Neuroplasticity and neuroimmune interactions in fatal asthma.

BACKGROUND: Alteration of airway neuronal function and density and bidirectional interaction between immune cells and sensory peripheral nerves have been proposed to trigger and perpetuate inflammation that contribute to asthma severity. To date, few studies analysed neuroplasticity and neuroinflammation in tissue of asthmatic individuals. We hypothesized that the presence of these phenomena would be a pathological feature in fatal asthma. METHODS: We have quantified the expression of the pan-neuronal marker PGP9.5 and the neuronal sensory-derived neuropeptide calcitonin gene-related peptide (CGRP) in the large airways of 12 individuals deceased due to an asthma attack and compared to 10 control lung samples. The proximity between nerve bundles to eosinophils, mast cells and CADM1+ cells was also quantified. We have additionally developed a hPSC-derived sensory neuron/mast cell co-culture model, from where mast cells were purified and differences in gene expression profile assessed. RESULTS: Fatal asthma patients presented a higher PGP9.5 and CGRP positive area in the airways, indicating sensory neuroplasticity. Eosinophils, mast cells and CADM1+ cells were observed in close contact or touching the airway nerve bundles, and this was found to be statistically higher in fatal asthma samples. In vitro co-culture model showed that human mast cells adhere to sensory neurons and develop a distinct gene expression profile characterized by upregulated expression of genes related to heterophilic adhesion, activation and differentiation markers, such as CADM4, PTGS2, C-KIT, GATA2, HDC, CPA3, ATXN1 and VCAM1. CONCLUSIONS: Our results support a significant role for neuroplasticity and neuroimmune interactions in fatal asthma, that could be implicated in the severity of the fatal attack. Accordingly, the presence of physical neuron and mast cell interaction leads to differential gene expression profile in the later cell type.

Lung tissue expression of epithelial injury markers is associated with acute lung injury severity but does not discriminate sepsis from ARDS.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a common cause of respiratory failure in critically ill patients, and diffuse alveolar damage (DAD) is considered its histological hallmark. Sepsis is one of the most common aetiology of ARDS with the highest case-fatality rate. Identifying ARDS patients and differentiate them from other causes of acute respiratory failure remains a challenge. To address this, many studies have focused on identifying biomarkers that can help assess lung epithelial injury. However, there is scarce information available regarding the tissue expression of these markers. Evaluating the expression of elafin, RAGE, and SP-D in lung tissue offers a potential bridge between serological markers and the underlying histopathological changes. Therefore, we hypothesize that the expression of epithelial injury markers varies between sepsis and ARDS as well as according to its severity. METHODS: We compared the post-mortem lung tissue expression of the epithelial injury markers RAGE, SP-D, and elafin of patients that died of sepsis, ARDS, and controls that died from non-pulmonary causes. Lung tissue was collected during routine autopsy and protein expression was assessed by immunohistochemistry. We also assessed the lung injury by a semi-quantitative analysis. RESULTS: We observed that all features of DAD were milder in septic group compared to ARDS group. Elafin tissue expression was increased and SP-D was decreased in the sepsis and ARDS groups. Severe ARDS expressed higher levels of elafin and RAGE, and they were negatively correlated with PaO2/FiO2 ratio, and positively correlated with bronchopneumonia percentage and hyaline membrane score. RAGE tissue expression was negatively correlated with mechanical ventilation duration in both ARDS and septic groups. In septic patients, elafin was positively correlated with ICU admission length, SP-D was positively correlated with serum lactate and RAGE was correlated with C-reactive protein. CONCLUSIONS: Lung tissue expression of elafin and RAGE, but not SP-D, is associated with ARDS severity, but does not discriminate sepsis patients from ARDS patients.

Airway wall extracellular matrix changes induced by bronchial thermoplasty in severe asthma.

BACKGROUND: Airway remodeling is a prominent feature of asthma, which involves increased airway smooth muscle mass and altered extracellular matrix composition. Bronchial thermoplasty (BT), a bronchoscopic treatment for severe asthma, targets airway remodeling. OBJECTIVE: We sought to investigate the effect of BT on extracellular matrix composition and its association with clinical outcomes. METHODS: This is a substudy of the TASMA trial. Thirty patients with severe asthma were BT-treated, of whom 13 patients were treated for 6 months with standard therapy (control group) before BT. Demographic data, clinical data including pulmonary function, and bronchial biopsies were collected. Biopsies at BT-treated and nontreated locations were analyzed by histological and immunohistochemical staining. Associations between histology and clinical outcomes were explored. RESULTS: Six months after treatment, it was found that the reticular basement membrane thickness was reduced from 7.28 µm to 5.74 µm (21% relative reduction) and the percentage area of tissue positive for collagen increased from 26.3% to 29.8% (13% relative increase). Collagen structure analysis revealed a reduction in the curvature frequency of fibers. The percentage area positive for fibulin-1 and fibronectin increased by 2.5% and 5.9%, respectively (relative increase of 124% and 15%). No changes were found for elastin. The changes in collagen and fibulin-1 negatively associated with changes in FEV1 reversibility. CONCLUSIONS: Besides reduction of airway smooth muscle mass, BT has an impact on reticular basement membrane thickness and the extracellular matrix arrangement characterized by an increase in tissue area occupied by collagen with a less dense fiber organization. Both collagen and fibulin-1 are negatively associated with the change in FEV1 reversibility.

COVID-19 induces more pronounced extracellular matrix deposition than other causes of ARDS.

BACKGROUND: Lung fibrosis is a major concern in severe COVID-19 patients undergoing mechanical ventilation (MV). Lung fibrosis frequency in post-COVID syndrome is highly variable and even if the risk is proportionally small, many patients could be affected. However, there is still no data on lung extracellular matrix (ECM) composition in severe COVID-19 and whether it is different from other aetiologies of ARDS. METHODS: We have quantified different ECM elements and TGF-ß expression in lung tissue of 28 fatal COVID-19 cases and compared to 27 patients that died of other causes of ARDS, divided according to MV duration (up to six days or seven days or more). In COVID-19 cases, ECM elements were correlated with lung transcriptomics and cytokines profile. RESULTS: We observed that COVID-19 cases presented significant increased deposition of collagen, fibronectin, versican, and TGF-ß, and decreased decorin density when compared to non-COVID-19 cases of similar MV duration. TGF-ß was precociously increased in COVID-19 patients with MV duration up to six days. Lung collagen was higher in women with COVID-19, with a transition of upregulated genes related to fibrillogenesis to collagen production and ECM disassembly along the MV course. CONCLUSIONS: Fatal COVID-19 is associated with an early TGF-ß expression lung environment after the MV onset, followed by a disordered ECM assembly. This uncontrolled process resulted in a prominent collagen deposition when compared to other causes of ARDS. Our data provides pathological substrates to better understand the high prevalence of pulmonary abnormalities in patients surviving COVID-19.

Evaluation of Structural Viability of Porcine Tracheal Scaffolds after 3 and 6 Months of Storage under Three Different Protocols.

Tracheal replacement with a bioengineered tracheal substitute has been developed for long-segment tracheal diseases. The decellularized tracheal scaffold is an alternative for cell seeding. It is not defined if the storage scaffold produces changes in the scaffold's biomechanical properties. We tested three protocols for porcine tracheal scaffold preservation immersed in PBS and alcohol 70%, in the fridge and under cryopreservation. Ninety-six porcine tracheas (12 in natura, 84 decellularized) were divided into three groups (PBS, alcohol, and cryopreservation). Twelve tracheas were analyzed after three and six months. The assessment included residual DNA, cytotoxicity, collagen contents, and mechanical properties. Decellularization increased the maximum load and stress in the longitudinal axis and decreased the maximum load in the transverse axis. The decellularization of the porcine trachea produced structurally viable scaffolds, with a preserved collagen matrix suitable for further bioengineering. Despite the cyclic washings, the scaffolds remained cytotoxic. The comparison of the storage protocols (PBS at 4 °C, alcohol at 4 °C, and slow cooling cryopreservation with cryoprotectants) showed no significant differences in the amount of collagen and in the biomechanical properties of the scaffolds. Storage in PBS solution at 4 °C for six months did not change the scaffold mechanics.

Inhibition of Macrophage-Specific CHIT1 as an Approach to Treat Airway Remodeling in Severe Asthma.

Chitotriosidase (CHIT1) is an enzyme produced by macrophages that regulates their differentiation and polarization. Lung macrophages have been implicated in asthma development; therefore, we asked whether pharmacological inhibition of macrophage-specific CHIT1 would have beneficial effects in asthma, as it has been shown previously in other lung disorders. CHIT1 expression was evaluated in the lung tissues of deceased individuals with severe, uncontrolled, steroid-naïve asthma. OATD-01, a chitinase inhibitor, was tested in a 7-week-long house dust mite (HDM) murine model of chronic asthma characterized by accumulation of CHIT1-expressing macrophages. CHIT1 is a dominant chitinase activated in fibrotic areas of the lungs of individuals with fatal asthma. OATD-01 given in a therapeutic treatment regimen inhibited both inflammatory and airway remodeling features of asthma in the HDM model. These changes were accompanied by a significant and dose-dependent decrease in chitinolytic activity in BAL fluid and plasma, confirming in vivo target engagement. Both IL-13 expression and TGFß1 levels in BAL fluid were decreased and a significant reduction in subepithelial airway fibrosis and airway wall thickness was observed. These results suggest that pharmacological chitinase inhibition offers protection against the development of fibrotic airway remodeling in severe asthma.

Bronchial eosinophils, neutrophils, and CD8 + T cells influence asthma control and lung function in schoolchildren and adolescents with severe treatment-resistant asthma.

BACKGROUND: Studies in adult severe treatment-resistant asthma (STRA) have demonstrated heterogeneous pathophysiology. Studies in the pediatric age group are still scarce, and few include bronchial tissue analysis. OBJECTIVE: We investigated 6-18-year-old patients diagnosed with STRA in Sao Paulo, Brazil, by characterizing the different lung compartments and their correlations with asthma control and lung function. METHODS: Inflammatory profiles of 13 patients with a confirmed diagnosis of STRA were analyzed using blood, induced sputum, bronchoalveolar lavage, viral and bacterial screens and endobronchial biopsy. Inflammatory cells, cytokines, and basement membrane thickening were tested for correlations with the asthma control test (ACT) and spirometry and plethysmography parameters. RESULTS: Endobronchial biopsy specimens from 11 patients were viable for analysis. All biopsies showed eosinophilic infiltration. Submucosal (SM) eosinophils and neutrophils were correlated with worse lung function (pre-BD FEV1), and SM neutrophils were correlated with fixed obstruction (post-BD FEV1). Intraepithelial (IE) neutrophils were positively correlated with lung function (pre-BD sGaw). CD8 + T cells had the highest density in the IE and SM layers and were positively correlated with ACT and negatively correlated with the cytokines IL1ß, IL2, IL5, IL7, IL10, IL12, IL17, GCSF, MCP-1, INF-δ, and TNFα in sputum supernatant. The ASM chymase + mast cell density correlated positively with quality-of-life score (pAQLQ) and ACT. CONCLUSION: Eosinophils and SM neutrophils correlated with worse lung function, while IE neutrophils correlated with better lung function. Most importantly, CD8 + T cells were abundant in bronchial biopsies of STRA patients and showed protective associations, as did chymase + mast cells.

Allergic sensitization and exposure to ambient air pollution beginning early in life lead to a COPD-like phenotype in young adult mice.

The perinatal period and early infancy are considered critical periods for lung development. During this period, adversities such as environmental exposures, allergic sensitization, and asthma are believed to impact lung health in adulthood. Therefore, we hypothesized that concomitant exposure to allergic sensitization and urban-derived fine particulate matter (PM2.5) in the early postnatal period of mice would cause more profound alterations in lung alveolarization and growth and differently modulate lung inflammation and gene expression than either insult alone in adult life. BALB/c mice were sensitized with ovalbumin (OVA) and exposed to PM2.5 from the fifth day of life. Then, we assessed lung responsiveness, inflammation in BALF, lung tissue, and alveolarization by stereology. In addition, we performed a transcriptomic analysis of lung tissue on the 40th day of life. Our results showed that young adult mice submitted to allergic sensitization and exposure to ambient PM2.5 since early life presented decreased lung growth with impaired alveolarization, a mixed neutrophilic-eosinophilic pattern of lung inflammation, increased airway responsiveness, and increased expression of genes linked to neutrophil recruitment when compared to animals that were OVA-sensitized or PM2.5 exposed only. Both, early life allergic sensitization and PM2.5 exposure, induced inflammation and impaired lung growth, but concomitant exposure was associated with worsened inflammation parameters and caused alveolar enlargement. Our experimental data provide pathological support for the hypothesis that allergic or environmental insults in early life have permanent adverse consequences for lung growth. In addition, combined insults were associated with the development of a COPD-like phenotype in young adult mice. Together with our data, current evidence points to the urgent need for healthier environments with fewer childhood disadvantage factors during the critical windows of lung development and growth.

Diffuse alveolar damage patterns reflect the immunological and molecular heterogeneity in fatal COVID-19.

BACKGROUND: Severe COVID-19 lung disease exhibits a high degree of spatial and temporal heterogeneity, with different histological features coexisting within a single individual. It is important to capture the disease complexity to support patient management and treatment strategies. We provide spatially decoded analyses on the immunopathology of diffuse alveolar damage (DAD) patterns and factors that modulate immune and structural changes in fatal COVID-19. METHODS: We spatially quantified the immune and structural cells in exudative, intermediate, and advanced DAD through multiplex immunohistochemistry in autopsy lung tissue of 18 COVID-19 patients. Cytokine profiling, viral, bacteria, and fungi detection, and transcriptome analyses were performed. FINDINGS: Spatial DAD progression was associated with expansion of immune cells, macrophages, CD8+ T cells, fibroblasts, and (lymph)angiogenesis. Viral load correlated positively with exudative DAD and negatively with disease/hospital length. In all cases, enteric bacteria were isolated, and Candida parapsilosis in eight cases. Cytokines correlated mainly with macrophages and CD8+T cells. Pro-coagulation and acute repair were enriched pathways in exudative DAD whereas intermediate/advanced DAD had a molecular profile of elevated humoral and innate immune responses and extracellular matrix production. INTERPRETATION: Unraveling the spatial and molecular immunopathology of COVID-19 cases exposes the responses to SARS-CoV-2-induced exudative DAD and subsequent immune-modulatory and remodeling changes in proliferative/advanced DAD that occur side-by-side together with secondary infections in the lungs. These complex features have important implications for disease management and the development of novel treatments. FUNDING: CNPq, Bill and Melinda Gates Foundation, HC-Convida, FAPESP, Regeneron Pharmaceuticals, and the Swedish Heart & Lung Foundation.

LPS Response Is Impaired by Urban Fine Particulate Matter.

Fine particulate matter (PM2.5) is a complex mixture of components with diverse chemical and physical characteristics associated with increased respiratory and cardiovascular diseases mortality. Our study aimed to investigate the effects of exposure to concentrated PM2.5 on LPS-induced lung injury onset. BALB/c male mice were exposed to either filtered air or ambient fine PM2.5 in an ambient particle concentrator for 5 weeks. Then, an acute lung injury was induced with nebulized LPS. The animals were euthanized 24 h after the nebulization to either LPS or saline. Inflammatory cells and cytokines (IL-1ß, IL-4, IL-5, IL-6, IL-10, IL-17, TNF) were assessed in the blood, bronchoalveolar lavage fluid (BALF), and lung tissue. In addition, lung morphology was assessed by stereological methods. Our results showed that the PM+LPS group showed histological evidence of injury, leukocytosis with increased neutrophils and macrophages, and a mixed inflammatory response profile, with increased KC, IL-6, IL-1ß, IL-4, and IL-17. Our analysis shows that there is an interaction between the LPS nebulization and PM2.5 exposure, differently modulating the inflammatory response, with a distinct response pattern as compared to LPS or PM2.5 exposure alone. Further studies are required to explain the mechanism of immune modulation caused by PM2.5 exposure.

Airborne microplastics and SARS-CoV-2 in total suspended particles in the area surrounding the largest medical centre in Latin America.

Microplastics (MPs) have been reported in the outdoor/indoor air of urban centres, raising health concerns due to the potential for human exposure. Since aerosols are considered one of the routes of Coronavirus disease 2019 (COVID-19) transmission and may bind to the surface of airborne MPs, we hypothesize that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could be associated with the levels of MPs in the air. Our goal was to quantify the SARS-CoV-2 RNA and MPs present in the total suspended particles (TSP) collected in the area surrounding the largest medical centre in Latin America and to elucidate a possible association among weather variables, MPs, and SARS-CoV-2 in the air. TSP were sampled from three outdoor locations in the areas surrounding a medical centre. MPs were quantified and measured under a fluorescence microscope, and their polymeric composition was characterized by Fourier transform infrared (FT-IR) microspectroscopy coupled with attenuated total reflectance (ATR). The viral load of SARS-CoV-2 was quantified by an in-house real-time PCR assay. A generalized linear model (GzLM) was employed to evaluate the effect of the SARS-CoV-2 quantification on MPs and weather variables. TSP samples tested positive for SARS-CoV-2 in 22 out of 38 samples at the three sites. Polyester was the most frequent polymer (80%) found in the samples. The total amount of MPs was positively associated with the quantification of SARS-CoV-2 envelope genes and negatively associated with weather variables (temperature and relative humidity). Our findings show that SARS-CoV-2 aerosols may bind to TSP, such as MPs, and facilitate virus entry into the human body.

Quantification of airborne SARS-CoV-2 genomic particles in different hospital settings.

We quantified the presence of SARS-CoV-2 RNA in the air of different hospital settings and the autopsy room of the largest medical centre in Sao Paulo, Brazil. Real-time reverse-transcription PCR was used to determine the presence of the envelope protein of SARS-CoV-2 and the nucleocapsid protein genes. The E-gene was detected in 5 out of 6 samples at the ICU-COVID-19 ward and in 5 out of 7 samples at the ward-COVID-19. Similarly, in the non-dedicated facilities, the E-gene was detected in 5 out of 6 samples collected in the ICU and 4 out of 7 samples in the ward. In the necropsy room, 6 out of 7 samples were positive for the E-gene. When both wards were compared, the non-COVID ward presented a significantly higher concentration of the E-gene than in the COVID-19 ward (p = 0.003). There was no significant difference in E-gene concentration between the ICU-COVID-19 and the ICU (p = 0.548). Likewise, there was no significant difference among E-gene concentrations found in the autopsy room versus the ICUs and wards (dedicated or not) (p = 0.245). Our results show the widespread presence of aerosol contamination in different hospital units.

Inflammation response, oxidative stress and DNA damage caused by urban air pollution exposure increase in the lack of DNA repair XPC protein.

Air pollution represents a considerable threat to health worldwide. The São Paulo Metropolitan area, in Brazil, has a unique composition of atmospheric pollutants with a population of nearly 20 million people and 9 million passenger cars. It is long known that exposure to particulate matter less than 2.5 µm (PM2.5) can cause various health effects such as DNA damage. One of the most versatile defense mechanisms against the accumulation of DNA damage is the nucleotide excision repair (NER), which includes XPC protein. However, the mechanisms by which NER protects against adverse health effects related to air pollution are largely unknown. We hypothesized that reduction of XPC activity may contribute to inflammation response, oxidative stress and DNA damage after PM2.5 exposure. To address these important questions, XPC knockout and wild type mice were exposed to PM2.5 using the Harvard Ambient Particle concentrator. Results from one-single exposure have shown a significant increase in the levels of anti-ICAM, IL-1ß, and TNF-α in the polluted group when compared to the filtered air group. Continued chronic PM2.5 exposure increased levels of carbonylated proteins, especially in the lung of XPC mice, probably as a consequence of oxidative stress. As a response to DNA damage, XPC mice lungs exhibit increased γ-H2AX, followed by severe atypical hyperplasia. Emissions from vehicles are composed of hazardous substances, with polycyclic aromatic hydrocarbons (PAHs) and metals being most frequently cited as the major contributors to negative health impacts. This analysis showed that benzo[b]fluoranthene, 2-nitrofluorene and 9,10-anthraquinone were the most abundant PAHs and derivatives. Taken together, these findings demonstrate the participation of XPC protein, and NER pathway, in the protection of mice against the carcinogenic potential of air pollution. This implicates that DNA is damaged directly (forming adducts) or indirectly (Reactive Oxygen Species) by the various compounds detected in urban PM2.5.

Air pollution impairs recovery and tissue remodeling in a murine model of acute lung injury.

Evidence regarding the impact of air pollution on acute respiratory distress syndrome (ARDS) is limited, and most studies focus on ARDS onset. Our study aimed to evaluate whether exposure to fine particulate matter interferes with lung recovery and remodeling in a murine model of acute lung injury. Forty-eight mice received nebulized LPS or the vehicle (controls). Blood, BALF, lungs and spleen were collected after 5 weeks of exposure to either PM2.5 (PM and LPS + PM group) or filtered air (control and LPS5w groups). Inflammatory cells and cytokines were assessed in the blood, BALF, lungs and spleen. Stereological analyses and remodeling assessments were performed by histology. The LPS + PM group showed increased BALF leukocytes, characterized by increased macrophages, increased IL-1ß and IL-6 levels, anemia and thrombocytopenia. Moreover, we also observed septal thickening, decreased alveolar air space total volume and, septa surface density. Finally, regarding tissue remodeling, we observed elastosis of the lung parenchyma, and unlike in the LPS5w group, we did not observe fibrosis in the LPS + PM group. In conclusion, the delayed inflammation resolution due to subchronic exposure to PM2.5 could be influenced by low systemic and local lymphocyte counts, which lead to impaired lung injury recovery and tissue remodeling.

Diesel exhaust exposure intensifies inflammatory and structural changes associated with lung aging in mice.

Life expectancy is increasing worldwide. Lung aging is a process marked by changes in multiple morphological, physiological and age-related biomarkers (e.g., sirtuins) and is influenced by external factors, such as air pollution. Hence, the elderly are considered more vulnerable to the air pollution hazards. We hypothesized that diesel exhaust (DE) exposure intensifies changes in lung inflammatory and structural parameters in aging subjects. Two- and fifteen-month-old mice were exposed to DE for 30 days. Lung function was measured using the forced oscillation method. The inflammatory profile was evaluated in the bronchoalveolar lavage fluid (BALF) and blood, and lung volumes were estimated by stereology. Antioxidant enzyme activity was evaluated by spectrophotometry, sirtuin 1 (SIRT1), sirtuin 2 (SIRT2) and sirtuin 6 (SIRT6) expression was assessed by reverse transcription polymerase chain reaction (RT-PCR), and levels of the sirtuin proteins were evaluated by immunohistochemical staining in lung tissues. Older mice presented decreased pulmonary resistance and elastance, increased macrophage infiltration and decreased tumor necrosis factor (TNF) and interleukin 10 (IL-10) levels in the BALF, reduced activities of the antioxidant enzymes glutathione peroxidase (GPx) and glutathione reductase (GR), and increased activity glutathione S-transferase (GST); increased lung volumes with decreased elastic fiber and increased airway collagen content. SIRT1 gene expression was decreased in older animals, but protein levels were increased. DE exposure increased macrophage infiltration and oxidative stress in the lungs of animals of both ages. SIRT6 gene expression was decreased by DE exposure, with increased protein levels. In older animals, DE affected lung structure and collagen content. Lung aging features, such as decreased antioxidant reserves, lower IL-10 expression, and decreased SIRT1 levels may predispose subjects to exacerbated responses after DE exposure. Our data support the hypothesis that strategies designed to reduce ambient air pollution are an important step towards healthy aging.

Pre- and postnatal exposure of mice to concentrated urban PM2.5 decreases the number of alveoli and leads to altered lung function at an early stage of life.

Gestational exposure to air pollution is associated with negative outcomes in newborns and children. In a previous study, we demonstrated a synergistic negative effect of pre- and postnatal exposure to PM2.5 on lung development in mice. However, the means by which air pollution affects development of the lung have not yet been identified. In this study, we exposed pregnant BALB/c mice and their offspring to concentrated urban PM2.5 (from São Paulo, Brazil; target dose 600 µg/m3 for 1 h daily). Exposure was started on embryonic day 5.5 (E5.5, time of placental implantation). Lung tissue of fetuses and offspring was submitted to stereological and transcriptomic analyses at E14.5 (pseudoglandular stage of lung development), E18.5 (saccular stage) and P40 (postnatal day 40, alveolarized lung). Additionally, lung function and cellularity of bronchoalveolar lavage (BAL) fluid were studied in offspring animals at P40. Compared to control animals that were exposed to filtered air throughout gestation and postnatal life, PM-exposed mice exhibited higher lung elastance and a lower alveolar number at P40 whilst the total lung volume and cellularity of BAL fluid were not affected. Glandular and saccular structures of fetal lungs were not altered upon gestational exposure; transcriptomic signatures, however, showed changes related to DNA damage and its regulation, inflammation and regulation of cell proliferation. A differential expression was validated at E14.5 for the candidates Sox8, Angptl4 and Gas1. Our data substantiate the in utero biomolecular effect of gestational exposure to air pollution and provide first-time stereological evidence that pre- and early life-postnatal exposure compromise lung development, leading to a reduced number of alveoli and an impairment of lung function in the adult mouse.

Early and late pulmonary effects of nebulized LPS in mice: An acute lung injury model.

BACKGROUND AND OBJECTIVE: Acute respiratory distress syndrome (ARDS) has a high mortality rate of 35-46% depending on its severity. Animal models are crucial to better understand the pathophysiology of diseases, including ARDS. This study presents a feasible animal model of acute lung injury (ALI) using nebulized lipopolysaccharide (LPS) in a non-invasive approach, focusing on its short and long-term effects. METHODS: Mice received nebulized LPS or vehicle only (control group). Blood, BALF and lung tissue were collected 24 hours (LPS 24h) or 5 weeks (LPS 5w) after the nebulized LPS-induced lung injury. Inflammatory cytokines were assessed in the blood serum, BALF and lung tissue. Stereological analyses and remodeling changes were assessed by histology and immunohistochemistry at the specified time points. RESULTS: The LPS 24h group showed increased pro-inflammatory cytokine levels, intense cell influx, increased total septal volume, septal thickening and decreased surface density of the alveolar septa. The LPS 5w group showed persistent lung inflammation, septal thickening, increased total lung volume, accentuated collagen deposition, especially of collagen type I, and decreased MMP-2 protein expression. CONCLUSION: We present a feasible, reproducible and non-invasive nebulized-LPS animal model that allows the assessment of both the acute and late phases of acute lung injury. The presence of lung remodeling with collagen deposition after 5 weeks makes it useful to study the pathophysiology, complications, and possible therapeutic intervention studies that aim to understand and reduce pulmonary fibrosis in the late phases of ALI.