Thilo Jakob

Genome-wide, large-scale production of mutant mice by ENU mutagenesis

In the post-genome era, the mouse will have a major role as a model system for functional genome analysis. This requires a large number of mutants similar to the collections available from other model organisms such as Drosophila melanogaster and Caenorhabditis elegans. Here we report on a systematic, genome-wide, mutagenesis screen in mice. As part of the German Human Genome Project, we have undertaken a large-scale ENU-mutagenesis screen for dominant mutations and a limited screen for recessive mutations. In screening over 14,000 mice for a large number of clinically relevant parameters, we recovered 182 mouse mutants for a variety of phenotypes. In addition, 247 variant mouse mutants are currently in genetic confirmation testing and will result in additional new mutant lines. This mutagenesis screen, along with the screen described in the accompanying paper, leads to a significant increase in the number of mouse models available to the scientific community. Our mutant lines are freely accessible to non-commercial users (for information, see http://www.gsf.de/ieg/groups/enu-mouse.html).

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.

Omalizumab in patients with symptomatic chronic idiopathic/spontaneous urticaria despite standard combination therapy

BACKGROUND Patients with chronic idiopathic urticaria/chronic spontaneous urticaria (CIU/CSU) often continue to experience symptoms despite receiving standard-of-care therapy with H1-antihistamines along with 1 or more add-on therapies. OBJECTIVES We sought to evaluate the safety and efficacy of 24 weeks of treatment with omalizumab in patients with persistent CIU/CSU despite treatment with H₁-antihistamines at up to 4 times the approved dose plus H₂-antihistamines, leukotriene receptor antagonists, or both. METHODS In this phase III study patients were randomized to receive 6 subcutaneous injections at 4-week intervals of either 300 mg of omalizumab or placebo, followed by a 16-week observation period. The primary objective of the study was to evaluate the overall safety of omalizumab compared with placebo. Efficacy (itch severity, hive, and urticaria activity scores) was evaluated at weeks 12 and 24. RESULTS The overall incidence and severity of adverse events and serious adverse events were similar between omalizumab and placebo recipients; the safety profile was consistent with omalizumab in patients with allergic asthma. At week 12, the mean change from baseline in weekly itch severity score was -8.6 (95% CI, -9.3 to -7.8) in the omalizumab group compared with -4.0 (95% CI, -5.3 to -2.7) in the placebo group (P < .001). Significant improvements were seen for additional efficacy end points at week 12; these benefits were sustained to week 24. CONCLUSION Omalizumab was well tolerated and reduced the signs and symptoms of CIU/CSU in patients who remained symptomatic despite the use of H₁-antihistamines (up to 4 times the approved dose) plus H₂-antihistamines, leukotriene receptor antagonists, or both.

Pollen-associated phytoprostanes inhibit dendritic cell interleukin-12 production and augment T helper type 2 cell polarization

Pollen grains induce allergies in susceptible individuals by release of allergens upon contact with mucosal membranes of the upper respiratory tract. We recently demonstrated that pollen not only function as allergen carriers but also as rich sources of bioactive lipids that attract cells involved in allergic inflammation such as neutrophils and eosinophils. Here we demonstrate that soluble factors from birch (Betula alba L.) pollen activate human dendritic cells (DCs) as documented by phenotypical and functional maturation and altered cytokine production. Betula alba L. aqueous pollen extracts (Bet.-APE) selectively inhibited interleukin (IL)-12 p70 production of lipopolysaccharide (LPS)- or CD40L-activated DC, whereas IL-6, IL-10, and TNFα remained unchanged. Presence of Bet.-APE during DC activation resulted in DC with increased T helper type 2 (Th2) cell and reduced Th1 cell polarizing capacity. Chemical analysis of Bet.-APE revealed the presence of phytoprostanes (dinor isoprostanes) with prostaglandin E1-, F1-, A1-, or B1-ring systems of which only E1-phytoprostanes dose dependently inhibited the LPS-induced IL-12 p70 release and augmented the Th2 cell polarizing capacity of DC. These results suggest that pollen-derived E1-phytoprostanes not only resemble endogenous prostaglandin E2 structurally but also functionally in that they act as regulators that modulate human DC function in a fashion that favors Th2 cell polarization.

Mechanisms of chemical-induced innate immunity in allergic contact dermatitis

To cite this article:  Martin SF, Esser PR, Weber FC, Jakob T, Freudenberg MA, Schmidt M, Goebeler M. Invited review for the journal allergy mechanisms of chemical‐induced innate immunity in allergic contact dermatitis. Allergy 2011; 66: 1152–1163.

Toll-like receptor and IL-12 signaling control susceptibility to contact hypersensitivity

Allergic contact hypersensitivity (CHS) is a T cell–mediated inflammatory skin disease. Interleukin (IL)-12 is considered to be important in the generation of the allergen-specific T cell response. Loss of IL-12 function in IL-12Rβ2–deficient mice, however, did not ameliorate the allergic immune response, suggesting alternate IL-12–independent pathways in the induction of CHS. Because exposure to contact allergens always takes place in the presence of microbial skin flora, we investigated the potential role of Toll-like receptors (TLRs) in the induction of CHS. Using mice deficient in TLR4, the receptor for bacterial lipopolysaccharide (LPS), IL-12 receptor (R) β2, or both, we show that the concomitant absence of TLR4 and IL-12Rβ2, but not the absence of TLR4 or IL-12Rβ2 alone, prevented DC-mediated sensitization, generation of effector T cells, and the subsequent CHS response to 2,4,6-trinitro-1-chlorobenzene (TNCB), oxazolone, and fluorescein isothiocyanate. Introduction of the TLR4 transgene into the TLR4/IL-12Rβ2 mutant restored the CHS inducibility, showing a requirement for TLR4 in IL-12–independent CHS induction. Furthermore, the concomitant absence of TLR2 and TLR4 prevented the induction of CHS to TNCB in IL-12–competent mice. Finally, CHS was inducible in germ-free wild-type and IL-12Rβ2–deficient mice, but not in germ-free TLR4/IL-12Rβ2 double deficient mice, suggesting that the necessary TLR activation may proceed via endogenous ligands.

Lack of the purinergic receptor P2X7 results in resistance to contact hypersensitivity

Engagement of P2X7 on mouse dendritic cells, presumably by ATP released in response to contact allergen, is needed for IL-1β production and the sensitization phase of contact hypersensitivity.

Health Effects of Ambient Particulate Matter—Biological Mechanisms and Inflammatory Responses to In Vitro and In Vivo Particle Exposures

In this article, we review and analyze different modes of exposure to ultrafine particles in order to assess particle-induced inflammatory responses and the underlying mechanisms in vitro and in vivo. Based on results from monocytic cells cultured under submerged conditions, we discuss (1) the impact of particle properties such as surface area and oxidative potential on lipid metabolism as a highly sensitive regulatory pathway and (2) the interference of diesel exhaust particles with toll-like receptor-mediated inflammatory responses. Furthermore, new developments of air–liquid interface exposure used as an alternative approach to simulate cell particle interactions are presented. In addition to the in vitro approaches, animal exposure studies are described that apply selected mouse models to elucidate potential allergic and inflammatory pulmonary responses and mast-cell-related mechanisms after particle exposure. Long-term inhalation of ultrafine particles might lead to irreversible changes in lung structure and function. Clinical studies addressing the characteristics of inflammatory airway cells are a promising approach to understand underlying pathophysiological mechanisms in chronic obstructive pulmonary disease. Finally, a potential outcome of human particle exposure is chronic cough in children. Here, discrimination between asthmatic and nonasthmatic cough by means of immunological parameters appears to be an important step toward improving diagnosis and therapy.

Bacterial DNA and CpG-containing oligodeoxynucleotides activate cutaneous dendritic cells and induce IL-12 production : Implications for the augmentation of Th1 responses

Background: Unmethylated CpG sequences in bacterial DNA act as adjuvants selectively inducing Th1 predominant immune responses during genetic vaccination or when used in conjunction with protein Ag. The precise mechanism of this adjuvant effect is unknown. Because dendritic cells (DC) are thought to be crucially involved in T cell priming and Th1/Th2 education during vaccination via skin, we characterized the effects of bacterial DNA and CpG–containing oligodeoxynucleotides (CpG ODN) on cutaneous DC. Methods and Results: Stimulation with CpG ODN 1826 (6 μg/ml) induced activation of immature Langerhans cell (LC)–like DC as determined by an increased expression of MHC class II and costimulatory molecules, loss of E–cadherin–mediated adhesion and increased ability to stimulate allogeneic T cells. Composition–matched control ODN 1911 lacking CpG sequences at equal concentrations was without effect. In comparison to LPS and ODN 1911, CpG ODN 1826 selectively stimulated DC to release large amounts of IL–12 (p40) and little IL–6 or TNF–α within 18 h and detectable levels of IL–12 p70 within 72 h. Stimulation with Escherichia coli DNA, but not calf thymus DNA, similarly induced DC maturation and IL–12 p40 production. Injection of CpG ODN into murine dermis induced enhanced expression of MHC class II and CD86 by LC in the overlying epidermis and intracytoplasmic IL–12 p40 accumulation in a subpopulation of activated LC. Conclusion: Bacterial DNA and CpG ODN stimulate DC in vitro and in vivo and may preferentially elicit Th1–predominant immune responses because they can activate and mobilize DC, inducing them to produce IL–12.

A Potential Role for P2X7R in Allergic Airway Inflammation in Mice and Humans

P2X₇R deficiency is associated with a less severe outcome in acute and chronic inflammatory disorders. Recently, we demonstrated that extracellular adenosine triphosphate is involved in the pathogenesis of asthma by modulating the function of dendritic cells (DCs). However, the role of the purinergic receptor subtype P2X₇ is unknown. To elucidate the role of P2X₇R in allergic airway inflammation (AAI) in vitro and in vivo, P2X₇R expression was measured in lung tissue and immune cells of mice or in humans with allergic asthma. By using a specific P2X₇R-antagonist and P2X₇R-deficient animals, the role of this receptor in acute and chronic experimental asthma was explored. P2X₇R was found to be up-regulated during acute and chronic asthmatic airway inflammation in mice and humans. In vivo experiments revealed the functional relevance of this finding because selective P2X₇R inhibition or P2X₇R deficiency was associated with reduced features of acute and chronic asthma in the ovalbumin-alum or HDM model of AAI. Experiments with bone marrow chimeras emphasized that P2X₇R expression on hematopoietic cells is responsible for the proasthmatic effects of P2X₇R signaling. In the DC-driven model of AAI, P2X₇R-deficient DCs showed a reduced capacity to induce Th2 immunity in vivo. Up-regulation of P2X₇R on BAL macrophages and blood eosinophils could be observed in patients with chronic asthma. Our data suggest that targeting P2X₇R on hematopoietic cells (e.g., DCs or eosinophils) might be a new therapeutic option for the treatment of asthma.