Immune Dysfunction from Radiation Exposure

Abstract

Exposure to ionizing radiation causes acute damage and loss of bone marrow and peripheral immune cells that can result in high mortality due to reduced resistance to infections and hemorrhage. Besides these acute effects, tissue damage from radiation can trigger inflammatory responses, leading to progressive and chronic tissue damage by radiation-induced loss of immune cell types that are required for resolving tissue injuries. Understanding the mechanisms involved in radiation-induced immune system injury and repair will provide new insights for developing medical countermeasures that help restore immune homeostasis. For these reasons, The Radiation and Nuclear Countermeasures Program (RNCP) and the Basic Immunology Branch (BIB) under the Division of Allergy, Immunology, and Transplantation (DAIT) within the National Institute of Allergy and Infectious Diseases (NIAID) convened a two-day workshop, along with partners from the Biomedical Advanced Research and Development Authority (BARDA), and the Radiation Injury Treatment Network (RITN). This workshop, titled “Immune Dysfunction from Radiation Exposure,” was held virtually on September 9–10, 2020; this Commentary provides a high-level overview of what was discussed at the meeting.

INTRODUCTION

Ionizing radiation can cause damage to many tissue types, with the most sensitive tissues being those with cells that are actively dividing, such as the bone marrow. Bone marrow damage from radiation exposure markedly reduces replenishment of leukocytes, leading to reductions in systemic leukocytes, most notably neutrophils and platelets, resulting in an inability to effectively respond to infections and hemorrhage (1). Furthermore, radiation-induced tissue damage and infection initiate inflammatory responses that can lead to further systemic complications and tissue damage. Normally, this inflammatory response resolves as reactivity to multiple danger signals transition to tissue repair processes; however, these processes can be impaired due to damage to the immune system, or the repair could lead to some loss of function. Thus, radiation-induced immune dysfunction can range from incomplete recovery of hematopoietic function, permanent loss of immune regulatory cell types, persistent tissue damage, or a generalized state of immune suppression. Researchers have been studying various aspects of radiation-induced immune dysfunction to gain mechanistic insights and to develop medical countermeasures. A workshop that included scientists from the radiation biology and immunology disciplines was convened on September 9–10, 2020, to evaluate the latest research into radiation-induced immune dysfunction and to examine possible ways that this dysfunction could be treated. The planning committee included program staff from the Radiation and Nuclear Countermeasures Program (RNCP) and the Basic Immunology Branch (BIB) within the Division of Allergy, Immunology, and Transplantation (DAIT), along with partners at the Biomedical Advanced Research and Development Authority (BARDA), and staff from the Radiation Injury Treatment Network (RITN). This workshop featured scientific presentations from members of the research community, and discussion. The workshop examined the role of the immune system in maintaining homeostasis and covered the topics of aging, mechanisms of injury and repair, and inflammation. Despite the separation of topics, the various talks demonstrated the connectedness among these subject matter areas. This Commentary is a summary of the full meeting report (2).

SESSIONS

Keynote and Session I: Aging and the Immune System

The Keynote talk and Session I covered homeostasis and the long-term effects of ionizing radiation on animals as they age. The immune system, the body’s main defense mechanism, distinguishes between non-self and self, including sensing danger-associated molecular patterns (DAMPs), ultimately to restore injured tissue and maintain homeostasis (3, 4). Radiation exposure via intentional or unintentional means activates the cells of the innate and adaptive arms of the host immune system; however, during such exposure, the cells of the immune system undergo damage and subsequent loss. Inflammation, a host defense mechanism to manage an infection or non-infectious tissue injury, may become damaging or destructive when prolonged. An enhanced inflammatory state has been observed both in animal models and in atomic bomb survivors as they aged (5, 6). Inflammatory mediators such as IL-6, C-reactive protein, alpha-1 and alpha-2 globulins have been associated with radiation dose decades after exposure (7); however, the molecular and cellular processes that underlie the chronic inflammation observed post-exposure remain to be defined.

Studies delineating the response to radiation injury [total-body irradiation (TBI)] in mouse models demonstrated a blunted adaptive immune response in peripheral blood lymphocytes, lymph nodes and spleen, particularly in T and B cell compartments, except for the regulatory T cell (Treg) population whose levels increased (8). Losses remained substantial up to one month after radiation injury. On the contrary, innate immune cells, including macrophages, NK cells, and myeloid-derived suppressor cells (GR-1⁺) increased in number, while no significant changes were observed in dendritic cell numbers. Changes in the number and type of immune cells that persist after radiation exposure result in a variety of consequences that include reduced anti-microbial immunity, loss of barrier function at mucosal surfaces (e.g., gastrointestinal injury), uncontrolled opportunistic infections (e.g., bacterial sepsis, candidiasis, aspergillosis, reactivation of latent viruses), and chronic inflammation (e.g., failure to resolve injury, activation of the inflammasome pathway) contributing to persistent innate system activation (9). Understanding and elucidating mechanisms by which radiation exposure affects host immune function will provide insights and improved approaches for the development of novel medical countermeasures (MCM) such as trained immunity inducers (e.g. CpG-DNA) to restore immune health, increase resistance to infections, and enhance survival (10, 11).

The progressive manifestation of lifelong accumulation of cellular, molecular, tissue, and organ damage is associated with biological aging. Alterations in the immune response and increased susceptibility to infectious, chronic, and autoimmune diseases have been well-documented. Systemic low-grade inflammation in the absence of pathogens is characterized by the production of pro-inflammatory mediators such as tumor-necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) leading to the term “inflammaging” (11). Multiple mechanisms (e.g., thymic involution, cellular senescence, mitochondrial dysfunction, defects in autophagy) contribute to age-related diseases and accompanying morbidity and mortality in older individuals. In addition, epidemiological studies have provided valuable data on the association between radiation exposure and age-related diseases. Because of the similarity between age-related and radiation exposure-related alterations in the immune system, it is critical to determine the biological mechanisms underlying age-related radiation-induced effects as life expectancy rises in the population.

Radiation-induced damage after TBI or partial-body irradiation may result in death due to infection and/or hemorrhage from the loss of neutrophils, erythrocytes, lymphocytes, and platelets. Furthermore, defects in thymic development and B cell development have been shown to be exacerbated in mice (12), with skewed differentiation to the myeloid lineage in acute radiation syndrome (ARS) survivors compared to normal aging. Prior exposure to radiation also resulted in worsened thymic architecture. In hematopoietic stem cells (HSC) from hematopoietic-ARS (H-ARS) survivors, lymphopoiesis-related genes were down-regulated; in contrast, genes that normally decrease with aging were mainly increased. Gene categories that decreased in HSC from H-ARS survivors were enriched for aging-associated genes. In contrast, survivors of acute, high-dose radiation exposure are subject to the delayed effects of acute radiation exposure (DEARE) (13). DEARE may manifest as damage and progressive fibrosis in multiple tissues and organ systems. They include residual bone marrow damage (RBMD), malignancy, injury to the cardiovascular, pulmonary, renal, and gastrointestinal systems as well as the central nervous system.

Non-human primates (NHP) serve as a useful and relevant model for studying long-term effects of radiation exposure due to the length of their lifespan as well as their genetic and physiologic similarity to most human responses. The availability of a NHP colony of over 200 animals at Wake Forest University² and the ability to follow them over their lifespan to assess late effects of acute irradiation has contributed greatly to the knowledge gained after exposure of these animals to various doses of radiation. This NHP cohort has been monitored daily with regular urine collection, physical examinations, leukocyte phenotyping, and imaging. A subset of animals is administered additional tests that include cardiac MRI, gastrointestinal (GI) endoscopies, and cognitive testing. The main morbidities observed in the NHP cohort were in the GI tract (diarrhea), heart (murmur, valvular insufficiency, mean arterial pressure >120), diabetes, and malignancy. Circulating monocytes demonstrated an intermediate and non-classical phenotype, expressing CD14 and CD16 or CD16 only, respectively. Persistent systemic inflammation was observed in the radiation survivor late effects cohort as evidenced by increased circulating MCP-1 and LPS-binding protein (LBP) levels (5). Moreover, diverse neoplasms developed in multiple tissues in this NHP cohort in a radiation exposure dose-dependent manner. Antibody responses to Streptococcus pneumoniae vaccine were impaired. Thymic tissue in aged NHP after irradiation revealed decreased cellularity, non-distinct architecture, and limited diversity of the T-cell repertoire (5) and diminished thymic output as measured by T-cell receptor excision circles (TREC) (14). Thus, the NHP model is an excellent model for studying immune system homeostasis and function including the assessment of radiation counter-measures. Additional studies with longer follow-up times are necessary to determine the functional activity of antibodies, their ability to protect against disease, and mechanisms by which pathologies become more evident with increasing age.

Aging, radiation, and persistent infections are potential drivers of immunosenescence that can result in the damage of the tissue microenvironment and contribute to impaired renewal of crucial cell populations. Because cytomegalovirus (CMV) infects the majority of individuals over the age of 65, a mouse model of murine cytomegalovirus (MCMV) infection was used to reflect the impact of radiation and aging on the response to infection. Three-month-old mice were infected with MCMV, irradiated at 5 months with different doses, aged, vaccinated at 19 months with a West Nile Virus (WNV) vaccine, challenged subsequently with WNV and compared to controls. A 25–28% reduction in survival was noted in mice that were challenged post-vaccination. Waning of host immune responses against MCMV was observed and included increased PD-1 expression on CD4 T cells with reduced granzyme B expression on MCMV+ T cells. Defining the immune mechanisms involved in the activation of latent infection after irradiation may potentially enable the design and development of MCMs to mitigate virus proliferation and minimize the consequences of radiation injury in older individuals.

Session II: Mechanisms of Immune System Development and Recovery from Damage

Besides its function as a defense against pathogens, the immune system is vital for maintenance of homeostasis (15). How the immune system recovers from damage in the context of immune system development, and how it recovers in response to radiation injury, as well as possible interventions that might facilitate recovery, were addressed in Session II. The critical and evolving role of the thymus in establishing a T-cell repertoire in early life and its ongoing function of generating naïve T cells to maintain the T-cell repertoire and support immune responses to neoantigens was examined considering establishing immunity and responding to external stressors, including radiation. Thymic function is adversely impacted by both extrinsic and intrinsic factors. Examples of extrinsic effectors include infection, stress, and DNA damaging agents such as chemotherapy and radiation, whereas normal aging is the best recognized intrinsic factor negatively impacting thymic size and function (16). Among Hiroshima atomic bomb survivors, markers of decreased thymopoiesis were noted as a function of increased age and radiation dose in comparison to unexposed individuals (17). Although this effect was more prevalent in irradiated males than females, data from unirradiated controls indicated that age-related reduction of lymphoid areas, thymic epithelium, and cortex also occurs. Researchers studying this cohort ultimately concluded that acute exposure to a single sublethal dose of ionizing radiation accelerates thymic aging, decreases thymopoiesis, causes more rapid thymic involution among males with respect to females among those less than 60 years of age, and that thymopoietic potential is amenable to being modeled as a function of age and radiation exposure (17). Although acute radiation exposure may result in accelerated thymic aging, studies of atomic bomb survivors and the NHP radiation survivor cohort suggest this does not adversely affect vaccine responses later in life, such as the seasonal influenza vaccination response (14, 18).

In addition to the impact of radiation exposure on thymic aging and function, the potential for employing stem cells to fully reconstitute the hematopoietic system and immunity after irradiation was presented. It was noted that homogeneously purified mouse SCA1⁺c-kit⁺Lin⁻CD34⁻ stem cells saved lethally-irradiated mice when transplanted in place of whole bone marrow, and that the reconstituted marrow served as a source of stem cells, when transplanted, to save a separate irradiated cohort of mice (19). The ability of purified stem cells to support disease-free hematopoietic reconstitution was further indicated by several studies in cancer patients that resulted in dramatic improvements in overall survival, as well as in mouse studies to induce tolerance for autoimmune disorders. These studies, and others that were discussed, demonstrate the potential power of purified allogenic stem cells transplantation to reconstitute the hematopoietic system and restore functional immunity (20, 21).

Session II also addressed genetically engineered mouse models to assess the role of p53 and other genes on ARS, as well as radiation effects on the thymus. In this work it was observed that blocking p53-mediated radiation-induced apoptosis by knocking out the Bak and Bax genes resulted in survival and bone marrow protection after TBIs that were lethal in wild-type mice. Further studies in a p53 knockdown model (shp53 mice) were conducted to address the concern that transient p53 knockdown during irradiation may enhance lymphomagenesis. Notably, not only were there fewer thymic lymphomas in shp53 mice than in control mice after irradiation, the shp53 mice also exhibited significant protection from H-ARS (22). Possible mechanisms underlying these results were investigated and the results discussed in the session suggested that p53 can act to promote lymphoma after TBI by inducing cell death in the thymus that reduces clonal competition during regeneration and permits tumor-initiating cells to expand. While p53 knockdown mice still exhibit cell death and thymic atrophy after irradiation, cell death and thymic atrophy are not as severe as in wild-type mice, and regeneration in the knockdown mice occurs more rapidly, resulting in more clonal competition in the thymus and a reduction in radiation-induced lymphomas. Further studies are underway to evaluate transient p53 knockdown in other tissues and tumor types for mice irradiated with higher doses. Preliminary data from these studies suggest that p53 knockdown enhances tumor development and decreases survival.

The previous work raises questions regarding potential immune functions that may affect radiation resilience. This concept was discussed with respect to how radiation-damaged tissues interact with the immune system. Various insults and injuries ranging from trauma and infection to radiation damage result in the expression or release of danger signals that are perceived by resident cells which in-turn release pro-inflammatory agents that activate the vasculature and feedback systemically to recruit responder cells to the tissue. Over time, successful resolution of the instigating lesion is achieved as the acute inflammatory response shifts toward an anti-inflammatory phase in which M2-type macrophages remove pro-oxidant mediators and produce anti-inflammatory cytokines; however, if the initial injury is not satisfactorily resolved, inflammation can become chronic or begin cycling. One factor that could instigate this condition is unresolved DNA damage. This concept was supported by work that identified DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS) that persist in irradiated tissues long after the acute inflammation phase and may act to keep the immune system engaged. Furthermore, continuous type I IFN responses may prevent maturation of macrophages to the M2 phenotype and perpetuate the proinflammatory state. This may, in part, also be in response to the cGAS-STING pathway that is activated when cells with DNA damage divide, leading to heightened type I IFN responses and STAT1 pathway activation. There are many pathways that respond to these signals, including the signals resulting from the senescence-associated secretory phenotype associated with DNA-SCARS. These responding pathways can activate common powerful downstream proinflammatory pathways like NFκB, AP-1 and JAK-STAT responses that lead to immune activation (23, 24). This work suggests possible targets for intervention to mitigate persistent radiation-induced pro-inflammatory responses.

Discussion also addressed the observation that ionizing radiation exposure in animal models and in humans appears to result in immunological aging. Findings supporting this include a T-cell-senescent phenotype with a diminished population of naive cells, an increase in memory cells, clonal aberration, and myeloid bias. Like radiation-associated changes, aging is associated with thymic involution and decreased naïve T cell production, reduced antibody response capacity and innate immune cell function, along with reduced phagocytosis and antigen presentation. As seen in aging, even at low doses the T-cell repertoire is skewed towards differentiated effector cells producing pro-inflammatory cytokines, a myeloid bias in HSC differentiation potential, and a greater baseline micronuclei frequency (25, 26).

As noted above, the thymus is sensitive to damage and depletion of cellularity from many exogenous damaging agents and exhibits a chronic decline as a function of age that may be mimicked by radiation exposure. Although the thymus has a robust capacity for regeneration and recovery of immunocompetence after such insults, this capacity also declines with age. Consequently, interventions that could act to reverse these effects may not only restore a more youthful immune system but may also be effective mitigators of exogenous damaging agents. Two distinct thymic regenerative pathways have been identified that may permit development of such interventions. The first involves activation of thymic epithelial cells to mediate regeneration in response to IL-22 expressed by innate lymphoid cells that have been stimulated by dendritic cell (DC) produced IL-23, which is triggered by DCs responding to thymic damage. The second pathway is mediated by bone morphogenetic protein 4 (BMP4) produced by endothelial cells responding to thymic damage. BMP4 also stimulates thymic epithelial cells to mediate regeneration (27, 28). Production of both IL-23 by DCs and BMP4 by endothelial cells is inhibited by the presence and prevalence of apoptotic double positive CD4⁺CD8⁺ thymocytes, the most numerous cell type in the thymus and the precursor of single positive T cells. Approaches that alter the prevalence of apoptotic double positives or shift their mode of death from immunogenically silent apoptosis toward immunogenic necrotic cell death, can trigger the IL-23 and BMP4 dependent regenerative pathways. Similarly, intracellular to extracellular zinc translocation after thymic damage has also been observed to trigger the regenerative response via BMP4 production (29). These studies suggest there may be pathways that are amenable to interventions to enhance thymic function and to support thymic regeneration after injury.

The discussions for this session centered around the role of the immune system in maintaining homeostasis and tissue repair. It was noted that injuries, such as radiation exposure or chemotherapy, which result in a loss of immune cells, lead to diminished regenerative signaling to stem cells, which in turn impedes recovery. An ongoing theme of the work discussed here is the importance of thymus function in recovery from radiation injury, while also being particularly sensitive to radiation-induced damage. Development of interventions that can enhance thymic function or support T cell regeneration after injury or loss may benefit recovery from the effects of radiation exposure, chemotherapy, transplantation, or aging.

Session III: Radiation-Induced Inflammation

An important aspect of the return to homeostasis after radiation injury is the immune system’s ability to facilitate the recovery of damaged tissues. Session III focused on the use of animal models which allow for the elucidation of the progression of acute injury and the inflammatory response, injury recovery and the resolution of the inflammatory response, and the mechanisms that drive these processes.

Radiation-induced lung injury, which occurs after thoracic irradiation, is characterized by an inflammatory response (pneumonitis) and scarring (fibrosis) (30). Animal models used to examine radiation-induced lung injury include mice (31) and NHPs (32). Although radiation-induced lung injury is a latent effect of irradiation, inflammatory responses that could lead to these late effects occur in the first two weeks after injury. Immediate cellular damage includes the apoptosis and necroptosis pathways, cytokine cascades, and vascular damage (33). In mice, radiation injury can vary among strains. The immune response of pneumonitis-prone mice (C57L/J) tends towards a T-cell-mediated inflammatory response (34), whereas fibrosis-prone mice (C57BL/6) have increased TGF-β in the lung one week after irradiation (35). Although cytokine profiles may differ from the human experience, the histopathology and manifestation of the disease for mice and humans are similar. For example, one cytokine that is associated with pneumonitis in animal models and in people is IL-6 (36), which could be a target for intervention.

Delayed effects of acute exposure to radiation, recovery from radiation-induced tissue damage, and the reconstitution of the immune system have been advanced through studies in the NHP in which 2.5% or 5% of the bone marrow was shielded (32). In these bone marrow-sparing models, the immune system is suppressed, but the sparing of some of the bone marrow allows for hematopoietic recovery. In the NHPs, the recovery of different types of leukocytes, in particular lymphocyte subsets, has been studied. By day 75 postirradiation, CD20⁺ B cells, whose numbers declined rapidly after irradiation, had recovered to pre-irradiation baseline levels (37). In contrast, CD3⁺ T cell numbers showed some recovery by day 75 but not to pre-irradiation levels. This lack of T cell recovery was due to diminished recovery of CD4⁺ T cells, which only reached 50% of baseline by day 75 and was characterized by reduced numbers of naïve and memory T cells. Thymic recovery, as shown by reduced TREC levels, was also impaired.

Cell death by pyroptosis can occur after radiation-induced tissue damage and is triggered by two signals: toll-like receptor engagement and inflammasome activation. A key component for inflammasome pore formation is gasdermin D (GSDMD) cleavage by caspase-1 or 11. Screenings performed to identify potential pyroptosis inhibitors led to the discovery of a molecule, necrosulfonamide, that inhibited pore formation, cell death, and sepsis (38). Being an alkylating agent, necrosulfonamide would be too toxic to be a viable pharmaceutical product; therefore, other products that target GSDMD are being tested. One overarching issue that arises is determining the consequences of inhibiting pyroptosis. Instead of pyroptosis, other mechanisms of cell death, such as apoptosis or necroptosis, may be activated; this activation may result in a different set of issues as various components of the inflammatory response are engaged.

Another approach to dampening the inflammatory response is calorie restriction. When the body is in negative energy balance, fatty acids, rather than glucose, are used as fuel, and the immune system is suppressed (39). Although in most cases immunosuppression can lead to harm, immunosuppression can restore balance when one is in a persistent inflammatory state. One subset of the inflammasome family, the NLRP (leucine-rich repeat-containing protein) inflammasome, has been studied in both aging and radiation injury. For example, NLRP inflammasome knockout mice displayed less age-related inflammation damage (12) and also faster immune reconstitution after radiation injury and bone marrow transplantation (40). A link between calorie restriction and inflammation could be the short fatty acid ketone body, beta hydroxybutyrate (BHB), which can provide energy via the citric acid cycle and reduce NLRP3 inflammasome activation (41). On the human side, the Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy (CALERIE) clinical trial has also shown improved immune reconstitution (39). One possible mediator of the anti-inflammatory response is PLA2G7, which is a platelet activating factor acetylhydrolase, suggesting a potential target.

The immune system is intimately tied to the body’s microbiome and the gut microbiome has also been a subject of study in radiation biology. In a series of mouse studies, the gut microbiome was examined to characterize its contribution to the heterologous responses to lethal doses of radiation. It was found that mice that were relatively radioresistant (so-called “elite survivors,” or ES) had gut microbiomes that were distinct from more susceptible mice (42). This distinction was probed further in the comparison between specific-pathogen-free (SPF) mice and so-called dirty mice (wild-caught or from pet stores), which tend to be more radioresistant than standard SPF laboratory mice. These two sets of animals were found to have distinct gut microbiomes. To test whether the microbiome was responsible for this difference, SPF mice were housed with soiled bedding from dirty mice. This microbial transfer allowed for the recipient mice to be more radiation resistant, with greater survival, lower clinical severity scores, and greater bone marrow cellularity. Comparison of the gut microbiomes showed enriched Lachnospiraceae and Enterococcaceae levels in the ES mice, and this improvement appeared to be related to higher levels of short-chain fatty acid (SCFA) metabolites produced by the bacteria. In fact, survival of irradiated mice improved when SCFAs were added to drinking water. A link to patient survival has been shown in a small clinical study in which patients with less diarrhea after radiation therapy also had higher levels of Lachnospiraceae and Enterococcaceae in their feces.

CONCLUSION

In this workshop, researchers explored various aspects of radiation-induced immune dysfunction, which is an essential aspect of radiation-induced injury and recovery from radiation injury. From following the health over time of the survivors of the atomic bombings in Japan, to studying aspects of the natural history of animals exposed to lethal radiation doses, much has been learned about the role that the immune system plays in tissue damage and recovery. This knowledge has led to research into interventions that could aid in the recovery of the immune system and improve tissue repair, in turn leading to increased survival and reduced immune system-related morbidities. The hope for the organizers is that this workshop will lead to further insight and collaborations among researchers in the immunology and radiation biology communities to improve understanding and advance the development of possible MCMs.