Martin Mempel

Various members of the Toll-like receptor family contribute to the innate immune response of human epidermal keratinocytes

Toll‐like receptors (TLRs) are important pattern recognition molecules that activate the nuclear factor (NF)‐κB pathway leading to the production of antimicrobial immune mediators. As keratinocytes represent the first barrier against exogenous pathogens in human skin, we investigated their complete functional TLR1–10 expression profile. First, reverse transcription–polymerase chain reaction (PCR) analysis revealed a very similar pattern of TLR mRNA expression when comparing freshly isolated human epidermis and cultured primary human keratinocytes. Thus, further experiments were carried out with primary keratinocytes in comparison with the spontaneously immortalized human keratinocyte cell line HaCaT. The quantitative expression of TLR1–10 mRNA in real‐time PCR of primary human keratinocytes and HaCaT cells was analysed. Both cell types constitutively expressed TLR2, TLR3, TLR5, and to a lesser extent TLR10. TLR4 was only found in HaCaT cells, TLR1 to a higher degree in primary keratinocytes. In line with this, LPS induced mRNA expression of CD14 and TLR4 only in HaCaT cells. After stimulation with various TLR ligands, the NF‐κB‐activated chemokine interleukin‐8 (IL‐8) was measured. In primary keratinocytes and HaCaT cells the TLR3 ligand poly (I:C) was the most potent stimulator of IL‐8 secretion. The TLR ligands peptidoglycan, Pam3Cys and flagellin which bind to TLR2, TLR1/TLR2 heterodimer, and TLR5, respectively, also induced IL‐8 secretion, whereas no IL‐8 was induced by LPS, R‐848, loxoribine and cytosine guanine dinucleotide‐containing oligodeoxynucleotide. A corresponding pattern was found in the RelA NF‐κB translocation assay after ligand stimulation of primary keratinocytes. These studies provide substantial evidence for a functional TLR expression and signalling profile of normal human keratinocytes contributing to the antimicrobial defence barrier of human skin.

Genome-Wide Scan on Total Serum IgE Levels Identifies FCER1A as Novel Susceptibility Locus

High levels of serum IgE are considered markers of parasite and helminth exposure. In addition, they are associated with allergic disorders, play a key role in anti-tumoral defence, and are crucial mediators of autoimmune diseases. Total IgE is a strongly heritable trait. In a genome-wide association study (GWAS), we tested 353,569 SNPs for association with serum IgE levels in 1,530 individuals from the population-based KORA S3/F3 study. Replication was performed in four independent population-based study samples (total n = 9,769 individuals). Functional variants in the gene encoding the alpha chain of the high affinity receptor for IgE (FCER1A) on chromosome 1q23 (rs2251746 and rs2427837) were strongly associated with total IgE levels in all cohorts with P values of 1.85×10−20 and 7.08×10−19 in a combined analysis, and in a post-hoc analysis showed additional associations with allergic sensitization (P = 7.78×10−4 and P = 1.95×10−3). The “top” SNP significantly influenced the cell surface expression of FCER1A on basophils, and genome-wide expression profiles indicated an interesting novel regulatory mechanism of FCER1A expression via GATA-2. Polymorphisms within the RAD50 gene on chromosome 5q31 were consistently associated with IgE levels (P values 6.28×10−7−4.46×10−8) and increased the risk for atopic eczema and asthma. Furthermore, STAT6 was confirmed as susceptibility locus modulating IgE levels. In this first GWAS on total IgE FCER1A was identified and replicated as new susceptibility locus at which common genetic variation influences serum IgE levels. In addition, variants within the RAD50 gene might represent additional factors within cytokine gene cluster on chromosome 5q31, emphasizing the need for further investigations in this intriguing region. Our data furthermore confirm association of STAT6 variation with serum IgE levels.

Stratum corneum lipids, skin barrier function and filaggrin mutations in patients with atopic eczema

To cite this article: Jungersted JM, Scheer H, Mempel M, Baurecht H, Cifuentes L, Høgh JK, Hellgren LI, Jemec GBE, Agner T, Weidinger S. Stratum corneum lipids, skin barrier function and filaggrin mutations in patients with atopic eczema. Allergy 2010; 65: 911–918.

Antigen-specific T regulatory-1 cells are associated with immunosuppression in a chronic helminth infection (onchocerciasis).

Different mechanisms underlie the phenomenon of peripheral tolerance. Recently, a new subset of CD4+ T cells, called T regulatory-1 (Tr1) cells, was described which show suppressor functions in vitro and in vivo and are characterized by a predominant production of IL-10 and/or TGF-beta. Tr1 cells have so far been generated experimentally in an IL-10-rich environment and hold promise for exploitation in the suppression of alloreactions and inflammatory or allergic dispositions. However, these cells have not been characterized in infectious diseases. Here we show that in the chronic helminth infection onchocerciasis (river blindness), where patients have relatively little sign of dermatitis despite the presence of millions of small worms in the skin, T cells can be obtained which bear characteristics of Tr1 cells, producing no IL-2 or IL-4 but substantial amounts of IL-10, variable amounts of IL-5, and some IFN-gamma. These cells display elevated amounts of CTLA-4 after stimulation and are able to inhibit other T cells in coculture, in contrast to Th1 and Th2 clones. This is the first time that this type of suppressor T cell has been cloned as naturally occurring during an infectious disease.

Gender difference, sex hormones, and immediate type hypersensitivity reactions

Gender differences in the development and prevalence of human diseases have long been recognized. Immense interest grows in the understanding of the role of sex hormones in the homeostasis of immunity. Asthma predominates in boys before puberty and this gender preference reverses after puberty and in adulthood, when adult women tend to have a more severe disease, often recalcitrant to treatment. Atopic eczema in preschool children shows insignificant gender difference or male preponderance in different studies, with more adult females suffering from atopic eczema. The limited data on the prevalence of immediate hypersensitivity to hymenoptera venom show controversial results. Discrepancy exists regarding the gender difference in food allergy, with females reporting significantly more allergic reactions in questionnaire studies. In general, adverse reactions to nonionic iodinated radiocontrast media are more commonly observed in females. The course of allergic diseases varies unpredictably during pregnancy, whereas hormone replacement therapy in postmenopausal women usually has a favorable influence on the course of asthma. Experiments in rodents confirm an effect of estrogens on mast cell activation and allergic sensitization, while progesterone is shown to suppress histamine release but potentiate IgE induction. Dehydroepiandrosterone may antagonize the production of Th2 cytokines but the effect of testosterone and the other androgens remains less defined. Actual data from human studies are lacking.

Double-Stranded RNA Induces an Antiviral Defense Status in Epidermal Keratinocytes through TLR3-, PKR-, and MDA5/RIG-I-Mediated Differential Signaling

Emerging evidence suggests an important role for human epidermal keratinocytes in innate immune mechanisms against bacterial and viral skin infections. The proinflammatory effect of viral infections can be mimicked by double-stranded RNA (dsRNA). Herein, we demonstrate that keratinocytes express all known dsRNA sensing receptors at a constitutive and inducible level, and that they use several downstream signaling pathways leading to a broad pattern of gene expression, not only proinflammatory and immune response genes under the control of NF-κB, but also genes under transcriptional control of IRF3. As a consequence, dsRNA, a stimulus for TLR3, protein kinase R (PKR), and the RNA helicases retinoic acid-inducible gene I (RIG-I) and MDA5, induces a status of antiviral defense in keratinocytes. Using inhibitors for the various dsRNA signaling pathways and specific small interfering RNA for TLR3, RIG-I, and MDA5, we demonstrated that in human keratinocytes, TLR3 seems to be necessary for NF-κB but not for IRF3 activation, whereas RIG-I and MDA5 are crucial for IRF3 activation. PKR is essential for the dsRNA response in both signaling pathways and thus represents the central antiviral receptor for dsRNA stimulation. Moreover, human keratinocytes up-regulate TLR7, the receptor for single-stranded RNA, in response to stimulation with dsRNA, which renders keratinocytes functionally responsive to the TLR7 agonist gardiquimod, a member of the imidazoquinoline antiviral immune response modifier family. Thus, in addition to building a physical barrier against infectious pathogens, keratinocytes are specially equipped with a full antiviral defense program that enables them to efficiently target viral infections of the skin.

Mutual Antagonism of T Cells Causing Psoriasis and Atopic Eczema

The simultaneous occurrence of psoriasis driven by type 1 helper T (Th1) cells and type 17 helper T (Th17) cells and atopic eczema dominated by type 2 helper T (Th2) cells is rare. Here, we describe three patients with co-occurring psoriasis and atopic eczema with an antagonistic course and distinct T-cell infiltrates in lesions from psoriasis and those from atopic eczema. Sensitized patients with psoriasis had a reaction to epicutaneous allergen challenge, with clinically and histologically verified eczema lesions containing a large number of allergen-reactive T cells. These findings support a causative role for T cells triggered by specific antigens in both psoriasis and atopic eczema. (Supported by the German Research Foundation and others.)

Invasion of human keratinocytes by Staphylococcus aureus and intracellular bacterial persistence represent haemolysin-independent virulence mechanisms that are followed by features of necrotic and apoptotic keratinocyte cell death.

Summary Background Colonization of human skin by Staphylococcus aureus is a characteristic feature of several inflammatory skin diseases, which is often followed by tissue invasion and severe cell damage. A crucial role has been attributed to staphylococcal haemolysins in the cytotoxicity to epidermal structures.

Silver-Coated Textiles Reduce Staphylococcus aureus Colonization in Patients with Atopic Eczema

Background: In atopic eczema (AE), skin colonization with Staphylococcus aureus is known to play a major triggering and possibly pathophysiological role. Methods: In this open-labeled controlled side-to-side comparative trial, affected sites (flexures of both elbows) in 15 patients diagnosed as having generalized or localized AE were evaluated regarding S. aureus colonization and clinical severity of AE over a 2-week period. Flexures of the elbows were covered with silver-coated textiles on one arm and cotton on the other for 7 days followed by a 7-day control period. Results: A highly significant decrease in S. aureus colonization could be seen on the site covered by the silver-coated textile already 2 days after initiation lasting until the end of the treatment. Seven days after cessation, S. aureus density remained significantly lower compared to baseline. In addition, significantly lower numbers of S. aureus were observed on the silver-coated textile site in comparison to cotton at the end of treatment as well as at the time point of control. Clinical improvement correlated with the reduction of S. aureus colonization. Conclusion: A superior improvement achieved by silver-coated compared to cotton textiles paralleled a potent anti-S.-aureus effect.

IL-22 and TNF-α represent a key cytokine combination for epidermal integrity during infection with Candida albicans

T cells exercise their full impact on target cells through a combination of secreted cytokines. The recently described T helper cell subset Th22 is characterized by a combinatorial secretion of IL‐22 and TNF‐α. Here, we demonstrate that IL‐22 increases the TNF‐α‐dependent induction and secretion of several immune‐modulatory molecules such as initial complement factors C1r and C1s, antimicrobial peptides S100A7 and HBD‐2 (human β defensin 2), and antimicrobial chemokines CXCL‐9/‐10/‐11 in primary human keratinocytes. The synergism of IL‐22 and TNF‐α is transmitted intracellularly by MAP kinases and downstream by transcription factors of the AP‐1 family. The induction of innate immunity is relevant in an in vitro infection model, where keratinocytes stimulated with Th22 supernatants or recombinant IL‐22 plus TNF‐α effectively inhibit the growth of Candida albicans and maintain survival of epithelia. Accordingly, the combinatorial stimulation of keratinocytes with IL‐22 and TNF‐α most efficiently conserves the integrity of the epidermal barrier in a three‐dimensional skin infection model as compared with IFN‐γ, IL‐17, IL‐22 or TNF‐α alone. In summary, we demonstrate that IL‐22 and TNF‐α represent a potent, synergistic cytokine combination for cutaneous immunity.