Anja Fritsch

A hypomorphic mouse model of dystrophic epidermolysis bullosa reveals mechanisms of disease and response to fibroblast therapy

Dystrophic epidermolysis bullosa (DEB) is a severe skin fragility disorder associated with trauma-induced blistering, progressive soft tissue scarring, and increased risk of skin cancer. DEB is caused by mutations in type VII collagen. In this study, we describe the generation of a collagen VII hypomorphic mouse that serves as an immunocompetent animal model for DEB. These mice expressed collagen VII at about 10% of normal levels, and their phenotype closely resembled characteristics of severe human DEB, including mucocutaneous blistering, nail dystrophy, and mitten deformities of the extremities. The oral blistering experienced by these mice resulted in growth retardation, and repeated blistering led to excessive induction of tissue repair, causing TGF-beta1-mediated contractile fibrosis generated by myofibroblasts and pseudosyndactyly in the extremities. Intradermal injection of WT fibroblasts resulted in neodeposition of collagen VII and functional restoration of the dermal-epidermal junction. Treated areas were also resistant to induced frictional stress. In contrast, untreated areas of the same mouse showed dermal-epidermal separation following induced stress. These data demonstrate that fibroblast-based treatment can be used to treat DEB in a mouse model and suggest that this approach may be effective in the development of clinical therapeutic regimens for patients with DEB.

Supramolecular Interactions in the Dermo-epidermal Junction Zone: ANCHORING FIBRIL-COLLAGEN VII TIGHTLY BINDS TO BANDED COLLAGEN FIBRILS*

The dermis and the epidermis of normal human skin are functionally separated by a basement membrane but, together, form a stable structural continuum. Anchoring fibrils reinforce this connection by insertion into the basement membrane and by intercalation with banded collagen fibrils of the papillary dermis. Structural abnormalities in collagen VII, the major molecular constituent of anchoring fibrils, lead to a congenital skin fragility condition, dystrophic epidermolysis bullosa, associated with skin blistering. Here, we characterized the molecular basis of the interactions between anchoring fibrils and banded collagen fibrils. Suprastructural fragments of the dermo-epidermal junction zone were generated by mechanical disruption and by separation with magnetic Immunobeads. Anchoring fibrils were tightly attached to banded collagen fibrils. In vitro binding studies demonstrated that a von Willebrand factor A-like motif in collagen VII was essential for binding of anchoring fibrils to reconstituted collagen I fibrils. Since collagen I and VII molecules reportedly undergo only weak interactions, the attachment of anchoring fibrils to collagen fibrils depends on supramolecular organization of their constituents. This complex is stabilized in situ and resists dissociation by strong denaturants.

PI3Kβ Plays a Critical Role in Neutrophil Activation by Immune Complexes

The β isoform of phosphoinositide 3-kinase may be an effective therapeutic target in inflammatory diseases. The Integrating Isoform The class I phosphoinositide 3-kinases (PI3Ks) are implicated in processes such as growth factor signaling and inflammation. PI3Kγ is activated by G protein–coupled receptors (GPCRs), whereas PI3Kα and PI3Kδ are activated by protein tyrosine kinase–coupled receptors. PI3Kβ is unusual in that it appears to respond to signals from both types of receptors, depending on the cellular context. Kulkarni et al. investigated the responses of mouse neutrophils to immune complexes of antibody and antigen, which trigger chronic inflammation in conditions such as autoimmune arthritis. Genetic and pharmacological evidence suggested that immune complexes stimulated PI3Kβ in a process involving activation of FcγR, a tyrosine kinase–coupled low-affinity antibody receptor, and autocrine signaling by a proinflammatory lipid (LTB4) through its GPCR. Mice deficient in PI3Kβ fared better than did controls in models of arthritis and inflammatory skin disease. These data implicate PI3Kβ in the integration of signals from tyrosine kinase–coupled receptors and GPCRs—and suggest that this isoform may be an effective therapeutic target in inflammatory diseases. Neutrophils are activated by immunoglobulin G (IgG)–containing immune complexes through receptors that recognize the Fc portion of IgG (FcγRs). Here, we used genetic and pharmacological approaches to define a selective role for the β isoform of phosphoinositide 3-kinase (PI3Kβ) in FcγR-dependent activation of mouse neutrophils by immune complexes of IgG and antigen immobilized on a plate surface. At low concentrations of immune complexes, loss of PI3Kβ alone substantially inhibited the production of reactive oxygen species (ROS) by neutrophils, whereas at higher doses, similar suppression of ROS production was achieved only by targeting both PI3Kβ and PI3Kδ, suggesting that this pathway displays stimulus strength–dependent redundancy. Activation of PI3Kβ by immune complexes involved cooperation between FcγRs and BLT1, the receptor for the endogenous proinflammatory lipid leukotriene B4. Coincident activation by a tyrosine kinase–coupled receptor (FcγR) and a heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptor (BLT1) may provide a rationale for the preferential activation of the β isoform of PI3K. PI3Kβ-deficient mice were highly protected in an FcγR-dependent model of autoantibody-induced skin blistering and were partially protected in an FcγR-dependent model of inflammatory arthritis, whereas combined deficiency of PI3Kβ and PI3Kδ resulted in near-complete protection in the latter case. These results define PI3Kβ as a potential therapeutic target in inflammatory disease.

Mechanisms of Fibroblast Cell Therapy for Dystrophic Epidermolysis Bullosa: High Stability of Collagen VII Favors Long-term Skin Integrity

Here, we report on the first systematic long-term study of fibroblast therapy in a mouse model for recessive dystrophic epidermolysis bullosa (RDEB), a severe skin-blistering disorder caused by loss-of-function of collagen VII. Intradermal injection of wild-type (WT) fibroblasts in >50 mice increased the collagen VII content at the dermal-epidermal junction 3.5- to 4.7-fold. Although the active biosynthesis lasted <28 days, collagen VII remained stable and dramatically improved skin integrity and resistance to mechanical forces for at least 100 days, as measured with a digital 3D-skin sensor for shear forces. Experiments using species-specific antibodies, collagen VII-deficient fibroblasts, gene expression analyses, and cytokine arrays demonstrated that the injected fibroblasts are the major source of newly deposited collagen VII. Apart from transitory mild inflammation, no adverse effects were observed. The cells remained within an area <or=10 mm of the injection site, and did not proliferate, form tumors, or cause fibrosis. Instead, they became gradually apoptotic within 28 days. These data on partial restoration of collagen VII in the skin demonstrate the excellent ratio of clinical effects to biological parameters, support suitability of fibroblast-based therapy approaches for RDEB, and, as a preclinical test, pave way to human clinical trials.

Collagen VII plays a dual role in wound healing

Although a host of intracellular signals is known to contribute to wound healing, the role of the cell microenvironment in tissue repair remains elusive. Here we employed 2 different mouse models of genetic skin fragility to assess the role of the basement membrane protein collagen VII (COL7A1) in wound healing. COL7A1 secures the attachment of the epidermis to the dermis, and its mutations cause a human skin fragility disorder coined recessive dystrophic epidermolysis bullosa (RDEB) that is associated with a constant wound burden. We show that COL7A1 is instrumental for skin wound closure by 2 interconnected mechanisms. First, COL7A1 was required for re-epithelialization through organization of laminin-332 at the dermal-epidermal junction. Its loss perturbs laminin-332 organization during wound healing, which in turn abrogates strictly polarized expression of integrin α6β4 in basal keratinocytes and negatively impacts the laminin-332/integrin α6β4 signaling axis guiding keratinocyte migration. Second, COL7A1 supported dermal fibroblast migration and regulates their cytokine production in the granulation tissue. These findings, which were validated in human wounds, identify COL7A1 as a critical player in physiological wound healing in humans and mice and may facilitate development of therapeutic strategies not only for RDEB, but also for other chronic wounds.

Extracellular Phosphorylation of Collagen XVII by Ecto-Casein Kinase 2 Inhibits Ectodomain Shedding

Ecto-phosphorylation is emerging as an important mechanism to regulate cellular ligand interactions and signal transduction. Here we show that extracellular phosphorylation of the cell surface receptor collagen XVII regulates shedding of its ectodomain. Collagen XVII, a member of the novel family of collagenous transmembrane proteins and component of the hemidesmosomes, mediates adhesion of the epidermis to the dermis in the skin. The ectodomain is constitutively shed from the cell surface by metalloproteinases of the ADAM (a disintegrin and metalloproteinase) family, mainly by tumor necrosis factor-α converting enzyme (TACE). We used biochemical, mutagenesis, and structural modeling approaches to delineate mechanisms controlling ectodomain cleavage. A standard assay for extracellular phosphorylation, incubation of intact keratinocytes with cell-impermeable [γ-32P]ATP, led to collagen XVII labeling. This was significantly diminished by both broad-spectrum extracellular kinase inhibitor K252b and a specific casein kinase 2 (CK2) inhibitor. Collagen XVII peptides containing a putative CK2 recognition site were phosphorylated by CK2 in vitro, disclosing Ser542 and Ser544 in the ectodomain as phosphate group acceptors. Phosphorylation of Ser544 in vivo and in vitro was confirmed by immunoblotting of epidermis and HaCaT keratinocyte extracts with phosphoepitope-specific antibodies. Functionally, inhibition of CK2 kinase activity or mutation of the phosphorylation acceptor Ser544 to Ala significantly increased ectodomain shedding, whereas overexpression of CK2α inhibited cleavage of collagen XVII. Structural modeling suggested that the phosphorylation of serine residues prevents binding of TACE to its substrate. Thus, extracellular phosphorylation of collagen XVII by ecto-CK2 inhibits its shedding by TACE and represents novel mechanism to regulate adhesion and motility of epithelial cells.

Dominant-negative Effects of COL7A1 Mutations Can be Rescued by Controlled Overexpression of Normal Collagen VII

Dominant-negative interference by glycine substitution mutations in the COL7A1 gene causes dominant dystrophic epidermolysis bullosa (DDEB), a skin fragility disorder with mechanically induced blistering. Although qualitative and quantitative alterations of the COL7A1 gene product, collagen VII, underlie DDEB, the lack of direct correlation between mutations and the clinical phenotype has rendered DDEB less amenable to therapeutic targeting. To delineate the molecular mechanisms of DDEB, we used recombinant expression of wild-type (WT) and mutant collagen VII, which contained a naturally occurring COL7A1 mutation, G1776R, G2006D, or G2015E, for characterization of the triple helical molecules. The mutants were co-expressed with WT in equal amounts and could form heterotrimeric hybrid triple helices, as demonstrated by affinity purification and mass spectrometry. The thermal stability of the mutant molecules was strongly decreased, as evident in their sensitivity to trypsin digestion. The helix-to-coil transition, Tm, of the mutant molecules was 31–34 °C, and of WT collagen VII 41 °C. Co-expression of WT with G1776R- or G2006D-collagen VII resulted in partial intracellular retention of the collagen, and mutant collagen VII had reduced ability to support cell adhesion. Intriguingly, controlled overexpression of WT collagen VII gradually improved the thermal stability of the collective of collagen VII molecules. Co-expression in a ratio of 90% WT:10% mutant increased the Tm to 41 °C for G1776R-collagen VII and to 39 °C for G2006D- and G2015E-collagen VII. Therefore, increasing the expression of WT collagen VII in the skin of patients with DDEB can be considered a valid therapeutic approach.

Some epidermolysis bullosa acquisita sera react with epitopes within the triple-helical collagenous domain as indicated by immunoelectron microscopy.

Background  Epidermolysis bullosa acquisita (EBA) autoantibodies recognize epitopes predominantly within the N‐terminal noncollagenous (NC)‐1 domain of type VII collagen. Recently, some EBA cases with reactivity to other domains, i.e. the triple‐helical (T‐H) collagenous domain and the NC‐2 domain, have been reported.

A case of epidermolysis bullosa acquisita with clinical features of Brunsting-Perry pemphigoid showing an excellent response to colchicine.

BACKGROUND Brunsting-Perry pemphigoid is a rare subepidermal blistering disease characterized by scarring blisters on the head and neck. However, the identity of the responsible autoantigens is still unresolved. METHODS We reported a patient with epidermolysis bullosa acquisita who had clinical features typical of Brunsting-Perry pemphigoid and investigated the involved type VII collagen epitopes. The patient was a 65-year-old Japanese woman with a 20-month history of recurrent subepidermal bullae on her head, face, and neck. RESULTS Immunoblot studies revealed that the serum of this patient reacted with type VII collagen, specifically with the noncollagenous domain 1 and the triple-helical domain. The patient responded completely to colchicine monotherapy. LIMITATIONS This study was performed on only one case. CONCLUSION This study suggests that Brunsting-Perry pemphigoid may be a clinical variant of epidermolysis bullosa acquisita.

Cross-reactivity of autoantibodies from patients with epidermolysis bullosa acquisita with murine collagen VII.

The pathomechanism of antibody-mediated tissue damage in autoimmune diseases can be best studied in experimental models by passively transferring specific autoantibodies into animals. The reproduction of the disease in animals depends on several factors, including the cross-reactivity of patient autoantibodies with the animal tissue. Here, we show that autoantibodies from patients with epidermolysis bullosa acquisita (EBA), a subepidermal autoimmune blistering disease, recognize multiple epitopes on murine collagen VII. Indirect immunofluorescence microscopy revealed that EBA patients’ IgG cross-reacts with mouse skin. Overlapping, recombinant fragments of murine collagen VII were used to characterize the reactivity of EBA sera and to map the epitopes on the murine antigen by ELISA and immunoblotting. The patients’ autoantibody binding to murine collagen VII triggered pathogenic events as demonstrated by a complement fixing and an ex vivo granulocyte-dependent dermal–epidermal separation assay. These findings should greatly facilitate the development of improved disease models and novel therapeutic strategies.