RESUMEN
Rosacea is a chronic inflammatory condition that predominantly affects the skin of the face. Sera from rosacea patients display elevated reactivity to proteins from a bacterium (Bacillus oleronius) originally isolated from a Demodex mite from a rosacea patient suggesting a possible role for bacteria in the induction and persistence of this condition. This work investigated the ability of B. oleronius proteins to activate neutrophils and demonstrated activation via the IP3 pathway. Activated neutrophils displayed increased levels of IP1 production, F-actin formation, chemotaxis, and production of the pro-inflammatory cytokines IL-1ß and IL-6 following stimulation by pure and crude B. oleronius protein preparations (2 µg/ml), respectively. In addition, neutrophils exposed to pure and crude B. oleronius proteins (2 µg/ml) demonstrated increased release of internally stored calcium (Ca(2+)), a hallmark of the IP3 pathway of neutrophil activation. Neutrophils play a significant role in the inflammation associated with rosacea, and this work demonstrates how B. oleronius proteins can induce neutrophil recruitment and activation.
Asunto(s)
Proteínas Bacterianas/inmunología , Inositol 1,4,5-Trifosfato/metabolismo , Ácaros/microbiología , Activación Neutrófila/inmunología , Infiltración Neutrófila/inmunología , Rosácea/inmunología , Animales , Bacillus/inmunología , Calcio/metabolismo , Humanos , Fosfatos de Inositol/metabolismo , Interleucina-1beta/inmunología , Interleucina-6/inmunología , Neutrófilos/inmunología , Rosácea/microbiología , Piel/microbiología , Piel/patologíaRESUMEN
Leukotriene B4 (LTB4) contributes to many inflammatory diseases, including genetic and nongenetic forms of chronic obstructive pulmonary disease. α-1 Antitrypsin (AAT) deficiency (AATD) is characterized by destruction of lung parenchyma and development of emphysema, caused by low AAT levels and a high neutrophil burden in the airways of affected individuals. In this study we assessed whether AATD is an LTB4-related disease and investigated the ability of serum AAT to control LTB4 signaling in neutrophils. In vitro studies demonstrate that neutrophil elastase is a key player in the LTB4 inflammatory cycle in AATD, causing increased LTB4 production, and associated BLT1 membrane receptor expression. AATD patients homozygous for the Z allele were characterized by increased neutrophil adhesion and degranulation responses to LTB4. We demonstrate that AAT can bind LTB4 and that AAT/LTB4 complex formation modulates BLT1 engagement and downstream signaling events, including 1,4,5-triphosphate production and Ca(2+) flux. Additionally, treatment of ZZ-AATD individuals with AAT augmentation therapy decreased plasma LTB4 concentrations and reduced levels of membrane-bound neutrophil elastase. Collectively, these results provide a mechanism by which AAT augmentation therapy impacts on LTB4 signaling in vivo, and not only reinforces the utility of this therapy for resolving inflammation in AATD, but supports useful future clinical applications in treatment of other LTB4-related diseases.
Asunto(s)
Señalización del Calcio/inmunología , Degranulación de la Célula/inmunología , Leucotrieno B4/inmunología , Neutrófilos/inmunología , Receptores de Leucotrieno B4/inmunología , Deficiencia de alfa 1-Antitripsina/inmunología , alfa 1-Antitripsina/inmunología , Adulto , Femenino , Humanos , Elastasa de Leucocito/inmunología , Pulmón/inmunología , Pulmón/patología , Masculino , Neutrófilos/patología , alfa 1-Antitripsina/uso terapéutico , Deficiencia de alfa 1-Antitripsina/tratamiento farmacológico , Deficiencia de alfa 1-Antitripsina/patologíaRESUMEN
Lymphangioleiomyomatosis (LAM) is a rare and progressive cystic lung condition affecting approximately 3.4-7.5/million women, with an average lag time between symptom onset and diagnosis of upwards of 4 years. The aim of this work was to identify altered proteins in LAM serum which may be potential biomarkers of disease. Serum from LAM patient volunteers and healthy control volunteers were pooled and analysis carried out using quantitative 4-plex iTRAQ technology. Differentially expressed proteins were validated using ELISAs and pathway analysis was carried out using Ingenuity Pathway Analysis. Fourteen proteins were differentially expressed in LAM serum compared to control serum (p<0.05). Further screening validated the observed differences in extracellular matrix remodelling proteins including fibronectin (30% decrease in LAM, pâ=â0.03), von Willebrand Factor (40% reduction in LAM, pâ=â0.03) and Kallikrein III (25% increase in LAM, pâ=â0.03). Pathway networks elucidated the relationships between the ECM and cell trafficking in LAM. This study was the first to highlight an imbalance in networks important for remodelling in LAM, providing a set of novel potential biomarkers. These understandings may lead to a new effective treatment for LAM in the future.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Linfangioleiomiomatosis/sangre , Linfangioleiomiomatosis/metabolismo , Mapas de Interacción de Proteínas , Adulto , Anciano , Proteínas Sanguíneas/análisis , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteómica , Transducción de SeñalRESUMEN
The T cell Ig and mucin domain-containing molecule (TIM) family of receptors have emerged as potential therapeutic targets to correct abnormal immune function in chronic inflammatory conditions. TIM-3 serves as a functional receptor in structural cells of the airways and via the ligand galectin-9 (Gal-9) can modulate the inflammatory response. The aim of this study was to investigate TIM-3 expression and function in neutrophils, focusing on its potential role in cystic fibrosis (CF) lung disease. Results revealed that TIM-3 mRNA and protein expression values of circulating neutrophils were equal between healthy controls (n = 20) and people with CF (n = 26). TIM-3 was detected on resting neutrophil membranes by FACS analysis, and expression levels significantly increased post IL-8 or TNF-α exposure (p < 0.05). Our data suggest a novel role for TIM-3/Gal-9 signaling involving modulation of cytosolic calcium levels. Via TIM-3 interaction, Gal-9 induced neutrophil degranulation and primed the cell for enhanced NADPH oxidase activity. Killing of Pseudomonas aeruginosa was significantly increased upon bacterial opsonization with Gal-9 (p < 0.05), an effect abrogated by blockade of TIM-3 receptors. This mechanism appeared to be Gram-negative bacteria specific and mediated via Gal-9/ LPS binding. Additionally, we have demonstrated that neutrophil TIM-3/Gal-9 signaling is perturbed in the CF airways due to proteolytic degradation of the receptor. In conclusion, results suggest a novel neutrophil defect potentially contributing to the defective bacterial clearance observed in the CF airways and suggest that manipulation of the TIM-3 signaling pathway may be of therapeutic value in CF, preferably in conjunction with antiprotease treatment.
Asunto(s)
Fibrosis Quística/inmunología , Galectinas/inmunología , Pulmón/inmunología , Proteínas de la Membrana/inmunología , Neutrófilos/inmunología , Pseudomonas aeruginosa/inmunología , Fibrosis Quística/microbiología , Fibrosis Quística/patología , Femenino , Receptor 2 Celular del Virus de la Hepatitis A , Humanos , Lipopolisacáridos/inmunología , Pulmón/microbiología , Pulmón/patología , Masculino , Neutrófilos/microbiología , Transducción de Señal/inmunologíaRESUMEN
Secretory leukoprotease inhibitor (SLPI) is an anti-inflammatory protein present in respiratory secretions. Whilst epithelial cell SLPI is extensively studied, neutrophil associated SLPI is poorly characterised. Neutrophil function including chemotaxis and degranulation of proteolytic enzymes involves changes in cytosolic calcium (Ca(2+)) levels which is mediated by production of inositol 1,4,5-triphosphate (IP3) in response to G-protein-coupled receptor (GPCR) stimuli. The aim of this study was to investigate the intracellular function of SLPI and the mechanism-based modulation of neutrophil function by this antiprotease. Neutrophils were isolated from healthy controls (n = 10), individuals with cystic fibrosis (CF) (n = 5) or chronic obstructive pulmonary disease (COPD) (n = 5). Recombinant human SLPI significantly inhibited fMet-Leu-Phe (fMLP) and interleukin(IL)-8 induced neutrophil chemotaxis (P < 0.05) and decreased degranulation of matrix metalloprotease-9 (MMP-9), hCAP-18, and myeloperoxidase (MPO) (P < 0.05). The mechanism of inhibition involved modulation of cytosolic IP3 production and downstream Ca(2+) flux. The described attenuation of Ca(2+) flux was overcome by inclusion of exogenous IP3 in electropermeabilized cells. Inhibition of IP3 generation and Ca(2+) flux by SLPI may represent a novel anti-inflammatory mechanism, thus strengthening the attractiveness of SLPI as a potential therapeutic molecule in inflammatory airway disease associated with excessive neutrophil influx including CF, non-CF bronchiectasis, and COPD.
Asunto(s)
Antiinflamatorios/metabolismo , Fibrosis Quística/patología , Inositol 1,4,5-Trifosfato/biosíntesis , Espacio Intracelular/metabolismo , Neutrófilos/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/patología , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismo , Adulto , Antiinflamatorios/farmacología , Calcio/metabolismo , Degranulación de la Célula/efectos de los fármacos , Quimiotaxis/efectos de los fármacos , Fibrosis Quística/metabolismo , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Citosol/efectos de los fármacos , Citosol/metabolismo , Femenino , Humanos , Factores Inmunológicos/metabolismo , Factores Inmunológicos/farmacología , Espacio Intracelular/efectos de los fármacos , Masculino , Modelos Biológicos , Activación Neutrófila/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Neutrófilos/fisiología , Oxidación-Reducción/efectos de los fármacos , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Proteínas Recombinantes/farmacologíaRESUMEN
Larvae of Galleria mellonella are widely used to study the virulence of microbial pathogens and for assessing the potency of antimicrobial agents. This work examined the effect of nutritional deprivation on the ability of larvae to withstand infection in order to establish standardized conditions for the treatment of larvae for in vivo testing. Larvae deprived of food for seven days demonstrated an increased susceptibility to infection by the yeast Candida albicans. These larvae displayed a lower density of hemocytes compared with controls but hemocytes from starved and control larvae demonstrated the same ability to kill yeast cells. Hemolymph from starved larvae demonstrated reduced expression of a range of antimicrobial peptides (e.g., lipocalin) and immune proteins (e.g., apolipophorin and arylphorin). Deprivation of G. mellonella larvae of food leads to a reduction in the cellular and immune responses and an increased susceptibility to infection. Researchers utilizing these larvae should ensure adequate food is provided to larvae in order to allow valid comparisons to be made between results from different laboratories.
Asunto(s)
Candida albicans/inmunología , Candidiasis/inmunología , Proteínas de Insectos/metabolismo , Larva/inmunología , Mariposas Nocturnas/inmunología , Animales , Péptidos Catiónicos Antimicrobianos/biosíntesis , Apolipoproteínas/biosíntesis , Candida albicans/patogenicidad , Candidiasis/microbiología , Privación de Alimentos , Hemocitos/inmunología , Hemolinfa/inmunología , Proteínas de Insectos/biosíntesis , Larva/microbiología , Lipocalinas/biosíntesisRESUMEN
Administration of non-toxic concentrations (10 µM) of cytochalasin b and nocodazole to larvae of Galleria mellonella increased their susceptibility to infection by the yeast Candida albicans. These agents were found to inhibit the process of phagocytosis and to reduce the killing ability of haemocytes. In addition, both cytochalasin b and nocodazole reduced the release of antimicrobial peptides (e.g. apolipophorin 3) and enzymes (e.g. serine protease) from PMA stimulated haemocytes. Rhodamine coupled phalloidin staining revealed reduced F-actin formation in haemocytes treated with nocodazole or cytochalasin b. By disrupting the formation of F-actin cytochalasin b and nocodazole have the ability to retard the function of haemocytes, in the same manner as they affect mammalian neutrophils, and thus increase the susceptibility of larvae to infection. The results presented here demonstrate that haemocytes are sensitive to inhibition by nocodazole and cytochalasin b, in a similar manner to neutrophils, thus highlighting another similarity between both cell types and so increasing the attractiveness of using insects as alternative models to the use of mammals for in vivo pathogen or drug screening.
Asunto(s)
Candida albicans/inmunología , Hemocitos/inmunología , Larva/inmunología , Mariposas Nocturnas/inmunología , Nocodazol/farmacología , Fagocitosis/efectos de los fármacos , Actinas/antagonistas & inhibidores , Actinas/inmunología , Animales , Apolipoproteínas/inmunología , Apolipoproteínas/metabolismo , Candida albicans/efectos de los fármacos , Células Cultivadas , Citocalasina B/farmacología , Hemocitos/efectos de los fármacos , Hemocitos/microbiología , Humanos , Proteínas de Insectos/inmunología , Proteínas de Insectos/metabolismo , Larva/efectos de los fármacos , Larva/microbiología , Modelos Animales , Mariposas Nocturnas/efectos de los fármacos , Mariposas Nocturnas/microbiología , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Neutrófilos/microbiología , Fagocitosis/inmunología , Serina Proteasas/inmunología , Serina Proteasas/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Moduladores de Tubulina/farmacologíaRESUMEN
Carnitine has beneficial vitamin-like qualities and functions as a compatible solute. The presence of L-carnitine in infant formula significantly promoted the growth of Listeria monocytogenes at 7 degrees C, whereas disruption of the genes for carnitine and glycine betaine uptake compromised growth of the L. monocytogenes mutant in formula containing carnitine that was stored at 7 degrees C. Thus, addition of carnitine to baby formula may pose a potential food safety risk.