class="html-italic">p</span> &lt; 0.05, Kruskal-Wallis test vs. 0 Gy). (<b>C</b>) Intracellular ROS content, relative to non-irradiated cells, after C-ion irradiation of HMVEC with indicated doses (mean + SEM; <span class="html-italic">n</span> = 7–8). (<b>D</b>) Experimental setup of adhesion assays, performed 24 h after C-ion exposure. (<b>E</b>) Relative mRNA expression of indicated adhesion molecules after irradiation with C-ions (mean + SEM; <span class="html-italic">n</span> = 8; * <span class="html-italic">p</span> &lt; 0.05, Kruskal-Wallis test vs. 0 Gy). (<b>F</b>) Relative PBL adhesion at 24 h after irradiation of HMVEC cells with C-ions (mean + SEM; <span class="html-italic">n</span> = 2–6).</p>
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<p>Oxidative stress induces leukocyte adhesion to HMVEC under shear stress and Nrf2 activation decreases adhesion molecule surface expression. (<b>A</b>) Experimental scheme: HMVEC were plated and laminar shear stress was applied 24 h before TNF-α stimulation. After 22.5 h, cells were either treated with the ROS scavenger N-acetyl-L-cysteine (NAC) or left untreated. Oxidative stress was induced 1 h later by addition of hydrogen peroxide either without (H<sub>2</sub>O<sub>2</sub>) or with pretreatment with NAC (N+H<sub>2</sub>O<sub>2</sub>). (<b>B</b>) ROS measurement was subsequently acquired by flow cytometry (Mean + SEM; <span class="html-italic">n</span> = 6; * <span class="html-italic">p</span> &lt; 0.05, one-way ANOVA vs. 0 Gy). (<b>C</b>) For adhesion experiments, Cell Tracker Green-stained PBL were added to the medium reservoir of the flow chamber and adhesion to HMVEC was allowed for 30 min under laminar flow (Mean + SEM; <span class="html-italic">n</span> = 6; * <span class="html-italic">p</span> &lt; 0.05, one-way ANOVA vs. 0 Gy). (<b>D</b>) Exemplary pictures, depicting adhered PBL (CellTracker Green CMFDA (5-chloromethylfluorescein diacetate)), either alone or merged with nuclei (DAPI, blue) and F-actin staining of HMVEC cells (phalloidin-TRITC, red). Bar, 100 µm. (<b>E</b>) To analyze the impact of Nrf2 activation on adhesion molecule expression, HMVEC were plated, medium was changed after 24 h and cells were treated with TNF-α and DMSO or the Nrf2 activator AI-1. After 24 h, cells were fixed, stained for Nrf2 (AF488, green) and DAPI (blue). (<b>F</b>) Depicted are exemplary pictures, showing Nrf2 staining alone (green channel) or merged with nuclei (DAPI, blue). (<b>G</b>) Samples were evaluated microscopically for Nrf2 translocation, or (<b>H</b>) adhesion molecules ICAM-1, VCAM-1 and E-Selectin were measured by flow cytometry and values calculated as MFI + SEM relative to DMSO-treated cells. <span class="html-italic">n</span> = 4; * <span class="html-italic">p</span> &lt; 0.05 (Mann-Whitney U test vs. DMSO).</p>
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<p>Irradiation of primary endothelial cells (HMVEC) with X-ray and C-ions induces shear stress-dependently the expression of anti-oxidative factors, reduces ROS and decreases PBL adhesion at doses between 0.1 and 0.5 Gy only under physiological shear stress. (<b>A</b>) X-ray or carbon ion (C-ion) irradiation with low doses induces under laminar conditions the expression of the transcription factor Nrf2, expression of anti-oxidative enzymes (Anti-ox. enzymes), decreases adhesion molecule expression and PBL adhesion, thereby probably contributing to anti-inflammatory effects of low-dose radiation therapy. (<b>B</b>) In contrast, low-dose irradiation of HMVEC cells under normal, static cell culture conditions does not modulate the anti-oxidative system or leukocyte adhesion.</p>
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16 pages, 2965 KiB  
Article
Dose and Dose Rate-Dependent Effects of Low-Dose Irradiation on Inflammatory Parameters in ApoE-Deficient and Wild Type Mice
by Annegret Glasow, Ina Patties, Nicholas D. Priest, Ronald E. J. Mitchel, Guido Hildebrandt and Katrin Manda
Cells 2021, 10(11), 3251; https://doi.org/10.3390/cells10113251 - 20 Nov 2021
Cited by 3 | Viewed by 2650
Abstract
Anti-inflammatory low-dose therapy is well established, whereas the immunomodulatory impact of doses below 0.1 Gy is much less clear. In this study, we investigated dose, dose rate and time-dependent effects in a dose range of 0.005 to 2 Gy on immune parameters after [...] Read more.
Anti-inflammatory low-dose therapy is well established, whereas the immunomodulatory impact of doses below 0.1 Gy is much less clear. In this study, we investigated dose, dose rate and time-dependent effects in a dose range of 0.005 to 2 Gy on immune parameters after whole body irradiation (IR) using a pro-inflammatory (ApoE−/−) and a wild type mouse model. Long-term effects on spleen function (proliferation, monocyte expression) were analyzed 3 months, and short-term effects on immune plasma parameters (IL6, IL10, IL12p70, KC, MCP1, INFγ, TGFβ, fibrinogen, sICAM, sVCAM, sE-selectin/CD62) were analyzed 1, 7 and 28 days after Co60 γ-irradiation (IR) at low dose rate (LDR, 0.001 Gy/day) and at high dose rate (HDR). In vitro measurements of murine monocyte (WEHI-274.1) adhesion and cytokine release (KC, MCP1, IL6, TGFβ) after low-dose IR (150 kV X-ray unit) of murine endothelial cell (EC) lines (H5V, mlEND1, bEND3) supplement the data. RT-PCR revealed significant reduction of Ki67 and CD68 expression in the spleen of ApoE−/− mice after 0.025 to 2 Gy exposure at HDR, but only after 2 Gy at LDR. Plasma levels in wild type mice, showed non-linear time-dependent induction of proinflammatory cytokines and reduction of TGFβ at doses as low as 0.005 Gy at both dose rates, whereas sICAM and fibrinogen levels changed in a dose rate-specific manner. In ApoE−/− mice, levels of sICAM increased and fibrinogen decreased at both dose rates, whereas TGFβ increased mainly at HDR. Non-irradiated plasma samples revealed significant age-related enhancement of cytokines and adhesion molecules except for sICAM. In vitro data indicate that endothelial cells may contribute to systemic IR effects and confirm changes of adhesion properties suggested by altered sICAM plasma levels. The differential immunomodulatory effects shown here provide insights in inflammatory changes occurring at doses far below standard anti-inflammatory therapy and are of particular importance after diagnostic and chronic environmental exposures. Full article
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Figure 1
<p>IR dose-dependent expression of Ki67 and CD68 in the spleen of ApoE−/− mice, <span class="html-italic">n</span> = 4. (<b>A</b>,<b>B</b>) Relative expression levels of CD68 and Ki67 normalized on 18S rRNA in the spleen, mean-SEM, 3 months after IR at low dose rate (LDR) or high dose rate (HDR) are shown. Significant changes are indicated compared to 0 Gy control (=1) by asterisks (*, <span class="html-italic">p</span> ≤ 0.05; **, <span class="html-italic">p</span> ≤ 0.01; ***, <span class="html-italic">p</span> ≤ 0.001). (<b>C</b>) Mean Cycle threshold (Ct) values of house keeping genes, β2M and 18 S, are presented for all samples, mean ± SEM.</p>
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<p>IR dose-dependent effects on plasma parameters in mice, <span class="html-italic">n</span> = 10. Relative plasma levels on day 1, 7, 28 after IR are presented in wild type ApoE+/+ and ApoE−/− mice after IR at low dose rate (LDR) and high dose rate (HDR). Values are normalized on individual levels 7 days before IR and are presented relative to 0 Gy control of the corresponding time point (=1). Significant changes compared to 0 Gy group at the corresponding time point are indicated by asterisks (*, <span class="html-italic">p</span> ≤ 0.05; **, <span class="html-italic">p</span> ≤ 0.01; ***, <span class="html-italic">p</span> ≤ 0.001). Plasma levels for dose 0.5 and 2 Gy LDR, day 1, and 2 Gy LDR, day 7 are not available because these doses could not be delivered till the indicated time points due to the low dose rate. Missing bars in ApoE−/− mice represent plasma parameters below detection limit.</p>
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<p>Age-dependent changes of plasma parameters in non-irradiated mice. Relative concentration of 11 plasma markers is presented in 7, 8, 9 and 12-week-old, sham-irradiated 0 Gy control ApoE−/− and ApoE+/+ mice. Significant changes compared to 7-week group (=1) are indicated by asterisks (*, <span class="html-italic">p</span> ≤ 0.05; **, <span class="html-italic">p</span> ≤ 0.01; ***, <span class="html-italic">p</span> ≤ 0.001). Numbers in the bars represent the number of animals (sample size). Absolute plasma values of ApoE−/− and wild type ApoE+/+ mice at 12 weeks of life are given in the table (b.d. = below detection limit).</p>
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<p>Time dependent analysis of IL6 release in mlEnd1 cells after 16 h-prestimulation (<b>A</b>) and after costimulation (<b>B</b>) with TNFα. Relative IL6 values are presented (mean of duplicates ± SEM, <span class="html-italic">n</span> = 1) versus untreated control (=1). Relative adhesion values of monocytes (WEHI-274.1) on H5V, mlEND1, bEND3 ECs in (<b>C</b>) nonstimulated (0 Gy control = 100%) and in (<b>D</b>) TNFα costimulated cells, (TNFα-treated 0 Gy control = 100%), <span class="html-italic">n</span> = 3. Asterisks shows significant enhancement vs. control in the bEND3 cell line (**, <span class="html-italic">p</span> ≤ 0.01). <span class="html-italic">p</span>-values of combined analysis of all three cell lines are shown in blue.</p>
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<p>Concentration of TGFβ, KC, IL6 and MCP1 in supernatants of H5V, mlEnd1 and bEND3 endothelial cell lines. (<b>A</b>) Effect of TNFα (5 ng/mL) compared to untreated control, absolute values. (<b>B</b>,<b>C</b>) Dose-dependent effects of IR without (<b>B</b>) TNFα costimulation, relative to 0 Gy control samples (=1), and with TNFα costimulation (<b>C</b>) relative to TNFα-stimulated, 0 Gy control samples (=1) 24 h after IR. Asterisks indicate significant differences for each cell line (*, <span class="html-italic">p</span> ≤ 0.05; **, <span class="html-italic">p</span> ≤ 0.01; 3 experiments per cell line, each in quadruplicates).</p>
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17 pages, 3755 KiB  
Article
Cisplatin Reduces the Frequencies of Radiotherapy-Induced Micronuclei in Peripheral Blood Lymphocytes of Patients with Gynaecological Cancer: Possible Implications for the Risk of Second Malignant Neoplasms
by Aneta Węgierek-Ciuk, Anna Lankoff, Halina Lisowska, Piotr Kędzierawski, Pamela Akuwudike, Lovisa Lundholm and Andrzej Wojcik
Cells 2021, 10(10), 2709; https://doi.org/10.3390/cells10102709 - 9 Oct 2021
Cited by 4 | Viewed by 2665
Abstract
Gynaecologic cancers are common among women and treatment includes surgery, radiotherapy or chemotherapy, where the last two methods induce DNA damage in non-targeted cells like peripheral blood lymphocytes (PBL). Damaged normal cells can transform leading to second malignant neoplasms (SMN) but the level [...] Read more.
Gynaecologic cancers are common among women and treatment includes surgery, radiotherapy or chemotherapy, where the last two methods induce DNA damage in non-targeted cells like peripheral blood lymphocytes (PBL). Damaged normal cells can transform leading to second malignant neoplasms (SMN) but the level of risk and impact of risk modifiers is not well defined. We investigated how radiotherapy alone or in combination with chemotherapy induce DNA damage in PBL of cervix and endometrial cancer patients during therapy. Blood samples were collected from nine endometrial cancer patients (treatment with radiotherapy + chemotherapy—RC) and nine cervical cancer patients (treatment with radiotherapy alone—R) before radiotherapy, 3 weeks after onset of radiotherapy and at the end of radiotherapy. Half of each blood sample was irradiated ex vivo with 2 Gy of gamma radiation in order to check how therapy influenced the sensitivity of PBL to radiation. Analysed endpoints were micronucleus (MN) frequencies, apoptosis frequencies and cell proliferation index. The results were characterised by strong individual variation, especially the MN frequencies and proliferation index. On average, despite higher total dose and larger fields, therapy alone induced the same level of MN in PBL of RC patients as compared to R. This result was accompanied by a higher level of apoptosis and stronger inhibition of cell proliferation in RC patients. The ex vivo dose induced fewer MN, more apoptosis and more strongly inhibited proliferation of PBL of RC as compared to R patients. These results are interpreted as evidence for a sensitizing effect of chemotherapy on radiation cytotoxicity. The possible implications for the risk of second malignant neoplasms are discussed. Full article
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Graphical abstract

Graphical abstract
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<p>Timeline of treatment and blood collection. Blue and red boxes represent treatments, green boxes represent blood collection. Numbers 0–44 represent days of treatment. Chemotherapy was always given concomitantly to radiotherapy. One row represents one patient. Numbers in the patient rows represent white blood cell counts in 10<sup>9</sup>/L.</p>
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<p>Individual micronucleus frequencies in lymphocytes of RC (<b>A</b>) and R (<b>B</b>) patients. Each line represents a single donor. Lines are shown to facilitate identification of donors and not to represent a time response. Boxes show 95% confidence intervals of the respective mean value. Blue boxes: values induced by therapy alone. Red boxes: values induced by therapy plus 2 Gy ex vivo irradiation. Dashed lines represent patients RC1, RC6 and RC8 who received colony stimulating factors (see <a href="#cells-10-02709-f001" class="html-fig">Figure 1</a>). 2 Gy designates samples irradiated under ex vivo conditions. Numbers above each treatment represent coefficients of variation. RC: patients receiving radiotherapy + chemotherapy; R: patients receiving radiotherapy alone; W: week of treatment. Patient numbers are given on the right Y axes in the order of the last treatment (5/6W + 2Gy). (<b>C</b>): exemplary images of binucleated cells with micronuclei.</p>
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<p>Individual values of cell proliferation marker in lymphocytes of RC (<b>A</b>) and R (<b>B</b>) patients. Each line represents a single donor. Lines are shown to facilitate identification of donors and not to represent a time response. Boxes show 95% confidence intervals of the respective mean value. Blue boxes: values induced by Therapy alone. Red boxes: values induce by therapy plus 2 Gy ex vivo irradiation. Dashed lines represent patients RC1, RC6 and RC8 who received colony stimulating factors (see <a href="#cells-10-02709-f001" class="html-fig">Figure 1</a>). 2 Gy designates samples irradiated under ex vivo conditions. Numbers above each treatment represent coefficients of variation. RC: patients receiving radiotherapy + chemotherapy; R: patients receiving radiotherapy alone; W: week of treatment. Patient numbers are given on the right Y axes in the order of the last treatment (5/6W + 2Gy).</p>
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<p>Individual levels of apoptosis in lymphocytes of RC (<b>A</b>) and R (<b>B</b>) patients. Each line represents a single donor. Lines are shown to facilitate identification of donors and not to represent a time response. Boxes show 95% confidence limits of the respective mean value. Blue boxes: values induced by Therapy alone. Red boxes: values induce by therapy plus 2 Gy ex vivo irradiation. Dashed lines represent patients RC1, RC6 and RC8 who received colony stimulating factors (see <a href="#cells-10-02709-f001" class="html-fig">Figure 1</a>). 2 Gy designates samples irradiated under ex vivo conditions. Numbers above each treatment represent coefficients of variation. RC: patients receiving radiotherapy + chemotherapy; R: patients receiving radiotherapy alone; W: week of treatment. Patient numbers are given on the right Y axes in the order of the last treatment (5/6W + 2Gy). (<b>C</b>): exemplary images of apoptotic cells.</p>
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<p>Mean results for RC and R patients. (<b>A</b>) panels: micronucleus frequencies, (<b>B</b>) panels: proliferation marker, (<b>C</b>) panels: apoptosis levels. Right panels (marked 2) show the net or relative fits to facilitate following the kinetics of response. Round symbols and solid lines demonstrate the effect of therapy alone. Square symbols and dashed lines demonstrate the effect of therapy plus 2 Gy ex vivo irradiation. RC: patients receiving radiotherapy + chemotherapy; R: patients receiving radiotherapy alone; W: week of treatment. Asterisks indicate significant differences between the respective curves.</p>
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<p>Correlations. Panels (<b>A</b>–<b>D</b>): correlations between individual micronucleus frequencies observed in all 18 donors at different blood collection time points. Panels A and B: micronuclei induced by therapy + 2 Gy. Panels (<b>C</b>) and (<b>D</b>): micronuclei induced by therapy alone. Panels (<b>E</b>–<b>L</b>): correlations between the mean frequencies of MN, percent BNC and percent apoptosis. Panels (<b>E</b>–<b>H</b>): results after 2 Gy ex vivo irradiation (RC patients: (<b>E</b>,<b>F</b>), R patients (<b>G</b>,<b>H</b>)), panels (<b>I</b>,<b>L</b>): results after therapy alone (RC patients: (<b>E</b>,<b>F</b>,<b>I</b>,<b>J</b>), R patients (<b>K</b>,<b>L</b>)). W: week of treatment. Dashed lines symbolise 95% confidence intervals.</p>
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Review

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28 pages, 1988 KiB  
Review
COVID-19: The Disease, the Immunological Challenges, the Treatment with Pharmaceuticals and Low-Dose Ionizing Radiation
by Jihang Yu, Edouard I. Azzam, Ashok B. Jadhav and Yi Wang
Cells 2021, 10(9), 2212; https://doi.org/10.3390/cells10092212 - 27 Aug 2021
Cited by 4 | Viewed by 4471
Abstract
The year 2020 will be carved in the history books—with the proliferation of COVID-19 over the globe and with frontline health workers and basic scientists worldwide diligently fighting to alleviate life-threatening symptoms and curb the spread of the disease. Behind the shocking prevalence [...] Read more.
The year 2020 will be carved in the history books—with the proliferation of COVID-19 over the globe and with frontline health workers and basic scientists worldwide diligently fighting to alleviate life-threatening symptoms and curb the spread of the disease. Behind the shocking prevalence of death are countless families who lost loved ones. To these families and to humanity as a whole, the tallies are not irrelevant digits, but a motivation to develop effective strategies to save lives. However, at the onset of the pandemic, not many therapeutic choices were available besides supportive oxygen, anti-inflammatory dexamethasone, and antiviral remdesivir. Low-dose radiation (LDR), at a much lower dosage than applied in cancer treatment, re-emerged after a 75-year silence in its use in unresolved pneumonia, as a scientific interest with surprising effects in soothing the cytokine storm and other symptoms in severe COVID-19 patients. Here, we review the epidemiology, symptoms, immunological alterations, mutations, pharmaceuticals, and vaccine development of COVID-19, summarizing the history of X-ray irradiation in non-COVID diseases (especially pneumonia) and the currently registered clinical trials that apply LDR in treating COVID-19 patients. We discuss concerns, advantages, and disadvantages of LDR treatment and potential avenues that may provide empirical evidence supporting its potential use in defending against the pandemic. Full article
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Figure 1

Figure 1
<p>Weekly death analysis of COVID-19 and influenza in the USA. Data may lag by an average of 1–2 weeks, so the trend of decline in recent weeks may not accurately reflect the current situation. Available online: <a href="https://data.cdc.gov/NCHS/Provisional-COVID-19-Death-Counts-by-Week-Ending-D/r8kw-7aab" target="_blank">https://data.cdc.gov/NCHS/Provisional-COVID-19-Death-Counts-by-Week-Ending-D/r8kw-7aab</a> (accessed on 24 August 2021).</p>
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<p>SARS-CoV-2 structure. Similar to other coronaviruses, SARS-CoV-2 contains a membrane (M) glycoprotein, an envelop (E) protein, a spike (S) protein, and a nucleocapsid (N) protein (not shown at the outer layer).</p>
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<p>SARS-CoV-2 infection route. Infected COVID-19 patients can spread the virus by sneezing or coughing, or by any other route that drops the infectious particles onto another surface. When a person contracts the virus, the virus recognizes its binding target, ACE2, which is expressed on type II alveolar cells (AT2) in the lower respiratory tract. In the fusion step, the S protein on SARS-CoV-2 binds to ACE2, followed by cleavage of the S glycoprotein-ACE2 complex at polybasic motif (PPAR) at S1/S2 by type 2 transmembrane serine protease (TMPRSS2) and other proteases, leading to conformational alteration and activation of the S-glycoprotein. After fusion with the host cell membrane, the virus releases its genomic material into the cytoplasm.</p>
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<p>Remdesivir versus remdesivir plus baricitinib clinical trials. Comparison of mortality rate and serious adverse events of two clinical trials regarding remdesivir (R) compared with a placebo, and remdesivir in combination with baricitinib (R+B) compared with remdesivir alone (R alone). Data source: Remdesivir for the Treatment of COVID-19—Final Report [<a href="#B139-cells-10-02212" class="html-bibr">139</a>], Baricitinib plus Remdesivir for Hospitalized Adults with COVID-19 [<a href="#B143-cells-10-02212" class="html-bibr">143</a>].</p>
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