3-carene supresses inflammatory cytokine interleukin-4, interleukin-5 and interleukin-13 in a murine model of asthma.

Asthma is a common airway disease associated with allergic inflammation. Environmental factors, such as pollens, pollution, insect-borne antigens, or commercial chemicals, cause this disease. The common symptoms of this airway allergic reaction are increasing mucus, narrowing of the airway wall, coughing, and chest tightness. Medications, such as steroids, alleviate the disease but with severe side effects. Several studies have reported the anti-inflammatory effects of tree-based essential oil components, particularly 3-carene. Therefore, this study used 3-carene to determine if it alleviates asthmatic symptoms in the murine model. First, BALB/c mice were sensitized to an ovalbumin and aluminium hydroxide mixture on day 7th and 14th. From days 21st to 23rd, the mice were challenged with 3-carene and budesonide. The lung trachea, plasma, and bronchiolar lavage fluid (BAL fluid) were collected on day 24. The 3-carene treatment suppressed the cytokine gene expression, such as interleukin-4 (IL-4), IL-5, and IL-13, reducing the lung epithelial cell thickness in the asthmatic model. These results suggest that essential oil 3-carene has an anti-asthmatic effect.

A single dose of angiotensin-(1-7) resolves eosinophilic inflammation and protects the lungs from a secondary inflammatory challenge.

OBJECTIVE: Angiotensin-(1-7) [Ang-(1-7)] is a pro-resolving mediator. It is not known whether the pro-resolving effects of Ang-(1-7) are sustained and protect the lung from a subsequent inflammatory challenge. This study sought to investigate the impact of treatment in face of a second allergic or lipopolysaccharide (LPS) challenge. METHODS: Mice, sensitized and challenged with ovalbumin (OVA), received a single Ang-(1-7) dose at the peak of eosinophilic inflammation, 24 h after the final OVA challenge. Subsequently, mice were euthanized at 48, 72, 96, and 120 h following the OVA challenge, and cellular infiltrate, inflammatory mediators, lung histopathology, and macrophage-mediated efferocytic activity were evaluated. The secondary inflammatory stimulus (OVA or LPS) was administered 120 h after the last OVA challenge, and subsequent inflammatory analyses were performed. RESULTS: Treatment with Ang-(1-7) resulted in elevated levels of IL-10, CD4+Foxp3+, Mres in the lungs and enhanced macrophage-mediated efferocytic capacity. Moreover, in allergic mice treated with Ang-(1-7) and then subjected to a secondary OVA challenge, inflammation was also reduced. Similarly, in mice exposed to LPS, Ang-(1-7) effectively prevented the lung inflammation. CONCLUSION: A single dose of Ang-(1-7) resolves lung inflammation and protect the lung from a subsequent inflammatory challenge highlighting its potential therapeutic for individuals with asthma.

Involvement of necroptotic cell death in macrophages in progression of bleomycin and LPS-induced acute exacerbation of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is the severe form of interstitial pneumonias. Acute exacerbation (AE) of IPF is characterized by progressive lung fibrosis with the irreversible lung function decline and inflammation, and is often fatal with poor prognosis. However, the physiological and molecular mechanisms in AE of IPF are still not fully understood. In this study, we investigated the mechanism underlying AE of IPF, using bleomycin (BLM) and lipopolysaccharide (LPS) (BLM + LPS)-treated mice. The mice were treated with a single dose of 1.5 mg/kg BLM (on day 0) and/or 0.5 mg/kg LPS (on day 14), and maintained for another 7 days (total 21 days). Administration of BLM + LPS more severely aggravated the respiratory function, fibrosis, and inflammation in the lungs, together with the elevated interleukin-6 level in bronchoalveolar lavage fluid, than the control or BLM alone-treated mice. Moreover, the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay demonstrated that subsequent treatment with LPS elevated cell death in the lungs of BLM-administered mice. Furthermore, the expression levels of mixed lineage kinase domain-like protein (MLKL), a marker of necroptotic cell death, and CD68-positive macrophages were increased, and most of them were co-stained in the lungs of BLM + LPS-treated mice. These results, taken together, indicate that BLM + LPS treatment showed more exacerbated the respiratory function with extensive fibrosis and inflammation than treatment with BLM alone in mice. Fibrosis and inflammation in AE of IPF seen in BLM + LPS-administered mice included an increase in macrophages and their necroptotic cell death.

Xin-Yi-Qing-Fei-Tang and its critical components reduce asthma symptoms by suppressing GM-CSF and COX-2 expression in RBL-2H3 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: The traditional Chinese medicine (TCM) XYQFT is composed of 10 herbs. According to the NHIRD, XYQFT is one of the top ten most commonly used TCM prescriptions for asthma treatment. AIM OF THE STUDY: The aim of this study was to explore whether XYQFT reduces asthma symptoms in a mouse model of chronic asthma and determine the immunomodulatory mechanism of mast cells. MATERIALS AND METHODS: BALB/c mice were intratracheally (it) stimulated with 40 µL (2.5 µg/µL) of Dermatophagoides pteronyssinus (Der p) once a week for 6 consecutive weeks and orally administered XYQFT at 1 g/kg 30 min before Der p stimulation. Airway hypersensitivity, inflammatory cells in the BALF and total IgE in the blood were assessed in mice. In addition, RBL-2H3 cells (mast cells) were stimulated with DNP-IgE, after which different concentrations of XYQFT were added for 30 min to evaluate the effect of XYQFT on the gene expression and degranulation of DNP-stimulated RBL-2H3 cells. After the compounds in XYQFT were identified using LC‒MS/MS, the PBD method was used to identify the chemical components that inhibited the expression of the GM-CSF and COX-2 genes in mast cells. RESULTS: The airway hypersensitivity assay demonstrated that XYQFT significantly alleviated Der p-induced airway hypersensitivity. Moreover, cell counting and typing of bronchoalveolar lavage fluid revealed a significant reduction in Der p-induced inflammatory cell infiltration with XYQFT treatment. ELISA examination further indicated a significant decrease in Der p-induced total IgE levels in serum following XYQFT administration. In addition, XYQFT inhibited the degranulation and expression of genes (IL-3, IL-4, ALOX-5, IL-13, GM-CSF, COX-2, TNF-α, and MCP-1) in RBL-2H3 cells after DNP stimulation. The compounds timosaponin AIII and genkwanin in XYQFT were found to be key factors in the inhibition of COX-2 and GM-CSF gene expression in mast cells. CONCLUSION: By regulating mast cells, XYQFT inhibited inflammatory cell infiltration, airway hypersensitivity and specific immunity in a mouse model of asthma. In addition, XYQFT synergistically inhibited the expression of the GM-CSF and COX-2 genes in mast cells through timosaponin AIII and genkwanin.

Small extracellular vesicles derived from human mesenchymal stem cells prevent Th17-dominant neutrophilic airway inflammation via immunoregulation on Th17 cells.

Type 17 helper T cells (Th17)-dominant neutrophilic airway inflammation is critical in the pathogenesis of steroid-resistant airway inflammation such as severe asthma. Small extracellular vesicles (sEV) derived from human mesenchymal stem cells (MSCs) display extensive therapeutic effects and advantages in many diseases. However, the role of MSC-sEV in Th17-dominant neutrophilic airway inflammation and the related mechanisms are still poorly studied. Here we found that MSC-sEV significantly alleviated the infiltration of inflammatory cells in peribronchial interstitial tissues and reduced levels of inflammatory cells, especially neutrophils, in bronchoalveolar lavage fluids (BALF) of mice with neutrophilic airway inflammation. Consistently, MSC-sEV significantly decreased levels of IL-17A in BALF and Th17 in lung tissues. Furthermore, we found that labelled MSC-sEV were taken up by human CD4+ T cells most obviously at 12 h after incubation, and distributed mostly in mouse lungs. More importantly, potential signaling pathways involved in the MSC-sEV mediated inhibition of Th17 polarization were found using RNA sequencing. Using Western blot, JAK2-STAT3 pathway was identified as an important role in the inhibition of Th17 polarization by MSC-sEV. We found that proteins in MSC-sEV were mostly involved in the therapeutic effects of MSC-sEV. In total, our study suggested that MSC-sEV could be a potential therapeutic strategy for the treatment of neutrophilic airway inflammation.

Differential cell analysis in ovine bronchoalveolar lavage fluid: Effect of different sampling methods and seasons.

Background: Bronchoalveolar lavage (BAL) is a method for collecting the cellular and fluid components of the airway surface in the lungs. The assessment of differential cell profiles is potentially valuable in the diagnosis of pulmonary diseases, but there is no information about the normal BAL profiles in the Gezel breed. Aim: This study aimed to characterize the normal cryptologic findings of BAL with Gezel sheep. Methods: Twenty healthy sheep (15 females: 5 males, bodyweight: 55-65 kg) were sedated with xylazine (0.02-0.04 mg/kg IV). Two methods; the transtracheal bronchoalveolar lavage technique and the bronchoscopic bronchoalveolar lavage using a scope were evaluated. Sampling was performed in the summer and winter seasons. Results: Normal value (Mean ± SEM) for total cell, macrophage, lymphocyte, neutrophil, epithelial, and bronchial cells in BAL sampled in summer were (343.75 ± 30.23), ) 24.50 ± 2.62), (2.81 ± 0.51), (1.43 ± 0.88), and (3.12 ± 0.32), respectively. The normal values for the total cell, macrophage, lymphocyte, neutrophil, epithelial, and bronchial cells in BAL sampled in winter were (355.55 ± 37.67), (59.11 ± 4.30), (21.33 ± 3.10), (3.88 ± 1.07), (8.88 ± 3.78), and (6.33 ± 1.44), respectively. Conclusion: No significant change in the percentage of neutrophils was detected between seasons, although the percentages of bronchial and epithelial cells in winter were significantly high (p < 0.05). Except for the mentioned cases, neither the total cell number nor the percentage differential cell populations of BAL changed significantly (p < 0.05) in different sampling methods and seasons. Normal BAL profiles in the Gezel breed were determined and could be used in result interpretations. Also, both sampling methods can be used without significantly affecting the results.

Studying of anti-inflammatory and antioxidant effects of tectorigenin in ovalbumin-induced asthma mice models.

BACKGROUND: Allergic asthma is an important respiratory system problem characterized by airway inflammation, breathlessness, and bronchoconstriction. Allergic asthma and its outcomes are triggered by type 2 allergic immune responses. Tectorigenin is a methoxy-isoflavone with anti-inflammatory effects. In this study, we investigated the effects of tectorigenin on the pathophysiology of allergic asthma in an animal model. METHODS: Asthmatic mice were treated with tectorigenin. Then airway hyperresponsiveness (AHR), eosinophil percentage, levels of interleukin (IL)-33, IL-25, IL-13, IL-5, IL-4, total and ovalbumin (OVA)-specific immunoglobulin (Ig)E, and lung histopathology were evaluated. RESULT: Tectorigenin significantly (P 〈 0.05) reduced eosinophil infiltration (41 ± 7%) in the broncho-alveolar lavage fluid (BALF), serum IL-5 level (41 ± 5, pg/mL), and bronchial and vascular inflammation (scores of 1.3 ± 0.2 and 1.1 ± 0.3, respectively) but had no significant effects on AHR, serum levels of IL-33, -25, -13, and -4 (403 ± 24, 56 ± 7, 154 ± 11, and 89 ± 6 pg/mL, respectively), total and OVA-specific IgE (2684 ± 265 and 264 ± 19 ng/mL, respectively), goblet cell hyperplasia, and mucus production. CONCLUSION: Tectorigenin could control inflammation and the secretion of inflammatory mediators of asthma, so it can be regarded as a potential antiasthma treatment with the ability to control eosinophilia-related problems.

Clinical findings and outcome predictors for multinodular pulmonary fibrosis in horses: 46 cases (2009-2019).

BACKGROUND: Prognostic indicators for equine multinodular pulmonary fibrosis (EMPF), an interstitial fibrosing lung disease, are poorly described. HYPOTHESIS/OBJECTIVES: Describe diagnostic findings and outcome predictors for EMPF. ANIMALS: Forty-six adult horses with EMPF. METHODS: Retrospective multicenter case series from 2009 to 2019. Radiographic (n = 27) and ultrasonographic studies (n = 19) from EMPF horses and bronchoalveolar lavage fluid (BALF) cytology from 6 EMPF and 13 asthma cases were independently reviewed and blinded to diagnosis and outcome. Associations between predictor variables and survival were assessed by predictor screening followed by Fisher's exact and Wilcoxon rank sum tests. RESULTS: Primary clinical findings were weight loss (36/46, 78%), increased respiratory effort (33/46, 72%), tachypnea (32/46, 70%), and fever (18/46, 39%). Macrophage atypia was seen in more EMPF than asthmatic horse BALF (67% vs. 8%; P = .02). Equine herpesvirus 5 (EHV-5) was detected in 24 of 30 (80%) and hyperfibrinogenemia in 25 of 28 (89%) cases. Twenty-seven of 46 horses (59%) and 11 of 45 (24%) survived to discharge and to 3 months, respectively. Three-month survival was associated with lower median (range) respiratory rates (30 [24-36] vs. 41 [30-60] breaths per minute; P = .04), and higher BALF lymphocyte:neutrophil ratios (4.7 [1.4-22] vs. 0.47 [0.11-1.9]; P = .01) and blood lymphocyte counts (1.25 [0.93-2.55] vs. 0.90 [0.70-1.24] × 109/L; P = .03). Imaging findings, EHV-5 detection, and corticosteroid treatment were not associated with survival. CONCLUSIONS AND CLINICAL IMPORTANCE: Fever is not a sensitive clinical sign of EMPF. Diagnostic testing should be pursued for horses with increased respiratory rate and effort and weight loss. The prognosis for EMPF horses is poor. Corticosteroid treatment does not improve 3-month survival.

Blocking group 2 innate lymphoid cell activation and macrophage M2 polarization: potential therapeutic mechanisms in ovalbumin-induced allergic asthma by calycosin.

BACKGROUND: Calycosin, a flavonoid compound extracted from Astragalus membranaceus, has shown anti-asthma benefits in house dust mite-induced asthma. Recent studies have suggested that innate-type cells, including group 2 innate lymphoid cells (ILC2s) and macrophages, serve as incentives for type 2 immunity and targets for drug development in asthma. This work focuses on the effects of calycosin on the dysregulated ILC2s and macrophages in allergic asthma. METHODS: In vivo, the asthmatic mouse model was established with ovalbumin (OVA) sensitization and challenge, and calycosin was intraperitoneally administered at doses of 20 and 40 mg/kg. In vivo, mouse primary ILC2s were stimulated with interleukin (IL)-33 and mouse RAW264.7 macrophages were stimulated with IL-4 and IL-13 to establish the cell models. Cells were treated with calycosin at doses of 5 and 10 µM. RESULTS: In vivo, we observed significantly reduced numbers of eosinophils, neutrophils, monocyte macrophages and lymphocytes in the bronchoalveolar lavage fluid (BALF) of OVA-exposed mice with 40 mg/kg calycosin. Histopathological assessment showed that calycosin inhibited the airway inflammation and remodeling caused by OVA. Calycosin markedly decreased the up-regulated IL-4, IL-5, IL-13, IL-33, and suppression tumorigenicity 2 (ST2) induced by OVA in BALF and/or lung tissues of asthmatic mice. Calycosin repressed the augment of arginase 1 (ARG1), IL-10, chitinase-like 3 (YM1) and mannose receptor C-type 1 (MRC1) levels in the lung tissues of asthmatic mice. In vivo, calycosin inhibited the IL-33-induced activation as well as the increase of IL-4, IL-5, IL-13 and ST2 in ILC2s. Calycosin also repressed the increase of ARG1, IL-10, YM1 and MRC1 induced by IL-4 and IL-13 in RAW264.7 macrophages. In addition, we found that these changes were more significant in 40 mg/kg calycosin treatment than 20 mg/kg calycosin. CONCLUSIONS: Collectively, this study showed that calycosin might attenuate OVA-induced airway inflammation and remodeling in asthmatic mice via preventing ILC2 activation and macrophage M2 polarization. Our study might contribute to further study of asthmatic therapy.

The effect of Bruton's tyrosine kinase (BTK) inhibitor in the eosinophilic asthma model of mouse.

Bruton's Tyrosine kinase (BTK) plays a pivotal role as the key mediator in B cell signaling. Recent research has revealed that it is also expressed in cells critical to asthma development, such as T cells, and eosinophils. This study aims to investigate the potential of BTK inhibitor in eosinophilic asthma mouse model. BALB/c mice were sensitized with ovalbumin (OVA) via intraperitoneal injections and followed by OVA nebulizations. The mice were treated with 250 µg/ml or 500 µg/ml of ibrutinib before the second intraperitoneal injection and the first nebulization. Two days after the last OVA challenge, airway hyperresponsiveness (AHR) was assessed with methacholine, and differential cell count in bronchoalveolar lavage fluid (BALF) was performed. The cytokines were measured in BALF, and serum OVA-specific IgE and IgG antibody levels were evaluated by ELISA. The inhibitory effect of ibrutinib was also evaluated in splenic mononuclear cells, mast cells, eosinophils, and T cells in vitro. Treatment with ibrutinib significantly attenuated AHR and airway inflammation, compared to the OVA-induced positive control. The treatment also reduced IL-4, IL-5, IL-13 and IFN-γ cytokine levels and suppressed OVA-specific IgE and IgG production compared to the OVA-induced positive control. Additionally, ibrutinib decreased beta-hexosaminidase release from mast cells, type 2 cytokine productions from mononuclear cells and T cells, and eosinophilic activation markers in vitro. The results of this study suggest that ibrutinib treatment could exert anti-allergic effects by inactivating B cells and other BTK-expressing cells. Further studies are needed to investigate the potential therapeutic effect of ibrutinib on allergic diseases.

Prolonged exposure to lung-derived cytokines is associated with activation of microglia in patients with COVID-19.

BACKGROUNDSurvivors of pneumonia, including SARS-CoV-2 pneumonia, are at increased risk for cognitive dysfunction and dementia. In rodent models, cognitive dysfunction following pneumonia has been linked to the systemic release of lung-derived pro-inflammatory cytokines. Microglia are poised to respond to inflammatory signals from the circulation, and their dysfunction has been linked to cognitive impairment in murine models of dementia and in humans.METHODSWe measured levels of 55 cytokines and chemokines in bronchoalveolar lavage fluid and plasma from 341 patients with respiratory failure and 13 healthy controls, including 93 unvaccinated patients with COVID-19 and 203 patients with other causes of pneumonia. We used flow cytometry to sort neuroimmune cells from postmortem brain tissue from 5 patients who died from COVID-19 and 3 patients who died from other causes for single-cell RNA-sequencing.RESULTSMicroglia from patients with COVID-19 exhibited a transcriptomic signature suggestive of their activation by circulating pro-inflammatory cytokines. Peak levels of pro-inflammatory cytokines were similar in patients with pneumonia irrespective of etiology, but cumulative cytokine exposure was higher in patients with COVID-19. Treatment with corticosteroids reduced expression of COVID-19-specific cytokines.CONCLUSIONProlonged lung inflammation results in sustained elevations in circulating cytokines in patients with SARS-CoV-2 pneumonia compared with those with pneumonia secondary to other pathogens. Microglia from patients with COVID-19 exhibit transcriptional responses to inflammatory cytokines. These findings support data from rodent models causally linking systemic inflammation with cognitive dysfunction in pneumonia and support further investigation into the role of microglia in pneumonia-related cognitive dysfunction.FUNDINGSCRIPT U19AI135964, UL1TR001422, P01AG049665, P01HL154998, R01HL149883, R01LM013337, R01HL153122, R01HL147290, R01HL147575, R01HL158139, R01ES034350, R01ES027574, I01CX001777, U01TR003528, R21AG075423, T32AG020506, F31AG071225, T32HL076139.

Retrospective application of WHO reporting system for lung cytopathology with assessment of risk of malignancy.

INTRODUCTION: The recently introduced World Health Organization (WHO) Reporting System for Lung Cytopathology presents 5 diagnostic categories with corresponding risk of malignancy (ROM) and management protocols. This study uses the system to categorize our institutional respiratory tract cytology specimens, evaluating ROM and diagnostic accuracy for each category. MATERIALS AND METHODS: In a retrospective analysis (May 2020 to August 2021), the following respiratory cytology specimens were classified based on the WHO categories: bronchoalveolar lavage (BAL), bronchial wash/bronchial brushings (BB/BW), endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA), fine-needle aspiration cytology (FNAC), sputum, biopsy imprint (BI), and endotracheal wash. Exclusions comprised pleural effusions and EBUS-TBNA from mediastinal and hilar lymph nodes. Correlation of cytologic and histopathologic diagnoses was performed to assess ROM collectively and individually. RESULTS: A total of 1518 respiratory samples (BAL [968], BW/BB [380], EBUS-TBNA [42], FNAC [32], sputum [80], BI [11] and endotracheal wash [5]) of 1410 patients were screened, of which 522 cases (34.3%) had histopathologic correlation. One hundred forty-one cases (9.3%) were Insufficient/Inadequate/Non-Diagnostic (ND), 1221 (80.4%) were Benign (B), 3 (0.2%) were Atypical (A), 32 (2.1%) were Suspicious for malignancy (SM) and 121 (8.0%) were Malignant (M). The estimated ROM for each category was 49.2% for ND, 13.3% for B, 66.6% for A, 81.5% for SM and 92.7% for M. FNAC and EBUS-TBNA exhibited the highest sensitivity (100%) compared with BW/BB (66.3%). Specificity ranged from 96.8% to 100% across the samples, while diagnostic accuracy varied from 58.8% to 100%. CONCLUSIONS: Application of the WHO reporting system enhances standardized terminology, aiding clinicians in informed decision-making and improving patient care through accurate risk assessment of malignancy.

Bakuchicin alleviates ovalbumin-induced allergic asthma by regulating M2 macrophage polarization.

OBJECTIVE: Asthma is an airway inflammatory disease caused by activation of numerous immune cells including macrophages. Bakuchicin (BKC) is known to exhibit anti-inflammatory effects and type 2 T helper (Th2) regulation, but has not been investigated for airway inflammation. This study aimed to evaluate the effects of BKC on airway inflammation and demonstrate the mechanisms of macrophage polarization. METHODS: The anti-inflammatory effects were determined using lipopolysaccharide (LPS)-stimulated macrophages. The ovalbumin (OVA)-induced asthma mouse model was used to evaluate the effects of BKC on airway inflammation and Th2 responses. Moreover, the effect of BKC on macrophage polarization was confirmed in bone marrow-derived macrophages (BMDMs) differentiation. RESULTS: BKC suppressed nitric oxide production and expression of pro-inflammatory cytokines by inhibiting signaling pathway in LPS-stimulated macrophages. In an OVA-induced asthma model, BKC treatment alleviated histological changes and mast cell infiltration and reduced the levels of eosinophil peroxidase, ß-hexosaminidase, and immunoglobulin levels. In addition, BKC alleviated Th2 responses and M2 macrophage populations in bronchoalveolar fluid. In BMDMs, BKC suppressed IL-4-induced M2 macrophage polarization and the expression of M2 markers such as arginase-1 and Fizz-1 through inhibiting sirtuin 2 levels. CONCLUSION: BKC could be a drug candidate for the treatment of allergic asthma.

Strictosamide alleviates acute lung injury via regulating T helper 17 cells, regulatory T cells, and gut microbiota.

BACKGROUND: Nauclea officinalis (Pierre ex Pit.) Merr. & Chun (Rubiaceae) is widely used to treat respiratory diseases in China. Strictosamide is its main active component and has significant anti-inflammatory activity. However, the effects and molecular mechanisms of strictosamide in the treatment of acute lung injury (ALI) remain largely unknown. PURPOSE: This study aimed to examine the regulatory effects of strictosamide on T helper 17 cells (Th17 cells)/Regulatory T cells (Treg cells) and gut microbiota in ALI-affected mice. MATERIALS AND METHODS: The ALI model was induced using lipopolysaccharide (LPS) intraperitoneal injection. Hematoxylin-eosin (H&E) staining, the number of inflammatory cells in broncho-alveolar lavage fluid (BALF), the Wet/Dry (W/D) ratio, and myeloperoxidase (MPO) activity were utilized as evaluation indices for the therapeutic efficacy of strictosamide on ALI. Flow cytometry (FCM), enzyme-linked immune sorbent assay (ELISA), quantitative reverse transcription polymerase chain reaction (qRT-PCR), and western blotting were used to determine the regulation of strictosamide on the Th17/Treg cells and the STAT3/STAT5 signaling pathway. The analysis of gut microbiota was conducted using 16S rDNA sequencing. The verification of the relationship between the gut microbiome and immune function was conducted using Spearman analysis. RESULTS: Strictosamide attenuated inflammation on ALI induced by LPS, which reduced the levels of Th17-related factors interleukin (IL)-6 and IL-17 and increased Treg-related factors IL-10 and transforming growth factor (TGF)-ß. In the spleens and whole blood, strictosamide reduced the proportion of Th17 cells and increased the proportion of Treg cells. Furthermore, strictosamide increased Forkhead/winged helix transcription factor 3 (Foxp3) and p-STAT5 protein expression while inhibiting Retinoid-related orphan nuclear receptors-γt (RORγt) and p-STAT3 expression. Moreover, strictosamide reshaped the diversity and structure of the gut microbiota, and influence the associations between immune parameters and gut microbiota in ALI mice. CONCLUSIONS: In summary, the results of the current investigation showed that strictosamide has a therapeutic impact on LPS-induced ALI. The mechanism of action of this effect may be associated with the modulation of Th17 and Treg cells differentiation via the SATA signaling pathway, as well as the impact of the gut microbiota.

Endoplasmic reticulum stress drives macrophages to produce IL-33 to favor Th2 polarization in the airways.

Interleukin (IL)-33 is a key driver of T helper 2 (Th2) cell polarization. Endoplasmic reticulum (ER) stress plays a role in the skewed T cell activation. The objective of this project is to elucidate the role of IL-33 derived from macrophages in inducing Th2 polarization in the airways. In this study, bronchoalveolar lavage fluids (BALF) were collected from patients with asthma and healthy control subjects. Macrophages were isolated from the BALF by flow cytometry cell sorting. An asthmatic mouse model was established using the ovalbumin/alum protocol. The results showed that increased IL33 gene activity and ER stress-related molecules in BALF-derived M2a macrophages was observed in asthmatic patients. Levels of IL33 gene activity in M2a cells were positively correlated with levels of asthma response in asthma patients. Sensitization exacerbated the ER stress in the airway macrophages, which increased the expression of IL-33 in macrophages of airway in sensitized mice. Conditional ablation of Il33 or Perk or Atf4 genes in macrophages prevented induction of airway allergy in mice. In conclusion, asthma airway macrophages express high levels of IL-33 and at high ER stress status. Inhibition of IL-33 or ER stress in macrophages can effectively alleviate experimental asthma.

Th2-skewed peripheral T-helper cells drive B-cells in allergic bronchopulmonary aspergillosis.

INTRODUCTION: Patients with allergic bronchopulmonary aspergillosis (ABPA) suffer from repeated exacerbations. The involvement of T-cell subsets remains unclear. METHODS: We enrolled ABPA patients, asthma patients and healthy controls. T-helper type 1 (Th1), 2 (Th2) and 17 (Th17) cells, regulatory T-cells (Treg) and interleukin (IL)-21+CD4+T-cells in total or sorted subsets of peripheral blood mononuclear cells and ABPA bronchoalveolar lavage fluid (BALF) were analysed using flow cytometry. RNA sequencing of subsets of CD4+T-cells was done in exacerbated ABPA patients and healthy controls. Antibodies of T-/B-cell co-cultures in vitro were measured. RESULTS: ABPA patients had increased Th2 cells, similar numbers of Treg cells and decreased circulating Th1 and Th17 cells. IL-5+IL-13+IL-21+CD4+T-cells were rarely detected in healthy controls, but significantly elevated in the blood of ABPA patients, especially the exacerbated ones. We found that IL-5+IL-13+IL-21+CD4+T-cells were mainly peripheral T-helper (Tph) cells (PD-1+CXCR5-), which also presented in the BALF of ABPA patients. The proportions of circulating Tph cells were similar among ABPA patients, asthma patients and healthy controls, while IL-5+IL-13+IL-21+ Tph cells significantly increased in ABPA patients. Transcriptome data showed that Tph cells of ABPA patients were Th2-skewed and exhibited signatures of follicular T-helper cells. When co-cultured in vitro, Tph cells of ABPA patients induced the differentiation of autologous B-cells into plasmablasts and significantly enhanced the production of IgE. CONCLUSION: We identified a distinctly elevated population of circulating Th2-skewed Tph cells that induced the production of IgE in ABPA patients. It may be a biomarker and therapeutic target for ABPA.

Monotropein Alleviates Ovalbumin-Induced Asthma in Mouse Model by Inhibiting AKT/NF-κB Pathway.

INTRODUCTION: Clinical management of asthma remains as a prevalent challenge. Monotropein (MON) is a naturally occurring cyclic enol ether terpene glycoside with medical application potential. This study aims to evaluate the potential therapeutic effects of MON in the mouse model of chronic asthma. METHODS: An ovalbumin (OVA)-induced asthmatic mouse model was established to evaluate the therapeutic effect of MON at different doses (20, 40, and 80 mg/kg). The potential involvement of protein kinase B (AKT)/nuclear factor kappa B (NF-κB) pathway in the effect of MON was investigated by the administration of an AKT activator SC79. Histological changes in pulmonary tissues were examined by hematoxylin and eosin staining. The profiles of inflammatory cytokines (interleukin [IL]-4, IL-5, IL-13, and tumor necrosis factor [TNF]-α) in bronchoalveolar lavage fluid (BALF), and OVA-specific IgE in blood samples were analyzed by enzyme-linked immunosorbent assay (ELISA). The oxidative stress in the lung tissues was determined by measuring malondialdehyde level. The phosphorylation activation of AKT and NF-κB was examined by immunoblotting in the lung tissues. RESULTS: MON treatment suppressed the infiltration of inflammatory cells in the airways of OVA-induced asthma mice and reduced the thickness of the bronchial wall and smooth muscle layer in a dose-dependent manner. MON treatment also reduced the levels of OVA-specific IgE in serum and cytokines in BALF in asthma-induced mice, and attenuated the oxidative stress in the lung tissues. OVA induced the phosphorylation of AKT and NF-κB proteins in the lung tissues of asthmatic mice, which was significantly suppressed by MON treatment. The co-administration of AKT activator SC79 impaired the therapeutic effect of MON on asthma-induced mice. CONCLUSION: Our data demonstrated the potential therapeutic effect of MON on asthmatic mouse model, suggesting that MON attenuated the inflammatory and oxidative damages in ling tissues by dampening the AKT/NF-κB signaling pathway.

Inter-species cell detection - datasets on pulmonary hemosiderophages in equine, human and feline specimens.

Pulmonary hemorrhage (P-Hem) occurs among multiple species and can have various causes. Cytology of bronchoalveolar lavage fluid (BALF) using a 5-tier scoring system of alveolar macrophages based on their hemosiderin content is considered the most sensitive diagnostic method. We introduce a novel, fully annotated multi-species P-Hem dataset, which consists of 74 cytology whole slide images (WSIs) with equine, feline and human samples. To create this high-quality and high-quantity dataset, we developed an annotation pipeline combining human expertise with deep learning and data visualisation techniques. We applied a deep learning-based object detection approach trained on 17 expertly annotated equine WSIs, to the remaining 39 equine, 12 human and 7 feline WSIs. The resulting annotations were semi-automatically screened for errors on multiple types of specialised annotation maps and finally reviewed by a trained pathologist. Our dataset contains a total of 297,383 hemosiderophages classified into five grades. It is one of the largest publicly available WSIs datasets with respect to the number of annotations, the scanned area and the number of species covered.

Early life exposure to nicotine modifies lung gene response after elastase-induced emphysema.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is among the top 5 causes of mortality in the world and can develop as a consequence of genetic and/or environmental factors. Current efforts are focused on identifying early life insults and how these contribute to COPD development. In line with this, our study focuses on the influence of early life nicotine exposure and its potential impact on (a) lung pulmonary functions, and (b) elastase-induced emphysema in adulthood. METHODS: To address this hypothesis, we developed a model of 2 hits, delivered at different time points: mouse pups were first exposed to nicotine/placebo in utero and during lactation, and then subsequently received elastase/placebo at the age of 11 weeks. The effect of nicotine pretreatment and elastase instillation was assessed by (a) measurement of pulmonary function at post-elastase day (ped) 21, and (b) transcriptomic profiling at ped3 and 21, and complementary protein determination. Statistical significance was determined by 3- and 2-way ANOVA for pulmonary functions, and RNAseq results were analyzed using the R project. RESULTS: We did not observe any impact of nicotine pre- and early post-natal exposure compared to control samples on lung pulmonary functions in adulthood, as measured by FLEXIVENT technology. After elastase instillation, substantial lung damage was detected by x-ray tomography and was accompanied by loss in body weight at ped3 as well as an increase in cell numbers, inflammatory markers in BAL and lung volume at ped21. Lung functions showed a decrease in elastance and an increase in deep inflation volume and pressure volume (pv) loop area in animals with emphysema at ped21. Nicotine had no effect on elastance and deep inflation volume, but did affect the pv loop area in animals with emphysema at ped21. Extensive transcriptomic changes were induced by elastase at ped3 both in the nicotine-pretreated and the control samples, with several pathways common to both groups, such as for cell cycle, DNA adhesion and DNA damage. Nicotine pretreatment affected the number of lymphocytes present in BAL after elastase instillation and some of the complement pathway related proteins, arguing for a slight modification of the immune response, as well as changes related to general body metabolism. The majority of elastase-induced transcriptomic changes detected at ped3 had disappeared at ped21. In addition, transcriptomic profiling singled out a common gene pool that was independently activated by nicotine and elastase. CONCLUSIONS: Our study reports a broad spectrum of transient transcriptomic changes in mouse emphysema and identifies nicotine as influencing the emphysema-associated immune system response.

Anti-inflammatory effect of flavonoids from chestnut flowers in lipopolysaccharide-stimulated RAW 264.7 macrophages and acute lung injury in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Chestnut flowers were one of the by-products during chestnut industrial processing. Chestnut (Castanea mollissima Blume) flower is rich in flavonoids and has been used as a traditional medicine to treat a variety of diseases including respiratory disorders for a long history. AIM OF THE STUDY: The present study aims to investigate the potential anti-inflammatory effect of flavonoids from chestnut flower (FCF) in lipopolysaccharide (LPS)-treated RAW 264.7 cells and stimulated acute lung injury (ALI) in mice. MATERIALS AND METHODS: HPLC-ESI-MS/MS was applied to identify flavonoids from Chestnut flower. The ROS content in cells and lung tissue was measured by flow cytometry. The malondialdehyde (MDA) content, superoxide dismutase (SOD) activity and glutathione (GSH) content in cells and bronchoalveolar lavage fluid (BALF) was analyzed by photometry. Furthermore, the level of pro-inflammatory factors was analyzed by ELISA, and the expression of inflammatory gene mRNA by fluorescence quantitative PCR. H&E staining was used to evaluate the degree of lung tissue injury in mice. MPO activity was used to measure the degree of neutrophil infiltration. Total protein content was detected by BCA method. RESULTS: A total of forty-nine flavonoids compounds were tentatively identified in FCF by mass spectrometry analysis. The results of cell experiment suggested that FCF could alleviate oxidative injury via increasing SOD activity and GSH content, as well as inhibiting the production of intracellular ROS and MDA. FCF exerted its protective effect by suppressing the expression of both inducible nitric oxide synthase (iNOS) and cycooxygenase 2 (COX-2) to inhibit the synthesis of pro-inflammatory factors and cytokines, including NO, PGE2, TNF-α, IL-6 and IL-1ß. Besides, FCF treatment could alleviate the thickening of alveolar wall and pulmonary congestion in LPS-treated ALI mice, and significantly inhibit the activity of myeloperoxidas (MPO) and the expression of cytokines in BALF. CONCLUSIONS: FCF could ameliorate inflammation and oxidative stress in LPS-treated inflammation, resulting in an overall improvement in both macroscopic and histological parameters.