The clinical impacts of lung microbiome in bronchiectasis with fixed airflow obstruction: a prospective cohort study.

BACKGROUND: Airflow obstruction is a hallmark of disease severity and prognosis in bronchiectasis. The relationship between lung microbiota, airway inflammation, and outcomes in bronchiectasis with fixed airflow obstruction (FAO) remains unclear. This study explores these interactions in bronchiectasis patients, with and without FAO, and compares them to those diagnosed with chronic obstructive pulmonary disease (COPD). METHODS: This prospective observational study in Taiwan enrolled patients with either bronchiectasis or COPD. To analyze the lung microbiome and assess inflammatory markers, bronchoalveolar lavage (BAL) samples were collected for 16S rRNA gene sequencing. The study cohort comprised 181 patients: 86 with COPD, 46 with bronchiectasis, and 49 with bronchiectasis and FAO, as confirmed by spirometry. RESULTS: Patients with bronchiectasis, with or without FAO, had similar microbiome profiles characterized by reduced alpha diversity and a predominance of Proteobacteria, distinctly different from COPD patients who exhibited more Firmicutes, greater diversity, and more commensal taxa. Furthermore, compared to COPD and bronchiectasis without FAO, bronchiectasis with FAO showed more severe disease and a higher risk of exacerbations. A significant correlation was found between the presence of Pseudomonas aeruginosa and increased airway neutrophilic inflammation such as Interleukin [IL]-1ß, IL-8, and tumor necrosis factor-alpha [TNF]-α, as well as with higher bronchiectasis severity, which might contribute to an increased risk of exacerbations. Moreover, in bronchiectasis patients with FAO, the ROSE (Radiology, Obstruction, Symptoms, and Exposure) criteria were employed to classify individuals as either ROSE (+) or ROSE (-), based on smoking history. This classification highlighted differences in clinical features, inflammatory profiles, and slight microbiome variations between ROSE (-) and ROSE (+) patients, suggesting diverse endotypes within the bronchiectasis with FAO group. CONCLUSION: Bronchiectasis patients with FAO may exhibit two distinct endotypes, as defined by ROSE criteria, characterized by greater disease severity and a lung microbiome more similar to bronchiectasis without FAO than to COPD. The significant correlation between Pseudomonas aeruginosa colonization and increased airway neutrophilic inflammation, as well as disease severity, underscores the clinical relevance of microbial patterns. This finding reinforces the potential role of these patterns in the progression and exacerbations of bronchiectasis with FAO.

Nrf2 Deficiency Accelerates IL-17-Dependent Neutrophilic Airway Inflammation in Asthmatic Mice.

Asthma is a heterogeneous disease that can be broadly classified into type 2, which is primarily steroid-sensitive and eosinophilic, and non-type 2, which is primarily steroid-resistant and neutrophilic. While the mechanisms leading to the development of molecular-targeted therapies for type 2 asthma are being elucidated, much remains to be learned about non-type 2 asthma. To investigate the role of oxidative stress in refractory allergic airway inflammation, we compared asthma models generated by immunizing wild-type and nuclear factor erythroid-2-related factor 2 (Nrf2)-deficient mice with the house dust mite antigen. Both asthma models had similar levels of airway inflammation and hyperresponsiveness, but the Nrf2-deficient mice had increased oxidative stress and exacerbated neutrophilic airway inflammation compared with the wild-type mice. Type 2 cytokines and the expression of GATA3, a transcription factor that is important for Th2 cell differentiation, had decreased in Nrf2-deficient mice compared with the wild-type mice, whereas helper T (Th) 17 cytokines and the expression of RORγt, which is important for Th17 cell differentiation, had increased. Furthermore, the neutrophilic airway inflammation caused by Nrf2 deficiency was ameliorated by interleukin (IL)-17 neutralization. We have concluded that the disruption of the Nrf2-mediated antioxidant defense system contributed to the induction of Th17 differentiation and exacerbated allergic neutrophilic airway inflammation.

Immuno-Nanocomplexes Target Heterogenous Network of Inflammation and Immunity in Myocardial Infarction.

Despite the proceeds in the management of acute myocardial infarction (AMI), the current therapeutic landscape still suffers from limited success in the clinic. Exaggerated inflammatory immune response and excessive oxidative stress are key pathological features aggravating myocardium damage. Herein, catalytic immunomodulatory nanocomplexes as anti-AMI therapeutics to resolve reactive oxygen species (ROS)-proinflammatory neutrophils-specific-inflammation is engineered. The nanocomplexes contain lyophilic S100A8/9 inhibitor ABR2575 in the core of nanoemulsions, which effectively disrupts the neutrophils-S100A8/A9-inflammation signaling pathway in the AMI microenvironment. Additionally, ROS scavenger ultrasmall CuxO nanoparticles are incorporated into the nanoemulsions via coordinating with SH groups of poly(ethylene glycol) (PEG)-conjugated lipids, which mimic multiple enzymes, dramatically alleviating the oxidative stress damage to myocardial tissue. This combination strategy significantly suppresses the infiltration of pro-inflammatory monocytes, macrophages, and neutrophils, as well as the secretion of inflammatory cytokines. Additionally, it potentially triggers cardiac Tert activation, which promotes myocardial function and decreases infarction size in preclinical murine AMI models. This approach offers a new nanomedicine for treating AMI, resulting in a dramatically enhanced therapeutic outcome.

Endotypes of severe neutrophilic and eosinophilic asthma from multi-omics integration of U-BIOPRED sputum samples.

BACKGROUND: Clustering approaches using single omics platforms are increasingly used to characterise molecular phenotypes of eosinophilic and neutrophilic asthma. Effective integration of multi-omics platforms should lead towards greater refinement of asthma endotypes across molecular dimensions and indicate key targets for intervention or biomarker development. OBJECTIVES: To determine whether multi-omics integration of sputum leads to improved granularity of the molecular classification of severe asthma. METHODS: We analyzed six -omics data blocks-microarray transcriptomics, gene set variation analysis of microarray transcriptomics, SomaSCAN proteomics assay, shotgun proteomics, 16S microbiome sequencing, and shotgun metagenomic sequencing-from induced sputum samples of 57 severe asthma patients, 15 mild-moderate asthma patients, and 13 healthy volunteers in the U-BIOPRED European cohort. We used Monti consensus clustering algorithm for aggregation of clustering results and Similarity Network Fusion to integrate the 6 multi-omics datasets of the 72 asthmatics. RESULTS: Five stable omics-associated clusters were identified (OACs). OAC1 had the best lung function with the least number of severe asthmatics with sputum paucigranulocytic inflammation. OAC5 also had fewer severe asthma patients but the highest incidence of atopy and allergic rhinitis, with paucigranulocytic inflammation. OAC3 comprised only severe asthmatics with the highest sputum eosinophilia. OAC2 had the highest sputum neutrophilia followed by OAC4 with both clusters consisting of mostly severe asthma but with more ex/current smokers in OAC4. Compared to OAC4, there was higher incidence of nasal polyps, allergic rhinitis, and eczema in OAC2. OAC2 had microbial dysbiosis with abundant Moraxella catarrhalis and Haemophilus influenzae. OAC4 was associated with pathways linked to IL-22 cytokine activation, with the prediction of therapeutic response to anti-IL22 antibody therapy. CONCLUSION: Multi-omics analysis of sputum in asthma has defined with greater granularity the asthma endotypes linked to neutrophilic and eosinophilic inflammation. Modelling diverse types of high-dimensional interactions will contribute to a more comprehensive understanding of complex endotypes. KEY POINTS: Unsupervised clustering on sputum multi-omics of asthma subjects identified 3 out of 5 clusters with predominantly severe asthma. One severe asthma cluster was linked to type 2 inflammation and sputum eosinophilia while the other 2 clusters to sputum neutrophilia. One severe neutrophilic asthma cluster was linked to Moraxella catarrhalis and to a lesser extent Haemophilus influenzae while the second cluster to activation of IL-22.

Identification of novel biomarkers related to neutrophilic inflammation in COPD.

Background: Chronic obstructive pulmonary disease (COPD) is one of the most prevalent chronic respiratory diseases and the fourth cause of mortality globally. Neutrophilic inflammation has a vital role in the occurrence and progression of COPD. This study aimed to identify the novel hub genes involved in neutrophilic inflammation in COPD through bioinformatic prediction and experimental validation. Methods: Both the single-cell RNA sequencing (scRNA-seq) dataset (GSE173896) and the RNA sequencing (RNA-seq) dataset (GSE57148) were downloaded from the Gene Expression Omnibus (GEO) database. The Seurat package was used for quality control, dimensions reduction, and cell identification of scRNA-seq. The irGSEA package was used for scoring individual cells. The Monocle2 package was used for the trajectory analysis of neutrophils. The CIBERSORT algorithm was used for analysis of immune cell infiltration in the lungs of COPD patients and controls in RNA-seq dataset, and weighted gene co-expression network analysis (WGCNA) correlated gene modules with neutrophil infiltration. The Mendelian randomization (MR) analysis explored the causal relationship between feature DEGs and COPD. The protein-protein interaction (PPI) network of novel hub genes was constructed, and real-time quantitative polymerase chain reaction (qRT-PCR) was used to validate novel hub genes in clinical specimens. Results: In scRNA-seq, the gene sets upregulated in COPD samples were related to the neutrophilic inflammatory response and TNF-α activation of the NF-κB signaling pathway. In RNA-seq, immune infiltration analysis showed neutrophils were upregulated in COPD lung tissue. We combined data from differential and modular genes and identified 51 differential genes associated with neutrophilic inflammation. Using MR analysis, 6 genes were explored to be causally associated with COPD. Meanwhile, 11 hub genes were identified by PPI network analysis, and all of them were upregulated. qRT-PCR experiments validated 9 out of 11 genes in peripheral blood leukocytes of COPD patients. Furthermore, 5 genes negatively correlated with lung function in COPD patients. Finally, a network of transcription factors for NAMPT and PTGS2 was constructed. Conclusion: This study identified nine novel hub genes related to the neutrophilic inflammation in COPD, and two genes were risk factors of COPD, which may serve as potential biomarkers for the clinical severity of COPD.

Cooperation of immune regulators Tollip and surfactant protein A inhibits influenza A virus infection in mice.

BACKGROUND: Influenza A virus (IAV) infection is a significant risk factor for respiratory diseases, but the host defense mechanisms against IAV remain to be defined. Immune regulators such as surfactant protein A (SP-A) and Toll-interacting protein (Tollip) have been shown to be involved in IAV infection, but whether SP-A and Tollip cooperate in more effective host defense against IAV infection has not been investigated. METHODS: Wild-type (WT), Tollip knockout (KO), SP-A KO, and Tollip/SP-A double KO (dKO) mice were infected with IAV for four days. Lung macrophages were isolated for bulk RNA sequencing. Precision-cut lung slices (PCLS) from WT and dKO mice were pre-treated with SP-A and then infected with IAV for 48 h. RESULTS: Viral load was significantly increased in bronchoalveolar lavage (BAL) fluid of dKO mice compared to all other strains of mice. dKO mice had significantly less recruitment of neutrophils into the lung compared to Tollip KO mice. SP-A treatment of PCLS enhanced expression of TNF and reduced viral load in dKO mouse lung tissue. Pathway analysis of bulk RNA sequencing data suggests that macrophages from IAV-infected dKO mice reduced expression of genes involved in neutrophil recruitment, IL-17 signaling, and Toll-like receptor signaling. CONCLUSIONS: Our data suggests that both Tollip and SP-A are essential for the lung to exert more effective innate defense against IAV infection.

Impaired autophagy in myeloid cells aggravates psoriasis-like skin inflammation through the IL-1ß/CXCL2/neutrophil axis.

BACKGROUND: Psoriasis is an inflammatory skin disease characterized by the hyperproliferative epidermal keratinocytes and significant immune cells infiltration, leading to cytokines production such as IL-1ß, TNF-α, IL-23, and IL-17. Recent study highlights the critical role of IL-1ß in the induction and activation of pathogenic Th17 and IL-17-producing γδ T cells, contributing to psoriasis. However, the mechanism underlying IL-1ß dysregulation in psoriasis pathogenesis is unclear. Autophagy regulates IL-1ß production and has a pleiotropic effect on inflammatory disorders. Previous studies showed controversial role of autophagy in psoriasis pathogenesis, either pro-inflammatory in autophagy-deficient keratinocyte or anti-inflammatory in pharmacologically autophagy-promoting macrophages. Thus, the direct role of autophagy and its therapeutic potential in psoriasis remains unclear. METHODS: We used myeloid cell-specific autophagy-related gene 7 (Atg7)-deficient mice and determined the effect of autophagy deficiency in myeloid cells on neutrophilia and disease pathogenesis in an imiquimod-induced psoriasis mouse model. We then assessed the pathogenic mechanism focusing on immune cells producing IL-1ß and IL-17 along with gene expression profiles associated with psoriasis in mouse model and public database on patients. Moreover, therapeutic potential of IL-1ß blocking in such context was assessed. RESULTS: We found that autophagy deficiency in myeloid cells exacerbated neutrophilic inflammation and disease pathogenesis in mice with psoriasis. This autophagy-dependent effect was associated with a significant increase in IL-1ß production from myeloid cells, particularly macrophages, Cxcl2 expression, and IL-17 A producing T cells including γδ T cells. Supporting this, treatment with systemic IL-1 receptor blocking antibody or topical saccharin, a disaccharide suppressing pro-IL-1ß expression, led to the alleviation of neutrophilia and psoriatic skin inflammation linked to autophagy deficiency. The pathophysiological relevance of this finding was supported by dysregulation of autophagy-related genes and their correlation with Th17 cytokines in psoriatic skin lesion from patients with psoriasis. CONCLUSIONS: Our results suggest that autophagy dysfunction in myeloid cells, especially macrophages, along with IL-1ß dysregulation has a causal role in neutrophilic inflammation and psoriasis pathogenesis.

The Efficacy & Molecular Mechanisms of a Terpenoid Compound Ganoderic Acid C1 on Corticosteroid-Resistant Neutrophilic Airway Inflammation: In vivo and in vitro Validation.

Introduction: Neutrophil predominant airway inflammation is associated with severe and steroid-resistant asthma clusters. Previously, we reported efficacy of ASHMI, a three-herb TCM asthma formula in a steroid-resistant neutrophil-dominant murine asthma model and further identified Ganoderic Acid C1 (GAC1) as a key ASHMI active compound in vitro. The objective of this study is to investigate GAC1 effect on neutrophil-dominant, steroid-resistant asthma in a murine model. Methods: In this study, Balb/c mice were systematically sensitized with ragweed (RW) and alum and intranasally challenged with ragweed. Unsensitized/PBS challenged mice served as normal controls. Post sensitization, mice were given 4 weeks of oral treatment with GAC1 or acute dexamethasone (Dex) treatment at 48 hours prior to challenge. Pulmonary cytokines were measured by ELISA, and lung sections were processed for histology by H&E staining. Furthermore, GAC1 effect on MUC5AC expression and on reactive oxygen species (ROS) production in human lung epithelial cell line (NCI-H292) was determined by qRT-PCR and ROS assay kit, respectively. Computational analysis was applied to select potential targets of GAC1 in steroid-resistant neutrophil-dominant asthma. Molecular docking was performed to predict binding modes between GAC1 and Dex with TNF-α. Results: The result of the study showed that chronic GAC1 treatment, significantly reduced pulmonary inflammation (P < 0.01-0.001 vs Sham) and airway neutrophilia (P < 0.01 vs Sham), inhibited TNF-α, IL-4 and IL-5 levels (P < 0.05-0.001 vs Sham). Acute Dex treatment reduced eosinophilic inflammation and IL-4, IL-5 levels, but had no effect on neutrophilia and TNF-α production. GAC1 treated H292 cells showed decreased MUC5AC gene expression and production of ROS (P < 0.001 vs stimulated/untreated cells). Molecular docking results showed binding energy of complex GAC1-TNF was -10.8 kcal/mol. Discussion: GAC1 may be a promising anti-asthma botanical drug for treatment of steroid-resistant asthma.

Ribociclib leverages phosphodiesterase 4 inhibition in the treatment of neutrophilic inflammation and acute respiratory distress syndrome.

INTRODUCTION: Overwhelming neutrophil activation and oxidative stress significantly contribute to acute respiratory distress syndrome (ARDS) pathogenesis. However, the potential of repurposing ribociclib, a cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor used clinically in cancer treatment, for treating neutrophilic ARDS remains uncertain. This study illustrated the ability and underlying mechanism of ribociclib for treating ARDS and neutrophilic inflammation. METHODS: Primary human neutrophils were used to determine the therapeutic effects of ribociclib on respiratory bursts, chemotactic responses, and inflammatory signaling. In vitro and silico analyses were performed to determine the underlying molecular mechanisms. The potential of ribociclib repurposing was evaluated using an in vivo ARDS model in lipopolysaccharide (LPS)-primed mice. RESULTS: We found that treatment using ribociclib markedly limited overabundant oxidative stress (reactive oxygen species [ROS]) production and chemotactic responses (integrin levels and adhesion) in activated human neutrophils. Ribociclib was also shown to act as a selective inhibitor of phosphodiesterase 4 (PDE4), thereby promoting the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway, leading to the inhibition of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) phosphorylation, and calcium influx. Notably, prophylactic administration and post-treatment with ribociclib ameliorated neutrophil infiltration, lung inflammation, accumulation of oxidative stress, pulmonary destruction, and mortality in mice with LPS-induced ARDS. CONCLUSION: We demonstrated for the first time that ribociclib serves as a novel PDE4 inhibitor for treating neutrophilic inflammation and ARDS. The repurposing ribociclib and targeting neutrophilic PDE4 offer a potential off-label alternative for treating lung lesions and other inflammatory conditions.

Tackling Severe Neutrophilic Inflammation in Airway Disorders with Functionalized Nanosheets.

Severe airway inflammatory disorders impose a significant societal burden, and the available treatments are unsatisfactory. High levels of neutrophil extracellular trap (NET) and cell-free DNA (cfDNA) were detected in the inflammatory microenvironment of these diseases, which are closely associated with persistent uncontrolled neutrophilic inflammation. Although DNase has proven to be effective in mitigating neutrophilic airway inflammation in mice by reducing cfDNA and NET levels, its clinical use is hindered by severe side effects. Here, we synthesized polyglycerol-amine (PGA) with a series of hydroxyl/amine ratios and covered them with black phosphorus (BP) nanosheets. The BP nanosheets functionalized with polyglycerol-50% amine (BP-PGA50) efficiently lowered cfDNA levels, suppressed toll-like receptor 9 (TLR9) activation and inhibited NET formation in vitro. Importantly, BP-PGA50 nanosheets demonstrated substantial accumulation in inflamed airway tissues, excellent biocompatibility, and potent inflammation modulation ability in model mice. The 2D sheet-like structure of BP-PGA50 was identified as a crucial factor for the therapeutic efficacy, and the hydroxyl/amine ratio was revealed as a significant parameter to regulate the protein resistance, cfDNA-binding efficacy, and cytotoxicity. This study shows the promise of the BP-PGA50 nanosheet for tackling uncontrolled airway inflammation, which is also significant for the treatment of other neutrophilic inflammatory diseases. In addition, our work also highlights the importance of proper surface functionalization, such as hydroxyl/amine ratio, in therapeutic nanoplatform construction for inflammation modulation.

Pathogenesis-Guided Rational Engineering of Nanotherapies for the Targeted Treatment of Abdominal Aortic Aneurysm by Inhibiting Neutrophilic Inflammation.

Abdominal aortic aneurysm (AAA) remains a fatal disease in the elderly. Currently, no drugs can be clinically used for AAA therapy. Considering the pivotal role of neutrophils in the pathogenesis of AAA, herein we propose the targeted therapy of AAA by site-specifically regulating neutrophilic inflammation. Based on a luminol-conjugated α-cyclodextrin material (LaCD), intrinsically anti-inflammatory nanoparticles (NPs) were engineered by simple nanoprecipitation, which were examined as a nanotherapy (defined as LaCD NP). After efficient accumulation in the aneurysmal aorta and localization in pathologically relevant inflammatory cells in rats with CaCl2-induced AAA, LaCD NP significantly alleviated AAA progression, as implicated by the decreased aortic expansion, suppressed elastin degradation, inhibited calcification, and improved structural integrity of the abdominal aorta. By functionalizing LaCD NP with alendronate, a calcification-targeting moiety, the in vivo aneurysmal targeting capability of LaCD NP was considerably enhanced, thereby affording significantly potentiated therapeutic outcomes in AAA rats. Mechanistically, LaCD NP can effectively inhibit neutrophil-mediated inflammatory responses in the aneurysmal aorta. Particularly, LaCD NP potently attenuated the formation of neutrophil extracellular traps (NETs), thereby suppressing NETs-mediated pro-inflammatory events and NETosis-associated negative effects responsible for AAA progression. Consequently, we demonstrated the effectiveness and underlying mechanisms of anti-NETosis nanotherapies for the targeted treatment of AAA. Our findings provide promising insights into discovering precision therapies for AAA and other inflammatory vascular diseases.

Tobacco introduced Perilla frutescens and Ocimum basilicum genes attenuates neutrophilic inflammation in lung tissues of COPD rats.

The new-type tobacco varieties "Zisu" and "Luole" were obtained by distant hybridization between N. tabacum L. var. HHY and Perilla frutescens and Ocimum basilicum, with obviously different chemical composition. Smoking is the major risk factor for COPD, characterized by neutrophil-dominant inflammation. In the present study, rat COPD model was established by cigarette exposure, and the health hazard of three varieties was compared by general condition observation, pathological and morphological evaluation, total and differential cell numeration, and characterization of major inflammatory mediators and MAPK/NF-κB pathway, etc. Rats in "HHY" group developed obvious symptoms such as cough, dyspnea, mental fatigue, etc., but these symptoms were obviously mitigated in "Zisu" and "Luole" groups. H&E staining analysis, including score, MLI, MAN, wt% and WA%, showed that "Zisu" and "Luole" significantly alleviated lung injury and the degree of airway remodeling and emphysema compared to "HHY". In BALF, the number of total leukocyte and the percent neutrophils in "Zisu" and "Luole" groups were evidently lower than "HHY" group. The levels of inflammatory mediators, such as IL-8, MPO, MIP-2, LTB4, TNF-α and neutrophil elastase, in "HHY" group were obviously higher than "Zisu" and "Luole" groups. The ROS-mediated NF-κB p65 and p38MAPK pathways may play an important role. Results indicated that tobacco introduced perilla and basil genes could remarkably attenuate recruitment, infiltration and activation of neutrophils and intervene in airway inflammation, retarding disease progression, especially "Zisu". Changes in chemical composition via breeding techniques may be a novel way for tobacco harm reduction.

Unlocking the Potential of Octocoral-Derived Secondary Metabolites against Neutrophilic Inflammatory Response.

Inflammation is a critical defense mechanism that is utilized by the body to protect itself against pathogens and other noxious invaders. However, if the inflammatory response becomes exaggerated or uncontrollable, its original protective role is not only demolished but it also becomes detrimental to the affected tissues or even to the entire body. Thus, regulating the inflammatory process is crucial to ensure that it is resolved promptly to prevent any subsequent damage. The role of neutrophils in inflammation has been highlighted in recent decades by a plethora of studies focusing on neutrophilic inflammatory diseases as well as the mechanisms to regulate the activity of neutrophils during the overwhelmed inflammatory process. As natural products have demonstrated promising effects in a wide range of pharmacological activities, they have been investigated for the discovery of new anti-inflammatory therapeutics to overcome the drawbacks of current synthetic agents. Octocorals have attracted scientists as a plentiful source of novel and intriguing marine scaffolds that exhibit many pharmacological activities, including anti-inflammatory effects. In this review, we aim to provide a summary of the neutrophilic anti-inflammatory properties of these marine organisms that were demonstrated in 46 studies from 1995 to the present (April 2023). We hope the present work offers a comprehensive overview of the anti-inflammatory potential of octocorals and encourages researchers to identify promising leads among numerous compounds isolated from octocorals over the past few decades to be further developed into anti-inflammatory therapeutic agents.

Canakinumab leads to rapid reduction of neutrophilic inflammation and long-lasting response in Schnitzler syndrome.

Interleukin-1 (IL-1)-blocking therapies are effective in reducing disease severity and inflammation in Schnitzler syndrome. Here, we present a patient with Schnitzler syndrome treated successfully using canakinumab for over 10 years. Complete clinical response was associated with a decrease in dermal neutrophil number and expression of the pro-inflammatory cytokines IL-1ß, IL-8, and IL-17 as assessed by immunohistochemical studies.

Endocrine-disrupting chemical exposure augments neutrophilic inflammation in severe asthma through the autophagy pathway.

Corticosteroid resistance, progressive lung function decline, and frequent asthma exacerbations are the hallmarks of neutrophilic asthma (NA). However, the potential contributors and their mechanisms of NA aggravation have not yet been fully clarified. This study was conducted to assess the precise mechanism and inflammatory effects of endocrine-disrupting chemicals using mono-n-butyl phthalate (MnBP) on an NA model. BALB/c mice from normal control and LPS/OVA-induced NA groups were treated with or without MnBP. The effects of MnBP on the airway epithelial cells (AECs), macrophages (Mφ), and neutrophils were investigated in vitro and in vivo. NA mice exposed to MnBP had significantly increased airway hyperresponsiveness, total and neutrophil cell counts in the bronchoalveolar lavage fluid, and the percentage of M1Mφ in the lung tissues compared to those non-exposed to MnBP. In in vitro study, MnBP induced the human neutrophil activation to release neutrophil DNA extracellular traps, Mφ polarizing toward M1Mφ, and AEC damage. Treatment with hydroxychloroquine (an autophagy inhibitor) reduced the effects of MnBP in vivo and in vitro. The results of our study suggest that MnBP exposure may increase the risk of neutrophilic inflammation in severe asthma and autophagy pathway-targeted therapeutics can help control MnBP-induced harmful effects in asthma.

Characterization of acute lung injury in the bleomycin rat model.

The aim of this study was to describe and characterize the pathophysiological changes occurring during the early inflammatory phase (first 3 days) in the rat bleomycin model of lung injury preceding the development of fibrosis. Further, we wanted to understand the kinetics and factors contributing to bleomycin-induced acute lung injury (ALI) and provide a robust, reliable and reproducible framework of features of ALI readouts to assess effects of therapeutics on bleomycin-induced ALI in rats. We induced ALI in rats with intratracheal (i.t.) installation of bleomycin. The animals were sacrificed on predetermined time points, that is, Day 0, 1, 2, and 3 post the bleomycin challenge. We analyzed bronchoalveolar lavage fluid (BALF) and lung tissue to establish and assess relevant experimental features of ALI. We demonstrated that bleomycin induced key features of experimental ALI including a profound increase in neutrophils in BALF (50-60%), pulmonary edema, and lung pathology on Day 3 after challenge. Furthermore, we showed that TGF-ß1, IL-1ß, TNF-α, IL-6, CINC-1, TIMP-1, and WISP-1 were induced by studying their kinetic profile during the first 3 days after bleomycin injury consistent with their known role ALI. We also confirmed that detectable fibrogenesis occurs at the earliest on Day 3 after injury based on collagen content, along with changes in the TGF-ß/Smad signaling pathway and increased expression of Galectin-3, Vimentin, and Fibronectin in lung homogenate. Our report presents robust features and contributing mediators/factors to the pathology of bleomycin-induced ALI in rats on Day 3. The kinetic data provide insights on the progression of ALI and a detailed understanding of early events before actual fibrosis development. This set of experimental endpoints is very appropriate and invaluable for efficacy testing of potential novel therapeutic treatments (single or combined) in ALI and understanding their mechanism of action.

Group 3 innate lymphoid cells secret neutrophil chemoattractants and are insensitive to glucocorticoid via aberrant GR phosphorylation.

BACKGROUND: Patients with neutrophil-mediated asthma have poor response to glucocorticoids. The roles and mechanisms of group 3 innate lymphoid cells (ILC3s) in inducing neutrophilic airway inflammation and glucocorticoid resistance in asthma have not been fully clarified. METHODS: ILC3s in peripheral blood were measured by flow cytometry in patients with eosinophilic asthma (EA) and non-eosinophilic asthma (NEA). ILC3s were sorted and cultured in vitro for RNA sequencing. Cytokines production and signaling pathways in ILC3s after IL-1ß stimulation and dexamethasone treatment were determined by real-time PCR, flow cytometry, ELISA and western blot. RESULTS: The percentage and numbers of ILC3s in peripheral blood was higher in patients with NEA compared with EA, and negatively correlated with blood eosinophils. IL-1ß stimulation significantly enhanced CXCL8 and CXCL1 production in ILC3s via activation of p65 NF-κB and p38/JNK MAPK signaling pathways. The expression of neutrophil chemoattractants from ILC3s was insensitive to dexamethasone treatment. Dexamethasone significantly increased phosphorylation of glucocorticoid receptor (GR) at Ser226 but only with a weak induction at Ser211 residues in ILC3s. Compared to human bronchial epithelial cell line (16HBE cells), the ratio of p-GR S226 to p-GR S211 (p-GR S226/S211) was significantly higher in ILC3s at baseline and after dexamethasone treatment. In addition, IL-1ß could induce Ser226 phosphorylation and had a crosstalk effect to dexamethasone via NF-κB pathway. CONCLUSIONS: ILC3s were elevated in patients with NEA, and associated with neutrophil inflammation by release of neutrophil chemoattractants and were glucocorticoid (GC) resistant. This paper provides a novel cellular and molecular mechanisms of neutrophil inflammation and GC-resistance in asthma. Trial registration The study has been prospectively registered in the World Health Organization International Clinical Trials Registry Platform (ChiCTR1900027125).

TNFR2+ regulatory T cells protect against bacteremic pneumococcal pneumonia by suppressing IL-17A-producing γδ T cells in the lung.

Streptococcus pneumoniae is a pathogen of global morbidity and mortality. Pneumococcal pneumonia can lead to systemic infections associated with high rates of mortality. We find that, upon pneumococcal infection, pulmonary Treg cells are activated and have upregulated TNFR2 expression. TNFR2-deficient mice have compromised Treg cell responses and highly activated IL-17A-producing γδ T cell (γδT17) responses, resulting in significantly enhanced neutrophil infiltration, tissue damage, and rapid development of bacteremia, mirroring responses in Treg cell-depleted mice. Deletion of total Treg cells predominantly activate IFNγ-T cell responses, whereas adoptive transfer of TNFR2+ Treg cells specifically suppress the γδT17 response, suggesting a targeted control of γδT17 activation by TNFR2+ Treg cells. Blocking IL-17A at early stage of infection significantly reduces bacterial blood dissemination and improves survival in TNFR2-deficient mice. Our results demonstrate that TNFR2 is critical for Treg cell-mediated regulation of pulmonary γδT17-neutrophil axis, with impaired TNFR2+ Treg cell responses increasing susceptibility to disease.