Macrophages In Bronchoalveolar Lavage Fluid Research Articles

Statin administration or blocking PCSK9 alleviates airway hyperresponsiveness and lung fibrosis in high-fat diet-induced obese mice

BackgroundObesity is associated with airway hyperresponsiveness and lung fibrosis, which may reduce the effectiveness of standard asthma treatment in individuals suffering from both conditions. Statins and proprotein convertase subtilisin/kexin-9 inhibitors not only reduce serum cholesterol, free fatty acids but also diminish renin-angiotensin system activity and exhibit anti-inflammatory effects. These mechanisms may play a role in mitigating lung pathologies associated with obesity.MethodsMale C57BL/6 mice were induced to develop obesity through high-fat diet for 16 weeks. Conditional TGF-β1 transgenic mice were fed a normal diet. These mice were given either atorvastatin or proprotein convertase subtilisin/kexin-9 inhibitor (alirocumab), and the impact on airway hyperresponsiveness and lung pathologies was assessed.ResultsHigh-fat diet-induced obesity enhanced airway hyperresponsiveness, lung fibrosis, macrophages in bronchoalveolar lavage fluid, and pro-inflammatory mediators in the lung. These lipid-lowering agents attenuated airway hyperresponsiveness, macrophages in BALF, lung fibrosis, serum leptin, free fatty acids, TGF-β1, IL-1β, IL-6, and IL-17a in the lung. Furthermore, the increased RAS, NLRP3 inflammasome, and cholecystokinin in lung tissue of obese mice were reduced with statin or alirocumab. These agents also suppressed the pro-inflammatory immune responses and lung fibrosis in TGF-β1 over-expressed transgenic mice with normal diet.ConclusionsLipid-lowering treatment has the potential to alleviate obesity-induced airway hyperresponsiveness and lung fibrosis by inhibiting the NLRP3 inflammasome, RAS and cholecystokinin activity.Graphical abstract

Aprepitant Alleviates Poststroke Pneumonia in a Mouse Model of Middle Cerebral Artery Occlusion.

Elevated substance P can be utilized to predict early mortality during the first week of cerebral infarction. Whether aprepitant, a substance P receptor blocker could be utilized to alleviate poststroke pneumonia which is investigated in this study. Intraluminal monofilament model of middle cerebral artery occlusion (MCAO) was constructed in C57BL/6J male mice, and the relative expression of substance P was detected in collected bronchoalveolar lavage fluid (BALF) and lung tissue homogenate at 24 hours, 48 hours, and 72 hours poststroke. On the other hand, different concentrations of aprepitant (0.5, 1, and 2 mg/kg) were atomized and inhaled into MCAO mice. Inflammation cytokines and bacterial load were detected in collected BALF and lung tissue homogenate at 72-hour poststroke, and lung injury was revealed by histological examination. Aprepitant administration decreased total proteins, total cells, neutrophils, and macrophages in BALF. The concentrations of interleukin (IL)-6, IL-1β, tumor necrosis factor-α, interferon γ, monocyte chemoattractant protein-1, and IL-10 in lung tissue homogenates were also diminished by the administration of aprepitant. In conclusion, aprepitant could attenuate poststroke pneumonia in mice suggesting its potential therapeutic use in the clinic.

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.

Changes in Cellular Morphology in Bronchoalveolar Lavage Fluid of Children with Mycoplasma Pneumoniae Pneumonia

Objective To study changes of cell morphology in BALF in children with Mycoplasma pneumoniae pneumonia (MPP). Methods From December 2021 to May 2022, a group of 32 children diagnosed with Acute MPP and admitted for treatment in the Pediatrics Department and PICU of the First Affiliated Hospital of Xinxiang Medical University were selected for our study. These patients underwent bronchoalveolar lavage as part of their clinical assessment. For comparison, we included a control group comprising 10 children who were not infected but had bronchial foreign bodies. We investigated the cellular composition in the bronchoalveolar lavage fluid (BALF) using Wright-Giemsa staining and microscopic evaluation, aiming to understand the relationship between shifts in cell proportions and extra-pulmonary symptoms associated with MPP. Results In this study, a total of 42 cases were enrolled, with 32 cases in the study group and 10 cases in the control group. There were no statistically significant differences in gender, age, height, weight, and BMI between the two groups (p > 0.05). The study group exhibited significantly higher levels of neutrophil percentage (GRA%), CRP, D-dimer, and LDH in blood routine tests compared to the control group (p < 0.05). Furthermore, the proportions of neutrophils (%) and macrophages (%) in BALF were significantly higher in the study group compared to the control group (p< 0.05), while the proportion of lymphocytes (%) in BALF showed no statistically significant difference between the two groups (p> 0.05). Conclusion In the acute phase of MPP in children, BALF is predominantly composed of neutrophils. A lower proportion of lymphocytes in BALF is associated with a higher incidence of extra-pulmonary manifestations and longer hospitalization duration.

<i>Avena sativa’s</i> Therapeutic Potential: Exploring Antiasthmatic Effects in Guinea Pig Asthma Model

Avena sativa (oat) has emerged as a potential therapeutic candidate for asthma, a global health challenge characterized by chronic airway inflammation. This research investigates the anti-asthmatic potential of the Hydro-Alcoholic Extract of Avena sativa (HAEAS) in a guinea pig asthma model induced by histamine and ovalbumin. The study explores the influence of HAEAS on oxidative stress markers, leucocytes, eosinophils, and histopathological changes in lung tissues. Results reveal that HAEAS, particularly at 400 mg/kg, significantly increases the latent period and percentage protection in histamine induced bronchospasm. In ovalbumin-sensitized guinea pigs, HAEAS demonstrates a notable reduction in total leucocyte count, eosinophils, neutrophils, and macrophages in bronchoalveolar lavage fluid. Moreover, HAEAS exhibits antioxidative effects by increasing superoxide dismutase levels and decreasing malondialdehyde levels. Histopathological analysis demonstrates a decrease in inflammatory cell infiltration, hyperplasia, and bronchoconstriction. This study highlights the potential of Avena sativa as a novel therapeutic avenue for asthma, offering anti-inflammatory and antioxidant benefits.

S100 Calcium-Binding Protein A9, a Potential Novel Diagnostic Biomarker for Idiopathic Pulmonary Fibrosis.

Neutrophilic inflammation is a characteristic feature of idiopathic pulmonary fibrosis (IPF). S100 calcium-binding protein A9 (S100A9) is a neutrophil-derived protein involved in the development of neutrophil-related chronic inflammatory disorders. However, the role of S100A9 in IPF remains unclear. We used enzyme-linked immunosorbent assays to measure S100A9 levels in bronchoalveolar lavage fluid (BALF) and serum obtained from healthy controls (HCs) and patients with IPF, non-specific interstitial pneumonia, hypersensitivity pneumonitis, and sarcoidosis. Compared with HCs, BALF S100A9 levels were significantly higher in IPF patients (P < 0.001), patients with hypersensitivity pneumonitis (P = 0.043), and patients with nonspecific interstitial pneumonia (P < 0.001). The S100A9 level in BALF of 0.093 ng/mL could distinguish IPF patients from HCs, with a specificity of 78.8% and a sensitivity of 81.6%. Similarly, the S100A9 level in BALF of 0.239 ng/mL had a specificity of 64.7% and a sensitivity of 66.7% for distinguishing IPF patients from patients with other interstitial lung diseases. Additionally, BALF S100A9 levels were significantly correlated with neutrophil counts (r = 0.356, P < 0.001) in BALF. IPF patients with S100A9 levels in BALF > 0.533 ng/mL had lower survival rates, compared with patients who had levels ≤ 0.553 ng/mL (n = 49; hazard ratio [HR], 3.62; P = 0.021). Combination analysis revealed that IPF patients with S100A9 levels in BALF> 0.553 ng/mL or neutrophil percentages > 49.1% (n = 43) had significantly lower survival rates than patients with S100A9 levels in BALF ≤ 0.553 ng/mL and neutrophil percentages ≤ 49.1% (n = 41) (HR, 3.91; P = 0.014). Additionally, patients with serum S100A9 levels > 0.077 ng/mL (n = 29) had significantly lower survival rates than patients with levels ≤ 0.077 ng/mL (n = 53, HR, 2.52; P = 0.013). S100A9 was expressed on neutrophils and macrophages in BALF from IPF patients as well as α-smooth muscle actin positive cells in the lung tissues. S100A9 is involved in the development and progression of IPF. Moreover, S100A9 levels in BALF and serum may be surrogate markers for IPF diagnosis and survival prediction, particularly when analyzed in combination with neutrophil percentages.

Effects of imperatorin on airway remodeling in bronchial asthma through S1PR2/STAT3 signaling pathway.

In this study, we analyzed the effect of Imperatorin (IMP) on airway remodeling in bronchial asthma (BA) through the S1PR2/STAT3 signaling pathway. First, 30 BALB/c mice were randomized into control, model, and intervention groups. The control group was left untreated; the model and intervention groups were BA modeled and; the intervention group was further intraperitoneally injected with IMP following modeling. Lung tissue pathological changes, inflammatory cell deposition in bronchoalveolar lavage fluid (BALF), expression of inflammatory factors, and oxidative stress (OS) were detected in three groups of mice. We found that the intervention group had reduced macrophage and lymphocyte counts in BALF and ameliorated pathological damage of lung tissue than the control group after intervention. In addition, the post-interventional inflammatory factors and malonaldehyde (MDA) in the intervention group were elevated compared with the control group but reduced versus the model group, while the superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were lower than those in the control group and higher compared with the model group (P<0.05). In addition, the expression of S1PR2/STAT3 pathway in three groups of mice showed that S1PR2/STAT3 signaling was activated in the model group, while the expression of S1PR2 and STAT3 in the intervention group was lower than that in the model group (P<0.05). These results demonstrate that IMP reverses pathological injury in BA and alleviates airway remodeling by inhibiting the S1PR2/STAT3 axis.

Acacetin protects against sepsis-induced acute lung injury by facilitating M2 macrophage polarization via TRAF6/NF-κB/COX2 axis.

Acute lung injury (ALI) is the leading cause of death in patients with sepsis syndrome and without effective protective or therapeutic treatments. Acacetin, a natural dietary flavonoid, reportedly exerts several biological effects, such as anti-tumor, anti-inflammatory, and anti-oxidative effects. However, acacetin's effect and underlying mechanism on sepsis-induced ALI remain unclear. Here, the mouse model was established to explore the impact of acacetin on sepsis-induced ALI. Acacetin significantly increased ALI murine survival and attenuated lung injury in histological examinations. Additionally, acacetin down-regulated myeloperoxidase activity, protein concentration, and number of neutrophils and macrophages in bronchoalveolar lavage fluid. Subsequently, inflammatory cytokines, including TNF-α, IL-1β, and IL-6, were examined. Results showed that acacetin dramatically suppressed the production of TNF-α, IL-1β, and IL-6. These above results indicated that acacetin attenuated sepsis-induced ALI by inhibiting the inflammatory response. Moreover, acacetin inhibited the expression of markers for M1-type (iNOS, CD86) macrophages and promoted the expression of markers for M2-type (CD206, Arg1) macrophages by western blot. In addition, acacetin down-regulated the expression TRAF6, NF-κB, and Cyclooxygenase-2 (COX2) by western blot. The high concentration of acacetin had a better effect than the low concentration. Besides, over-expression of TRAF6 up-regulated the expression of COX2, CD86, and iNOS, and the ratio of p-NF-κB to NF-κB increased the mRNA levels of TNF-α, IL-1β, and IL-6, down-regulated the expression of CD206 and Arg1. The effects of TRAF6 were the opposite of acacetin. And TRAF6 could offset the impact of acacetin. This study demonstrated that acacetin could prevent sepsis-induced ALI by facilitating M2 macrophage polarization via TRAF6/NF-κB/COX2 axis.

Flow Cytometric Analysis of Macrophages and Cytokines Profile in the Bronchoalveolar Lavage Fluid in Patients with Lung Cancer.

Macrophages play an important role in the suppression and activation of immune anti-cancer response, but little is known about dominant macrophage phenotype in the lung cancer environment, evaluated by bronchoalveolar lavage fluid (BALF). The aim of this study was to characterize macrophages in BALF from a lung affected by cancer (cBALF) and a healthy lung (hBALF) of the same patient regarding their individual macrophage polarization and selected cytokines profile. A total of 36 patients with confirmed lung cancer were investigated. Macrophages markers: CD206 CD163 CD80 CD86 CD40 CD45, Arginase-1, and CD68 were evaluated by flow cytometry. Cytokines (IL-1 RA, IL-6, IL-10, TNF-α, IL-1β, IL-12, IL-23, and TGF-β) profile was analyzed. There was higher median proportion of macrophages in Cbalf than in Hbalf. The population of macrophages presented immunophenotype: Ccd68+bright CD206+bright CD163+bright CD80+ CD86+ CD40+bright CD45+ cArginase+. We observed some trends in the expression of the analyzed antigens in clBALF and hlBLAF. The highest concentrations of IL-1RA and IL-6 were in Cbalf and Hbalf supernatant. There were the correlations between pro- and anti-inflammatory cytokines. The findings showed that macrophages include a diverse and plastic group with the presence of different antigens and cytokines, and determining the target phenotype is a complex and variable process.

Daqing formula ameliorated allergic asthma and airway dysbacteriosis in mice challenged with ovalbumin and ampicillin

Ethnopharmacological relevanceAsthma is a chronic airway inflammatory disease that can lead to several complications caused by bacterial infections. However, recurrent attacks of the disease require long-term use of antibiotics, resulting in lung dysbiosis and poor outcomes. Daqing Formula (DQF) is a well-known herbal medicine in Pharmacopoeia of China, which is widely used for various stimuli-induced lower respiratory diseases, including asthma, bronchitis, and pneumonia. Thus, it has been demonstrated to be a plant-derived broad-spectrum antibiotic for treating and preventing various acute and chronic respiratory diseases. Aim of the studyThis study evaluated the efficacy and possible mechanism of DQF on allergic asthma and airway dysbiosis. Methods and materialsThe mice were co-challenged with ovalbumin and ampicillin to induce allergic asthma combined with airway dysbacteriosis. The populations of lung microbiota were detected by using 16s DNA sequencing. The levels of asthmatic markers in BALF were detected by ELISA. The levels of Th1/Th2 cytokines in splenic CD4+ cells of mice were analyzed by flow cytometry. The expressions of the GSK-3β signaling pathway in the lung tissues of asthmatic mice and eosinophils were detected by western blotting assay. The inhibition of DQF on the production of pro-inflammatory cytokines in eosinophils of asthmatic mice. ResultsThe results showed that treatment with DQF at 200–800 mg/kg doses significantly reduced the frequency of nasal rubbing and lung inflammation as well as the number of total cells, eosinophils, and macrophages in bronchoalveolar lavage fluid. It decreased the relative abundances of Streptococcus, Cuoriavidus, and Moraxella, increased Akkermansia and Prevotella_6 in lung tissues of asthmatic mice, and inhibited the growth of Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae and their resistant strains in vitro. Furthermore, DQF reduced the levels of eotaxin, TSLP, IL-4, IL-5, IL-25, and IL-33, but enhanced IFN-γ and IL-12 in BALF. It elevated the population of Th1 cells, inhibited eosinophil activation, and downregulated the expressions of p-GSK-3β, p-p65, nuclear β-catenin, and p-STAT3 in the lung tissues of asthmatic mice. ConclusionsThe results revealed that DQF reduced airway inflammation, ameliorated lung dysbiosis, shifted the Th1/Th2 balance, and inhibited eosinophil activation in asthmatic mice, indicating its potential for severe asthma treatment.

Clinical characteristics and risk factors for bronchoscopic airway mucus hypersecretion in childhood pneumonia infected by different pathogens

Objective: To investigate the risk factors for airway mucus hypersecretion in childhood pneumonia infected by different pathogens. Method: A retrospective cohort included 968 children who were hospitalized for Mycoplasma pneumoniae pneumonia (MPP), respiratory syncytial virus (RSV) pneumonia, adenovirus pneumonia and underwent bronchoscopy in Respiratory Department of Children's Hospital of Chongqing Medical University from January 2019 to December 2021 was conducted. The children were divided into two groups distinguished by airway mucus secretion according to the airway mucus hypersecretion score which were scored according to the mucus secretion under the bronchoscope. The demographic characteristics, clinical characteristics, laboratory tests and disease severity of the two groups were compared. And the risk factors for the development of airway mucus hypersecretion in two groups were analyzed. Chi square test, Mann-Whithey U test and Fisher exact test were used to analyze the differences between the two groups, and multivariate Logistic regression was used to analyze the influencing factors. Result: There were 559 males and 409 females in the 968 children, with an age of 4.0 (1.4, 6.0) years. Among the 642 children with MPP, 185 cases were in the hypersecretion group and 457 cases were in the non-hypersecretion group. There were 41 cases in the hypersecretion group and 160 cases in the non-hypersecretion group of 201 children with RSV pneumonia. In the 125 children with adenovirus pneumonia, there were 39 cases in the hypersecretion group and 86 cases in the non-hypersecretion group. In these children, the age of children in the hypersecretion group was older than that in the non-hypersecretion group (6.0 (4.0, 7.0) vs. 5.0 (3.0, 7.0) years old, 1.5 (0.5, 3.6) vs. 0.8 (0.4, 1.6) years old, 2.0 (1.2, 4.5) vs. 1.3 (0.8, 2.0) years old, U=35 295.00, 2 492.00, 1 101.00, all P<0.05). Through multivariate Logistic regression analysis it found that increased risk of airway mucus hypersecretion was present in childhood MPP with increase in peripheral blood white blood cell count (OR=3.30, 95%CI 1.51-7.93, P=0.004) or increase in neutrophil ratio (OR=2.24, 95%CI 1.16-4.33, P=0.016) or decrease in lymphocyte count (OR=3.22, 95%CI 1.66-6.31, P<0.001) or decrease in serum albumin (OR=2.00, 95%CI 1.01-3.98, P=0.047). The risk of airway mucus hypersecretion was increased in children with RSV pneumonia combined with elevated peripheral blood eosinophils (OR=3.04, 95%CI 1.02-8.93, P=0.043). Meanwhile, airway mucus hypersecretion was associated with severe pneumonia (OR=2.46, 95%CI 1.03-6.15, P=0.047) in children with RSV pneumonia. Older age was associated with increased risk of airway mucus hypersecretion in children with adenovirus pneumonia (OR=1.02, 95%CI 1.00-1.04, P=0.026). In these children with occurrence of pulmonary rales, wheezes or sputum sounds (OR=3.65, 95%CI 1.22-12.64, P=0.028) had an increased risk of airway mucus hypersecretion. Neutrophils in bronchoalveolar lavage fluid (BALF) demonstrated higher ratio in hypersecretion group from children with MPP (0.65 (0.43, 0.81) vs. 0.59 (0.34, 0.76), U=24 507.00, P<0.01), while the proportion of macrophages in BALF was lower (0.10 (0.05, 0.20) vs. 0.12 (0.06, 0.24), U=33 043.00, P<0.05). Nucleated cell count and neutrophil ratio in BALF were higher in hypersecretion group of children with RSV pneumonia (1 210 (442, 2 100)×106 vs. 490 (210, 1 510)×106/L, 0.43 (0.26, 0.62) vs. 0.30 (0.13, 0.52), U=2 043.00, 2 064.00, all P<0.05). Conclusions: The increase in peripheral blood white blood cell count, neutrophil ratio and decrease in lymphocyte count, serum albumin in children with MPP is related to the development of airway mucus hypersecretion. In children with RSV pneumonia, the abnormal increase of eosinophils in peripheral blood has relationship with hypersecretion. The appearance of lung rale, wheezing, and sputum rale are associated with airway mucus hypersecretion in children with adenovirus pneumonia. In addition, local neutrophil infiltration in the respiratory tract is closely related to the occurrence of airway mucus hypersecretion caused by Mycoplasma pneumoniae and RSV infection.

CXCL17 Attenuates Diesel Exhaust Emissions Exposure-Induced Lung Damage by Regulating Macrophage Function.

Exposure to diesel exhaust emissions (DEE) is strongly linked to innate immune injury and lung injury, but the role of macrophage chemoattractant CXCL17 in the lung damage caused by DEE exposure remains unclear. In this study, whole-body plethysmography (WBP), inflammatory cell differential count, and histopathological analysis were performed to assess respiratory parameters, airway inflammation, and airway injury in C57BL/6 male mice exposed to DEE for 3 months. qRT-PCR, IHC (immunohistochemistry), and ELISA were performed to measure the CXCL17 expression in airway epithelium or BALF (bronchoalveolar lavage fluid) following DEE/Diesel exhaust particle (DEP) exposure. Respiratory parameters, airway inflammation, and airway injury were assessed in CXCL17-overexpressing mice through adeno-associated virus vector Type 5 (AAV5) infection. Additionally, an in vitro THP-1 and HBE co-culture system was constructed. Transwell assay was carried out to evaluate the effect of rh-CXCL17 (recombinant human protein-CXCL17) on THP-1 cell migration. Flow cytometry and qRT-PCR were conducted to assess the impacts of rh-CXCL17 on apoptosis and inflammation/remodeling of HBE cells. We found that the mice exposed to DEE showed abnormal respiratory parameters, accompanied by airway injury and remodeling (ciliary injury in airway epithelium, airway smooth muscle hyperplasia, and increased collagen deposition). Carbon content in airway macrophages (CCAM), but not the number of macrophages in BALF, increased significantly. CXCL17 expression significantly decreased in mice airways and HBE after DEE/DEP exposure. AAV5-CXCL17 enhanced macrophage recruitment and clearance of DEE in the lungs of mice, and it improved respiratory parameters, airway injury, and airway remodeling. In the THP-1/HBE co-culture system, rh-CXCL17 increased THP-1 cell migration while attenuating HBE cell apoptosis and inflammation/remodeling. Therefore, CXCL17 might attenuate DEE-induced lung damage by recruiting and activating pulmonary macrophages, which is expected to be a novel therapeutic target for DEE-associated lung diseases.

Deficiency of interleukin-6 receptor ameliorates PM2.5 exposure-induced pulmonary dysfunction and inflammation but not abnormalities in glucose homeostasis

BackgroundAmbient fine particulate matter (PM2.5) exposure increases local and systemic interleukin-6 (IL-6). However, the pathogenic role of IL-6 signalling following PM2.5 exposure, particularly in the development of pulmonary dysfunction and abnormal glucose homeostasis, has hardly been investigated. ResultsIn the study, IL-6 receptor (IL-6R)-deficient (IL-6R-/-) and wildtype littermate (IL-6R+/+) mice were exposed to concentrated ambient PM2.5 (CAP) or filtered air (FA), and their pulmonary and metabolic responses to these exposures were analyzed. Our results demonstrated that IL-6R deficiency markedly alleviated PM2.5 exposure-induced increases in lung inflammatory markers including the inflammation score of histological analysis, the number of macrophages in bronchoalveolar lavage fluid (BALF), and mRNA expressions of TNFα, IL-1β and IL-6 and abnormalities in lung function test. However, IL-6R deficiency did not reduce the hepatic insulin resistance nor systemic glucose intolerance and insulin resistance induced by PM2.5 exposure. ConclusionOur findings support the crucial role of IL-6 signalling in the development of pulmonary inflammation and dysfunction due to PM2.5 exposure but question the putative central role of pulmonary inflammation for the extra-pulmonary dysfunctions following PM2.5 exposure, providing a deep mechanistic insight into the pathogenesis caused by PM2.5 exposure.

Anti-inflammatory effect of Gyeji-tang in a chronic obstructive pulmonary disease mouse model induced by cigarette smoke and lipopolysaccharide

Context Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory lung disease associated with respiratory symptoms and narrowing of airways. Gyeji-tang (GJT) is a traditional Asian medicine that has been used to relieve early-stage cold symptoms, headache, and chills. Objective We examined the effect and potential molecular action mechanism of GJT in a mouse model of COPD induced by cigarette smoke (CS) plus lipopolysaccharide (LPS). Materials and methods COPD was induced in C57BL/6J mice via daily exposure to CS for 1 h for 8 weeks and intranasal administration of LPS on weeks 1, 3, 5, and 7. GJT (100 or 200 mg/kg) or roflumilast (5 mg/kg) was administrated daily for the final 4 weeks of COPD induction. Results Administration of GJT significantly suppressed the CS/LPS-induced increases in: the numbers of total cells and macrophages in bronchoalveolar lavage fluid; the expression levels of tumour necrosis factor-α, interleukin (IL)-6, IL-1β, and IL-8; the activities (phosphorylation) of nuclear factor kappa B and signal transducer and activator of transcription 3; and the expression levels of the structural remodelling markers, transforming growth factor beta, matrix metallopeptidase (MMP)-7, and MMP-9. Discussion and conclusions These results demonstrate that GJT prevents the lung inflammation and airway remodelling induced by CS plus LPS exposure in mice, suggesting that GJT may have therapeutic potential for the treatment of COPD.

Key factors to establish the ovalbumin-induced atopic dermatitis minipig model: age and body weight

BackgroundGiven its similar structure and immune response to the human skin, porcine is a good model for dermal studies. Here, we sensitized ovalbumin (Ova) on minipig back skin for 2–4 weeks to induce chronic atopic dermatitis (AD).ResultsGross observation, serum cytokine level, epidermal thickness, and epidermal integrity did not change after 4 weeks of Ova induction compared with the control, indicating AD modeling failure. Only the neutrophils in the blood and macrophages in bronchoalveolar lavage fluid changed slightly until 3 or 2 weeks after Ova sensitization, respectively. The successful and failed Ova-induced AD minipig models only differ in age and body weight of the minipigs. The minipigs, 12 months old with a 30-kg median weight, had a two-fold thicker dermis than minipigs 8–10 months old, with an 18.97-kg median weight, resulting in impaired Ova permeability and immune response.ConclusionAge and body weight are key factors that should be considered when developing an Ova-induced AD minipig model.

Oil Red O Staining of Pulmonary Macrophages in Bronchoalveolar Lavage Specimens Is Not Specific for Vaping-Associated Lung Injury.

Oil Red O (ORO) positivity in bronchoalveolar lavage (BAL) fluid macrophages in the setting of e-cigarette, or vaping, product use-associated acute lung injury (EVALI) has been frequently requested by clinicians based on rare reports and subsequent US Centers for Disease Control and Prevention guidelines. The aim of this study was to determine the specificity of ORO staining in BAL specimens with disease states other than EVALI. Consecutive BAL specimens (October-December 2019) were stained with ORO. The lipid-laden macrophage index (LLMI) was calculated for each case. We studied BAL samples from 50 patients. Indications for BAL were surveillance bronchoscopy for lung transplantation (27/50), suspected infection (12/50), sarcoidosis/suspected sarcoidosis (3/50), nodules or ground-glass opacities (3/50), hemoptysis (2/50), asthma or eosinophilic pneumonia (2/50), and idiopathic pulmonary fibrosis (1/50). ORO staining was seen in BAL fluid macrophages in 45 of 50 cases (focal in 18, moderate in 23, diffuse in 4); LLMI ranged from 0 to 218. Using a threshold of LLMI of 85 or higher as positive, ORO was positive in 7 of 50 (14%) cases (range, 85-218). ORO staining in BAL fluid macrophages is not specific for EVALI. Even when an LLMI of 85 or higher is used as a threshold for positivity, ORO positivity occurs in a significant subset of non-vaping-related cases.

LncRNA H19 alleviates sepsis-induced acute lung injury by regulating the miR-107/TGFBR3 axis

ObjectiveAcute lung injury (ALI) increases sepsis morbidity and mortality. LncRNA H19 plays a critical role in sepsis. miR-107 is highly-expressed and TGFβ type III receptor (TGFBR3) is poorly-expressed in sepsis, yet their roles in sepsis development require further investigation. This study aimed to investigate the mechanism of H19 in alleviating sepsis-induced ALI through the miR-107/TGFBR3 axis.MethodsMice were intravenously injected with Ad-H19 adenovirus vector or control vector one week before establishing the mouse model of cecal ligation and puncture (CLP). Pulmonary microvascular endothelial cells (PMVECs) were transfected with oe-H19 or oe-NC plasmids and then stimulated by lipopolysaccharide (LPS). Lung injury was assessed via hematoxylin–eosin staining, measurement of wet-to-dry (W/D) ratio, and TUNEL staining. Levels of H19, miR-107, and TGFBR3 were determined by RT-qPCR. Apoptosis of PMVECs was evaluated by flow cytometry. Levels of Bax and Bcl-2 in lung tissues and PMVECs were measured using Western blot. Total protein concentration and the number of total cells, neutrophils, and macrophages in bronchoalveolar lavage fluid (BALF) were quantified. Levels of TNF-α, IL-1β, IL-6, and IL-10 in BALF, lung tissues, and PMVECs were measured by ELISA. Cross-linking relationships among H19, miR-107 and TGFBR3 were verified by dual-luciferase and RIP assays.ResultsH19 was poorly-expressed in CLP-operated mice. H19 overexpression attenuated sepsis-induced ALI, which was manifested with complete alveolar structure, decreased lung injury score and lung W/D ratio, and inhibited apoptosis in CLP-operated mice, which was manifested with decreased number of TUNEL-positive cells and Bax level and increased Bcl-2 level. CLP-operated mice had increased concentration of total protein and number of total cells, neutrophils, and macrophages in BALF, which was nullified by H19 overexpression. H19 overexpression declined levels of TNF-α, IL-1β, and IL-6 and elevated IL-10 levels. H19 inhibited LPS-induced PMVEC apoptosis and pro-inflammatory cytokine production. H19 targeted TGFBR3 as the ceRNA of miR-107. miR-107 overexpression or silencing TGFBR3 partially averted the inhibition of H19 overexpression on LPS-induced PMVEC apoptosis and pro-inflammatory cytokine production.ConclusionLncRNA H19 inhibited LPS-induced PMVEC apoptosis and pro-inflammatory cytokine production and attenuated sepsis-induced ALI by targeting TGFBR3 as the ceRNA of miR-107.

Immune cell composition of the bronchoalveolar lavage fluid in healthy and respiratory diseased dromedary camels

BackgroundRespiratory diseases are among the most common and expensive to treat diseases in camels with a great economic impact on camel health, welfare, and production. Bronchoalveolar lavage fluid (BALF) has been proven as a valuable sample for investigating the leukocyte populations in the respiratory tract of several species. In the present study, fluorescent antibody labeling and flow cytometry were used to study the immune cell composition of BALF in dromedary camels. Animals with clinical respiratory diseases (n = seven) were compared with apparently healthy animals (n = 10). In addition, blood leukocytes from the same animals were stained in parallel with the same antibodies and analyzed by flow cytometry.ResultsCamel BALF macrophages, granulocytes, monocytes, and lymphocytes were identified based on their forward and side scatter properties. The expression pattern of the cell markers CD172a, CD14, CD163, and MHCII molecules on BALF cells indicates a similar phenotype for camel, bovine, and porcine BALF myeloid cells. The comparison between camels with respiratory disease and healthy camels regarding cellular composition in their BALF revealed a higher total cell count, a higher fraction of granulocytes, and a lower fraction of macrophages in diseased than healthy camels. Within the lymphocyte population, the percentages of helper T cells and B cells were also higher in diseased than healthy camels. The elevated expression of the activation marker CD11a on helper T cells of diseased camels is an indication of the expansion of helper T cells population due to infection and exposure to respiratory pathogens. The higher abundance of MHCII molecules on BALF macrophages from diseased camels indicates a polarization toward an inflammatory macrophage phenotype (M1) in respiratory diseased camels. No significant differences were observed in the systemic leukogram between healthy and diseased animals.ConclusionsCollectively, the current study represents the first report on flow cytometric analysis of immune cell composition of bronchoalveolar lavage fluid (BALF) in dromedary camels.

Reprogramming alternative macrophage polarization by GATM-mediated endogenous creatine synthesis: A potential target for HDM-induced asthma treatment.

Cellular energy metabolism plays a crucial role in the regulation of macrophage polarization and in the execution of immune functions. A recent study showed that Slc6a8-mediated creatine uptake from exogenous supplementation modulates macrophage polarization, yet little is known about the role of the de novo creatine de novobiosynthesis pathway in macrophage polarization. Here, we observed that glycine amidinotransferase (GATM), the rate-limiting enzyme for creatine synthesis, was upregulated in alternative (M2) polarized macrophages, and was dependent on the transcriptional factor STAT6, whereas GATM expression was suppressed in the classical polarized (M1) macrophage. Next, we revealed that exogenous creatine supplementation enhanced IL-4-induced M2 polarization, confirming recent work. Furthermore, we revealed that genetic ablation of GATM did not affect expression of M1 marker genes (Nos2, IL1b, IL12b) or the production of nitric oxide in both peritoneal macrophages (PMs) and bone marrow-derived macrophages (BMDMs). By contrast, expression levels of M2 markers (Arg1, Mrc1, Ccl17 and Retnla) were lower following GATM deletion. Moreover, we found that deletion of GATM in resident alveolar macrophages (AMs) significantly blocked M2 polarization but with no obvious effect on the number of cells in knockout mice. Lastly, an upregulation of GATM was found in lung tissue and bronchoalveolar lavage fluid macrophages from HDM-induced asthmatic mice. Our study uncovers a previously uncharacterized role for the de novo creatine biosynthesis enzyme GATM in M2 macrophage polarization, which may be involved in the pathogenesis of related inflammatory diseases such as an T helper 2 (Th2)-associated allergic asthma.

Protective effect of Lindera aggregata on lipopolysaccharide-induced mice acute respiratory distress syndrome by regulating p38MAPK/ERK pathway

To explore the protective effect of Lindera aggregata on acute respiratory distress syndrome (ARDS) induced by lipopolysaccharide (LPS) in mice and its possible mechanism. Forty C57BL/6 mice were randomly divided into sham operation group, ARDS model group, low-dose Lindera aggregata (L-LA) group and high-dose Lindera aggregata (H-LA) group, with 10 mice in each group. ARDS model was established by injecting 5 mg/kg LPS through the trachea. The L-LA group and H-LA group were orally administrated 1 g/kg and 5 g/kg of the Lindera aggregate extract once a day, respectively, while the ARDS model group was given the same volume of normal saline, the sham group received no treatment. The Lindera aggregata was preadministered for 3 days before modeling, and continued for 2 days after modeling, then the animals were sacrificed, and bronchoalveolar lavage fluid (BALF) and lung tissue were collected. The pathological changes of lung tissue in each group of mice were observed under the microscope and the wet/dry weight ratio (W/D) of the lung were measured. Enzyme linked immunosorbent assay (ELISA) was used to examine the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in mice serum and BALF, and flow cytometry was used to detect the expression rate of CD40 on the surface of BALF macrophages. The phosphorylation levels of p38 and extracellular signal-regulated protein kinase 1/2 (ERK1/2) proteins in lung tissue were measured by Western blotting. Lung histopathology under light microscope showed that the damage of alveolar structure, thickening of alveolar septum and infiltration of inflammatory cells in the H-LA group were less severe than those in the ARDS model group, while the pathological characteristics of ARDS in the L-LA group were not significantly different from those in the ARDS model group. Compared with the sham operation group, the lung W/D ratio, TNF-α and IL-6 protein contents in serum and BALF, BALF macrophage CD40 expression rate and lung tissue p38 and ERK1/2 protein phosphorylation levels were significantly increased in ARDS model group. The W/D ratio, the concentrations of TNF-α and IL-6 in serum and BALF, the expression rate of CD40 in BALF macrophages, and the phosphorylation levels of p38 and ERK1/2 protein in lung tissue in the L-LA group were not significantly different from those in the ARDS model group. The above indexes in the H-LA group were significantly lower than those in the ARDS model group and the L-LA group [W/D ratio: 5.70±0.19 vs. 6.20±0.31, 6.01±0.17; serum TNF-α (ng/L): 83.63±15.04 vs. 111.75±18.45, 108.12±13.98; serum IL-6 (ng/L): 111.38±8.75 vs. 244.13±26.85, 227.50±9.37; BALF TNF-α (ng/L): 36.25±2.82 vs. 51.13±5.44, 47.50±5.78; BALF IL-6 (ng/L): 35.63±2.20 vs. 49.63±4.90, 46.38±3.50; CD40 expression rate (%): 23.28±2.45 vs. 30.32±2.40, 28.17±1.98; p-p38/p38: 0.50±0.04 vs. 0.74±0.07, 0.69±0.04; p-ERK1/2/ERK1/2: 0.47±0.07 vs. 0.72±0.07, 0.68±0.05; all P < 0.01]. Lindera aggregata can inhibit LPS-induced lung inflammation and alleviate lung injury in ARDS mice. The mechanism may be related to the inhibition of the activation of p38 mitogen activated protein kinase/ERK (p38MAPK/ERK) signaling pathway.