Updates on the controversial roles of regulatory lymphoid cells in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is the most common and severe form of pulmonary fibrosis, characterized by scar formation in the lung interstitium. Transforming growth factor beta (TGF-ß) is known as a key mediator in the fibrotic process, acting on fibroblasts and mediating their proliferation and differentiation into myofibroblasts. Although the immune system is not considered responsible for the initiation of IPF, markers of tolerogenic immunity define the pro-fibrotic microenvironment in the lungs. In homeostatic conditions, regulatory T cells (Tregs) constitute the main lymphoid population responsible for maintaining peripheral tolerance. Similar to Tregs, regulatory B cells (Bregs) represent a recently described subset of B lymphocytes with immunosuppressive functions. In the context of IPF, numerous studies have suggested a role for Tregs in enhancing fibrosis, mainly via the secretion of TGF-ß. In humans, most studies show increased percentages of Tregs associated with the severity of IPF, although their exact role remains unclear. In mice, the most commonly used model involves triggering acute lung inflammation with bleomycin, leading to a subsequent fibrotic process. Consequently, data are still conflicting, as Tregs may play a protective role during the inflammatory phase and a deleterious role during the fibrotic phase. Bregs have been less studied in the context of IPF, but their role appears to be protective in experimental models of lung fibrosis. This review presents the latest updates on studies exploring the implication of regulatory lymphoid cells in IPF and compares the different approaches to better understand the origins of conflicting findings.

Immune mRNA Expression and Fecal Microbiome Composition Change Induced by Djulis (Chenopodium formosanum Koidz.) Supplementation in Aged Mice: A Pilot Study.

Background and Objectives: The aging process has always been associated with a higher susceptibility to chronic inflammatory lung diseases. Several studies have demonstrated the gut microbiome's influence on the lungs through cross-talk or the gut-lungs axis maintaining nutrient-rich microenvironments. Taiwan djulis (Chenopodium formosanum Koidz.) provides antioxidant and anti-inflammatory characteristics that could modulate the gut microbiome. This could induce the gut-lung axis through microbial cross-talk, thus favoring the modulation of lung inflammation. Materials and Methods: Here, we investigate the immune mRNA expression in the spleen, fecal microbiome composition, and hyperplasia of the bronchial epithelium in aged 2-year-old BALB/c mice after 60 days of supplementation of djulis. Results: The pro-inflammatory cytokines IFN-γ, TNF-α, and IL-1ß, T; cells CD4 and CD8; and TLRs TLR3, TLR4, TLR5, TLR7, TLR8, and TLR9 were reduced in their mRNA expression levels, while the anti-inflammatory cytokines IL-2, IL-4, and IL-10 were highly expressed in the C. formosanum-treated group. Interestingly, the fecal microbiome composition analysis indicated higher diversity in the C. formosanum-treated group and the presence of butyrate-producing bacteria that are beneficial in the gut microbiome. The histopathology showed reduced hyperplasia of the bronchial epithelium based on the degree of lesions. Conclusions: Our findings suggest that Taiwan djulis can modulate the gut microbiome, leading to microbial cross-talk; reducing the mRNA expression of pro-inflammatory cytokines, T cells, and TLRs; and increasing anti-inflammatory cytokines in the spleen, as cytokines migrate in the lungs, preventing lung inflammation damage in aged mice or the gut-lung axis. Thus, Taiwan djulis could be considered a beneficial dietary component for the older adult population. The major limitation includes a lack of protein validation of cytokines and TLRs and quantification of the T cell population in the spleen as a marker of the gut-lung axis.

Chronic exposure to E-cigarette aerosols potentiates atherosclerosis in a sex-dependent manner.

Despite being designed for smoking cessation, e-cigarettes and their variety of flavors have become increasingly attractive to teens and young adults. This trend has fueled concerns regarding the potential role of e-cigarettes in advancing chronic diseases, notably those affecting the cardiovascular system. E-cigarettes contain a mixture of metals and chemical compounds, some of which have been implicated in cardiovascular diseases like atherosclerosis. Our laboratory has optimized in vivo exposure regimens to mimic human vaping patterns. Using these established protocols in an inducible (AAV-PCSK9) hyperlipidemic mouse model, this study tests the hypothesis that a chronic exposure to e-cigarette aerosols will increase atherosclerotic plaques. The exposures were conducted using the SCIREQ InExpose™ nose-only inhalation system and STLTH or Vuse products for 16 weeks. We observed that only male mice exposed to STLTH or Vuse aerosols had significantly increased plasma total cholesterol, triglycerides, and LDL cholesterol levels compared to mice exposed to system air. Moreover, these male mice also had a significant increase in aortic and sinus plaque area. Male mice exposed to e-cigarette aerosol had a significant reduction in weight gain over the exposure period. Our data indicate that e-cigarette use in young hyperlipidemic male mice increases atherosclerosis in the absence of significant pulmonary and systemic inflammation. These results underscore the need for extensive research to unravel the long-term health effects of e-cigarettes.

The storage mite Tyrophagus putrescentiae induces greater lung inflammation than house dust mites in mice.

Exposure to storage mite (SM) and house dust mite (HDM) allergens is a risk factor for sensitization and asthma development; however, the related immune responses and their pathology have not been fully investigated. The HDMs Dermatophagoides farinae and Dermatophagoides pteronyssinus and SM Tyrophagus putrescentiae are potent allergens that induce asthma. Most SM-related studies have focused on the allergic reactions of individuals by measuring their immunoglobulin (Ig)E expression. Considering the limited research on this topic, the present study aims to investigate the differences in the immune responses induced by HDMs and SMs and histologically analyze lung tissues in a mouse asthma model to understand the differential effects of HDM and SM. The results revealed that all mite species induced airway inflammation. Mice challenged with T. putrescentiae had the highest airway resistance and total cell, eosinophil, and neutrophil counts in the bronchoalveolar lavage fluid (BALF). The SM-sensitized groups showed more severe lesions and mucus hypersecretions than the HDM-sensitized groups. Although the degree of HDM and SM exposure was the same, the damage to the respiratory lung tissue was more severe in SM-exposed mice, which resulted in excessive mucin secretion and increased fibrosis. Furthermore, these findings suggest that SM sensitization induces a more significant hypersensitivity response in mucosal immunity than HDM sensitization in asthma models.

Endothelialized Bronchioalveolar Lung Organoids Model Endothelial Cell Responses to Injury.

Organoid 3D systems are powerful platforms to study development and disease. Recently, the complexity of lung organoid models derived from adult mouse and human stem cells has increased substantially in terms of cellular composition and structural complexity. However, a murine lung organoid system with a clear integrated endothelial compartment is still missing. Here, we describe a novel method that adds another level of intricacy to our published bronchioalveolar lung organoid (BALO) model by microinjection of FACS-sorted lung endothelial cells (ECs) into differentiated organoid cultures. Before microinjection, ECs obtained from the lung homogenate (LH) of young mice expressed typical ECs markers such as CD31 and vascular endothelial (VE)-Cadherin and showed tube formation capacity. Following microinjection, ECs surrounded BALO´s alveolar-like compartment aligning with both alveolar epithelial cells type I (AECI) and type II (AECII), as demonstrated by confocal and electron microscopy. Notably, expression of Car4 and Aplnr was as well detected, suggesting presence of EC microvascular phenotypes in the cultured ECs. Moreover, upon epithelial cell injury by lipopolysaccharides (LPS) and influenza A virus (IV), endothelialized BALO (eBALO) released proinflammatory cytokines leading to the upregulation of the intercellular adhesion molecule 1 (ICAM-1) in ECs. In summary, we characterized for the first time a organoid model that incorporates ECs into the alveolar structures of lung organoids, not only increasing our previous model ́s cellular and structural complexity but also providing a suitable niche to model lung endothelium responses to injury ex vivo.

First-in-Class Mitogen-Activated Protein Kinase p38α: MAPK-Activated Protein Kinase-2 (MK2) Dual Signal Modulator with Anti-inflammatory and Endothelial-stabilizing Properties.

We previously identified a small molecule, UM101, predicted to bind to the substrate-binding groove of p38aMitogen-activated Protein Kinase (MAPK) near the binding site of its proinflammatory substrate, MAPK-activated protein kinase (MK2). UM101 exhibited anti-inflammatory, endothelial-stabilizing, and lung-protective effects. To overcome its limited aqueous solubility and p38a binding affinity, we designed an analog of UM101, GEn-1124, with improved aqueous solubility, stability, and p38a binding affinity. Compared with UM101, GEn-1124 has 18-fold greater p38a-binding affinity as measured by Surface Plasmon Resonance (SPR), 11-fold greater aqueous solubility, enhanced barrier-stabilizing activity in thrombin-stimulated human pulmonary artery endothelial cells (hPAEC) in vitro, and greater lung protection in vivo GEn-1124 improved survival from 10% to 40% in murine acute lung injury (ALI) induced by combined exposure to intratracheal bacterial endotoxin lipopolysaccharide (LPS) instillation and febrile-range hyperthermia (FRH) and from 0% to 50% in a mouse influenza pneumonia model. Gene expression analysis by RNASeq in TNFa-treated hPAEC showed that the gene-modifying effects of GEn-1124 were much more restricted to TNFa-inducible genes than the catalytic site p38 inhibitor, SB203580. Gene expression pathway analysis, confocal immunofluorescence analysis of p38aand MK2 subcellular trafficking, and SPR analysis of phosphorylated p38a:MK2 binding affinity supports a novel mechanism of action. GEn-1124 destabilizes the activated p38a:MK2 complex, dissociates nuclear export of MK2 and p38a, thereby promoting intranuclear retention and enhanced intranuclear signaling by phosphorylated p38a retention, and accelerated inactivation of p38-free cytosolic MK2 by unopposed phosphatases. Significance Statement We describe an analog of our first-in-class small molecule modulator of p38a/MK2 signaling targeted to a pocket near the ED substrate binding domain of p38a, which destabilizes the p38a:MK2 complex without blocking p38 catalytic activity or ablating downstream signaling. The result is a rebalancing of downstream pro- and anti-inflammatory signaling, yielding anti-inflammatory, endothelial-stabilizing, and lung-protective effects with therapeutic potential in ARDS.

Zangsiwei prevents particulate matter-induced lung inflammation and fibrosis by inhibiting the TGF-ß/SMAD pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Zangsiwei(ZSW) is a traditional Tibetan medicine from China consisting of extracts of Rhododendron anthopogonoides Maxim, Gentiana Tourn, Corydalis hendersonii Hemsl and Berberis kansuensis C.K.Schneid. Traditionally, ZSW has been used by Tibetan physicians to treat chronic respiratory diseases. The role of ZSW in particulate matter-induced lung inflammation and fibrosis remains unclear. AIM OF THE STUDY: Combining non-targeted metabolomics, network pharmacology, and molecular docking to explore the mechanism of ZSW in the treatment of particulate matter-induced lung inflammation and fibrosis, and validated by in vivo and in vitro experiments. MATERIALS AND METHODS: The serum metabolite profile post-ZSW administration was first identified utilizing non-targeted metabolomics. Network pharmacology and molecular docking were employed to predict potential bioactive components and their corresponding targets. The in silico predictions were subsequently validated through in vivo studies in mice exposed to PM2.5 and silica dust, as well as in vitro studies utilizing human lung epithelial cells (A549) and lung fibroblasts (MRC5). RESULTS: Metabolomic analysis identified specific serum metabolites that were associated with ZSW treatment. Network pharmacology and molecular docking identified key targets involved in the Transforming growth factor-ß (TGF-ß)/SMAD pathway, which were subsequently validated through in vivo experiments demonstrating a reduction in lung inflammation and fibrosis in ZSW-treated mice. In vitro studies demonstrated that ZSW exerts protective effects against PM2.5-induced cytotoxicity and modulates fibrotic markers in a dose-dependent manner. This is consistent with the inhibition of the TGF-ß/SMAD pathway. CONCLUSION: Our integrated approach, which combines non-targeted metabolomics, network pharmacology, and molecular docking, followed by rigorous in vivo and in vitro validation, establishes ZSW as a potential therapeutic agent for particulate matter-induced lung inflammation and fibrosis.

siRNA Nanoparticle Dry Powder Formulation with High Transfection Efficiency and Pulmonary Deposition for Acute Lung Injury Treatment.

Acute lung injury (ALI) is a severe inflammatory syndrome, which was caused by diverse factors. The COVID-19 pandemic has resulted in a higher mortality rate of these conditions. Currently, effective treatments are lacking. Although siRNA nucleotide-based drugs are promising therapeutic approaches, their poor stability and inability to efficiently reach target cells limit their clinical translation. This study identified a peptide from known cell-penetrating peptides that can form an efficient anti-inflammatory complex with TNF-α siRNA, termed SAR6EW/TNF-α siRNA. This complex can effectively transport TNF-α siRNA into the cytoplasm and achieve potent gene silencing in vitro as well as in vivo. By using lactose and triarginine as coexcipients and optimizing the spray-drying process, a powder was produced with micrometer-scale spherical and porous structures, enhancing aerosol release and lung delivery efficiency. The dry powder formulation and process preserve the stability and integrity of the siRNA. When administered intratracheally to ALI model mice, the complex powder demonstrated specific pulmonary gene silencing activity and significantly reduced inflammation symptoms caused by ALI, suggesting a potential strategy for the clinical therapeutic approach of respiratory diseases.

Dietary Aflatoxin G1 exposure causes an imbalance between pulmonary tissue-resident alveolar macrophages and monocyte-derived macrophages in both mother and offspring mice.

Aflatoxin G1 (AFG1) is a mycotoxin commonly found in agricultural products, including dried fruits, meat, and milk products. Oral AFG1 administration induced tumor necrosis factor (TNF)-α-dependent chronic pulmonary inflammation, promoting AFG1-induced damage in alveolar epithelial cell, which is associated with lung adenocarcinoma. Pulmonary macrophages may be divided into tissue-resident alveolar macrophages (TRAMs) and monocyte-derived macrophages (MoMs), which involve in chronic lung inflammation. However, whether these macrophages contribute to AFG1-induced chronic pulmonary inflammation remains unknown. In this study, we found oral AFG1 administration disrupted the balance between TRAMs and MoMs, increasing MoMs infiltration and decreasing the number of TRAMs. AFG1 upregulated TNF-α expression in MoMs, but downregulated sialic acid binding Ig-like lectin F (Siglec-F) expression in TRAMs. Inhibition of TNF-α-dependent inflammation rescued the imbalance between TRAMs and MoMs in AFG1-treated lung tissues. Additionally, AFG1 stimulated MoMs differentiation to the proinflammatory M1 phenotype in vitro. Using a specific in vitro TRAM model, AFG1 downregulated Siglec-F and the M2 phenotypic markers arginase 1 and YM1, and upregulated the M1 phenotypic markers IL-6, iNOS and TNF-α, altering the TRAMs phenotype to the pro-inflammatory M1 phenotype in vitro. Additionally, mouse maternal dietary exposure to AFG1 caused an imbalance in pulmonary macrophages, decreasing TRAMs and increasing MoMs population in offspring, which was associated with proliferative lesions in the alveolar septa. Thus, dietary AFG1 exposure triggered an imbalance in pulmonary macrophages in both mother and offspring mice, and induced pro-inflammatory phenotypic alterations, which contributed to AFG1-induced chronic lung inflammation. These results provide clues to how AFG1-induced immunotoxicity and genotoxicity in humans might be prevented.

Cell-Specific Contribution of IL4 Receptor α Signaling Shapes the Overall Manifestation of Allergic Airway Disease.

IL-4 and IL-13 play a critical role in allergic asthma pathogenesis via their common receptor, i.e., IL4Rα. However, the cell-specific role of IL4Rα in mixed allergens (MA)-induced allergic asthma has remained unclear. Therefore, we aimed to identify the cell-specific contribution of IL4Rα signaling in the manifestation of various pathological outcomes in mice with allergic airway disease. We compared MA-induced pathological outcomes between hematopoietic progenitor cells (HPCs)- or non-HPCs-specific IL4Rα-deficient chimera, myeloid cell-specific IL4Rα-deficient (LysMcre+/+/IL4Rαfl/fl), and airway epithelial cell-specific IL4Rα-deficient (CCSP-Cre+ /IL4Rαfl/fl) mice. Chimeric mice with systemic IL4Rα sufficiency displayed hallmark features of allergic asthma, including eosinophilic and lymphocytic infiltration, type 2 (Th2) cytokine/chemokine production, IgE production, and lung pathology. These features were markedly reduced in chimeric mice with systemic IL4Rα deficiency. Non-HPCs-specific IL4Rα-deficient mice displayed typical inflammatory features of allergic asthma but with markedly reduced mucous cell metaplasia (MCM). Deletion of IL4Rα signaling on airway epithelial cells, a subpopulation within the non-HPC lineage, resulted in almost complete absence of MCM. In contrast, all features of allergic asthma except for MCM and mucin production were mitigated in HPCs-specific IL4Rα-deficient chimeric mice. Deleting IL4Rα signaling in myeloid cells, a subpopulation within the HPC lineage, significantly alleviated MA-induced allergic airway inflammatory responses, but similar to the HPCs-specific IL4Rα-deficient chimeric mice, these mice showed significant MCM and mucin production. Our findings demonstrate that the differential allergen responsiveness seen in mice with HPCs-specific and non-HPCs-specific IL4Rα deficiency is predominantly driven by the absence of IL4Rα in myeloid cells and airway epithelial cells, respectively. Our findings also highlight distinct and mutually exclusive roles of IL4Rα signaling in mediating pathological outcomes within the myeloid and airway epithelial cell compartments.

Lung-brain crosstalk: Behavioral disorders and neuroinflammation in septic survivor mice.

Although studies have suggested an association between lung infections and increased risk of neuronal disorders (e.g., dementia, cognitive impairment, and depressive and anxious behaviors), its mechanisms remain unclear. Thus, an experimental mice model of pulmonary sepsis was developed to investigate the relationship between lung and brain inflammation. Male Swiss mice were randomly assigned to either pneumosepsis or control groups. Pneumosepsis was induced by intratracheal instillation of Klebsiella pneumoniae, while the control group received a buffer solution. The model's validation included assessing systemic markers, as well as tissue vascular permeability. Depression- and anxiety-like behaviors and cognitive function were assessed for 30 days in sepsis survivor mice, inflammatory profiles, including cytokine levels (lungs, hippocampus, and prefrontal cortex) and microglial activation (hippocampus), were examined. Pulmonary sepsis damaged distal organs, caused peripheral inflammation, and increased vascular permeability in the lung and brain, impairing the blood-brain barrier and resulting in bacterial dissemination. After sepsis induction, we observed an increase in myeloperoxidase activity in the lungs (up to seven days) and prefrontal cortex (up to 24 h), proinflammatory cytokines in the hippocampus and prefrontal cortex, and percentage of areas with cells positive for ionized calcium-binding adaptor molecule 1 (IBA-1) in the hippocampus. Also, depression- and anxiety-like behaviors and changes in short-term memory were observed even 30 days after sepsis induction, suggesting a crosstalk between inflammatory responses of lungs and brain.

Glycolytic reprogramming governs crystalline silica-induced pyroptosis and inflammation through promoting lactylation modification.

Prolonged inhalation of environmental crystalline silica (CS) can cause silicosis, characterized by persistent pulmonary inflammation and irreversible fibrosis, but the mechanism has not been elucidated. To uncover the role and underlying mechanism of glycolytic reprogramming in CS-induced pulmonary inflammation, the mouse silicosis models and glycolysis inhibition models were established in vivo. And the CS-induced macrophage activation models were utilized to further explore the underlying mechanism in vitro. The results showed that CS induced lung inflammation accompanied by glycolytic reprogramming and pyroptosis. The application of glycolysis inhibitor (2-DG) suppressed CS-induced pyroptosis and alleviated lung inflammation. In vitro, 2-DG effectively impeded CS-induced macrophage pyroptosis and inflammatory response. Mechanistically, 2-DG suppressed pyroptosis by inhibiting NLRP3 inflammasome activation both in vivo and in vitro. Furtherly, metabolite lactate facilitated NLRP3-dependent pyroptosis synergistically with CS particles, while blocking the source of lactate largely alleviated NLRP3 inflammasome activation and subsequent pyroptosis triggered by CS. More profoundly, the increment of lactate induced by CS might drive NLRP3-dependent pyroptosis by increasing histone lactylation levels. In conclusion, our findings demonstrated inhibiting glycolytic reprogramming could alleviate CS-induced inflammatory response through suppressing NLRP3 -dependent pyroptosis. Increased glycolytic metabolite lactate and protein lactylation modifications might represent significant mechanisms during CS-induced NLRP3 activation and macrophage pyroptosis.

Investigating Pulmonary Neutrophil Responses to Inflammation in Mice via Flow Cytometry.

Neutrophils play a crucial role in maintaining lung health by defending against infections and participating in inflammation processes. Here we describe a detailed protocol for evaluating pulmonary neutrophil phenotype using a murine model of sterile inflammation induced by the fungal cell wall particle zymosan. We provide step-by-step instructions for the isolation of single cells from both lung tissues and airspaces, followed by comprehensive staining techniques for both cell surface markers and intracellular components. This protocol facilitates the sorting and detailed characterization of lung neutrophils via flow cytometry, making it suitable for downstream applications such as mRNA extraction, single-cell sequencing, and analysis of neutrophil heterogeneity. We also identify and discuss essential considerations for conducting successful neutrophil flow cytometry experiments. This work is aimed at researchers exploring the intricate functions of neutrophils in the lung under physiological and pathological conditions with the aid of flow cytometry.

Pycnogenol-Assisted Alleviation of Titanium Dioxide Nanoparticle-Induced Lung Inflammation via Thioredoxin-Interacting Protein Downregulation.

Titanium dioxide nanoparticles (TiO2NPs) are used in products that are applied to the human body, such as cosmetics and food, but their biocompatibility remains controversial. Pycnogenol (PYC), a natural extract of pine bark, exerts anti-inflammatory and antioxidant effects. In this study, we investigated whether PYC effectively alleviates pulmonary toxicity induced by airway exposure to TiO2NPs, and the beneficial effects of PYC were explained through the analysis of changes to the mechanism of cytotoxicity. TiO2NPs induced pulmonary inflammation and mucus production, increased the levels of malondialdehyde, and upregulated thioredoxin-interacting protein (TXNIP) and cleaved-caspase 3 (Cas3) in the lungs of mice. However, PYC treatment reduced the levels of all toxicity markers of TiO2NPs and restored glutathione levels. These antioxidant and anti-inflammatory effects of PYC were also demonstrated in TiO2NP-exposed human airway epithelial cells by increasing the mRNA levels of antioxidant enzymes and decreasing the expression of TXNIP, cleaved-Cas3, and inflammatory mediators. Taken together, our results showed that PYC attenuated TiO2NP-induced lung injury via TXNIP downregulation. Therefore, our results suggest the potential of PYC as an effective anti-inflammatory and antioxidant agent against TiO2NP-induced pulmonary toxicity.

The PARP inhibitor rucaparib blocks SARS-CoV-2 virus binding to cells and the immune reaction in models of COVID-19.

BACKGROUND AND PURPOSE: To date, there are limited options for severe Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2 virus. As ADP-ribosylation events are involved in regulating the life cycle of coronaviruses and the inflammatory reactions of the host; we have, here, assessed the repurposing of registered PARP inhibitors for the treatment of COVID-19. EXPERIMENTAL APPROACH: The effects of PARP inhibitors on virus uptake were assessed in cell-based experiments using multiple variants of SARS-CoV-2. The binding of rucaparib to spike protein was tested by molecular modelling and microcalorimetry. The anti-inflammatory properties of rucaparib were demonstrated in cell-based models upon challenging with recombinant spike protein or SARS-CoV-2 RNA vaccine. KEY RESULTS: We detected high levels of oxidative stress and strong PARylation in all cell types in the lungs of COVID-19 patients, both of which negatively correlated with lymphocytopaenia. Interestingly, rucaparib, unlike other tested PARP inhibitors, reduced the SARS-CoV-2 infection rate through binding to the conserved 493-498 amino acid region located in the spike-ACE2 interface in the spike protein and prevented viruses from binding to ACE2. In addition, the spike protein and viral RNA-induced overexpression of cytokines was down-regulated by the inhibition of PARP1 by rucaparib at pharmacologically relevant concentrations. CONCLUSION AND IMPLICATIONS: These results point towards repurposing rucaparib for treating inflammatory responses in COVID-19.

Effects of Bryophyllum pinnatum on Dysfunctional Autophagy in Rats Lungs Exposed to Zinc Oxide Nanoparticles.

Lung inflammation as a result of exposure to toxicants is a major pathological problem. Autophagy (AP) is a process of cell self-digestion and can be disrupted by environmental toxicants, leading to oxidative stress, inflammation and cellular damage. Bryophyllum pinnatum (Lam.) Oken has been used in folklore medicine to manage pathological abnormalities, including inflammation, but mechanisms remain unclear. This work investigated the effects of Bryophyllum pinnatum ethanol leaf extract (BP) on dysfunctional AP in the lungs of Wistar rats exposed to zinc oxide nanoparticles (ZONPs). The experimental rats were orally administered ZONPs for seven days (10 mg/kg). Some exposed rats were post-treated with BP (62.5 and 125 mg/kg) through oral gavage. Oxidative stress, inflammation, and apoptotic and autophagic parameters were assessed using biochemical assay and gene expression methods. Several indices of pulmonary damage were also evaluated. PCR analysis suggested that ZONP downregulated the expression of pro-autophagy-related genes (Beclin 2, ATG5, DAPK, and FOXP3) and upregulated the expression of the TNF-alpha, NF-Kb, LC3 and Bcl2 genes. In contrast, BP significantly (p < 0.0001) reversed ZONP-induced pulmonary toxicity and oxidative stress. It reduced MDA levels and increased SOD, CAT, GSH and GPxD activities. BP significantly (p < 0.0001) downregulated the expressions of proinflammatory genes (IL-6 and JNK) and upregulated the expressions of IL-10, CAT and SOD genes in ZONP-exposed rats. BP restored the lung's histoarchitectural structure after ZNOP-induced distortion. The results suggested that BP has antioxidant and anti-inflammatory properties, and could effectively restore ZNOP-induced dysfunctional AP in the lungs of Wistar rats.

MDM2 Is Essential to Maintain the Homeostasis of Epithelial Cells by Targeting p53.

INTRODUCTION: MDM2 is known as the primary negative regulator of p53, and MDM2 promotes lung cancer fibrosis and lung injury through p53-dependent and p53-independent pathways. However, the mechanism by which MDM2 influences the pathogenesis of asthma is unknown. In this study, we investigated the function of MDM2 in lung epithelial cells in type 2 lung inflammation. METHODS: We used type II alveolar epithelial cell-specific heterozygous knockout of Mdm2 mice to validate its function. Then papain-induced asthma model was established, and changes in inflammation were observed by measuring immunohistochemistry and flow cytometry analysis. RESULTS: In this study, we knockdown the mouse Mdm2 gene in type 2 alveolar epithelial cells. We demonstrated that heterozygous Mdm2 gene-deleted mice were highly susceptible to protease allergen papain-induced pulmonary inflammation characterized by increased ILC2 numbers, IL-5 and IL-13 cytokine levels, and lung pathology. A mechanistic study showed that following the decreased expression of Mdm2 in lung epithelial cells and A549 cell line, p53 was overactivated, and the expression of its downstream genes p21, Puma, and Noxa was elevated, which resulted in apoptosis. After Mdm2 knockdown, the mRNA expression of inflammation-related gene IL-25, HMGB1, and TNF-α were increased, which further amplified the downstream ILC2 response and lung inflammation. CONCLUSION: These results indicate that Mdm2 maintains the homeostasis of lung epithelial cells by targeting P53 and regulates the function of lung epithelial cells under type 2 lung inflammation.

Microbial intestinal dysbiosis drives long-term allergic susceptibility by sculpting an ILC2-B1 cell-innate IgE axis.

BACKGROUND: The abundance and diversity of intestinal commensal bacteria influence systemic immunity with impact on disease susceptibility and severity. For example, loss of short chain fatty acid (SCFA)-fermenting bacteria in early life (humans and mice) is associated with enhanced type 2 immune responses in peripheral tissues including the lung. OBJECTIVE: Our goal was to reveal the microbiome-dependent cellular and molecular mechanisms driving enhanced susceptibility to type 2 allergic lung disease. METHODS: We used low-dose vancomycin to selectively deplete SCFA-fermenting bacteria in wild-type mice. We then examined the frequency and activation status of innate and adaptive immune cell lineages with and without SCFA supplementation. Finally, we used ILC2-deficient and signal transducer and activator of transcription 6 (STAT6)-deficient transgenic mouse strains to delineate the cellular and cytokine pathways leading to enhanced allergic disease susceptibility. RESULTS: Mice with vancomycin-induced dysbiosis exhibited a 2-fold increase in lung ILC2 primed to produce elevated levels of IL-2, -5, and -13. In addition, upon IL-33 inhalation, mouse lung ILC2 displayed a novel ability to produce high levels of IL-4. These expanded and primed ILC2s drove B1 cell expansion and IL-4-dependent production of IgE that in turn led to exacerbated allergic inflammation. Importantly, these enhanced lung inflammatory phenotypes in mice with vancomycin-induced dysbiosis were reversed by administration of dietary SCFA (specifically butyrate). CONCLUSION: SCFAs regulate an ILC2-B1 cell-IgE axis. Early-life administration of vancomycin, an antibiotic known to deplete SCFA-fermenting gut bacteria, primes and amplifies this axis and leads to lifelong enhanced susceptibility to type 2 allergic lung disease.

Interleukin-1 receptor-associated kinase 4 (IRAK4) is a critical regulator of inflammatory signalling through toll-like receptors 4 and 7/8 in murine and human lungs.

BACKGROUND AND PURPOSE: Toll-like receptors 4 (TLR4) and TLR7/TLR8 play an important role in mediating the inflammatory effects of bacterial and viral pathogens. Interleukin-1 receptor-associated kinase 4 (IRAK4) is an important regulator of signalling by toll-like receptor (TLR) and hence is a potential therapeutic target in diseases characterized by increased lung inflammatory signalling. EXPERIMENTAL APPROACH: We used an established murine model of acute lung inflammation, and studied human lung tissue ex vivo, to investigate the effects of inhibiting IRAK4 on lung inflammatory pathways. KEY RESULTS: We show that TLR4 stimulation produces an inflammatory response characterized by neutrophil influx and tumour necrosis factor-α (TNF-α) production in murine lungs and that these responses are markedly reduced in IRAK4 kinase-dead mice. In addition, we characterize a novel selective IRAK4 inhibitor, BI1543673, and show that this compound can reduce lipopolysaccharide (LPS)-induced airway inflammation in wild-type mice. Additionally, BI1543673 reduced inflammatory responses to both TLR4 and TLR7/8 stimulation in human lung tissue studied ex vivo. CONCLUSION AND IMPLICATIONS: These data demonstrate a key role for IRAK4 signalling in lung inflammation and suggest that IRAK4 inhibition has potential utility to treat lung diseases characterized by inflammatory responses driven through TLR4 and TLR7/8.

Exosome miRNA profile and mitigating effect of miR-23a-3p/Cul3 axis on apoptosis in the pathogenesis of SiO2 dust-induced lung fibrosis.

Silicosis is an irreversible interstitial lung fibrosis resulting from persistent inflammation induced by long-term inhalation of SiO2 dust. Treatment and early diagnosis are extremely challenging due to the lack of specific targets and biomarkers. MiRNAs play an important role in the early diagnosis and treatment of various diseases, due to their stability, small variations, and easy detection. Exosomes have become fashionable candidates to deliver miRNAs. However, the specific role of exosomes-loaded miRNAs in silicosis inflammation and fibrosis remains unclear. In the present study, the expression profile of serum exosomal miRNAs in the peripheral blood of silicosis patients was determined by transcritome sequencing. MiR-23a-3p was recognized as a protector against silicosis by bioinformatic analysis. The expression and regulatory axis of miR-23a-3p and its predicted target gene CUL3 were then confirmed. The therapeutic role of the miR-23a-3p/CUL3 axis and its alleviating effect on SiO2-induced apoptosis were verified in mice and in epithelial cells. Furthermore, the communication of exosomes carrying miR-23a-3p between macrophages and epithelial cells was demonstrated using a cell co-culture model. Our results suggest that exosomal miR-23a-3p could be prospective as a biomarker in early diagnose for SiO2-induced lung fibrosis, and provided new threads for the treatment of silicosis.