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2.
Eur Respir J ; 51(4)2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29545277

RESUMO

It is currently unknown how cigarette smoke-induced airway remodelling affects highly expressed respiratory epithelial defence proteins and thereby mucosal host defence.Localisation of a selected set of highly expressed respiratory epithelial host defence proteins was assessed in well-differentiated primary bronchial epithelial cell (PBEC) cultures. Next, PBEC were cultured at the air-liquid interface, and during differentiation for 2-3 weeks exposed daily to whole cigarette smoke. Gene expression, protein levels and epithelial cell markers were subsequently assessed. In addition, functional activities and persistence of the cigarette smoke-induced effects upon cessation were determined.Expression of the polymeric immunoglobulin receptor, secretory leukocyte protease inhibitor and long and short PLUNC (palate, lung and nasal epithelium clone protein) was restricted to luminal cells and exposure of differentiating PBECs to cigarette smoke resulted in a selective reduction of the expression of these luminal cell-restricted respiratory host defence proteins compared to controls. This reduced expression was a consequence of cigarette smoke-impaired end-stage differentiation of epithelial cells, and accompanied by a significant decreased transepithelial transport of IgA and bacterial killing.These findings shed new light on the importance of airway epithelial cell differentiation in respiratory host defence and could provide an additional explanation for the increased susceptibility of smokers and patients with chronic obstructive pulmonary disease to respiratory infections.


Assuntos
Brônquios/citologia , Diferenciação Celular/efeitos dos fármacos , Células Epiteliais/citologia , Fumaça , Produtos do Tabaco/toxicidade , Brônquios/imunologia , Células Cultivadas , Células Epiteliais/imunologia , Expressão Gênica/efeitos dos fármacos , Humanos , Imunoglobulina A/imunologia , Microscopia Confocal
3.
Am J Respir Cell Mol Biol ; 56(6): 749-761, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28231019

RESUMO

Vitamin D is a regulator of host defense against infections and induces expression of the antimicrobial peptide hCAP18/LL-37. Vitamin D deficiency is associated with chronic inflammatory lung diseases and respiratory infections. However, it is incompletely understood if and how (chronic) airway inflammation affects vitamin D metabolism and action. We hypothesized that long-term exposure of primary bronchial epithelial cells to proinflammatory cytokines alters their vitamin D metabolism, antibacterial activity, and expression of hCAP18/LL-37. To investigate this, primary bronchial epithelial cells were differentiated at the air-liquid interface for 14 days in the presence of the proinflammatory cytokines, TNF-α and IL-1ß (TNF-α/IL-1ß), and subsequently exposed to vitamin D (inactive 25(OH)D3 and active 1,25(OH)2D3). Expression of hCAP18/LL-37, vitamin D receptor, and enzymes involved in vitamin D metabolism (CYP24A1 and CYP27B1) was determined using quantitative PCR, Western blot, and immunofluorescence staining. Furthermore, vitamin D-mediated antibacterial activity was assessed using nontypeable Haemophilus influenzae. We found that TNF-α/IL-1ß treatment reduced vitamin D-induced expression of hCAP18/LL-37 and killing of nontypeable H. influenzae. In addition, CYP24A1 (a vitamin D-degrading enzyme) was increased by TNF-α/IL-1ß, whereas CYP27B1 (that converts 25(OH)D3 to its active form) and vitamin D receptor expression remained unaffected. Furthermore, we have demonstrated that the TNF-α/IL-1ß-mediated induction of CYP24A1 was, at least in part, mediated by the transcription factor specific protein 1, and the epidermal growth factor receptor-mitogen-activated protein kinase pathway. These findings indicate that TNF-α/IL-1ß decreases vitamin D-mediated antibacterial activity and hCAP18/LL-37 expression via induction of CYP24A1 and suggest that chronic inflammation impairs protective responses induced by vitamin D.


Assuntos
Brônquios/citologia , Citocinas/metabolismo , Células Epiteliais/imunologia , Mediadores da Inflamação/metabolismo , Vitamina D/farmacologia , Lesão Pulmonar Aguda/patologia , Peptídeos Catiônicos Antimicrobianos , Calcifediol/farmacologia , Catelicidinas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Haemophilus influenzae/efeitos dos fármacos , Humanos , Interleucina-17/farmacologia , Interleucina-1beta/farmacologia , Viabilidade Microbiana/efeitos dos fármacos , Mucinas/metabolismo , Fator de Transcrição Sp1/metabolismo , Fatores de Tempo , Fator de Necrose Tumoral alfa/farmacologia , Vitamina D3 24-Hidroxilase/metabolismo
4.
J Innate Immun ; 9(4): 359-374, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28171878

RESUMO

Antimicrobial proteins and peptides (AMPs) are a central component of the antibacterial activity of airway epithelial cells. It has been proposed that a decrease in antibacterial lung defense contributes to an increased susceptibility to microbial infection in smokers and patients with chronic obstructive pulmonary disease (COPD). However, whether reduced AMP expression in the epithelium contributes to this lower defense is largely unknown. We investigated the bacterial killing activity and expression of AMPs by air-liquid interface-cultured primary bronchial epithelial cells from COPD patients and non-COPD (ex-)smokers that were stimulated with nontypeable Haemophilus influenzae (NTHi). In addition, the effect of cigarette smoke on AMP expression and the activation of signaling pathways was determined. COPD cell cultures displayed reduced antibacterial activity, whereas smoke exposure suppressed the NTHi-induced expression of AMPs and further increased IL-8 expression in COPD and non-COPD cultures. Moreover, smoke exposure impaired NTHi-induced activation of NF-κB, but not MAP-kinase signaling. Our findings demonstrate that the antibacterial activity of cultured airway epithelial cells induced by acute bacterial exposure was reduced in COPD and suppressed by cigarette smoke, whereas inflammatory responses persisted. These findings help to explain the imbalance between protective antibacterial and destructive inflammatory innate immune responses in COPD.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Fumar Cigarros/efeitos adversos , Infecções por Haemophilus/imunologia , Haemophilus influenzae/imunologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Mucosa Respiratória/imunologia , Peptídeos Catiônicos Antimicrobianos/genética , Bacteriólise , Células Cultivadas , Humanos , Imunidade , Imunomodulação , Interleucina-8/metabolismo , NF-kappa B/metabolismo , Mucosa Respiratória/microbiologia , Transdução de Sinais
5.
Respir Res ; 12: 59, 2011 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-21529380

RESUMO

The airway epithelium forms a barrier against infection but also produces antimicrobial peptides (AMPs) and other inflammatory mediators to activate the immune system. It has been shown that in allergic disorders, Th2 cytokines may hamper the antimicrobial activity of the epithelium. However, the presence of Th2 cytokines also affects the composition of the epithelial layer which may alter its function. Therefore, we investigated whether exposure of human primary bronchial epithelial cells (PBEC) to Th2 cytokines during mucociliary differentiation affects expression of the human cathelicidin antimicrobial protein (hCAP18)/LL-37 and human beta defensins (hBD), and antimicrobial activity.PBEC were cultured at an air-liquid interface (ALI) for two weeks in the presence of various concentrations of IL-4 or IL-13. Changes in differentiation and in expression of various AMPs and the antimicrobial proteinase inhibitors secretory leukocyte protease inhibitor (SLPI) and elafin were investigated as well as antimicrobial activity.IL-4 and IL-13 increased mRNA expression of hCAP18/LL-37 and hBD-2. Dot blot analysis also showed an increase in hCAP18/LL-37 protein in apical washes of IL-4-treated ALI cultures, whereas Western Blot analysis showed expression of a protein of approximately 4.5 kDa in basal medium of IL-4-treated cultures. Using sandwich ELISA we found that also hBD-2 in apical washes was increased by both IL-4 and IL-13. SLPI and elafin levels were not affected by IL-4 or IL-13 at the mRNA or protein level. Apical wash obtained from IL-4- and IL-13-treated cultures displayed increased antimicrobial activity against Pseudomonas aeruginosa compared to medium-treated cultures. In addition, differentiation in the presence of Th2 cytokines resulted in increased MUC5AC production as has been shown previously.These data suggest that prolonged exposure to Th2 cytokines during mucociliary differentiation contributes to antimicrobial defence by increasing the expression and release of selected antimicrobial peptides and mucus.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Brônquios/metabolismo , Diferenciação Celular , Células Epiteliais/metabolismo , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Depuração Mucociliar , Mucosa Respiratória/metabolismo , Peptídeos Catiônicos Antimicrobianos/genética , Western Blotting , Brônquios/imunologia , Brônquios/microbiologia , Catelicidinas/metabolismo , Células Cultivadas , Elafina/metabolismo , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Humanos , Mucina-5AC/metabolismo , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , RNA Mensageiro/metabolismo , Mucosa Respiratória/imunologia , Mucosa Respiratória/microbiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inibidor Secretado de Peptidases Leucocitárias/metabolismo , Fatores de Tempo , beta-Defensinas/metabolismo
6.
Respir Res ; 12: 34, 2011 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-21426578

RESUMO

BACKGROUND: Macrophages have been implicated in the pathogenesis of COPD. M1 and M2 macrophages constitute subpopulations displaying pro- and anti-inflammatory properties. We hypothesized that smoking cessation affects macrophage heterogeneity in the lung of patients with COPD. Our aim was to study macrophage heterogeneity using the M2-marker CD163 and selected pro- and anti-inflammatory mediators in bronchoalveolar lavage (BAL) fluid and induced sputum from current smokers and ex-smokers with COPD. METHODS: 114 COPD patients (72 current smokers; 42 ex-smokers, median smoking cessation 3.5 years) were studied cross-sectionally and underwent sputum induction (M/F 99/15, age 62 ± 8 [mean ± SD] years, 42 (31-55) [median (range)] packyears, post-bronchodilator FEV1 63 ± 9% predicted, no steroids past 6 months). BAL was collected from 71 patients. CD163+ macrophages were quantified in BAL and sputum cytospins. Pro- and anti-inflammatory mediators were measured in BAL and sputum supernatants. RESULTS: Ex-smokers with COPD had a higher percentage, but lower number of CD163+ macrophages in BAL than current smokers (83.5% and 68.0%, p = 0.04; 5.6 and 20.1 × 10(4)/ml, p = 0.001 respectively). The percentage CD163+ M2 macrophages was higher in BAL compared to sputum (74.0% and 30.3%, p < 0.001). BAL M-CSF levels were higher in smokers than ex-smokers (571 pg/ml and 150 pg/ml, p = 0.001) and correlated with the number of CD163+ BAL macrophages (Rs = 0.38, p = 0.003). No significant differences were found between smokers and ex-smokers in the levels of pro-inflammatory (IL-6 and IL-8), and anti-inflammatory (elafin, and Secretory Leukocyte Protease Inhibitor [SLPI]) mediators in BAL and sputum. CONCLUSIONS: Our data suggest that smoking cessation partially changes the macrophage polarization in vivo in the periphery of the lung towards an anti-inflammatory phenotype, which is not accompanied by a decrease in inflammatory parameters.


Assuntos
Citocinas/análise , Mediadores da Inflamação/análise , Macrófagos/imunologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Abandono do Hábito de Fumar , Fumar/imunologia , Idoso , Antígenos CD/análise , Antígenos de Diferenciação Mielomonocítica/análise , Biomarcadores/análise , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Distribuição de Qui-Quadrado , Estudos Transversais , Feminino , Humanos , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Países Baixos , Doença Pulmonar Obstrutiva Crônica/etiologia , Receptores de Superfície Celular/análise , Fumar/efeitos adversos , Prevenção do Hábito de Fumar , Escarro/citologia , Escarro/imunologia
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