Environment International 149 (2021) 106212 Contents lists available at ScienceDirect Environment International journal homepage: www.elsevier.com/locate/envint Low dose ionizing radiation effects on the immune system Katalin Lumniczky a, *, Nathalie Impens b, *, Gemma Armengol c, Serge Candéias d, Alexandros G. Georgakilas e, Sabine Hornhardt f, Olga A. Martin g, Franz Rödel h, Dörthe Schaue i a National Public Health Centre, Department of Radiation Medicine, Budapest, Albert Florian u. 2-6, 1097, Hungary b Belgian Nuclear Research Centre, Biosciences Expert Group, Boeretang 200, 2400 Mol, Belgium c Unit of Biological Anthropology, Department of Animal Biology, Plant Biology and Ecology, Faculty of Biosciences, Universitat Autònoma de Barcelona, 08193-Bel­ laterra, Barcelona, Catalonia, Spain d Université Grenoble-Alpes, CEA, CNRS, IRIG-LCBM, 38000 Grenoble, France e DNA Damage Laboratory, Physics Department, School of Applied Mathematical and Physical Sciences, National Technical University of Athens (NTUA), Zografou 15780, Athens, Greece f Federal Office for Radiation Protection (BfS), Ingolstaedter Landstr.1, 85764 Oberschleissheim, Germany g Peter MacCallum Department of Oncology, The University of Melbourne, Melbourne 3052, Victoria, Australia h Department of Radiotherapy and Oncology, University Hospital, Goethe University Frankfurt am Main, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany i Department of Radiation Oncology, David Geffen School of Medicine, University of California at Los Angeles (UCLA), Los Angeles, CA 90095-1714, USA A R T I C L E I N F O A B S T R A C T Handling Editor: Adrian Covaci Ionizing radiation interacts with the immune system in many ways with a multiplicity that mirrors the complexity of the immune system itself: namely the need to maintain a delicate balance between different Keywords: compartments, cells and soluble factors that work collectively to protect, maintain, and restore tissue function in Low-dose ionizing radiation the face of severe challenges including radiation damage. The cytotoxic effects of high dose radiation are less Immune system relevant after low dose exposure, where subtle quantitative and functional effects predominate that may go Epidemiological data unnoticed until late after exposure or after a second challenge reveals or exacerbates the effects. For example, DNA damage response Inflammation low doses may permanently alter immune fitness and therefore accelerate immune senescence and pave the way for a wide spectrum of possible pathophysiological events, including early-onset of age-related degenerative disorders and cancer. By contrast, the so called low dose radiation therapy displays beneficial, anti-inflammatory and pain relieving properties in chronic inflammatory and degenerative diseases. In this review, epidemiological, clinical and experimental data regarding the effects of low-dose radiation on the homeostasis and functional integrity of immune cells will be discussed, as will be the role of immune-mediated mechanisms in the systemic manifestation of localized exposures such as inflammatory reactions. The central conclusion is that ionizing radiation fundamentally and durably reshapes the immune system. Further, the importance of discovery of immunological pathways for modifying radiation resilience amongst other research directions in this field is implied. 1. Introduction made soon after the discovery of X-rays (Anderson and Warner, 1976; Radiation, 1972; Schaue, 2017). With the discovery of antibiotics and The immune system is the body’s main defence mechanism able to anti-inflammatory drugs, together with an increased awareness of ra­ distinguish between self and non-self as well as sensing danger. Its main diation carcinogenesis the initial enthusiasm to treat infections and function is to recognize and eliminate different pathogens and damaged benign diseases with radiation subsided for the most part and was or abnormal cells within the body (Fig. 1) (Murphy and Weaver, 2017). replaced with a sole focus on cancer radiotherapy (RT) using doses that Direct ionizing radiation (IR) effects on the immune system are well- can kill cancer cells (i.e. high doses delivered in multiple fractions, documented and were among the first radiobiological observations generally 2 Gy per daily fraction). As a result, the radiation literature * Corresponding authors. E-mail addresses: lumniczky.katalin@nnk.gov.hu, lumniczky.katalin@osski.hu (K. Lumniczky), nimpens@sckcen.be (N. Impens), gemma.armengol@uab.cat (G. Armengol), serge.candeias@cea.fr (S. Candéias), alexg@mail.ntua.gr (A.G. Georgakilas), shornhardt@bfs.de (S. Hornhardt), olga.martin@petermac.org (O.A. Martin), franz.roedel@kgu.de (F. Rödel), dschaue@mednet.ucla.edu (D. Schaue). https://doi.org/10.1016/j.envint.2020.106212 Received 29 June 2020; Received in revised form 20 August 2020; Accepted 3 September 2020 Available online 5 December 2020 0160-4120/© 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). K. Lumniczky et al. Environment International 149 (2021) 106212 Fig. 1. Schematic representation of the structure of the immune system and its major functional features. A molecule that is recognized by the immune system is called an antigen, which can be both self and non-self in origin. The immune system can be divided in two main compartments: the innate immune system and the adaptive immune system. The innate immune system is composed of a cellular compartment consisting of mononuclear cells (monocytes/macrophages, mast cells), polymorphonuclear cells (neutrophils, basophils, eosinophils), dendritic cells (DCs), innate immune cells (e.g. natural killer or NK cells) and the humoral complement system (Artis and Spits, 2015). Innate immune cells see danger through their germline-encoded pattern recognition receptors (PRRs), which recognize specific molecular structures present on pathogens (so-called pathogen-associated molecular patterns or PAMPs) or produced by damaged cells (so-called damage- associated molecular patterns or DAMPs) (Amarante-Mendes et al., 2018). Forming our first-line of defense, this recognition is relatively non-specific and quick, reaching its maximal intensity shortly after antigen encounter without yielding specific immunological memory. Phagocytosis is one of the main mechanisms for antigen elimination by innate immune cells. During danger recognition and antigen processing innate immune cells mature and release various soluble immune mediators called cytokines and chemokines, which drive inflammation and attract adaptive immune cells (Commins et al., 2010). In fact, an important role of the innate immune system is the activation of the adaptive arm. Macrophages and DCs in particular are professional antigen presenting cells with the unique ability to activate naïve cells of the adaptive immune system by displaying components of the processed antigens within the major histocompatibility complex (MHC) on their surface and present them to lymphocytes in the presence of necessary co-stimulatory signals (Wynn et al., 2013). Cells of the adaptive immune system include T lymphocytes (such as CD4 + helper, CD8 + cytotoxic and Foxp3 + regulatory) and B lymphocytes. T cells are responsible for cell-mediated immune response while B cells play role in humoral immune response (mediated by antibodies). In contrast to the innate immune system, the major features of the adaptive immune response are: high antigen specificity, latency of maximal response and development of immunological memory exemplified by faster and qualitatively different recall re­ sponses (Santana and Esquivel-Guadarrama, 2006). The first step in the activation of the adaptive immune system is antigen recognition by CD4 + or CD8 + cells through their highly antigen-specific T-cell receptors (TCRs). Professional antigen presenting cells present antigenic peptides conjugated either to MHCII, inducing CD4 + activation or to MHCI, contributing to CD8 + activation. Activated CD8 + T cells kill the antigen-presenting cells through the release of cytotoxic agents stored in intracellular granules, or directly by cell-to-cell contact engaging death receptors, or through the production of cytokines that trigger apoptosis. B cells, on the other hand, recognize extracellular antigens via their antigen-specific B cell receptor, which are essentially antibodies bound on the cell membrane forming a transmembrane receptor. Once activated with help from CD4 + T cells, B cells start to divide and differentiate into plasma cells which secrete huge numbers of soluble antibodies similar to the one that recognized the antigen in the first place (Hardy and Hayakawa, 2001). Circulating antibodies bind to their specific antigens and these antigen–antibody complexes induce activation of the complement system, which in turn leads to a rapid neutralisation by the proteolytic activity of the complement system and further phagocytosis by innate cells, i.e. antibody-dependent cellular cytotoxicity. Most of the intercellular communication in the immune system is guided through a complex system of chemokines, cytokines and interferons that affect trafficking, activation, differentiation and functional maturation (Turner et al., 2014). To prevent tissue damage from excessive immune activation multiple control mechanisms are in place that act through cell-to-cell contact or cytokines, involving among others regulatory T cells (Tregs) (Persa et al., 2015). Finally, to mount an effective response, immune components must circulate between the blood and lymph nodes, recognize antigens upon contact with presenting cells, and differentiate to effector T cells and plasma cells. Moreover, these cells must extravasate the lymph nodes, migrate to affected tissue to secure host-protective activities and to recircle to blood to counteract chronic activation (Germain et al., 2012). Accordingly, one has to consider a high degree of cellular motility and interaction dynamics of the immune system. 2 K. Lumniczky et al. Environment International 149 (2021) 106212 tends to be dominated for the most part by high dose exposure studies and what is “not yet known” based on existing epidemiological, clinical with single doses above 1 Gy (Schaue, 2017). Data from radiation and pre-clinical data (Table 1). oncology patients come with generally well-defined dosimetry and clinical monitoring, but the presence of a tumour is an important con­ 2. Human biomonitoring and epidemiological data on low dose founding factor from an immunological point of view. For instance, radiation-induced immunological changes many patients receive fractionated local tumor irradiation of up to 74 Gy total dose as part of an extensive combined modality treatment that can Data from genuine epidemiological studies on immune alterations in include chemotherapy, surgery and/or immunotherapy (Demaria et al., the context of low dose exposures remain scarce (Table 2). This may be 2015). Therefore immunological alterations during cancer treatment because symptoms of immune-related diseases do not appear in a form may not necessarily be due to radiation effects alone. Studies based on or along a time course that can easily be related to radiation exposure high dose IR applied to cancer patients indicated that radiation had apart from the well-known high radiation sensitivity of many resting immune suppressive properties and this paradigm prevailed in the sci­ lymphocytes. entific literature for decades (Anderson and Warner, 1976; Balogh et al., A substantial amount of data is available from different mass casu­ 2013; Hader et al., 2020; Kachikwu et al., 2011; Lumniczky and Safrany, alties implying acute or chronic exposure scenarios. Among the best 2015; McFarland et al., 2012; Merrick et al., 2005). On the other hand studied cohorts today are A-bomb survivors and clean-up workers (liq­ low (below 100 mGy) and intermediate dose (between 100 mGy and 1 uidators) of the Chernobyl nuclear power plant accident, subjected to Gy) exposure scenarios are much more relevant for the general popu­ single dose acute exposure. lation as they may have public health consequences. They are also much more difficult to study because toxicity and carcinogenesis are a lot less 2.1. A-bomb survivors and Chernobyl clean-up workers cohorts obvious while other, more subtle functional alterations gain in impor­ tance. DNA damage correlates with the dose and the probability of se­ Data regarding radiation effects on the immune system of A-bomb vere direct DNA damage after low doses and dose rates is low, thus other survivors started to accumulate in the 1980′ s and have been periodically mechanisms may prevail (Mothersill and Seymour, 2014). Within the updated ever since. The caveat is of course that there may be influences long time span between exposure and the onset of the clinically apparent of race and life style factors. Akiyama reported from the Life Span Study pathologies, likely persisting alterations in the functional integrity of the on about 120,000 people who had been exposed to an average of 0.16 Gy organism as a whole must be present leading to the development of a according to the dosimetry system revised in 1986 (DS86), which in­ pathology. Not only the DNA-damage response (DDR) related pathways, cludes “non-exposed” controls (Akiyama, 1995; Akiyama et al., 1983). but also other, immune-related pathways may contribute to both cancer Many long-term immune effects were observed that generally suggested and non-cancer health outcomes from exposure to IR. Hence, a holistic a shift in the peripheral lymphocyte balance in favor of B cells with approach that integrates these multiple mechanisms at all organiza­ increased serum immunoglobulin (Ig) levels at the expense of cluster of tional levels is needed to understand the complex response system differentiation 3 (CD3) + T cells which were reduced in both numbers (Mavragani et al., 2016). and functionality. A dose-dependent drop in naïve CD4+ and CD8+ T An accumulating amount of scientific evidence based on epidemio­ lymphocytes was recorded, while memory cells were less affected logical and pre-clinical studies indicate that low dose exposures might (Kusunoki et al., 1998; Kusunoki et al., 2003). That T cell differentiation directly impact immune functions and - although controversial -, these and development may be permanently altered was suggested by a rise in data indicate that IR may not only be immune suppressive (Cui et al., rare double negative CD4-CD8-, alpha/beta T cells (Kusunoki et al., 2017; Hellweg, 2015; Makinodan and James, 1990; Rodel et al., 2015; 2003; Kyoizumi et al., 2010). The appearance and persistence of TCR- Sambani et al., 1996; Xu et al., 1996). A comprehensive review of mutant T cells was detected mostly in the memory CD4+ T cell available data on IR effects on the immune system was published in the compartment in a dose-dependent manner in individuals aged 20 or UNSCEAR 2006 report (Radiation, 2008) including both high and low older at the time of bombing (Kusunoki et al., 2003). A relatively robust dose effects and highlighted complex functional changes within the dose-dependent readout for TCR mutant frequency was suggested after immune system in response to radiation. This was the first report studies on Chernobyl clean-up workers, even at doses of 0.25 Gy (Saenko released by an international organisation investigating radiation health et al., 2000). This would suggest the use of TCR mutation as a potential effects which abandoned the “classical” paradigm that IR is purely im­ biodosimeter relevant to T cell function. mune suppressive. Actually, this report proposes to consider IR as an In many ways, immune changes observed in A-bomb survivors immunomodulatory agent due to the multitude and sometimes opposing resemble those associated with aging. It was reported that the output of ways it can influence the immune system depending on various pa­ naïve T cells was reduced, the memory T cell pool was expanded while rameters such as dose, dose rate, genetic background, age, health status, the TCR repertoire became limited, all of which were associated with comorbidities, lifestyle, environmental co-stressors, etc (Radiation, low grade inflammation that involved myeloid cells known as inflam­ 2008). maging (Denkinger et al., 2015; Franceschi et al., 2019; Fulop et al., This review is part of a collection of papers summarizing discussions 2018; Kusunoki and Hayashi, 2008; Kusunoki et al., 2010). Doses in the of the MELODI workshop on non-cancer effects of low dose IR, organized range of 0.005–0.2 Gy drove accelerated thymic involution, which could in Sitges, Spain, 10–12 April 2019. Here we aim to provide an update on still be evident 30 years later when the natural, age-related process was low dose IR effects on the human immune system with the goal to well under way if not completed (Ito et al., 2017). The frequency and summarize what is “known”, what is suspected but still controversial counts of monocytes were dose-dependently increased by radiation exposure and this increase was more pronounced after 60 years showing a possible acceleration of age-dependent clonal haematopoiesis (Yosh­ Table 1 ida et al., 2019). However, the response to vaccination in elderly atomic Doses and dose ranges and main type of studies related to the relevant dose bomb survivors seemed not to be impaired by radiation exposure early ranges aprincipal type of studies discussed in this review addressing this dose in life (Hayashi et al., 2018). range. RT: radiotherapy; LD-RT: low-dose radiotherapy. The role of IR in promoting accelerated aging was further demon­ Low doses Intermediate doses High doses strated by looking at telomere length in leukocytes of A-bomb survivors. Dose <100 mGy 100 mGy–1 Gy >1 Gy Lustig et al. showed that circulating leukocytes in A-bomb survivors had range shorter than expected telomeres, and impaired function, which was Studiesa Epidemiological Epidemiological Clinical Clinical (RT) dependent on dose and age at exposure. It was more severe in the young Experimental (LD-RT) Experimental Experimental than in the elderly, showing a significant dose-dependency in 3 K. Lumniczky et al. Environment International 149 (2021) 106212 Table 2 Overview of studies describing immunological changes in people exposed to different scenarios of irradiation. Citation Population/Site Collective & Numbers Dose Endpoints Remarks & Conclusions Acute and chronic exposure (Ito et al., 2017) A-bomb Cohort, 165 human tissue no (<5 mGy), low (5–200 Immunohistology of thymus from Low dose is sufficient to result in survivors blocks from RERF archive mGy), moderate-to-high pathology archive decreased thymic function many (<200 mGy) exposure; 11 years after exposure (years from unexposed controls exposure 9–41), accelerated thymus aging (involution) (Kusunoki et al., A-bomb Cohort, 159 exposed, 234 1) <5 mGy; 2) >5 mGy to PBMCs by flow cytometry, subsets A-bomb radiation might have 1998) survivors controls (<0.05 Gy) >1.5 Gy of T, B, NK cells triggered dominant Th2-cell responses, stimulating B-cell lymphopoiesis for a long period. (Kusunoki et al., A-bomb Cohort, 1280 individuals 2 groups: <5 mGy and >5 Subsets of CD4+ T cells Memory CD4+ T-cells of 2003) survivors mGy individuals who received significant radiation doses in adulthood may have become dependent on a much less TCR Vß families than unexposed (Kusunoki et al., A-bomb Cohort, 1035 individuals dose categories: <5 mGy, 5 PBMCs by flow cytometry, plasma A-bomb survivors may have 2010) survivors between 2006 and 2008 mGy–0.5 Gy, 0.5 Gy–1.0 Gy, TNF-alpha level (only a subgroup) induced T-cell immunosenescence 1.0 Gy–4.0 Gy resulting in attenuation of T-cell- mediated immunity. (Kyoizumi et al., A-bomb 203 A-bomb survivors, 6 A-bomb survivors: 1) <5 mGy Mutation frequency of T-cell No significant dose effects in A- 1992) survivors Thorotrast patients, 18 (n = 125); 2) >1.5 Gy (n=78) receptor (TCR) in PBMCs: flow bomb survivors. thyroid disease patients; one cytometry with CD3 and CD4 Chernobyl person with high antibodies assuming that mutant accidental exposure CD4+ T cells have only a small fraction of CD3 expression (Kyoizumi et al., A-bomb Cohort, 916 individuals dose categories: <5 mGy, 5 PBMCs characterized by flow The steady state of the T-cell 2010) survivors mGy–0.5 Gy, 0.5 Gy–1.0 Gy, cytometry: subsets of memory T- memory, which is regulated by 1.0 Gy–4.0 Gy cells by CD43 level cell activation and/or cell survival processes in subsets may have been perturbed by prior radiation exposure. (Lustig et al., A-bomb Cohort, 415 individuals, 2 3 exposure groups: 157 with T cell counts, telomere length; Radiation damage drives changes 2016) survivors time points: 55 and 66 years no dose (<5 mGy), 123 with serum cytokines, c-reactive protein in telomere length that persist in after exposure low (5 mGy–700 mGy), 135 (CRP) the progeny over half a century with high (>700 mGy) dose and therefore likely derived from the initial lesion. Radiation damage seems more severe in the young than the old. Telomere shortening likely cause functional defects that in the case of lymphocytes would lead to less T cell immunity and less myeloid function (less inflammatory cytokines). (Yoshida et al., A-bomb Cohort, 620 participants dose range 0–1.736 Gy PBMCs: telomere length of naïve Radiation exposure perturbs T- 2016) survivors and memory CD4+ T cells, total cell homeostasis involving CD8+ T cells; metabolic status telomere length maintenance by multiple biological mechanisms, depending on dose, and that long- term radiation-induced effects on the maintenance of T-cell telomeres may be modified by the subsequent metabolic conditions of individuals. (Yoshida et al., A-bomb Cohort, 14,349 participants 3 dose groups; <1 Gy (n = Longitudinal statistical analysis of Radiation exposure might 2019) survivors 1616), >1 Gy (n = 9393), blood cell counts accelerate aging-associated clonal control (not-in-town, n = haematopoiesis, which could 3340) result in a long-lasting elevation of circulating monocytes. (Ilienko et al., Chernobyl 235 Chernobyl accident male Mean dose ± SD: 419.48 mSv Lymphoctes: gene expression of Cellular immunity, gene 2018) clean-up workers exposed in ± 654.60; range 0.10–3,500 candidate genes: BCL2, CDKN2A, expression, telomere length, 1986–1987; 45 matched non- mSv CLSTN2, GSTM1, IFNG, IL1B, intracellular protein parameters exposed controls MCF2L, SERPINB9, STAT3, TERF1, are shown to be among TERF2,TERT, TNF, TP53, CCND1; perspective biological markers at relative telomere length; immune a late period after radiation cell subsets, γ-H2AX and CyclinD1. exposure. (Kuzmenok Chernobyl Chernobyl healthy clean up Dose estimation: 150 PBMCs; isolated T-cells; mitogen An approach to a more accurate et al., 2003) workers from Belarus: 134 mGy–500mGy stimulation analysis of the immunological workers and 89 matched disorders found after exposure to controls radiation from Chernobyl-related activities. (continued on next page) 4 K. Lumniczky et al. Environment International 149 (2021) 106212 Table 2 (continued ) Citation Population/Site Collective & Numbers Dose Endpoints Remarks & Conclusions (Oradovskaia Chernobyl Liquidators, comparison of Differences by time and PBMC subpopulations; Specific features of changes in the et al., 2011a) different time points (1986, timing of liquidation work immunoglobulins immune system depend on dose of 1987) and working external gamma-irradiation. conditions However, distinctions in the age dynamics of the immune system in liquidators in the presence and in the absence of cancer manifested themselves in a stable level of CD3+, CD4+, CD8(+)-T- lymphocytes, immune regulation index, CD95+, serum IgA at the age between 40 and 70 years. (Saenko et al., Chernobyl 57 liquidators, 21 controls Physical dosimetry from erythrocyte variant cells bearing a In Chernobyl clean-up workers 2000) official records; Chernobyl mutated glycophorin A (GPA) the TCR mutant frequency was liquidators <0.25 Gy surface marker significantly higher than in control non-irradiated individuals. (Chang et al., Home 196 exposed residents with Protracted gamma-radiation, blood: lymphocyte subpopulations Significant immunological effects 1999a) environment, 2–13 years of exposure in mean excess cumulative dose: were observed in those who Taiwan their homes; 55 close 169 +/− 272 mSv; mean received chronic low-dose relatives non-exposed annual excess dose 24+/− radiation exposure. 29.9 mSv (Jain and Das, Kerala, India Cohort, 36 healthy male 5 dose groups based on Gene expression in PBMCs, gene Individuals exposed to 2017) individuals, age 28–52 living annual background dose ontology, pathway analysis background doses of >5 mGy/ in different level natural received; I (control): <1.5 year showed alterations in the background radiation areas mGy/year; II: 1.51–5.0 mGy/ expression of genes involved in year; III: 5.01–15.0 mGy/ immune system-related pathways. year; IV: >15 mGy/year; individual dosimetry (Takahashi Marshall Cohort, 4766 individuals No dose estimation provided Thyroid examination by Dietary intake of iodine needs to et al., 1999) Islands aged to be at risk from ultrasound, thyroid hormone be taken into account when exposure of radioactive determination, anti-thyroid looking at the link between fallout by the US nuclear antibodies, questionnaire, iodine radiation exposure and thyroid testing programme on Bikini status (urine samples) nodules. and Eniertah atoll (1946 and 1958) (Attar et al., Ramsar, Iran 100 individuals from villages 13 times higher than normal PBMCs for functional assays, Immune system adaptation in 2007) with high level natural in HLNBR area cytokines IL-2, IL-4, IL-10, IFN- individuals living in high natural background radiation gamma radiation background areas (HLNBR) and villages with low background radiation (Borzoueisileh Ramsar, Iran 50 individuals aged 25–35 estimated dose of 10.2–260 Flow cytometry of PBMC Multiple immune system et al., 2013) years, exposure duration mSv/year in Ramsar area subpopulations: CD4+/CD45+ (T- alterations 10–35 years, different level helper-cells), CD8+ (cytotoxic T- natural background radiation cells), NK cells and CD107a-cells areas (Ghiassi-nejad Ramsar, Iran Individuals from HLNBR Annual radiation absorbed chromosome aberrations after in An adative response in terms of et al., 2002) areas vs normal background dose from background vitro challenge dose with 1.5 Gy chromosomal aberrations induced radiation area radiation up to 260 mSv/year by chronic low dose exposure (Ghiassi-Nejad Ramsar, Iran 50 exposed individuals from Estimated annual effective Immunoglobulins IgM, IgG, IgA, Stimulation of Th2 response is et al., 2004) HLNBR area aged 40+/− 16 dose: 1.6–42 mSv/year; 2.3 IgE, complement (C3, C4, C1- discussed years; 30 matched controls mSv/year for controls inactivator), rheumatoid factor, CRP; flow cytometry of PHA stimulated and unstimulated PBMCs with CD3, CD4, CD5, CD69 markers; cytogenetic analysis (Molaie et al., Ramsar, Iran Subjects from high and low high and low natural Neutrophil chemotaxis, Nitro-Blue The level of IL-4 increased in 2012) level natural background background radiation Tetrazolium (NBT), antioxidant individuals who lived in area with radiation areas effects, cytokines (IL-2, IL-4) levels high levels of natural radiation, which could lead to Th2 pattern of immune response (Akleyev et al., Techa River, Cohort, 66 residents of the Dose estimation according to Number of CD19+, CD3+, Low dose exposure induced long 2019) Mayak area Techa River basin the Techa River Dosimetry CD3+CD4+, CD3+CD8+, term changes of the innate contaminated due to release system 2009 (TRDS-2009): CD3+CD4+/CD3+CD8+ cell ratio, immune system; immune system of liquid radioactive waste main group (TCR-mutations): immunglobulins (IgA, IgM, IgG); seems to react to DNA damage from the Mayak Production dose rate to bone marrow number of neutrophils, monocytes driving innate immune cell Association (Plutonium) in 0.21+/− 0.02 Gy/year 1951, and their phagocytotic, lysosomal activation in an effort to eliminate 1952; groups: 29 people with absorbed dose = 0.89+/ activity and intensity of TCR-mutated lymphocytes other vs 37 people without − 0.09 Gy (individual intracellular oxygen-dependent than by apoptosis increased TCR-mutations 0.09–1.96 Gy) comparison metabolism; eosinophils, group: dose rate to BM 0.25 basophils, CD16+CD56+ and +/− 0.02 Gy/year 1951; CD3+CD16+CD56+ lymphocytes; absorbed dose = 1.03 +/− cytokines; colony stimulating 0.07 Gy (range 0.03–2.34 Gy) factors: GM-CSF, G-CSF, TNF- alpha; IFN-alpha, IFN-gamma (Li et al., 2019) lymphocyte subsets; 30 cytokines (continued on next page) 5 K. Lumniczky et al. Environment International 149 (2021) 106212 Table 2 (continued ) Citation Population/Site Collective & Numbers Dose Endpoints Remarks & Conclusions Yangjiang 100 women exposed to estimated cumulative dose in Immune function was found to be district, China HLNBR, 100 matched exposed group: 58.5–249.13 affected in humans exposed to controls mSv long-term low dose radiation: increase in CD8+ T-cell numbers and upregulated inflammatory biomarkers like IFN-gamma, MCP-1, sIL6R, EGFR, CRP (Gyuleva et al., Nuclear power Nuclear Power Plant (NPP) Cumulative doses between Flow cytometry of lymphocyte Assumption that while the 2015a) plant workers “Kozloduy”, Bulgaria. 438 0.06 mSv and 766.36 mSv subpopulations, serum levels of adaptation processes are persons working in NPP; 10 and a control group with 65 IgG, IgA, IgM dominated with low prevalence of year survey persons T-helper 1 (Th1) immune response to cumulative doses <100 mSv, a switch to TH-2 response occured at doses >100 mSv. (Gyuleva et al., Nuclear power NPP “Kozloduy”, Bulgaria. Cumulative doses between Flow cytometry measurements of Some of the studied parameters 2015b) plant workers 438 persons working in NPP; 0.06 mSv and 766.36 mSv T, B, natural killer (NK) and natural could be interpreted in terms of 10 year survey and a control group with 65 killer T (NKT) cells adaptation processes at low doses. persons; At doses above 100–200 mSv, compensatory mechanisms might be involved to balance deviations in lymphocyte subsets. Some observed variations in some cases on the immune system might be due to other unknown factors. (Gyuleva et al., Nuclear power NPP “Kozloduy”, Bulgaria. control, 4 dose groups: <25 lymphocyte subpopulations; serum The observed even slight trends in 2018) plant workers 105 employees mSv; <100 mSv; <200 mSv; IgG, IgM, IgA; IL-2, IL-4, IFN- some lymphocyte populations and > 200 mSv gamma in cytokines profile allow to assume a possibility of a gradual polarization of Th1 to Th2 immune response at dose range 100 to 200 mSv. (Rees et al., NPP workers British Nuclear Fuels, Film badge dosimetry over PBMCs: T cell and B cell subsets No significant immunological 2004) Sellafield: 194 male radiation 30.6 years vs 23.9 years; effects in male radiation workers workers >200 mSv (mean cumulative exposure >200 at >200 mSv compared to <27.5 331.5 mSv); 131 workers mSv vs <27.5 mSv mSv; smoking is an important <27.5 mSv (mean 13.9 mSv) confounding variable. (Ahmad et al., Radiology 60 healthy individuals mean dose: 2.03 mSv/year; Superoxide, DNA oxidation, The data suggest a pro- 2016) workers working in different medical duration of radiation cytokines inflammatory response at doses diagnostic units: 20 exposed, exposure: 16 years above 17 mSv. A threshold and 40 matched controls non-linearity is discussed. (Godekmerdan Radiology 50 radiology workers vs 35 <3.5 mSv/year for 86%; the Subgroups of PBMCs; serum T helper cell and humoral et al., 2004) workers age-matched healthy rest received above that; complement and Igs immune components are controls, mean age 30.1 +/− exposure time >5 years in compromised. 7 vs 31.5 +/− 5.8 years; 48% 48% vs 0% smokers; (Karimi et al., Radiology 30 radiology workers vs 20 Exposure <50 mSv PBMCs, PHA stimulation assay; No dose response tested. A shift 2017) workers control laboratory workers serum cytokines towards Th1 responses by low dose radiation is discussed. (Klucinski et al., Radiology X-ray diagnostics units: 47 Period of employment: 1–33 Flow cytometry of B-cell subsets: B- Association of suppressive 2014) workers workers (14 men, 33 years with annual effective cells (CD19 + ), B1-cells (CD5+ influence of low level ionizing women); control group 38 dose <1 mSv CD19+ ), memory B-cells (CD27+ radiation on B and memory B-cells (10 men, 28 women) non- CD19+ ) is discussed. exposed (Rybkina et al., Mayak Mayak Production 14 workers exposed to Cytokines: TGF-beta1, TNF-alpha, Chronic occupational IR exposure 2014) production Association workers cohort; external gamma-rays (total IFN-gamma, IL-1beta, IL-8; of workers induced a depletion of workers 91 workers and 43 controls dose 05–3.0 Gy), 77 workers immunoglobulins: IgM, IgG, IgA, immune cells in peripheral blood with combined exposure IgE; p53, HSP70, MMP-9; (external gamma-rays and lymphocyte subsets internal alpha radiation from incorporated plutonium) (Zakeri et al., Interventional 37 interventional 8.14 mSv/year (range serum cytokines and Igs; cytokine No dose response observed due to 2010) cardiologists cardiologist vs 37 control; 1.2–27.8) for 12.1 +/− 6.6 release from activated low case numbers years and an accumulated lymphocytes, PBMC phenotypes dose over the last 5 years of 30.5 +/− 24.3 mSv Studies on radiation- exposed children (Imaizumi et al., A-bomb A-bomb survivors exposed in mean maternal uterine Thyroid: solid thyroid nodules and Antithyroid antibodies were not 2008) survivors utero; 328 persons (mean age radiation dose 0.256 Gy; <5 cysts; blood: antithyroid antibodies associated with dose or 55.2 year; 162 male); mGy, 5 mGy-0.1 Gy, 0.1–0.5 (ATAs): antithyroperoxidase (TPO- gestational week at exposure. No examination 55–58 year after Gy, 0.5–1 Gy, > 1 Gy Ab) and antithyroglobuline (TgAb) significant dose–response exposure in utero relationship for autoimmune thyroid disease in the in utero- (continued on next page) 6 K. Lumniczky et al. Environment International 149 (2021) 106212 Table 2 (continued ) Citation Population/Site Collective & Numbers Dose Endpoints Remarks & Conclusions exposed subjects (similar to exposed children). (Chang et al., Home 289 children exposed at High dose group estimated Blood draw for basic differential Persistent changes in 1999) environment, kindergarden in 1983–92 to 21–85 mSv in total (200-800 blood counts haematopoietic system following Taiwan continuous low dose Co-60 chest X-rays) compared to chronic low dose expsoures in the gamma irradiation vs 751 low dose group 2–5 mSv observed children. aged- and sex-matched (20–50 chest X-rays) exposed to lower dose, studied 5–7 years later (Agate et al., Chernobyl Cohort, 1433 sera from Contaminated areas included ATAs: TPO-Ab, TgAb; thyroid TPO-AB prevalence in adolescents 2008) adolescents 13-17 years Klintsy (Russia), Korosten function based on circulating levels exposed to radioactive fallout was (born 1982–1986); (Ukraine) and Lelchitsky of thyroid-stimulating hormone still increased in Belarus 13–15 additional 1441 control sera (Belarus) at 555–1480 kBq/ (TSH) and free triiodothyronine years but a lot less than at 6–8 from aged-matched and sex- m2; iodine deficiency (FT3) and thyroxine (FT4) years after the Chernobyl accident matched children in prevalent in both but normal thyorid function Denmark and Sardinia contaminated and non- possibly suggests a transient contamined areas; radiation-induced autoimmune reaction without triggering clinical thyorid autoimmune disease. (Chernyshov Chernobyl 120 children aged 6–13 years Exposed children from areas Major lymphocyte subsets analysed Long-time exposure to low et al., 1997) from 15 radiation- within a 40–75 km radius in whole blood by flow cytometry radiation doses may affect the contaminated areas in North from the reactor; estimated immune balance, especially in Ukraine after Chernobyl dose of Cs-137 and Sr-90 of vulnerable populations. accident with/withour 0.57–3.09 mSv over 3 years; recurrent respiratory disease two groups < or >1 mSv (RRDC); 87 children from non-contaminated areas with/without RRDC (Kasatkina et al., Chernobyl 89 children from Uritzky Average Cs-137 soil Thyroid dimension by clinical Autoimmune thyroid disease 1997) region (416 km north of contamination 1.71 Ci/km2 exam and ultrasound; thyroid markedly increased in children Chernobyl); 116 non- (range 0.18–3.97) function (hormones); with poor iodine nutrition who contaminated Kolpnyansky autoantibodies; fine needle were exposed to low level area; 2 age groups: age at aspiration radiation. Low level radiation exposure in utero (n = 89 and may induce thyroid gland changes n = 100 controls) or 8–9 in children who had inadequate years (n = 81 and n = 97 iodine intake. controls) (Pacini et al., Chernobyl 472 patients with thyroid Radioactive contamination I- Thyroid immunity and function: Young children (<5 year) are 1998) carcinoma from Belarus 131 in Belarus: ranging from T4, T3, TSH, thyroid ATAs: TPO- especially vulnerable to radiation- diagnosed at <21 year 185 to 37,000 kBq/m2 Ab, TgAb induced thyroid cancer that tend compared to aged-matched to be more aggressive in nature controls with thyroid and associated with signs of carcinoma from Italy and thyroid autoimmunity France: a) <14 year children (n = 372); b) adolescent 14–21 year (n = 100); (Sheikh Chernobyl Chernobyl area: children Internal whole body Lymphocyte subsets, cytokines: IL- Children with irritable bowel Sajjadieh aged 4–18 years with/ radioactivity due to Cs-137; 4, IFN-gamma disease had less CD4+ T-cells, a et al., 2012) without diagnosed irritable group1 (21 children aged higher level of IL-4 and a lower bowel disease 4–9): 1.9 Bq, group2 (26 level of IFN gamma, suggesting a children aged 10–13): 1.85 stronger polarization toward a Bq, group3 (28 children aged Th2 phenotype. There was no 14–18): 2.01 Bq, group4 (21 difference with age, suggesting healthy childen aged 5–15): that there was no radiation-dose 1.8 Bq effect. (Vykhovanets Chernobyl 6–14 year old children in Low doses of radiation to the Major lymphocyte subsets analysed Possibility that long-term et al., 2000) radiation-contaminated whole body from Cs-137 in whole blood by flow cytometry exposure to low doses of Cs-137 areas in North Ukraine after ranging from 1.79 to 53.7 may have altered the immune Chernobyl accident (n = 78; mSv (1991) and 2.17–29.33 balance in especially vulnerable 5 years after accident) and mSv (1994–96) and various children. The shifts in circulating 141 different children (8–10 doses of radiation to the lymphocyte subsets between years after accident); thyroid from I-131 as fallout healthy children and those with children with recurrent RRDC may be attributed to long- respiratory disease (RRDC, term low-dose exposure of the mean age 8.3 years) vs non- whole body to radiation from Cs- RRDC in contaminated areas; 137 and exposure of the thyroid to n = 61 (1991) and n = 87 radiation from I-131. aged-matched controls from non-contaminated areas individuals younger than 12-years at exposure. The authors concluded peripheral blood cell counts were lost in irradiated individuals indi­ that because this was measurable in the progeny decades later, the initial cating that radiation effects override those of aging. The authors hy­ lesion was the likely source. The association of telomere shortening with pothesized that the resultant functional defects may not necessarily be a circulating biomarkers for aging, such as cytokine production and disadvantage as myeloid cells may produce fewer inflammatory 7 K. Lumniczky et al. Environment International 149 (2021) 106212 cytokines, whereas this argument is difficult to make for reduced T cell showed that the innate immune system, NKT cells, and neutrophils in function (Lustig et al., 2016). Of special interest to the discussion here particular, might be activated by chronic exposure to approx. 0.9 Gy in a might be the observation by Yoshida el al. who showed a biphasic dose and dose-rate dependent manner in residents of Techa River alteration of CD4+ telomere length with irradiation dose: longer telo­ (Akleyev et al., 2019). Another study indicated a dose-dependent meres after low dose exposure and progressively decreasing telomere decrease in the concentration of T helper cells, reduced IFN-γ levels, length with doses above 0.5 Gy which correlated with the individual increased NKT lymphocyte numbers as well as transforming growth metabolic status (Yoshida et al., 2016). factor beta (TGF-β), matrix metalloproteinase 9 (MMP-9), IgA and IgM Further low-dose IR induced quantitative and functional alterations levels in Mayak workers exposed to external gamma rays with or in immune parameters in Chernobyl clean-up workers were reported by without internal alpha radiation. Authors concluded that changes Ilienko et al., who investigated cellular immune parameters of 235 in­ detected in the immune parameters of the investigated individuals fav­ dividuals exposed to doses between 0.1 and 3500 mSv. They found oured the maintenance of a chronic inflammatory status, which could decreased CD4+/CD8+ ratios and increased Tregs in 56% of the studied contribute to the development of radiation-related late pathologies such clean-up workers, especially in those exposed to low doses. Interest­ as cardiovascular and malignant diseases (Rybkina et al., 2014). Very ingly, the authors showed that the decrease in the level of B cells and similar conclusions were reached by Kiselev et al., who reviewed activated T cells correlated with the increase in interleukin (IL)-1β levels immunological changes in radiation workers at a Siberian chemical in individuals exposed to doses below 100 mSv (Ilienko et al., 2018). complex (over 4000 workers), at the Mayak nuclear facility and at the Oradovskaia et al. studied a cohort of clean-up workers who developed Chepetsk chemical and metallurgical plant. The common characteristic different malignant diseases and identified typical immune parameter of these facilities was the presence of a mixed-type exposure (external changes in these people 1–3 years before the manifestation of cancer. gamma rays and internal alpha radiation due to incorporated uranium). These changes include reduced CD3+ CD4+ T cell levels, increased Importantly, the study recorded not only basic laboratory changes in CD8+ T cell levels and hence a reduced CD4/CD8 ratio, and a preva­ immune parameters but also clinically relevant symptomatic immune lence for natural killer T (NKT) cells over conventional natural killer dysfunctions such as infections, allergies, autoimmunity and immuno­ (NK) cells (Oradovskaia et al., 2011b) confirming previous data of proliferative diseases and found an increased risk for immune deficiency increased NKT cells in clean-up workers (Kuzmenok et al., 2003). leading to an infectious syndrome in nuclear industry workers compared Though these markers seem to indicate a risk for cancer development, it to controls. Significantly elevated IgE levels in a certain group of is not clear to what extent these alterations were specific for radiation workers in the absence of a relevant allergic anamnesis was also re­ exposed individuals rather than the general population developing ported, which might indicate a low dose radiation induced imbalance in cancer at later times. humoral immunity (Kiselev et al., 2017). Gyuleva et al. basically confirmed most of these findings in Bulgarian nuclear power plant 2.2. Environmental or occupational radiation exposures workers with additional details on individual lymphocyte sub­ populations and their activation status. Namely, they showed a subtle Other exposure scenarios of interest are long-term chronic exposures but significant decrease in the proportion of naïve CD4+62L+ cells, affecting either residents living in regions with increased background CD4+CD25+ activated/regulator T cells and an increase in activated radiation (natural or human-made) or workers occupationally exposed CD8+CD28+ cytotoxic T cells, along with an increase in NKT cells in to radiation. Studies investigating individuals living in Ramsar, Iran at persons receiving doses below 200 mSv. Thus, they hypothesised a natural high background radioactivity of up to 260 mSv per year showed possible shift from a Th1 to Th2 response at doses above 200 mSv. The mild immunological alterations manifested in increased Ig levels (in main added value of this study compared to the previous ones was that particular IgE), increased levels of activated CD4+ cells and a tendency a) it took into account confounding factors such as smoking and alcohol towards T helper 2 (Th2) polarization without changes in innate im­ consumption and b) it discussed the influence of aging as another con­ mune parameters (in terms of neutrophil chemotaxis), while results founding factor on immune parameters, which can overlap with certain relating to cytogenetic damage in blood lymphocytes were contradictory changes seen following radiation. Though, they also showed that most of (Attar et al., 2007; Borzoueisileh et al., 2013; Ghiassi-nejad et al., 2002; the changes in the measured parameters remained within the normal Ghiassi-Nejad et al., 2004; Molaie et al., 2012). Studies investigating reference values illustrating the discrete effects of low doses (Gyuleva residents living in the Yangjian high background radiation area (with et al., 2018; Gyuleva et al., 2015a; Gyuleva et al., 2015b). Other occu­ cumulative doses up to 249 mSv) showed an increased tendency of both pational data are from interventional cardiologists, radiologists and CD4+ and CD8+ T cells in the peripheral blood mononuclear cells radiation workers. These studies generally report exposure doses well (PBMCs), the latter correlating with dose. Moreover, multiple below those found in nuclear industry workers mentioned above. At inflammation-related cytokines and blood proteins were significantly doses of 8.14 ± 7.81 mSv/year for at least 5 years, i.e. below 50 mSv in increased, such as soluble IL-6 receptor (sIL-6R), interferon (IFN)-γ, cumulative dose, drastic fluctuations in circulating immune cells are monocyte chemoattractant protein-1 (MCP-1) and C-reactive protein unlikely to occur, however more subtle changes such as priming the (CRP) (Li et al., 2019). Chang et al investigated home environments immune system towards stronger Th1 response upon a secondary chal­ giving off on average 169 mSv gamma radiation from building materials lenge were reported (Ahmad et al., 2016; Karimi et al., 2017; Zakeri to 196 individuals over 2–13 years. They found a change in lymphocyte et al., 2010). Occasional quantitative changes in the cellular and hu­ subsets in favour of CD8+ T cells while CD4+ T cell numbers and CD4/ moral immune system components have been reported even at standard CD8 ratio negatively correlated with dose when compared to 55 non- annual dose levels below 3.5 mSv/year with smoking being an impor­ exposed close relatives (Chang et al., 1999a). Despite the fact that the tant confounder (Godekmerdan et al., 2004; Klucinski et al., 2014; Rees cited studies focused on different immune parameters their conclusions et al., 2004). High variability amongst “normal” individuals is an were quite convergent, showing increased activity in the adaptive im­ important limitation, especially as pre-exposure levels are rarely if ever mune response and the presence of pro-inflammatory factors. A recent known in immune epidemiological studies. Diurnal rhythm has a pro­ study on gene expression profiles of PBMCs from inhabitants of the high nounced effect that is impossible to control and individual variation in level background radioactivity area in Kerala, India found that immune lymphocyte radiosensitivity is also large. response pathways were among the radiation-affected over-represented pathways (Jain and Das, 2017). 2.3. Studies on radiation-exposed children Immune markers were investigated in people living in radiation- contaminated areas such as around the Mayak nuclear complex, There is a limited number of studies investigating immune changes in including the Techa River in the Russian Federation. Akleyev et al. children exposed to chronic low dose exposures. In general, as reviewed 8 K. Lumniczky et al. Environment International 149 (2021) 106212 in the UNSCEAR 2013 report, children might be more at risk for a persistent alterations in CD4+ T cell numbers and function; b) a shift number of radiation-induced late effects both of stochastic and deter­ towards humoral immunity; c) contradictory conclusions on cytotoxic ministic nature. This is true for the incidence of certain tumors such as CD8+ T cell numbers and innate immunity, most probably with a shift leukaemias, skin, brain and thyroid cancer as well as deterministic towards activation of certain NK cell compartments; d) limited knowl­ health effects such as cognitive defects, cataracts and thyroid nodules edge on changes in granulocytes; e) likely accelerated immune aging (Radiation, 2014). For this reason and also for the fact that the immune (Table 3). Overall, there are big gaps in our understanding of functional system maturation in children is still ongoing and damage at this state alterations in immune parameters which are of particular interest at this might lead to different consequences in immune function than in adults dose range. or older individuals with various degrees of immune senescence, we Results from the diverse epidemiological or human biomonitoring considered discussing studies of radiation-exposed children separately studies are difficult to compare mainly due to gross heterogeneities in from those targeting adults. Chronic low dose exposure in children of the studied cohorts in terms of age (both in terms of age at exposure and kindergarden-age can have long-lasting effects on peripheral blood cell time elapsed from exposure), doses received, irradiation scenarios counts. Children exposed to 21–85 mSv gamma radiation over a 1–2 (acute or chronic, external, internal or mixed exposures), presence of year period experienced a drop in total leukocyte and neutrophil counts confounding factors (lifestyle, comorbidities, genetic background) and and an increase in eosinophils that lasted as long as 5–7 years after the studied endpoints. A major drawback to almost all of these studies is end of exposure, with total lymphocyte numbers not affected when their purely descriptive nature without much effort to link these to compared to children exposed to 2–5 mSv (Chang et al., 1999). Alter­ specific pathological conditions or diseases. Studies investigating im­ ations in T cell immunity were detected in children 6–13 year old living mune alterations in A-bomb survivors suggested a correlation between within a 40–75 km radius of radiation-contaminated areas around radiation-induced chronic inflammation and increased incidence of Chernobyl in North Ukraine and exposed to persistent low level radia­ chronic degenerative-type conditions (e.g. cardiovascular diseases, tion. These children had lower CD4 + T cell counts and a shift in the metabolic alterations). The few observations pointing to an increase in immune balance towards cytotoxic T cell and NK cell subsets especially the incidence or susceptibility to infections or towards an increased in vulnerable populations such as those who developed recurrent res­ predisposition for autoimmune disorders await confirmation by further piratory problems (Vykhovanets et al., 2000). The effects were more studies. So far, it has been difficult to discriminate between immune pronounced at doses above 1 mSv but were also seen below that dose alterations that are within the normal resilience capacity of an indi­ (Chernyshov et al., 1997), although confounding factors such as general vidual versus those that fall outside that range and might link directly to health status, individual immunogenetic make-up and exposure to certain diseases. The fact that immune system alterations are part of pathogens make radiation dose response patterns difficult to ascertain. many if not all pathophysiological processes leading to chronic diseases Though in children with irritable bowel disease living in the Chernobyl not traditionally considered as bona fide immune diseases (e.g. heart contaminated area no association with radiation was found (Sheikh conditions) only adds further to the complexity of the issue. A meta- Sajjadieh et al., 2012). analysis of all epidemiological and biomonitoring studies would be The link between childhood radiation exposure and thyroid cancer useful to evaluate relevant correlations between radiation effects, im­ was firmly established in the aftermath of the Chernobyl accident in mune system changes and related health consequences. A better char­ 1986 (Pacini et al., 1997), with non-cancer thyroid diseases including acterisation of the control group and the study design (including the autoimmune thyroid disorders emerging a few years after the accident. statistical analysis used), categorisation of the measured endpoints and This was less of a research focus in the A-bomb survivor cohorts, reported health outcomes would help identifying the most informative although thyroid pathologies were studied in middle aged adults from studies. the Hiroshima and Nagasaki cohorts who were exposed in utero, with a mean maternal uterine dose of 0.256 Gy (range 0.005 Gy to 1 Gy). It was 3. Experimental and pre-clinical data on low dose ionizing determined that they had similar risk estimates as those in the Chernobyl radiation effects on the immune system study but there was no significant linear dose response for thyroid nodules or autoimmune thyroid disease (Imaizumi et al., 2008). Simi­ The number of in vitro and in vivo data about low dose IR effects on larly, thyroid nodules tended to be much more prevalent in the the immune system is increasing in parallel with a progressively radiation-exposed Marshallese population, but without correlating to increasing concern of the medical and scientific community regarding abnormal thyroid function (Takahashi et al., 1999). A comprehensive long-term biological effects of low doses. These experimental evidences review on the initial epidemiological studies is provided by Saenko often confirm epidemiological observations and shed light on the et al., who concluded that the frequencies of abnormal haematological mechanisms how IR interacts with the immune system. In the following parameters and thyroid autoimmunity did not correlate with dose paragraphs, we will review the most pertinent experimental findings (Saenko et al., 2011). According to long-term studies, the rise in auto­ which complement epidemiological data, focusing on the link between antibodies is very common even at lower doses but it can be transient basic molecular mechanisms targeted by IR and the immune system, as and does not necessarily yield clinical thyroid autoimmune disease or well as direct low dose effects on quantitative and functional changes in thyroid dysfunction although well-designed long-term investigations are still needed (Agate et al., 2008; Eheman et al., 2003; Kasatkina et al., 1997; Ron and Brenner, 2010). The disconnection between the rise in Table 3 autoantibodies and the lack of clinical disease might be due to a radia­ Summary of described immune effects by radiation exposure. tion damage-driven release of thyroid antigen without subsequent Radiation effect Possible immune marker lymphocytic infiltration. One explanation is the possibility of a transient Imbalance of peripheral blood Changes in B cell count autoimmune reaction (Agate et al., 2008). It is also important to mononuclear cells Changes in T cell count consider the dietary iodine uptake. However, determining the associa­ Changes in T cell subpopulations tion between low dose exposures and their clinical significance in terms Changes in NKT count of benign thyroid diseases is not without challenges, which include Acceleration of immunoaging Reduction of naive T cells Expansion of memory T cells limited sample size, inadequate dose estimates, prevalence in healthy Thymus involution subsets, gender differences and technical problems that relate to the Reduction of telomere length of leukocytes variety of antibodies that can be studied (Eheman et al., 2003). Humoral immune response Changes in immunoglobulin level (IgA, Overall, epidemiological studies on long-term low dose irradiation IgM, IgG, IgE) Inflammation Pro-inflammatory response by cytokines effects on the immune system indicate a) a consensus regarding 9 K. Lumniczky et al. Environment International 149 (2021) 106212 the different immune compartments. Finally, we will present regulation clearance of damaged cells and tissue remodeling/regeneration (Ermo­ of the inflammatory response as an example of the differential mecha­ laeva and Schumacher, 2014). nisms and outputs initiated by low-dose versus high-dose irradiation. Thus, local RT can cause changes in tissues and organs outside the field of irradiation. This is called the abscopal effect (Siva et al., 2015) 3.1. Association between DNA damage response and immune response at where it can cause damage to unirradiated normal tissues, or to a low doses of radiation distant, non-irradiated tumor. Clinically, such effects are well recog­ nised, as patients often suffer from fatigue, diarrhea and weight loss DNA damage is considered a primary consequence of IR and the during local RT. Reports of spontaneous anti-tumour abscopal responses activation of the DDR pathway is a key factor in determining long-term are rare, since an immune tolerant state has already been established in cell fate after irradiation. IR at low doses already may induce a variety of the patient; Abuodeh et al reported 35 cases over 45 years, out of mil­ lesions like double-strand beaks (DSBs) accompanied by single-strand lions of RT patients treated during this period (Abuodeh et al., 2016). breaks (SSBs) and/or oxidized bases in a bistranded or unistranded Since the first report by Demaria et al that the immune system was an form. Manning et al showed increased frequency of micronucleated integral component of the abscopal response (Demaria et al., 2004), erythrocytes in the blood of mice treated in vivo with either external low there have been studies reported, in which activation of the immune dose X rays or with PET scan associated internal exposure with the system, commonly using checkpoint inhibitors against cytotoxic T radioisotope 18F-FDG (Manning et al., 2014). Rothkamm et al reported lymphocyte antigen 4 (CTLA-4), programmed cell death protein 1 (PD- that quantification of γH2AX foci in the leukocytes of patients subjected 1) and OX40 in combination with local RT, was able to induce growth to CT scans could reliably estimate the level of radiation exposure suppression in the second unirradiated primary tumor (Esposito et al., (Rothkamm et al., 2007). DNA damage after such low doses is not suf­ 2015). On the other hand, under optimal conditions (eg targeting a ficiently severe to induce cells death but can initiate danger signalling hypoxic core of the tumour with ablative RT), the anti-tumor effect can (Mavragani et al., 2016; Mavragani et al., 2017). Interestingly, both in be achieved with RT alone (Tubin et al., 2019). Exposure of blood and vitro and in vivo studies have shown a remarkable persistence of the DSBs normal tissues surrounding the tumor, including the bone marrow and (as evidenced by the slow resolution kinetics of γH2AX foci) even after thymus, to low-dose scatter radiation, may play a role in the abscopal doses in the range of computed tomography (CT) scans, well below 100 effects affecting normal non-irradiated tissues. After both external mGy. These changes were first detected and extensively studied in fi­ beam- and radionuclide RT, persistent and statistically significant broblasts (Grudzenski et al., 2010; Lobrich et al., 2005; Rothkamm and increased DNA damage was observed in circulating PBMCs, and their Lobrich, 2003) but actually more recent investigations showed a similar repair capacity was affected (Denoyer et al., 2015; Yin et al., 2019). This persistence and delayed repair kinetics of DNA damage in lymphocytes could be explained by protracted induction of DNA damage by abscopal as well (Beels et al., 2010; Lassmann et al., 2010). Plenty of evidence effects in susceptible progenitor cells that is revealed later as increased suggests a strong crosstalk between DDR activation and inflammatory unrepaired damage in PMBCs. response triggering primarily innate immune responses (Ermolaeva and Schumacher, 2013; Pateras et al., 2015) also in case of deficient DNA 3.3. Low dose radiation induced quantitative and functional changes in repair or persistent DNA damage (Karakasilioti et al., 2013). Damaged immune parameters cells can release a variety of stress or danger signals called damage associated molecular patterns (DAMPs) which act as mediators of innate Lymphocytes are very sensitive to high dose irradiation. Though, a immune responses (Heil and Land, 2014). These endogenous molecules more in-depth analysis reveals significant differences in the radiosensi­ released from damaged, stressed or dying cells, in analogy to the tivity of the different immune cell subsets even after high doses with B pathogen-associated molecular patterns - are recognised by the pattern and CD8+ T lymphocytes being more radiosensitive while dendritic recognition receptors located within and on innate immune system cells cells (DCs), monocytes, macrophages and regulatory T cells being more and instigate inflammatory responses. DAMPs can be different cyto­ radioresistant (Heylmann et al., 2014). Radiosensitivity of NK cells is kines, DNA, RNA, ATP, intra-cellular proteins or protein fragments, etc. controversial, with some studies reporting that NK cells are more radi­ It is remarkable, that this feature of stress response is common with oresistant than CD3+ cells (Bogdandi et al., 2010; Swanson et al., 2020), variations across organisms of different complexity and evolutionary while others reporting the opposite (Falcke et al., 2018). phylogeny i.e. from plants to mammals (Land, 2015; Pavlopoulou et al., Low dose radiation elicits a more heterogeneous immune response to 2019). radiation. Using murine models, it was shown that single low doses (up to 100 mGy) induced mild and transient (up to 21 days post irradiation) 3.2. Systemic and abscopal effects of local radiotherapy decrease in the pool of all the above mentioned splenic subpopulations (Bogdandi et al., 2010; Gridley et al., 2009). If similar doses were Although typical fraction doses for RT are 2 Gy to the tumor, delivered chronically over a period of 1–17 weeks, no changes were neighboring tissues can receive a wide range of low to medium doses up detected in the short-term (Shin et al., 2010) but a long-lasting increase to 0.5 Gy (Pouget et al., 2018). In many cases of RT, biological and in T and B cell numbers were registered in mice after antigen stimulation clinical systemic immune and inflammatory responses were found to be (Ina and Sakai, 2005). Much lower doses (100 mGy/year) on the other similar to responses of the irradiated tissues reflected in changes of key hand did not impact on T or B cell pools, indicating these doses were too modulators of the immune system like cytokines or chemokines. For low to induce immune effects (Courtade et al., 2001). TCR repertoire example, in a study by Mathias et al differential anti- and pro- profiling in mice showed that acute low/intermediate exposure could inflammatory responses were detected in the heart after local heart promote premature immune aging called immunosenescence. Exposure irradiation with low and high doses and these differential responses to a single dose of radiation was found to accelerate ageing of the pe­ were partly reflected at systemic level, in the plasma as well (Mathias ripheral TCR repertoire. The consequences of exposure were already et al., 2015). Several studies in human patients undergoing RT support a visible after 1 month and lasted for 6 months. Interestingly, they were time- and treatment-dependent modulation of specific cytokines at more pronounced in animals exposed to 0.1 Gy than in those exposed to systemic level, which in some cases can persist up to several years post- 1 Gy, where they were partially corrected with time, indicating that they treatment, indicating a definite systemic response to radiation mediated can be reversed/attenuated. Of note, some of these effects were due to by inflammatory pathways (Marconi et al., 2019). These systemic phe­ radiation-induced impairment of hematopoietic stem cells (Candeias nomena have also been observed in animals as reviewed in (Mavragani et al., 2017). et al., 2017). Of course, these excessive immune responses not only Chronic low dose rate irradiation of mice induced a) stimulation of contribute to chronic inflammation and tissue damage but also to innate immunity by enhancing the cytotoxicity of pre-stimulated NK 10 K. Lumniczky et al. Environment International 149 (2021) 106212 cells (Sonn et al., 2012; Yang et al., 2014), b) myeloid cell differentiation whole human blood exposed in vitro to 0.05 or 1 Gy suggested that low and activation, c) suppression of pro-inflammatory responses (Shin dose radiation rather induced pro-survival/anti-apoptotic and immune et al., 2010) and d) a shift towards a Th2-type T cell phenotype (Shin response pathways, whereas high dose exposure rather induced p53- et al., 2010) due in part to radiation-induced gene expression alterations dependent signaling pathways (El-Saghire et al., 2013b). A similar di­ in CD4+ T cells (Cho et al., 2018). Low dose effects on DCs are less well chotomy was found for the response to low and high dose radiation of characterized. Persa et al. showed that low and high dose acute irradi­ purified human monocytes exposed in vitro (El-Saghire et al., 2013a). ation induced qualitatively different functional changes in murine Low, but not high dose exposure induced the transcription of genes splenic DCs in vivo. By this, low doses stimulated antigen uptake and involved in TLR signaling, with a concomitant activation of NF-κB, p38, lowered antigen presentation while high doses did not influence these extracellular-signal-regulated kinase (ERK) and c-Jun N-terminal kinase parameters, on the other hand high doses increased the expression of T (JNK). In contrast, exposure to 1 Gy induced phosphorylation of p53 and cell costimulatory markers and enhanced the production of pro- transcription of p53-dependent genes (El-Saghire et al., 2013b). In vitro inflammatory cytokines (Persa et al., 2018). Other groups performing exposure of human PBMCs obtained from healthy donors to 0.05 Gy of X in vitro irradiation with doses ranging from 0.05 to 5 Gy of DC-precursors rays or 12C ions did not induce significant changes in the proportion of T or mature DCs stimulated by lipopolysaccharide (LPS) reported no in­ or NK cells 24 h later. Both types of radiation induced the transcription fluence of irradiation on surface marker (CD80, CD83, CD86), cytokine and the secretion of IL-2, IFN-γ and TNF-α, and an increase in the expression or the capacity of the DCs to stimulate T-cell proliferation cytotoxic activity of the irradiated PBMCs. These effects appeared (Jahns et al., 2011). Moreover, a co-incubation of bone marrow-derived stronger with high than with low LET radiation (Chen et al., 2010). DCs with supernatant of LPS stimulated and irradiated macrophages Altogether, experimental data do not indicate a linear dose response resulted in a diminished CD40 expression but did not impact on the DC- relationship of immunological parameters modulation. IR may have derived induction of T-cell proliferation (Wunderlich et al., 2019). qualitatively different effects at different doses/dose ranges. However, Toll-like receptors (TLR), appearing on innate immune cells induced some responses/endpoints display discontinuous characteristics shared DC maturation which primed T-cells. In the presence of costimulatory with non (DNA)-targeted properties of IR (Mothersill and Seymour, elements, cytokine release was established which lead to macrophage 2011). The molecular mechanisms responsible for these non-linear dose activation (Ratikan et al., 2015). Low dose fractionated irradiation of response relationships, however, remain elusive and may be associated mice lead to increased phagocytic activity of macrophages and increased with a connexion of multiple pathways initiated at different threshold CD8+ T cell cytotoxicity (Pandey et al., 2005). Another pathway doses and following different kinetics (Mothersill and Seymour, 2011; important in radiation response from the immune system is through Rodel et al., 2012). There is growing evidence for a mechanistic link nucleotide binding oligomerisation domain (NOD)-like receptors. These between transcription factor activity including p53 protein, activating are inflammasomes, which are important in case of damage from IR or protein 1 (AP-1), and NF-κB in the regulation of the DDR and that of from viruses. The redox status of cells may work as a rheostat, which immune response (Habraken and Piette, 2006; Magne et al., 2006; may lead to a vicious circle of harm in case of excess oxidation, or may Prasad et al., 1995). For example, a dose-dependent biphasic tran­ calm down the inflammation in reduced conditions. TLR agonists like scriptional activity of NF-κB has been shown in endothelial cells (ECs) LPS (especially for TLR4), may influence the response to ionizing radi­ and macrophages (Rodel et al., 2004; Wunderlich et al., 2015). ation (Ratikan et al., 2015). As previously mentioned, RT can induce long lasting systemic 3.4. Initiation of anti-inflammatory vs pro-inflammatory processes by IR changes in immune/inflammatory parameters (Marconi et al., 2019). These changes may appear gradually during the course of the treatment Functional changes in immune parameters after low dose exposure (Manning et al., 2017), and even further evolve in the weeks following show a significant degree of heterogeneity and are often qualitatively its completion (Balazs et al., 2019; Frey et al., 2020). Hence, the mod­ different from changes induced by high doses. Inflammatory response is ulation of the expression of some inflammatory (ARG1, BCL2L1, MYC) the best studied immune mechanism which is regulated differentially by genes in blood cells was not apparent after the first two fractions but low/medium dose and high dose irradiation. In the next paragraphs we became significant after the 25th in patients treated for endometrial summarize the current knowledge on the regulation of inflammatory cancer. Of note, RT did not modulate these same genes as strongly in processes by low versus high dose irradiation (Fig. 2). head and neck patients (Manning et al., 2017). This difference may be due to different volumes of blood being exposed at each fraction in 3.4.1. Modulation of basic inflammatory mechanisms by low/medium dose endometrial and head and neck patients, or to different effects of the radiation exposure different tumour entities on the immune system. The presence of a Inflammation is a basic immunological effector process in response carcinoma in the head and neck area have indeed been shown to pro­ to harmful stimuli, such as pathogens, damaged cells or irritants (Mur­ mote an immunosuppressive state in patients, which is even accentuated phy and Weaver, 2017) and is regulated by complex interactions be­ (increase in Tregs, increased expression of checkpoint molecules on tween a variety of immune components and soluble mediators. CD4+ T cells), during and after RT (Balazs et al., 2019). Systemic Among the initial step in the inflammatory pathways is the recruit­ modulation of cellular and humoral immune parameters can also ment of leukocytes to the site of damaged tissue mediated by local depend on the mode of radiation delivery (Frey et al., 2020). However, adhesion to the endothelium. By analysing PBMC and poly­ most of these studies addressed changes occurring after repeated local­ morphonuclear (PMN: granulocytes) cell attachment to cytokine TNF-α ized exposure, and it is therefore difficult to assess the exact role of low activated endothelium a significant reduction of adhesion in the dose dose in these observations. Radiation-induced gene expression changes range of 0.3–0.7 Gy was among the first mechanisms reported to were also investigated in peripheral blood cells of RT-treated prostate contribute to an anti-inflammatory effects of intermediate dose exposure cancer patients (El-Saghire et al., 2014) 18–24 hours after the first (Hildebrandt et al., 2002; Kern et al., 2000; Roedel et al., 2002). These fraction (2.09 Gy), resulting in an equivalent total body blood dose of findings functionally coincide with secretion of the anti-inflammatory around 31 mGy. Gene set enrichment analysis showed the activation of cytokine TGF-β1 from ECs, both in vitro and in a murine model several pathways involved in immune signaling: IFN secretion, CD28 (Arenas et al., 2006; Roedel et al., 2002). Subsequent studies further stimulation, antigen processing and presentation, B-cell receptor (BCR) indicate an impaired adhesion to be associated with a non-linear pro­ and TCR activation, inflammasomes, transcription factor nuclear factor duction of reactive oxygen species (ROS) in ECs, which is functionally kappa B (NF-κB), TLR and cytokine signalling indicating that local high based on a modulation of the transcription factor nuclear factor E2- dose resulted in systemic activation of pathways involved in immune related factor 2 (Nrf2) and anti-oxidative enzymes such as superoxide processes. Interestingly, the comparison of the response elicited in dismutase, catalase and glutathione peroxidase (Large et al., 2015). 11 K. Lumniczky et al. Environment International 149 (2021) 106212 Fig. 2. Schematic representation of the most important immune- and inflammation-related processes developing after low, intermediate and high dose irradiation based on available epidemiological, clinical and experimental data. Further, clearance of apoptotic cells by macrophages is reported to result reduced paw swelling and increased grip strength (Frey et al., 2009). in non– or even anti-inflammatory responses (Gaipl et al., 2005; Voll More recent findings further indicate a significant reduction of inflam­ et al., 1997). In line with that, a discontinuous appearance of apoptosis matory and erosive areas with a lowered detection of bone-resorbing was observed in PBMCs following 0.1–0.3 Gy irradiation (Kern et al., osteoclasts and neutrophils. In addition, starting at a dose of 0.5 Gy, 1999), which may foster a reduced adhesion. This is enhanced by a proliferation and expression of receptor activator of NF-kB ligand diminished surface expression of the adhesion molecule E-selectin on (RANK-L) in fibroblast-like synoviocytes decreased, numbers of differ­ ECs (Hildebrandt et al., 2002; Roedel et al., 2002) or by the proteolytic entiated osteoclasts and their bone resorbing activity also diminished in cleavage of L-selectin from apoptotic PBMCs (Kern et al., 1999). line with a discontinuous dose response relationship in osteoblast- A key mechanism in the subsequent effector phase of inflammation induced mineralization (Deloch et al., 2018a). By contrast, irradiation covers the accumulation of monocytes and their differentiation into DCs with a single dose of 0.5 Gy displayed no harmful effects on cells in and inflammatory macrophages (Valledor et al., 2010). The latter sup­ healthy joints (Deloch et al., 2018b). Thus, one can conclude that in this port the local inflammatory process by a variety of functions such as dose range irradiation may also act via osteo-immunological mechanism phagocytosis, presentation of antigens, cytotoxic activity and secretion with the anti-inflammatory properties depending on the initial inflam­ of cytokines, ROS and nitric oxide (NO) (Fujiwara and Kobayashi, matory status (Candeias and Testard, 2015; Frey et al., 2017). In 2005). NO in turn impacts on vascular permeability, promotes tissue conclusion, recent experimental in vitro data have characterized mech­ oedema and is involved in the development of inflammatory pain anisms and factors that may contribute to the anti-inflammatory effect (Holthusen, 1997). Notably, irradiation at 0.3–1.25 Gy of activated of a low to medium dose exposure while in vivo models have confirmed macrophages resulted in decreased expression of the enzyme inducible improvements in a variety of clinical symptoms and parameters. nitric oxide synthase (iNOS) and NO production (Hildebrandt et al., First clinical studies of patients that had been exposed to very low 1998) in line with a hampered release of ROS and superoxide production doses of high linear energy transfer (LET) radon gas (0.3 mSv) gave first (Schaue et al., 2002) that have mechanistically been linked to the anti- hints that even here systemic immune modulations took place. Espe­ inflammatory and analgesic effects of low-dose exposure. In addition, cially, activation status of immune cells was influenced as well as levels irradiation with doses up to 2 Gy significantly decreased major histo­ of TGF-β (Cucu et al., 2017; Kullmann et al., 2019; Ruhle et al., 2017). compatibility (MHC) II expression and transmigration of macrophages One of the most compelling observations in this patient population was a (Wunderlich et al., 2019) but fostered chemotaxis of LPS activated long-term shift within the CD4+ T cell compartment towards a higher macrophages without impacting viability and phagocytic functions proportion of circulating Tregs along with a fall in CD8+ T cells after (Wunderlich et al., 2015). cumulative doses of around 0.3 mSv (Cucu et al., 2017). Decreased In order to establish experimental conditions mimicking as close as levels of lymphocytes expressing the activation marker CD69 and a possible the patients situation, a human TNF-α transgenic mouse model temporary rise in systemic TGF-β and higher IL-18 serum levels were was adopted to investigate the therapeutic and cellular effects of low found to directly relate to better pain control in patients after radon spa dose irradiation. These animals overexpress the cytokine during their treatment (Kullmann et al., 2019). lifetime and develop a polyarthritis with joint swelling and deformation, synovial inflammation, cartilage damage, and bone erosion comparable 3.4.2. Lung injury during radiotherapy – A clinical example of radiation- to the human situation (Keffer et al., 1991). Irradiation of these mice induced pro-inflammatory pathology with five times 0.5 Gy at early stages (4–6 weeks) revealed significantly The lung is one of the most radiosensitive organs, and RT-associated 12 K. Lumniczky et al. Environment International 149 (2021) 106212 lung tissue inflammation can occur early (radiation pneumonitis, within radiation-induced fibrosis (Forrester et al., 2014). Vascular endothelial 6 months) and late (chronic lung fibrosis, later than 6 months) after RT growth factor, E-selectin, I-selectin, basic fibroblast growth factor (Giridhar et al., 2015). Currently, radiation pneumonitis develops in (bFGF), IL-1, IL-6, IL-8, IL-10, IL-12 and IL-18 have also been suggested about 30% of patients and is fatal in 2% (Palma et al., 2013). It is the as possible predictive biomarkers (Sprung et al., 2015). main dose-limiting toxicity and can limit quality of life of cancer sur­ Chronic inflammatory responses as predictors of radiation toxicity vivors. Genetic factors contribute to the severity of the radiation- have also been described in patients treated with RT for other malig­ induced pneumonitis and fibrosis (Gatti, 2001). Pathophysiology, mo­ nancies than lung cancer. A recent review by Marconi et al. included 15 lecular and cellular mechanisms of these injuries have been described publications from the last 5 years on breast cancer patients receiving RT, (Giridhar et al., 2015; Huang et al., 2017), however still remain not fully many with conventionally fractionated regimens to a total dose of 50 Gy. understood. Some radiation-driven changes in cytokine levels persisted in these pa­ After irradiation, as a consequence of radiation-induced oxidative tients for months, such as IFN-γ and TGF-β. Similarly to lung, both IL-6 stress the DDR is dysregulated in lung, resulting in a population of cells and TGF-β correlated with radiation toxicity, although tumour stage and with unrepaired DNA damage. This in turn leads to destruction of timing were important factors, too (Marconi et al., 2019). Moreover, in alveolar epithelial and vascular ECs inducing tissue hypoxia. Oxidative 37 patients with prostate cancer receiving RT, cytokines were reported stress and tissue hypoxia are important regulators of cellular signalling to appear in recurring waves with pro-inflammatory IL-1 and macro­ pathways controlling the production of various factors involved in the phage colony-stimulating factor (M-CSF) preceding TGF-β1 but more development of radiation-induced lung injury such as secretion of pro- importantly the magnitude of these waves correlated with doses up to 9 inflammatory and pro-fibrogenic cytokines, growth factors and pro­ Gy given in daily 1.8 Gy fractions (Kovacs et al., 2003). teases that destroy the extracellular matrix and the barrier of lung tissue. Altogether, these clinical observations indicate that chronic inflam­ Hypoxia-inducible factor 1α (HIF-1α) protein is an important tran­ matory reactions are key players in the pathophysiology of high dose scription factor involved in the development of radiation-induced lung irradiation-induced normal tissue toxicity. injury, which is expressed early after irradiation as a consequence of Although not directly linked to the topic of the paper, we would like oxidative stress and tissue hypoxia and its expression progressively in­ to briefly mention that a cancer developing in an inflammatory micro­ creases for months thereafter. Once induced, it regulates the expression environment is in general more radioresistant compared to a cancer of a series of genes involved in pathways like inflammation, oxidative where chronic inflammation is not present. The link between local stress response, proliferation, differentiation or angiogenesis (Imtiyaz inflammation and cancer radioresistance is thoroughly investigated and and Simon, 2010; Jackson et al., 2012; Rabbani et al., 2010). Blood several excellent reviews exist on this topic (Aggarwal et al., 2009; vessel dilation and increased permeability allows for accumulation of Jarosz-Biej et al., 2019; Multhoff and Radons, 2012; Woodward et al., inflammatory cells (with macrophages being the most prominent cell 2010). types) at the site of irradiated tissue and in the alveolar cavity and cause An important point to consider when comparing inflammatory re­ a strong immune response. This acute inflammatory response may lead sponses after cancer RT with low dose radiation treatment of benign to radiation pneumonitis that induces a vicious circle of further diseases is the different initial temporal and spatial states of the immune inflammation and finally fibrosis induction. IR leads to release of cyto­ system. Many cancer patients have a more immune-suppressive tumor kines as well. A first wave of cytokine release settles within two weeks. microenvironment, in contrast to the chronic inflammatory state in The second wave starts around 6–8 weeks post-irradiation (Rube et al., benign disease; both of which are far from the more balanced, neutral 2004) and is associated with accumulation of oxidative DNA damage, set-point in healthy people. As the baseline immune balance has shifted, ROS or reactive nitrogen species (RNS), hypoxia, TGF-β expression, and the effect of radiation will shift also. decreased lung perfusion. TGF-β, in particular, TGF-β1, is mainly generated by activated macrophages and is a key player in development 3.5. The role of genetic background in the variability of radiation effects of fibrosis. It triggers proliferation and conversion of fibroblasts to on the immune system myofibroblasts. Myofibroblasts in turn produce excessive collagen and other extracellular matrix components, and secrete angiotensin and Individual variation in response to IR is well documented in cancer hydrogen peroxide, which induce apoptosis in alveolar epithelial cells. patients receiving RT and radiosensitive patients can present therapy- Activin A is induced by TGF-β1 and it also stimulates production of related side effects such as fibrosis, necrosis, dermatitis, esophagitis, collagen (Forrester et al., 2017). Fibrosis induces hypoxia and conse­ neuropathological disorders, inflammation and vascular damage, quent release of pro-fibrotic and pro-angiogenic factors like TGF-β1 among others (Pavlopoulou et al., 2017). The mechanism of inflam­ (Vujaskovic et al., 2001). These cycles are the basis of RT-induced mation and fibrosis developing in lung after RT has been described in the chronic inflammatory lung disease. previous chapter and it is evident that many of the adverse effects Clinical predictors have been identified in an effort to minimize ra­ involve processes of the immune system. Heritability studies suggest diation pneumonitis. Since the inflammatory response underlies RT- that 58–82% of the individual variation in sensitivity to radiation and in related lung toxicities, immunological factors involved in this response the development of RT-related toxicity can be attributed to genetic may provide potential biomarkers to predict individual response to RT. factors (West and Barnett, 2011). Currently the role of the genetic Lierova et al. recently reviewed 56 articles that have evaluated the as­ background in individual radiosensitivity has almost exclusively been sociation between serum cytokines and pneumonitis in irradiated lung investigated for high doses and practically no data exist in the literature cancer patients, most of them receiving around 60 Gy (Lierova et al., regarding low dose effects. We aim to provide a short summary of the 2018). Serum IL-6 and TGF-β1 tended to stand out in predicting sus­ data available regarding the role of the genetic background in the im­ ceptibility to subacute pneumonitis, whereas circulating IL-4 and IL-13 mune system changes influencing individual radiation response. appeared more critical for delayed effects such as chronic inflamma­ Several studies with murine models have shown genotype-dependent tion and fibrosis. Higher grade pulmonary toxicity in lung cancer pa­ inflammatory-type responses after whole-body irradiation. For example, tients was associated with early changes during RT treatment in plasma macrophages from CBA/Ca mice exhibited a pro-inflammatory pheno­ levels of IP-10, MCP-1, eotaxin, IL-6, and tissue inhibitor of matrix type, whereas those from C57BL/6 showed an anti-inflammatory metalloproteinase 1 (TIMP1) (Siva et al., 2016; Siva et al., 2014). The phenotype after irradiation with 4 Gy (Coates et al., 2008; Rastogi expression levels of follistatin, the member of TGF-β superfamily, et al., 2013). Moreover, different immune-related outputs were obtained fibrillin-2, which stores the latent form of TGF-β in extracellular matrix after doses below 100 mGy in two mouse strains with different sensi­ and inhibits its activation, and dermatopontin, which increases cellular tivity to radiation-induced mammary carcinoma: the sensitive strain but response to TGF-β, predicted lung radiosensitivity manifested as not the resistant one showed transcriptional signatures of macrophage 13 K. Lumniczky et al. Environment International 149 (2021) 106212 activation, pattern recognition receptor signalling, NK cell and TGF-β Table 4 signalling. While changes in immune gene signatures were transient, List of genes reported to be significantly associated with immune-associated activation of various cancer pathways in the sensitive strain after irra­ endpoints after radiotherapy. Only studies with >100 samples were included. diation was a durable effect (Snijders et al., 2012). This would confirm *BC, breast cancer; HNC, head and neck cancer; LC, lung cancer; PC, prostate the importance of genetic background in low dose IR-induced effects. cancer. It is well known that rare mutations in DNA DSB repair genes can Endpoint Tumor Genes Genes with References cause heritable syndromes, such as ataxia telangiectasia, that show type* associated contradictory with the results extreme radiosensitivity. However, radiosensitivity is now considered endpoint not to rely only on a single rare gene mutation but to be an inherited Dermatitis BC ABCA1, (Isomura et al., complex trait that depends on the interaction of common risk alleles in a IL12RB2 2008) large number of genes (Andreassen et al., 2002). The different responses HNC GSK3B, (Borchiellini to RT in patients treated with the same radiation doses and not affected MDM2, et al., 2017; by one of the above-mentioned heritable syndromes, support the XRCC1 Chen et al., consensus of the involvement of common genetic factors in radiosensi­ 2017; Yu et al., 2016) tivity (Guo et al., 2015). Esophagitis LC BLM HSPB1, (Guerra et al., The degree of the association between specific genes and pathways PRKCE, 2012; Lopez and sensitivity to radiation probably depends on the specific tissue TGFB1, Guerra et al., involved and the particular endpoint analysed (Andreassen et al., 2002). TNFSF7 2011; Pu et al., 2014; Zhao Focusing on the immune system, some genetic variants (mainly single et al., 2016) nucleotide polymorphisms (SNPs)) have been found to be associated Fibrosis BC ATM (lung TGFB1, XRCC1 (Barnett et al., with inflammation and fibrosis after radiation and in particular after RT fibrosis), Class 2010; Barnett (Table 4). For example, in early studies with relatively small sample III MHC et al., 2012; sizes, polymorphisms in glutathione S-transferase alpha1 (GSTA1), region, Edvardsen GSTA1, et al., 2013; glutathione S-transferase pi1 (GSTP1), TGF-β1, thioredoxin reductase 2 GSTP1, Edvardsen (TXNRD2), and X-ray repair cross-complementing 1 (XRCC1) genes TXNRD2 et al., 2007; were found to be significantly associated with radiation-induced fibrosis Giotopoulos in breast cancer patients. On the other hand, polymorphisms in ataxia et al., 2007; Grossberg et al., telangiectasia mutated (ATM), ERCC excision repair 5, endonuclease 2018; Quarmby (ERCC5), mouse double minute 2 (MDM2), XRCC1 and XRCC3 were et al., 2003; associated with fibrosis in naso-pharyngeal cancer patients. This would Seibold et al., agree with the tissue-specificity of the associations in some cases. 2015; Talbot However, some studies failed to replicate the initial associations for et al., 2012; Terrazzino TGF-β1, XRCC1 and XRCC3 (see Table 4 for references). et al., 2012) Later, more-powered studies were carried out (and some are still HNC ATM, ERCC5, XRCC3 (Alsbeih et al., ongoing) in collaborative projects with thousands of patients, e.g. HDM2, 2014; RadGenomics (Iwakawa et al., 2002), GENEPI (Baumann et al., 2003), XRCC1 Borchiellini et al., 2017; Gene-PARE (Ho et al., 2006), RAPPER (Burnet et al., 2013), and Cheuk et al., REQUITE (Seibold et al., 2019; West et al., 2014). These collaborative 2014) projects have successfully identified association of alleles from the MHC Several ATM (Zhang et al., III region with fibrosis in 2036 breast cancer patients and of ribonu­ cancers 2016) clease L (RNASEL) gene with IL-6 levels in 2550 prostate cancer patients IL6 levels PC RNASEL (Meyer et al., 2010) (Meyer et al., 2010; Talbot et al., 2012). However, a large study with Mucositis HNC APC, ATG10, XRCC1 (Chen et al., 2782 patients reported no association of a SNP in TGF-β1 gene with RT- ATG16L2, 2017; Ma et al., induced breast fibrosis (Barnett et al., 2012). Similarly, results from Zhu EDN1, NBN 2017; Pratesi et al. with 2926 patients with several cancer types failed to find a cor­ et al., 2011; relation between TGF-β1 SNPs and fibrosis after RT (Zhu et al., 2013). Venkatesh et al., 2014; All these studies were focused on candidate genes suspected to be Yang and Liu, causally related to radiation toxicity, such as DNA damage recognition 2019; Yu et al., and repair, free radical scavenging or anti-inflammatory response. Be­ 2016) sides the candidate-gene approach, few genome-wide association Pneumonitis LC AKT2, APEX, ATM, (Chen et al., ATG16L2, CBLB, HSPB1, 2013; Du et al., studies (GWAS) have been carried out to unravel genes important in BMP2, IL1A, IL13, LIG4, 2018a; Du et al., radiosensitivity unbiasedly (Barnett et al., 2014; Fachal et al., 2014; DDX58, MIF, NOS3, 2018b; Kerns et al., 2016; Kerns et al., 2010; Kerns et al., 2013a; Kerns et al., ERCC1, TGFB1, Hildebrandt 2013b; Kerns et al., 2013c). Most of these GWAS provided evidence for GSTP1, TNFRSF1B, et al., 2010; Li associations of polymorphisms with overall toxicity after RT or with HIPK2, IL4, VEGF, XRCC1 et al., 2014; Li IL8, LIN28B, et al., 2016; individual endpoints other than inflammation or fibrosis, so candidate MTHFR, Mak et al., gene approach studies previously mentioned (Table 3) could not be MUS81, 2012; Niu et al., confirmed or refuted. However, some GWAS identified genetic variants NEIL1, 2012; Pang that may underlie the variability of radiation effects on the immune NFKBIA, et al., 2013; Pu PI3CA, et al., 2014; system, specifically in prostate cancer patients treated with RT and who RAD51, SP-D, Tang et al., presented rectal bleeding, which could be caused by proctitis or TNF, TOPBP1, 2016; Tang inflammation of the rectum (Kerns et al., 2016; Kerns et al., 2013c). TP53 et al., 2019; Recently, Kerns et al. performed a meta-analysis with 3871 prostate Tang et al., cancer patients and identified one SNP significantly associated with 2020; Voets et al., 2012; rectal bleeding (Kerns et al., 2020). (continued on next page) In conclusion the role of genetic background in shaping the 14 K. Lumniczky et al. Environment International 149 (2021) 106212 Table 4 (continued ) radiation effects on other components of the innate immune system Endpoint Tumor Genes Genes with References are either contradictory (DCs) or are too few (polymorphonuclear type* associated contradictory cells) to allow for any pertinent conclusion regarding their behaviour with the results post exposure. endpoint 4. There is an interesting dichotomy when it comes to inflammatory Wang and Bi, responses in the context of low dose radiation. We can surely state 2010; Wen that low-to-moderate doses of low and high LET radiation have anti- et al., 2018; inflammatory effects on individuals with local inflammatory condi­ Wen et al., 2014; Xiong tions and may result in improvement in a variety of clinical symp­ et al., 2013; Xu toms and parameters. On the other hand long-term observations of et al., 2019; cohorts exposed to very low doses of radiation (acute or chronic) Yang et al., indicate a pro-inflammatory immune profile, which might contribute 2017; Yang et al., 2011; Yin to increased incidence of chronic degenerative diseases with an in­ et al., 2011a; flammatory component. Yin et al., 5. Epidemiological studies were the first to raise the possibility of a 2011b; Yin radiation-induced acceleration of immune aging as well as radiation- et al., 2012a; induced metabolic perturbations in immune cells. Recently experi­ Yin et al., 2012b; Yuan mental data seem to validate these observations, though low dose et al., 2009; studies in these topics are extremely few. Zhang et al., 2010; Zhao 5. Recommendations for future research et al., 2016) Despite all the data presented here, there are still some research gaps individual immune response after high doses is well demonstrated even that need to be filled and we present some recommendations for future in the absence of identified consensus on the identity of the gene(s) or studies: gene cluster(s) responsible for this effect. While studies investigating the role of genetic background in individual immune parameters to low dose 1. Dysregulation of the immune system is thought to play a key role in IR are very scarce in the literature there is no doubt that an individual many adverse health outcomes following exposure to IR. These can genetic signature is crucial in low dose response as well, especially be cancerous or non-cancerous, such as cardiovascular, neuro­ taking into account the high inter-individual variability of low dose ef­ cognitive and metabolic diseases, as described by Tapio et al. in fects, which are most probably governed by genetic (and epigenetic) another paper of this special issue. Clearly one of the greatest gaps in factors. This highlights the future need to focused research deciphering epidemiological studies is that immune changes identified in the role of genetic background in low dose regulated immune responses. different cohorts are not linked to changes in the incidence of particular diseases. These are intricately linked and need to be 4. Conclusions studied together, i.e. the role of the immune system in radiation- induced chronic non-cancer diseases such as cardiovascular, neuro- There is a wealth of scientific evidence based on epidemiological cognitive, metabolic diseases or autoimmune disorders. A better data from various cohorts exposed to different radiation doses, dose understanding of the mechanisms would allow us to develop effec­ rates and radiation qualities as well as clinical studies and experimental tive countermeasures and to identify predictive biomarkers. Given in vitro and in vivo data, all collectively supporting the notion that low the nature of the immune system, immune parameters considered to dose IR affects the immune system in multiple ways. be in the normal range show very high variability in the population The most important of these findings can be summarized as follows: at large but are relatively constant at an individual level. Hence, looking at population means might easily mask individual changes. 1. Both studies on human subjects and experimental data indicate low Ideally, in order to correctly evaluate individual changes they should dose radiation-induced damage on T cell immunity, especially CD4+ be compared to pre-exposure values of the particular individual if T cells. There is a fair amount of consensus in the literature on low possible, in addition to mean values of control population. Since dose radiation doses driving changes in the functional profile of most immune monitoring is performed on blood samples, future CD4+ T cells, most often a shift towards a Th2 phenotype. CD8+ epidemiological studies should aim to include data from pre- damage (both quantitative and functional) is also reported in most exposure blood samples as well as include potential covariates/ studies but there are discrepancies as to their radiosensitivity and confounders such as additional stressors, lifestyle factors and genetic their functional impairment depending on whether acute or long background. term chronic exposures were performed. It is also known that T cell 2. Most evidence indicates that the relationship between IR exposure activation can drive radioresistancy, which may explain some of and immunological changes is anything but linear. In some instances, these discrepancies. low, intermediate and high IR doses of different radiation qualities 2. Data regarding low dose radiation effects on humoral immunity and applied in different dose rates induce discontinuous or biphasic mostly arise from epidemiological observations and relatively few effects on the immune system. However experimental data also show experimental studies have focused on this component of the immune that low/intermediate versus high doses (or dose ranges) induce system. While most studies show some degree of humoral immune qualitatively different shifts in immune responses (e. g. the shift from enhancement, these are in contradiction with sporadic reports on an Th1 to Th2 profile of T helper cells or a shift from a pro-inflammatory increased proneness to bacterial infections among radiation exposed towards an anti-inflammatory response or vice versa). The effects of individuals, although this may vary with the infectious agent. low/intermediate doses may depend on the initial inflammatory 3. Within the innate immune system, NK cells are perhaps the best state of the irradiated tissue, the dose rate and quality of radiation. studied lineage and there is a relative consensus both in epidemio­ Big gaps exist in our knowledge of the molecular events responsible logical and experimental data on the role of low dose radiation in for these different response patterns. A better understanding of these stimulating certain types of NK cells. Reports about low dose mechanisms would enable a better control of radiation-induced im­ mune changes. In line with that, one major characteristics of the 15 K. Lumniczky et al. Environment International 149 (2021) 106212 immune system is a high degree of cellular motility and interaction Agate, L., Mariotti, S., Elisei, R., Mossa, P., Pacini, F., Molinaro, E., Grasso, L., Masserini, L., Mokhort, T., Vorontsova, T., Arynchyn, A., Tronko, M.D., Tsyb, A., dynamics of the different components to activate host-protective Feldt-Rasmussen, U., Juul, A., Pinchera, A., 2008. Thyroid autoantibodies and activities and to terminate immune responses to counteract chronic thyroid function in subjects exposed to Chernobyl fallout during childhood: evidence and harmful activation. Thus, multiple sampling and the usage of for a transient radiation-induced elevation of serum thyroid antibodies without an multiplexed, high-resolution in situ examination is recommended to increase in thyroid autoimmune disease. J. Clin. Endocrinol. Metab. 93, 2729–2736. https://doi.org/10.1210/jc.2008-0060. unravel the complexity of immune interactions. 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Nathalie Impens: Concep­ inflammation parameters of head and neck cancer patients. Cancers (Basel) 11. tualization, Writing - original draft. Gemma Armengol: Writing - https://doi.org/10.3390/cancers11091324. original draft. Serge Candéias: Writing - original draft, Writing - review Balogh, A., Persa, E., Bogdandi, E.N., Benedek, A., Hegyesi, H., Safrany, G., Lumniczky, K., 2013. The effect of ionizing radiation on the homeostasis and & editing. Alexandros G. Georgakilas: Writing - original draft, Writing functional integrity of murine splenic regulatory T cells. Inflamm. Res. 62, 201–212. - review & editing. Sabine Hornhardt: Writing - original draft, Writing - https://doi.org/10.1007/s00011-012-0567-y. review & editing. Olga A. Martin: Writing - original draft, Writing - Barnett, G.C., Coles, C.E., Burnet, N.G., Pharoah, P.D., Wilkinson, J., West, C.M., review & editing. Franz Rödel: Writing - original draft, Writing - review Elliott, R.M., Baynes, C., Dunning, A.M., 2010. No association between SNPs regulating TGF-beta1 secretion and late radiotherapy toxicity to the breast: results & editing. Dörthe Schaue: Writing - original draft. from the RAPPER study. Radiother. Oncol. 97, 9–14. https://doi.org/10.1016/j. radonc.2009.12.006. Barnett, G.C., Elliott, R.M., Alsner, J., Andreassen, C.N., Abdelhay, O., Burnet, N.G., Declaration of Competing Interest Chang-Claude, J., Coles, C.E., Gutierrez-Enriquez, S., Fuentes-Raspall, M.J., Alonso- Munoz, M.C., Kerns, S., Raabe, A., Symonds, R.P., Seibold, P., Talbot, C.J., Wenz, F., The authors declare that they have no known competing financial Wilkinson, J., Yarnold, J., Dunning, A.M., Rosenstein, B.S., West, C.M., Bentzen, S. M., 2012. Individual patient data meta-analysis shows no association between the interests or personal relationships that could have appeared to influence SNP rs1800469 in TGFB and late radiotherapy toxicity. Radiother. 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