Koji Nishifuji

Use of autoantigen-knockout mice in developing an active autoimmune disease model for pemphigus

The development of experimental models of active autoimmune diseases can be difficult due to tolerance of autoantigens, but knockout mice, which fail to acquire tolerance to the defective gene product, provide a useful tool for this purpose. Using knockout mice lacking desmoglein 3 (Dsg3), the target antigen of pemphigus vulgaris (PV), we have generated an active disease model for this autoantibody-mediated disease. Dsg3(-/-) mice, but not Dsg3(+/-) littermates, produced anti-Dsg3 IgG that binds native Dsg3, when immunized with recombinant mouse Dsg3. Splenocytes from the immunized Dsg3(-/-) mice were then adoptively transferred into Rag-2(-/-) immunodeficient mice expressing Dsg3. Anti-Dsg3 IgG was stably produced in the recipient mice for more than 6 months without further boosting. This IgG bound to Dsg3 in vivo and disrupted the cell-cell adhesion of keratinocytes. Consequently, the recipient mice developed erosions in their oral mucous membranes with typical histologic findings of PV. In addition, the recipient mice showed telogen hair loss, as found in Dsg3(-/-) mice. Collectively, the recipient mice developed the phenotype of PV due to the pathogenic anti-Dsg3 IgG. This model will be valuable for developing novel therapeutic strategies. Furthermore, our approach can be applied broadly for the development of various autoimmune disease models.

Identification of the Staphylococcus aureus etd pathogenicity island which encodes a novel exfoliative toxin, ETD, and EDIN-B

ABSTRACT We identified a novel pathogenicity island in Staphylococcus aureus which contains open reading frames (ORFs) similar to the exfoliative toxin (ET) gene, glutamyl endopeptidase gene, and edin-B gene in tandem and the phage resistance gene, flanked by hsdM, hsdS (restriction and modification system), and IS256. The protein encoded by the ET-like gene showed 40, 59, and 68% amino acid sequence identities with exfoliative toxin A (ETA), exfoliative toxin B (ETB), and Staphylococcus hyicus ETB (ShETB), respectively. When injected into neonatal mice, the recombinant protein derived from the ET-like gene induced exfoliation of the skin with loss of cell-to-cell adhesion in the upper part of the epidermis as observed in histological examinations, just as was found in neonatal mice injected with ETA or ETB. Western blot analysis indicated that the recombinant protein is serologically distinct from ETA and ETB. Therefore, the product encoded by this new ORF is a new ET member produced by S. aureus and is termed ETD. ETD did not induce blisters in 1-day-old chickens. In the skins of mice injected with ETD, cell surface staining of desmoglein 1 (Dsg1), a cadherin type cell-to-cell adhesion molecule in desmosomes, was abolished without affecting that of desmoglein 3 (Dsg3). Furthermore, in vitro incubation of the recombinant extracellular domains of Dsg1 and Dsg3 with the recombinant protein demonstrated that both mouse and human Dsg1, but not Dsg3, were directly cleaved in a dose-dependent manner. These results demonstrate that ETD and ETA induce blister formation by identical pathophysiological mechanisms. Clinical strains positive for edin-B were suggested to be clonally associated, and all edin-B-positive strains tested were positive for etd. Among 18 etd-positive strains, 12 produced ETD extracellularly. Interestingly, these strains are mainly isolated from other sources of infections and not from patients with bullous impetigo or staphylococcal scalded-skin syndrome. This strongly suggests that ETD might play a pathogenic role in a broader spectrum of bacterial infections than previously considered.

Molecular mechanisms of blister formation in bullous impetigo and staphylococcal scalded skin syndrome

Bullous impetigo due to Staphylococcus aureus is one of the most common bacterial infections of man, and its generalized form, staphylococcal scalded skin syndrome (SSSS), is a frequent manifestation of staphylococcal epidemics in neonatal nurseries. Both diseases are mediated by exfoliative toxins (ETs), which show exquisite pathologic specificity in blistering only the superficial epidermis. We show that these toxins act as serine proteases with extremely focused molecular specificity to cleave mouse and human desmoglein 1 (Dsg1) once after glutamic acid residue 381 between extracellular domains 3 and 4. Mutation of the predicted catalytically active serine to alanine completely inhibits cleavage. The mutated ETs bind specifically to Dsg1 by immunofluorescence colocalization and by coimmunoprecipitation. Thus, ETs, through specific recognition and proteolytic cleavage of one structurally critical peptide bond in an adhesion molecule, cause its dysfunction and allow S. aureus to spread under the stratum corneum, the main barrier of the skin, explaining how, although they circulate through the entire body in SSSS, they cause pathology only in the superficial epidermis.

Defining the pathogenic involvement of desmoglein 4 in pemphigus and staphylococcal scalded skin syndrome

Desmogleins (Dsgs), cadherin-type cell adhesion molecules, are targeted in skin-blistering diseases such as pemphigus and staphylococcal scalded skin syndrome (SSSS). The role of Dsg4, a new isoform, was investigated in these diseases. Dsg4 was recognized by 30 (77%) of 39 pemphigus sera containing anti-Dsg1 IgG but not by 16 pemphigus sera containing no anti-Dsg1 IgG or by 34 normal control sera. The Dsg4 immunoreactivity of these sera was abolished by removal of anti-Dsg1 IgG. Conversely, the removal of anti-Dsg4 IgG from pemphigus sera reduced the immunoreactivity against Dsg1 only 13.8% +/- 8.8% (n = 23) and did not affect its ability to induce blisters in neonatal mice. IgG that was affinity-purified on Dsg4 recognized Dsg1 but failed to induce blisters, while IgG purified on Dsg1 from the same pemphigus foliaceus sera induced blisters. Thus, pemphigus sera show Dsg4 reactivity due to cross-reactivity of a subset of anti-Dsg1 IgG, and the Dsg4/Dsg1-cross-reacting IgG has no demonstrable pathogenic effect. In addition, Dsg4 was not cleaved by exfoliative toxins that induce blisters in SSSS. These findings suggest that Dsg4 may play a role other than adhesion and that the cross-reactivity of desmoglein autoantibodies should be factored into the framework of future studies of autoimmune mechanisms in pemphigus.

Epitope Spreading Is Rarely Found in Pemphigus Vulgaris by Large-Scale Longitudinal Study Using Desmoglein 2–Based Swapped Molecules

Epitope spreading is involved in inducing and maintaining self-reactivity. Epitope spreading in pemphigus vulgaris (PV), caused by IgG autoantibodies to desmoglein 3 (Dsg3) and Dsg1, was previously analyzed using Dsg3/Dsg1 extracellular domain-swapped molecules. However, precise identification of the responsible epitopes in each molecule by using only this method was problematic. In this study, we studied epitope spreading in PV by a novel immunoprecipitation-immunoblot method using Dsg3 (or Dsg1)/Dsg2 domain-swapped molecules, which overcomes the problems associated with the previous approaches. We analyzed the antigenic epitopes recognized by 212 sera collected from 53 PV patients at multiple disease stages. The major epitopes were present at the N-terminal region of Dsgs and were unchanged over the course of the disease in both anti-Dsg3 mucosal dominant-type PV and anti-Dsg3/Dsg1 mucocutaneous-type PV. These N-terminal epitopes were calcium dependent. Circulating antibodies in paraneoplastic pemphigus and pemphigus herpetiformis had unique epitope distributions, although the Dsg N-termini still contained the major epitopes. These results suggest that, after onset, intramolecular and intermolecular epitope spreading among extracellular domains on Dsg3 and Dsg1 is rare in PV and has no correlation with disease course.

Enzymatic and molecular characteristics of the efficiency and specificity of exfoliative toxin cleavage of desmoglein 1.

Exfoliative toxins (ETs) from Staphylococcus aureus blister the superficial epidermis by hydrolyzing a single peptide bond, Glu381–Gly382, located between extracellular domains 3 and 4 of desmoglein 1 (Dsg1). Enzyme activity is dependent on the calcium-stabilized structure of Dsg1. Here we further define the characteristics of this cleavage. Kinetic studies monitoring the cleavage of Dsg1 by ETA, ETB, and ETD demonstrated kcat/Km values of 2–6 × 104 m–1 s–1, suggesting very efficient proteolysis. Proteolysis by ETA was not efficiently inhibited by broad spectrum serine protease inhibitors, suggesting that the enzyme cleavage site may be inactive or inaccessible before specific binding to its substrate. Using truncated mutants of human Dsg1 and chimeric molecules between human Dsg1 and either human Dsg3 or canine Dsg1, we show that for cleavage, human-specific amino acids from Dsg1 are necessary in extracellular domain 3 upstream of the scissile bond. If these residues are canine rather than human, ETA binds, but does not cleave, canine Dsg1. These data suggest that the exquisite specificity and efficiency of ETA may depend on the enzymes binding upstream of the cleavage site with a very specific fit, like a key in a lock.

Alteration of stratum corneum ceramide profiles in spontaneous canine model of atopic dermatitis

Abstract:  Ceramides (CERs) in the stratum corneum (SC) are thought to play a key role in cutaneous barrier function. It has been reported that human SC contains 11 free CER classes and that their profiles are altered in humans with atopic dermatitis (AD). Although decreased proportions of free CERs or quantities of protein‐bound CERs in the SC have been reported in dogs with AD, the overall profile of CERs in the canine SC has not been fully elucidated. The aim of this study was thus to investigate the profile of free CERs in the canine SC and to identify alterations in the CER profiles in dogs with AD. Normal‐phase liquid chromatography–electrospray ionization–mass spectrometry indicated 11 clusters of peaks for free CER classes, similar to those recognized in the human SC. The fractions of free SC CER in dogs with AD and in breed‐ and age‐matched healthy dogs were quantitatively compared using high‐performance thin‐layer chromatography. CER[EOS], CER[EOP] and CER[NP], which are known to be decreased in the skin of humans with AD, were also decreased in the skin of dogs with AD. These findings highlight canine AD as a spontaneous animal model for investigating the disruption of CER‐associated cutaneous barrier functions in the corresponding human disease.

Identification of a novel Staphylococcus pseudintermedius exfoliative toxin gene and its prevalence in isolates from canines with pyoderma and healthy dogs.

Staphylococcal exfoliative toxins are involved in some cutaneous infections in mammals by targeting desmoglein 1 (Dsg1), a desmosomal cell-cell adhesion molecule. Recently, an exfoliative toxin gene (exi) was identified in Staphylococcus pseudintermedius isolated from canine pyoderma. The aim of this study was to identify novel exfoliative toxin genes in S. pseudintermedius. Here, we describe a novel orf in the genome of S. pseudintermedius isolated from canine impetigo, whose deduced amino acid sequence was homologous to that of the SHETB exfoliative toxin from Staphylococcus hyicus (70.4%). The ORF recombinant protein caused skin exfoliation and abolished cell surface staining of Dsg1 in canine skin. Moreover, the ORF protein degraded the recombinant extracellular domains of canine Dsg1, but not Dsg3, in vitro. PCR analysis revealed that the orf was present in 23.2% (23/99) of S. pseudintermedius isolates from dogs with superficial pyoderma exhibiting various clinical phenotypes, while the occurrence in S. pseudintermedius isolates from healthy dogs was 6.1% (3/49). In summary, this newly found orf in S. pseudintermedius encodes a novel exfoliative toxin, which targets a cell-cell adhesion molecule in canine epidermis and might be involved in a broad spectrum of canine pyoderma.

Canine hair-follicle keratinocytes enriched with bulge cells have the highly proliferative characteristic of stem cells

Homeostasis of the epidermis and skin appendages is maintained by tissue-specific stem cells. In mice and humans, two populations of epithelial stem cells have been identified: one in the basal layer of the interfollicular epidermis and another in the bulge area of hair follicles. However, our understanding of canine epithelial stem cells is extremely limited. In this study, in vitro colony-forming assays were performed to locate highly proliferative keratinocytes in canine skin. Their phenotypic resemblance to epithelial stem cells in other species was also assessed. When equal numbers of epidermal or hair-follicle keratinocytes were cultured, colonies derived from follicular keratinocytes were significantly larger both in total numbers and size, than those derived from epidermal keratinocytes. In addition, immunoreactivity for CD34, a putative bulge stem-cell marker in the mouse, was predominantly detected in follicular keratinocytes. Thus in dogs, follicular keratinocytes were distinct from epidermal keratinocytes in proliferative capacity and CD34 expression. Using microdissection, highly proliferative keratinocytes were located within the middle portion of hair follicles, including the bulge area. Immunohistochemical study revealed that keratin 15, an established marker of bulge stem cells in mice and humans, was also predominantly expressed in the canine bulge area. Flow cytometry analysis revealed high numbers of keratin-15-positive cells in the highly proliferative keratinocyte compartment. Of note, keratin 15(high) cells possessed the phenotypic characteristics of putative stem cells. This study represents the first in vitro identification and isolation of highly proliferative canine keratinocytes, which represent candidate epithelial stem cells.

Staphylococcus pseudintermedius exfoliative toxin EXI selectively digests canine desmoglein 1 and causes subcorneal clefts in canine epidermis.

Staphylococcal exfoliative toxins are known to digest desmoglein (Dsg) 1, a desmosomal cell-cell adhesion molecule, thus causing intraepidermal splitting in human bullous impetigo, staphylococcal scalded skin syndrome and swine exudative epidermitis. Recently, a novel exfoliative toxin gene (exi), whose sequence shares significant homology with previously identified exfoliative toxins, was isolated from Staphylococcus pseudintermedius. Little is known about the pathogenic involvement of this toxin in canine pustular diseases such as impetigo. The aim of this study was to determine whether EXI, the product of the exi gene, digests canine Dsg1 and causes intraepidermal splitting in canine skin. An exi gene was isolated from chromosomal DNA of an S. pseudintermedius strain obtained from a pustule of a dog with impetigo, and was used to produce a recombinant EXI by Escherichia coli expression. When purified recombinant EXI was injected intradermally into normal dogs, it caused the development of vesicles or erosions with superficial epidermal splitting. In addition, the EXI abolished immunofluorescence for Dsg1, but not for Dsg3, at the injection sites. Moreover, the EXI directly degraded baculovirus-secreted recombinant extracellular domains of canine Dsg1, but not that of canine Dsg3, in vitro. The EXI also degraded mouse Dsg1α and swine Dsg1, but not human Dsg1, mouse Dsg1β and Dsg1γ. Conversely, recombinant SIET, previously designated as S. intermedius exfoliative toxin, did not cause intraepidermal splitting or degradation of any Dsgs. These findings indicate that EXI has a proteolytic activity that digests canine Dsg1, and this characteristic might be involved in the pathogenesis of intraepidermal splitting in canine impetigo.