Lung Inflammation In Mouse Model Research Articles

Imaging polarity changes in pneumonia and lung cancer using a lipid droplet-targeted near-infrared fluorescent probe

As the main organ of gas exchange, the lungs are susceptible to various exogenous attacks, and pneumonia is one of the major inflammatory diseases that threaten human health. Generally, pneumonia is a disease that occurs in the alveoli and respiratory bronchioles induced by pathogens and further causes local and systemic inflammatory responses. The development of pneumonia can bring various serious complications, including lung abscess, sepsis, meningitis, brain damage and hearing loss. Over the past few decades, the mortality rate of pneumonia patients has remained high. While lung cancer is another lung-related malignant tumors worldwide, with a low 5 year survival rate. Exploring the mechanisms of their occurrence and interaction between pneumonia and lung cancer is a challenging and meaningful task. The abnormalities of lipid droplets (LDs) polarity have been found strongly accompanied by many diseases, especially cancer, inflammation, and metabolic diseases. However, their exact role is not yet clear. Hence, it is significant to develop a novel detection method to observe the polarity changes of LDs, which would help to reveal the development process of diseases pneumonia and lung cancer. In this work, a new polarity-sensitive LDs-targeted near-infrared probe BFZ up to 712 nm was designed, according to the intramolecular charge transfer mechanism, which displayed high fluorescence intensity in low polarity while showing decreased fluorescence intensity in high-polarity conditions with a significant redshift. The BFZ was successfully applied to the change of LDs polarity in lipopolysaccharide (LPS)-stimulated A549 cells, and a mouse model of lung inflammation. It also tells the polarity differences between normal and tumor cells and between normal and tumor tissues. Moreover, the correlations between pneumonia and polarity changes were observed through the imaging experiments, which may provide an insightful method for the early diagnosis of pneumonia and lung cancer.

Inhalation of panaxadiol alleviates lung inflammation via inhibiting TNFA/TNFAR and IL7/IL7R signaling between macrophages and epithelial cells

BackgroundLung inflammation occurs in many lung diseases, but has limited effective therapeutics. Ginseng and its derivatives have anti-inflammatory effects, but their unstable physicochemical and metabolic properties hinder their application in the treatment. Panaxadiol (PD) is a stable saponin among ginsenosides. Inhalation administration may solve these issues, and the specific mechanism of action needs to be studied. MethodsA mouse model of lung inflammation induced by lipopolysaccharide (LPS), an in vitro macrophage inflammation model, and a coculture model of epithelial cells and macrophages were used to study the effects and mechanisms of inhalation delivery of PD. Pathology and molecular assessments were used to evaluate efficacy. Transcriptome sequencing was used to screen the mechanism and target. Finally, the efficacy and mechanism were verified in a human BALF cell model. ResultsInhaled PD reduced LPS-induced lung inflammation in mice in a dose-dependent manner, including inflammatory cell infiltration, lung tissue pathology, and inflammatory factor expression. Meanwhile, the dose of inhalation was much lower than that of intragastric administration under the same therapeutic effect, which may be related to its higher bioavailability and superior pharmacokinetic parameters. Using transcriptome analysis and verification by a coculture model of macrophage and epithelial cells, we found that PD may act by inhibiting TNFA/TNFAR and IL7/IL7R signaling to reduce macrophage inflammatory factor-induced epithelial apoptosis and promote proliferation. ConclusionPD inhalation alleviates lung inflammation and pathology by inhibiting TNFA/TNFAR and IL7/IL7R signaling between macrophages and epithelial cells. PD may be a novel drug for the clinical treatment of lung inflammation.

Vitamin B6 regulates IL-33 homeostasis to alleviate type 2 inflammation.

Interleukin-33 (IL-33) is a crucial nuclear cytokine that induces the type 2 immune response and maintains immune homeostasis. The fine-tuned regulation of IL-33 in tissue cells is critical to control of the type 2 immune response in airway inflammation, but the mechanism is still unclear. Here, we found that healthy individuals had higher phosphate-pyridoxal (PLP, an active form of vitamin B6) concentrations in the serum than asthma patients. Lower serum PLP concentrations in asthma patients were strongly associated with worse lung function and inflammation. In a mouse model of lung inflammation, we revealed that PLP alleviated the type 2 immune response and that this inhibitory effect relied on the activity of IL-33. A mechanistic study showed that in vivo, pyridoxal (PL) needed to be converted into PLP, which inhibited the type 2 response by regulating IL-33 stability. In mice heterozygous for pyridoxal kinase (PDXK), the conversion of PL to PLP was limited, and IL-33 levels were increased in the lungs, aggravating type 2 inflammation. Furthermore, we found that the mouse double minute 2 homolog (MDM2) protein, an E3 ubiquitin-protein ligase, could ubiquitinate the N-terminus of IL-33 and sustain IL-33 stability in epithelial cells. PLP reduced MDM2-mediated IL-33 polyubiquitination and decreased the level of IL-33 through the proteasome pathway. In addition, inhalation of PLP alleviated asthma-related effects in mouse models. In summary, our data indicate that vitamin B6 regulates MDM2-mediated IL-33 stability to constrain the type 2 response, which might help develop a potential preventive and therapeutic agent for allergy-related diseases.

Antioxidative and anti-inflammatory effect of ethanol extracts from Magnolia kobusDC on LPS-induced in vitroand in vivomodel

Abstract Magnolia kobusDC (M. kobus) has biological effects such as antioxidant, anti-inflammatory and antibacterial properties in its flower, bud, and stem bark extracts. Despite of diverse effectiveness of M. kobus, the pharmacological effects supporting inhibition of oxidation and inflammation on acute lung inflammation states have not been studied yet. The purpose of this study was to evaluate the pharmacological activity of M. kobusleaf ethanol extract (MkDE) by antioxidant and anti-inflammatory effects on acute lung inflammatory diseases. The antioxidant and anti-inflammatory effects of MkDE was evaluated using ROS detection assay, NO assay, qPCR, ELISA, and Western blot on LPS-induced macrophages. The therapeutic effect of MkDE was investigated in vivooxidative and inflammatory responses by histological observations, qPCR, ELISA, and Western blot using the LPS-induced acute lung inflammation mouse model. MkDE decreased ROS production and protein of Nrf2. MkDE showed a protective effect against H 2O 2as well as a decrease in NO production. MkDE reduced the levels of pro-inflammatory cytokines and chemokine, the activation of COX-2 and iNOS, and NF-κB translocation in LPS-induced macrophage. The intraperitoneal injection of MkDE showed to decrease the lung/body weights, infiltrated inflammatory cells into the lung alveoli, pro-inflammatory mediators, and serum released proteins. Furthermore, MkDE inhibited the activation of Nrf2, COX-2 and NF-κB in LPS-induced acute lung tissue. MkDE exhibits the pharmacological effect by reducing oxidative and inflammatory mediators on LPS-induced macrophages and acute lung inflammation mouse model. Taken together, MkDE might be used to treat acute lung disease.

PHLIP Peptides Target Acidity in Activated Macrophages.

Acidity can be a useful alternative biomarker for the targeting of metabolically active cells in certain diseased tissues, as in acute inflammation or aggressive tumors. We investigated the targeting of activated macrophages by pH low insertion peptides (pHLIPs), an established technology for targeting cell-surface acidity. The uptake of fluorescent pHLIPs by activated macrophages was studied in cell cultures, in a mouse model of lung inflammation, and in a mouse tumor model. Fluorescence microscopy, whole-body and organ imaging, immunohistochemistry, and FACS analysis were employed. We find that cultured, activated macrophages readily internalize pHLIPs. The uptake is higher in glycolytic macrophages activated by LPS and INF-γ compared to macrophages activated by IL-4/IL-13. Fluorescent pHLIPs target LPS-induced lung inflammation in mice. In addition to marking cancer cells within the tumor microenvironment, fluorescent pHLIPs target CD45+, CD11b+, F4/80+, and CD206+ tumor-associated macrophages with no significant targeting of other immune cells. Also, fluorescent pHLIPs target CD206-positive cells found in the inguinal lymph nodes of animals inoculated with breast cancer cells in mammary fat pads. pHLIP peptides sense low cell surface pH, which triggers their insertion into the cell membrane. Unlike cancerous cells, activated macrophages do not retain inserted pHLIPs on their surfaces, instead their highly active membrane recycling moves the pHLIPs into endosomes. Targeting activated macrophages in diseased tissues may enable clinical visualization and therapeutic opportunities.

Machine learning identifies molecular regulators and therapeutics for targeting SARS‐CoV2‐induced cytokine release

Although 15–20% of COVID‐19 patients experience hyper‐inflammation induced by massive cytokine production, cellular triggers of this process and strategies to target them remain poorly understood. Here, we show that the N‐terminal domain (NTD) of the SARS‐CoV‐2 spike protein substantially induces multiple inflammatory molecules in myeloid cells and human PBMCs. Using a combination of phenotypic screening with machine learning‐based modeling, we identified and experimentally validated several protein kinases, including JAK1, EPHA7, IRAK1, MAPK12, and MAP3K8, as essential downstream mediators of NTD‐induced cytokine production, implicating the role of multiple signaling pathways in cytokine release. Further, we found several FDA‐approved drugs, including ponatinib, and cobimetinib as potent inhibitors of the NTD‐mediated cytokine release. Treatment with ponatinib outperforms other drugs, including dexamethasone and baricitinib, inhibiting all cytokines in response to the NTD from SARS‐CoV‐2 and emerging variants. Finally, ponatinib treatment inhibits lipopolysaccharide‐mediated cytokine release in myeloid cells in vitro and lung inflammation mouse model. Together, we propose that agents targeting multiple kinases required for SARS‐CoV‐2‐mediated cytokine release, such as ponatinib, may represent an attractive therapeutic option for treating moderate to severe COVID‐19.

Selective Imaging of Lung Macrophages Using [11C]PBR28-Based Positron Emission Tomography.

We tested whether the translocator protein (TSPO)-targeted positron emission tomography (PET) tracer, N-acetyl-N-(2-[11C]methoxybenzyl)-2-phenoxy-5-pyridinamine ([11C]PBR28), could distinguish macrophage dominant from neutrophilic inflammation better than 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) in mouse models of lung inflammation and assessed TSPO association with macrophages in lung tissue from the mouse models and in patients with chronic obstructive pulmonary disease (COPD). MicroPET imaging quantified [11C]PBR28 and [18F]FDG lung uptake in wild-type (Wt) C57BL/6J or heterozygous transgenic monocyte-deficient Wt/opT mice at 49 days after Sendai virus (SeV) infection, during macrophage-dominant inflammation, and in Wt mice at 3 days after SeV infection or 24 h after endotoxin instillation during neutrophilic inflammation. Immunohistochemical staining for TSPO in macrophages and neutrophils was performed using Mac3 and Ly6G for cell identification in mouse lung sections and CD68 and neutrophil elastase (NE) in human lung sections taken from explanted lungs from patients with COPD undergoing lung transplantation and donor lungs rejected for transplantation. Differences in tracer uptake among SeV-infected, endotoxin-treated, and uninfected/untreated control mice and in TSPO staining between neutrophils and macrophage populations in human lung sections were tested using analysis of variance. In Wt mice, [11C]PBR28 uptake (% injected dose/ml lung tissue) increased significantly with macrophage-dominant inflammation at 49 days (D49) after SeV infection compared to controls (p = <0.001) but not at 3 days (D49) after SeV infection (p = 0.167). [11C]PBR28 uptake was unchanged at 24 h after endotoxin instillation (p = 0.958). [18F]FDG uptake increased to a similar degree in D3 and D49 SeV-infected and endotoxin-treated Wt mice compared to controls with no significant difference in the degree of increase among the tested conditions. [11C]PBR28 but not [18F]FDG lung uptake at D49 post-SeV infection was attenuated in Wt/opT mice compared to Wt mice. TSPO localized predominantly to macrophages in mouse lung tissue by immunostaining, and TSPO staining intensity was significantly higher in CD68+ cells compared to neutrophils in the human lung sections. PET imaging with [11C]PBR28 can specifically detect macrophages versus neutrophils during lung inflammation and may be a useful biomarker of macrophage accumulation in lung disease.

Mesenchymal stromal cells reprogram macrophages with processing bodies

Background & Aim: Mesenchymal stromal cells (MSCs) are an investigational cell therapy for inflammatory diseases. Although they have robust anti-inflammatory properties, their success has been variable in clinical trials due to an unclear understanding of their mechanism. Once injected, a majority of MSCs traffic to the lung, where they are rapidly cleared, signifying an opportunity to target lung inflammatory [Figure presented] conditions such as acute respiratory distress syndrome (ARDS). ARDS is a catastrophic condition of the lungs, involving pulmonary inflammation that develops with severe SARS-CoV2 and other respiratory infections. MSCs are expected to prevent alveolar damage by suppressing the immune response and there is evidence that MSCs protect in phase I/IIa trials for ARDS associated with COVID-19. While these results are promising, understanding the mechanism is critical to determine dosing, maximize efficacy, and ultimately lead to an approved product. Methods, Results & Conclusion: We and others have demonstrated that within their short time in the lung, MSCs interact with monocytes and macrophages. Through direct cell contact, MSCs transfer cytoplasmic components, notably cytoplasmic processing-bodies (p-bodies) to monocytes and macrophages. P-bodies are membrane-less organelles that contain RNA binding proteins, microRNAs, and mRNAs enriched for genes that regulate the transcriptional landscape of cells. MSC interactions result in long-term transcriptional reprogramming of monocytes and macrophages to suppress a helper T cell response and upregulate tissue repair pathways. To investigate the mechanisms of MSCs in vivo, we utilized 2 mouse models of lung inflammation: 1. intranasal lipopolysaccharide (LPS) to study general acute inflammation and 2. an engineered vesicular stomatitis virus (VSV) with a SARS-CoV2 Spike protein. Using these models, we demonstrated that during inflammation cytoplasm of MSCs transferred to lung macrophages to decrease activation and the expression of MHC-II. Further, MSCs prevented a decrease in resident alveolar macrophages, suppressed proinflammatory macrophages, and blocked an influx in infiltrating monocytes (Fig 1). Depleting p-bodies from MSCs abolished the beneficial effects, despite transfer cytoplasmic component to macrophages at similar levels of control MSCs. Overall, our data suggest a novel form of cell communication that could explain how MSCs could lead to long-term beneficial effects on lung inflammation despite being rapidly cleared. [Figure presented]

IL36 is a critical upstream amplifier of neutrophilic lung inflammation in mice

IL-36, which belongs to the IL-1 superfamily, is increasingly linked to neutrophilic inflammation. Here, we combined in vivo and in vitro approaches using primary mouse and human cells, as well as, acute and chronic mouse models of lung inflammation to provide mechanistic insight into the intercellular signaling pathways and mechanisms through which IL-36 promotes lung inflammation. IL-36 receptor deficient mice exposed to cigarette smoke or cigarette smoke and H1N1 influenza virus had attenuated lung inflammation compared with wild-type controls. We identified neutrophils as a source of IL-36 and show that IL-36 is a key upstream amplifier of lung inflammation by promoting activation of neutrophils, macrophages and fibroblasts through cooperation with GM-CSF and the viral mimic poly(I:C). Our data implicate IL-36, independent of other IL-1 family members, as a key upstream amplifier of neutrophilic lung inflammation, providing a rationale for targeting IL-36 to improve treatment of a variety of neutrophilic lung diseases.

Analysis of T-Cells in Inflamed Nonlymphoid Tissues.

Under chronic inflammatory conditions, T and B cells are frequently found in nonlymphoid tissues. We recently identified a follicular helper-like T cell population in inflamed lung tissue, which drives the local differentiation of antigen-specific B cells into germinal center-like cells and plasma blasts. Here, we describe a lung inflammation mouse model, which is ideally suited to analyze antigen-specific T and B cells in secondary lymphoid organs and inflamed nonlymphoid tissue in parallel.

Imperatorin ameliorates mast cell-mediated allergic airway inflammation by inhibiting MRGPRX2 and CamKII/ERK signaling pathway

BackgroundAllergic asthma is a common inflammatory lung disease associated with complex pathogenesis. Mast cell (MC) is one of the key drivers of allergic asthma, Mas-related G protein-coupled receptor X2 (MRGPRX2) on the MC could mediate MC activation and trigger a pseudo-allergic reaction. Imperatorin (IMP), the main active compound of Radix Angelicae Dahuricae, has been reported to exert various pharmacological effects. In this study, we focused on the therapeutical mechanism of IMP on MRGPRX2-induced pseudo-allergy and allergic asthma. MethodsWe examined the effect of IMP on MRGPRX2 related mast cell activation in mouse peritoneal MC (MPMC), Human Laboratory of Allergic Disease 2 MCs (LAD2 cells) and Mrgprx2-expressing HEK293 cells. Molecular docking and Surface plasmon resonance (SPR) were taken to reveal the binding character between IMP and MRPGRX2. MRGPRX2 downstream proteins were also detected by western blotting. IgE-independent responses was evaluated by using passive cutaneous anaphylaxis (PCA) and active systemic anaphylaxis (ASA) models. The therapeutic effect of IMP on asthma was evaluated by a lung inflammation mouse model which was induced by ovalbumin (OVA). ResultsIMP was found to reduce substance P (SP) induced calcium flux and suppressed degranulation of MC. SP can promote the phosphorylation of ERK and CamKII, which regulates the synthesis of inflammatory factors such as MIP-2 and TNF-α in MC. In vivo assays revealed that IMP can mitigate SP-induced mouse PCA and ASA. IMP could also mitigate lung inflammation in an OVA induced mice model by inhibiting MC activation in the lung tissue. Furthermore, IMP binds well to MRGPRX2 protein. The binding constant (KD) is 4.48 ± 0.49 × 10−7 M. The data suggeste that IMP is a novel inhibitor of MRGPRX2 to treat allergic asthma.

Saponin Micelles Lead to High Mucosal Permeation and In Vivo Efficacy of Solubilized Budesonide.

Due to fast nasal mucociliary clearance, only the dissolved drug content can effectively permeate the mucosa and be pharmaceutically active after intranasal application of suspensions. Therefore, the aim of this study was to increase the budesonide concentration in solution of a nasal spray formulation. Budesonide, a highly water-insoluble corticosteroid, was successfully solubilized using a micellar formulation comprising escin, propylene glycol and dexpanthenol in an aqueous buffered environment (“Budesolv”). A formulation based on this micellar system was well-tolerated in the nasal cavity as shown in a good laboratory practice (GLP) local tolerance study in rabbits. Ex vivo permeation studies into porcine nasal mucosa revealed a faster and more efficient absorption. Budesolv with 300 µg/mL solubilized budesonide resulted in a budesonide concentration of 42 µg/g tissue after only 15 min incubation. In comparison, incubation with the marketed product Rhinocort® aqua 64 (1.28 mg/mL budesonide as suspension) led to 15 µg/g tissue. The in vivo tumor-necrosis-factor (TNF)-α secretion in an acute lung inflammation mouse model was significantly reduced (p < 0.001) following a prophylactic treatment with Budesolv compared to Rhinocort® aqua 64. Successful treatment 15 min after the challenge was only possible with Budesolv (40% reduction of TNF-α, p = 0.0012) suggesting a faster onset of action. The data reveal that solubilization based on saponin micelles presents an opportunity for the development of products containing hardly soluble substances that result in a faster onset and a better topical treatment effect.

Heterogeneity of regulatory T cells during lung inflammation

Abstract Pulmonary inflammation is the leading cause of respiratory illness worldwide. Although strong T cell responses are desired for defending the hosts against infections, the cytotoxic effector function of the innate and adaptive immune responses can lead to the development of pulmonary immunopathology, which may lead to death. While T cells produce inflammatory cytokines during infections and allergies, they can also produce the immunomodulatory cytokine IL-10, which is critical for limiting the immunopathology caused by the excessive effector immune responses. However, the composition of IL-10-producing lymphocyte population and their molecular signatures are unclear. Using mouse models that report the production of IL-10 by GFP and expression of T regulatory cell marker Foxp3 by RFP, mouse models of lung inflammation (including allergic asthma, hypersensitivity pneumonitis, and flu), transcriptomic analyses at the population and single cell levels, and transgenic mouse models that are impaired in T cell-derived IL-10 production, we found that, in the disease models used, regulatory T cells in the mouse airway are mainly comprised of CD4+ Foxp3+, CD4+ Foxp3− and CD8+ subsets, and they differ under different disease conditions. Furthermore, within each subset, IL-10-producing T cells exhibit significant molecular heterogeneity. Information gained from this dataset provides insights into the T cell subset heterogeneity and signature markers, and shed light for future strategic design for therapeutic development utilizing the immunomodulatory features of T cells for the treatment of pulmonary immunopathology.

Versican-A Critical Extracellular Matrix Regulator of Immunity and Inflammation.

The extracellular matrix (ECM) proteoglycan, versican increases along with other ECM versican binding molecules such as hyaluronan, tumor necrosis factor stimulated gene-6 (TSG-6), and inter alpha trypsin inhibitor (IαI) during inflammation in a number of different diseases such as cardiovascular and lung disease, autoimmune diseases, and several different cancers. These interactions form stable scaffolds which can act as “landing strips” for inflammatory cells as they invade tissue from the circulation. The increase in versican is often coincident with the invasion of leukocytes early in the inflammatory process. Versican interacts with inflammatory cells either indirectly via hyaluronan or directly via receptors such as CD44, P-selectin glycoprotein ligand-1 (PSGL-1), and toll-like receptors (TLRs) present on the surface of immune and non-immune cells. These interactions activate signaling pathways that promote the synthesis and secretion of inflammatory cytokines such as TNFα, IL-6, and NFκB. Versican also influences inflammation by interacting with a variety of growth factors and cytokines involved in regulating inflammation thereby influencing their bioavailability and bioactivity. Versican is produced by multiple cell types involved in the inflammatory process. Conditional total knockout of versican in a mouse model of lung inflammation demonstrated significant reduction in leukocyte invasion into the lung and reduced inflammatory cytokine expression. While versican produced by stromal cells tends to be pro-inflammatory, versican expressed by myeloid cells can create anti-inflammatory and immunosuppressive microenvironments. Inflammation in the tumor microenvironment often contains elevated levels of versican. Perturbing the accumulation of versican in tumors can inhibit inflammation and tumor progression in some cancers. Thus versican, as a component of the ECM impacts immunity and inflammation through regulating immune cell trafficking and activation. Versican is emerging as a potential target in the control of inflammation in a number of different diseases.

Fractions of Boswellia Serrata Suppress LTA4, LTC4, Cyclooxygenase-2 Activities and mRNA in HL-60 Cells and Reduce Lung Inflammation in BALB/c Mice.

Purified fractions from a Boswellia serrata Roxb. Ex. Colebr. (Burseraceae) extract (ETOH and DCM) contain biologically active compounds that are well known for having inflammation inhibitory properties. In this work, the purified fractions were tested in-vitro for LTC4, LTA4 and COX-2 activities using ELISA and qPCR was performed to determine gene regulation in human leukemia (HL-60) Cells. Two D-imaging tomography was performed to determine the anti-inflammatory activities of the fractions in BALB/c mouse model of lung inflammation. To evaluate anti-inflammatory activities of bioactive compounds of Boswellia serrata purified fractions. In-vitro MTT assay was performed in HL-60 cell lines for measuring the toxicity/ viability of the cells. ELISA tests were performed for evaluating LTA4, LTC4 and COX-2 activities. qPCR was performed to evaluate the expression of mRNA in HL-60 cells. In-vivo experiments were performed in OVA sensitized and challenged BALB/c mice at two doses of Boswellia serrata purified fraction containing 6% Boswellic acid of 50 and 100mg/kg body weight were given orally and the standard drug dexamethasone (DXA, 4 mg/kg body weight) and reduction in lung inflammation was assessed by using an IVIS Xenogen in-vivo fluorescence imaging system. A purified fraction of Boswellia serrata ETOH extracts reduced leukotriene-C4-synthase activity by 52%, leuktotriene-A4-hydrolase activity by 22% and COX-2 activity by 99% with an IC50 of 12.5μg/ml. Intragastric administration of the purified fraction of Boswellia serrata at two doses of 50mg/kg b.w. and 100mg/kg b.w., respectively along with 2-3% HPMC resulted in a ~51% (P value <0.01) reduction in OVA induced lung inflammation in BALB/c mice as observed by imaging tomography. Treatment of the OVA challenged mice with standard drug dexamethasone (DXA) reduced inflammation by ~66% with significant value (P<0.0001). The present study describes that Boswellia serrata ethanolic extracts purified fraction (ETOH-BS) possess significant anti-inflammatory activities in HL-60 and in BALB/c and further supports for its use as Ayurvedic medicines traditionally in the treatment of lung disorders including allergy and asthma.

Benzo(a)pyrene facilitates dermatophagoides group 1 (Der f 1)-induced epithelial cytokine release through aryl hydrocarbon receptor in asthma.

BackgroundEnvironmental pollutants, which coexist with allergens, have been associated with the exacerbation of asthma. However, the underlying molecular mechanisms remain elusive. We sought to determine whether benzo(a)pyrene (BaP) co‐exposure with dermatophagoides group 1 allergen (Der f 1) can potentiate Der f 1‐induced asthma and its underlying mechanisms.MethodsThe effect of BaP was investigated in Der f 1‐induced mouse model of asthma, including airway hyper‐responsiveness, allergic inflammation, and epithelial‐derived cytokines. The impact of BaP on Der f 1‐induced airway epithelial cell oxidative stress (ROS) and cytokine release was further analyzed. The role of aryl hydrocarbon receptor (AhR) signaling in BaP‐promoted Der f 1‐induced ROS, cytokine production, and allergic inflammation was also investigated.ResultsCompared with Der f 1, BaP co‐exposure with Der f 1 led to airway hyper‐responsiveness and increased lung inflammation in mouse model of asthma. Increased expression of TSLP, IL‐33, and IL‐25 was also found in the airways of these mice. Moreover, BaP co‐exposure with Der f 1 activated AhR signaling with increased expression of AhR and CYP1A1 and promoted airway epithelial ROS generation and TSLP and IL‐33, but not IL‐25, expression. Interestingly, AhR antagonist CH223191 or cells with AhR knockdown abrogated the increased expression of ROS, TSLP, and IL‐33. Furthermore, ROS inhibitor N‐acetyl‐L‐cysteine (NAC) also suppressed BaP co‐exposure‐induced expression of epithelial TSLP, IL‐33, and IL‐25. Finally, AhR antagonist CH223191 and NAC inhibited BaP co‐exposure with Der f 1‐induced lung inflammation.ConclusionsOur findings suggest that BaP facilitates Der f 1‐induced epithelial cytokine release through the AhR‐ROS axis.

Sea snake cathelicidin (Hc-cath) exerts a protective effect in mouse models of lung inflammation and infection

We investigated the anti-inflammatory and antibacterial activities of Hc-cath, a cathelicidin peptide derived from the venom of the sea snake, Hydrophis cyanocyntus, using in vivo models of inflammation and infection. Hc-cath function was evaluated in in vitro, in vivo in the wax moth, Galleria mellonella, and in mouse models of intraperitoneal and respiratory Pseudomonas aeruginosa infection. Hc-Cath downregulated LPS-induced pro-inflammatory responses in macrophages and significantly improved the survival of P. aeruginosa infected G. mellonella over a 5-day period. We also demonstrated, for the first time, that Hc-cath can modulate inflammation in a mouse model of LPS-induced lung inflammation by significantly reducing the release of the pro-inflammatory cytokine and neutrophil chemoattractant, KC, resulting in reduced cellular infiltration into the lungs. Moreover, Hc-cath treatment significantly reduced the bacterial load and inflammation in mouse models of P. aeruginosa intraperitoneal and respiratory infection. The effect of Hc-cath in our studies highlights the potential to develop this peptide as a candidate for therapeutic development.

Evaluating sex differences in miR‐106a expression in a mouse model of lung inflammation and asthma

MicroRNAs (miRNAs) are oligonucleotides involved in various processes in the cell, including gene expression regulation. MicroRNA‐106a is involved in transcription regulation of the gene coding for interleukin 10 (IL‐10), an anti‐inflammatory cytokine, and has been implicated in asthma exacerbation in mice. However, despite the well‐known sex differences in human asthma incidence, no studies have been done evaluating sex differences on the impact of miR‐106a in asthma. In this study, we hypothesized that there are sex differences in miR‐106a expression in the lungs of asthmatic mice, under normal conditions and in response to air pollution challenge. To test this hypothesis, both male and female mice were exposed to house dust mites (HDM) or phosphate‐buffered saline (control) for 5 weeks to trigger the asthma phenotype. Mice were subsequently subjected to ozone challenge (2ppm, 3 hours) to model asthma exacerbations. The asthma phenotype was confirmed by lung histology, lung function testing using a rodent ventilator, and expression of inflammatory markers by Real Time PCR. To measure miRNA expression, lungs were harvested, total RNA was extracted and converted to cDNA. We performed Real Time PCR with specific primers for miR‐106a and the small RNA U6 (normalization control) using the miRCURY LNA® RT® kit. Our results show sex differences in expression of miR‐106a upon HDM and ozone challenge. Ozone exposure increased miR‐106a expression in non‐asthmatic females and asthmatic males when compared to filtered air exposed mice (p&lt;0.0001 and p&lt;0.05, respectively). Male asthmatic mice had higher miR‐106a expression than non‐asthmatic male mice (p&lt;0.0001). However, the interaction between ozone exposure and sex was significant in non‐asthmatic mice only. We conclude that the observed differences in miR‐106a expression may be a result of different lung immune cells recruited to the male and female asthmatic lung. Understanding the mechanisms behind these sex differences may lead to future development of novel treatments for asthma.Support or Funding InformationThanks to the American Physiological Society Undergraduate Summer Research Fellowship for allowing me to do this research and to the NIH grants HL133520 and HL141618 for supporting this work.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Suppression of lung inflammation by the ethanol extract of Chung-Sang and the possible role of Nrf2

BackgroundAsian traditional herbal remedies are typically a concoction of a major and several complementary herbs. While balancing out any adverse effect of the major herb, the complementary herbs could dilute the efficacy of the major herb, resulting in a suboptimal therapeutic effect of an herbal remedy. Here, we formulated Chung-Sang (CS) by collating five major herbs, which are used against inflammatory diseases, and tested whether an experimental formula composed of only major herbs is effective in suppressing inflammation without significant side effects.MethodsThe 50% ethanol extract of CS (eCS) was fingerprinted by HPLC. Cytotoxicity to RAW 264.7 cells was determined by an MTT assay and a flow cytometer. Nuclear NF-κB and Nrf2 were analyzed by western blot. Ubiquitinated Nrf2 was similarly analyzed following immunoprecipitation of Nrf2. Acute lung inflammation and sepsis were induced in C57BL/6 mice. The effects of eCS on lung disease were measured by HE staining of lung sections, a differential cell counting of bronchoalveolar lavage fluid, a myeloperoxidase (MPO) assay, a real-time qPCR, and Kaplan-Meier survival of mice.ResultseCS neither elicited cytotoxicity nor reactive oxygen species. While not suppressing NF-κB, eCS activated Nrf2, reduced the ubiquitination of Nrf2, and consequently induced the expression of Nrf2-dependent genes. In an acute lung inflammation mouse model, an intratracheal (i.t.) eCS suppressed neutrophil infiltration, the expression of inflammatory cytokine genes, and MPO activity. In a sepsis mouse model, a single i.t. eCS was sufficient to significantly decrease mouse mortality.ConclusionseCS could suppress severe lung inflammation in mice. This effect seemed to associate with eCS activating Nrf2. Our findings suggest that herbal remedies consisting of only major herbs are worth considering.

The catalytic subunit of DNA-PK has a unique function in inflammation independently of Ku70 and DNA repair: a new opportunity to target the enzyme without interfering with DNA repair

Abstract Our laboratory demonstrated a critical role for DNA-dependent protein kinase (DNA-PK) in asthma pathogenesis via modulating the pertinent immune responses. DNA-PK is DNA repair enzyme composed of a catalytic subunit (DNA-PKcs) and two DNA-binding subunits (Ku70 and Ku80). Human cells express high levels of DNA-PK, surprisingly such high levels do not confer increased ability to repair DNA damage. Here we show that the role of DNA-PK in promoting inflammatory responses is independent of its function in the DNA repair. We examined the effect(s) of partial depletion of Ku70 on Ovalbumin (OVA) induced lung inflammation in mouse model of the disease. Of note, depletion of Ku70 by gene heterozygosity causes deficiency in DNA-PK-dependent DNA repair. Unlike the protective effects provided by DNA-PKcs gene heterozygosity, Ku70 heterozygosity did not alter the OVA-induced eosinophilia, mucus hypersecretion, Th2 cytokines production, or OVA-specific IgE upon OVA challenge. Interestingly, Ku70 heterozygosity enhanced methacholine-induced AHR over that of WT mice. Using a cell culture system, we demonstrate that while IL-4 and TNF-α are potent inducers of DNA-PKcs, such activation did not coincide with any detectable DNA damage or repair responses. Remarkably, while Ku70−/− blocked DNA-PKcs autophosphorylation in response to the DNA damage agent, etoposide, it did not affect the kinase in response to TNFα. Our findings suggest that the mechanism by which DNA-PK functions in inflammation is completely unrelated to its role in DNA repair, thus, unraveling a completely novel function for the kinase. More importantly, this provides a window of opportunity to target DNA-PK in inflammatory diseases without interfering with DNA repair processes.