RESUMEN
Rationale: CC16 (Club Cell Secretory Protein) is a protein produced by club cells and other non-ciliated epithelial cells within the lungs. CC16 has been shown to protect against the development of obstructive lung diseases and attenuate pulmonary pathogen burden. Despite recent advances in understanding CC16 effects in circulation, the biological mechanisms of CC16 in pulmonary epithelial responses have not been elucidated. Objectives: We sought to determine if CC16 deficiency impairs epithelial-driven host responses and identify novel receptors expressed within the pulmonary epithelium through which CC16 imparts activity. Methods: We utilized mass spectrometry and quantitative proteomics to investigate how CC16 deficiency impacts apically secreted pulmonary epithelial proteins. Mouse tracheal epithelial cells (MTECS), human nasal epithelial cells (HNECs) and mice were studied in naïve conditions and after Mp challenge. Measurements and main results: We identified 8 antimicrobial proteins significantly decreased by CC16-/- MTECS, 6 of which were validated by mRNA expression in Severe Asthma Research Program (SARP) cohorts. Short Palate Lung and Nasal Epithelial Clone 1 (SPLUNC1) was the most differentially expressed protein (66-fold) and was the focus of this study. Using a combination of MTECs and HNECs, we found that CC16 enhances pulmonary epithelial-driven SPLUNC1 expression via signaling through the receptor complex Very Late Antigen-2 (VLA-2) and that rCC16 given to mice enhances pulmonary SPLUNC1 production and decreases Mycoplasma pneumoniae (Mp) burden. Likewise, rSPLUNC1 results in decreased Mp burden in mice lacking CC16 mice. The VLA-2 integrin binding site within rCC16 is necessary for induction of SPLUNC1 and the reduction in Mp burden. Conclusion: Our findings demonstrate a novel role for CC16 in epithelial-driven host defense by up-regulating antimicrobials and define a novel epithelial receptor for CC16, VLA-2, through which signaling is necessary for enhanced SPLUNC1 production.
Asunto(s)
Asma , Integrina alfa2beta1 , Animales , Humanos , Ratones , Asma/metabolismo , Integrina alfa2beta1/metabolismo , Pulmón/metabolismo , Mycoplasma pneumoniae , Transducción de SeñalRESUMEN
Nasopharyngeal carcinoma (NPC) is one of the aggressive malignant tumors with high mortality, and the proliferation of myeloid-derived suppressor cells (MDSCs) could promote the metastasis of NPC through inhibiting the function of T cells. Meanwhile, SPLUNC1 was known to inhibit the malignant behavior of NPC cells, while the detailed function of SPLUNC1 in LPS-modified immune microenvironment of NPC remains unclear. To assess the impact of SPLUNC1 in immune microenvironment during the progression of NPC, NPC cells were exposed to LPS and then co-cultured with MDSCs for 48 h. RT-qPCR and western blot were performed to evaluate the mRNA and protein level of SPLUNC1, CXCL-2 and CXCR-2, respectively. The level of IL-1ß, IL-6, TNF-α, PD-L1, Arg-1 and iNOS were tested by ELISA. Meanwhile, the expression of CD33+ was tested by flow cytometry. The expression of CXCL-2 and CXCR-2 in NPC cells was higher, compared to that in NP69 cells. In contrast, SPLUNC1 level in NPC cells was much lower than that in NP69 cells. SPLUNC1 level was negatively correlated with CXCL-2 and CXCR-2. Overexpression of SPLUNC1 reversed LPS-induced inflammatory responses and proliferation in NPC cells. In addition, SPLUNC1 upregulation could reverse LPS-induced proliferation of MDSCs in tumor microenvironment. Meanwhile, SPLUNC1 overexpression could regulate CXCL-2/CXCR-2 axis through decreasing CXCL-2 and CXCR-2 protein and mRNA expression. SPLUNC1 regulates LPS-induced progression of nasopharyngeal carcinoma and proliferation of MDSCs. Thus, our study might provide a theoretical basis for discovering new strategies against NPC.
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Glicoproteínas/metabolismo , Células Supresoras de Origen Mieloide , Neoplasias Nasofaríngeas , Fosfoproteínas/metabolismo , Antígeno B7-H1 , Proliferación Celular , Humanos , Interleucina-6 , Lipopolisacáridos/farmacología , Carcinoma Nasofaríngeo , Microambiente Tumoral , Factor de Necrosis Tumoral alfaRESUMEN
Asthma and its heterogeneity change with age. Increased airspace neutrophil numbers contribute to severe steroid-resistant asthma exacerbation in the elderly, which correlates with the changes seen in adults with asthma. However, whether that resembles the same disease mechanism and pathophysiology in aged and adults is poorly understood. Here, we sought to address the underlying molecular mechanism of steroid-resistant airway inflammation development and response to corticosteroid (Dex) therapy in aged mice. To study the changes in inflammatory mechanism, we used a clinically relevant treatment model of house-dust mite (HDM)-induced allergic asthma and investigated lung adaptive immune response in adult (20-22 wk old) and aged (80-82 wk old) mice. Our result indicates an age-dependent increase in airway hyperresponsiveness (AHR), mixed granulomatous airway inflammation comprising eosinophils and neutrophils, and Th1/Th17 immune response with progressive decrease in frequencies and numbers of HDM-bearing dendritic cells (DC) accumulation in the draining lymph node (DLn) of aged mice as compared with adult mice. RNA-Seq experiments of the aged lung revealed short palate, lung, and nasal epithelial clone 1 (SPLUNC1) as one of the steroid-responsive genes, which progressively declined with age and further by HDM-induced inflammation. Moreover, we found increased glycolytic reprogramming, maturation/activation of DCs, the proliferation of OT-II cells, and Th2 cytokine secretion with recombinant SPLUNC1 (rSPLUNC1) treatment. Our results indicate a novel immunomodulatory role of SPLUNC1 regulating metabolic adaptation/maturation of DC. An age-dependent decline in the SPLUNC1 level may be involved in developing steroid-resistant airway inflammation and asthma heterogeneity.
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Envejecimiento/patología , Glicoproteínas/metabolismo , Inflamación/patología , Fosfoproteínas/metabolismo , Sistema Respiratorio/patología , Esteroides/farmacología , Animales , Presentación de Antígeno/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Dendríticas/efectos de los fármacos , Células Dendríticas/patología , Dermatophagoides pteronyssinus/efectos de los fármacos , Dexametasona/farmacología , Eosinófilos/efectos de los fármacos , Eosinófilos/patología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Glucólisis/efectos de los fármacos , Granuloma/patología , Ganglios Linfáticos/patología , Mediastino/patología , Modelos Biológicos , Sistema Respiratorio/parasitologíaRESUMEN
Orai1 is a plasma membrane Ca2+ channel that mediates store-operated Ca2+ entry (SOCE) and regulates inflammation. Short palate lung and nasal epithelial clone 1 (SPLUNC1) is an asthma gene modifier that inhibits Orai1 and SOCE via its C-terminal α6 region. SPLUNC1 levels are diminished in asthma patient airways. Thus, we hypothesized that inhaled α6 peptidomimetics could inhibit Orai1 and reduce airway inflammation in a murine asthma model. To evaluate α6-Orai1 interactions, we used fluorescent assays to measure Ca2+ signaling, Förster resonance energy transfer, fluorescent recovery after photobleaching, immunostaining, total internal reflection microscopy, and Western blotting. To test whether α6 peptidomimetics inhibited SOCE and decreased inflammation in vivo, wild-type and SPLUNC1-/- mice were exposed to house dust mite (HDM) extract with or without α6 peptide. We also performed nebulization, jet milling, and scanning electron microscopy to evaluate α6 for inhalation. SPLUNC1-/- mice had an exaggerated response to HDM. In BAL-derived immune cells, Orai1 levels increased after HDM exposure in SPLUNC1-/- but not wild-type mice. Inhaled α6 reduced Orai1 levels in mice regardless of genotype. In HDM-exposed mice, α6 dose-dependently reduced eosinophilia and neutrophilia. In vitro, α6 inhibited SOCE in multiple immune cell types, and α6 could be nebulized or jet milled without loss of function. These data suggest that α6 peptidomimetics may be a novel, effective antiinflammatory therapy for patients with asthma.
Asunto(s)
Asma , Peptidomiméticos , Animales , Asma/tratamiento farmacológico , Calcio/metabolismo , Glicoproteínas , Humanos , Inflamación , Pulmón/metabolismo , Ratones , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , FosfoproteínasRESUMEN
Short palate, lung, and nasal epithelium clone 1 (SPLUNC1) is a kind of secretory protein, and gets expressed abundantly in normal respiratory epithelium of humans. As a natural immune molecule, SPLUNC1 is proved to be involved in inflammatory response and airway host defense. This review focuses on summarizing and discussing the role of SPLUNC1 in regulating airway surface liquid (ASL) and participating in airway host defense. PubMed and MEDLINE were used for searching and identifying the data in this review. The domain of bactericidal/permeability-increasing protein in SPLUNC1 and the α-helix, α4, are essential for SPLUNC1 to exert biological activities. As a natural innate immune molecule, SPLUNC1 plays a significant role in inflammatory response and airway host defense. Its special expression patterns are not only observed in physiological conditions, but also in some respiratory diseases. The mechanisms of SPLUNC1 in airway host defense include modulating ASL volume, acting as a surfactant protein, inhibiting biofilm formation, as well as regulating ASL compositions, such as LL-37, mucins, Neutrophil elastase, and inflammatory cytokines. Besides, potential correlations are found among these different mechanisms, especially among different ASL compositions, which should be further explored in more systematical frameworks. In this review, we summarize the structural characteristics and expression patterns of SPLUNC1 briefly, and mainly discuss the mechanisms of SPLUNC1 exerted in host defense, aiming to provide a theoretical basis and a novel target for future studies and clinical treatments.
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Regulación de la Expresión Génica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Mucosa Respiratoria/metabolismo , Fenómenos Fisiológicos Respiratorios , Animales , Antiinfecciosos/metabolismo , Biomarcadores , Secreciones Corporales/inmunología , Secreciones Corporales/metabolismo , Citocinas/metabolismo , Glicoproteínas/química , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Elastasa de Leucocito/metabolismo , Mucinas/metabolismo , Especificidad de Órganos , Fosfoproteínas/química , Surfactantes Pulmonares/inmunología , Surfactantes Pulmonares/metabolismo , Mucosa Respiratoria/inmunologíaRESUMEN
Clinical therapeutic and immunoregulatory effects of recombinant SPLUNC1 protein (rSPLUNC1) were evaluated in Mycoplasma ovipneumoniae (Mo)-infected Argali hybrid sheep (AHS). Group A contained six Bashibai sheep (BS) and groups B-D contained six AHS each. All sheep were manually infected with Mo. Five days post-infection, rSPLUNC1 from BS and AHS was injected intratracheally into group C and D animals; physiological saline was administered to groups A and B. Serum IL-5, IL-6, and IL-9 were quantified by ELISA. After sacrificing the sheep, lung tissues were extracted for pathological examination. The qPCR was used to quantify Mo load in the lungs and evaluate therapeutic efficacy. Serum IL-5, IL-6, and IL-9 concentrations increased during early infection stages in all groups but were significantly lower in groups A, C, and D than in group B on days 14 and 21. On day 21, IL-5 concentrations were lower in group A than in groups C and D. IL-6 concentration in groups A, C, and D was significantly lower than that in group B, and that in groups C and D was significantly lower than that in group A. Mean mycoplasma pneumonia histopathology scores were significantly lower in groups C and D than in group B, and Mo load in group C and D lung tissue decreased significantly compared to that in group B. Intratracheal injection of rSPLUNC1 into Mo-infected sheep decreased the cytokine levels and alleviated clinical symptoms with no mortality. rSPLUNC1 had significant therapeutic effects on Mo-infected AHS and can regulate pro-inflammatory cytokines.
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Glicoproteínas/uso terapéutico , Neumonía por Mycoplasma/veterinaria , Proteínas Recombinantes/uso terapéutico , Enfermedades de las Ovejas/tratamiento farmacológico , Animales , Ensayo de Inmunoadsorción Enzimática/veterinaria , Glicoproteínas/genética , Interleucinas/sangre , Pulmón/microbiología , Pulmón/patología , Mycoplasma ovipneumoniae , Neumonía por Mycoplasma/tratamiento farmacológico , Neumonía por Mycoplasma/patología , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Ovinos , Enfermedades de las Ovejas/patología , Resultado del TratamientoRESUMEN
BACKGROUND: ENaC inhibition has been investigated as a CF treatment; however, small molecule inhibitors of ENaC lack efficacy and/or have shown dose-limiting hyperkalemia. SPX-101 is a novel, investigational small peptide (SPLUNC1 mimetic) that regulates ENaC density with the potential for efficacy without systemic effects. METHODS: Two trials are presented: The first was a Phase 1, 2-part, randomized, double-blind, placebo-controlled, ascending-dose study of nebulized SPX-101 in healthy adults. Part 1 evaluated 4 single doses of SPX-101 ranging from 20 to 240â¯mg. Part 2 evaluated a 14-day regimen of SPX-101 at 4 doses of SPX-101 ranging from 10 to 120â¯mg BID. Pharmacokinetics, adverse events, spirometry, vital signs, electrocardiograms, pulse oximetry, and clinical laboratory values were assessed. The second trial was a tolerability-confirming, Phase 1b, open-label study conducted in 5 adult subjects with CF. Ascending doses of SPX-101 inhalation solution (10â¯mg-120â¯mg BID) were administered for 7 days. Safety was assessed as described above. RESULTS: All 64 healthy volunteers (32 in each Part) completed the single and multiple dose study. SPX-101 was well tolerated with little/no systemic exposure and with no hyperkalemia. Adverse events were generally mild with reported respiratory events associated with the purported pharmacological activity of SPX-101. Tolerability of SPX-101 was similarly observed in adults with CF; all 5 subjects treated with SPX-101 completed the study. CONCLUSIONS: SPX-101 was well-tolerated across a range of doses and had little/no systemic exposure in healthy adults and adults with CF, thus supporting further study in patients with CF. CLINICALTRIAL. GOV REGISTRATION: NCT03056989.
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Fibrosis Quística/tratamiento farmacológico , Bloqueadores del Canal de Sodio Epitelial/farmacocinética , Bloqueadores del Canal de Sodio Epitelial/uso terapéutico , Administración por Inhalación , Adulto , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Bloqueadores del Canal de Sodio Epitelial/efectos adversos , Canales Epiteliales de Sodio , Femenino , Glicoproteínas/metabolismo , Voluntarios Sanos , Humanos , Masculino , Persona de Mediana Edad , Fosfoproteínas/metabolismoRESUMEN
Multidrug resistance (MDR) by bacterial pathogens constitutes a global health crisis, and resistance to treatment displayed by biofilm-associated infections (e.g., cystic fibrosis, surgical sites, and medical implants) only exacerbates a problem that is already difficult to overcome. Antimicrobial peptides (AMPs) are a promising class of therapeutics that may be useful in the battle against antibiotic resistance, although certain limitations have hindered their clinical development. The goal of this study was to examine the therapeutic potential of novel AMPs derived from the multifunctional respiratory host defense protein SPLUNC1. Using standard growth inhibition and antibiofilm assays, we demonstrated that a novel structurally optimized AMP, α4-short, was highly effective against the most common group of MDR bacteria while showing broad-spectrum bactericidal and antibiofilm activities. With negligible hemolysis and toxicity to white blood cells, the new peptide also demonstrated in vivo efficacy when delivered directly into the airway in a murine model of Pseudomonas aeruginosa-induced respiratory infection. The data warrant further exploration of SPLUNC1-derived AMPs with optimized structures to assess the potential application to difficult-to-cure biofilm-associated infections.IMPORTANCE The rise of superbugs underscores the urgent need for novel antimicrobial agents. Antimicrobial peptides (AMPs) have the ability to kill superbugs regardless of resistance to traditional antibiotics. However, AMPs often display a lack of efficacy in vivo. Sequence optimization and engineering are promising but may result in increased host toxicity. We report here the optimization of a novel AMP (α4-short) derived from the multifunctional respiratory protein SPLUNC1. The AMP α4-short demonstrated broad-spectrum activity against superbugs as well as in vivo efficacy in the P. aeruginosa pneumonia model. Further exploration for clinical development is warranted.
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Antiinfecciosos/uso terapéutico , Péptidos Catiónicos Antimicrobianos/uso terapéutico , Productos Biológicos/uso terapéutico , Glicoproteínas/metabolismo , Fosfoproteínas/metabolismo , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones del Sistema Respiratorio/tratamiento farmacológico , Animales , Antiinfecciosos/aislamiento & purificación , Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Péptidos Catiónicos Antimicrobianos/farmacología , Bacterias/efectos de los fármacos , Productos Biológicos/aislamiento & purificación , Productos Biológicos/farmacología , Modelos Animales de Enfermedad , Ratones , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Resultado del TratamientoRESUMEN
INTRODUCTION: Cystic fibrosis is an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that codes for the CFTR anion channel. In the absence of functional CFTR, the epithelial Na+ channel is also dysregulated. Airway surface liquid (ASL) hydration is maintained by a balance between epithelial sodium channel (ENaC)-led Na+ absorption and CFTR-dependent anion secretion. This finely tuned homeostatic mechanism is required to maintain sufficient airway hydration to permit the efficient mucus clearance necessary for a sterile lung environment. In CF airways, the lack of CFTR and increased ENaC activity lead to ASL/mucus dehydration that causes mucus obstruction, neutrophilic infiltration, and chronic bacterial infection. Rehydration of ASL/mucus in CF airways can be achieved by inhibiting Na+ absorption with pharmacological inhibitors of ENaC. Areas covered: In this review, we discuss ENaC structure and function and its role in CF lung disease and focus on ENaC inhibition as a potential therapeutic target to rehydrate CF mucus. We also discuss the failure of the first generation of pharmacological inhibitors of ENaC and recent alternate strategies to attenuate ENaC activity in the CF lung. Expert opinion: ENaC is an attractive therapeutic target to rehydrate CF ASL that may serve as a monotherapy or function in parallel with other treatments. Given the increased number of strategies being employed to inhibit ENaC, this is an exciting and optimistic time to be in this field.
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Fibrosis Quística/tratamiento farmacológico , Canales Epiteliales de Sodio/metabolismo , Enfermedades Pulmonares/tratamiento farmacológico , Animales , Fibrosis Quística/genética , Fibrosis Quística/fisiopatología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Desarrollo de Medicamentos/métodos , Bloqueadores del Canal de Sodio Epitelial/farmacología , Canales Epiteliales de Sodio/efectos de los fármacos , Humanos , Enfermedades Pulmonares/etiología , Enfermedades Pulmonares/fisiopatología , Terapia Molecular DirigidaRESUMEN
The secreted airway mucus cell protein chloride channel regulator, calcium-activated 1, CLCA1, plays a role in inflammatory respiratory diseases via as yet unidentified pathways. For example, deficiency of CLCA1 in a mouse model of acute pneumonia resulted in reduced cytokine expression with less leukocyte recruitment and the human CLCA1 was shown to be capable of activating macrophages in vitro. Translation of experimental data between human and mouse models has proven problematic due to several CLCA species-specific differences. We therefore characterized activation of macrophages by CLCA1 in detail in solely murine ex vivo and in vitro models. Only alveolar but not bone marrow-derived macrophages freshly isolated from C57BL6/J mice increased their expression levels of several pro-inflammatory and leukotactic cytokines upon CLCA1 stimulation. Among the most strongly regulated genes, we identified the host-protective and immunomodulatory airway mucus component BPIFA1, previously unknown to be expressed by airway macrophages. Furthermore, evidence from an in vivo Staphylococcus aureus pneumonia mouse model suggests that CLCA1 may also modify BPIFA1 expression in airway epithelial cells. Our data underscore and specify the role of mouse CLCA1 in inflammatory airway disease to activate airway macrophages. In addition to its ability to upregulate cytokine expression which explains previous observations in the Clca1-deficient S. aureus pneumonia mouse model, modulation of BPIFA1 expression expands the role of CLCA1 in airway disease to involvement in more complex downstream pathways, possibly including liquid homeostasis, airway protection, and antimicrobial defense.
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Células de la Médula Ósea/metabolismo , Canales de Cloruro/metabolismo , Citocinas/genética , Glicoproteínas/genética , Leucocitos/metabolismo , Macrófagos Alveolares/metabolismo , Fosfoproteínas/genética , Animales , Células de la Médula Ósea/citología , Diferenciación Celular , Células Cultivadas , Canales de Cloruro/deficiencia , Citocinas/metabolismo , Modelos Animales de Enfermedad , Glicoproteínas/metabolismo , Leucocitos/patología , Macrófagos Alveolares/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfoproteínas/metabolismo , SolubilidadRESUMEN
The short palate, lung, and nasal epithelium clone 1 (SPLUNC1) protein is an important innate material in the upper airway, and lactoferrin (LF) aids the innate functions in humans. In this study, a nasal epithelial model was used to investigate how LF modulates SPLUNC1 to reduce the inflammatory process mediated by lipopolysaccharide (LPS). The inflammation of human RPMI-2650 cells was induced with LPS to evaluate SPLUNC1 expression after treating the cells with bovine LF (bLF). The interaction pathway between LF and SPLUNC1 in LPS-induced inflammation was further investigated. Our study reveals that the addition of bLF results in the recovery of SPLUNC1 expression in nasal epithelial cells under LPS-induced inflammation. MAPK is involved in the main pathway for the SPLUNC1 and bLF interaction. Decreased SPLUNC1 function could be recovered by addition of bLF. The MEK1/2-MAPK signaling pathway is crucial for the SPLUNC1 and bLF interaction. Therefore, LF could support SPLUNC1 in the innate immunity recovery process.
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Glicoproteínas/metabolismo , Inflamación/prevención & control , Lactoferrina/metabolismo , Lipopolisacáridos/efectos adversos , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 2/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mucosa Nasal/efectos de los fármacos , Fosfoproteínas/metabolismo , Animales , Bovinos , Células Cultivadas , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Inflamación/inducido químicamente , Inflamación/metabolismo , Mucosa Nasal/citología , Mucosa Nasal/metabolismo , Transducción de SeñalRESUMEN
OBJECTIVE: Experiments were conducted to clone the sequence of Wild Argali short palate, lung and nasal epithelium clone 1 (SPLUNC1) cDNA, and to lay the foundation for further study the biological function of Wild Argali SPLUNC1. METHODS: The complete sequence of Wild Argali SPLUNC1 cDNA was generated by rapid amplification of cDNA ends. The entire coding sequence was inserted into the pPIC9K vector and expressed in Pichia pastoris (P. pastoris) GS115. The recombinant SPLUNC1 protein was detected by Western blot and purified by Ni2+ chelate affinity chromatography. The test of effect of the protein on Mycoplasma ovipneumoniae (MO) was performed with real-time polymerase chain reaction. RESULTS: The Wild Argali SPLUNC1 cDNA was 1,076 bp with an open reading frame of 768 bp, which encoded a 26.49 kDa protein composed of 255 amino acids. Its amino acid sequence shared 98.4%, 96.9%, 94.5%, 90.2%, 80.8%, 78.4%, 78.3%, 72.5%, 72.3%, 68.8% identity with those of SPLUNC1 cDNA from Ovis aries (accession no. NP_001288334.1), Capra hircus (accession no. XP_005688516.1), Pantholops hodgsonii (accession no. XP_005979709.1), Bos taurus (accession no. NP_776851.1), Felis catus (accession no. XP_006929910.1), Homo sapiens (accession no. NP_001230122.1), Sus scrofa (accession no. NP_001005727.1), Chinchilla lanigera (accession no. NP_001269294.1), Mus musculus (accession no. NP_035256.2), and Rattus norvegicus (accession no. NP_742028.1), respectively. The recombinant protein corresponded to the expected molecular mass of 25.47 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and it was detected in the supernatant of P. pastoris, and it could be purified. The results from the test of inhibition effect of argali recombinant SPLUNC1 protein on MO showed that the product could inhibit MO very well (p<0.01). CONCLUSION: The amino acid sequence of Wild Argali SPLUNC1 was different from other organisms. The recombinant SPLUNC1 protein has good biological activity.
RESUMEN
Upper airway irritation is common among individuals working in moldy and damp buildings. The aim of this study was to investigate effects on the protein composition of the nasal lining fluid. The prevalence of symptoms in relation to work environment was examined in 37 individuals working in two damp buildings. Microbial growth was confirmed in one of the buildings. Nasal lavage fluid was collected from 29 of the exposed subjects and 13 controls, not working in a damp building. Protein profiles were investigated with a proteomic approach and evaluated by multivariate statistical models. Subjects from both workplaces reported upper airway and ocular symptoms. Based on protein profiles, symptomatic subjects in the two workplaces were discriminated from each other and separated from healthy controls. The groups differed in proteins involved in inflammation and host defense. Measurements of innate immunity proteins showed a significant increase in protein S100-A8 and decrease in SPLUNC1 in subjects from one workplace, while alpha-1-antitrypsin was elevated in subjects from the other workplace, compared with healthy controls. The results show that protein profiles in nasal lavage fluid can be used to monitor airway mucosal effects in personnel working in damp buildings and indicate that the profile may be separated when the dampness is associated with the presence of molds.
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Líquido del Lavado Nasal/química , Enfermedades Profesionales/metabolismo , Exposición Profesional/análisis , Enfermedades Respiratorias/metabolismo , Síndrome del Edificio Enfermo/metabolismo , Adulto , Contaminación del Aire Interior/efectos adversos , Biomarcadores/metabolismo , Calgranulina A/análisis , Estudios de Casos y Controles , Estudios Transversales , Femenino , Hongos/crecimiento & desarrollo , Glicoproteínas/análisis , Humanos , Humedad , Masculino , Persona de Mediana Edad , Análisis Multivariante , Enfermedades Profesionales/etiología , Fosfoproteínas/análisis , Proteómica , Enfermedades Respiratorias/etiología , Síndrome del Edificio Enfermo/etiología , alfa 1-Antitripsina/análisisRESUMEN
The palate, lung, and nasal epithelium clone (PLUNC) proteins are intricate immune molecules and arisen questions from them are still unresolved. In order to identify the role of PLUNC family proteins, we had analyzed its homolog protein YH1/SPLUNC1, which highly expresses in nontumor nasopharyngeal epithelium while expresses weakly in nasopharyngeal carcinoma (NPC) tissues. It is found that YH1/SPLUNC1 protein expression level was higher in chronic normal nasopharynx inflammatory cells compared to NPC tissue cells. An approach to produce active YH1/SPLUNC1 protein had been established and recombinant YH1/SPLUNC1 protein could bind to all four Gram-positive and four Gram-negative bacteria we tested, and triggered the aggregation of those bacteria. Interestingly, YH1/SPLUNC1 protein has antimicrobial activity, and it can directly kill Escherichia coli and Acinetobacter haemolyticus. The microorganism cell showed morphological changes in cell wall such as cell damage and cytoplasmic leakage after exposure to YH1/SPLUNC1 protein, indicating that YH1/SPLUNC1 directly killed the microorganisms by cell wall permeabilization. All these results indicated that YH1/SPLUNC1 might be an important antimicrobial protein involved in innate immunity defense.
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Carcinoma/metabolismo , Glicoproteínas/metabolismo , Mucosa Nasal/metabolismo , Neoplasias Nasofaríngeas/metabolismo , Nasofaringe/metabolismo , Fosfoproteínas/metabolismo , Acinetobacter/efectos de los fármacos , Acinetobacter/metabolismo , Acinetobacter/ultraestructura , Secuencia de Aminoácidos , Antibacterianos/metabolismo , Antibacterianos/farmacología , Western Blotting , Carcinoma/patología , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Escherichia coli/ultraestructura , Glicoproteínas/genética , Glicoproteínas/farmacología , Humanos , Inmunohistoquímica , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/patología , Fosfoproteínas/genética , Fosfoproteínas/farmacología , Unión Proteica , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologíaRESUMEN
Otitis media (inflammation of the middle ear) is one of the most common diseases of early childhood. Susceptibility to otitis is influenced by a number of factors, including the actions of innate immune molecules secreted by the epithelia lining the nasopharynx, middle ear and Eustachian tube. The SPLUNC1 (short palate, lung, nasal epithelial clone 1) protein is a highly abundant secretory product of the mammalian nasal, oral and respiratory mucosa that is thought to play a multifunctional role in host defense. In this study we investigated Splunc1 expression in the ear of the mouse, and examined whether this protein contributes to overall host defense in the middle ear and/or Eustachian tube. We found that Splunc1 is highly expressed in both the surface epithelium and in submucosal glands in these regions in wild-type mice. In mice lacking Splunc1, we noted histologically an increased frequency of otitis media, characterized by the accumulation of leukocytes (neutrophils with scattered macrophages), proteinaceous fluid and mucus in the middle ear lumens. Furthermore, many of these mice had extensive remodeling of the middle ear wall, suggesting a chronic course of disease. From these observations, we conclude that loss of Splunc1 predisposes mice to the development of otitis media. The Splunc1(-/-) mouse model should help investigators to better understand both the biological role of Splunc1 as well as host defense mechanisms in the middle ear.
Asunto(s)
Glicoproteínas/deficiencia , Otitis Media/patología , Fosfoproteínas/deficiencia , Animales , Bacterias/metabolismo , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Oído Medio/metabolismo , Oído Medio/microbiología , Oído Medio/patología , Trompa Auditiva/patología , Hongos/fisiología , Glicoproteínas/metabolismo , Ratones Endogámicos C3H , Penetrancia , Fosfoproteínas/metabolismoRESUMEN
Bactericidal/permeability-increasing protein fold-containing family member A1 (BPIFA1), formerly known as SPLUNC1, is one of the most abundant proteins in respiratory secretions and has been identified with increasing frequency in studies of pulmonary disease. Its expression is largely restricted to the respiratory tract, being highly concentrated in the upper airways and proximal trachea. BPIFA1 is highly responsive to airborne pathogens, allergens, and irritants. BPIFA1 actively participates in host protection through antimicrobial, surfactant, airway surface liquid regulation, and immunomodulatory properties. Its expression is modulated in multiple lung diseases, including cystic fibrosis, chronic obstructive pulmonary disease, respiratory malignancies, and idiopathic pulmonary fibrosis. However, the role of BPIFA1 in pulmonary pathogenesis remains to be elucidated. This review highlights the versatile properties of BPIFA1 in antimicrobial protection and its roles as a sensor of environmental exposure and regulator of immune cell function. A greater understanding of the contribution of BPIFA1 to disease pathogenesis and activity may clarify if BPIFA1 is a biomarker and potential drug target in pulmonary disease.
Asunto(s)
Glicoproteínas/metabolismo , Enfermedades Pulmonares/microbiología , Fosfoproteínas/metabolismo , Mucosa Respiratoria/metabolismo , Animales , Regulación de la Expresión Génica , Glicoproteínas/genética , Interacciones Huésped-Patógeno , Humanos , Inmunidad Innata , Mediadores de Inflamación/metabolismo , Enfermedades Pulmonares/genética , Enfermedades Pulmonares/inmunología , Enfermedades Pulmonares/virología , Fosfoproteínas/genética , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/microbiología , Mucosa Respiratoria/virología , Transducción de SeñalRESUMEN
Human SPLUNC1 can suppress nasopharyngeal carcinoma (NPC) tumor formation; however, the correlation between SPLUNC1expression and NPC patient prognosis has not been reported. In the present study, we used a large-scale sample of 1015 tissue cores to detect SPLUNC1 expression and its association with patient prognosis. SPLUNC1 expression was reduced in NPC samples compared to nontumor nasopharyngeal epithelium tissues. Positive expression of SPLUNC1 in NPC predicted a better prognosis (disease-free survival, P = 0.034; overall survival, P = 0.048). Cox's proportional hazards model revealed that SPLUNC1 could be a significant prognostic factor affecting disease-free survival (P = 0.027). A cDNA micro-array analyzed by significant analysis of micro-array (SAM) and ingenuity pathway analysis (IPA) revealed that an indirect interaction existed between SPLUNC1 and retinoic acid (RA) in the cancer regulatory network. To further investigate the molecular mechanisms involved, we utilized several bioinformatics tools and identified 12 retinoid X receptors heterodimer binding sites in the promoter region of the SPLUNC1 gene. The transcriptional activity of the SPLUNC1 promoter was up-regulated significantly by all-trans-retinoic acid (ATRA). SPLUNC1 and retinoic acid receptor expression were induced significantly by ATRA, and removal of ATRA led to a progressive loss of SPLUNC1 and retinoic acid receptor expression. ATRA inhibited proliferation and induced the differentiation of NPC cells. Interestingly, over-expression of SPLUNC1 sensitized NPC cells to ATRA, whereas knockdown of SPLUNC1 in HNE1 cells increased cell viability. Under SPLUNC1 knockdown conditions, differentiation was reversed by ATRA treatment. We concluded that SPLUNC1 could potentially predict prognosis for NPC patients and play an important role in ATRA-induced growth inhibition and differentiation in NPC cells.
Asunto(s)
Carcinoma/metabolismo , Glicoproteínas/fisiología , Neoplasias Nasofaríngeas/metabolismo , Proteínas de Neoplasias/fisiología , Fosfoproteínas/fisiología , Tretinoina/farmacología , Regiones no Traducidas 5'/genética , Adolescente , Adulto , Anciano , Sitios de Unión , Carcinoma/tratamiento farmacológico , Carcinoma/mortalidad , Carcinoma/patología , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Glicoproteínas/biosíntesis , Glicoproteínas/genética , Humanos , Metástasis Linfática , Masculino , Persona de Mediana Edad , Neoplasias Nasofaríngeas/tratamiento farmacológico , Neoplasias Nasofaríngeas/mortalidad , Neoplasias Nasofaríngeas/patología , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Fosfoproteínas/biosíntesis , Fosfoproteínas/genética , Pronóstico , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Receptores X Retinoide/biosíntesis , Receptores X Retinoide/genética , Receptores X Retinoide/fisiología , Adulto JovenRESUMEN
OBJECTIVE: Palate lung nasal epithelial clone (PLUNC) is a family of proteins, which are proposed to participate in the innate immune defense against infections in the upper aero-digestive tract. The aim of this study was to investigate the expression of SPLUNC1 in allergic rhinitis subjects with considerations taken to the mucosal function and smoking habits. METHODS: The participants, recruited from a cohort followed from infancy, were re-examined at the age of 18 years regarding allergy development. Based on medical histories and skin prick tests the participants were classified into groups with persistent allergic rhinitis (n=18), intermittent allergic rhinitis (n = 8) and healthy controls (n = 13). Seven subjects (3, 2 and 2 in each group, respectively) reported smoking habits. The SPLUNC1 levels in nasal lavage fluids were analyzed by Western blot. Changes in the volume of the proper nasal cavity before and after physical exercise (Vol2(increase)) were analyzed by acoustic rhinometry. RESULTS: Compared to the control group the SPLUNC1 level was significantly lower in the persistent allergy group (3.8 ± 3.4 OD vs. 1.3 ± 1.5 OD; p = 0.02), but not in the intermittent allergy group without current exposure to allergens (3.6 ± 4.7 OD). No differences were found in Vol2(increase) between any of the allergy groups and controls. In smokers Vol2(increase) was significantly reduced (p < 0.01) and the SPLUNC1 levels were lower compared to non-smokers. A significant correlation was found between SPLUNC1 and Vol2(increase) (p < 0.01; r = 0.53) in non-smokers. CONCLUSIONS: Current allergen exposure has an impact on SPLUNC1 expression in nasal lavage fluid, why allergy ought to be considered in study populations where analyses of SPLUNC1 levels are included in the reports. The normal nasal decongestion after exercise was not affected by allergy in contrast to smoking habits. The correlation between SPLUNC1 levels and Vol2(increase) in non-smokers may indicate involvement of SPLUNC1in the regulation of the normal function of the nasal mucosa. Complementary studies are needed to confirm the smoke-related reduction of SPLUNC1 expression and to analyze the possible participation of SPLUNC1 in the nasal mucosa regulation.
Asunto(s)
Ejercicio Físico/fisiología , Glicoproteínas/metabolismo , Fosfoproteínas/metabolismo , Rinitis Alérgica/genética , Rinitis Alérgica/inmunología , Fumar/epidemiología , Adolescente , Adulto , Biomarcadores/metabolismo , Estudios de Casos y Controles , Estudios de Cohortes , Femenino , Expresión Génica , Glicoproteínas/genética , Humanos , Masculino , Persona de Mediana Edad , Líquido del Lavado Nasal/inmunología , Fosfoproteínas/genética , Valores de Referencia , Rinitis Alérgica/diagnóstico , Rinometría Acústica , Índice de Severidad de la Enfermedad , Fumar/efectos adversos , Estadísticas no Paramétricas , Adulto JovenRESUMEN
Interactions between Candida albicans, saliva and saliva-coated oral surfaces are initial events in the colonization of the oral cavity by this commensal yeast, which can cause oral diseases such as candidiasis and denture stomatitis. Candida albicans also colonizes silicone voice prostheses, and the microbial biofilm formed can impair valve function, necessitating frequent prosthesis replacement. We have previously shown that saliva promoted binding of C. albicans cells to silicone in vitro, and that the selective binding of specific salivary proteins to voice prosthesis silicone mediated attachment of C. albicans cells. The C. albicans cells adhered to a polypeptide (or polypeptides) of ~36 kDa eluted from saliva-treated silicone. We show here that a protein of similar size was identified in replicate blots of the eluate from saliva-treated silicone when the blots were probed with antibodies to human SPLUNC2, a salivary protein with reported microbial agglutination properties. In addition, SPLUNC2 was depleted from saliva that had been incubated with silicone coupons. To determine whether SPLUNC2 is a yeast-binding protein, SPLUNC2 cDNA was expressed in Escherichia coli. Purified recombinant His-tagged protein (SPLUNC2r) bound to silicone as demonstrated by immunoblot analysis of an eluate from SPLUNC2r-treated silicone coupons and (35) S-radiolabelled C. albicans cells adhered in a dose-dependent manner to SPLUNC2r-coated silicone. We conclude that SPLUNC2 binds to silicone and acts as a receptor for C. albicans adherence to, and subsequent colonization of, voice prosthesis silicone.