Immune system and tissue radiobiology

Group leader: Prof. Dr. Claudia Fournier

Impact of low dose radon exposure on the organism

Beside photons used in LD-RT (low dose radiotherapy), densely ionizing radiation, e.g. in radon spa, is also used to treat rheumatoid arthritis, ankolysing spondylitis, musculosceletal disorders, psoriasis and others (Figure 1). The cellular and molecular mechanisms underlying the observed alleviation from pain in radon spa are widely unknown.

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Figure 1: Patients in a radon gallery. Copyright: Gasteinertal Tourismus GmbH.
Gasteinertal Tourismus GmbH

GREWIS Consortium

Therefore, we set out to investigate potentially beneficial and harmful aspects of low dose radon exposure. To achieve this, we work together in the frame of the „GREWIS“-project with experts covering the fields of particle physics, cytogenetics and DNA repair, immunology, molecular cell biology and physiology, s. Helmholtz Association. The consortium includes 8 working groups from 4 institutions and associated medical collaborators (Figure 2).

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Figure 2: Organization Charts of the GREWIS and GREWIS-alpha Consortiums.
Prof. Dr. Claudia Fournier, GSI Helmholtzzentrum für Schwerionenforschung GmbH

The research groups of the GREWIS project have established an integrated strategy to study several aspects of risk and the potential immune-modulating effect of low radiation doses.

We work on the molecular and cellular level, in tissues and animals (Figure 3A). We have access to human bone marrow (hematopoietic and mesenchymal stem cells) and skin from healthy donors, and blood from radon patients.

Furthermore, we have developed a unique tool for radon exposure: A chamber allowing radon exposure of cells and mice, operating under controlled conditions including humidity, temperature and radon concentration, which is comparable to radon therapy and beyond  (Figure 4, click to enlarge) [1]. Diffusion and solubility measurements of radon and an analysis algorithm were developed. In addition, α-particle irradiation set ups have been constructed and are available at GSI and TU Darmstadt, i.e. Americium-241 source with comparable energies to the α-particles emitted by radon. Heavy ions with similar characteristics as α-particles (helium and carbon ions) are used (Figure 3B).

Biodosimetry based on DNA damage markers after radon exposure has been established for different organs and X-ray reference curves have been measured. As blood vessels as well as the interaction of endothelial cells of the blood vessel wall and hematopoietic cells have a crucial role in the first steps of immune reactions, a “flow chamber” was constructed to mimic the shear stress caused by the blood flow. Experiments are performed in vitro using both primary mouse and human cells (hematopoietic, skin, bone, synovial and fat cells; differentiated from stem and progenitor cells from healthy donors), also as 3D tissue equivalents. An in vivo approach takes advantage of the hTNFα-transgenic mouse model for polyarthritis to investigate maturation and activity of immune and bone cells. In addition, a first explorative study RAD-0N01 using blood samples from 100 patients who underwent radon spa treatment has been conducted for detailed immune monitoring, and the investigation of markers of the bone metabolism and inflammation before and up to 30 weeks after therapy. An LD-RT study to compare these results to photon exposure is in progress. Overall, our results obtained up to now reveal that the accumulation of radon is more pronounced in fat compared to muscle tissue, pointing to an unequal distribution in the organism. A decrease of the activation status of immune cells, inflammatory factors and marker proteins indicating bone resorption was detected. This is consistent with functional changes observed for low dose photon exposure in the polyarthritis mouse model and results obtained in vitro on the molecular level (Figure 5, click to enlarge) [2-10, 20]. 

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Figure 3: Experimental systems (A) and radiation facilities (B) used by all GREWIS partners.
Figure 4: Radon chamber
Development of a radon chamber to simulate treatment in a radon spa. The chamber can be used to irradiate cell cultures or small animals.
Prof. Dr. Claudia Fournier, GSI Helmholtzzentrum für Schwerionenforschung GmbH
Dr. Andreas Mayer, GSI Helmholtzzentrum für Schwerionenforschung GmbH

GREWIS-related research activities in our group

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Figure 4: Radon chamber.
Figure 5: Effects of low-dose-irradiation on bone progenitor/joint cells.
Figure 6: Low-dose-radiation response of fat cells.
Figure 7: Adhesion of leukocytes.
Development of a radon chamber to simulate treatment in a radon spa. The chamber can be used to irradiate cell cultures or small animals.
Shown here are terminally differentiated osteoclasts, cultivated on bone slices.
Low-dose-radiation response of fat cells, i.e. in the presence of inflammatory factors, is investigated in human primary cells.
Adhesion of leukocytes, i.e. lymphocytes on endothelial cells is measured after low-dose irradiation. Human primary endothelial cells are cultivated statically or under physiological laminar flow.
Dr. Andreas Mayer, GSI Helmholtzzentrum für Schwerionenforschung GmbH
Dr. Aljona Cucu, GSI Helmholtzzentrum für Schwerionenforschung GmbH
Vanessa Rzeznik, GSI Helmholtzzentrum für Schwerionenforschung GmbH
Dr. Felicitas Rapp, GSI Helmholtzzentrum für Schwerionenforschung GmbH

Immunomodulation of radiation effects

Consistent with the putative anti-inflammatory and immune suppressive effects  of low doses on the organism, higher doses, as applied in tumor radiotherapy, elicit immune reactions which might impair the outcome of the treatment [7]. In one project we started to investigate the response of radioresistant tumor cells on low and high LET irradiation, with the goal to improve the tumor cell killing effect by activating an anti-tumor immune response. First in vitro results with murine cells from colon cancer, breast cancer and melanoma (CT26.WT, 4T1, B16-F10, respectively) indicate that in the presence of specific drugs in combination with carbon ion irradiation, the expression of some surface molecules is more pronounced compared to radiation treatment alone. In vivo, the surface expression of these molecules renders tumor cells more susceptible to immune mediated cell killing. Our ongoing research activities are dedicated to verify these promising results in a mouse model.

Radiation response of human hematopoietic stem and progenitor cells

Secondary cancer occurs months to years after exposure and is one important issue in radiation protection for radiotherapy and manned space missions. The interference in the process of self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPC) via radiation exposure is considered to increase the risk for radiation induced leukemia (rAML). However, risk assessment for radiation induced leukemogenesis needs an improved knowledge about the radiation response of HSPC and, in particular their genetic stability. Therefore we investigated the transmission of chromosomal changes in HSPC exposed to low and higher LET irradiation (up to 85 keV/µm). In view of known differences in genetic stability and DNA repair between murine and human HSPC, we used human HSPC, provided in collaboration with Prof. H. Bönig (Frankfurt University) (Figure 8, click to enlarge). Our results showed, independent of LET, no chromosomal instability, but a considerable frequency of clonal chromosomal aberrations in the descendants of irradiated HSPC [11, 12]. To elucidate the capacity for correct DNA repair in HSPC, we assessed in collaboration with Prof. L. Wiesmüller (Ulm University), the quality and molecular components of DSB (double strand break-) repair compared to mature peripheral blood lymphocytes using an EGF plasmid reporter systems (developed by Prof. L. Wiesmüller). We could show that HSPC bear a deficiency for DSB repair (homologous recombination) due to diminished NF-κB signaling, which was confirmed for heavy ion exposure and could explain the occurrence of clonal aberrations in the following generations after exposure [13, 14]. More recently, we started to investigate the combined influence of micro-gravity and high LET exposure in HSPC.

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Figure 8: Human CD34+ HSPC were irradiated and cultured for 14 days in a Colony-forming-unit assay (CFU-assay).
HSPC differentiate into cells of the myeloid lineage (CFU-GEMM, BFUe, CFU-G/M/GM) (click to enlarge). Here the growth and differentiation of a BFUe colony with predominantly hemoglobinized erythroblasts cells (>200) is shown. Single colonies can be isolated and chromosomal and other changes can be studied.
Dr. Daniela Kraft, GSI Helmholtzzentrum für Schwerionenforschung GmbH

Heavy ion induced late effects in tissue

In other projects, our research group studies late effects induced by exposure to heavy ions. Radiation induced non-cancer effects include impact on the functionality of tissues and organs, which is often caused by loss of functional cells or modifications in the differentiation process and subsequent reorganisation of the tissue. The resulting changes can evoke inflammation, a physiological “repair” process, which may turn into a chronic process, as in the case of fibrosis. The increasing application of charged particles in radiotherapy requires a better understanding of these processes for charged particles, in particular for carbon ions. In a collaborative project with Prof. R. Coppes and Dr. P.v. Luijk (Groningen University), we investigate functional and molecular changes in the lung of carbon ion irradiated rats.

Especially skin is exposed in all scenarios of radiation exposure. We investigate in another project the early skin response after carbon ion exposure, whose molecular and cellular basis is not well understood. In order to complement previous RBE data sets obtained in a pig model [15] by cellular and molecular studies, we use mono- and co-cultures of epidermal cells, a full skin 3D tissue equivalent, and ex vivo exposed human skin from healthy donors, provided by our collaboration partners PD Dr. M. Podda and M. Kovacs (Darmstadt Hospital) (Figure 9, click to enlarge). Similar to photon exposure, epidermal tissue organization was modified at low doses and differentiation at high doses of carbon ions, with a moderate release of inflammatory cytokines [16]. In ongoing experiments, the results are verified in carbon ion irradiated pig skin.

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Figure 9: Human skin equivalent (HSE) after irradiation with 2 Gy of carbon ions.
A cobblestoned basal layer (arrow head) shows changes in the morphology, whereas hyperkeratosis (thickening of the stratum corneum, double arrows) and parakeratosis (nuclei in the stratum corneum; closed arrows) are a sign for an accelearted and changed differntiation process (modified after Simoniello 2016).
Dr. Julia Wiedemann, GSI Helmholtzzentrum für Schwerionenforschung GmbH

Theses pig skin samples were obtained in experiments performed in collaboration with MD I. Lehmann and MD D. Packer (Mayo Clinics, Rochester), dedicated to investigate the possibility to treat cardiac arrhythmias by targeted charged particle irradiation. In our current activities in collaboration with Dr. P. Simoniello (Parthenope University of Naples), we characterize the cellular and molecular changes in the targeted volumes of the exposed hearts occurring concomitantly to electrophysiological changes in conductivity (Figure 10, click to enlarge), so far demonstrated in ex vivo irradiated pig hearts [17].

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Figure 10: Histology with haematoxylin and eosin staining, inflammation and haemorrhage.
Histology with haematoxylin and eosin staining, inflammation and haemorrhage occur 3 months after irradiation with 40 Gy 12C in left ventricle (LV) tissue (B). Normal structure of cardiomyocytes (A).
Dr. P. Simoniello, GSI Helmholtzzentrum für Schwerionenforschung GmbH

References:

[1] Maier et al., Nucl. Instr. Meth. Phys. Res. B, 362:187-193 (2015)

[2] Large et al., Rad. Oncology, 9:80 (2014)

[3] Large et al., Strahlenther Onkol, 191:742-749 (2015)

[4] Thangaraj et al., Chemico-Biological Interactions, in press (2016)

[5] Roth et al., Pflügers Archive-European. J. Physiol. 467:1835-1849 (2014)

[6] Gibhardt et al., Scientific reports, 5:13861 (2015)

[7] Rühle et al., Autoimmunity. 50(2):133-140 (2017)

[8] Erbeldinger et al., Front Immunol. 8:627 (2017)

[9] Cucu et al., Front Immunol. 8:882 (2017)

[10] Maier et al., Nucl. Instr. Meth. Phys. Res. B, 416:119-127 (2018)

[11] Kraft et al., Mutat. Res., 777:43-51 (2015)

[12] Becker et al., IJRB, 85(11):1051-1059 (2009)

[13] Kraft et al., Leukemia, 29:1543-1554 (2015)

[14] Rall et al., Frontiers in Oncology, 5:250 (2015)

[15] Zacharias et al., Acta Oncologica, 36(6):637–642 (1997)

[16] Simoniello et al., Frontiers in Oncology, 5:294 (2016)

[17] Lehmann et al., Circ Arrhythm Electrophysiol. 8(2):429-38 (2015)

[18] Rödel et al., Front Immunol. 8:519 (2017)

[19] Durante et al., Trends Mol. Med., 19(9):565–82 (2013)

[20] Rühle et al., Modern Rheumatology, published online (2018)