Distinctive CD39+CD9+ lung interstitial macrophages suppress IL-23/Th17-mediated neutrophilic asthma by inhibiting NETosis.

The IL-23-Th17 axis is responsible for neutrophilic inflammation in various inflammatory diseases. Here, we discover a potential pathway to inhibit neutrophilic asthma. In our neutrophil-dominant asthma (NDA) model, single-cell RNA-seq analysis identifies a subpopulation of CD39+CD9+ interstitial macrophages (IMs) suppressed by IL-23 in NDA conditions but increased by an IL-23 inhibitor αIL-23p19. Adoptively transferred CD39+CD9+ IMs suppress neutrophil extracellular trap formation (NETosis), a representative phenotype of NDA, and also Th17 cell activation and neutrophilic inflammation. CD39+CD9+ IMs first attach to neutrophils in a CD9-dependent manner, and then remove ATP near neutrophils that contribute to NETosis in a CD39-dependent manner. Transcriptomic data from asthmatic patients finally show decreased CD39+CD9+ IMs in severe asthma than mild/moderate asthma. Our results suggest that CD39+CD9+ IMs function as a potent negative regulator of neutrophilic inflammation by suppressing NETosis in the IL-23-Th17 axis and can thus serve as a potential therapeutic target for IL-23-Th17-mediated neutrophilic asthma.

Permissive lung neutrophils facilitate tuberculosis immunopathogenesis in male phagocyte NADPH oxidase-deficient mice.

NADPH oxidase 2 (NOX2) is an enzyme responsible for generating reactive oxygen species, primarily found in phagocytes. Chronic Granulomatous Disease (CGD), along with bacterial infections such as Mycobacterium tuberculosis (Mtb), is a representative NOX2-deficient X-linked disease characterized by uncontrolled inflammation. However, the precise roles of host-derived factors that induce infection-mediated hyperinflammation in NOX2-deficient condition remain incompletely understood. To address this, we compared Mtb-induced pathogenesis in Nox2-/- and wild type (WT) mice in a sex-dependent manner. Among age- and sex-matched mice subjected to Mtb infection, male Nox2-/- mice exhibited a notable increase in bacterial burden and lung inflammation. This was characterized by significantly elevated pro-inflammatory cytokines such as G-CSF, TNF-α, IL-1α, IL-1ß, and IL-6, excessive neutrophil infiltration, and reduced pulmonary lymphocyte levels as tuberculosis (TB) progressed. Notably, lungs of male Nox2-/- mice were predominantly populated with CD11bintLy6GintCXCR2loCD62Llo immature neutrophils which featured mycobacterial permissiveness. By diminishing total lung neutrophils or reducing immature neutrophils, TB immunopathogenesis was notably abrogated in male Nox2-/- mice. Ultimately, we identified G-CSF as the pivotal trigger that exacerbates the generation of immature permissive neutrophils, leading to TB immunopathogenesis in male Nox2-/- mice. In contrast, neutralizing IL-1α and IL-1ß, which are previously known factors responsible for TB pathogenesis in Nox2-/- mice, aggravated TB immunopathogenesis. Our study revealed that G-CSF-driven immature and permissive pulmonary neutrophils are the primary cause of TB immunopathogenesis and lung hyperinflammation in male Nox2-/- mice. This highlights the importance of quantitative and qualitative control of pulmonary neutrophils to alleviate TB progression in a phagocyte oxidase-deficient condition.

Airway epithelial CD47 plays a critical role in inducing influenza virus-mediated bacterial super-infection.

Respiratory viral infection increases host susceptibility to secondary bacterial infections, yet the precise dynamics within airway epithelia remain elusive. Here, we elucidate the pivotal role of CD47 in the airway epithelium during bacterial super-infection. We demonstrated that upon influenza virus infection, CD47 expression was upregulated and localized on the apical surface of ciliated cells within primary human nasal or bronchial epithelial cells. This induced CD47 exposure provided attachment sites for Staphylococcus aureus, thereby compromising the epithelial barrier integrity. Through bacterial adhesion assays and in vitro pull-down assays, we identified fibronectin-binding proteins (FnBP) of S. aureus as a key component that binds to CD47. Furthermore, we found that ciliated cell-specific CD47 deficiency or neutralizing antibody-mediated CD47 inactivation enhanced in vivo survival rates. These findings suggest that interfering with the interaction between airway epithelial CD47 and pathogenic bacterial FnBP holds promise for alleviating the adverse effects of super-infection.

Proteomic analysis-based discovery of a novel biomarker that differentiates intestinal Behçet's disease from Crohn's disease.

Intestinal Behçet's disease (BD) and Crohn's disease (CD) present similar manifestations, but there are no specific diagnostic tests to differentiate them. We used a proteomic approach to discover novel diagnostic biomarkers specific to intestinal BD. Colon mucosa tissue samples were obtained from patients with intestinal BD or CD using colonoscopy-guided biopsy of the affected bowel. Peptides from seven intestinal BD and seven CD patients were extracted and labeled using tandem mass tag (TMT) reagents. The labeled peptides were identified and quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The proteins were further validated using immunohistochemical (IHC) analysis with tissue samples and an ELISA test with serum samples from 20 intestinal BD and 20 CD patients. Using TMT/LC-MS/MS-based proteomic quantification, we identified 39 proteins differentially expressed between intestinal BD and CD. Beta-2 glycoprotein 1 (APOH) and maltase-glucoamylase (MGAM) showed higher intensity in the IHC staining of intestinal BD tissues than in CD tissues. The serum MGAM level was higher in intestinal BD patients. Proteomic analysis revealed that some proteins were differentially expressed in patients with intestinal BD compared with those with CD. Differential MGAM expression in intestinal BD suggests its role as a potential novel diagnostic biomarker.

Quantitative Proteomic Analysis of the Expression of SARS-CoV-2 Receptors in the Gut of Patients with Chronic Enterocolitis.

The cellular entry of severe respiratory syndrome coronavirus-2 (SARS-CoV-2) is mediated by interaction with the human angiotensin-converting enzyme 2 (ACE2), a receptor that is expressed on both lung and intestinal epithelial cells. We performed a quantitative proteomic analysis to investigate the expression of possible receptors for SARS-CoV-2 in the intestinal mucosa of 23 patients with chronic colitis. ACE2 expression was low and remained unaltered in the gut of patients with ulcerative colitis (UC), Crohn's disease (CD), intestinal Behcet's disease (BD), and intestinal tuberculosis (TB), when compared with that of healthy individuals. Additionally, the expression levels of some probable co-receptors, including dipeptidyl peptidase 4 (DPP4), aminopeptidase N (AMPN), and glutamyl aminopeptidase (AMPE), were unchanged in the affected UC, CD, intestinal BD, and intestinal TB colon mucosa samples. In conclusion, gut inflammation associated with chronic colitis does not mediate a further increase in the cellular entry of SARS-CoV-2.

NOX4 Deficiency Exacerbates the Impairment of Cystatin C-Dependent Hippocampal Neurogenesis by a Chronic High Fat Diet.

Hippocampal neurogenesis is linked with a cognitive process under a normal physiological condition including learning, memory, pattern separation, and cognitive flexibility. Hippocampal neurogenesis is altered by multiple factors such as the systemic metabolic changes. NADPH oxidase 4 (NOX4) has been implicated in the regulation of brain function. While the role of NOX4 plays in the brain, the mechanism by which NOX4 regulates hippocampal neurogenesis under metabolic stress is unclear. In this case, we show that NOX4 deficiency exacerbates the impairment of hippocampal neurogenesis by inhibiting neuronal maturation by a chronic high fat diet (HFD). NOX4 deficiency resulted in less hippocampal neurogenesis by decreasing doublecortin (DCX)-positive neuroblasts, a neuronal differentiation marker, and their branched-dendrites. Notably, NOX4 deficiency exacerbates the impairment of hippocampal neurogenesis by chronic HFD. Moreover, NOX4 deficiency had a significant reduction of Cystatin C levels, which is critical for hippocampal neurogenesis, under chronic HFD as well as normal chow (NC) diet. Furthermore, the reduction of Cystatin C levels was correlated with the impairment of hippocampal neurogenesis in NOX4 deficient and wild-type (WT) mice under chronic HFD. Our results suggest that NOX4 regulates the impairment of Cystatin C-dependent hippocampal neurogenesis under chronic HFD.

Apolipoprotein E and Periostin Are Potential Biomarkers of Nasal Mucosal Inflammation. A Parallel Approach of In Vitro and In Vivo Secretomes.

No previously suggested biomarkers of nasal mucosal inflammation have been practically applied in clinical fields, and nasal epithelium-derived secreted proteins as biomarkers have not specifically been investigated. The goal of this study was to identify secreted proteins that dynamically change during the differentiation from basal cells to fully differentiated cells and examine whether nasal epithelium-derived proteins can be used as biomarkers of nasal mucosal inflammation, such as chronic rhinosinusitis. To achieve this goal, we analyzed two secretomes using the isobaric tag for relative and absolute quantification technique. From in vitro secretomes, we identified the proteins altered in apical secretions of primary human nasal epithelial cells according to the degree of differentiation; from in vivo secretomes, we identified the increased proteins in nasal lavage fluids obtained from patients 2 weeks after endoscopic sinus surgery for chronic sinusitis. We then used a parallel approach to identify specific biomarkers of nasal mucosal inflammation; first, we selected apolipoprotein E as a nasal epithelial cell-derived biomarker through screening proteins that were upregulated in both in vitro and in vivo secretomes, and verified highly secreted apolipoprotein E in nasal lavage fluids of the patients by Western blotting. Next, we selected periostin as an inflammatory mediator-inducible biomarker from in vivo secretomes, the secretion of which was not induced under in vitro culture conditions. We demonstrated that those two nasal epithelium-derived proteins are possible biomarkers of nasal mucosal inflammation.

Naphthalene-based fluorescent probes for glutathione and their applications in living cells and patients with sepsis.

Rationale: Among the biothiols-related diseases, sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection and can result in severe oxidative stress and damage to multiple organs. In this study, we aimed to develop a fluorescence chemosensor that can both detect GSH and further predict sepsis. Methods: In this study, two new naphthalene dialdehyde compounds containing different functional groups were synthesized, and the sensing abilities of these compounds towards biothiols and its applications for prediction of sepsis were investigated. Results: Our study revealed that the newly developed probe 6-methoxynaphthalene-2, 3-dicarbaldehyde (MNDA) has two-photon is capable of detecting GSH in live cells with two-photon microscopy (TPM) under the excitation at a wavelength of 900 nm. Furthermore, two GSH detection probes naphthalene-2,3-dicarboxaldehyde (NDA) and 6-fluoronaphthalene-2,3-dicarbaldehyde (FNDA) not only can detect GSH in living cells, but also showed clinical significance for the diagnosis and prediction of mortality in patients with sepsis. Conclusions: These results open up a promising direction for further medical diagnostic techniques.

A Far-Red-Emitting Fluorescence Probe for Sensitive and Selective Detection of Peroxynitrite in Live Cells and Tissues.

In this study, the far-red-emitting fluorescence probe 1, containing a rhodamine derivative and a hydrazide reactive group, was developed for peroxynitrite detection and imaging. This probe, which is cell permeable and shows high sensitivity and selectivity in fluorometric detection of peroxynitrite over other ROS/RNS, was successfully utilized to detect exogenous and endogenous peroxynitrite in HeLa and RAW 264.7 cells, respectively. More importantly, 1 can also be used to detect endogenous peroxynitrite generated in Pseudomonas aeruginosa (PAO1)-infected mouse bone marrow-derived neutrophils. We anticipate that the new probe will serve as a powerful molecular imaging tool in investigations of the role(s) played by peroxynitrite in a variety of physiological and pathological contexts.

SESN2/sestrin2 suppresses sepsis by inducing mitophagy and inhibiting NLRP3 activation in macrophages.

Proper regulation of mitophagy for mitochondrial homeostasis is important in various inflammatory diseases. However, the precise mechanisms by which mitophagy is activated to regulate inflammatory responses remain largely unknown. The NLRP3 (NLR family, pyrin domain containing 3) inflammasome serves as a platform that triggers the activation of CASP1 (caspase 1) and secretion of proinflammatory cytokines. Here, we demonstrate that SESN2 (sestrin 2), known as stress-inducible protein, suppresses prolonged NLRP3 inflammasome activation by clearance of damaged mitochondria through inducing mitophagy in macrophages. SESN2 plays a dual role in inducing mitophagy in response to inflammasome activation. First, SESN2 induces "mitochondrial priming" by marking mitochondria for recognition by the autophagic machinery. For mitochondrial preparing, SESN2 facilitates the perinuclear-clustering of mitochondria by mediating aggregation of SQSTM1 (sequestosome 1) and its binding to lysine 63 (Lys63)-linked ubiquitins on the mitochondrial surface. Second, SESN2 activates the specific autophagic machinery for degradation of primed mitochondria via an increase of ULK1 (unc-51 like kinase 1) protein levels. Moreover, increased SESN2 expression by extended LPS (lipopolysaccharide) stimulation is mediated by NOS2 (nitric oxide synthase 2, inducible)-mediated NO (nitric oxide) in macrophages. Thus, Sesn2-deficient mice displayed defective mitophagy, which resulted in hyperactivation of inflammasomes and increased mortality in 2 different sepsis models. Our findings define a unique regulatory mechanism of mitophagy activation for immunological homeostasis that protects the host from sepsis.

Preparation of a cyanine-based fluorescent probe for highly selective detection of glutathione and its use in living cells and tissues of mice.

Glutathione (GSH) is a major endogenous antioxidant that has a central role in cellular defense against toxins and free radicals. This protocol describes the preparation of CPDSA, a cyanine-based near-infrared (NIR) fluorescent probe for the detection of GSH in cells and in vivo. CPDSA is prepared with high yield through a simple two-step process. The first step is to react commercially available IR-780 iodide with excess anhydrous piperazine in anhydrous N,N-dimethyl formamide at 85 °C to form cyanine-piperazine (CP). The second step is the sulfonylation of CP with dansyl chloride in anhydrous dichloromethane. CPDSA selectively detects GSH in cells, and it has been shown to not react with other biothiols such as cysteine (Cys) and homocysteine (Hcy). This probe can also be used to monitor the GSH level of mouse bone marrow-derived neutrophils (BMDNs). The preparation of probe CPDSA takes 2 d, and experiments in cells and mice take 12-13 d.

The Induction of Pattern-Recognition Receptor Expression against Influenza A Virus through Duox2-Derived Reactive Oxygen Species in Nasal Mucosa.

We studied the relative roles of Duox2-derived reactive oxygen species (ROS) in host defense against influenza A virus (IAV) infection in normal human nasal epithelial cells and mouse nasal mucosa. We found that Duox2 primarily generated ROS rapidly after IAV infection in normal human nasal epithelial cells and that knockdown of Duox2 aggravated IAV infection. In addition, Duox2-derived ROS enhancement significantly suppressed IAV infection in nasal epithelium. In particular, Duox2-derived ROS were required for the induction of retinoic acid-inducible gene (RIG)-I and melanoma differentiation-associated protein 5 (MDA5) transcription. After intranasal IAV inoculation into mice, viral infection was significantly aggravated from 3 days postinoculation (dpi) in the nasal mucosa, and the IAV viral titer was highest at 7 dpi. Both RIG-I and MDA5 messenger RNA levels increased dominantly in mouse nasal mucosa from 3 dpi; consistent with this, RIG-I and MDA5 proteins were also induced after IAV infection. RIG-I and MDA5 messenger RNA levels were induced to a lower extent in the nasal mucosa of the mice that were inoculated with Duox2 short hairpin RNA, and the IAV viral titer was significantly higher in nasal lavage. Taken together, Duox2-derived ROS are necessary for the innate immune response and trigger the induction of RIG-I and MDA5 to resist IAV infection in human nasal epithelium and mouse nasal mucosa.

Dual oxidase 2 in lung epithelia is essential for hyperoxia-induced acute lung injury in mice.

AIMS: Acute lung injury (ALI) induced by excessive hyperoxia has been employed as a model of oxidative stress imitating acute respiratory distress syndrome. Under hyperoxic conditions, overloading quantities of reactive oxygen species (ROS) are generated in both lung epithelial and endothelial cells, leading to ALI. Some NADPH oxidase (NOX) family enzymes are responsible for hyperoxia-induced ROS generation in lung epithelial and endothelial cells. However, the molecular mechanisms of ROS production in type II alveolar epithelial cells (AECs) and ALI induced by hyperoxia are poorly understood. RESULTS: In this study, we show that dual oxidase 2 (DUOX2) is a key NOX enzyme that affects hyperoxia-induced ROS production, particularly in type II AECs, leading to lung injury. In DUOX2 mutant mice (DUOX2(thyd/thyd)) or mice in which DUOX2 expression is knocked down in the lungs, hyperoxia-induced ALI was significantly lower than in wild-type (WT) mice. DUOX2 was mainly expressed in type II AECs, but not endothelial cells, and hyperoxia-induced ROS production was markedly reduced in primary type II AECs isolated from DUOX2(thyd/thyd) mice. Furthermore, DUOX2-generated ROS are responsible for caspase-mediated cell death, inducing ERK and JNK phophorylation in type II AECs. INNOVATION: To date, no role for DUOX2 has been defined in hyperoxia-mediated ALI despite it being a NOX homologue and major ROS source in lung epithelium. CONCLUSION: Here, we present the novel finding that DUOX2-generated ROS induce AEC death, leading to hyperoxia-induced lung injury.

Cyanine-based fluorescent probe for highly selective detection of glutathione in cell cultures and live mouse tissues.

Glutathione (GSH) plays a crucial role in human pathologies. Near-infrared fluorescence-based sensors capable of detecting intracellular GSH in vivo would be useful tools to understand the mechanisms of diseases. In this work, two cyanine-based fluorescent probes, 1 and 2, containing sulfonamide groups were prepared. Evaluation of the fluorescence changes displayed by probe 1, which contains a 2,4-dinitrobenzenesulfonamide group, shows that it is cell-membrane-permeable and can selectively detect thiols such as GSH, cysteine (Cys), and homocysteine (Hcy) in living cells. The response of 1 to thiols can be reversed by treatment with N-methylmaleimide (NMM). Probe 2, which possesses a 5-(dimethylamino)naphthalenesulfonamide group, displays high selectivity for GSH over Cys and Hcy, and its response can be reversed using NMM. The potential biological utility of 2 was shown by its use in fluorescence imaging of GSH in living cells. Furthermore, probe 2 can determine changes in the intracellular levels of GSH modualated by H2O2. The properties of 2 enable its use in monitoring GSH in vivo in a mouse model. The results showed that intravenous injection of 2 into a mouse generates a dramatic image in which strong fluorescence is emitted from various tissues, including the liver, kidney, lung, and spleen. Importantly, 2 can be utilized to monitor the depletion of GSH in mouse tissue cells promoted by excessive administration of the painkiller acetaminophen. The combined results coming from this effort suggest that the new probe will serve as an efficient tool for detecting cellular GSH in animals.

Reactive oxygen species induce antiviral innate immune response through IFN-λ regulation in human nasal epithelial cells.

This study sought to explore the role of the IFN-related innate immune responses (IFN-ß and IFN-λ) and of reactive oxygen species (ROS) after influenza A virus (IAV) infection for antiviral innate immune activity in normal human nasal epithelial (NHNE) cells that are highly exposed to IAV. Passage-2 NHNE cells were inoculated with the IAV WSN/33 for 1, 2, and 3 days to assess the capacity of IFN and the relationship between ROS generation and IFN-λ secretion for controlling IAV infection. Viral titers and IAV mRNA levels increased after infection. In concert with viral titers, we found that the generation of IFNs, such as IFN-ß, IFN-λ1, and IFN-λ2/3, was induced after IAV infection until 3 days after infection. The induction of IFN-λ gene expression and protein secretion may be predominant after IAV infection. Similarly, we observed that intracellular ROS generation increased 60 minutes after IAV infection. Viral titers and mRNA levels of IAV were significantly higher in cases with scavenging ROS, in cases with an induced IFN-λ mRNA level, or where the secreted protein concentration of IFN-λ was attenuated after the suppression of ROS generation. Both mitochondrial and dual oxidase (Doux)2-generated ROS were correlated with IAV mRNA and viral titers. The inhibition of mitochondrial ROS generation and the knockdown of Duox2 gene expression highly increased IAV viral titers and decreased IFN-λ secretion. Our findings suggest that the production of ROS may be responsible for IFN-λ secretion to control IAV infection. Both mitochondria and Duox2 are possible sources of ROS generation, which is required to initiate an innate immune response in NHNE cells.

Distinct TLR-mediated pathways regulate house dust mite-induced allergic disease in the upper and lower airways.

BACKGROUND: Allergic rhinitis (AR) and asthma are 2 entities of allergic airway diseases that frequently occur together, which is referred to as united airways. In contrast to this general concept, we hypothesized that innate immunity of the upper and lower airways is respectively distinctive, because the immunologic conditions of the nasal and lung mucosa as well as the functions of the immune cells within their epithelia are different. OBJECTIVE: We wanted to identify distinctive mechanisms of innate immunity in the nose and lung mucosa, which are responsible for house dust mite (HDM)-induced AR and allergic asthma (AA), respectively. METHODS: We constructed a mouse model of AR or AA induced by sensitization and consequent provocation with HDM extracts. RESULTS: HDM-derived ß-glucans, rather than LPS, were proven to be essential to activating innate immunity in the nasal mucosa and triggering AR, which depended on Toll-like receptor 2 (TLR2), but not on TLR4; however, the LPS/TLR4 signaling axis, rather than ß-glucans/TLR2, was critical to HDM-induced AA. These differences were attributed to the specific role of ß-glucans and LPS in inducing the surface expression of TLR2 and TLR4 and their translocation to lipid rafts in nasal and bronchial epithelial cells, respectively. We also showed that dual oxidase 2-generated reactive oxygen species mediate both ß-glucan-induced TLR2 activation and LPS-induced TLR4 activation. CONCLUSIONS: We describe a novel finding of distinctive innate immunity of the nose and lungs, respectively, which trigger AR and AA, by showing the critical role of HDM-induced TLR activation via dual oxidase 2-mediated reactive oxygen species.

Dual oxidase 2 is essential for the toll-like receptor 5-mediated inflammatory response in airway mucosa.

AIMS: Airway mucosa is constantly exposed to various airborne microbes, and epithelial host defense requires a robust innate immunity. Recently, it has been suggested that NADPH oxidase (NOX) isozymes serve functional roles in toll-like receptor (TLR)-mediated innate immune responses. However, the molecular mechanism between TLR and NOX-mediated reactive oxygen species (ROS) production in human airway mucosa has been poorly understood. RESULTS: Here, we show that flagellin-induced ROS generation is dependent on dual oxidase 2 (DUOX2) activation, which is regulated by [Ca(2+)](i) mobilization in primary normal human nasal epithelial (NHNE) cells. Interestingly, we observed that silencing of DUOX2 expression in NHNE cells and nasal epithelium of Duox2 knockout mice failed to trigger mucin and MIP-2? production upon challenging flagellin. INNOVATION: Our observation in this study reveals that flagellin-induced hydrogen peroxide (H(2)O(2)) generation is critical for TLR5-dependent innate immune responses, including IL-8 production and MUC5AC expression in the nasal epithelium. Furthermore, DUOX2-mediated H(2)O(2) generation activated by the flagellin-TLR5 axis might serve as a novel therapeutic target for infectious inflammation diseases in the airway tract. CONCLUSION: Taken together, we propose that DUOX2 plays pivotal roles in TLR5-dependent inflammatory response of nasal airway epithelium.

MyD88-dependent Toll-like receptor signaling is required for murine macrophages response to IS2.

IS2, a soluble ß-glucan isolated from the cell wall of mutated Saccharomyces cerevisiae (S. cerevisiae) enhances the immune response compared to the wild type (WT) ß-glucan. In the present investigation we report that Toll-like receptor (TLR)/MyD88 signaling pathway was responsible in IS2 ß-glucan-mediated cellular response in RAW264.7 murine macrophages. Data revealed that IS2 ß-glucan significantly up-regulated the TLR2/TLR4 expression. Moreover, TLR2/TLR4 responds to IS2 resulting in murine macrophage activation. In addition, the IS2 signal led to cytokine secretions of IL-6 and TNF-α. In the case of thioglycolate-elicited peritoneal macrophages from MyD88-deficient mice, the decrease in cytokines was observed. Further the mitogen-activated protein kinases (MAPKs) phosphorylation was evident and degradation of IκB-α was increased after stimulation with IS2 ß-glucan. Further examination with MyD88-deficient mice revealed that the MyD88 pathway might play an important role for IS2 ß-glucan-mediated activation of macrophages.

Crosstalk between platelet-derived growth factor-induced Nox4 activation and MUC8 gene overexpression in human airway epithelial cells.

Reactive oxygen species (ROS) contribute to chronic airway inflammation, and NADPH oxidase (Nox) is an important source of ROS. However, little is known of the role that ROS play in chronic upper respiratory tract inflammation. We investigated the mechanism of ROS generation and its association with mucin gene overexpression in the nasal epithelium. The level of platelet-derived growth factor (PDGF) expression was increased in sinusitis mucosa, and high-level PDGF expression induced intracellular ROS, followed by MUC8 gene overexpression in normal human nasal epithelial cells. Knockdown of Nox4 expression with Nox4 siRNA decreased PDGF-induced intracellular ROS and MUC8 expression. Infection with an adenovirus containing Nox4 cDNA resulted in Nox4 overexpression and increased intracellular levels of ROS and MUC8 expression. PDGF and Nox4 overexpression are essential components of intracellular ROS generation and may contribute to chronic inflammation in the nasal epithelium through induction of MUC8 overexpression.

α-Melanocyte-stimulating hormone inhibits tumor necrosis factor α-stimulated MUC5AC expression in human nasal epithelial cells.

Mucin hypersecretion is an important clinical feature of several respiratory diseases, including asthma, cystic fibrosis, nasal allergy, rhinitis, and sinusitis. It has been shown that α-melanocyte-stimulating hormone (α-MSH), a proopiomelanocortin (POMC)-derived peptide, has immunomodulatory activities by inhibiting NF-κB activation induced by proinflammatory cytokines such as TNF-α. Because MUC5AC expression is known to be up-regulated by TNF-α via NF-κB activation, we evaluated the inhibitory effect of α-MSH on MUC5AC gene expression induced by TNF-α in normal human nasal epithelial (NHNE) cells. Melanocortin-1-receptor (MC-1R) was detected by RT-PCR, Western blotting, and immunofluorescent labeling in NHNE cells. α-MSH suppressed NF-κB/p65 phosphorylation induced by TNF-α as well as IkB-α degradation in a dose-dependent manner, as assessed by Western blotting. In addition, α-MSH inhibited TNF-α-induced nuclear translocation of NF-κB and NF-κB luciferase activity. Real-time quantitative PCR data showed that α-MSH inhibited TNF-α-induced expression of MUC5AC, and this effect of α-MSH was neutralized by knockdown of MC-1R using MC-1R shRNA lentivirus. Analyses using RT-PCR and Western blotting showed the expression of POMC and two key enzymes in the POMC processing, proprotein convertases (PC)1 and PC2, and 7B2, which is required for enzymatic activity of PC2, in normal human nasal mucosa. We conclude that α-MSH down-regulates MUC5AC expression by inhibiting TNF-α-induced NF-κB activity through MC-1R stimulation in NHNE cells and that normal human nasal mucosa possesses the POMC processing machinery. Therefore, α-MSH may be a promising candidate to decrease mucin overproduction initiated by NF-κB activation.