Christoph Beisswenger

Microbial DNA Induces a Host Defense Reaction of Human Respiratory Epithelial Cells

Epithelial cells represent the initial site of bacterial colonization in the respiratory tract. TLR9 has been identified in B cells and CD 123+ dendritic cells and found to be involved in the recognition of microbial DNA. It was the aim of the study to investigate the role of TLR9 in the host defense reactions of the respiratory epithelium. Respiratory epithelial cell lines (IHAEo−, Calu-3) or fully differentiated primary human cells as air-liquid interface cultures were stimulated with bacterial DNA or synthetic oligonucleotides containing CpG motifs (CpG oligodeoxynucleotides). Expression of TLR9, cytokines, and human β-defensin 2 was determined by quantitative RT-PCR or by ELISA. We found that TLR9 is expressed by respiratory epithelial cell lines and fully differentiated primary epithelial cells at low levels. Stimulation of the above-mentioned cells with bacterial DNA or CpG oligodeoxynucleotide resulted in an inflammatory reaction characterized by a dose-dependent up-regulation of cytokines (IL-6, IL-8) and human β-defensin 2. Up-regulation of NF-κB in epithelial cells in response to the CpG motif containing DNA was inhibited by overexpression of a dominant negative form of MyD88. These results provide clear evidence that the human respiratory epithelium is capable of detecting microbial DNA by TLR9. The respiratory epithelium has an important function in triggering innate immune responses and therefore represents an interesting target for anti-inflammatory therapy.

Allergic Airway Inflammation Inhibits Pulmonary Antibacterial Host Defense

The innate immune system of the lung is a multicomponent host defense system and in addition has an instructing role in regulating the quality and quantity of the adaptive immune response. When the interaction between innate and adaptive immunity is disturbed, pathological conditions such as asthma can develop. It was the aim of the study to investigate the effect of the allergic inflammation of the lung on the innate host defense during bacterial infection. Human bronchial epithelial cells were preincubated with Th2 cytokines and infected with Pseudomonas aeruginosa. The effect of the Th2 cytokines on the mRNA levels of antimicrobial peptides and the antimicrobial activity of HBEC was determined. To investigate the influence of an allergic inflammation on pulmonary host defense in vivo, mice sensitized and challenged with OVA were infected with P. aeruginosa, and the number of viable bacteria in the lungs was determined together with markers of inflammation like cytokines and antimicrobial peptides. Exposure of airway epithelial cells to Th2 cytokines resulted in a significantly decreased antimicrobial activity of the cells and in suppressed mRNA levels of the antimicrobial peptide human β-defensin 2. Furthermore, mice with allergic airway inflammation had significantly more viable bacteria in their lungs after infection. This was consistent with reduced levels of proinflammatory cytokines and of the antimicrobial peptide cathelin-related antimicrobial peptide. These results show that an allergic airway inflammation suppresses the innate antimicrobial host defense. The adaptive immune system modulates the functions of the pulmonary innate immune system.

Functions of Antimicrobial Peptides in Host Defense and Immunity

Antimicrobial peptides (AMPs) are effector molecules of the innate immune system. AMPs have a broad antimicrobial spectrum and lyse microbial cells by interaction with biomembranes. Besides their direct antimicrobial function, they have multiple roles as mediators of inflammation with impact on epithelial and inflammatory cells influencing diverse processes such as cytokine release, cell proliferation, angiogenesis, wound healing, chemotaxis, immune induction, and protease-antiprotease balance. Furthermore, AMPs qualify as prototypes of innovative drugs that may be used as antibiotics, anti-lipopolysaccharide drugs, or modifiers of inflammation. This review summarizes the current knowledge about the basic and applied biology of antimicrobial peptides and discusses features of AMPs in host defense and inflammation.

Exposure of differentiated airway epithelial cells to volatile smoke in vitro.

Background: Cigarette smoke (CS) is the predominant pathogenetic factor in the development of chronic bronchitis and chronic obstructive pulmonary disease. The knowledge about the cellular and molecular mechanisms underlying the smoke-induced inflammation in epithelial cells is limited. Objectives: The aim of this study was to develop an in vitro model to monitor the effects of volatile CS on differentiated airway epithelial cells. Methods: The airway epithelial cell line MM-39 and primary human bronchial epithelial cells were cultivated as air-liquid interface cultures and exposed directly to volatile CS. We used two types of exposure models, one using ambient air, the other using humidified and warm air. Cytokine levels were measured by quantitative PCR and ELISA. Phosphorylation of p38 MAP kinase was assessed by Western blot analysis. To reduce the smoke-induced inflammation, antisense oligonucleotides directed against the p65 subunit of NF-ĸB were applied. Results: Exposure of epithelia to cold and dry air resulted in a significant inflammatory response. In contrast, exposure to humidified warm air did not elicit a cellular response. Stimulation with CS resulted in upregulation of mRNA for IL-6 and IL-8 and protein release. Exposure to CS combined with heat-inactivated bacteria synergistically increased levels of the cytokines. Reactions of differentiated epithelial cells to smoke are mediated by the MAP kinase p38 and the transcription factor NF-ĸB. Conclusions: We developed an exposure model to examine the consequences of direct exposure of differentiated airway epithelial cells to volatile CS. The model enables to measure the cellular reactions to smoke exposure and to determine the outcome of therapeutic interventions.

The antimicrobial peptide cathelicidin interacts with airway mucus

Antimicrobial peptides (AMPs) and mucins are components of airway secretions and both contribute to the innate host defense system. At neutral pH, AMPs are positively charged, mucins negatively. It was the aim of the study to test whether these opposite charges result in interactions between AMPs and mucins. We measured binding of mucins isolated from porcine gastric mucosa to the cathelicidin LL-37 coated to multiwell plates and found that LL-37 electrostatically interacts with mucins. Circular dichroism spectra of the peptide revealed the induction of alpha-helical conformation by mucins. Addition of mucins to solutions of LL-37 significantly decreased the antimicrobial activity of the peptide against Pseudomonas aeruginosa and Streptococcus pneumoniae. We then tested whether LL-37 is bound to mucins in airway secretions from human subjects and found that a significant proportion of the peptide and its propeptide are bound to high molecular weight components. Together these data show that cationic AMPs interact with anionic mucins in airway secretions. Functions of AMPs are modulated by this interaction.

Antimicrobial Peptides in Lung Inflammation

Antimicrobial peptides (AMPs) are expressed in the respiratory tract and act as effector substances of the innate immune system. A variety of cells synthesize and secrete AMPs including epithelial and professional host defense cells such as neutrophils, macrophages, and NK cells. In the human lung, beta-defensins originate from epithelial cells, macrophages and lymphocytes. alpha-defensins are synthesized by neutrophils. LL-37/hCAP-18 is produced by epithelial cells, neutrophils, lymphocytes, and macrophages. AMPs act as endogenous antibiotics by direct destruction of microorganisms. Recently, it became clear that AMPs bind to cellular receptors and activate a variety of cell types such as airway epithelial cells, endothelial cells, mast cells, macrophages, dendritic cells, and neutrophils amongst others. Concentrations of AMPs in lung secretions are altered in several pulmonary diseases. This chapter describes the basic and applied biology of AMPs in the human lung and their potential role in pulmonary disease.

Myeloid RelA regulates pulmonary host defense networks

The pulmonary innate immune system in the respiratory tract eliminates inhaled pathogens. Several cell types contribute to host defense within a complex network. The aim of this study was to evaluate the role of macrophages during pneumonia and in the regulation of the epithelial response to microorganisms. We performed lung infection models in mice lacking myeloid RelA/p65. To study the mechanistic relationships between individual cell types, we applied co-culture models composed of airway epithelial cells (AECs) and macrophages. Mice lacking myeloid RelA/p65 showed significantly decreased bacterial clearance, cytokine expression and neutrophil influx. In addition, the induction of epithelial keratinocyte chemoattractant expression was blunted in these animals. In vitro, AECs were largely insensitive to ligands of Toll-like receptor (TLR)2 or TLR5. Exposure to secretory products of macrophages results in an increased release of pro-inflammatory cytokines and augmented antimicrobial activity. This was associated with increased expression of TLR genes and surface expression of the proteins. Experiments with blocking antibodies showed that the effect of macrophages depends on secreted mediators, including tumour necrosis factor-α. In conclusion, the present data show that myeloid RelA is critical for pulmonary host defense. One important mechanism is that macrophages induce the sensitivity of AECs to microbial patterns.

Aspergillus fumigatus conidia induce interferon-β signalling in respiratory epithelial cells

Aspergillus fumigatus is a fungal pathogen of major clinical importance. However, little is known about the role of human bronchial epithelial cells (HBECs) during A. fumigatus conidia induced inflammation. Here, we show that differentiated respiratory epithelial cells recognise inactivated resting conidia but not swollen conidia or hyphae, resulting in the induction of the interferon (IFN)-&bgr; signalling pathway and the expression of IFN-&bgr;-inducible genes, such as IFN-&ggr;-inducible protein (IP)-10. This induction was internalisation dependent. We identified double-stranded conidial RNA recognised by Toll-like receptor-3 as a factor responsible for the expression of IFN-&bgr; and IP-10. Inhibition of receptor-interacting protein-1/TANK-binding kinase-1, known to mediate IFN-&bgr; signalling, was sufficient to inhibit the induction of IFN-&bgr; and IP-10 expression by conidia. Even though conidia induced the activation of nuclear factor (NF)-&kgr;B in HBECs, IP-10 expression was only partially dependent on NF-&kgr;B signalling. These results provide evidence that respiratory cells are activated by the double-stranded RNA of resting conidia and initiate a first immune response to inhaled conidia in an IFN-&bgr;-dependent manner.

Regulation and Function of Antimicrobial Peptides in Immunity and Diseases of the Lung

Cationic antimicrobial peptides (AMPs) are among the best studied antimicrobial factors expressed in the respiratory tract. AMPs are released by epithelial cells and immune cells into the airway surface liquid covering the epithelial surfaces of the lung where they act as endogenous antibiotics. Plenty of studies showed that AMPs possess additional, often immunomodulatory functions besides their antimicrobial activities. AMPs are chemotactic for immune cells and modulate cellular mechanisms, such as proliferation of epithelial cells, epithelial regeneration, and angiogenesis. The expression and activity of AMPs are impacted by lung diseases and AMPs can have adverse effects in lung diseases. In this review, we discuss the regulation and functions of AMPs in host defense and respiratory tract diseases.

IL-17A attracts inflammatory cells in murine lung infection with P. aeruginosa

IL-17A-dependent immunity is of importance in the protection against extracellular bacterial pathogens. However, IL-17A is also suggested to mediate the pathogenesis of lung diseases, such as acute respiratory distress syndrome. Here, we studied the role of IL-17A in a mouse model of acute pneumonia. IL-17A mediated the expression of keratinocyte-derived chemokine (KC) and the recruitment of inflammatory cells in mice infected with a sub-lethal dose of Pseudomonas aeruginosa. IL-17A deficiency protected mice from lethal P. aeruginosa lung infection. A sub-lethal infection with Streptococcus pneumoniae resulted in increased bacterial burden associated with increased pulmonary inflammation. Thus, the type of infectious bacteria seemed to influence the way in which IL-17A functions during pulmonary infection. Reducing pulmonary inflammation by targeting IL-17A may be a therapeutic option in acute P. aeruginosa pneumonia.