Bronchoalveolar Lavage Fluid Total Cell Research Articles

Guggulsterone protects against cigarette smoke-induced COPD linked lung inflammation.

Recently, we have shown that guggulsterone is the principal constituent responsible for protective effects of Commiphora wightii against elastase-induced chronic obstructive pulmonary disease (COPD)-linked inflammation/emphysema. Given that cigarette smoke (CS) exposure is a primary risk factor for COPD and beneficial effects of guggulsterone have not been investigated in CS-induced COPD-linked lung inflammation. The present work was designed to validate the potential of guggulsterone in amelioration of COPD-linked lung inflammation by using a CS-based mouse model of the condition. Male BALB/c mice were exposed to 9 cigarettes/day with 1 h interval for 4 days daily. Guggulsterone was administered daily at a dose of 10 mg/kg orally for 4 consecutive days, 1 h before initiation of CS exposure. Mice were subjected to measurement of lung function followed by procurement of bronchoalveolar lavage fluid (BALF)/lung tissue. BALF was analyzed for inflammatory cells and pro-inflammatory cytokines. Lung tissue was subjected to RT-PCR for gene expression analysis. Data showed that CS exposure resulted in a significant increase in total BALF cells, predominantly neutrophils, and macrophages. Interestingly, guggulsterone administration significantly blunted CS-induced inflammation as reflected by reduced neutrophil and macrophage count. Further, the compound inhibited CS-induced gene expression of pro-inflammatory mediators TNF-α/ IL-1β/ G-CSF/and KC in lungs along with the production of pro-inflammatory mediators TNF-α/ IL-1β/ IL-6/ G-CSF/ KC/and MCP-1 in BALF. Further, guggulsterone improved the lung function parameters upon CS exposure. Analysis of mRNA expression of matrix metalloproteinase (MMP)-9 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 suggests that guggulsterone may restore the fine balance between matrix-degrading proteases and its inhibitor in lung tissue upon CS exposure, which may contribute in the development of emphysema at later stages. Overall, our data show that guggulsterone protects against CS-induced COPD-linked lung inflammation by modulating relevant molecular players. Based on the potential effects of guggulsterone in the amelioration of CS-induced lung inflammation, we speculate that guggulsterone might alter chronic CS-induced emphysema.

Mesenchymal Stem Cell-Derived Extracellular Vesicle Therapy for Asthma in Murine Models: A Systematic Review and Meta-analysis.

Asthma is a common disease, and among the most predominant causes of the years lived with disability. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have emerged as a promising avenue for asthma management. The objective of this study is to perform a systematic review and meta-analysis of pre-clinical studies investigating the therapeutic use of MSC-EVs in murine models of asthma. A systematic search of electronic databases was performed. Meta-analyses were conducted on broncho-alveolar lavage fluid (BALF) cells and cytokines, as well as airway hyper-responsiveness Penh values and histological staining scores to determine the efficacy of MSC-EVs-based therapy, comparing treated rodents with untreated ones. BALF IL-4, BALF total cells, and BALF eosinophils were chosen as the primary outcomes, while airway hyper-responsiveness Penh values, BALF cytokines excluding IL-4, and histological staining scores were chosen as secondary outcomes. A total of 19 eligible studies were included in the current systematic review, with 9 assessing BALF IL-4, 11 assessing BALF total cells, and 10 assessing BALF eosinophils. Pooled Hedges' g(p-value) for each outcome was - 4.407 (< 0.001), -4.976 (< 0.001), and - 4.071 (< 0.001), showing that MSC-EVs therapy inhibits asthma pathology. Changes in secondary outcomes also indicated a reduction in inflammation, goblet cell hyperplasia, and airway hyper-responsiveness. Subgroup analyses did not reveal significant disparities between the type of rodents and administration routes, and meta-regressions were only significant for MSC-EVs source and dose in the IL-4 meta-analysis, and for administration frequency and time from the last challenge to sacrifice in the BALF total cell meta-analysis. This review highlights the current pre-clinical evidence of MSC-EVs therapy for asthma and finds its application ameliorates multiple aspects of asthma's pathology. We further underline the importance of MSC-EVs source, dose, administration frequency, and timing on the therapeutic effect and warrant further investigation and clinical translation to assess the best treatment regimen and to gauge the efficacy of EV therapy in human asthma cases.

Urolithin A exerts a protective effect on lipopolysaccharide-induced acute lung injury by regulating HMGB1-mediated MAPK and NF-κB signaling pathways.

Acute lung injury (ALI) is a severe inflammatory disorder that has a high morbidity and mortality rate. Urolithin A (UA) is reported to have anti-inflammatory and anti-oxidative effects in ALI. However, its molecular mechanisms in ALI remain to be explored. Mice and BEAS-2B cells were administrated with lipopolysaccharide (LPS) to mimic the ALI model in vivo and in vitro. Hematoxylin-eosin (HE) staining was used to detect the pathological injury of lung tissues. The levels of proinflammatory cytokines in bronchoalveolar lavage fluid (BALF) and culture supernatant and the levels of oxidative stress markers in lung tissues were measured using ELISA. DCFH-DA probe was used to assess the reactive oxygen species (ROS) level. TUNEL staining and flow cytometry were performed to determine cell apoptosis. The key targets and pathways were confirmed by immunohistochemistry (IHC) and western blot. UA suppressed the pathologic damage, wet/dry weight ratio, and total protein and inflammatory cells in BALF. UA decreased neutrophil infiltration and proinflammatory cytokines production. UA reduced the level of malondialdehyde (MDA) and increased the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in pulmonary tissues. UA also inhibited cell apoptosis in lung tissues by decreasing Bax expression and increasing Bcl-2 expression. In addition, UA suppressed LPS-induced inflammatory factor production, ROS level, and cell apoptosis in BEAS-2B. Importantly, UA decreased the expression of HMGB1 in LPS-treated mice and BEAS-2B cells. HMGB1 overexpression greatly abrogated the inhibition of UA on inflammation, ROS, and cell apoptosis in LPS-administrated BEAS-2B. Furthermore, UA treatment suppressed the phosphorylated levels of p38, JNK, ERK, and p65 in LPS-administrated mice and BEAS-2B cells. UA alleviated lung inflammation, oxidative stress, and apoptosis in ALI by targeting HMGB1 to inactivate the MAPK/NF-κB signaling, suggesting the potential of UA to treat ALI.

Kaempferitrin alleviates LPS-induced septic acute lung injury in mice through downregulating NF-κB pathway.

Acute lung injury (ALI) causes severe and uncontrolled pulmonary inflammation and has high morbidity in dying patients. This study aimed to evaluate the potential function of Kaempferitrin (Kae) and uncover its mechanisms in ALI. We evaluated the role of Kae in ALI through the lipopolysaccharide (LPS)-induced histopathological changes, lung wet/dry (W/D) ratio, total bronchoalveolar lavage fluid (BALF) cells count, pulmonary inflammation, and the levels of interleukin (IL)-6, tumor necrosis factor-α (TNF-α), and IL-1β. The effect of Kae on NF-κB signaling pathway was discovered through the protein expression levels of transcription factors p65, p-p65, IκBα, and p-IκBα by Western blot analysis. The results showed that Kae could improve lung injury by reducing apoptosis, histopathological changes, and lung W/D ratio; more importantly, Kae enhanced the survival of ALI mice. Moreover, Kae relieved inflammation, as it reduced total BALF cells count, and deceased the levels of TNF-α, IL-6, and IL-1β in serum. In addition, Western blot analysis data suggested that Kae could decrease the protein expression levels of transcription factors p65, p-p65, IκB-α, and p-IκB-α, which were promoted by LPS. The results of this study suggested that Kae could relieve LPS-induced ALI in mice and reduce inflammation and apoptosis through NF-κB pathway.

Effects of caspase recruitment domain protein 9 gene knockout on airway inflammation in C57BL/6 mice with steroid resistant asthma

Objective: To investigate the role of caspase recruitment domain protein 9 (CARD9) in airway injury and inflammation of steroid resistant asthma in C57BL/6 mice. Methods: C57BL/6 mice were divided into A group (control group), B group (model group) and C group (dexamethasone treatment group), with 6 mouse in each group using random number table. The mouse asthma model was established in B and C group by subcutaneous injection of ovalbumin (OVA)/complete Freund adjuvant (CFA) in the abdomen and OVA aerosol challenge, the pathological change and cell count in broncho alveolar lavage fluid (BALF) were detected in order to confirm the model as steroid resistant asthma, and the lung tissue inflammatory infiltration was scored. Western blot was used to detect the changes of CARD9 protein between the group A and B; then wild-type and CARD9 knockout mice were divided into D group (wild-type control group), E group (wild-type model group), F group (CARD9 knockout control group) and G group (CARD9 knockout model group), the following indicators were observed and compared after establishing steroid resistant asthma model separately: HE staining was used to observe the pathological changes of lung tissue, ELISA was used to detect the protein levels of interleukin-4 (IL-4), interleukin-5 (IL-5) and interleukin-17(IL-17) in BALF, and RT-PCR was used to detect the mRNA levels of CXC motif chemokine ligand-10 (CXCL-10) and IL-17 in lung. Results: The inflammatory score (3.33±0.82 vs 0.67±0.52) and BALF total cell count [(10.13±4.83) ×105/ml vs (3.76±0.84) ×105/ml] in B group were higher than those in the A group with statistical significance (P<0.05). There was no significant difference between group C and group B in inflammatory infiltration score (2.83±0.75 vs 3.33±0.82) and BALF total cell count [(9.80±3.19) ×105/ml vs (10.13±4.83) ×105/ml] (P>0.05). Moreover the protein level of CARD9 was increased in the B group than A group (0.245±0.090 vs 0.047±0.014, P=0.004). Compared to E group and F group, more obviously inflammatory cells, neutrophils, eosinophils infiltration and tissue injury were observed in G group (P<0.05), so did the expression of IL-4 (P<0.05), IL-5 and IL-17. Meanwhile the mRNA expression levels of IL-17 and CXCL-10 also increased in lung tissue (P<0.05) of G group. Conclusion: CARD9 gene deletion may aggravate the steroid resistant of asthma by increasing neutrophil chemokines, such as IL-17 and CXCL-10, therefore increasing infiltration of neutrophils in C57BL/6 mice asthma model.

Allergen-specific immunotherapy with Alutard SQ improves allergic inflammation in house-dust mites-induced allergic asthma rats through inactivation of the HMGB1/TLR4/NF-κB pathway.

Allergen-specific immunotherapy (AIT) is the only available safe, effective, and long-term treatment for allergic airway diseases, including allergic asthma. However, the potential molecular mechanism of AIT in ameliorating airway inflammation remains unknown. Rats were sensitized and challenged with house dust mite (HDM) and administered with Alutard SQ or/and high mobility group box 1 (HMGB1) inhibitor, ammonium glycyrrhizinate (AMGZ) or HMGB1 lentivirus. The total and differential cell counts in rat bronchoalveolar lavage fluid (BALF) were detected. Hematoxylin and eosin staining (H&E) was performed to examine the pathological lesions in lung tissues. Enzyme-linked immunosorbent assay (ELISA) was performed to assess the expression of inflammatory factors in lungs, BALF, and serum. Quantitative real-time PCR (qRT-PCR) was used to measure the levels of inflammatory factors in the lungs. Western blot assay was used to evaluate the expression of HMGB1, Τoll-like receptor 4 (TLR4), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the lungs. Consequently, AIT with Alutard SQ attenuated airway inflammation, the total and differential cells in BALF, and expression of Th (T helper)2 related cytokines and transforming growth factor beta 1 (TGF-β1). The regimen also upregulated Th-1-related cytokine expression by inhibiting the HMGB1/TLR4/NF-κB pathway in HDM-induced asthmatic rats. Furthermore, AMGZ, a HMGB1 antagonist, amplified the functions of AIT with Alutard SQ in the asthma rat model. Nevertheless, overexpression of HMGB1 reversed the functions of AIT with Alutard SQ in the asthma rat model. In summary, this work demonstrates the role of AIT with Alutard SQ, which inhibits the HMGB1/TLR4/NF-κB signaling pathway in allergic asthma management.

Quantitative Transcriptome Analysis of Purified Equine Mast Cells Identifies a Dominant Mucosal Mast Cell Population with Possible Inflammatory Functions in Airways of Asthmatic Horses.

Asthma is a chronic inflammatory airway disease and a serious health problem in horses as well as in humans. In humans and mice, mast cells (MCs) are known to be directly involved in asthma pathology and subtypes of MCs accumulate in different lung and airway compartments. The role and phenotype of MCs in equine asthma has not been well documented, although an accumulation of MCs in bronchoalveolar lavage fluid (BALF) is frequently seen. To characterize the phenotype of airway MCs in equine asthma we here developed a protocol, based on MACS Tyto sorting, resulting in the isolation of 92.9% pure MCs from horse BALF. We then used quantitative transcriptome analyses to determine the gene expression profile of the purified MCs compared with total BALF cells. We found that the MCs exhibited a protease profile typical for the classical mucosal MC subtype, as demonstrated by the expression of tryptase (TPSB2) alone, with no expression of chymase (CMA1) or carboxypeptidase A3 (CPA3). Moreover, the expression of genes involved in antigen presentation and complement activation strongly implicates an inflammatory role for these MCs. This study provides a first insight into the phenotype of equine MCs in BALF and their potential role in the airways of asthmatic horses.

Role of TRPA1/TRPV1 in acute ozone exposure induced murine model of airway inflammation and bronchial hyperresponsiveness.

BackgroundTransient receptor potential (TRP) ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) mediate the development of lung injury and inflammation. This study investigated the role and mechanism of the TRPA1/TRPV1 pathway in airway inflammation and bronchial hyperresponsiveness (BHR) induced by acute ozone exposure.MethodsC57BL/6 mice (8–10 weeks) were intraperitoneally injected with phosphate buffered saline (PBS), A967079 (TRPA1 inhibitor) or AMG9810 (TRPV1 inhibitor) 1 h before or after ozone exposure (2.5 ppm, 3 h). BHR, cell counts in bronchoalveolar lavage (BAL) fluid, oxidative stress biomarkers, inflammatory cytokines, TRPA1 and TPRV1 protein levels, mitochondrial dynamics- and mitophagy-related protein levels, and activities of mitochondrial respiratory chain (MRC) in lung were measured.ResultsThe preventive treatment effect was similar to the therapeutic treatment effect. Both A967079 and AMG9810 intervention suppressed BHR, inflammatory cytokines, total BAL fluid cells, malondialdehyde (MDA) levels and inflammatory cytokines mRNA including Substance P (SP), Keratinocyte-Derived Chemokine (KC), interleukin-18 (IL-18) and chemokine (C-X-C motif) ligand 8 (CXCL8) expression, and enhanced reduced glutathione (GSH)/oxidized glutathione (GSSG) levels compared with ozone-exposed mice. A967079 and AMG9810 intervention inhibited dynamin-related protein (DRP1), mitochondrial fission factor (MFF), Parkinson protein 2 E3 ubiquitin protein ligase (PARK2) and Sequestosome 1 (SQSTM1)/p62 expression, increased Optic atrophy 1 (OPA1), mitofusin 2 (MFN2) and PTEN-induced putative kinase 1 (PINK1) expression, and up-regulated the activities of MRC complex III and V in lung tissue.ConclusionsThe results show that both TRPA1 and TRPV1 pathways are involved in acute ozone exposure-induced airway inflammation and BHR and influence oxidative stress, mitochondrial quality control and MRC activity, which could be a potential target for clinical therapy of respiratory diseases.

Association between quantitative bacterial culture of bronchoalveolar lavage fluid and antibiotic requirement in dogs with lower respiratory tract signs.

BackgroundHistorically, positive bacterial cultures from the lower respiratory tract (LRT) have been considered clinically relevant when quantitative bacterial cultures of bronchoalveolar lavage fluid (BALF) were >1700 colony forming units (cfu)/mL. However, this threshold might not accurately predict a requirement for antibiotics.ObjectivesTo study whether quantitative BALF bacterial culture results were predictive of antibiotic requirement in dogs with LRT signs.AnimalsThirty‐three client‐owned dogs.MethodsCross‐sectional study. Dogs with positive quantitative bacterial culture of BALF were included. Dogs were divided into 2 groups, depending on whether they had a LRT infection requiring antibiotics (LRTI‐RA) or LRT disease not requiring antibiotics (LRTD‐NRA), based on thoracic imaging features, presence of intracellular bacteria on BALF cytology, and response to treatment. Predictive effect of cfu/mL and BALF total nucleated cell count (TNCC) on antibiotic requirement, adjusting for ongoing or prior antibiotic therapy and age, were studied using logistic regression.ResultsTwenty‐two and 11 dogs were included in the LRTI‐RA and LRTD‐NRA groups, respectively. The cfu/mL was not significantly predictive of antibiotic requirement, independent of ongoing or prior antibiotic treatment and age (LRTI‐RA: median, 10 000 cfu/mL; range, 10‐3 × 108; LRTD‐NRA: median, 10 000 cfu/mL; range, 250‐1.3 × 109; P = .27). The TNCC was not significantly predictive of antibiotic requirement when only dogs with bronchial disease were considered (LRTI‐RA: median, 470 cells/μL; range, 240‐2260; LRTD‐NRA: median, 455 cells/μL; range, 80‐4990; P = .57).Conclusion and Clinical ImportanceThe cfu/mL is an inappropriate measure for determining whether antibiotics are of benefit in dogs with LRT signs.

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.

Salidroside Attenuates Airway Inflammation and Remodeling via the miR-323-3p/SOCS5 Axis in Asthmatic Mice

Introduction: Salidroside (Sal) a bioactive component extracted from Rhodiola rosea is remarkable for its anti-asthmatic effects. The study aimed to explore the molecular mechanism of Sal in airway inflammation and remodeling in asthmatic mice and provide a novel theoretical basis for asthma treatment. Methods: An asthmatic mouse model was established via ovalbumin (OVA) treatment, followed by injection of Sal and transfection of miR-323-3p-mimic and sh- suppressor of cytokine signaling 5 (SOCS5). Expressions of miR-323-3p, SOCS5 mRNA, collagen (COL)-I, and COL-III were detected via reverse transcription quantitative polymerase chain reaction. SOCS5 protein level was detected via Western blot. Levels of IgE, IL-13, IL-4, and IL-5 were detected via enzyme-linked immunosorbent assay. Inflammatory cell infiltration was observed via hematoxylin-eosin staining. Collagen disposition was observed via Masson staining. Resistance index (RI) of airway hyperresponsiveness, and the number of total cells, inflammatory cells (eosinophil, macrophage, neutrophil, and lymphocyte) in bronchoalveolar lavage fluid (BALF) were observed. The binding relationship between miR-323-3p and SOCS5 was predicted through the RNA22 website and verified via dual-luciferase reporter assay. Results: miR-323-3p was highly expressed in OVA-treated mice. Sal treatment reduced inflammatory cell infiltration, COL disposition, miR-323-3p expression, and IgE, IL-13, IL-4, IL-5, COL-I, and COL-III levels, RI value, and the number of total cells and inflammatory cells in BALF. miR-323-3p inhibited SOCS5 transcription. miR-323-3p overexpression or SOCS5 downregulation reversed the protecting role of Sal in asthmatic mice. Conclusion: Sal inhibited miR-323-3p expression to promote SOCS5 transcription, thereby attenuating airway inflammation and remodeling in asthmatic mice.

Enhancing Extracellular Adenosine Levels Restores Barrier Function in Acute Lung Injury Through Expression of Focal Adhesion Proteins.

Background: Acute respiratory distress syndrome (ARDS) is a clinical presentation of acute lung injury (ALI) with often fatal lung complication. Adenosine, a nucleoside generated following cellular stress provides protective effects in acute injury. The levels of extracellular adenosine can be depleted by equilibrative nucleoside transporters (ENTs). ENT inhibition by pharmaceutical agent dipyridamole promotes extracellular adenosine accumulation and is protective in ARDS. However, the therapeutic potential of dipyridamole in acute lung injury has not yet been evaluated. Methods: Adenosine acts on three adenosine receptors, the adenosine A1 (Adora1), A2a (Adora2a), the A2b (Adora2b) or the adenosine A3 (Adora 3) receptor. Accumulation of adenosine is usually required to stimulate the low-affinity Adora2b receptor. In order to investigate the effect of adenosine accumulation and the contribution of epithelial-specific ENT2 or adora2b expression in experimental ALI, dipyridamole, and epithelial specific ENT2 or Adora2b deficient mice were utilized. MLE12 cells were used to probe downstream Adora2b signaling. Adenosine receptors, transporters, and targets were determined in ARDS lungs. Results: ENT2 is mainly expressed in alveolar epithelial cells and is negatively regulated by hypoxia following tissue injury. Enhancing adenosine levels with ENT1/ENT2 inhibitor dipyridamole at a time when bleomycin-induced ALI was present, reduced further injury. Mice pretreated with the ADORA2B agonist BAY 60-6583 were protected from bleomycin-induced ALI by reducing vascular leakage (558.6 ± 50.4 vs. 379.9 ± 70.4, p < 0.05), total bronchoalveolar lavage fluid cell numbers (17.9 ± 1.8 to 13.4 ± 1.4 e4, p < 0.05), and neutrophil infiltration (6.42 ± 0.25 vs. 3.94 ± 0.29, p < 0.05). While mice lacking Adora2b in AECs were no longer protected by dipyridamole. We also identified occludin and focal adhesion kinase as downstream targets of ADORA2B, thus providing a novel mechanism for adenosine-mediated barrier protection. Similarly, we also observed similar enhanced ADORA2B (3.33 ± 0.67 to 16.12 ± 5.89, p < 0.05) and decreased occludin (81.2 ± 0.3 to 13.3 ± 0.4, p < 0.05) levels in human Acute respiratory distress syndrome lungs. Conclusion: We have highlighted a role of dipyridamole and adenosine signaling in preventing or treating ALI and identified Ent2 and Adora2b as key mediators in important for the resolution of ALI.

Hypoxic hUCMSC-derived extracellular vesicles attenuate allergic airway inflammation and airway remodeling in chronic asthma mice

BackgroundAs one of the main functional forms of mesenchymal stem cells (MSCs), MSC-derived extracellular vesicles (MSC-EVs) have shown an alternative therapeutic option in experimental models of allergic asthma. Oxygen concentration plays an important role in the self-renewal, proliferation, and EV release of MSCs and a recent study found that the anti-asthma effect of MSCs was enhanced by culture in hypoxic conditions. However, the potential of hypoxic MSC-derived EVs (Hypo-EVs) in asthma is still unknown.MethodsBALB/c female mice were sensitized and challenged with ovalbumin (OVA), and each group received PBS, normoxic human umbilical cord MSC-EVs (Nor-EVs), or Hypo-EVs weekly. After treatment, the animals were euthanized, and their lungs and bronchoalveolar lavage fluid (BALF) were collected. With the use of hematoxylin and eosin (HE), periodic acid-Schiff (PAS) and Masson’s trichrome staining, enzyme-linked immune sorbent assay (ELISA), Western blot analysis, and real-time PCR, the inflammation and collagen fiber content of airways and lung parenchyma were investigated.ResultsHypoxic environment can promote human umbilical cord MSCs (hUCMSCs) to release more EVs. In OVA animals, the administration of Nor-EVs or Hypo-EVs significantly ameliorated the BALF total cells, eosinophils, and pro-inflammatory mediators (IL-4 and IL-13) in asthmatic mice. Moreover, Hypo-EVs were generally more potent than Nor-EVs in suppressing airway inflammation in asthmatic mice. Compared with Nor-EVs, Hypo-EVs further prevented mouse chronic allergic airway remodeling, concomitant with the decreased expression of pro-fibrogenic markers α-smooth muscle actin (α-SMA), collagen-1, and TGF-β1-p-smad2/3 signaling pathway. In vitro, Hypo-EVs decreased the expression of p-smad2/3, α-SMA, and collagen-1 in HLF-1 cells (human lung fibroblasts) stimulated by TGF-β1. In addition, we showed that miR-146a-5p was enriched in Hypo-EVs compared with that in Nor-EVs, and Hypo-EV administration unregulated the miR-146a-5p expression both in asthma mice lung tissues and in TGF-β1-treated HLF-1. More importantly, decreased miR-146a-5p expression in Hypo-EVs impaired Hypo-EV-mediated lung protection in OVA mice.ConclusionOur findings provided the first evidence that hypoxic hUCMSC-derived EVs attenuated allergic airway inflammation and airway remodeling in chronic asthma mice, potentially creating new avenues for the treatment of asthma.

The Effect of Bronchoconstriction by Methacholine Inhalation in a Murine Model of Asthma

Introduction: Bronchoconstriction was recently shown to cause airway remodeling and induce allergic airway inflammation in asthma. However, the mechanisms how mechanical stress via bronchoconstriction could induce airway inflammation and remodeling remain unclear. Objective: We investigated the effect of bronchoconstriction induced by methacholine inhalation in a murine model of asthma. Methods: BALB/c female mice were sensitized and challenged with ovalbumin (OVA), followed by treatment with methacholine by a nebulizer twice a day for 7 days. Twenty-four hours after the last methacholine treatment, the bronchoalveolar lavage fluid (BALF) and lung tissues were collected. The BALF was analyzed for total and differential cell counts and cytokine levels. The lung tissues were analyzed for goblet cell metaplasia, thickness of the smooth muscle, and lung fibrosis. The expression of cytokines in the lung was also examined. Results: OVA sensitization and challenge induced infiltration of total cells, macrophages, and eosinophils in the BALF along with goblet cell metaplasia and increased airway smooth muscle hypertrophy. Seven days after the last OVA challenge, untreated mice achieved reduction in airway inflammation, while methacholine maintained the number of BALF total cells, macrophages, and eosinophils. The percentage of goblet cells and the thickness of airway smooth muscle were also maintained by methacholine. Moreover, the treatment of methacholine induced the expression of transforming growth factor (TGF)-β in the lung. This result indicates that the production of TGF-β is involved in induction of airway remodeling caused by bronchoconstriction with methacholine. Conclusions: Repeated bronchoconstriction caused by methacholine inhalation elicited allergic airway inflammation and airway remodeling.

Exhaled volatile organic compounds analysis by e-nose can detect idiopathic pulmonary fibrosis

The current diagnostic work-up and monitoring of idiopathic pulmonary fibrosis (IPF) is often invasive and time consuming. Breath analysis by e-nose technology has shown potential in the diagnosis of numerous respiratory diseases. In this pilot study, we investigated whether exhaled breath analysis by an e-nose could discriminate among patients with IPF, healthy controls and COPD. Second, we verified whether these classification could be repeated in a set of newly recruited patients as external validation. Third, we evaluated any significant relationships between exhaled VOCs and Bronchoalveolar lavage fluid (BALF) in IPF patients. We enrolled 32 patients with well-characterized IPF, 33 individuals with COPD and 36 healthy controls. An electronic nose (Cyranose 320) was used to analyze exhaled breath samples. Raw data were processed by Principal component reduction and linear discriminant analysis. External validation in newly recruited patients (10 IPF, 10 COPD and 10 controls) was tested using the previous training set. Exhaled VOC-profiles of patients with IPF were distinct from those of healthy controls (CVA = 98.5%) as well as those with COPD (CVA = 80.0%). External validation confirmed the above findings (IPF vs COPD vs healthy controls, CVA 96.7%). Moreover, a significant inversely proportional correlation was shown between BALF total cell count and both Principal Components 1 and 2 (r = 0.543, r2 = 0.295, p < 0.01; r = 0.501, r2 = 0.251; p < 0.01, respectively). The exhaled breath Volatile Organic Compounds- profile of patients with IPF can be detected by an electronic nose. This suggests that breath analysis has potential for diagnosis and/or monitoring of IPF.

Glyburide attenuates ozone-induced pulmonary inflammation and injury by blocking the NLRP3 inflammasome.

Glyburide is a classic antidiabetic drug that is dominant in inflammation regulation, but its specific role in ozone-induced lung inflammation and injury remains unclear. In order to investigate whether glyburide prevents ozone-induced pulmonary inflammation and its mechanism, C57BL/6 mice were intratracheally pre-instilled with glyburide or the vehicle 1 hour before ozone (1 ppm, 3 hours) or filtered air exposure. After 24 hours, the total inflammatory cells and total protein in bronchoalveolar lavage fluid (BALF) were detected. The pathological alternations in lung tissues were evaluated by HE staining. The expression of NLRP3, interleukin-1β (IL-1β), and IL-18 protein in lung tissues was detected by immunohistochemistry. Western blotting was used to examine the levels of caspase-1 p10 and active IL-1β protein. Levels of IL-1β and IL-18 in BALF were measured using ELISA kits. Glyburide treatment decreased the total cells in BALF, the inflammatory score, and the mean linear intercept induced by ozone in lung tissues. In addition, glyburide inhibited the expression of NLRP3, IL-18, and IL-1β protein in lung tissues, and also suppressed NLRP3 inflammasome activation, including caspase-1 p10, active IL-1β protein in lung tissues, IL-1β, and IL-18 in BALF. These results demonstrate that glyburide effectively attenuates ozone-induced pulmonary inflammation and injury via blocking the NLRP3 inflammasome.

Losartan does not inhibit cigarette smoke-induced lung inflammation in mice

Chronic Obstructive Pulmonary Disease (COPD) is a progressive lung disease largely caused by cigarette smoking (CS) and is characterized by lung inflammation and airflow limitation that is not fully reversible. Approximately 50% of people with COPD die of a cardiovascular comorbidity and current pharmacological strategies provide little benefit. Therefore, drugs that target the lung and the cardiovascular system concurrently may be an advantageous therapeutic strategy. The aim of this study was to see whether losartan, an angiotensin-II AT1a receptor antagonist widely used to treat hypertension associated with cardiovascular disease, protects against CS-induced lung inflammation in mice. Male BALB/c mice were exposed to CS for 8 weeks and treated with either losartan (30 mg/kg) or vehicle daily. Mice were euthanized and bronchoalveolar lavage fluid (BALF) inflammation, and whole lung cytokine, chemokine and protease mRNA expression assessed. CS caused significant increases in BALF total cells, macrophages, neutrophils and whole lung IL-6, TNF-α, CXCL-1, IL-17A and MMP12 mRNA expression compared to sham-exposed mice. However, losartan only reduced CS-induced increases in IL-6 mRNA expression. Angiotensin-II receptor expression was reduced in lung tissue from CS-exposed mice. In conclusion, losartan did not inhibit CS-induced BALF cellularity despite reducing whole lung IL-6 mRNA and Ang-II receptor expression.

Epithelial Expression of an Interstitial Lung Disease-Associated Mutation in Surfactant Protein-C Modulates Recruitment and Activation of Key Myeloid Cell Populations in Mice.

Patients with idiopathic pulmonary fibrosis (IPF) often experience precipitous deteriorations, termed "acute exacerbations" (AE), marked by diffuse alveolitis and altered gas exchange, resulting in a significant loss of lung function or mortality. The missense isoleucine to threonine substitution at position 73 (I73T) in the alveolar type 2 cell-restricted surfactant protein-C (SP-C) gene (SFTPC) has been linked to clinical IPF. To better understand the sequence of events that impact AE-IPF, we leveraged a murine model of inducible SP-CI73T (SP-CI73T/I73TFlp+/- ) expression. Following administration of tamoxifen to 8-12-wk-old mice, an upregulation of SftpcI73T initiated a diffuse lung injury marked by increases in bronchoalveolar lavage fluid (BALF) protein and histochemical evidence of CD45+ and CD11b+ cell infiltrates. Flow cytometry of collagenase-digested lung cells revealed a transient, early reduction in SiglecFhiCD11blowCD64hiCD11chi macrophages, countered by the sequential accumulation of SiglecFloCD11b+CD64-CD11c-CCR2+Ly6C+ immature macrophages (3 d), Ly6G+ neutrophils (7 d), and SiglecFhiCD11bhiCD11clo eosinophils (2 wk). By mRNA analysis, BALF cells demonstrated a time-dependent phenotypic shift from a proinflammatory (3 d) to an anti-inflammatory/profibrotic activation state, along with serial elaboration of monocyte and eosinophil recruitment factors. The i.v. administration of clodronate effectively reduced total BALF cell numbers, CCR2+ immature macrophages, and eosinophil influx while improving survival. In contrast, resident macrophage depletion from the intratracheal delivery of clodronate liposomes enhanced SftpcI73T -induced mortality. These results using SftpcI73T mice provide a detailed ontogeny for AE-IPF driven by alveolar epithelial dysfunction that induces a polycellular inflammation initiated by the early influx of proinflammatory CCR2+Ly6Chi immature macrophages.

Human albumin augmented airway inflammation induced by PM2.5 in NC/Nga mice.

The synergic allergic inflammatory effects of particulate matter (PM) 2.5 and human albumin were investigated in NC/Nga mice, which are hypersensitive to mite allergens. PM2.5 or PM2.5 plus human albumin with aluminum oxide was injected twice intraperitoneally for sensitization. After 7 days, PM2.5 or PM2.5 plus human albumin was administered five times intranasally to mice for further sensitization. Subsequently, PM2.5 was administered as a challenge on the 11th day. On the 12th day, mice were examined for airway hyperresponsiveness (AHR), bronchoalveolar lavage fluid (BALF) cell count, mRNA expression of Th1 , Th2 cytokines, chemokines, and mucus proteins (MUC5AC and MUC5B) in the lung tissue and histopathology. Although PM2.5 or human albumin alone did not induce allergic airway inflammation, simultaneous inoculation of PM2.5 and human albumin-induced airway inflammation showing increase in AHR, total BALF cell numbers, mRNA levels of IL-13, eotaxin 1, eotaxin 2, and MUC5AC, and anti-IG against human serum albumin. Inflammation was observed around the bronchus in PM2.5 plus human albumin-induced lungs. These results demonstrate that PM2.5 can induce allergic airway inflammation through the synergistic action with human albumin in NC/Nga mice.

Prior cigarette smoke exposure does not affect acute post-stroke outcomes in mice.

Chronic obstructive pulmonary disease (COPD) is currently the third leading cause of death globally and is characterized by airflow limitation that is progressive and not fully reversible. Cigarette smoking is the major cause of COPD. Fifty percent of deaths in the COPD population are due to a cardiovascular event and it is now recognised that COPD is a risk factor for stroke. Whether COPD increases stroke severity has not been explored. The aim of this study was to investigate whether functional and histological endpoints of stroke outcomes in mice after transient middle cerebral artery occlusion (tMCAo) were more severe in mice exposed to cigarette smoke (CS). 7-week-old male C57BL/6 mice were exposed to room air or CS generated from 9 cigarettes/day, 5 days/week for 2, 8 and 12 weeks. Following air or CS exposure, mice underwent tMCAO surgery with an ischaemic period of 30–40 min or sham surgery. Mice were euthanised 24 h following the induction of ischaemia and bronchoalveolar lavage fluid (BALF), lungs and brains collected. Mice exposed to CS for 2 weeks and subjected to a stroke had similar BALF macrophages to air-exposed and stroke mice. However, CS plus stroke mice had significantly more BALF total cells, neutrophils and lymphocytes than air plus stroke mice. Mice exposed to CS for 8 and 12 weeks had significantly greater BALF total cells, macrophages, neutrophils and lymphocytes than air-exposed mice, but stroke did not affect CS-induced BALF cellularity. Prior CS exposure did not worsen stroke-induced neurological deficit scores, reduced foregrip strength, infarct and oedema volumes. Collectively, we found that although CS exposure caused significant BALF inflammation, it did not worsen acute post-stroke outcomes in mice. This data suggests that while patients with COPD are at increased risk of stroke, it may not translate to COPD patients having more severe stroke outcomes.