Ken Natsuga

Paper-based ELISA for the detection of autoimmune antibodies in body fluid-the case of bullous pemphigoid

Bullous pemphigoid (BP), a common autoimmune blistering disease, is increasing in incidence and conveys a high mortality. Detection of autoantibodies targeting the noncollagenous 16A (NC16A) domain of type XVII collagen using enzyme-linked immunosorbent assay (ELISA) has demonstrated high sensitivity and specificity for diagnosing BP. We have developed a rapid, low-cost, and widely applicable ELISA-based system to detect the NC16A autoimmune antibody and then diagnose and monitor BP disease activity using a piece of filter paper, a wax-printer, and NC16A antigens. Both sera and/or blister fluids from 14 untreated BP patients were analyzed. The control group included healthy volunteers and patients with other blistering disorders such as pemphigus vulgaris. In our established paper-based ELISA (P-ELISA) system, only 2 μL of serum or blister fluid and 70 min were required to detect anti-NC16A autoimmune antibodies. The relative color intensity was significantly higher in the BP group than in the control groups when using either serum (P < 0.05) or blister fluid (P < 0.001) specimens from BP patients. The results of P- ELISA were moderately correlated with the titer of the commercial ELISA kit (MBL, Japan) (rho = 0.5680, P = 0.0011). This newly developed system allows for rapid and convenient diagnosis and/or monitoring of BP disease activity.

Autoantibody Profile Differentiates between Inflammatory and Noninflammatory Bullous Pemphigoid

Bullous pemphigoid (BP) is a major autoimmune blistering skin disorder, in which a majority of the autoantibodies (autoAbs) target the juxtamembranous extracellular noncollagenous 16A domain (NC16A) domain of hemidesmosomal collagen XVII. BP-autoAbs may target regions of collagen XVII other than the NC16A domain; however, correlations between epitopes of BP-autoAbs and clinical features have not been fully elucidated. To address correlations between the clinical features and specific epitopes of BP-autoAbs, we evaluated the epitope profiles of BP-autoAbs in 121 patients. A total of 87 patients showed a typical inflammatory phenotype with erythema and autoAbs targeting the anti-NC16A domain, whereas 14 patients showed a distinct noninflammatory phenotype, in which autoAbs specifically targeted the midportion of collagen XVII, but not NC16A. Interestingly, this group clinically showed significantly reduced erythema associated with scant lesional infiltration of eosinophils. Surprisingly, 7 of the 14 cases (50.0%) received dipeptidyl peptidase-IV inhibitors for the treatment of diabetes. Dipeptidyl peptidase-IV inhibitors were used in 3 of 76 (3.9%) typical cases of BP with autoAbs targeting NC16A; thus, dipeptidyl peptidase-IV inhibitors are thought to be involved in the development of atypical noninflammatory BP. This study shows that the autoAb profile differentiates between inflammatory and noninflammatory BP, and that noninflammatory BP may be associated with dipeptidyl peptidase-IV inhibitors.

Epidermal Wnt/β-catenin signaling regulates adipocyte differentiation via secretion of adipogenic factors

Significance The synchronized patterns of hair follicle growth and expansion of the dermal adipocyte layer have long been recognized. Although factors secreted by adipocytes are known to regulate the hair growth cycle, it is unclear whether, conversely, the epidermis can regulate adipogenesis. Our study now demonstrates that activation of epidermal Wnt/β-catenin signaling stimulates adipocyte differentiation in vivo and in vitro. The effect can be mediated by secreted factors, including insulin-like growth factor 2 and bone morphogenetic proteins 2 and 6. It has long been recognized that the hair follicle growth cycle and oscillation in the thickness of the underlying adipocyte layer are synchronized. Although factors secreted by adipocytes are known to regulate the hair growth cycle, it is unclear whether the epidermis can regulate adipogenesis. We show that inhibition of epidermal Wnt/β-catenin signaling reduced adipocyte differentiation in developing and adult mouse dermis. Conversely, ectopic activation of epidermal Wnt signaling promoted adipocyte differentiation and hair growth. When the Wnt pathway was activated in the embryonic epidermis, there was a dramatic and premature increase in adipocytes in the absence of hair follicle formation, demonstrating that Wnt activation, rather than mature hair follicles, is required for adipocyte generation. Epidermal and dermal gene expression profiling identified keratinocyte-derived adipogenic factors that are induced by β-catenin activation. Wnt/β-catenin signaling-dependent secreted factors from keratinocytes promoted adipocyte differentiation in vitro, and we identified ligands for the bone morphogenetic protein and insulin pathways as proadipogenic factors. Our results indicate epidermal Wnt/β-catenin as a critical initiator of a signaling cascade that induces adipogenesis and highlight the role of epidermal Wnt signaling in synchronizing adipocyte differentiation with the hair growth cycle.

Bone marrow transplantation restores epidermal basement membrane protein expression and rescues epidermolysis bullosa model mice

Attempts to treat congenital protein deficiencies using bone marrow-derived cells have been reported. These efforts have been based on the concepts of stem cell plasticity. However, it is considered more difficult to restore structural proteins than to restore secretory enzymes. This study aims to clarify whether bone marrow transplantation (BMT) treatment can rescue epidermolysis bullosa (EB) caused by defects in keratinocyte structural proteins. BMT treatment of adult collagen XVII (Col17) knockout mice induced donor-derived keratinocytes and Col17 expression associated with the recovery of hemidesmosomal structure and better skin manifestations, as well improving the survival rate. Both hematopoietic and mesenchymal stem cells have the potential to produce Col17 in the BMT treatment model. Furthermore, human cord blood CD34+ cells also differentiated into keratinocytes and expressed human skin component proteins in transplanted immunocompromised (NOD/SCID/γcnull) mice. The current conventional BMT techniques have significant potential as a systemic therapeutic approach for the treatment of human EB.

Prevalent LIPH founder mutations lead to loss of P2Y5 activation ability of PA-PLA1α in autosomal recessive hypotrichosis†

Autosomal recessive hypotrichosis (ARH) is characterized by sparse hair on the scalp without other abnormalities. Three genes, DSG4, LIPH, and LPAR6 (P2RY5), have been reported to underlie ARH. We performed a mutation search for the three candidate genes in five independent Japanese ARH families and identified two LIPH mutations: c.736T>A (p.Cys246Ser) in all five families, and c.742C>A (p.His248Asn) in four of the five families. Out of 200 unrelated control alleles, we detected c.736T>A in three alleles and c.742C>A in one allele. Haplotype analysis revealed each of the two mutant alleles is derived from a respective founder. These results suggest the LIPH mutations are prevalent founder mutations for ARH in the Japanese population. LIPH encodes PA‐PLA1α (LIPH), a membrane‐associated phosphatidic acid‐preferring phospholipase A1α. Two residues, altered by these mutations, are conserved among PA‐PLA1α of diverse species. Cys246 forms intramolecular disulfide bonds on the lid domain, a crucial structure for substrate recognition, and His248 is one amino acid of the catalytic triad. Both p.Cys246Ser‐ and p.His248Asn‐PA‐PLA1α mutants showed complete abolition of hydrolytic activity and had no P2Y5 activation ability. These results suggest defective activation of P2Y5 due to reduced 2‐acyl lysophosphatidic acid production by the mutant PA‐PLA1α is involved in the pathogenesis of ARH. Hum Mutat 31:1–9, 2010.

Antibodies to Pathogenic Epitopes on Type XVII Collagen Cause Skin Fragility in a Complement-Dependent and -Independent Manner

In bullous pemphigoid (BP), the most prevalent autoimmune blistering disease, type XVII collagen (COL17) is targeted by circulating autoantibodies. BP is thought to be an autoantibody-mediated complement-fixing blistering disease, and a juxtamembranous noncollagenous 16A (NC16A) domain spanning Glu490 to Arg566 was proved to be the main pathogenic region on COL17, although precise pathogenic epitopes within NC16A have not been elucidated. In this study, we showed that injection of rabbit IgG Abs targeting Asp522 to Gln545 induced skin fragility associated with in vivo deposition of IgG and complement in neonatal COL17-humanized mice. Notably, immunoadsorption of rabbit anti-NC16A IgG Ab with this epitope (Asp522 to Gln545) or the anti-NC16A IgG administered together with the peptides of this epitope as a decoy ameliorated skin fragility in the injected neonatal COL17-humanized mice compared with the anti-NC16A IgG alone even though all of the mice showed both IgG and complement deposition. These results led us to investigate an additional, complement-independent mechanism of skin fragility in the mice injected with anti-COL17 Abs. The rabbit anti-NC16A IgG depleted the expression of COL17 in cultured normal human keratinocytes, whereas immunoadsorption of the same IgG with this epitope significantly suppressed the depletion effect. Moreover, passive transfer of F(ab′)2 fragments of the human BP or rabbit IgG Abs against COL17 demonstrated skin fragility in neonatal COL17-humanized mice. In summary, this study reveals the importance of Abs directed against distinct epitopes on COL17, which induce skin fragility in complement-dependent as well as complement-independent ways.

Bullous Pemphigoid Autoantibodies Directly Induce Blister Formation without Complement Activation

Complement activation and subsequent recruitment of inflammatory cells at the dermal/epidermal junction are thought to be essential for blister formation in bullous pemphigoid (BP), an autoimmune blistering disease induced by autoantibodies against type XVII collagen (COL17); however, this theory does not fully explain the pathological features of BP. Recently, the involvement of complement-independent pathways has been proposed. To directly address the question of the necessity of the complement activation in blister formation, we generated C3-deficient COL17-humanized mice. First, we show that passive transfer of autoantibodies from BP patients induced blister formation in neonatal C3-deficient COL17-humanized mice without complement activation. By using newly generated human and murine mAbs against the pathogenic noncollagenous 16A domain of COL17 with high (human IgG1, murine IgG2), low (murine IgG1), or no (human IgG4) complement activation abilities, we demonstrate that the deposition of Abs, and not complements, is relevant to the induction of blister formation in neonatal and adult mice. Notably, passive transfer of BP autoantibodies reduced the amount of COL17 in lesional mice skin, as observed in cultured normal human keratinocytes treated with the same Abs. Moreover, the COL17 depletion was associated with a ubiquitin/proteasome pathway. In conclusion, the COL17 depletion induced by BP autoantibodies, and not complement activation, is essential for the blister formation under our experimental system.

Human IgG1 Monoclonal Antibody against Human Collagen 17 Noncollagenous 16A Domain Induces Blisters via Complement Activation in Experimental Bullous Pemphigoid Model

Bullous pemphigoid (BP) is an autoimmune blistering disease caused by IgG autoantibodies targeting the noncollagenous 16A (NC16A) domain of human collagen 17 (hCOL17), which triggers blister formation via complement activation. Previous in vitro analysis demonstrated that IgG1 autoantibodies showed much stronger pathogenic activity than IgG4 autoantibodies; however, the exact pathogenic role of IgG1 autoantibodies has not been fully demonstrated in vivo. We constructed a recombinant IgG1 mAb against hCOL17 NC16A from BP patients. In COL17-humanized mice, this mAb effectively reproduced a BP phenotype that included subepidermal blisters, deposition of IgG1, C1q and C3, neutrophil infiltration, and mast cell degranulation. Subsequently, alanine substitutions at various C1q binding sites were separately introduced to the Fc region of the IgG1 mAb. Among these mutated mAbs, the one that was mutated at the P331 residue completely failed to activate the complement in vitro and drastically lost pathogenic activity in COL17-humanized mice. These findings indicate that P331 is a key residue required for complement activation and that IgG1-dependent complement activation is essential for blister formation in BP. This study is, to our knowledge, the first direct evidence that IgG1 Abs to hCOL17 NC16A can induce blister formation in vivo, and it raises the possibility that IgG1 mAbs with Fc modification may be used to block pathogenic epitopes in autoimmune diseases.

Plectin expression patterns determine two distinct subtypes of epidermolysis bullosa simplex

Plectin is a cytoskeletal linker protein that has a dumbbell‐like structure with a long central rod and N‐ and C‐terminal globular domains. Mutations in the gene encoding plectin (PLEC1) cause two distinct autosomal recessive subtypes of epidermolysis bullosa (EB): EB simplex with muscular dystrophy (EBS‐MD), and EB simplex with pyloric atresia (EBS‐PA). Here, we demonstrate that normal human fibroblasts express two different plectin isoforms including full‐length and rodless forms of plectin. We performed detailed analysis of plectin expression patterns in six EBS‐MD and three EBS‐PA patients. In EBS‐PA, expression of all plectin domains was found to be markedly attenuated or completely lost; in EBS‐MD, the expression of the N‐ and C‐terminal domains of plectin remained detectable, although the expression of rod domains was absent or markedly reduced. Our data suggest that loss of the full‐length plectin isoform with residual expression of the rodless plectin isoform leads to EBS‐MD, and that complete loss or marked attenuation of full‐length and rodless plectin expression underlies the more severe EBS‐PA phenotype. These results also clearly account for the majority of EBS‐MD PLEC1 mutation restriction within the large exon 31 that encodes the plectin rod domain, whereas EBS‐PA PLEC1 mutations are generally outside exon 31. Hum Mutat 30:1–9, 2010.

Epidermal barrier defects link atopic dermatitis with altered skin cancer susceptibility

Atopic dermatitis can result from loss of structural proteins in the outermost epidermal layers, leading to a defective epidermal barrier. To test whether this influences tumour formation, we chemically induced tumours in EPI−/− mice, which lack three barrier proteins—Envoplakin, Periplakin, and Involucrin. EPI−/− mice were highly resistant to developing benign tumours when treated with 7,12-dimethylbenz(a)anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). The DMBA response was normal, but EPI−/− skin exhibited an exaggerated atopic response to TPA, characterised by abnormal epidermal differentiation, a complex immune infiltrate and elevated serum thymic stromal lymphopoietin (TSLP). The exacerbated TPA response could be normalised by blocking TSLP or the immunoreceptor NKG2D but not CD4+ T cells. We conclude that atopy is protective against skin cancer in our experimental model and that the mechanism involves keratinocytes communicating with cells of the immune system via signalling elements that normally protect against environmental assaults. DOI: http://dx.doi.org/10.7554/eLife.01888.001