Determining the toxicological effects of indoor air pollution on both a healthy and an inflammatory-comprised model of the alveolar epithelial barrier in vitro.

Exposure to indoor air pollutants (IAP) has increased recently, with people spending more time indoors (i.e. homes, offices, schools and transportation). Increased exposures of IAP on a healthy population are poorly understood, and those with allergic respiratory conditions even less so. The objective of this study, therefore, was to implement a well-characterised in vitro model of the human alveolar epithelial barrier (A549 + PMA differentiated THP-1 incubated with and without IL-13, IL-5 and IL-4) to determine the effects of a standardised indoor particulate (NIST 2583) on both a healthy lung model and one modelling a type-II (stimulated with IL-13, IL-5 and IL-4) inflammatory response (such as asthma).Using concentrations from the literature, and an environmentally appropriate exposure we investigated 232, 464 and 608ng/cm2 of NIST 2583 respectively. Membrane integrity (blue dextran), viability (trypan blue), genotoxicity (micronucleus (Mn) assay) and (pro-)/(anti-)inflammatory effects (IL-6, IL-8, IL-33, IL-10) were then assessed 24 h post exposure to both models. Models were exposed using a physiologically relevant aerosolisation method (VitroCell Cloud 12 exposure system).No changes in Mn frequency or membrane integrity in either model were noted when exposed to any of the tested concentrations of NIST 2583. A significant decrease (p < 0.05) in cell viability at the highest concentration was observed in the healthy model. Whilst cell viability in the "inflamed" model was decreased at the lower concentrations (significantly (p < 0.05) after 464ng/cm2). A significant reduction (p < 0.05) in IL-10 and a significant increase in IL-33 was seen after 24 h exposure to NIST 2583 (464, 608ng/cm2) in the "inflamed" model.Collectively, the results indicate the potential for IAP to cause the onset of a type II response as well as exacerbating pre-existing allergic conditions. Furthermore, the data imposes the importance of considering unhealthy individuals when investigating the potential health effects of IAP. It also highlights that even in a healthy population these particles have the potential to induce this type II response and initiate an immune response following exposure to IAP.

Mechanical and morphological characterization of the emphysematous lung tissue.

Irreversible alveolar airspace enlargement is the main characteristic of pulmonary emphysema, which has been extensively studied using animal models. While the alterations in lung mechanics associated with these morphological changes have been documented in the literature, the study of the mechanical behavior of parenchymal tissue from emphysematous lungs has been poorly investigated. In this work, we characterize the mechanical and morphological properties of lung tissue in elastase-induced emphysema rat models under varying severity conditions. We analyze the non-linear tissue behavior using suitable hyperelastic constitutive models that enable to compare different non-linear responses in terms of hyperelastic material parameters. We further analyze the effect of the elastase dose on alveolar morphology and tissue material parameters and study their connection with respiratory-system mechanical parameters. Our results show that while the lung mechanical function is not significantly influenced by the elastase treatment, the tissue mechanical behavior and alveolar morphology are markedly affected by it. We further show a strong association between alveolar enlargement and tissue softening, not evidenced by respiratory-system compliance. Our findings highlight the importance of understanding tissue mechanics in emphysematous lungs, as changes in tissue properties could detect the early stages of emphysema remodeling. STATEMENT OF SIGNIFICANCE: Gas exchange is vital for life and strongly relies on the mechanical function of the lungs. Pulmonary emphysema is a prevalent respiratory disease where alveolar walls are damaged, causing alveolar enlargement that induces harmful changes in the mechanical response of the lungs. In this work, we study how the mechanical properties of lung tissue change during emphysema. Our results from animal models show that tissue properties are more sensitive to alveolar enlargement due to emphysema than other mechanical properties that describe the function of the whole respiratory system.

HIF3A gene disruption causes abnormal alveoli structure and early neonatal death.

Transcriptional response to changes in oxygen concentration is mainly controlled by hypoxia-inducible transcription factors (HIFs). Besides regulation of hypoxia-responsible gene expression, HIF-3α has recently been shown to be involved in lung development and in the metabolic process of fat tissue. However, the precise mechanism for such properties of HIF-3α is still largely unknown. To this end, we generated HIF3A gene-disrupted mice by means of genome editing technology to explore the pleiotropic role of HIF-3α in development and physiology. We obtained adult mice carrying homozygous HIF3A gene mutations with comparable body weight and height to wild-type mice. However, the number of litters and ratio of homozygous mutation carriers born from the mating between homozygous mutant mice was lower than expected due to sporadic deaths on postnatal day 1. HIF3A gene-disrupted mice exhibited abnormal configuration of the lung such as a reduced number of alveoli and thickened alveolar walls. Transcriptome analysis showed, as well as genes associated with lung development, an upregulation of stearoyl-Coenzyme A desaturase 1, a pivotal enzyme for fatty acid metabolism. Analysis of fatty acid composition in the lung employing gas chromatography indicated an elevation in palmitoleic acid and a reduction in oleic acid, suggesting an imbalance in distribution of fatty acid, a constituent of lung surfactant. Accordingly, administration of glucocorticoid injections during pregnancy resulted in a restoration of normal alveolar counts and a decrease in neonatal mortality. In conclusion, these observations provide novel insights into a pivotal role of HIF-3α in the preservation of critically important structure and function of alveoli beyond the regulation of hypoxia-mediated gene expression.

Effects of sevoflurane on lung alveolar epithelial wound healing and survival in a sterile in vitro model of acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is a serious lung condition that often leads to hospitalization in intensive care units and a high mortality rate. Sevoflurane is a volatile anesthetic with growing interest for sedation in ventilated patients with ARDS. It has been shown to have potential lung-protective effects, such as reduced inflammation and lung edema, or improved arterial oxygenation. In this study, we investigated the effects of sevoflurane on lung injury in cultured human carcinoma-derived lung alveolar epithelial (A549) cells. We found that sevoflurane was associated with improved wound healing after exposure to inflammatory cytokines, with preserved cell proliferation but no effect on cell migration properties. Sevoflurane exposure was also associated with enhanced cell viability and active autophagy in A549 cells exposed to cytokines. These findings suggest that sevoflurane may have beneficial effects on lung epithelial injury by promoting alveolar epithelial wound healing and by influencing the survival and proliferation of A549 epithelial cells in vitro. Further research is needed to confirm these findings and to investigate the key cellular mechanisms explaining sevoflurane's potential effects on lung epithelial injury.

Progressive changes in the protein expression profile of alveolar septa in early-stage lung adenocarcinoma.

BACKGROUND: Adenocarcinomas show a stepwise progression from atypical adenomatous hyperplasia (AAH) through adenocarcinoma in situ (AIS) to invasive adenocarcinoma (IA). Immunoglobulin superfamily containing leucine-rich repeat (ISLR) is a marker of tumor-restraining cancer-associated fibroblasts (CAFs), which are distinct from conventional, strongly α-smooth muscle actin (αSMA)-positive CAFs. Fibroblast activation protein (FAP) has been focused on as a potential therapeutic and diagnostic target of CAFs. METHODS: We investigated the changes in protein expression during adenocarcinoma progression in the pre-existing alveolar septa by assessing ISLR, αSMA, and FAP expression in normal lung, AAH, AIS, and IA. Fourteen AAH, seventeen AIS, and twenty IA lesions were identified and randomly sampled. Immunohistochemical analysis was performed to evaluate cancer-associated changes and FAP expression in the pre-existing alveolar structures. RESULTS: Normal alveolar septa expressed ISLR. The ISLR level in the alveolar septa decreased in AAH and AIS tissues when compared with that in normal lung tissue. The αSMA-positive area gradually increased from the adjacent lung tissue (13.3% ± 15%) to AIS (87.7% ± 14%), through AAH (70.2% ± 21%). Moreover, the FAP-positive area gradually increased from AAH (1.69% ± 1.4%) to IA (11.8% ± 7.1%), through AIS (6.11% ± 5.3%). Protein expression changes are a feature of CAFs in the pre-existing alveolar septa that begin in AAH. These changes gradually progressed from AAH to IA through AIS. CONCLUSIONS: FAP-positive fibroblasts may contribute to tumor stroma formation in early-stage lung adenocarcinoma, and this could influence the development of therapeutic strategies targeting FAP-positive CAFs for disrupting extracellular matrix formation.

Repair and regeneration of the alveolar epithelium in lung injury.

Considerable progress has been made in understanding the function of alveolar epithelial cells in a quiescent state and regeneration mechanism after lung injury. Lung injury occurs commonly from severe viral and bacterial infections, inhalation lung injury, and indirect injury sepsis. A series of pathological mechanisms caused by excessive injury, such as apoptosis, autophagy, senescence, and ferroptosis, have been studied. Recovery from lung injury requires the integrity of the alveolar epithelial cell barrier and the realization of gas exchange function. Regeneration mechanisms include the participation of epithelial progenitor cells and various niche cells involving several signaling pathways and proteins. While alveoli are damaged, alveolar type II (AT2) cells proliferate and differentiate into alveolar type I (AT1) cells to repair the damaged alveolar epithelial layer. Alveolar epithelial cells are surrounded by various cells, such as fibroblasts, endothelial cells, and various immune cells, which affect the proliferation and differentiation of AT2 cells through paracrine during alveolar regeneration. Besides, airway epithelial cells also contribute to the repair and regeneration process of alveolar epithelium. In this review, we mainly discuss the participation of epithelial progenitor cells and various niche cells involving several signaling pathways and transcription factors.

Bronchioalveolar organoids: A preclinical tool to screen toxicity associated with antibody-drug conjugates.

Despite extensive preclinical testing, cancer therapeutics can result in unanticipated toxicity to non-tumor tissue in patients. These toxicities may pass undetected in preclinical experiments due to modeling limitations involving poor biomimicry of 2-dimensional in vitro cell cultures and due to lack of interspecies translatability in in vivo studies. Instead, primary cells can be grown into miniature 3-dimensional structures that recapitulate morphological and functional aspects of native tissue, termed "organoids." Here, human bronchioalveolar organoids grown from primary alveolar epithelial cells were employed to model lung epithelium and investigate off-target toxicities associated with antibody-drug conjugates (ADCs). ADCs with three different linker-payload combinations (mafodotin, vedotin, and deruxtecan) were tested in bronchioalveolar organoids generated from human, rat, and nonhuman primate lung cells. Organoids demonstrated antibody uptake and changes in viability in response to ADC exposure that model in vivo drug sensitivity. RNA sequencing identified inflammatory activation in bronchioalveolar cells in response to deruxtecan. Future studies will explore specific cell populations involved in interstitial lung disease and incorporate immune cells to the culture.

Inhibition the ubiquitination of ENaC and Na,K-ATPase with erythropoietin promotes alveolar fluid clearance in sepsis-induced acute respiratory distress syndrome.

Sepsis-induced acute respiratory distress syndrome (ARDS) causes significant fatalities worldwide and lacks pharmacological intervention. Alveolar fluid clearance (AFC) plays a pivotal role in the remission of ARDS and is markedly impaired in the pathogenesis of ARDS. Here, we demonstrated that erythropoietin could effectively ameliorate lung injury manifestations and lethality, restore lung function and promote AFC in a rat model of lipopolysaccharide (LPS)-induced ARDS. Moreover, it was proven that EPO-induced restoration of AFC occurs through triggering the total protein expression of ENaC and Na,K-ATPase channels, enhancing their protein abundance in the membrane, and suppressing their ubiquitination for degeneration. Mechanistically, the data indicated the possible involvement of EPOR/JAK2/STAT3/SGK1/Nedd4-2 signaling in this process, and the pharmacological inhibition of the pathway markedly eliminated the stimulating effects of EPO on ENaC and Na,K-ATPase, and subsequently reversed the augmentation of AFC by EPO. Consistently, in vitro studies of alveolar epithelial cells paralleled with that EPO upregulated the expression of ENaC and Na,K-ATPase, and patch-clamp studies further demonstrated that EPO substantially strengthened sodium ion currents. Collectively, EPO could effectively promote AFC by improving ENaC and Na,K-ATPase protein expression and abundance in the membrane, dependent on inhibition of ENaC and Na,K-ATPase ubiquitination, and resulting in diminishing LPS-associated lung injuries.

Mucopolysaccharidosis (MPS IIIA) mice have increased lung compliance and airway resistance, decreased diaphragm strength, and no change in alveolar structure.

Mucopolysaccharidosis type IIIA (MPS IIIA) is characterized by neurological and skeletal pathologies caused by reduced activity of the lysosomal hydrolase, sulfamidase, and the subsequent primary accumulation of undegraded heparan sulfate (HS). Respiratory pathology is considered secondary in MPS IIIA and the mechanisms are not well understood. Changes in the amount, metabolism, and function of pulmonary surfactant, the substance that regulates alveolar interfacial surface tension and modulates lung compliance and elastance, have been reported in MPS IIIA mice. Here we investigated changes in lung function in 20-wk-old control and MPS IIIA mice with a closed and open thoracic cage, diaphragm contractile properties, and potential parenchymal remodeling. MPS IIIA mice had increased compliance and airway resistance and reduced tissue damping and elastance compared with control mice. The chest wall impacted lung function as observed by an increase in airway resistance and a decrease in peripheral energy dissipation in the open compared with the closed thoracic cage state in MPS IIIA mice. Diaphragm contractile forces showed a decrease in peak twitch force, maximum specific force, and the force-frequency relationship but no change in muscle fiber cross-sectional area in MPS IIIA mice compared with control mice. Design-based stereology did not reveal any parenchymal remodeling or destruction of alveolar septa in the MPS IIIA mouse lung. In conclusion, the increased storage of HS which leads to biochemical and biophysical changes in pulmonary surfactant also affects lung and diaphragm function, but has no impact on lung or diaphragm structure at this stage of the disease.NEW & NOTEWORTHY Heparan sulfate storage in the lungs of mucopolysaccharidosis type IIIA (MPS IIIA) mice leads to changes in lung function consistent with those of an obstructive lung disease and includes an increase in lung compliance and airway resistance and a decrease in tissue elastance. In addition, diaphragm muscle contractile strength is reduced, potentially further contributing to lung function impairment. However, no changes in parenchymal lung structure were observed in mice at 20 wk of age.

Time-Controlled Adaptive Ventilation (TCAV): a personalized strategy for lung protection.

Acute respiratory distress syndrome (ARDS) alters the dynamics of lung inflation during mechanical ventilation. Repetitive alveolar collapse and expansion (RACE) predisposes the lung to ventilator-induced lung injury (VILI). Two broad approaches are currently used to minimize VILI: (1) low tidal volume (LVT) with low-moderate positive end-expiratory pressure (PEEP); and (2) open lung approach (OLA). The LVT approach attempts to protect already open lung tissue from overdistension, while simultaneously resting collapsed tissue by excluding it from the cycle of mechanical ventilation. By contrast, the OLA attempts to reinflate potentially recruitable lung, usually over a period of seconds to minutes using higher PEEP used to prevent progressive loss of end-expiratory lung volume (EELV) and RACE. However, even with these protective strategies, clinical studies have shown that ARDS-related mortality remains unacceptably high with a scarcity of effective interventions over the last two decades. One of the main limitations these varied interventions demonstrate to benefit is the observed clinical and pathologic heterogeneity in ARDS. We have developed an alternative ventilation strategy known as the Time Controlled Adaptive Ventilation (TCAV) method of applying the Airway Pressure Release Ventilation (APRV) mode, which takes advantage of the heterogeneous time- and pressure-dependent collapse and reopening of lung units. The TCAV method is a closed-loop system where the expiratory duration personalizes VT and EELV. Personalization of TCAV is informed and tuned with changes in respiratory system compliance (CRS) measured by the slope of the expiratory flow curve during passive exhalation. Two potentially beneficial features of TCAV are: (i) the expiratory duration is personalized to a given patient's lung physiology, which promotes alveolar stabilization by halting the progressive collapse of alveoli, thereby minimizing the time for the reopened lung to collapse again in the next expiration, and (ii) an extended inspiratory phase at a fixed inflation pressure after alveolar stabilization gradually reopens a small amount of tissue with each breath. Subsequently, densely collapsed regions are slowly ratcheted open over a period of hours, or even days. Thus, TCAV has the potential to minimize VILI, reducing ARDS-related morbidity and mortality.

Histopathological changes in lungs of patients with fatal COVID 19 infection: A series of 15 cases.

Introduction: The predominant pathology noted in the lungs of patients dying of COVID-19 is reported to be diffuse alveolar damage (DAD). Other studies have identified microthrombi to be a prominent finding of lung injury in patients affected by COVID-19. We describe the lung histopathological findings in fifteen cases of COVID-19 who died from the disease with the aim of reporting the microscopic changes in the lungs of patients dying from this disease. Materials and Methods: Lung tissues from fifteen consecutive autopsy cases of COVID-19 were studied for gross and microscopic features. The case history of the deaths was noted, and the information was analyzed. The lung damage seen was graded on a semiquantitative scale on the basis of the percentage of tissue involved. Results: Gross examination of the lungs showed multiple foci of consolidation mainly in the lower lobes of the lungs as the most commonly encountered finding. The other significant pattern was congested and edematous lungs with areas of consolidation. Microscopic assessment of lung sections showed 8 out of the 15 cases showing changes of the exudative phase of diffuse alveolar damage, whereas two cases were in the proliferative phase. Hyaline membranes were one of the common findings along with intra-alveolar edema and interstitial edema. Four cases showed changes in organizing phase. Other findings were microthrombi formation, fungal abscesses, dilated and collapsed alveoli, intra-alveolar hemorrhage, and acute neutrophilic pneumonia. Conclusion: DADand interstitial pneumonitis were the most striking features in our autopsy study. Features of different phases of diffuse alveolar damage were seen to coexist in the same patient indicating the temporal heterogeneity of the ongoing lung injury in these patients.

Rescue of alveolar wall liquid secretion blocks fatal lung injury due to influenza-staphylococcal coinfection.

Secondary lung infection by inhaled Staphylococcus aureus (SA) is a common and lethal event for individuals infected with influenza A virus (IAV). How IAV disrupts host defense to promote SA infection in lung alveoli, where fatal lung injury occurs, is not known. We addressed this issue using real-time determinations of alveolar responses to IAV in live, intact, perfused lungs. Our findings show that IAV infection blocked defensive alveolar wall liquid (AWL) secretion and induced airspace liquid absorption, thereby reversing normal alveolar liquid dynamics and inhibiting alveolar clearance of inhaled SA. Loss of AWL secretion resulted from inhibition of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel in the alveolar epithelium, and airspace liquid absorption was caused by stimulation of the alveolar epithelial Na+ channel (ENaC). Loss of AWL secretion promoted alveolar stabilization of inhaled SA, but rescue of AWL secretion protected against alveolar SA stabilization and fatal SA-induced lung injury in IAV-infected mice. These findings reveal a central role for AWL secretion in alveolar defense against inhaled SA and identify AWL inhibition as a critical mechanism of IAV lung pathogenesis. AWL rescue may represent a new therapeutic approach for IAV-SA coinfection.

Clinical Relevance of Rapid FOXF1-Targeted Sequencing in Patients Suspected of Alveolar Capillary Dysplasia With Misalignment of Pulmonary Veins.

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal congenital lung disorder that presents shortly after birth with respiratory failure and therapy-resistant pulmonary hypertension. It is associated with heterozygous point mutations and genomic deletions that involve the FOXF1 gene or its upstream regulatory region. Patients are unresponsive to the intensive treatment regimens and suffer unnecessarily because ACDMPV is not always timely recognized and histologic diagnosis is invasive and time consuming. Here, we demonstrate the usefulness of a noninvasive, fast genetic test for FOXF1 variants that we previously developed to rapidly diagnose ACDMPV and reduce the time of hospitalization.

Histologic features and decreased lung FOXF1 gene expression in severe bronchopulmonary dysplasia without a genetic diagnosis of alveolar capillary dysplasia.

We report the case of a preterm infant who died at 10 months of age with severe bronchopulmonary dysplasia (sBPD) with refractory pulmonary hypertension and respiratory failure who had striking histologic features compatible with the diagnosis of alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) but without genetic confirmation of the diagnosis. We further demonstrate dramatic reductions in lung FOXF1 and TMEM100 content in sBPD, suggesting common mechanistic links between ACDMPV and sBPD with impaired FOXF1 signaling.

CT Approach to Lung Injury.

Diffuse alveolar damage (DAD), which represents the pathologic changes seen after acute lung injury, is caused by damage to all three layers of the alveolar wall and can ultimately result in alveolar collapse with loss of the normal pulmonary architecture. DAD has an acute phase that predominantly manifests as airspace disease at CT owing to filling of the alveoli with cells, plasma fluids, and hyaline membranes. DAD then evolves into a heterogeneous organizing phase, with mixed airspace and interstitial disease characterized by volume loss, architectural distortion, fibrosis, and parenchymal loss. Patients with DAD have a severe clinical course and typically require prolonged mechanical ventilation, which may result in ventilator-induced lung injury. In those patients who survive DAD, the lungs will remodel over time, but most will have residual findings at chest CT. Organizing pneumonia (OP) is a descriptive term for a histologic pattern characterized by intra-alveolar fibroblast plugs. The significance and pathogenesis of OP are controversial. Some authors regard it as part of a spectrum of acute lung injury, while others consider it a marker of acute or subacute lung injury. At CT, OP manifests with various forms of airspace disease that are most commonly bilateral and relatively homogeneous in appearance at individual time points. Patients with OP most often have a mild clinical course, although some may have residual findings at CT. In patients with DAD and OP, imaging findings can be combined with clinical information to suggest the diagnosis in many cases, with biopsy reserved for difficult cases with atypical findings or clinical manifestations. To best participate in the multidisciplinary approach to patients with lung injury, radiologists must not only recognize these entities but also describe them with consistent and meaningful terminology, examples of which are emphasized in the article. © RSNA, 2023 See the invited commentary by Kligerman et al in this issue. Quiz questions for this article are available in the supplemental material.

Injury activated alveolar progenitors (IAAPs): the underdog of lung repair.

Alveolar epithelial type II cells (AT2s) together with AT1s constitute the epithelial lining of lung alveoli. In contrast to the large flat AT1s, AT2s are cuboidal and smaller. In addition to surfactant production, AT2s also serve as prime alveolar progenitors in homeostasis and play an important role during regeneration/repair. Based on different lineage tracing strategies in mice and single-cell transcriptomic analysis, recent reports highlight the heterogeneous nature of AT2s. These studies present compelling evidence for the presence of stable or transitory AT2 subpopulations with distinct marker expression, signaling pathway activation and functional properties. Despite demonstrated progenitor potentials of AT2s in maintaining homeostasis, through self-renewal and differentiation to AT1s, the exact identity, full progenitor potential and regulation of these progenitor cells, especially in the context of human diseases remain unclear. We recently identified a novel subset of AT2 progenitors named "Injury-Activated Alveolar Progenitors" (IAAPs), which express low levels of Sftpc, Sftpb, Sftpa1, Fgfr2b and Etv5, but are highly enriched for the expression of the surface receptor programmed cell death-ligand 1 (Pd-l1). IAAPs are quiescent during lung homeostasis but activated upon injury with the potential to proliferate and differentiate into AT2s. Significantly, a similar population of PD-L1 positive cells expressing intermediate levels of SFTPC are found to be expanded in human IPF lungs. We summarize here the current understanding of this newly discovered AT2 progenitor subpopulation and also try to reconcile the relationship between different AT2 stem cell subpopulations regarding their progenitor potential, regulation, and relevance to disease pathogenesis and therapeutic interventions.

Pulmonary tuberculosis associated acute fibrinous and organizing pneumonia: A case report and literature review.

BACKGROUND: Acute fibrinous and organizing pneumonia (AFOP) is a rare histological interstitial pneumonia pattern characterized by patches of "fibrin balls" distributed within the alveoli and organizing pneumonia. Currently, there is no consensus on the diagnosis and treatment of this disease. METHODS: We present the case of a 44-year-old male with AFOP secondary to Mycobacterium tuberculosis. We have further reviewed organizing pneumonia (OP) and AFOP caused by tuberculosis. CONCLUSION: Tuberculosis secondary to OP or AFOP is rare and challenging to diagnose. We need to constantly adjust the treatment plan based on the patient's symptoms, test results, and response to treatment in order to arrive at an accurate diagnosis and maximize treatment efficacy.

Bystander CD8 + T cells may be involved in the acute phase of diffuse alveolar damage.

Acute respiratory distress syndrome (ARDS) is a serious complication of systemic inflammatory response syndrome, and diffuse alveolar damage (DAD) is a histological manifestation of ARDS. Endothelial cell injury is mainly responsible for ARDS. Many neutrophils and macrophages/monocytes, which are inflammatory cells that play a role in innate immunity, infiltrate the lung tissue in DAD. In recent years, it has become clear that CD8 plays an important role not only in the acquired immune system, but also in the innate immune system. Non-antigen-activated bystander CD8 + T cells express the unique granzyme B (GrB) + /CD25-/programmed cell death-1 (PD-1)-phenotype. The involvement of bystander CD8 + T cells in lung tissue in DAD is an unexplored field. This study aimed to determine whether bystander CD8 is involved in DAD. Twenty-three consecutive autopsy specimens were retrieved from patients with DAD, and the phenotypes of infiltrating lymphocytes in the DAD lesions were evaluated using immunohistochemistry. In most cases, the number of CD8 + T cells was higher than that of CD4 + T cells, and many GrB + cells were also observed. However, the number of CD25 + and PD-1 + cells was low. We conclude that bystander CD8 + T cells may be involved in cell injury during the development of DAD.