Martin Hafner

TNF-mediated inflammatory skin disease in mice with epidermis-specific deletion of IKK2

The IκB kinase (IKK), consisting of the IKK1 and IKK2 catalytic subunits and the NEMO (also known as IKKγ) regulatory subunit, phosphorylates IκB proteins, targeting them for degradation and thus inducing activation of NF-κB (reviewed in refs 1, 2). IKK2 and NEMO are necessary for NF-κB activation through pro-inflammatory signals. IKK1 seems to be dispensable for this function but controls epidermal differentiation independently of NF-κB. Previous studies suggested that NF-κB has a function in the growth regulation of epidermal keratinocytes. Mice lacking RelB or IκBα, as well as both mice and humans with heterozygous NEMO mutations, develop skin lesions. However, the function of NF-κB in the epidermis remains unclear. Here we used Cre/loxP-mediated gene targeting to investigate the function of IKK2 specifically in epidermal keratinocytes. IKK2 deficiency inhibits NF-κB activation, but does not lead to cell-autonomous hyperproliferation or impaired differentiation of keratinocytes. Mice with epidermis-specific deletion of IKK2 develop a severe inflammatory skin disease, which is caused by a tumour necrosis factor-mediated, αβ T-cell-independent inflammatory response that develops in the skin shortly after birth. Our results suggest that the critical function of IKK2-mediated NF-κB activity in epidermal keratinocytes is to regulate mechanisms that maintain the immune homeostasis of the skin.

Integrin alpha 2-deficient mice develop normally, are fertile, but display partially defective platelet interaction with collagen.

The integrin α2-subunit was ablated in mice by targeted deletion of the ITGA2 gene. α2-Deficient animals develop normally, are fertile, and reproduce. Surprisingly, no obvious anatomical or histological differences were observed in mutant mice. Besides its significance in tissue morphogenesis, integrin α2β1 has been reported to play a major role in hemostasis by mediating platelet adhesion and activation on subendothelial collagen. To define its role in hemostasis, α2-deficient platelets were analyzed for their capacity to adhere to and aggregate in response to fibrillar or soluble collagen type I. We show that aggregation of α2-deficient platelets to fibrillar collagen is delayed but not reduced, whereas aggregation to enzymatically digested soluble collagen is abolished. Furthermore, α2-deficient platelets normally adhere to fibrillar collagen. However, in the presence of an antibody against GPVI (activating platelet collagen receptor), adhesion of α2-deficient but not wild type platelets is abrogated. These results demonstrate that integrin α2β1 significantly contributes to platelet adhesion to (fibrillar) collagen, which is further confirmed by the abolished adhesion of α2-deficient platelets to soluble collagen. Thus, α2β1 plays a supportive rather than an essential role in platelet-collagen interactions. These results are in agreement with the observation that α2β1-deficient animals suffer no bleeding anomalies.

Novel reporter mouse reveals constitutive and inflammatory expression of IFN-beta in vivo.

Type I IFN is a major player in innate and adaptive immune responses. Besides, it is involved in organogenesis and tumor development. Generally, IFN responses are amplified by an autocrine loop with IFN-β as the priming cytokine. However, due to the lack of sensitive detection systems, where and how type I IFN is produced in vivo is still poorly understood. In this study, we describe a luciferase reporter mouse, which allows tracking of IFN-β gene induction in vivo. Using this reporter mouse, we reveal strong tissue-specific induction of IFN-β following infection with influenza or La Crosse virus. Importantly, this reporter mouse also allowed us to visualize that IFN-β is expressed constitutively in several tissues. As suggested before, low amounts of constitutively produced IFN might maintain immune cells in an activated state ready for a timely response to pathogens. Interestingly, thymic epithelial cells were the major source of IFN-β under noninflammatory conditions. This relatively high constitutive expression was controlled by the NF Aire and might influence induction of tolerance or T cell development.

Sequence and Characterization of the Ig Heavy Chain Constant and Partial Variable Region of the Mouse Strain 129S1

Although the entire mouse genome has been sequenced, there remain challenges concerning the elucidation of particular complex and polymorphic genomic loci. In the murine Igh locus, different haplotypes exist in different inbred mouse strains. For example, the Ighb haplotype sequence of the Mouse Genome Project strain C57BL/6 differs considerably from the Igha haplotype of BALB/c, which has been widely used in the analyses of Ab responses. We have sequenced and annotated the 3′ half of the Igha locus of 129S1/SvImJ, covering the CH region and approximately half of the VH region. This sequence comprises 128 VH genes, of which 49 are judged to be functional. The comparison of the Igha sequence with the homologous Ighb region from C57BL/6 revealed two major expansions in the germline repertoire of Igha. In addition, we found smaller haplotype-specific differences like the duplication of five VH genes in the Igha locus. We generated a VH allele table by comparing the individual VH genes of both haplotypes. Surprisingly, the number and position of DH genes in the 129S1 strain differs not only from the sequence of C57BL/6 but also from the map published for BALB/c. Taken together, the contiguous genomic sequence of the 3′ part of the Igha locus allows a detailed view of the recent evolution of this highly dynamic locus in the mouse.

Synthetic Mimetics of the gp130 Binding Site for Viral Interleukin-6 as Inhibitors of the vIL-6-gp130 Interaction

The transmembrane protein gp130 acts as the signal transducing receptor subunit for interleukin‐6 type cytokines, including viral interleukin‐6, which is encoded by the Kaposi’s sarcoma‐associated herpes virus. Viral interleukin‐6 has been shown to mimic human IL‐6 functions, including activation of the JAK1 and STAT1/3 signaling pathways. Based on the crystal structure of three extracellular domains of gp130 in complex with viral interleukin‐6, we have designed and synthesized a range of assembled peptides that mimic the sequentially discontinuous binding site of gp130 for viral interleukin‐6. These peptides, which present the three binding site fragments of gp130 in a nonlinear, discontinuous fashion, were shown to inhibit the interaction of gp130 with viral interleukin‐6, as well as the stimulation of viral interleukin‐6‐induced cell proliferation. These results validate the concept of synthetic mimicry of discontinuous protein‐binding sites through assembled peptides, and the use of such molecules as modulators of protein–ligand interactions.

Das humane Genomprojekt: Folgen für die Dermatologie

Erkrankungen des Menschen konnen entweder durch den Kontakt des Organismus mit exogenen Faktoren (pathogene Keime, Allergene, Kanzerogene) verursacht oder erblich bedingt sein. Die Ursachen solcher angeborenen Erkrankungen sind dann in den Genomen der betroffenen Patienten zu suchen (Abb. 1). Neben vielen anderen konnten in den letzten Jahren auch zahlreiche Mutationen identifiziert werden, die ursachlich fur eine Reihe von genetisch bedingten Erkrankungen der Haut verantwortlich sind, zum Beispiel palmoplantare Hyperkeratosen, Neurofibromatose und Epidermolysis bullosa. Komplexer als diese monogenetischen Erkrankungen, bei denen die Mutationen jeweils nur ein Gen betreffen, sind polygenetische Erkrankungen, die auf Defekten in mehreren Genen beruhen, und Krankheiten, die auf genetische Pradispositionen der betroffenen Patienten zuruckzufuhren sind. Letztere werden unter anderem als ursachlich fur die Pathogenese von Krankheiten wie der Psoriasis oder Autoimmunerkrankungen angesehen. Wie genetische Erkrankungen im engeren Sinne beruhen genetische Pradispositonen auf dem Besitz bestimmter Allele, die in den Genomen nicht erkrankender Personen nicht vorkommen. Im Gegensatz zu den erstgenannten be dingen genetische Pradispositionen jedoch nicht zwangslaufig ein Erkranken des Merkmalstragers, da zur Auslosung einer Erkrankung weitere im Lauf des Lebens erworbene Mutationen oder exogene pathogene Stimuli notwendig sind.