Cranial irradiation disrupts homeostatic microglial dynamic behavior.

Cranial irradiation causes cognitive deficits that are in part mediated by microglia, the resident immune cells of the brain. Microglia are highly reactive, exhibiting changes in shape and morphology depending on the function they are performing. Additionally, microglia processes make dynamic, physical contacts with different components of their environment to monitor the functional state of the brain and promote plasticity. Though evidence suggests radiation perturbs homeostatic microglia functions, it is unknown how cranial irradiation impacts the dynamic behavior of microglia over time. Here, we paired in vivo two-photon microscopy with a transgenic mouse model that labels cortical microglia to follow these cells and determine how they change over time in cranial irradiated mice and their control littermates. We show that a single dose of 10 Gy cranial irradiation disrupts homeostatic cortical microglia dynamics during a 1-month time course. We found a lasting loss of microglial cells following cranial irradiation, coupled with a modest dysregulation of microglial soma displacement at earlier timepoints. The homogeneous distribution of microglia was maintained, suggesting microglia rearrange themselves to account for cell loss and maintain territorial organization following cranial irradiation. Furthermore, we found cranial irradiation reduced microglia coverage of the parenchyma and their surveillance capacity, without overtly changing morphology. Our results demonstrate that a single dose of radiation can induce changes in microglial behavior and function that could influence neurological health. These results set the foundation for future work examining how cranial irradiation impacts complex cellular dynamics in the brain which could contribute to the manifestation of cognitive deficits.

IL-17A neutralization fails to attenuate airway remodeling and potentiates a proinflammatory lung microenvironment in diacetyl-exposed rats.

Bronchiolitis obliterans (BO) is a devastating lung disease that can develop following inhalation exposure to certain chemicals. Diacetyl (DA) is one chemical commonly associated with BO development when inhaled at occupational levels. Previous studies in rats have shown that repetitive DA vapor exposures increased lung CD4+CD25+ T cells and bronchoalveolar (BAL) interleukin-17A (IL-17A) concentrations concurrent with the development of airway remodeling. We hypothesized that IL-17A neutralization would attenuate the severity of airway remodeling after repetitive DA vapor exposures. Sprague-Dawley rats were exposed to 200 parts-per-million DA vapor or filtered air (RA) for 6 h/day × 5 days and monitored for 2 wk postexposure. Treatment with IL-17A neutralization (αIL-17A) or IgG (control) began immediately following exposures and continued twice weekly until study's end. Lungs were harvested for histology, flow cytometry, and BAL analyses. Survival, oxygen saturations, and percent weight change decreased significantly in DA-exposed versus RA-exposed rats, but did not differ significantly between DA + αIL-17A versus DA + IgG. Similarly, the number nor severity of airway lesions did not differ significantly between DA + αIL-17A versus DA + IgG rats despite the percentage of lung regulatory T cells increasing with decreased BAL IL-17A concentrations. Ashcroft scoring of the distal lung parenchyma suggested worse parenchymal remodeling in DA + αIL-17A versus DA + IgG rats with increased expression of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), and nuclear factor-kappa B (NF-κB). Collectively, IL-17A neutralization in DA-exposed rats failed to attenuate airway remodeling with increased expression of pro-inflammatory cytokines TNF-α, IL-1ß, and NF-κB.NEW & NOTEWORTHY Interleukin-17A (IL-17A) neutralization has shown benefit previously in preclinical models of transplant-associated bronchiolitis obliterans (BO), yet it remains unknown whether IL-17A neutralization has similar benefit for other forms of BO. Here, IL-17A neutralization fails to prevent severe airway remodeling in rats exposed repetitively to the flavoring chemical diacetyl, and instead, promotes a proinflammatory microenvironment with increased expression of TNF-α, IL-1ß, and NF-κB within the lung.

Influence of the irradiated pulmonary microenvironment on macrophage and T cell dynamics.

BACKGROUND: The lung is sensitive to radiation, increasing normal tissue toxicity risks following radiation therapy. Adverse outcomes include pneumonitis and pulmonary fibrosis, which result from dysregulated intercellular communication within the pulmonary microenvironment. Although macrophages are implicated in these pathogenic outcomes, the impact of their microenvironment is not well understood. MATERIALS AND METHODS: C57BL/6J mice received 6Gyx5 irradiation to the right lung. Macrophage and T cell dynamics were investigated in ipsilateral right lungs, contralateral left lungs and non-irradiated control lungs 4-26wk post exposure. Lungs were evaluated by flow cytometry, histology and proteomics. RESULTS: Following uni-lung irradiation, focal regions of macrophage accumulation were noted in both lungs by 8wk, however by 26wk fibrotic lesions were observed only in ipsilateral lungs. Infiltrating and alveolar macrophages populations expanded in both lungs, however transitional CD11b + alveolar macrophages persisted only in ipsilateral lungs and expressed lower CD206. Concurrently, arginase-1 + macrophages accumulated in ipsilateral but not contralateral lungs at 8 and 26wk post exposure, while CD206 + macrophages were absent from these accumulations. While radiation expanded CD8 + T cells in both lungs, T regulatory cells only increased in ipsilateral lungs. Unbiased proteomics analysis of immune cells revealed a substantial number of differentially expressed proteins in ipsilateral lungs when compared to contralateral lungs and both differed from non-irradiated controls. CONCLUSIONS: Pulmonary macrophage and T cell dynamics are impacted by the microenvironmental conditions that develop following radiation exposure, both locally and systemically. While macrophages and T cells infiltrate and expand in both lungs, they diverge phenotypically depending on their environment.

Prevention of Radiation-Induced Bladder Injury: A Murine Study Using Captopril.

PURPOSE: Pelvic radiation therapy (RT) can cause debilitating bladder toxicities but few clinical interventions exist to prevent injury or alleviate symptoms. From a large genome-wide association study in patients with prostate cancer it was previously reported that SNPs tagging AGT, part of the renin-angiotensin system (RAS), correlated with patient-reported late hematuria, identifying a potential targetable pathway to prevent RT-induced bladder injury. To investigate this association, we performed a preclinical study to determine whether RAS modulation protected the bladder against RT injury. METHODS AND MATERIALS: C57BL/6 male mice were treated with an oral angiotensin converting enzyme inhibitor (ACEi: 0.3g/L captopril) 5 days before focal bladder X-irradiation with either single dose (SD) 30 Gy or 3 fractions of 8 Gy (8 Gy × 3 in 5 days). RT was delivered using XStrahl SARRP Muriplan CT-image guidance with parallel-opposed lateral beams. ACEi was maintained for 20 weeks post RT. Bladder toxicity was assessed using assays to identify local injury that included urinalysis, functional micturition, bladder-released exosomes, and histopathology, as well as an assessment of systemic changes in inflammatory-mediated circulating immune cells. RESULTS: SD and fractionated RT increased urinary frequency and reduced the volume of individual voids at >14 weeks, but not at 4 weeks, compared with nonirradiated animals. Urothelial layer width was positively correlated with mean volume of individual voids (P = .0428) and negatively correlated with number of voids (P = .028), relating urothelial thinning to changes in RT-mediated bladder dysfunction. These chronic RT-induced changes in micturition patterns were prevented by captopril treatment. Focal bladder irradiation significantly increased the mean particle count of urine extracellular vesicles and the monocyte and neutrophil chemokines CCL2 and MIP-2, and the proportions of circulating inflammatory-mediated neutrophils and monocytes, which was also prevented by captopril. Exploratory transcriptomic analysis of bladder tissue implicated inflammatory and erythropoietic pathways. CONCLUSIONS: This study demonstrated that systemic modulation of the RAS protected against and alleviated RT-induced late bladder injury but larger confirmatory studies are needed.

Short-term and bystander effects of radiation on murine submandibular glands.

Many patients treated for head and neck cancers experience salivary gland hypofunction due to radiation damage. Understanding the mechanisms of cellular damage induced by radiation treatment is important in order to design methods of radioprotection. In addition, it is crucial to recognize the indirect effects of irradiation and the systemic responses that may alter saliva secretion. In this study, radiation was delivered to murine submandibular glands (SMGs) bilaterally, using a 137Cs gamma ray irradiator, or unilaterally, using a small-animal radiation research platform (SARRP). Analysis at 3, 24 and 48 h showed dynamic changes in mRNA and protein expression in SMGs irradiated bilaterally. Unilateral irradiation using the SARRP caused similar changes in the irradiated SMGs, as well as significant off-target, bystander effects in the non-irradiated contralateral SMGs.

Endurance exercise attenuates juvenile irradiation-induced skeletal muscle functional decline and mitochondrial stress.

BACKGROUND: Radiotherapy is commonly used to treat childhood cancers and can have adverse effects on muscle function, but the underlying mechanisms have yet to be fully elucidated. We hypothesized that endurance exercise following radiation treatment would improve skeletal muscle function. METHODS: We utilized the Small Animal Radiation Research Platform (SARRP) to irradiate juvenile male mice with a clinically relevant fractionated dose of 3× (every other day over 5 days) 8.2 Gy X-ray irradiation locally from the knee to footpad region of the right hindlimb. Mice were then singly housed for 1 month in cages equipped with either locked or free-spinning voluntary running wheels. Ex vivo muscle contractile function, RT-qPCR analyses, resting cytosolic and sarcoplasmic reticulum (SR) store Ca2+ levels, mitochondrial reactive oxygen species levels (MitoSOX), and immunohistochemical and biochemical analyses of muscle samples were conducted to assess the muscle pathology and the relative therapeutic impact of voluntary wheel running (VWR). RESULTS: Irradiation reduced fast-twitch extensor digitorum longus (EDL) muscle-specific force by 27% compared to that of non-irradiated mice, while VWR post-irradiation improved muscle-specific force by 37%. Radiation treatment similarly reduced slow-twitch soleus muscle-specific force by 14% compared to that of non-irradiated mice, while VWR post-irradiation improved specific force by 18%. We assessed intracellular Ca2+ regulation, oxidative stress, and mitochondrial homeostasis as potential mechanisms of radiation-induced pathology and exercise-mediated rescue. We found a significant reduction in resting cytosolic Ca2+ concentration following irradiation in sedentary mice. Intriguingly, however, SR Ca2+ store content was increased in myofibers from irradiated mice post-VWR compared to mice that remained sedentary. We observed a 73% elevation in the overall protein oxidization in muscle post-irradiation, while VWR reduced protein nitrosylation by 35% and mitochondrial reactive oxygen species (ROS) production by 50%. Finally, we found that VWR significantly increased the expression of PGC1α at both the transcript and protein levels, consistent with an exercise-dependent increase in mitochondrial biogenesis. CONCLUSIONS: Juvenile irradiation stunted muscle development, disrupted proper Ca2+ handling, damaged mitochondria, and increased oxidative and nitrosative stress, paralleling significant deficits in muscle force production. Exercise mitigated aberrant Ca2+ handling, mitochondrial homeostasis, and increased oxidative and nitrosative stress in a manner that correlated with improved skeletal muscle function after radiation.

Diacetyl Vapor Inhalation Induces Mixed, Granulocytic Lung Inflammation with Increased CD4+CD25+ T Cells in the Rat.

Diacetyl (DA) is a highly reactive alpha diketone associated with flavoring-related lung disease. In rodents, acute DA vapor exposure can initiate an airway-centric, inflammatory response. However, this immune response has yet to be fully characterized in the context of flavoring-related lung disease progression. The following studies were designed to characterize the different T cell populations within the lung following repetitive DA vapor exposures. Sprague-Dawley rats were exposed to 200 parts-per-million DA vapor for 5 consecutive days × 6 h/day. Lung tissue and bronchoalveolar lavage fluid (BALF) were analyzed for changes in histology by H&E and Trichrome stain, T cell markers by flow cytometry, total BALF cell counts and differentials, BALF IL17a and total protein immediately, 1 and 2 weeks post-exposure. Lung histology and BALF cell composition demonstrated mixed, granulocytic lung inflammation with bronchial lymphoid aggregates at all time points in DA-exposed lungs compared to air controls. While no significant change was seen in percent lung CD3+, CD4+, or CD8+ T cells, a significant increase in lung CD4+CD25+ T cells developed at 1 week that persisted at 2 weeks post-exposure. Further characterization of this CD4+CD25+ T cell population identified Foxp3+ T cells at 1 week that failed to persist at 2 weeks. Conversely, BALF IL-17a increased significantly at 2 weeks in DA-exposed rats compared to air controls. Lung CD4+CD25+ T cells and BALF IL17a correlated directly with BALF total protein and inversely with rat oxygen saturations. Repetitive DA vapor exposure at occupationally relevant concentrations induced mixed, granulocytic lung inflammation with increased CD4+CD25+ T cells in the rat lung.

Repetitive diacetyl vapor exposure promotes ubiquitin proteasome stress and precedes bronchiolitis obliterans pathology.

Bronchiolitis obliterans (BO) is a devastating lung disease seen commonly after lung transplant, following severe respiratory tract infection or chemical inhalation exposure. Diacetyl (DA; 2,3-butanedione) is a highly reactive alpha-diketone known to cause BO when inhaled, however, the mechanisms of how inhalation exposure leads to BO development remains poorly understood. In the current work, we combined two clinically relevant models for studying the pathogenesis of DA-induced BO: (1) an in vivo rat model of repetitive DA vapor exposures with recovery and (2) an in vitro model of primary human airway epithelial cells exposed to pure DA vapors. Rats exposed to 5 consecutive days 200 parts-per-million DA 6 h per day had worsening survival, persistent hypoxemia, poor weight gain, and histologic evidence of BO 14 days after DA exposure cessation. At the end of exposure, increased expression of the ubiquitin stress protein ubiquitin-C accumulated within DA-exposed rat lung homogenates and localized primarily to the airway epithelium, the primary site of BO development. Lung proteasome activity increased concurrently with ubiquitin-C expression after DA exposure, supportive of significant proteasome stress. In primary human airway cultures, global proteomics identified 519 significantly modified proteins in DA-exposed samples relative to controls with common pathways of the ubiquitin proteasome system, endosomal reticulum transport, and response to unfolded protein pathways being upregulated and cell-cell adhesion and oxidation-reduction pathways being downregulated. Collectively, these two models suggest that diacetyl inhalation exposure causes abundant protein damage and subsequent ubiquitin proteasome stress prior to the development of chemical-induced BO pathology.

Chemoradiation impairs myofiber hypertrophic growth in a pediatric tumor model.

Pediatric cancer treatment often involves chemotherapy and radiation, where off-target effects can include skeletal muscle decline. The effect of such treatments on juvenile skeletal muscle growth has yet to be investigated. We employed a small animal irradiator to administer fractionated hindlimb irradiation to juvenile mice bearing implanted rhabdomyosarcoma (RMS) tumors. Hindlimb-targeted irradiation (3 × 8.2 Gy) of 4-week-old mice successfully eliminated RMS tumors implanted one week prior. After establishment of this preclinical model, a cohort of tumor-bearing mice were injected with the chemotherapeutic drug, vincristine, alone or in combination with fractionated irradiation (5 × 4.8 Gy). Single myofiber analysis of fast-contracting extensor digitorum longus (EDL) and slow-contracting soleus (SOL) muscles was conducted 3 weeks post-treatment. Although a reduction in myofiber size was apparent, EDL and SOL myonuclear number were differentially affected by juvenile irradiation and/or vincristine treatment. In contrast, a decrease in myonuclear domain (myofiber volume/myonucleus) was observed regardless of muscle or treatment. Thus, inhibition of myofiber hypertrophic growth is a consistent feature of pediatric cancer treatment.

Radiation-Induced Damage to Prepubertal Pax7+ Skeletal Muscle Stem Cells Drives Lifelong Deficits in Myofiber Size and Nuclear Number.

During prepubertal development, muscle stem cells (satellite cells, SCs) actively contribute to myofiber growth. Because some SCs are active during this time, they may be particularly susceptible to damage. Using a Small Animal Radiation Research Platform (SARRP), we investigated the effects of local fractionated radiation treatment on prepubertal SCs. Immediately after this regimen, there was a reduction in SC number. Although surviving SCs had deficiencies in function, some myogenic potential remained. Indeed, some muscle regenerative capacity persisted immediately after irradiation. Lastly, we assessed the long-term consequences of radiation-induced SC loss during prepuberty. We observed a reduction of myofiber size and corresponding loss of nuclei in both fast- and slow-contracting muscles 14 months post-irradiation. Notably, prepubertal SC depletion mimicked these lifelong deficits. This work highlights the susceptibility of prepubertal SCs to radiation exposure. We also reveal the importance of prepubertal SC contribution to the lifelong maintenance of skeletal muscle.

Airway basal cell injury after acute diacetyl (2,3-butanedione) vapor exposure.

RATIONALE: Diacetyl (DA; 2,3-butanedione) is a chemical found commonly in foods and e-cigarettes. When inhaled, DA causes epithelial injury, though the mechanism of repair remain poorly understood. The objective of this study was to evaluate airway basal cell repair after DA vapor exposure. METHODS: Primary human bronchial epithelial cells were exposed to DA or PBS for 1 h. Lactate dehydrogenase, cleaved caspase 3/7 and trans-epithelial electrical resistance were measured prior to and following exposure. Exposed cultures were analyzed for the airway basal cell markers keratin 5 and p63 as well as ubiquitin and proteasome activity. Cultures were also treated with a proteasome inhibitor (MG132). RESULTS: DA vapor exposure caused a transient decrease in trans-epithelial electrical resistance in all DA-exposed cultures. Supernatant lactate dehydrogenase and cleaved caspase 3/7 increased significantly at the highest DA concentration but not at lower DA concentrations. Increased keratin 5 ubiquitination occurred after DA exposure but resolved by day 3. Damage to airway basal cells persisted at day 3 in the presence of MG132. CONCLUSIONS: Diacetyl exposure results in airway basal cell injury with keratin 5 ubiquitination and decreased p63 expression. The ubiquitin-proteasome-pathway partially mediates airway basal cell repair after acute DA exposure.

Apoptosis Resistance in Fibroblasts Precedes Progressive Scarring in Pulmonary Fibrosis and Is Partially Mediated by Toll-Like Receptor 4 Activation.

Inhalation of environmental toxicants such as cigarette smoke, metal or wood dust, silica, or asbestos is associated with increased risk for idiopathic pulmonary fibrosis (IPF). IPF involves progressive scarring of lung tissue, which interferes with normal respiration and is ultimately fatal; however, the complex cellular mechanisms of IPF pathogenesis remain unclear. Fibroblast apoptosis is essential in normal wound healing but is dysregulated in IPF. Recent studies suggest that Toll-like receptor 4 (TLR4) is key in the onset of IPF. Here, radiation-induced PF was used as a model for IPF because it very closely mimics the progressive and intractable nature of IPF. Female C57BL/6J (C57) and C57BL/6J TLR4-/- mice were exposed to a single dose of 13 Gy whole-thorax ionizing radiation. Although both strains showed similar levels of immediate radiation-induced damage, C57 mice exhibited more extensive fibrosis at 22-week postirradiation (PI) than TLR4-/- mice. Isolated C57 primary 1° MLFs showed decreased apoptosis susceptibility as early as 8-week postirradiation, a phenotype that persisted for the remainder of the radiation response. TLR4-/- 1° mouse lung fibroblasts did not exhibit significant apoptosis resistance at any point. Systemic release of high mobility group box 1, a TLR4 agonist, during the pneumonitis phase of the radiation response may act through TLR4 to contribute to fibroblast apoptosis resistance and thus interfere with wound resolution. These findings demonstrate that apoptosis resistance occurs earlier in pulmonary fibrosis pathogenesis than previously assumed, and that TLR4 signaling is a key mediator in this process.

A Potential Biomarker for Predicting the Risk of Radiation-Induced Fibrosis in the Lung.

Biomarkers could play an essential role during triage in the aftermath of a radiological event, where exposure to radiation will be heterogeneous and complicated by concurrent trauma. Used alongside biodosimetry, biomarkers can identify victims in need of treatment for acute radiation effects, and might also provide valuable information on later developing consequences that need to be addressed as part of a treatment strategy. Indeed, because the lung is particularly sensitive to radiation and resultant late effects not only affect quality of life, but can also lead to morbidity, the risk of developing downstream pulmonary complications in exposed individuals requires assessment. In this study, analyses of changes in pulmonary and circulating content of club cell secretory protein (CCSP) and surfactant protein D (SP-D), expressed by epithelial club cells and type II pneumocytes in the lung, respectively, were used to evaluate pulmonary epithelial damage in several lung injury models. Using a combined radiation exposure model, fibrosis-susceptible C57BL/6J (C57) and alveolitis-prone C3H/HeJ (C3H) mice received 5 Gy total-body irradiation plus 2.5-10 Gy whole-lung irradiation, and lung and plasma samples were collected throughout the course of the radiation response, at time points ranging from 24 h to 26 weeks postirradiation. Radiation significantly reduced bronchiole CCSP coverage in C57 mice at 26 weeks, a response that varied in extent among animals, but correlated with the severity of fibrosis in each animal. Interestingly, plasma CCSP content was elevated in C57 mice at multiple time points preceding and during the fibrotic period; this response that was not observed in C3H mice. Circulating CCSP/SP-D ratios, calculated as an index of lung integrity, were similarly increased throughout the time course in C57, but not C3H, mice. Furthermore, when the thoracic doses were reduced to subthreshold levels for fibrosis induction (2.5 or 7.5 Gy), although the CCSP/SP-D ratio in lung homogenates demonstrated dose-responsive changes, this was not reflected in the plasma ratios at acute and late time points. Importantly, plasma CCSP/SP-D ratios also were not significantly altered in C57 mice exposed to LPS, and only transiently decreased in influenza-exposed mice, demonstrating a level of specificity for radiation-induced lung injury. These results indicate that the CCSP/SP-D ratio, measured in plasma, is sensitive to individual variation in radiation sensitivity, correlates with fibrosis development, can be detected early after exposure and is specific to radiation-induced injury. This suggests that the CCSP/SP-D ratio may be useful as a biomarker of radiation-induced pulmonary fibrosis.

The Lactate Dehydrogenase Inhibitor Gossypol Inhibits Radiation-Induced Pulmonary Fibrosis.

Exposure of the lung to ionizing radiation that occurs in radiotherapy, as well as after accidental or intentional mass casualty incident can result in pulmonary fibrosis, which has few treatment options. Pulmonary fibrosis is characterized by an accumulation of extracellular matrix proteins that create scar tissue. Although the mechanisms leading to radiation-induced pulmonary fibrosis remain poorly understood, one frequent observation is the activation of the profibrotic cytokine transforming growth factor-beta (TGF-ß). Our laboratory has shown that the metabolite lactate activates latent TGF-ß by a reduction in extracellular pH. We recently demonstrated that lactate dehydrogenase-A (LDHA), the enzyme that produces lactate, is upregulated in patients with radiation-induced pulmonary fibrosis. Furthermore, genetic silencing of LDHA or pharmacologic inhibition using the LDHA inhibitor gossypol prevented radiation-induced extracellular matrix secretion in vitro through inhibition of TGF-ß activation. In the current study, we hypothesized that LDHA inhibition in vivo prevents radiation-induced pulmonary fibrosis. To test this hypothesis, C57BL/6 mice received 5 Gy total-body irradiation plus 10 Gy thoracic irradiation from a 137Cs source to induce pulmonary fibrosis. Starting at 4 weeks postirradiation, mice were treated with 5 mg/kg of the LDHA inhibitor gossypol or vehicle daily until sacrifice at 26 weeks postirradiation. Exposure to radiation resulted in pulmonary fibrosis, characterized by an increase in collagen content, fibrosis area, extracellular matrix gene expression and TGF-ß activation. Irradiated mice treated with gossypol had significantly reduced fibrosis outcomes, including reduced collagen content in the lungs, reduced expression of active TGF-ß, LDHA and the transcription factor hypoxia-inducible factor-1 alpha (HIF-1α). These findings suggest that inhibition of LDHA protects against radiation-induced pulmonary fibrosis, and may be a novel therapeutic strategy for radiation-induced pulmonary fibrosis.

Long-Lasting Impact of Neonatal Exposure to Total Body Gamma Radiation on Secondary Lymphoid Organ Structure and Function.

The acute period after total body irradiation (TBI) is associated with an increased risk of infection, principally resulting from the loss of hematopoietic stem cells, as well as disruption of mucosal epithelial barriers. Although there is a return to baseline infection control coinciding with the apparent progressive recovery of hematopoietic cell populations, late susceptibility to infection in radiation-sensitive organs such as lung and kidney is known to occur. Indeed, pulmonary infections are particularly prevalent in hematopoietic cell transplant (HCT) survivors, in both adult and pediatric patient populations. Preclinical studies investigating late outcomes from localized thoracic irradiation have indicated that the mechanisms underlying pulmonary delayed effects are multifactorial, including exacerbated and persistent production of pro-inflammatory molecules and abnormal cross-talk among parenchymal and infiltrating immune and inflammatory cell populations. However, in the context of low-dose TBI, it is not clear whether the observed exacerbated response to infection remains contingent on these same mechanisms. It is possible instead, that after systemic radiation-induced injury, the susceptibility to infection may be independently related to defects in alternative organs that are revealed only through the challenge itself; indeed, we have hypothesized that this defect may be due to radiation-induced chronic effects in the structure and function of secondary lymphoid organs (SLO). In this study, we investigated the molecular and cellular alterations in SLO (i.e., spleen, mediastinal, inguinal and mesenteric lymph nodes) after TBI, and the time points when there appears to be immune competence. Furthermore, due to the high incidence of pulmonary infections in the late post-transplantation period of bone marrow transplant survivors, particularly in children, we focused on outcomes in mice irradiated as neonates, which served as a model for a pediatric population, and used the induction of adaptive immunity against influenza virus as a functional end point. We demonstrated that, in adult animals irradiated as neonates, high endothelial venule (HEV) expansion, generation of follicular helper T cells (TFH) and formation of splenic germinal centers (GC) were rapidly and, more importantly, persistently impaired in SLO, suggesting that the early-life exposure to sublethal radiation had long-lasting effects on the induction of humoral immunity. Although the neonatal TBI did not affect the overall outcome from influenza infection in the adults at the earlier time points assessed, we believe that they nonetheless contribute significantly to the increased mortality observed at subsequent late time points. Furthermore, we speculate that the detrimental and persistent impact on the induction of CD4 T- and B-cell responses in the mediastinal lymph nodes will decrease the animals' ability to respond to other aerial pathogens. Since many of these pathogens are normally cleared by antibodies, our findings provide an explanation for the susceptibility of survivors of childhood HCT to life-threatening respiratory tract infections. These findings have implications regarding the need for increased monitoring in pediatric hematopoietic cell transplant patients, since they indicate that there are ongoing and cumulative defects in SLO, which, importantly, develop during the immediate and early postirradiation period when patients may appear immunologically competent. The identification of changes in immune-related signals may offer bioindicators of progressive dysfunction, and of potential mechanisms that could be targeted so as to reduce the risk of infection from extracellular pathogens. Furthermore, these results support the potential susceptibility of the pediatric population to infection after sublethal irradiation in the context of a nuclear or radiological event.

Examining the Effects of External or Internal Radiation Exposure of Juvenile Mice on Late Morbidity after Infection with Influenza A.

A number of investigators have suggested that exposure to low-dose radiation may pose a potentially serious health risk. However, the majority of these studies have focused on the short-term rather than long-term effects of exposure to fixed source radiation, and few have examined the effects of internal contamination. Additionally, very few studies have focused on exposure in juveniles, when organs are still developing and could be more sensitive to the toxic effects of radiation. To specifically address whether early-life radiation injury may affect long-term immune competence, we studied 14-day-old juvenile pups that were either 5 Gy total-body irradiated or injected internally with 50 µCi soluble (137)Cs, then infected with influenza A virus at 26 weeks after exposure. After influenza infection, all groups demonstrated immediate weight loss. We found that externally irradiated, infected animals failed to recover weight relative to age-matched infected controls, but internally (137)Cs contaminated and infected animals had a weight recovery with a similar rate and degree as controls. Externally and internally irradiated mice demonstrated reduced levels of club cell secretory protein (CCSP) message in their lungs after influenza infection. The externally irradiated group did not recover CCSP expression even at the two-week time point after infection. Although the antibody response and viral titers did not appear to be affected by either radiation modality, there was a slight increase in monocyte chemoattractant protein (MCP)-1 expression in the lungs of externally irradiated animals 14 days after influenza infection, with increased cellular infiltration present. Notably, an increase in the number of regulatory T cells was seen in the mediastinal lymph nodes of irradiated mice relative to uninfected mice. These data confirm the hypothesis that early-life irradiation may have long-term consequences on the immune system, leading to an altered antiviral response.

Exacerbation of lung radiation injury by viral infection: the role of Clara cells and Clara cell secretory protein.

Viral infections have been associated with exacerbation of disease in human cases of idiopathic pulmonary fibrosis. Since pulmonary fibrosis is a common outcome after irradiation to the lung, we hypothesized that viral infection after radiation exposure would exacerbate radiation-induced lung injury. Epithelial injury, a frequent outcome after infection, has been hypothesized to contribute to the pathogenesis of pulmonary fibrosis and bronchiolar epithelial Clara cells participate in epithelial repair. Therefore, it was further hypothesized that altered responses after irradiation involve the bronchiolar epithelial Clara cells. C57BL/6J or CCSP(-/-) mice were irradiated with 0 (sham), 5, 10 or 15 Gy to the whole thorax. At ten weeks post-irradiation, animals were mock infected or infected with influenza A virus and body weight and survival were monitored. Pulmonary function was assessed by whole-body plethysmography. The Clara cell markers, CCSP and Cyp2f2, were measured in the lung by qRT-PCR, and protein expression was visualized in the lung by immunofluorescence. Following pulmonary function tests, mice were sacrificed and tissues were collected for pathological analysis. In 15 Gy irradiated animals infected with influenza A virus, accelerated respiratory rates, reduced pulmonary function, and exacerbated lung pathology occurred earlier post-irradiation than previously observed after irradiation alone, suggesting infection accelerates the development of radiation injury. After irradiation alone, CCSP and Cyp2f2 mRNA levels were reduced, correlating with reductions in the number of Clara cells lining the airways. When combined with infection, these markers further declined and an apparent delay in recovery of mRNA expression was observed, suggesting that radiation injury leads to a chronic reduction in the number of Clara cells that may potentiate the epithelial injury observed after influenza A virus infection. This novel finding may have considerable therapeutic implications with respect to both thoracic tumor patients and recipients of bone marrow transplants.

Neonatal irradiation sensitizes mice to delayed pulmonary challenge.

Significant differences exist between the physiology of the immature, neonatal lung compared to that of the adult lung that may affect acute and late responses to irradiation. Identifying these differences is critical to developing successful mitigation strategies for this special population. Our current hypothesis proposes that irradiation during the neonatal period will alter developmental processes, resulting in long-term consequences, including altered susceptibility to challenge with respiratory pathogens. C57BL/6J mice, 4 days of age, received 5 Gy whole-body irradiation. At subsequent time points (12, 26 and 46 weeks postirradiation), mice were intranasally infected with 120 HAU of influenza A virus. Fourteen days later, mice were sacrificed and tissues were collected for examination. Morbidity was monitored following changes in body weight and survival. The magnitude of the pulmonary response was determined by bronchoalveolar lavage, histological examination and gene expression of epithelial and inflammatory markers. Viral clearance was assessed 7 days post-influenza infection. Following influenza infection, irradiated animals that were infected at 26 and 46 weeks postirradiation lost significantly more weight and demonstrated reduced survival compared with those infected at 12 weeks postirradiation, with the greatest deleterious effect seen at the late time point. The results of these experiments suggest that radiation injury during early life may affect the lung's response to a subsequent pathogenic aerial challenge, possibly through a chronic and progressive defect in the immune system. This finding may have implications for the development of countermeasures in the context of systemic radiation exposure.

Pro-apoptotic gene knockdown mediated by nanocomplexed siRNA reduces radiation damage in primary salivary gland cultures.

A critical issue in the management of head and neck tumors is radioprotection of the salivary glands. We have investigated whether siRNA-mediated gene knock down of pro-apoptotic mediators can reduce radiation-induced cellular apoptosis in salivary gland cells in vitro. We used novel, pH-responsive nanoparticles to deliver functionally active siRNAs into cultures of salivary gland cells. The nanoparticle molecules are comprised of cationic micelles that electrostatically interact with the siRNA, protecting it from nuclease attack, and also include pH-responsive endosomolytic constituents that promote release of the siRNA into the target cell cytoplasm. Transfection controls with Cy3-tagged siRNA/nanoparticle complexes showed efficiently internalized siRNAs in more than 70% of the submandibular gland cells. We found that introduction of siRNAs specifically targeting the Pkcδ or Bax genes significantly blocked the induction of these pro-apoptotic proteins that normally occurs after radiation in cultured salivary gland cells. Furthermore, the level of cell death from subsequent radiation, as measured by caspase-3, TUNEL, and mitochondrial disruption assays, was significantly decreased. Thus, we have successfully demonstrated that the siRNA/nanoparticle-mediated knock down of pro-apoptotic genes can prevent radiation-induced damage in submandibular gland primary cell cultures.

Effect of total body irradiation on late lung effects: hidden dangers.

PURPOSE: In our ongoing investigation into the consequences of a radiological terrorism or nuclear dispersion event, we assessed whether a dose range that is believed to be sub-threshold for the development of lung endpoints results in late pathological changes and, secondarily, whether those late changes affect the lung's ability to respond to subsequent challenge. MATERIALS AND METHODS: C57BL/6J mice received total body irradiation (0.5-10 Gy) and were followed for 6-18 months after irradiation. At 12 and 15 months, a subset of mice was exposed to a second challenge (aerosolised lipopolysaccharide [LPS]). RESULTS: Cytokines shown to be upregulated early (hours) following irradiation (interleukin [IL]6, keratinocyte chemoattractant [KC], IL1B, and IL1R2) demonstrated increases in messenger ribose nucleic acid (mRNA) expression at late time points, beginning at nine months. Although persistent, dose-dependent increases in T cell counts were seen, no other overt changes in pathophysiology were observed. Nonetheless, animals that were exposed to a secondary challenge at late time points demonstrated an increased inflammatory cell recruitment and persistence in response relative to controls. CONCLUSIONS: We propose that, following doses that elicit little change in pathophysiology, sub-clinical radiation-induced injury increases the lungs' susceptibility to a secondary challenge, possibly through a radiation-induced alteration in the immune defense system.