Robert Bals

Toll-like receptor expression reveals CpG DNA as a unique microbial stimulus for plasmacytoid dendritic cells which synergizes with CD40 ligand to induce high amounts of IL-12.

Human plasmacytoid dendritic cells (DC) (PDC, CD123+) and myeloid DC (MDC, CD11c+) may be able to discriminate between distinct classes of microbial molecules based on a different pattern of Toll‐like receptor (TLR) expression. TLR1–TLR9 were examined in purified PDC and MDC. TLR9, which is critically involved in the recognition of CpG motifs in mice, was present in PDCbut not in MDC. TLR4, which is required for the response to LPS, was selectively expressed on MDC. Consistent with TLR expression, PDC were susceptible to stimulation by CpG oligodeoxynucleotide (ODN) but not by LPS, while MDC responded to LPS but not to CpG ODN. In PDC, CpG ODN supported survival, activation (CD80, CD86, CD40, MHC class II), chemokine production (IL‐8, IP‐10) and maturation (CD83). CD40 ligand (CD40L) and CpG ODN synergized to activate PDC and to stimulate the production of IFN‐α and IL‐12 including bioactive IL‐12 p70. Previous incubation of PDC with IL‐3 decreased the amount of CpG‐induced IFN‐α and shifted the cytokine response in favor of IL‐12. CpG ODN‐activated PDC showed an increased ability to stimulate proliferation of naive allogeneic CD4 T cells, butTh1 polarization of developing T cells required simultaneous activation of PDC by CD40 ligation and CpG ODN. CpG ODN‐stimulated PDC expressed CCR7, which mediates homing to lymph nodes. In conclusion, our studies reveal that IL‐12 p70 production by PDC is under strict control of two signals, an adequate exogenous microbial stimulus such as CpG ODN, and CD40L provided endogenously by activatedT cells. Thus, CpG ODN acts as an enhancer of T cell help, while T cell‐controlled restriction to foreign antigens is maintained.

An angiogenic role for the human peptide antibiotic LL-37/hCAP-18

Antimicrobial peptides are effector molecules of the innate immune system and contribute to host defense and regulation of inflammation. The human cathelicidin antimicrobial peptide LL-37/hCAP-18 is expressed in leukocytes and epithelial cells and secreted into wound and airway surface fluid. Here we show that LL-37 induces angiogenesis mediated by formyl peptide receptor-like 1 expressed on endothelial cells. Application of LL-37 resulted in neovascularization in the chorioallantoic membrane assay and in a rabbit model of hind-limb ischemia. The peptide directly activates endothelial cells, resulting in increased proliferation and formation of vessel-like structures in cultivated endothelial cells. Decreased vascularization during wound repair in mice deficient for CRAMP, the murine homologue of LL-37/hCAP-18, shows that cathelicidin-mediated angiogenesis is important for cutaneous wound neovascularization in vivo. Taken together, these findings demonstrate that LL-37/hCAP-18 is a multifunctional antimicrobial peptide with a central role in innate immunity by linking host defense and inflammation with angiogenesis and arteriogenesis.

Innate immunity in the lung: how epithelial cells fight against respiratory pathogens

The human lung is exposed to a large number of airborne pathogens as a result of the daily inhalation of 10,000 litres of air. The observation that respiratory infections are nevertheless rare is testimony to the presence of an efficient host defence system at the mucosal surface of the lung. The airway epithelium is strategically positioned at the interface with the environment, and thus plays a key role in this host defence system. Recognition systems employed by airway epithelial cells to respond to microbial exposure include the action of the toll-like receptors. The airway epithelium responds to such exposure by increasing its production of mediators such as cytokines, chemokines and antimicrobial peptides. Recent findings indicate the importance of these peptides as effector molecules of innate immunity by killing microorganisms, but also as regulators of inflammation, immunity and wound repair. Finally, the clinical relevance of the functions of the airway epithelium in innate immunity is discussed.

Human beta-defensin 4: a novel inducible peptide with a specific salt-sensitive spectrum of antimicrobial activity.

β‐Defensins are antibiotic peptides involved in host defense at the epithelial surface. Three human β‐defensins (hBDs)‐‐hBD‐1, hBD‐2, and hBD‐3‐‐have been identified so far. We have characterized a new member of the β‐defensin family, hBD‐4, based on screening of genomic sequences and subsequent functional analysis. In contrast to hBD‐1, hBD‐2, and hBD‐3, which are diffusely expressed throughout many organs, hBD‐4 mRNA expression is confined to testis, stomach, uterus, neutrophils, thyroid, lung, and kidney. hBD‐4 expression was up‐regulated by infection with gram‐positive and gram‐negative bacteria in human respiratory epithelial cells, and in response to phorbol 12‐myristate 13‐acetate, but not in response to other inflammatory factors that up‐regulate the expression of hBD‐2 or hBD‐3. Synthetic hBD‐4 exhibits a selective, salt‐sensitive spectrum of antimicrobial activity, and it represents one of the most active antimicrobial peptides against Pseudomonas aeruginosa (minimal inhibitory concentration: 4.1 μg/ml) known to date. This new defensin is chemotactic for human blood monocytes, but it is inactive on neutrophils and eosinophils. These findings demonstrate the existence of a family of β‐defensin genes with different functions against diverse classes of microorganisms, regulated by different stimuli, and specific signal pathways, and confirm the relevance of antimicrobial peptides in host defense.

Epithelial antimicrobial peptides in host defense against infection

One component of host defense at mucosal surfaces seems to be epithelium-derived antimicrobial peptides. Antimicrobial peptides are classified on the basis of their structure and amino acid motifs. Peptides of the defensin, cathelicidin, and histatin classes are found in humans. In the airways, α-defensins and the cathelicidin LL-37/hCAP-18 originate from neutrophils. β-Defensins and LL-37/hCAP-18 are produced by the respiratory epithelium and the alveolar macrophage and secreted into the airway surface fluid. Beside their direct antimicrobial function, antimicrobial peptides have multiple roles as mediators of inflammation with effects on epithelial and inflammatory cells, influencing such diverse processes as proliferation, immune induction, wound healing, cytokine release, chemotaxis, protease-antiprotease balance, and redox homeostasis. Further, antimicrobial peptides qualify as prototypes of innovative drugs that might be used as antibiotics, anti-lipopolysaccharide drugs, or modifiers of inflammation.

Identification of a novel, multifunctional beta-defensin (human beta-defensin 3) with specific antimicrobial activity. Its interaction with plasma membranes of Xenopus oocytes and the induction of macrophage chemoattraction.

Abstract. Previous studies have shown the implication of β-defensins in host defense of the human body. The human β-defensins 1 and 2 (hBD-1, hBD-2) have been isolated by biochemical methods. Here we report the identification of a third human β-defensin, called human β-defensin 3 (hBD-3; cDNA sequence, Genbank accession no. AF295370), based on bioinformatics and functional genomic analysis. Expression of hBD-3 is detected throughout epithelia of many organs and in non-epithelial tissues. In contrast to hBD-2, which is upregulated by microorganisms or tumor necrosis factor-α (TNF-α), hBD-3 expression is increased particularly after stimulation by interferon-γ. Synthetic hBD-3 exhibits a strong antimicrobial activity against gram-negative and gram-positive bacteria and fungi, including Burkholderia cepacia. In addition, hBD-3 activates monocytes and elicits ion channel activity in biomembranes, specifically in oocytes of Xenopus laevis. This paper also shows that screening of genomic sequences is a valuable tool with which to identify novel regulatory peptides. Human β-defensins represent a family of antimicrobial peptides differentially expressed in most tissues, regulated by specific mechanisms, and exerting physiological functions not only related to direct host defense.

Antimicrobial peptides: current status and therapeutic potential.

Antimicrobial peptides (AMPs) are effector molecules of the innate immune system. A variety of AMPs have been isolated from species of all kingdoms and are classified based on their structure and amino acid motifs. 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 cell proliferation, immune induction, wound healing, cytokine release, chemotaxis and protease-antiprotease balance. AMPs qualify as prototypes of innovative drugs that may be used as antimicrobials, anti-lipopolysaccharide drugs or modifiers of inflammation. Several strategies have been followed to identify lead candidates for drug development, to modify the peptides’ structures, and to produce sufficient amounts for pre-clinical and clinical studies. This review summarises the current knowledge about the basic and applied biology of AMPs.

Differential Recognition of TLR-Dependent Microbial Ligands in Human Bronchial Epithelial Cells

Bronchial epithelial cells represent the first line of defense against invading airborne pathogens. They are important contributors to innate mucosal immunity and provide a variety of antimicrobial effectors. However, mucosal surfaces are prone to contact with pathogenic, as well as nonpathogenic microbes, and therefore, immune recognition principles have to be tightly controlled to avoid uncontrolled permanent activation. TLRs have been shown to recognize conserved microbial patterns and to mediate inducible activation of innate immunity. Our experiments demonstrate that bronchial epithelial cells express functional TLR1–6 and TLR9 and thus make use of a common principle of professional innate immune cells. Although it was observed that TLR2 ligands dependent on heterodimeric signaling either with TLR1 or TLR6 were functional, other ligands like lipoteichoic acid were not. Additionally, it was found that bronchial epithelial cells could be stimulated only marginally by Gram-positive bacteria bearing known TLR2 ligands while Gram-negative bacteria were easily recognized. This correlated with low expression of TLR2 and the missing expression of the coreceptor CD36. Transgenic expression of both receptors restored responsiveness to the complete set of TLR2 ligands and Staphylococcus aureus. Additional gene-array experiments confirmed hyporesponsiveness to this bacterium while Pseudomonas aeruginosa and respiratory syncytial virus induced common, as well as pathogen-specific, sets of genes. The findings indicate that bronchial epithelium regulates its sensitivity to recognize microbes by managing receptor expression levels. This could serve the special needs of controlled microbial recognition in mucosal compartments.

The discovery of α1-antitrypsin and its role in health and disease

α1-Antitrypsin (AAT) is the archetype member of the serine protease inhibitor (SERPIN) supergene family. The AAT deficiency is most often associated with the Z mutation, which results in abnormal Z AAT folding in the endoplasmic reticulum of hepatocytes during biogenesis. This causes intra-cellular retention of the AAT protein rather than efficient secretion with consequent deficiency of circulating AAT. The reduced serum levels of AAT contribute to the development of chronic obstructive pulmonary disease (COPD) and the accumulation of abnormally folded AAT protein increases risk for liver diseases. In this review we show that with the discovery of AAT deficiency in the early 60s as a genetically determined predisposition to the development of early-onset emphysema, intensive investigations of enzymatic mechanisms that produce lung destruction in COPD were pursued. To date, the role of AAT in other than lung and liver diseases has not been extensively examined. Current findings provide new evidence that, in addition to protease inhibition, AAT expresses anti-inflammatory, immunomodulatory and antimicrobial properties, and highlight the importance of this protein in health and diseases. In this review co-occurrence of several diseases with AAT deficiency is discussed.

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.