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1.
Am J Physiol Lung Cell Mol Physiol ; 324(2): L228-L242, 2023 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-36625485

RESUMO

More than 50% of people with asthma in the United States are obese, and obesity often worsens symptoms of allergic asthma and impairs response to treatment. Based on previously established roles of the epithelial NADPH oxidase DUOX1 in allergic airway inflammation, we addressed the potential involvement of DUOX1 in altered allergic inflammation in the context of obesity. Intranasal house dust mite (HDM) allergen challenge of subjects with allergic asthma induced rapid secretion of IL-33, then IL-13, into the nasal lumen, responses that were significantly enhanced in obese asthmatic subjects (BMI >30). Induction of diet-induced obesity (DIO) in mice by high-fat diet (HFD) feeding similarly enhanced acute airway responses to intranasal HDM challenge, particularly with respect to secretion of IL-33 and type 2/type 3 cytokines, and this was associated with enhanced epithelial DUOX1 expression and was avoided in DUOX1-deficient mice. DIO also enhanced DUOX1-dependent features of chronic HDM-induced allergic inflammation. Although DUOX1 did not affect overall weight gain by HFD feeding, it contributed to glucose intolerance, suggesting a role in glucose metabolism. However, glucose intolerance induced by short-term HFD feeding, in the absence of adiposity, was not sufficient to alter HDM-induced acute airway responses. DIO was associated with enhanced presence of the adipokine leptin in the airways, and leptin enhanced DUOX1-dependent IL-13 and mucin production in airway epithelial cells. In conclusion, augmented inflammatory airway responses to HDM in obesity are associated with increases in airway epithelial DUOX1, and by increased airway epithelial leptin signaling.


Assuntos
Asma , Intolerância à Glucose , Animais , Camundongos , Alérgenos , Asma/metabolismo , Dieta , Modelos Animais de Doenças , Oxidases Duais , Inflamação , Interleucina-13 , Interleucina-33 , Leptina , Obesidade , Pyroglyphidae
2.
J Immunol ; 206(12): 2989-2999, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34088769

RESUMO

The respiratory epithelium forms the first line of defense against inhaled pathogens and acts as an important source of innate cytokine responses to environmental insults. One critical mediator of these responses is the IL-1 family cytokine IL-33, which is rapidly secreted upon acute epithelial injury as an alarmin and induces type 2 immune responses. Our recent work highlighted the importance of the NADPH oxidase dual oxidase 1 (DUOX1) in acute airway epithelial IL-33 secretion by various airborne allergens associated with H2O2 production and reduction-oxidation-dependent activation of Src kinases and epidermal growth factor receptor (EGFR) signaling. In this study, we show that IL-33 secretion in response to acute airway challenge with house dust mite (HDM) allergen critically depends on the activation of Src by a DUOX1-dependent oxidative mechanism. Intriguingly, HDM-induced epithelial IL-33 secretion was dramatically attenuated by small interfering RNA- or Ab-based approaches to block IL-33 signaling through its receptor IL1RL1 (ST2), indicating that HDM-induced IL-33 secretion includes a positive feed-forward mechanism involving ST2-dependent IL-33 signaling. Moreover, activation of type 2 cytokine responses by direct airway IL-33 administration was associated with ST2-dependent activation of DUOX1-mediated H2O2 production and reduction-oxidation-based activation of Src and EGFR and was attenuated in Duox1 -/- and Src +/- mice, indicating that IL-33-induced epithelial signaling and subsequent airway responses involve DUOX1/Src-dependent pathways. Collectively, our findings suggest an intricate relationship between DUOX1, Src, and IL-33 signaling in the activation of innate type 2 immune responses to allergens, involving DUOX1-dependent epithelial Src/EGFR activation in initial IL-33 secretion and in subsequent IL-33 signaling through ST2 activation.


Assuntos
Alérgenos/imunologia , Oxidases Duais/imunologia , Interleucina-33/imunologia , Mucosa Respiratória/imunologia , Quinases da Família src/imunologia , Doença Aguda , Animais , Células Cultivadas , Proteína 1 Semelhante a Receptor de Interleucina-1/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucosa Respiratória/patologia , Transdução de Sinais/imunologia , Quinases da Família src/deficiência
3.
Am J Physiol Lung Cell Mol Physiol ; 321(1): L144-L158, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33951398

RESUMO

Aging is associated with a gradual loss of lung function due to increased cellular senescence, decreased regenerative capacity, and impaired innate host defense. One important aspect of innate airway epithelial host defense to nonmicrobial triggers is the secretion of alarmins such as IL-33 and activation of type 2 inflammation, which were previously found to depend on activation of the NADPH oxidase (NOX) homolog DUOX1, and redox-dependent signaling pathways that promote alarmin secretion. Here, we demonstrate that normal aging of C57BL/6J mice resulted in markedly decreased lung innate epithelial type 2 responses to exogenous triggers such as the airborne allergen Dermatophagoides pteronyssinus, which was associated with marked downregulation of DUOX1, as well as DUOX1-mediated redox-dependent signaling. DUOX1 deficiency was also found to accelerate age-related airspace enlargement and decline in lung function but did not consistently affect other features of lung aging such as senescence-associated inflammation. Intriguingly, observations of age-related DUOX1 downregulation and enhanced airspace enlargement due to DUOX1 deficiency in C57BL/6J mice, which lack a functional mitochondrial nicotinamide nucleotide transhydrogenase (NNT), were much less dramatic in C57BL/6NJ mice with normal NNT function, although the latter mice also displayed impaired innate epithelial injury responses with advancing age. Overall, our findings indicate a marked aging-dependent decline in (DUOX1-dependent) innate airway injury responses to external nonmicrobial triggers, but the impact of aging on DUOX1 downregulation and its significance for age-related senile emphysema development was variable between different C57BL6 substrains, possibly related to metabolic alterations due to differences in NNT function.


Assuntos
Lesão Pulmonar Aguda/patologia , Envelhecimento/patologia , Oxidases Duais/fisiologia , Inflamação/patologia , Enfisema Pulmonar/patologia , Mucosa Respiratória/patologia , Lesão Pulmonar Aguda/etiologia , Lesão Pulmonar Aguda/metabolismo , Animais , Feminino , Inflamação/etiologia , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Enfisema Pulmonar/etiologia , Enfisema Pulmonar/metabolismo , Mucosa Respiratória/metabolismo
4.
Am J Physiol Lung Cell Mol Physiol ; 320(3): L356-L367, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33325804

RESUMO

Idiopathic pulmonary fibrosis (IPF) is characterized by a disturbed redox balance and increased production of reactive oxygen species (ROS), which is believed to contribute to epithelial injury and fibrotic lung scarring. The main pulmonary sources of ROS include mitochondria and NADPH oxidases (NOXs), of which the NOX4 isoform has been implicated in IPF. Non-receptor SRC tyrosine kinases (SFK) are important for cellular homeostasis and are often dysregulated in lung diseases. SFK activation by the profibrotic transforming growth factor-ß (TGF-ß) is thought to contribute to pulmonary fibrosis, but the relevant SFK isoform and its relationship to NOX4 and/or mitochondrial ROS in the context of profibrotic TGF-ß signaling is not known. Here, we demonstrate that TGF-ß1 can rapidly activate the SRC kinase FYN in human bronchial epithelial cells, which subsequently induces mitochondrial ROS (mtROS) production, genetic damage shown by the DNA damage marker γH2AX, and increased expression of profibrotic genes. Moreover, TGF-ß1-induced activation of FYN involves initial activation of NOX4 and direct cysteine oxidation of FYN, and both FYN and mtROS contribute to TGF-ß-induced induction of NOX4. NOX4 expression in lung tissues of IPF patients is positively correlated with disease severity, although FYN expression is down-regulated in IPF and does not correlate with disease severity. Collectively, our findings highlight a critical role for FYN in TGF-ß1-induced mtROS production, DNA damage response, and induction of profibrotic genes in bronchial epithelial cells, and suggest that altered expression and activation of NOX4 and FYN may contribute to the pathogenesis of pulmonary fibrosis.


Assuntos
Brônquios/metabolismo , Células Epiteliais/metabolismo , Mitocôndrias/metabolismo , NADPH Oxidase 4/metabolismo , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismo , Brônquios/patologia , Células Epiteliais/patologia , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Mitocôndrias/patologia , Oxirredução , Espécies Reativas de Oxigênio/metabolismo
5.
Am J Respir Cell Mol Biol ; 63(2): 198-208, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32182090

RESUMO

The airway epithelium plays a critical role in innate responses to airborne allergens by secreting IL-1 family cytokines such as IL-1α and IL-33 as alarmins that subsequently orchestrate appropriate immune responses. Previous studies revealed that epithelial IL-33 secretion by allergens such as Alternaria alternata or house dust mite involves Ca2+-dependent signaling, via initial activation of ATP-stimulated P2YR2 (type 2 purinoceptor) and subsequent activation of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase DUOX1. We sought to identify proximal mechanisms by which epithelial cells sense these allergens and here highlight the importance of PAR2 (protease-activated receptor 2) and TRP (transient receptor potential) Ca2+ channels such as TRPV1 (TRP vanilloid 1) in these responses. Combined studies of primary human nasal and mouse tracheal epithelial cells, as well as immortalized human bronchial epithelial cells, indicated the importance of both PAR2 and TRPV1 in IL-33 secretion by both Alternaria alternata and house dust mite, based on both pharmacological and genetic approaches. TRPV1 was also critically involved in allergen-induced ATP release, activation of DUOX1, and redox-dependent activation of EGFR (epidermal growth factor receptor). Moreover, genetic deletion of TRPV1 dramatically attenuated allergen-induced IL-33 secretion and subsequent type 2 responses in mice in vivo. TRPV1 not only contributed to ATP release and P2YR2 signaling but also was critical in downstream innate responses to ATP, indicating potentiating effects of P2YR2 on TRPV1 activation. In aggregate, our studies illustrate a complex relationship between various receptor types, including PAR2 and P2YR2, in epithelial responses to asthma-relevant airborne allergens and highlight the central importance of TRPV1 in such responses.


Assuntos
Alérgenos/imunologia , Células Epiteliais/imunologia , Imunidade Inata/imunologia , Peptídeo Hidrolases/imunologia , Canais de Cátion TRPV/imunologia , Animais , Asma/imunologia , Brônquios/imunologia , Células Cultivadas , Epitélio/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Pyroglyphidae/imunologia , Receptor PAR-2/imunologia , Mucosa Respiratória/imunologia , Transdução de Sinais/imunologia
6.
J Biol Chem ; 291(44): 23282-23293, 2016 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-27650496

RESUMO

The epidermal growth factor receptor (EGFR) plays a critical role in regulating airway epithelial homeostasis and responses to injury. Activation of EGFR is regulated by redox-dependent processes involving reversible cysteine oxidation by reactive oxygen species (ROS) and involves both ligand-dependent and -independent mechanisms, but the precise source(s) of ROS and the molecular mechanisms that control tyrosine kinase activity are incompletely understood. Here, we demonstrate that stimulation of EGFR activation by ATP in airway epithelial cells is closely associated with dynamic reversible oxidation of cysteine residues via sequential sulfenylation and S-glutathionylation within EGFR and the non-receptor-tyrosine kinase Src. Moreover, the intrinsic kinase activity of recombinant Src or EGFR was in both cases enhanced by H2O2 but not by GSSG, indicating that the intermediate sulfenylation is the activating modification. H2O2-induced increase in EGFR tyrosine kinase activity was not observed with the C797S variant, confirming Cys-797 as the redox-sensitive cysteine residue that regulates kinase activity. Redox-dependent regulation of EGFR activation in airway epithelial cells was found to strongly depend on activation of either the NADPH oxidase DUOX1 or the homolog NOX2, depending on the activation mechanism. Whereas DUOX1 and Src play a primary role in EGFR transactivation by wound-derived signals such as ATP, direct ligand-dependent EGFR activation primarily involves NOX2 with a secondary role for DUOX1 and Src. Collectively, our findings establish that redox-dependent EGFR kinase activation involves a dynamic and reversible cysteine oxidation mechanism and that this activation mechanism variably involves DUOX1 and NOX2.


Assuntos
Receptores ErbB/metabolismo , Glicoproteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Transdução de Sinais , Animais , Oxidases Duais , Células Epiteliais/enzimologia , Células Epiteliais/metabolismo , Receptores ErbB/genética , Feminino , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , NADPH Oxidase 2 , NADPH Oxidases/genética , Oxirredução , Espécies Reativas de Oxigênio/metabolismo
7.
J Allergy Clin Immunol ; 137(5): 1545-1556.e11, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26597162

RESUMO

BACKGROUND: The IL-1 family member IL-33 plays a critical role in type 2 innate immune responses to allergens and is an important mediator of allergic asthma. The mechanisms by which allergens provoke epithelial IL-33 secretion are still poorly understood. OBJECTIVE: Based on previous findings indicating involvement of the NADPH oxidase dual oxidase 1 (DUOX1) in epithelial wound responses, we explored the potential involvement of DUOX1 in allergen-induced IL-33 secretion and potential alterations in airways of asthmatic patients. METHODS: Cultured human or murine airway epithelial cells or mice were subjected to acute challenge with Alternaria alternata or house dust mite, and secretion of IL-33 and activation of subsequent type 2 responses were determined. The role of DUOX1 was explored by using small interfering RNA approaches and DUOX1-deficient mice. Cultured nasal epithelial cells from healthy subjects or asthmatic patients were evaluated for DUOX1 expression and allergen-induced responses. RESULTS: In vitro or in vivo allergen challenge resulted in rapid airway epithelial IL-33 secretion, which depended critically on DUOX1-mediated activation of epithelial epidermal growth factor receptor and the protease calpain-2 through a redox-dependent mechanism involving cysteine oxidation within epidermal growth factor receptor and the tyrosine kinase Src. Primary nasal epithelial cells from patients with allergic asthma were found to express increased DUOX1 and IL-33 levels and demonstrated enhanced IL-33 secretion in response to allergen challenge compared with values seen in nasal epithelial cells from nonasthmatic subjects. CONCLUSION: Our findings implicate epithelial DUOX1 as a pivotal mediator of IL-33-dependent activation of innate airway type 2 immune responses to common airborne allergens and indicate that enhanced DUOX1 expression and IL-33 secretion might present important contributing features of allergic asthma.


Assuntos
Alérgenos/imunologia , Interleucina-33/imunologia , NADPH Oxidases/imunologia , Rinite Alérgica/imunologia , Alternaria/imunologia , Animais , Líquido da Lavagem Broncoalveolar , Linhagem Celular , Células Cultivadas , Oxidases Duais , Células Epiteliais/imunologia , Receptores ErbB/imunologia , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidases/genética , Pyroglyphidae/imunologia , RNA Interferente Pequeno/genética
8.
Am J Physiol Lung Cell Mol Physiol ; 311(5): L913-L923, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27612966

RESUMO

Acrolein is a major thiol-reactive component of cigarette smoke (CS) that is thought to contribute to increased asthma incidence associated with smoking. Here, we explored the effects of acute acrolein exposure on innate airway responses to two common airborne allergens, house dust mite and Alternaria alternata, and observed that acrolein exposure of C57BL/6 mice (5 ppm, 4 h) dramatically inhibited innate airway responses to subsequent allergen challenge, demonstrated by attenuated release of the epithelial-derived cytokines IL-33, IL-25, and IL-1α. Acrolein and other anti-inflammatory thiol-reactive electrophiles, cinnamaldehyde, curcumin, and sulforaphane, similarly inhibited allergen-induced production of these cytokines from human or murine airway epithelial cells in vitro. Based on our previous observations indicating the importance of Ca2+-dependent signaling, activation of the NADPH oxidase DUOX1, and Src/EGFR-dependent signaling in allergen-induced epithelial secretion of these cytokines, we explored the impact of acrolein on these pathways. Acrolein and other thiol-reactive electrophiles were found to dramatically prevent allergen-induced activation of DUOX1 as well as EGFR, and acrolein was capable of inhibiting EGFR tyrosine kinase activity via modification of C797. Biotin-labeling strategies indicated increased cysteine modification and carbonylation of Src, EGFR, as well as DUOX1, in response to acrolein exposure in vitro and in vivo, suggesting that direct alkylation of these proteins on accessible cysteine residues may be responsible for their inhibition. Collectively, our findings indicate a novel anti-inflammatory mechanism of CS-derived acrolein and other thiol-reactive electrophiles, by directly inhibiting DUOX1- and EGFR-mediated airway epithelial responses to airborne allergens.


Assuntos
Acroleína/farmacologia , Alérgenos/efeitos adversos , Brônquios/patologia , Células Epiteliais/metabolismo , Receptores ErbB/metabolismo , NADPH Oxidases/antagonistas & inibidores , Compostos de Sulfidrila/farmacologia , Acroleína/química , Administração por Inalação , Animais , Cálcio/metabolismo , Cisteína/metabolismo , Oxidases Duais , Ativação Enzimática/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/metabolismo , Imunidade Inata/efeitos dos fármacos , Interleucina-33/metabolismo , Camundongos Endogâmicos C57BL , NADPH Oxidases/metabolismo , Carbonilação Proteica/efeitos dos fármacos , Pyroglyphidae/efeitos dos fármacos , Pyroglyphidae/fisiologia , Compostos de Sulfidrila/química , Quinases da Família src/metabolismo
9.
Redox Biol ; 71: 103102, 2024 05.
Artigo em Inglês | MEDLINE | ID: mdl-38430684

RESUMO

Peroxidasin (PXDN) is a secreted heme peroxidase that catalyzes the oxidative crosslinking of collagen IV within the extracellular matrix (ECM) via intermediate hypobromous acid (HOBr) synthesis from hydrogen peroxide and bromide, but recent findings have also suggested alternative ECM protein modifications by PXDN, including incorporation of bromide into tyrosine residues. In this work, we sought to identify the major target proteins for tyrosine bromination by HOBr or by PXDN-mediated oxidation in ECM from mouse teratocarcinoma PFHR9 cells. We detected 61 bromotyrosine (BrY)-containing peptides representing 23 proteins in HOBr-modified ECM from PFHR9 cells, among which laminins displayed the most prominent bromotyrosine incorporation. Moreover, we also found that laminin α1, laminin ß1, and tubulointerstitial nephritis antigen-like (TINAGL1) contained BrY in untreated PFHR9 cells, which depended on PXDN. We extended these analyses to lung tissues from both healthy mice and mice with experimental lung fibrosis, and in lung tissues obtained from human subjects. Analysis of ECM-enriched mouse lung tissue extracts showed that 83 ECM proteins were elevated in bleomycin-induced fibrosis, which included various collagens and laminins, and PXDN. Similarly, mRNA and protein expression of PXDN and laminin α/ß1 were enhanced in fibrotic mouse lung tissues, and also in mouse bone-marrow-derived macrophages or human fibroblasts stimulated with transforming growth factor ß1, a profibrotic growth factor. We identified 11 BrY-containing ECM proteins, including collagen IV α2, collagen VI α1, TINAGL1, and various laminins, in both healthy and mouse fibrotic lung tissues, although the relative extent of tyrosine bromination of laminins was not significantly increased during fibrosis. Finally, we also identified 7 BrY-containing ECM proteins in human lung tissues, again including collagen IV α2, collagen VI α1, and TINAGL1. Altogether, this work demonstrates the presence of several bromotyrosine-modified ECM proteins, likely involving PXDN, even in normal lung tissues, suggesting a potential biological function for these modifications.


Assuntos
Bromatos , Proteínas da Matriz Extracelular , Fibrose Pulmonar , Humanos , Animais , Camundongos , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Brometos/efeitos adversos , Brometos/metabolismo , Laminina/genética , Laminina/metabolismo , Matriz Extracelular/metabolismo , Pulmão/metabolismo , Peroxidasina , Colágeno Tipo IV/metabolismo , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/genética , Fibrose Pulmonar/metabolismo , Tirosina/metabolismo
10.
Am J Respir Cell Mol Biol ; 48(3): 337-45, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23239498

RESUMO

The respiratory epithelium plays a critical role in innate defenses against airborne pathogens and pollutants, and alterations in epithelial homeostasis and repair mechanisms are thought to contribute to chronic lung diseases associated with airway remodeling. Previous studies implicated the nicotinamide adenine dinucleotide phosphate-reduced oxidase dual oxidase-1 (DUOX1) in redox signaling pathways involved in in vitro epithelial wound responses to infection and injury. However, the importance of epithelial DUOX1 in in vivo epithelial repair pathways has not been established. Using small interfering (si)RNA silencing of DUOX1 expression, we show the critical importance of DUOX1 in wound responses in murine tracheal epithelial (MTE) cells in vitro, as well as its contribution to epithelial regeneration in vivo in a murine model of epithelial injury induced by naphthalene, a selective toxicant of nonciliated respiratory epithelial cells (club cells [Clara]). Whereas naphthalene-induced club-cell injury is normally followed by epithelial regeneration after 7 and 14 days, such airway reepithelialization was significantly delayed after the silencing of airway DUOX1 by oropharyngeal administration of DUOX1-targeted siRNA. Wound closure in MTE cells was related to DUOX1-dependent activation of the epidermal growth factor receptor (EGFR) and the transcription factor signal transducer and activator of transcription-3 (STAT3), known mediators of epithelial cell migration and wound responses. Moreover, in vivo DUOX1 silencing significantly suppressed naphthalene-induced activation of STAT3 and EGFR during early stages of epithelial repair. In conclusion, these experiments demonstrate for the first time an important function for epithelial DUOX1 in lung epithelial regeneration in vivo, by promoting EGFR-STAT3 signaling and cell migration as critical events in initial repair.


Assuntos
Bronquíolos/fisiologia , Movimento Celular/fisiologia , Células Epiteliais/fisiologia , NADPH Oxidases/metabolismo , Reepitelização/fisiologia , Mucosa Respiratória/fisiologia , Cicatrização/fisiologia , Animais , Bronquíolos/citologia , Bronquíolos/enzimologia , Células Cultivadas , Oxidases Duais , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Células Epiteliais/metabolismo , Receptores ErbB/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mucosa Respiratória/citologia , Mucosa Respiratória/enzimologia , Mucosa Respiratória/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/fisiologia , Traqueia/metabolismo
11.
Respir Res ; 14: 107, 2013 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-24131734

RESUMO

BACKGROUND: Adverse health effects of tobacco smoke arise partly from its influence on innate and adaptive immune responses, leading to impaired innate immunity and host defense. The impact of smoking on allergic asthma remains unclear, with various reports demonstrating that cigarette smoke enhances asthma development but can also suppress allergic airway inflammation. Based on our previous findings that immunosuppressive effects of smoking may be largely attributed to one of its main reactive electrophiles, acrolein, we explored the impact of acrolein exposure in a mouse model of ovalbumin (OVA)-induced allergic asthma. METHODS: C57BL/6 mice were sensitized to ovalbumin (OVA) by intraperitoneal injection with the adjuvant aluminum hydroxide on days 0 and 7, and challenged with aerosolized OVA on days 14-16. In some cases, mice were also exposed to 5 ppm acrolein vapor for 6 hrs/day on days 14-17. Lung tissues or brochoalveolar lavage fluids (BALF) were collected either 6 hrs after a single initial OVA challenge and/or acrolein exposure on day 14 or 48 hrs after the last OVA challenge, on day 18. Inflammatory cells and Th1/Th2 cytokine levels were measured in BALF, and lung tissue samples were collected for analysis of mucus and Th1/Th2 cytokine expression, determination of protein alkylation, cellular thiol status and transcription factor activity. RESULTS: Exposure to acrolein following OVA challenge of OVA-sensitized mice resulted in markedly attenuated allergic airway inflammation, demonstrated by decreased inflammatory cell infiltrates, mucus hyperplasia and Th2 cytokines. Acrolein exposure rapidly depleted lung tissue glutathione (GSH) levels, and induced activation of the Nrf2 pathway, indicated by accumulation of Nrf2, increased alkylation of Keap1, and induction of Nrf2-target genes such as HO-1. Additionally, analysis of inflammatory signaling pathways showed suppressed activation of NF-κB and marginally reduced activation of JNK in acrolein-exposed lungs, associated with increased carbonylation of RelA and JNK. CONCLUSION: Acrolein inhalation suppresses Th2-driven allergic inflammation in sensitized animals, due to direct protein alkylation resulting in activation of Nrf2 and anti-inflammatory gene expression, and inhibition of NF-κB or JNK signaling. Our findings help explain the paradoxical anti-inflammatory effects of cigarette smoke exposure in allergic airways disease.


Assuntos
Acroleína/uso terapêutico , Asma/induzido quimicamente , Asma/prevenção & controle , Imunoglobulina G/metabolismo , Ovalbumina/efeitos adversos , Acroleína/farmacologia , Animais , Asma/metabolismo , Modelos Animais de Doenças , Glutationa/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Compostos de Sulfidrila/metabolismo , Equilíbrio Th1-Th2/efeitos dos fármacos , Fatores de Transcrição/metabolismo
12.
Mucosal Immunol ; 15(5): 977-989, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35654836

RESUMO

The NADPH oxidase DUOX1 contributes to epithelial production of alarmins, including interleukin (IL)-33, in response to injurious triggers such as airborne protease allergens, and mediates development of mucus metaplasia and airway remodeling in chronic allergic airways diseases. DUOX1 is also expressed in non-epithelial lung cell types, including macrophages that play an important role in airway remodeling during chronic lung disease. We therefore conditionally deleted DUOX1 in either lung epithelial or monocyte/macrophage lineages to address its cell-specific actions in innate airway responses to acute airway challenge with house dust mite (HDM) allergen, and in chronic HDM-driven allergic airway inflammation. As expected, acute responses to airway challenge with HDM, as well as type 2 inflammation and related features of airway remodeling during chronic HDM-induced allergic inflammation, were largely driven by DUOX1 with the respiratory epithelium. However, in the context of chronic HDM-driven inflammation, DUOX1 deletion in macrophages also significantly impaired type 2 cytokine production and indices of mucus metaplasia. Further studies revealed a contribution of macrophage-intrinsic DUOX1 in macrophage recruitment upon chronic HDM challenge, as well as features of macrophage activation that impact on type 2 inflammation and remodeling.


Assuntos
Remodelação das Vias Aéreas , Hipersensibilidade , Alérgenos , Animais , Antígenos de Dermatophagoides , Oxidases Duais , Inflamação , Pulmão , Macrófagos , Metaplasia , Muco , Pyroglyphidae
13.
JCI Insight ; 6(2)2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33301419

RESUMO

Chronic obstructive pulmonary disease (COPD) is a chronic respiratory disease characterized by small airway remodeling and alveolar emphysema due to environmental stresses such as cigarette smoking (CS). Oxidative stress is commonly implicated in COPD pathology, but recent findings suggest that one oxidant-producing NADPH oxidase homolog, dual oxidase 1 (DUOX1), is downregulated in the airways of patients with COPD. We evaluated lung tissue sections from patients with COPD for small airway epithelial DUOX1 protein expression, in association with measures of lung function and small airway and alveolar remodeling. We also addressed the impact of DUOX1 for lung tissue remodeling in mouse models of COPD. Small airway DUOX1 levels were decreased in advanced COPD and correlated with loss of lung function and markers of emphysema and remodeling. Similarly, DUOX1 downregulation in correlation with extracellular matrix remodeling was observed in a genetic model of COPD, transgenic SPC-TNF-α mice. Finally, development of subepithelial airway fibrosis in mice due to exposure to the CS-component acrolein, or alveolar emphysema induced by administration of elastase, were in both cases exacerbated in Duox1-deficient mice. Collectively, our studies highlight that downregulation of DUOX1 may be a contributing feature of COPD pathogenesis, likely related to impaired DUOX1-mediated innate injury responses involved in epithelial homeostasis.


Assuntos
Oxidases Duais/metabolismo , Doença Pulmonar Obstrutiva Crônica/enzimologia , Idoso , Animais , Estudos de Casos e Controles , Modelos Animais de Doenças , Regulação para Baixo , Oxidases Duais/genética , Matriz Extracelular/patologia , Matriz Extracelular/fisiologia , Feminino , Humanos , Pulmão/patologia , Pulmão/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/patologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Mucosa Respiratória/patologia , Mucosa Respiratória/fisiopatologia
14.
Sci Rep ; 9(1): 4844, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30890751

RESUMO

Lung cancers are frequently characterized by inappropriate activation of epidermal growth factor receptor (EGFR)-dependent signaling and epigenetic silencing of the NADPH oxidase (NOX) enzyme DUOX1, both potentially contributing to worse prognosis. Based on previous findings linking DUOX1 with redox-dependent EGFR activation, the present studies were designed to evaluate whether DUOX1 silencing in lung cancers may be responsible for altered EGFR regulation. In contrast to normal epithelial cells, EGF stimulation of lung cancer cell lines that lack DUOX1 promotes EGF-induced EGFR internalization and nuclear localization, associated with induction of EGFR-regulated genes and related tumorigenic outcomes. Each of these outcomes could be reversed by overexpression of DUOX1 or enhanced by shRNA-dependent DUOX1 silencing. EGF-induced nuclear EGFR localization in DUOX1-deficient lung cancer cells was associated with altered dynamics of cysteine oxidation of EGFR, and an overall reduction of EGFR cysteines. These various outcomes could also be attenuated by silencing of glutathione S-transferase P1 (GSTP1), a mediator of metabolic alterations and drug resistance in various cancers, and a regulator of cysteine oxidation. Collectively, our findings indicate DUOX1 deficiency in lung cancers promotes dysregulated EGFR signaling and enhanced GSTP1-mediated turnover of EGFR cysteine oxidation, which result in enhanced nuclear EGFR localization and tumorigenic properties.


Assuntos
Nucléolo Celular/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Células A549 , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Oxidases Duais/metabolismo , Receptores ErbB/metabolismo , Humanos , NADPH Oxidases/metabolismo , Oxirredução , Transdução de Sinais/fisiologia
15.
Diabetes ; 55(12): 3289-98, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17130472

RESUMO

beta-Cell mass is determined by a dynamic balance of proliferation, neogenesis, and apoptosis. The precise mechanisms underlying compensatory beta-cell mass (BCM) homeostasis are not fully understood. To evaluate the processes that maintain normoglycemia and regulate BCM during pancreatic regeneration, C57BL/6 mice were analyzed for 15 days following 60% partial pancreatectomy (Px). BCM increased in Px mice from 2 days onwards and was approximately 68% of the shams by 15 days, partly due to enhanced beta-cell proliferation. A transient approximately 2.8-fold increase in the prevalence of beta-cell clusters/small islets at 2 days post-Px contributed substantially to BCM augmentation, followed by an increase in the number of larger islets at 15 days. To evaluate the signaling mechanisms that may regulate this compensatory growth, we examined key intermediates of the insulin signaling pathway. We found insulin receptor substrate (IRS)2 and enhanced-activated Akt immunoreactivity in islets and ducts that correlated with increased pancreatic duodenal homeobox (PDX)1 expression. In contrast, forkhead box O1 expression was decreased in islets but increased in ducts, suggesting distinct PDX1 regulatory mechanisms in these tissues. Px animals acutely administered insulin exhibited further enhancement in insulin signaling activity. These data suggest that the IRS2-Akt pathway mediates compensatory beta-cell growth by activating beta-cell proliferation with an increase in the number of beta-cell clusters/small islets.


Assuntos
Células Secretoras de Insulina/fisiologia , Pancreatectomia , Actinas/metabolismo , Animais , Glicemia , Divisão Celular , Ciclina D2 , Ciclinas/metabolismo , Immunoblotting , Células Secretoras de Insulina/citologia , Ilhotas Pancreáticas/anatomia & histologia , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , RNA/genética , RNA/isolamento & purificação , Regeneração
16.
Diabetes ; 54(8): 2294-304, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16046294

RESUMO

The physiological mechanisms underlying the compensatory growth of beta-cell mass in insulin-resistant states are poorly understood. Using the insulin-resistant Zucker fatty (fa/fa) (ZF) rat and the corresponding Zucker lean control (ZLC) rat, we investigated the factors contributing to the age-/obesity-related enhancement of beta-cell mass. A 3.8-fold beta-cell mass increase was observed in ZF rats as early as 5 weeks of age, an age that precedes severe insulin resistance by several weeks. Closer investigation showed that ZF rat pups were not born with heightened beta-cell mass but developed a modest increase over ZLC rats by 20 days that preceded weight gain or hyperinsulinemia that first developed at 24 days of age. In these ZF pups, an augmented survival potential of beta-cells of ZF pups was observed by enhanced activated (phospho-) Akt, phospho-BAD, and Bcl-2 immunoreactivity in the postweaning period. However, increased beta-cell proliferation in the ZF rats was only detected at 31 days of age, a period preceding massive beta-cell growth. During this phase, we also detected an increase in the numbers of small beta-cell clusters among ducts and acini, increased duct pancreatic/duodenal homeobox-1 (PDX-1) immunoreactivity, and an increase in islet number in the ZF rats suggesting duct- and acini-mediated heightened beta-cell neogenesis. Interestingly, in young ZF rats, specific cells associated with ducts, acini, and islets exhibited an increased frequency of PDX-1+/phospho-Akt+ staining, indicating a potential role for Akt in beta-cell differentiation. Thus, several adaptive mechanisms account for the compensatory growth of beta-cells in ZF rats, a combination of enhanced survival and neogenesis with a transient rise in proliferation before 5 weeks of age, with Akt serving as a potential mediator in these processes.


Assuntos
Envelhecimento , Resistência à Insulina , Ilhotas Pancreáticas/patologia , Obesidade/patologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Animais , Diferenciação Celular , Divisão Celular , Sobrevivência Celular , Proteínas de Homeodomínio/análise , Marcação In Situ das Extremidades Cortadas , Ilhotas Pancreáticas/química , Masculino , Proteínas Serina-Treonina Quinases/análise , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Ratos , Ratos Zucker , Transdução de Sinais , Transativadores/análise
17.
J Immunotoxicol ; 13(2): 191-7, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-25875327

RESUMO

Acrolein (ACR), an α,ß-unsaturated aldehyde and a major component of tobacco smoke, is a highly reactive electrophilic respiratory irritant implicated in asthma pathogenesis and severity. However, few studies have directly investigated the influence of ACR exposure on allergen sensitization and pulmonary inflammation. The present study was designed to examine the impact of ACR inhalation on allergic sensitization to the inhaled antigen ovalbumin (OVA), as well as pulmonary inflammation during subsequent OVA challenge. Adult male C57BL/6 mice were exposed to inhaled OVA (1%, 30 min/day, 4 days/week) and/or ACR (5 ppm, 4 h/day, 4 days/week) over 2 weeks and subsequently challenged with aerosolized OVA (1%, 30 min/day) over three consecutive days. Serum anti-OVA IgG1 levels were increased significantly in animals exposed to both OVA and ACR, compared to animals exposed to either OVA or ACR alone. In addition, differential cell counts and histological analysis revealed an increase in BAL neutrophils in animals exposed to both OVA and ACR. However, exposure to both OVA and ACR did not influence mRNA expression of the cytokines il5, il10, il13 or tnfa, but significantly increased mRNA expression of ccl20. Moreover, ACR exposure enhanced lung mRNA levels of il17f and tgfb1, suggesting development of enhanced inhalation tolerance to OVA. Overall, the findings indicate that ACR inhalation can promote airway-mediated sensitization to otherwise innocuous inhaled antigens, such as OVA, but also enhances immune tolerance, thereby favoring neutrophilic airway inflammation.


Assuntos
Acroleína/toxicidade , Asma , Citocinas/imunologia , Imunoglobulina G/imunologia , Pulmão/imunologia , Neutrófilos/imunologia , Ovalbumina/imunologia , Administração por Inalação , Animais , Asma/induzido quimicamente , Asma/imunologia , Asma/patologia , Inflamação/induzido quimicamente , Inflamação/imunologia , Pulmão/patologia , Masculino , Camundongos , Neutrófilos/patologia
18.
JCI Insight ; 1(18): e88811, 2016 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-27812543

RESUMO

Chronic inflammation with mucous metaplasia and airway remodeling are hallmarks of allergic asthma, and these outcomes have been associated with enhanced expression and activation of EGFR signaling. Here, we demonstrate enhanced expression of EGFR ligands such as amphiregulin as well as constitutive EGFR activation in cultured nasal epithelial cells from asthmatic subjects compared with nonasthmatic controls and in lung tissues of mice during house dust mite-induced (HDM-induced) allergic inflammation. EGFR activation was associated with cysteine oxidation within EGFR and the nonreceptor tyrosine kinase Src, and both amphiregulin production and oxidative EGFR activation were diminished by pharmacologic or genetic inhibition of the epithelial NADPH oxidase dual oxidase 1 (DUOX1). DUOX1 deficiency also attenuated several EGFR-dependent features of HDM-induced allergic airway inflammation, including neutrophilic inflammation, type 2 cytokine production (IL-33, IL-13), mucous metaplasia, subepithelial fibrosis, and central airway resistance. Moreover, targeted inhibition of airway DUOX1 in mice with previously established HDM-induced allergic inflammation, by intratracheal administration of DUOX1-targeted siRNA or pharmacological NADPH oxidase inhibitors, reversed most of these outcomes. Our findings indicate an important function for DUOX1 in allergic inflammation related to persistent EGFR activation and suggest that DUOX1 targeting may represent an attractive strategy in asthma management.


Assuntos
Remodelação das Vias Aéreas , Asma/patologia , Oxidases Duais/metabolismo , Receptores ErbB/metabolismo , Células Caliciformes/citologia , Anfirregulina/metabolismo , Animais , Citocinas/metabolismo , Feminino , Humanos , Inflamação , Masculino , Metaplasia/patologia , Camundongos , Camundongos Endogâmicos C57BL
19.
Redox Biol ; 2: 436-46, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24624333

RESUMO

The NADPH oxidase homolog dual oxidase 1 (DUOX1) plays an important role in innate airway epithelial responses to infection or injury, but the precise molecular mechanisms are incompletely understood and the cellular redox-sensitive targets for DUOX1-derived H2O2 have not been identified. The aim of the present study was to survey the involvement of DUOX1 in cellular redox signaling by protein S-glutathionylation, a major mode of reversible redox signaling. Using human airway epithelial H292 cells and stable transfection with DUOX1-targeted shRNA as well as primary tracheal epithelial cells from either wild-type or DUOX1-deficient mice, DUOX1 was found to be critical in ATP-stimulated transient production of H2O2 and increased protein S-glutathionylation. Using cell pre-labeling with biotin-tagged GSH and analysis of avidin-purified proteins by global proteomics, 61 S-glutathionylated proteins were identified in ATP-stimulated cells compared to 19 in untreated cells. Based on a previously established role of DUOX1 in cell migration, various redox-sensitive proteins with established roles in cytoskeletal dynamics and/or cell migration were evaluated for S-glutathionylation, indicating a critical role for DUOX1 in ATP-stimulated S-glutathionylation of ß-actin, peroxiredoxin 1, the non-receptor tyrosine kinase Src, and MAPK phosphatase 1. Overall, our studies demonstrate the importance of DUOX1 in epithelial redox signaling through reversible S-glutathionylation of a range of proteins, including proteins involved in cytoskeletal regulation and MAPK signaling pathways involved in cell migration.


Assuntos
Glutationa/metabolismo , Pulmão/metabolismo , NADPH Oxidases/metabolismo , Oxirredução , Traqueia/metabolismo , Animais , Movimento Celular , Células Cultivadas , Oxidases Duais , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Humanos , Pulmão/citologia , Sistema de Sinalização das MAP Quinases , Camundongos , NADPH Oxidases/genética , Proteômica , Transdução de Sinais , Traqueia/citologia
20.
Endocrinology ; 152(4): 1300-13, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21285317

RESUMO

Circulating levels of matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitor of metalloproteinases (TIMPs), are altered in human obesity and may contribute to its pathology. TIMP-2 exerts MMP-dependent (MMP inhibition and pro-MMP-2 activation) and MMP-independent functions. To assess the role of TIMP-2 in a murine model of nutritionally induced obesity, weight gain in wild-type and TIMP-2 deficient [knockout (KO)] mice fed a chow or high-fat diet (HFD) was determined. The effects of diet on glucose tolerance and insulin sensitivity, as well as pancreatic ß-cell and adipocyte physiology, were assessed. Chow-fed TIMP-2 KO mice of both sexes became obese but maintained relatively normal glucose tolerance and insulin sensitivity. Obesity was exacerbated on the HFD. However, HFD-fed male, but not female, TIMP-2 KO mice developed insulin resistance with reduced glucose transporter 2 and pancreatic and duodenal homeobox 1 levels, despite increased ß-cell mass and hyperplasia. Thus, although ß-cell mass was increased, HFD-fed male TIMP-2 KO mice develop diabetes likely due to ß-cell exhaustion and failure. TIMP-2 mRNA, whose expression was greatest in sc adipose tissue, was down-regulated in HFD-fed wild-type males, but not females. Furthermore, HFD increased membrane type 1-MMP (MMP-14) expression and activity in male, but not female, sc adipose tissue. Strikingly, MMP-14 expression increased to a greater extent in TIMP-2 KO males and was associated with decreased adipocyte collagen. Taken together, these findings demonstrate a role for TIMP-2 in maintaining extracellular matrix integrity necessary for normal ß-cell and adipocyte physiology and that loss of extracellular matrix integrity may underlie diabetic and obesogenic phenotypes.


Assuntos
Obesidade/metabolismo , Inibidor Tecidual de Metaloproteinase-2/deficiência , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Animais , Western Blotting , Gorduras na Dieta/efeitos adversos , Feminino , Técnicas Imunoenzimáticas , Resistência à Insulina/genética , Resistência à Insulina/fisiologia , Células Secretoras de Insulina/metabolismo , Leptina/metabolismo , Masculino , Metaloproteinase 14 da Matriz/metabolismo , Camundongos , Camundongos Knockout , Microscopia Confocal , Microscopia de Fluorescência , Obesidade/induzido quimicamente , Reação em Cadeia da Polimerase , Fatores Sexuais , Inibidor Tecidual de Metaloproteinase-2/genética , Aumento de Peso/genética
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