The role of toll-like receptors in acute and chronic lung inflammation

By virtue of its direct contact with the environment, the lung is constantly challenged by infectious and non-infectious stimuli that necessitate a robust yet highly controlled host response coordinated by the innate and adaptive arms of the immune system. Mammalian Toll-like receptors (TLRs) function as crucial sentinels of microbial and non-infectious antigens throughout the respiratory tract and mediate host innate immunity. Selective induction of inflammatory responses to harmful environmental exposures and tolerance to innocuous antigens are required to maintain tissue homeostasis and integrity. Conversely, dysregulated innate immune responses manifest as sustained and self-perpetuating tissue damage rather than controlled tissue repair. In this article we review aspects of Toll-like receptor function that are relevant to the development of acute lung injury and chronic obstructive lung diseases as well as resistance to frequently associated microbial infections

High Na+ Environments Impair Phagocyte Oxidase-Dependent Antibacterial Activity of Neutrophils

Infection and inflammation can augment local Na+ abundance. These increases in local Na+ levels boost proinflammatory and antimicrobial macrophage activity and can favor polarization of T cells towards a proinflammatory Th17 phenotype. Although neutrophils play an important role in fighting intruding invaders, the impact of increased Na+ on the antimicrobial activity of neutrophils remains elusive. Here we show that, in neutrophils, increases in Na+ (high salt, HS) impair the ability of human and murine neutrophils to eliminate Escherichia coli and Staphylococcus aureus. High salt caused reduced spontaneous movement, degranulation and impaired production of reactive oxygen species (ROS) while leaving neutrophil viability unchanged. High salt enhanced the activity of the p38 mitogen-activated protein kinase (p38/MAPK) and increased the interleukin (IL)-8 release in a p38/MAPK-dependent manner. Whereas inhibition of p38/MAPK did not result in improved neutrophil defense, pharmacological blockade of the phagocyte oxidase (PHOX) or its genetic ablation mimicked the impaired antimicrobial activity detected under high salt conditions. Stimulation of neutrophils with phorbol-12-myristate-13-acetate (PMA) overcame high salt-induced impairment in ROS production and restored antimicrobial activity of neutrophils. Hence, we conclude that high salt-impaired PHOX activity results in diminished antimicrobial activity. Our findings suggest that increases in local Na+ represent an ionic checkpoint that prevents excessive ROS production of neutrophils, which decreases their antimicrobial potential and could potentially curtail ROS-mediated tissue damage

Untersuchungen zur Induktion von Adhäsion bei Escherichia coli und Salmonella Typhimurium durch murines SPLUNC1 und LPLUNC1

Die überwiegend in den Atemwegen und im Nasenrachenraum exprimierten PLUNCs (palate, lung and nasal epithelium clone) gehören zur Familie der LT (lipid transfer)/LBP (Lipopolysaccharide binding protein)-Proteine und werden in long (L)- und short (S)-PLUNCs (LPLUNC bzw. SPLUNC) unterteilt (Bingle und Craven 2002, Bingle et al. 2004). SPLUNC1 und LPLUNC1 sind die beiden einzigen Mitglieder der PLUNC-Proteinfamilie, die in den oberen Atemwegen (Trachea und Bronchien) exprimiert sind (Bingle und Bingle 2000, Bingle et al. 2010). In dieser Arbeit wurde der Einfluss dieser beiden Proteine auf die Adhäsion verschiedener gramnegativer Bakterien untersucht. Hierzu wurden rekombinant in Insektenzellen exprimiertes murines SPLUNC1 und LPLUNC1 in einem Kristallviolett-Adhäsionstest eingesetzt. Es konnte gezeigt werden, dass die getesteten PLUNC-Proteine bei den meisten Klebsiella pneumoniae-Isolaten sowie bei Serratia marcescens Adhäsion inhibieren. Bei Salmonella Typhimurium und Escherichia coli kommt es dagegen in Anwesenheit von mSPLUNC1 oder mLPLUNC1 zu einer Induktion von Adhäsion über Typ 1 Pili. Ein möglicher Rezeptor für die PLUNC-Proteine auf der Oberfläche der Bakterien ist LPS, da eine Zugabe von exogenem LPS im Adhäsionstest die Induktion von Adhäsion inhibiert. Weiterhin wurde durch Verwendung verkürzter Proteinvarianten gezeigt, dass die Adhäsion-induzierende Domäne von mSPLUNC1 am C-Terminus des Proteins lokalisiert ist. Zur Identifikation von bakteriellen Genen, die an der Induktion von Adhäsion durch die PLUNC-Proteine beteiligt sind, wurde eine Sammlung von S. Typhimurium-Zufallsmutanten im Kristallviolett-Adhäsionstest anlaysiert. Die Ergebnisse dieses Screenings sprechen für eine wichtige Rolle der Sensorkinase ArcB bei der Ausbildung von Typ 1 Pili. Um in zukünftigen Studien mSPLUNC1 auf Proteinebene zu detektieren und zu quantifizieren, wurden ein monoklonaler α-mSPLUNC1-Antikörper und ein polyklonales α-mSPLUNC1-Antiserum erzeugt. Auf Grundlage dieser beiden Antikörper wurde dann ein Sandwich-ELISA etabliert, der eine Quantifizierung von mSPLUNC1 bis zu einer Nachweisgrenze von 500 pg/ml ermöglicht

Of Men not Mice: Bactericidal/Permeability-increasing Protein expressed in human Macrophages acts as a Phagocytic receptor and Modulates entry and replication of gram-negative Bacteria

Macrophages as immune cells prevent the spreading of pathogens by means of active phagocytosis and killing. We report here the presence of an antimicrobial protein, bactericidal/permeability-increasing protein (BPI) in human macrophages, which actively participates in engulfment and killing of Gram-negative pathogens. Our studies revealed increased expression of BPI in human macrophages during bacterial infection and upon stimulation with various pathogen-associated molecular patterns, viz., LPS and flagellin. Furthermore, during the course of an infection, BPI interacted with Gram-negative bacteria, resulting in enhanced phagocytosis and subsequent control of the bacterial replication. However, it was observed that bacteria which can maintain an active replicating niche (Salmonella Typhimurium) avoid the interaction with BPI during later stages of infection. On the other hand, Salmonella mutants, which cannot maintain a replicating niche, as well as Shigella flexneri, which quit the endosomal vesicle, showed interaction with BPI. These results propose an active role of BPI in Gram-negative bacterial clearance by human macrophages

Toll-Like Receptors: Sentinels of Host Defence against Bacterial Infection

Innate immunity provides a fi rst line of host defence against infection through microbial recognition and killing while simultaneously activating a defi nitive adaptive immune response. Toll-like receptors (TLRs) are principal mediators of rapid microbial recognition and function mainly by detection of structural patterns that do not exist in the host. TLR2 and TLR4 were the fi rst members of this innate immune receptor family to be strongly implicated in antibacterial host defence. Following the initial description of the mammalian TLR family, susceptibility to infection with numerous human microbial pathogens has been intensively studied using mice with engineered deletions of each of these molecules. While it has become quite clear that TLR activation is necessary for optimal host defence, a comprehensive understanding of the mechanisms by which this family of pattern recognition receptors engages protective immunity, particularly the adaptive response, is still evolving

Die Aktivierung und Funktion von Bactericidal / Permeability - Increasing Protein (BPI) bei entzündlichen Darmprozessen

Das Antimikrobielle Protein (AMP) Bactericidal / Permeability - Increasing Protein (BPI), welches in der Lage ist, Lipopolysaccharide (LPS) effektiv zu binden und damit zu neutralisieren, spielt bei Erkrankungen wie z.B cystischer Fibrose eine entscheidende Rolle. Allerdings ist bisher unklar, welche Bedeutung BPI für Chronisch-entzündliche-Darmerkrankungen (CED) hat. Im Rahmen dieser Arbeit konnte erstmals gezeigt werden, dass BPI eine wichtige Funktion bei der DSS-induzierten Kolitis einnimmt. Dabei konnte in Wildtyp (WT) - Mäusen eine gesteigerte BPI-Expression während einer DSS-induzierten Kolitis nachgewiesen werden. Beim Vergleich der Phänotypen von WT- und BPI defizienten (BPI-/-) - Mäusen zeigten sich deutliche Unterschiede im Krankheitsverlauf. Die Defizienz von BPI führte zu einer gesteigerten Suzseptibilität in Mäusen nach DSSGabe. Nähere Untersuchungen dazu zeigten, dass BPI-/- - Mäuse mit einer stärkeren Immunantwort reagieren. Diese ist durch eine gesteigerte Zytokinfreisetzung von IL-1ß und eine signifikant erhöhte Anzahl von Entzündungszellen im Darm gekennzeichnet. Bei den Entzündungszellen im Darm handelt es sich um T- und BZellen, Makrophagen und neutrophile Granulozyten. Erste mechanistische Analysen zeigten, dass BPI weder Einfluss auf intrinsische zytoprotektive Eigenschaften des Epithels hat, noch einen direkten Einfluss auf den IL17C Signalweg nimmt. IL-17C wird eine entscheidende Funktion bei der mukosalen Immunabwehr zugeschrieben. Auch die Analysen zur Zusammensetzung der Mikroflora im Darm von WT- als auch BPI-/- - Mäusen mittels quantitativer PCR lieferten keine signifikanten Unterschiede zwischen den Genotypen. BPI scheint demzufolge keinen direkten Einfluss auf die Zusammensetzung der untersuchten Mikrobiota zu haben, was jedoch durch weitere detaillierte Analysen der Bakterienarten klarifiziert werden sollte

Expression and Antimicrobial Function of Bactericidal Permeability-Increasing Protein in Cystic Fibrosis Patients

In cystic fibrosis (CF), the condition limiting the prognosis of affected children is the chronic obstructive lung disease accompanied by chronic and persistent infection with mostly mucoid strains of Pseudomonas aeruginosa. The majority of CF patients have antineutrophil cytoplasmic antibodies (ANCA) primarily directed against the bactericidal permeability-increasing protein (BPI) potentially interfering with antimicrobial effects of BPI. We analyzed the expression of BPI in the airways of patients with CF. In their sputum samples or bronchoalveolar lavage specimens, nearly all patients expressed BPI mRNA and protein, which were mainly products of neutrophil granulocytes as revealed by intracellular staining and subsequent flow cytometry. Repeated measurements revealed consistent individual BPI expression levels during several months quantitatively correlating with interleukin-8. In vitro, P. aeruginosa isolates from CF patients initiated the rapid release of BPI occurring independently of protein de novo syntheses. Furthermore, purified natural BPI as well as a 27-mer BPI-derived peptide displayed antimicrobial activity against even patient-derived mucoid P. aeruginosa strains and bacteria resistant against all antibiotics tested. Thus, BPI that is functionally active against mucoid P. aeruginosa strains is expressed in the airways of CF patients but may be hampered by autoantibodies, resulting in chronic infection

The Human Antimicrobial Protein Bactericidal/Permeability-Increasing Protein (BPI) Inhibits the Infectivity of Influenza A Virus

In addition to their well-known antibacterial activity some antimicrobial peptides and proteins (AMPs) display also antiviral effects. A 27 aa peptide from the N-terminal part of human bactericidal/permeability-increasing protein (BPI) previously shown to harbour antibacterial activity inhibits the infectivity of multiple Influenza A virus strains (H1N1, H3N2 and H5N1) the causing agent of the Influenza pneumonia. In contrast, the homologous murine BPI-peptide did not show activity against Influenza A virus. In addition human BPI-peptide inhibits the activation of immune cells mediated by Influenza A virus. By changing the human BPIpeptide to the sequence of the mouse homologous peptide the antiviral activity was completely abolished. Furthermore, the human BPI-peptide also inhibited the pathogenicity of the Vesicular Stomatitis Virus but failed to interfere with HIV and measles virus. Electron microscopy indicate that the human BPI-peptide interferes with the virus envelope and at high concentrations was able to destroy the particles completely