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.

High neutrophils and low lymphocytes percentages in bronchoalveolar lavage fluid are prognostic factors of higher in-hospital mortality in diffuse alveolar hemorrhage

Background Diffuse alveolar hemorrhage (DAH) is a syndrome resulting from bleeding in the microcirculation of the lung, with a poor prognosis. The study aim was to identify prognostic factors of DAH, especially bronchoalveolar lavage fluids (BALF) cell pattern. Methods We conducted a single-center retrospective cohort study of patients diagnosed as having DAH and hospitalized at our hospital between October 2008 and July 2020. We performed univariate logistic regressions to identify variables associated with in-hospital death. Results Sixty-eight patients were included in our analysis. In-hospital mortality was 26.5%. Variables associated with in-hospital death were neutrophils percentage in BALF ≥ 44.5% [Odds Ratio (OR) 16.0, 95% confidence interval (CI) 4.33–58.9)], lymphocytes percentage in BALF < 14% (OR 7.44, 95% CI 2.11–26.2), idiopathic DAH (OR 0.31, 95% CI 0.10–0.95), oxygen flow ≥ 4L/min (OR 3.90, 95% CI 1.20–12.6), and estimated glomerular filtration rate < 60 mL/min (OR 5.00, 95%CI 1.29–19.4). Conclusions High neutrophils and low lymphocytes percentages in BALF were associated with poor prognosis.

Ghrelin Protects Lipopolysaccharide-Induced Acute Lung Injury Rats against Pulmonary Vascular Dysfunction by Inhibiting Inflammation

Objective. To determine the effect and mechanism of the anti-inflammatory agent ghrelin on pulmonary vascular dysfunction (PVD) in lipopolysaccharide- (LPS-) induced acute lung injury (ALI) rat models. Methods. Thirty-two adult male Sprague Dawley rats (n = 16/group) were randomly divided into ghrelin and saline groups, wherein ghrelin (10 nmol/kg) or saline was subcutaneously administered. After 30 min, eight rats from each group were randomly selected, and LPS (5 mg/kg) or saline was administered by intratracheal instillation to induce ALI. Four hours after establishing the ALI rat model, the mean pulmonary arterial pressure (mPAP), mean right ventricular systolic pressure (RVSP), levels of proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the bronchoalveolar lavage fluid (BALF), BALF cell count, wet-to-dry (W/D) lung weight ratios, and myeloperoxidase (MPO) activity in lung tissue for all four groups (ghrelin, ghrelin + ALI, saline, and saline + ALI) were measured. Immunohistochemical staining to detect alpha-smooth muscle actin (α-SMA) and proliferating cell nuclear antigen (PCNA) expression was performed to assess the intrapulmonary arterial wall thickness and the proliferation of smooth muscle cells, respectively. Results. The ghrelin-pretreated ALI rats showed lower mPAP, RVSP, PCNA expression, MPO activity, W/D lung weight ratio, TNF-α and IL-6 levels, and BALF cell count than the saline-pretreated ALI rats, but ghrelin had no effect on the intrapulmonary arterial wall thickness of ALI rats. Conclusion. Our results confirmed the association between inflammation and PVD in ALI and suggested that the suppression of inflammation by ghrelin pretreatment could protect LPS-induced ALI rats against PVD.

R-spondin 2 mediates neutrophil egress into the alveolar space through increased lung permeability.

Objective: R-spondin 2 (RSPO2) is required for proper lung morphogenesis. Our objective was to investigate whether RSPO2 is similarly important in homeostasis of the adult lung. Unexpectedly, we observed changes in neutrophil migration and lung vascular permeability in RSPO2-deficient (RSPO2-/-) mice compared to RSPO2 control (RSPO2+/+) mice, independent of experimental injury/challenge. Here we use multiple methods to quantify these observations to further understand how tonic RSPO2 expression regulates lung homeostasis. Results: Quantitative PCR (qPCR) analysis demonstrated significantly higher myeloperoxidase (MPO) expression in bronchoalveolar lavage fluid (BALF) cell content from RSPO2-/- mice compared to RSPO2+/+ mice. Immunocytochemical (ICC) analysis likewise demonstrated significantly more MPO+ cells in BALF from RSPO2-/- mice compared to RSPO2+/+ mice, confirming the increase of infiltrated neutrophils. We then assessed lung permeability/barrier disruption via Fluorescein isothiocyanate (FITC)-dextran instillation and found a significantly higher dextran concentration in the plasma of RSPO2-/- mice compared to identically treated RSPO2+/+ mice. These data demonstrate that RSPO2 may be crucial for lung barrier integrity and can facilitate an increase in neutrophil migration from circulation into alveolar spaces due to increased lung permeability/barrier disruption. Our studies suggest additional research is needed to evaluate RSPO2 in scenarios exhibiting either pulmonary edema or neutrophilia.

Active vs. sedentary lifestyle from weaning to adulthood and susceptibility to ozone in rats

The prevalence of a sedentary (SED) life style combined with calorically rich diets has spurred the rise in childhood obesity, which, in turn, translates to adverse health effects in adulthood. Obesity and lack of active (ACT) lifestyle may increase susceptibility to air pollutants. We housed 22-day-old female Long-Evans rats in a cage without (SED) or with a running wheel (ACT). After 10 wk the rats ran 310 ± 16.3 km. Responses of SED and ACT rats to whole-body O3 (0, 0.25, 0.5, or 1.0 ppm; 5 h/day for 2 days) was assessed. Glucose tolerance testing (GTT) was performed following the first day of O3. ACT rats had less body fat and an improved glucose GTT. Ventilatory function (plethysmography) of SED and ACT groups was similarly impaired by O3. Bronchoalveolar lavage fluid (BALF) was collected after the second O3 exposure. SED and ACT rats were hyperglycemic following 1.0 ppm O3. GTT was impaired by O3 in both groups; however, ACT rats exhibited improved recovery to 0.25 and 1.0 ppm O3. BALF cell neutrophils and total cells were similarly increased in ACT and SED groups exposed to 1.0 ppm O3. O3-induced increase in eosinophils was exacerbated in SED rats. Chronic exercise from postweaning to adulthood improved some of the metabolic and pulmonary responses to O3 (GTT and eosinophils) but several other parameters were unaffected. The reduction in O3-induced rise in BALF eosinophils in ACT rats suggests a possible link between a SED lifestyle and incidence of asthma-related symptoms from O3.

NLRP3 inflammasome expression in idiopathic pulmonary fibrosis and rheumatoid lung

In this study we investigated the implication of NLRP3 inflammasomes in the pathogenesis of idiopathic pulmonary fibrosis (IPF) and rheumatoid arthritis–usual interstitial pneumonia (RA-UIP).NLRP3 inflammasome activation at baseline and following stimulation with lipopolysaccharide/ATP was evaluated by measuring interleukin (IL)-1β and IL-18 levels released in the bronchoalveolar lavage fluid (BALF) fluid and by cultures of BALF cells. IL-1β and IL-18 levels were significantly elevated in the BALF and BALF macrophage cultures from RA-UIP patients, consistent with pre-existing inflammasome activation in these patients. In contrast, in IPF, BALF levels of IL-1β were significantly less elevated relative to RA-UIP and IL-18 was lower than controls. Furthermore, upon inflammasome stimulation, IPF BALF macrophage cultures failed to upregulate IL-1β and partly IL-18 secretion, in contrast to controls, which showed robust IL-1β and IL-18 upregulation. Interestingly, RA-UIP BALF cell cultures treated with lipopolysaccharide/ATP showed a potent stimulation of IL-18 secretion but not IL-1β, the latter being already elevated in the unstimulated cultures, while examination of the intracellular IL-1β levels in RA-UIP BALF cells upon NLRP3 inflammasome stimulation showed a significant upregulation of IL-1β suggesting the NLRP3 pathway could be further activated.Taken together, our results suggest distinct inflammasome activation profiles between autoimmune and idiopathic lung fibrosis.

Altered lymphocyte trafficking and diminished airway reactivity in transgenic mice expressing human MMP-9 in a mouse model of asthma

Matrix metalloproteinase-9 (MMP-9) is hypothesized to facilitate leukocyte extravasation and extracellular remodeling in asthmatic airways. Careful descriptive studies have shown that MMP-9 levels are higher in the sputum of asthmatics; however, the consequence of increased MMP-9 activity has not been determined in this disease. We induced asthma in transgenic mice that express human MMP-9 in the murine lung tissue macrophage to determine the direct effect of human MMP-9 expression on airway inflammation. Transgenic (TG) and wild-type (WT) mice were immunized and challenged with ovalbumin. Forty-eight hours after the ovalbumin challenge, airway hyperresponsiveness (AHR) was measured, and inflammatory cell infiltration was evaluated in bronchoalveolar lavage fluid (BALF) and lung tissue. Baseline levels of inflammation were similar in the TG and WT groups of mice, and pulmonary eosinophilia was established in both groups by sensitization and challenge with ovalbumin. There was a significant reduction in AHR in sensitized and challenged trangenics compared with WT controls. Although total BALF cell counts were similar in both groups, the lymphocyte number in the lavage of the TG group was significantly diminished compared with the WT group (0.25 ± 0.08 vs. 0.89 ± 0.53; P = 0.0032). In addition, the draining lymphocytes were found to be larger in the TG animals compared with the WT mice. Equal numbers of macrophages, eosinophils, and neutrophils were seen in both groups. IL-13 levels were found to be lower in the sensitized TG compared with the WT mice. These results demonstrate an inverse relationship between human MMP-9 and AHR and suggest that MMP-9 expression alters leukocyte extravasation by reducing lymphocyte accumulation in the walls of asthmatic airways.

Inflammatory and Haematotoxic Potential of Indoor Stachybotrys chartarum (Ehrenb.) Hughes Metabolites

Inflammatory and Haematotoxic Potential of Indoor Stachybotrys chartarum (Ehrenb.) Hughes MetabolitesMould Stachybotrys chartarum (Ehrenb.) Hughes is known to pose a health risk in indoor environments. Most of its strains can produce several intra- and extracellular trichothecene mycotoxins. Complex secondary metabolites of stachybotrys isolates from mouldy dwellings/public buildings in Slovakia were intratracheally instilled in Wistar male rats (4 μg in 0.2 mL of 0.2 % dimethylsulphoxide; diacetoxyscirpenol as the positive control). After three days, haematological parameters were measured in peripheral blood and inflammatory response biomarkers in bronchoalveolar lavage fluid (BALF), and the results were statistically analysed. Exometabolites proved to suppress red blood cell (RBC), decreasing the total RBC count, haemoglobin, and haematocrit. The exposed rats showed significantly higher total BALF cell count, indicating inflammation, lower alveolar macrophage counts, and increased granulocyte count related to the BALF cells. Due to haematotoxic and inflammation-inducing properties, metabolites of S. chartarum can cause damage to the airways and haematological disorders in occupants of mouldy buildings.

Mucosal injury and eicosanoid kinetics during hyperventilation-induced bronchoconstriction

Bronchoalveolar lavage (BAL) of canine peripheral airways was performed at various times after hyperventilation, and BAL fluid (BALF) cell and mediator data were used to evaluate two hypotheses: 1) hyperventilation-induced mucosal injury stimulates mediator production, and 2) mucosal damage is correlated with the magnitude of hyperventilation-induced bronchoconstriction. We found that epithelial cells increased in BALF immediately after a 2- and a 5-min dry air challenge (DAC). Prostaglandins D2 and F2α and thromboxane B2 were unchanged immediately after a 2-min DAC but were significantly increased after a 5-min DAC. Leukotriene C4, D4, and E4 did not increase until 5 min after DAC. Hyperventilation with warm moist air did not alter BALF cells or mediators and caused less airway obstruction that occurred earlier than DAC. BALF epithelial cells were correlated with mediator release, and mediator release and epithelial cells were correlated with hyperventilation-induced bronchoconstriction. These observations are consistent with the hypothesis that hyperventilation-induced mucosal damage initiates peripheral airway constriction via the release of biochemical mediators.