SLPI deficiency alters airway protease activity and induces cell recruitment in a model of muco-obstructive lung disease.

Secretory leukocyte protease inhibitor (SLPI) is an important cationic protein involved in innate airway immunity and highly expressed in mucosal secretions, shown to target and inhibit neutrophil elastase (NE), cathepsin G and trypsin activity to limit proteolytic activity. In addition to the potent anti-protease activity, SLPI has been demonstrated to exert a direct anti-inflammatory effect, which is mediated via increased inhibition and competitive binding of NF-κB, regulating immune responses through limiting transcription of pro-inflammatory gene targets. In muco-obstructive lung disorders, such as Chronic Obstructive Pulmonary Disease (COPD) and Cystic Fibrosis (CF), there is an observed elevation in airway SLPI protein concentrations as a result of increased lung inflammation and disease progression. However, studies have identified COPD patients presenting with diminished SLPI concentrations. Furthermore, there is a decrease in SLPI concentrations through cleavage and subsequent inactivation by NE degradation in Pseudomonas aeruginosa infected people with CF (pwCF). These observations suggest reduced SLPI protein levels may contribute to the compromising of airway immunity indicating a potential role of decreased SLPI levels in the pathogenesis of muco-obstructive lung disease. The Beta Epithelial Na+ Channel transgenic (ENaC-Tg) mouse model phenotype exhibits characteristics which replicate the pathological features observed in conditions such as COPD and CF, including mucus accumulation, alterations in airway morphology and increased pulmonary inflammation. To evaluate the effect of SLPI in muco-obstructive pulmonary disease, ENaC-Tg mice were crossed with SLPI knock-out (SLPI-/-) mice, generating a ENaC-Tg/SLPI-/- colony to further investigate the role of SLPI in chronic lung disease and determine the effect of its ablation on disease pathogenesis.

PTEN Depletion Increases Radiosensitivity in Response to Ataxia Telangiectasia-Related-3 (ATR) Inhibition in Non-Small Cell Lung Cancer (NSCLC).

Radiotherapy (RT) treatment is an important strategy for the management of non-small cell lung cancer (NSCLC). Local recurrence amongst patients with late-stage NSCLC remains a challenge. The loss of PTEN has been associated with radio-resistance. This study aimed to examine the efficacy of RT combined with ataxia telangiectasia-mutated Rad3-related (ATR) inhibition using Ceralasertib in phosphatase and tensin homolog (PTEN)-depleted NSCLC cells and to assess early inflammatory responses indicative of radiation pneumonitis (RP) after combined-modality treatment. Small hairpin RNA (shRNA) transfections were used to generate H460 and A549 PTEN-depleted models. Ceralasertib was evaluated as a single agent and in combination with RT in vitro and in vivo. Histological staining was used to assess immune cell infiltration in pneumonitis-prone C3H/NeJ mice. Here, we report that the inhibition of ATR in combination with RT caused a significant reduction in PTEN-depleted NSCLC cells, with delayed DNA repair and reduced cell viability, as shown by an increase in cells in Sub G1. Combination treatment in vivo significantly inhibited H460 PTEN-depleted tumour growth in comparison to H460 non-targeting PTEN-expressing (NT) cell-line-derived xenografts (CDXs). Additionally, there was no significant increase in infiltrating macrophages or neutrophils except at 4 weeks, whereby combination treatment significantly increased macrophage levels relative to RT alone. Overall, our study demonstrates that ceralasertib and RT combined preferentially sensitises PTEN-depleted NSCLC models in vitro and in vivo, with no impact on early inflammatory response indicative of RP. These findings provide a rationale for evaluating ATR inhibition in combination with RT in NSCLC patients with PTEN mutations.

In vitro evaluation of the potential use of snake-derived peptides in the treatment of respiratory infections using inhalation therapy: A proof of concept study.

Inhalation therapy using nebulisers is an attractive non-invasive route for drug delivery, particularly for the treatment of lung infections with anti-inflammatory and anti-microbial compounds. This study evaluated the suitability of three snake-derived peptides (termed Sn1b, SnE1 and SnE1-F), which we have recently shown have potent anti-inflammatory and bacteriostatic activities, for nebulisation using a vibrating mesh nebuliser (VMN). The effect of nebulisation on peptide concentration, stability and function were assessed, prior to progression to aerodynamic particle size distribution, and in vitro drug delivery in simulated adult spontaneous breathing and mechanical ventilated patient models. When nebulised, all three peptides exhibited similar functions to their non-nebulised counterparts and were found to be respirable during simulated mechanical ventilation. Based on the assessment of the droplet distributions of nebulised peptides using a Next Generation Impactor (NGI) demonstrated that if administered in vivo each peptide would likely be delivered to the lower airways. These data suggest that nebulisation using a VMN is a viable means of anti-microbial / anti-inflammatory peptide delivery targeting microbial respiratory infections, and possibly even systemic infections.

The Effect of CFTR Modulators on Airway Infection in Cystic Fibrosis.

The advent of Cystic fibrosis transmembrane receptor (CFTR) modulators in 2012 was a critical event in the history of cystic fibrosis (CF) treatment. Unlike traditional therapies that target downstream effects of CFTR dysfunction, CFTR modulators aim to correct the underlying defect at the protein level. These genotype-specific therapies are now available for an increasing number of CF patients, transforming the way we view the condition from a life-limiting disease to one that can be effectively managed. Several studies have demonstrated the vast improvement CFTR modulators have on normalization of sweat chloride, CFTR function, clinical endpoints, and frequency of pulmonary exacerbation. However, their impact on other aspects of the disease, such as pathogenic burden and airway infection, remain under explored. Frequent airway infections as a result of increased susceptibility and impaired innate immune response are a serious problem within CF, often leading to accelerated decline in lung function and disease progression. Current evidence suggests that CFTR modulators are unable to eradicate pathogenic organisms in those with already established lung disease. However, this may not be the case for those with relatively low levels of disease progression and conserved microbial diversity, such as young patients. Furthermore, it remains unknown whether the restorative effects exerted by CFTR modulators extend to immune cells, such as phagocytes, which have the potential to modulate the response of people with CF (pwCF) to infection. Throughout this review, we look at the potential impact of CFTR modulators on airway infection in CF and their ability to shape impaired pulmonary defences to pathogens.

Cathepsin S Contributes to Lung Inflammation in Acute Respiratory Distress Syndrome.

Rationale: Although the cysteine protease cathepsin S has been implicated in the pathogenesis of several inflammatory lung diseases, its role has not been examined in the context of acute respiratory distress syndrome, a condition that still lacks specific and effective pharmacological treatments. Objectives: To characterize the status of cathepsin S in acute lung inflammation and examine the role of cathepsin S in disease pathogenesis. Methods: Human and mouse model BAL fluid samples were analyzed for the presence and activity of cathepsin S and its endogenous inhibitors. Recombinant cathepsin S was instilled directly into the lungs of mice. The effects of cathepsin S knockout and pharmacological inhibition were examined in two models of acute lung injury. Protease-activated receptor-1 antagonism was used to test a possible mechanism for cathepsin S-mediated inflammation. Measurements and Main Results: Pulmonary cathepsin S concentrations and activity were elevated in acute respiratory distress syndrome, a phenotype possibly exacerbated by the loss of the endogenous antiprotease cystatin SN. Direct cathepsin S instillation into the lungs induced key pathologies of acute respiratory distress syndrome, including neutrophilia and alveolar leakage. Conversely, in murine models of acute lung injury, genetic knockdown and prophylactic or therapeutic inhibition of cathepsin S reduced neutrophil recruitment and protein leakage. Cathepsin S may partly mediate its pathogenic effects via protease-activated receptor-1, because antagonism of this receptor abrogated cathepsin S-induced airway inflammation. Conclusions: Cathepsin S contributes to acute lung injury and may represent a novel therapeutic target for acute respiratory distress syndrome.

The relationship between plasma cystatin C, mortality and acute respiratory distress syndrome subphenotype in the HARP-2 trial.

Objective: To evaluate the performance of cystatin C as a prognostic and predictive marker in a trial of patients with acute respiratory distress syndrome (ARDS). Design, patients and setting: A retrospective analysis was performed on plasma samples from patients included in the HARP-2 (hydroxymethylglutaryl-CoA reductase inhibition with simvastatin in acute lung injury to reduce pulmonary dysfunction) trial - a multicentre, phase 2b trial carried out in general intensive care units across 40 hospitals in the United Kingdom and Ireland. Cystatin C concentrations in plasma obtained from 466 patients with ARDS (before they were randomly assigned in the trial) were quantified by ELISA (enzyme-linked immunosorbent assay). Results: In a univariate analysis, plasma cystatin C concentrations were significantly higher in patients with ARDS who did not survive past 28 days (odds ratio [OR], 1.39 [95% CI, 1.12-1.72]; P = 0.002). In a multivariate model adjusted for selected covariates, cystatin C concentrations remained higher among patients with ARDS who did not survive, although this did not reach statistical significance (OR, 1.28 [95% CI, 0.96-1.71]; P = 0.090). Cystatin C concentration was also significantly associated with hyperinflammatory ARDS (OR, 2.64 [95% CI, 1.83-3.89]; P < 0.001). In multivariate models adjusted for both cystatin C concentration and ARDS subphenotype, hyperinflammatory ARDS was prognostic for mortality (OR, 2.06 [95% CI, 1.16-3.64]; P = 0.013) but cystatin C concentration was not (OR, 1.16 [95% CI, 0.85-1.57]; P = 0.346). In a multivariate analysis, hyperinflammatory ARDS was predictive of a beneficial effect of simvastatin on mortality (OR, 2.05 [95% CI, 1.16-3.62]; P = 0.014) but cystatin C concentration was not (OR, 1.10 [95% CI, 0.77-1.56]; P = 0.614). Conclusion: The association between cystatin C concentration and mortality in ARDS may be dependent on inflammatory subphenotype. Cystatin C concentration does not appear to add to existing prognostic or predictive approaches.

The Impact of Lung Proteases on Snake-Derived Antimicrobial Peptides.

Respiratory infections are a leading cause of global morbidity and mortality and are of significant concern for individuals with chronic inflammatory lung diseases. There is an urgent need for novel antimicrobials. Antimicrobial peptides (AMPs) are naturally occurring innate immune response peptides with therapeutic potential. However, therapeutic development has been hindered by issues with stability and cytotoxicity. Availing of direct drug delivery to the affected site, for example the lung, can reduce unwanted systemic side effects and lower the required dose. As cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) lungs typically exhibit elevated protease levels, the aim of this study was to assess their impact on snake-derived AMPs. Peptide cleavage was determined using SDS-PAGE and antimicrobial and anti-inflammatory activities of neutrophil elastase (NE)-incubated peptides were assessed using a radial diffusion assay (RDA) and an in vitro LPS-induced inflammation model, respectively. Although the snake-derived AMPs were found to be susceptible to cleavage by lung proteases including NE, several retained their function following NE-incubation. This facilitated the design of novel truncated derivatives that retained functionality following NE incubation. Snake-derived AMPs are tractable candidate treatments for use in environments that feature elevated NE levels, such as the CF airways.

Proteases, Mucus, and Mucosal Immunity in Chronic Lung Disease.

Dysregulated protease activity has long been implicated in the pathogenesis of chronic lung diseases and especially in conditions that display mucus obstruction, such as chronic obstructive pulmonary disease, cystic fibrosis, and non-cystic fibrosis bronchiectasis. However, our appreciation of the roles of proteases in various aspects of such diseases continues to grow. Patients with muco-obstructive lung disease experience progressive spirals of inflammation, mucostasis, airway infection and lung function decline. Some therapies exist for the treatment of these symptoms, but they are unable to halt disease progression and patients may benefit from novel adjunct therapies. In this review, we highlight how proteases act as multifunctional enzymes that are vital for normal airway homeostasis but, when their activity becomes immoderate, also directly contribute to airway dysfunction, and impair the processes that could resolve disease. We focus on how proteases regulate the state of mucus at the airway surface, impair mucociliary clearance and ultimately, promote mucostasis. We discuss how, in parallel, proteases are able to promote an inflammatory environment in the airways by mediating proinflammatory signalling, compromising host defence mechanisms and perpetuating their own proteolytic activity causing structural lung damage. Finally, we discuss some possible reasons for the clinical inefficacy of protease inhibitors to date and propose that, especially in a combination therapy approach, proteases represent attractive therapeutic targets for muco-obstructive lung diseases.

Therapeutic Inhibition of Cathepsin S Reduces Inflammation and Mucus Plugging in Adult ßENaC-Tg Mice.

BACKGROUND: Elevated levels of the cysteine protease cathepsin S (CatS) are associated with chronic mucoobstructive lung diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). We have previously demonstrated that prophylactic treatment with a CatS inhibitor from birth reduces inflammation, mucus plugging, and lung tissue damage in juvenile ß-epithelial Na+ channel-overexpressing transgenic (ßENaC-Tg) mice with chronic inflammatory mucoobstructive lung disease. In this study, we build upon this work to examine the effects of therapeutic intervention with a CatS inhibitor in adult ßENaC-Tg mice with established disease. METHODS: ßENaC-Tg mice and wild-type (WT) littermates were treated with a CatS inhibitor from 4 to 6 weeks of age, and CatS-/- ßENaC-Tg mice were analysed at 6 weeks of age. Bronchoalveolar lavage (BAL) fluid inflammatory cell counts were quantified, and lung tissue destruction and mucus obstruction were analysed histologically. RESULTS: At 6 weeks of age, ßENaC-Tg mice developed significant airway inflammation, lung tissue damage, and mucus plugging when compared to WT mice. CatS-/- ßENaC-Tg mice and ßENaC-Tg mice receiving inhibitor had significantly reduced airway mononuclear and polymorphonuclear (PMN) cell counts as well as mucus plugging. However, in contrast to CatS-/- ßENaC-Tg mice, therapeutic inhibition of CatS in ßENaC-Tg mice had no effect on established emphysema-like lung tissue damage. CONCLUSIONS: These results suggest that while early CatS targeting may be required to prevent the onset and progression of lung tissue damage, therapeutic CatS targeting effectively inhibited airway inflammation and mucus obstruction. These results indicate the important role CatS may play in the pathogenesis and progression of mucoobstructive lung disease.

Airway Inflammation and Host Responses in the Era of CFTR Modulators.

The arrival of cystic fibrosis transmembrane conductance regulator (CFTR) modulators as a new class of treatment for cystic fibrosis (CF) in 2012 represented a pivotal advance in disease management, as these small molecules directly target the upstream underlying protein defect. Further advancements in the development and scope of these genotype-specific therapies have been transformative for an increasing number of people with CF (PWCF). Despite clear improvements in CFTR function and clinical endpoints such as lung function, body mass index (BMI), and frequency of pulmonary exacerbations, current evidence suggests that CFTR modulators do not prevent continued decline in lung function, halt disease progression, or ameliorate pathogenic organisms in those with established lung disease. Furthermore, it remains unknown whether their restorative effects extend to dysfunctional CFTR expressed in phagocytes and other immune cells, which could modulate airway inflammation. In this review, we explore the effects of CFTR modulators on airway inflammation, infection, and their influence on the impaired pulmonary host defences associated with CF lung disease. We also consider the role of inflammation-directed therapies in light of the widespread clinical use of CFTR modulators and identify key areas for future research.

Mechanisms of Virus-Induced Airway Immunity Dysfunction in the Pathogenesis of COPD Disease, Progression, and Exacerbation.

Chronic obstructive pulmonary disease (COPD) is the integrated form of chronic obstructive bronchitis and pulmonary emphysema, characterized by persistent small airway inflammation and progressive irreversible airflow limitation. COPD is characterized by acute pulmonary exacerbations and associated accelerated lung function decline, hospitalization, readmission and an increased risk of mortality, leading to huge social-economic burdens. Recent evidence suggests ~50% of COPD acute exacerbations are connected with a range of respiratory viral infections. Nevertheless, respiratory viral infections have been linked to the severity and frequency of exacerbations and virus-induced secondary bacterial infections often result in a synergistic decline of lung function and longer hospitalization. Here, we review current advances in understanding the cellular and molecular mechanisms underlying the pathogenesis of COPD and the increased susceptibility to virus-induced exacerbations and associated immune dysfunction in patients with COPD. The multiple immune regulators and inflammatory signaling pathways known to be involved in host-virus responses are discussed. As respiratory viruses primarily target airway epithelial cells, virus-induced inflammatory responses in airway epithelium are of particular focus. Targeting virus-induced inflammatory pathways in airway epithelial cells such as Toll like receptors (TLRs), interferons, inflammasomes, or direct blockade of virus entry and replication may represent attractive future therapeutic targets with improved efficacy. Elucidation of the cellular and molecular mechanisms of virus infections in COPD pathogenesis will undoubtedly facilitate the development of these potential novel therapies that may attenuate the relentless progression of this heterogeneous and complex disease and reduce morbidity and mortality.

Schistosoma mansoni immunomodulatory molecule Sm16/SPO-1/SmSLP is a member of the trematode-specific helminth defence molecules (HDMs).

BACKGROUND: Sm16, also known as SPO-1 and SmSLP, is a low molecular weight protein (~16kDa) secreted by the digenean trematode parasite Schistosoma mansoni, one of the main causative agents of human schistosomiasis. The molecule is secreted from the acetabular gland of the cercariae during skin invasion and is believed to perform an immune-suppressive function to protect the invading parasite from innate immune cell attack. METHODOLOGY/PRINCIPAL FINDINGS: We show that Sm16 homologues of the Schistosomatoidea family are phylogenetically related to the helminth defence molecule (HDM) family of immunomodulatory peptides first described in Fasciola hepatica. Interrogation of 69 helminths genomes demonstrates that HDMs are exclusive to trematode species. Structural analyses of Sm16 shows that it consists predominantly of an amphipathic alpha-helix, much like other HDMs. In S. mansoni, Sm16 is highly expressed in the cercariae and eggs but not in adult worms, suggesting that the molecule is of importance not only during skin invasion but also in the pro-inflammatory response to eggs in the liver tissues. Recombinant Sm16 and a synthetic form, Sm16 (34-117), bind to macrophages and are internalised into the endosomal/lysosomal system. Sm16 (34-117) elicited a weak pro-inflammatory response in macrophages in vitro but also suppressed the production of bacterial lipopolysaccharide (LPS)-induced inflammatory cytokines. Evaluation of the transcriptome of human macrophages treated with a synthetic Sm16 (34-117) demonstrates that the peptide exerts significant immunomodulatory effects alone, as well as in the presence of LPS. Pathways most significantly influenced by Sm16 (34-117) were those involving transcription factors peroxisome proliferator-activated receptor (PPAR) and liver X receptors/retinoid X receptor (LXR/RXR) which are intricately involved in regulating the cellular metabolism of macrophages (fatty acid, cholesterol and glucose homeostasis) and are central to inflammatory responses. CONCLUSIONS/SIGNIFICANCE: These results offer new insights into the structure and function of a well-known immunomodulatory molecule, Sm16, and places it within a wider family of trematode-specific small molecule HDM immune-modulators with immuno-biotherapeutic possibilities.

Cathepsin S: investigating an old player in lung disease pathogenesis, comorbidities, and potential therapeutics.

Dysregulated expression and activity of cathepsin S (CTSS), a lysosomal protease and a member of the cysteine cathepsin protease family, is linked to the pathogenesis of multiple diseases, including a number of conditions affecting the lungs. Extracellular CTSS has potent elastase activity and by processing cytokines and host defense proteins, it also plays a role in the regulation of inflammation. CTSS has also been linked to G-coupled protein receptor activation and possesses an important intracellular role in major histocompatibility complex class II antigen presentation. Modulated CTSS activity is also associated with pulmonary disease comorbidities, such as cancer, cardiovascular disease, and diabetes. CTSS is expressed in a wide variety of immune cells and is biologically active at neutral pH. Herein, we review the significance of CTSS signaling in pulmonary diseases and associated comorbidities. We also discuss CTSS as a plausible therapeutic target and describe recent and current clinical trials examining CTSS inhibition as a means for treatment.

Sea snake cathelicidin (Hc-cath) exerts a protective effect in mouse models of lung inflammation and infection.

We investigated the anti-inflammatory and antibacterial activities of Hc-cath, a cathelicidin peptide derived from the venom of the sea snake, Hydrophis cyanocyntus, using in vivo models of inflammation and infection. Hc-cath function was evaluated in in vitro, in vivo in the wax moth, Galleria mellonella, and in mouse models of intraperitoneal and respiratory Pseudomonas aeruginosa infection. Hc-Cath downregulated LPS-induced pro-inflammatory responses in macrophages and significantly improved the survival of P. aeruginosa infected G. mellonella over a 5-day period. We also demonstrated, for the first time, that Hc-cath can modulate inflammation in a mouse model of LPS-induced lung inflammation by significantly reducing the release of the pro-inflammatory cytokine and neutrophil chemoattractant, KC, resulting in reduced cellular infiltration into the lungs. Moreover, Hc-cath treatment significantly reduced the bacterial load and inflammation in mouse models of P. aeruginosa intraperitoneal and respiratory infection. The effect of Hc-cath in our studies highlights the potential to develop this peptide as a candidate for therapeutic development.

Targeting of cathepsin S reduces cystic fibrosis-like lung disease.

Cathepsin S (CatS) is upregulated in the lungs of patients with cystic fibrosis (CF). However, its role in CF lung disease pathogenesis remains unclear.In this study, ß-epithelial Na+ channel-overexpressing transgenic (ßENaC-Tg) mice, a model of CF-like lung disease, were crossed with CatS null (CatS-/-) mice or treated with the CatS inhibitor VBY-999.Levels of active CatS were elevated in the lungs of ßENaC-Tg mice compared with wild-type (WT) littermates. CatS-/-ßENaC-Tg mice exhibited decreased pulmonary inflammation, mucus obstruction and structural lung damage compared with ßENaC-Tg mice. Pharmacological inhibition of CatS resulted in a significant decrease in pulmonary inflammation, lung damage and mucus plugging in the lungs of ßENaC-Tg mice. In addition, instillation of CatS into the lungs of WT mice resulted in inflammation, lung remodelling and upregulation of mucin expression. Inhibition of the CatS target, protease-activated receptor 2 (PAR2), in ßENaC-Tg mice resulted in a reduction in airway inflammation and mucin expression, indicating a role for this receptor in CatS-induced lung pathology.Our data indicate an important role for CatS in the pathogenesis of CF-like lung disease mediated in part by PAR2 and highlight CatS as a therapeutic target.

Protein Phosphatase 2A Reduces Cigarette Smoke-induced Cathepsin S and Loss of Lung Function.

Rationale: CTSS (cathepsin S) is a cysteine protease that is observed at higher concentrations in BAL fluid and plasma of subjects with chronic obstructive pulmonary disease (COPD). Objectives: To investigate whether CTSS is involved in the pathogenesis of cigarette smoke-induced COPD and determine whether targeting upstream signaling could prevent the disease. Methods: CTSS expression was investigated in animal and human tissue and cell models of COPD. Ctss-/- mice were exposed to long-term cigarette smoke and forced oscillation and expiratory measurements were recorded. Animals were administered chemical modulators of PP2A (protein phosphatase 2A) activity. Measurements and Main Results: Here we observed enhanced CTSS expression and activity in mouse lungs after exposure to cigarette smoke. Ctss-/- mice were resistant to cigarette smoke-induced inflammation, airway hyperresponsiveness, airspace enlargements, and loss of lung function. CTSS expression was negatively regulated by PP2A in human bronchial epithelial cells isolated from healthy nonsmokers and COPD donors and in monocyte-derived macrophages. Modulating PP2A expression or activity, with silencer siRNA or a chemical inhibitor or activator, during acute smoke exposure in mice altered inflammatory responses and CTSS expression and activity in the lung. Enhancement of PP2A activity prevented chronic smoke-induced COPD in mice. Conclusions: Our study indicates that the decrease in PP2A activity that occurs in COPD contributes to elevated CTSS expression in the lungs and results in impaired lung function. Enhancing PP2A activity represents a feasible therapeutic approach to reduce CTSS activity and counter smoke-induced lung disease.

Preclinical Evaluation of Dose-Volume Effects and Lung Toxicity Occurring In and Out-of-Field.

PURPOSE: The aim of this study was to define the dose and dose-volume relationship of radiation-induced pulmonary toxicities occurring in and out-of-field in mouse models of early inflammatory and late fibrotic response. MATERIALS AND METHODS: Early radiation-induced inflammation and fibrosis were investigated in C3H/NeJ and C57BL/6J mice, respectively. Animals were irradiated with 20 Gy delivered to the upper region of the right lung as a single fraction or as 3 consecutive fractions using the Small Animal Radiation Research Platform (Xstrahl Inc, Camberley, UK). Cone beam computed tomography was performed for image guidance before irradiation and to monitor late toxicity. Histologic sections were examined for neutrophil and macrophage infiltration as markers of early inflammatory response and type I collagen staining as a marker of late-occurring fibrosis. Correlation was evaluated with the dose-volume histogram parameters calculated for individual mice and changes in the observed cone beam computed tomography values. RESULTS: Mean lung dose and the volume receiving over 10 Gy (V10) showed significant correlation with late responses for single and fractionated exposures in directly targeted volumes. Responses observed outside the target volume were attributed to nontargeted effects and showed no dependence on either mean lung dose or V10. CONCLUSIONS: Quantitative assessment of normal tissue response closely correlates early and late pulmonary response with clinical parameters, demonstrating this approach as a potential tool to facilitate clinical translation of preclinical studies. Out-of-field effects were observed but did not correlate with dosimetric parameters, suggesting that nontargeted effects may have a role in driving toxicities outside the treatment field.