Bullous pemphigoid induced by IgG targeting type XVII collagen non-NC16A/NC15A extracellular domains is driven by Fc gamma receptor- and complement-mediated effector mechanisms and is ameliorated by neonatal Fc receptor blockade.

Bullous pemphigoid (BP) is an autoimmune blistering disease characterized by autoantibodies targeting type XVII collagen (Col17) with the noncollagenous 16A (NC16A) ectodomain representing the immunodominant site. The role of additional extracellular targets of Col17 outside NC16A has not been unequivocally demonstrated. In this study, we showed that Col17 ectodomain-reactive patient sera depleted in NC16A IgG induced dermal-epidermal separation in a cryosection model indicating the pathogenic potential of anti-Col17 non-NC16A extracellular IgG. Moreover, injection of IgG targeting the murine Col17 NC14-1 domains (downstream of NC15A, the murine homologue of human NC16A) into C57BL/6J mice resulted in erythematous skin lesions and erosions. Clinical findings were accompanied by IgG/C3 deposits along the basement membrane and subepidermal blistering with inflammatory infiltrates. Disease development was significantly reduced in either Fc-gamma receptor (FcγR)- or complement-5a receptor-1 (C5aR1)-deficient mice. Inhibition of the neonatal FcR (FcRn), an atypical FcγR regulating IgG homeostasis, with the murine Fc fragment IgG2c-ABDEG, a derivative of efgartigimod, reduced anti-NC14-1 IgG levels, resulting in ameliorated skin inflammation compared with isotype-treated controls. These data demonstrate that the pathogenic effects of IgG targeting the Col17 domain outside human NC16A/murine NC15A are partly attributable to antibody-mediated FcγR- and C5aR1 effector mechanisms while pharmacological inhibition of the FcRn represents a promising treatment for BP. The mouse model of BP will be instrumental in further investigating the role of Col17 non-NC16A/NC15A extracellular epitopes and validating new therapies for this disease. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Levels of type XVII collagen (BP180) ectodomain are elevated in circulation from patients with multiple cancer types and is prognostic for patients with metastatic colorectal cancer.

BACKGROUND: Collagens are the major components of the extracellular matrix (ECM) and are known to contribute to tumor progression and metastasis. There are 28 different types of collagens each with unique functions in maintaining tissue structure and function. Type XVII collagen (BP180) is a type II transmembrane protein that provides stable adhesion between epithelial cells and the underlying basement membrane. Aberrant expression and ectodomain shedding of type XVII collagen have been associated with epithelial damage, tumor invasiveness, and metastasis in multiple tumor types and may consequently be used as a potential (non-invasive) biomarker in cancer and treatment target. METHOD: An ELISA targeting the type XVII collagen ectodomain (PRO-C17) was developed for use in serum. PRO-C17 was measured in a cohort of patients with 11 different cancer types (n = 214) and compared to healthy controls (n = 23) (cohort 1). Based on the findings from cohort 1, PRO-C17 and its association with survival was explored in patients with metastatic colorectal cancer (mCRC) treated with bevacizumab in combination with chemotherapy (n = 212) (cohort 2). RESULTS: PRO-C17 was robust and specific towards the ectodomain of type XVII collagen. In cohort 1, PRO-C17 levels were elevated (p < 0.05) in serum from patients with CRC, kidney, ovarian, bladder, breast, and head and neck cancer compared to healthy controls. PRO-C17 was especially good at discriminating between CRC patients and healthy controls with an AUROC of 0.904. In cohort 2, patients with mCRC and high levels (tertile 3) of PRO-C17 had shorter overall survival (OS) with a median OS of 390 days compared to 539 days for patients with low levels of PRO-C17. When evaluated by multivariate Cox regression analysis, high PRO-C17 was predictive for poor OS independent of risk factors and the tumor fibrosis biomarker PRO-C3. CONCLUSION: PRO-C17 measures the ectodomain of type XVII collagen in serum and is a promising non-invasive biomarker that can aid in understanding tumor heterogeneity as well as elaborate on the role of collagen XVII in tumor progression. Moreover, the findings in the study proposes PRO-C17 as novel biomarker of epithelial damage in specific cancer types including CRC.

Autoimmune bullous skin diseases, pemphigus and pemphigoid.

Autoimmune bullous skin diseases, such as pemphigus and pemphigoid, may enable clarification of the mechanisms of immune regulation in the skin. Pemphigus and pemphigoid are mediated by essentially IgG autoantibodies against structural proteins of the desmosomes at cell-cell junctions and hemidesmosomes at epidermal-dermal junctions, respectively, and are characterized by blisters and erosions in the skin and/or mucous membranes. Intensive investigation over the last 3 decades has identified their target antigens and developed serological diagnostic tools as well as mouse models to help us understand their pathophysiology. Based on these advances, several new therapeutic approaches have become available, and more effective and less toxic targeted approaches are under development.

Personalized Development of Antisense Oligonucleotides for Exon Skipping Restores Type XVII Collagen Expression in Junctional Epidermolysis Bullosa.

Intermediate junctional epidermolysis bullosa caused by mutations in the COL17A1 gene is characterized by the frequent development of blisters and erosions on the skin and mucous membranes. The rarity of the disease and the heterogeneity of the underlying mutations renders therapy developments challenging. However, the high number of short in-frame exons facilitates the use of antisense oligonucleotides (AON) to restore collagen 17 (C17) expression by inducing exon skipping. In a personalized approach, we designed and tested three AONs in combination with a cationic liposomal carrier for their ability to induce skipping of COL17A1 exon 7 in 2D culture and in 3D skin equivalents. We show that AON-induced exon skipping excludes the targeted exon from pre-mRNA processing, which restores the reading frame, leading to the expression of a slightly truncated protein. Furthermore, the expression and correct deposition of C17 at the dermal-epidermal junction indicates its functionality. Thus, we assume AON-mediated exon skipping to be a promising tool for the treatment of junctional epidermolysis bullosa, particularly applicable in a personalized manner for rare genotypes.

The intercellular expression of type-XVII collagen, laminin-332, and integrin-ß1 promote contact following during the collective invasion of a cancer cell population.

Cancer cells can invade as a population in various cancer tissues. This phenomenon is called collective invasion, which is associated with the metastatic potential and prognosis of cancer patients. The collectiveness of cancer cells is necessary for collective invasion. However, the mechanism underlying the generation of collectiveness by cancer cells is not well known. In this study, the phenomenon of contact following, where neighboring cells move in the same direction via intercellular adhesion, was investigated. An experimental system was created to observe the two-dimensional invasion using a collagen gel overlay to study contact following in collective invasion. The role of integrin-ß1, one of the major extracellular matrix (ECM) receptors, in contact following was examined through the experimental system. Integrin-ß1 was localized to the intercellular site in squamous carcinoma cells. Moreover, the intercellular adhesion and contact following were suppressed by treatment of an integrin-ß1 inhibitory antibody. ECM proteins such as laminin-332 and type-XVII collagen were also localized to the intercellular site and critical for contact following. Collectively, it was demonstrated that the activity of integrin-ß1 and expression of ECM proteins in the intercellular site promote contact following in the collective invasion of a cancer cell population.

Regulatory T-cell dysfunction induces autoantibodies to bullous pemphigoid antigens in mice and human subjects.

BACKGROUND: Regulatory T (Treg) cells play a crucial role in peripheral immune tolerance in multiple organs, including the skin. Thus far, the effect of peripheral immune tolerance failure on autoantibody-related autoimmune reactions to the skin is unclear. OBJECTIVE: We sought to elucidate the target autoantigens in the skin under the condition of Treg cell dysfunction caused by forkhead box P3 (Foxp3) gene mutations in scurfy mice and patients with immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome. METHODS: Sera and skin from scurfy mice and sera from patients with IPEX syndrome were analyzed to detect target autoantigens by using immunofluorescence studies, ELISAs, and immunoblotting. The pathogenicity of scurfy IgG was examined by using a passive transfer experiment. CD4+ T cells from scurfy mice were transferred to immunodeficient mice to examine their pathogenicity. Signal transducer and activator of transcription 6 (Stat6)-/- scurfy mice were analyzed to further clarify the molecular pathway of autoantibody production. Follicular helper T-cell counts are measured in Stat6-/- scurfy mice and scurfy mice. RESULTS: Scurfy mice spontaneously generated IgG autoantibodies to the dermal-epidermal junction, which had been class-switched from IgM within 12 days after birth. The target autoantigens were murine BP230 and type XVII collagen (COL17). The scurfy polyclonal autoantibodies did not induce skin fragility in neonatal mice. Autoantibody production was induced by CD4+ T cells from scurfy mice and was ameliorated by Stat6 gene knockout in association with a decrease of follicular helper T cells. We also identified autoantibodies to COL17 and BP230 in patients with IPEX syndrome and found an association between production of autoantibodies to COL17 and an eczematous skin phenotype. CONCLUSIONS: Dysregulation of Treg cells generates autoantibodies to COL17 and BP230 in vivo.

Loss of interaction between plectin and type XVII collagen results in epidermolysis bullosa simplex.

Plectin is a linker protein that interacts with intermediate filaments and ß4 integrin in hemidesmosomes of the epidermal basement membrane zone (BMZ). Type XVII collagen (COL17) has been suggested as another candidate plectin binding partner in hemidesmosomes. Here, we demonstrate that plectin-COL17 binding helps to maintain epidermal BMZ organization. We identified an epidermolysis bullosa (EB) simplex patient as having markedly diminished expression of plectin and COL17 in skin. The patient is compound heterozygous for sequence variants in the plectin gene (PLEC); one is a truncation and the other is a small in-frame deletion sequence variant. The in-frame deletion is located in the putative COL17-binding domain of plectin and abolishes the plectin-COL17 interaction in vitro. These results imply that disrupted interaction between plectin and COL17 is involved in the development of EB. Our study suggests that protein-protein binding defects may underlie EB in patients with unidentified disease-causing sequence variants.

Autoantibodies of non-inflammatory bullous pemphigoid hardly deplete type XVII collagen of keratinocytes.

Type XVII collagen (COL17) and the non-collagenous 16A (NC16A) domain is regarded as the major pathogenic domains for bullous pemphigoid (BP). Some patients with BP have autoantibodies against parts of COL17 outside the NC16A domain (hereinafter the non-NC16A domain) and show less inflammatory manifestations. There were no significant differences in titres and IgG subclasses between NC16A-BP and non-NC16A-BP as determined by indirect immunofluorescent microscopy. The neutrophil activation capacities determined by ROS release did not differ between NC16A-BP and non-NC16A-BP. However, NC16A-BP IgG depleted COL17 in a dose-dependent manner. Treatment with NC16A-BP IgG, but not with non-NC16A-BP IgG, significantly decreased the adhesion strength. We speculate that the differences in clinical severity between NC16A-BP and non-NC16A-BP relate to the degree of COL17 depletion.

Bullous Pemphigoid With a Dual Pattern of Glomerular Immune Complex Disease.

A 75-year-old man presented with a blistering skin disease and nephrotic syndrome. Bullous pemphigoid was diagnosed by linear immunoglobulin G (IgG) and C3 staining along the basement membrane zone of a skin biopsy specimen and by the presence of circulating IgG recognizing the 180-kDa bullous pemphigoid antigen (BP180; type XVII collagen). A kidney biopsy specimen showed endocapillary inflammation without crescents. Direct immunofluorescence showed strong IgG and C3 staining in a combined granular and linear pattern along the glomerular basement membrane. Electron microscopy showed subepithelial deposits. In serum, no antibodies against the Goodpasture antigen (type IV collagen) or phospholipase A2 receptor were detected. Indirect immunofluorescence studies using the patient's serum showed a strikingly linear but not granular IgG pattern along the epithelial basement membranes of monkey esophagus and kidney. Although type XVII collagen was recently identified in the glomerulus, the patient's serum did not produce a 180-kDa band on immunoblot of kidney tissue and still stained glomeruli of BP180 knockout mice by indirect immunofluorescence. The patient was treated with prednisone and azathioprine, which resulted in complete remission of skin and kidney manifestations. Although bullous pemphigoid has been reported previously in association with anti-glomerular basement membrane disease or membranous nephropathy, this case demonstrates both elements in 1 patient. This concurrence and the linear pattern on indirect immunofluorescence support the possibility of cross-reactive or parallel autoantibodies to basement membranes with a secondary membranous component.

From bedside to bench--reverse translational medicine. Scientific lessons from revertant mosaicism in 'knockout' humans.

While the transition from basic life science research to clinical applications is often much more cumbersome than promised, Gostynski et al. took the opposite approach and demonstrated how a scholarly, biology-guided perspective on human skin disease can reveal basic principles of human biology. Hereditary blistering diseases represent not only a disastrous fate for affected patients, but also an opportunity for understanding human molecular physiology and pathophysiology. On the basis of their clinical expertise, Gostynski et al. have elegantly used differences between lesional and non-lesional as a unique opportunity to dissect the role of structural skin proteins--namely type XVII collagen, laminin ß3 and type VII collagen--in melanocyte biology, thus elucidating a new concept in melanocyte biology by transferring knowledge in reverse direction from bedside to bench.

Pigmentation and melanocyte supply to the epidermis depend on type XVII collagen.

Genetic deficiency of type XVII collagen (C17), laminin-332 or type VII collagen causes epidermolysis bullosa (EB). Spontaneous correction of the deficiency, also known as revertant mosaicism, is caused by a second somatic mutation that restores protein expression resulting in clinically healthy (revertant) patches surrounded by fragile (mutant) skin. Interestingly, in some patients, patches of revertant skin show hyperpigmentation. To study the possible role of affected proteins in pigmentation and melanocyte distribution, we investigated clinical documentation and skin biopsy specimens of 13 revertant EB patients having correcting mutations in the COL17A1, LAMB3 or COL7A1 genes. Analysis revealed that lack of C17 led to decreased melanin intensity and melanocyte density in the epidermis when compared with the revertant patches. Reversions of LAMB3 and COL7A1 in keratinocytes did not influence clinical pigmentation or density of melanocytes. We conclude that in human skin, melanocyte supply to the epidermis depends on C17 expression in keratinocytes.

Identification of a human type XVII collagen fragment with high capacity for maintaining skin health.

Collagen XVII (COL17) is a transmembrane protein that mediates skin homeostasis. Due to expression of full length collagen was hard to achieve in microorganisms, arising the needs for selection of collagen fragments with desired functions for microbial biosynthesis. Here, COL17 fragments (27-33 amino acids) were extracted and replicated 16 times for recombinant expression in Escherichia coli. Five variants were soluble expressed, with the highest yield of 223 mg/L. The fusion tag was removed for biochemical and biophysical characterization. Circular dichroism results suggested one variant (sample-1707) with a triple-helix structure at >37 °C. Sample-1707 can assemble into nanofiber (width, 5.6 nm) and form hydrogel at 3 mg/mL. Sample-1707 was shown to induce blood clotting and promote osteoblast differentiation. Furthermore, sample-1707 exhibited high capacity to induce mouse hair follicle stem cells differentiation and osteoblast migration, demonstrating a high capacity to induce skin cell regeneration and promote wound healing. A strong hydrogel was prepared from a chitosan and sample-1707 complex with a swelling rate of >30 % higher than simply using chitosan. Fed-batch fermentation of sample-1707 with a 5-L bioreactor obtained a yield of 600 mg/L. These results support the large-scale production of sample-1707 as a biomaterial for use in the skin care industry.

[Immunopathogenesis of mucous membrane pemphigoid]. / Immunpathogenese des Schleimhautpemphigoids.

A detailed understanding of the immunopathogenesis of mucous membrane pemphigoid (MMP) is of particular importance in view of the mostly difficult diagnostics and treatment of this blistering autoimmune dermatosis. A still unknown disturbance of the body's own immune tolerance leads to the formation of autoreactive cells. As the disease progresses these produce autoantibodies which are directed against structural proteins in the basement membrane zone (BMZ). After they bind to the target antigen, complement factors are deposited along the BMZ and inflammatory cells invade the underlying tissue and produce the characteristic subepithelial blistering. This inflammatory response is associated with fibrosis and scarring in many affected tissues. Most phases of MMP pathogenesis are poorly understood; however, the last few years have shed more light on this processes. These advances are mostly the result of animal and cell culture models. Typical clinical and immunopathological characteristics of MMP, such as oral, conjunctival and skin lesions, are reflected, for example, in an antibody transfer-induced mouse model for anti-laminin 332 MMP in adult mice. Dapsone, as first-line treatment for MMP patients, significantly reduced the severity of these symptoms, and fibrosis in the skin and mucous membranes was also found histologically, which makes the model well-suited for testing new therapeutic approaches for MMP patients and might be of help for further elucidation of the immunopathogenesis of MMP.

Expression of anti-aging type-XVII collagen (COL17A1/BP180) in hair follicle-associated pluripotent (HAP) stem cells during differentiation.

Hair-follicle-associated pluripotent (HAP) stem cells reside in the upper part of the bulge area of the the hair follicle. HAP stem cells are nestin-positive and keratin 15-negative and have the capacity to differentiate into various types of cells in vitro. HAP stem cells are also involved in nerve and spinal cord regeneration in mouse models. Recently, it was shown that the DNA-damage response in non-HAP hair follicle stem cells induces proteolysis of type-XVII collagen (COL17A1/BP180), which is involved in hair-follicle stem-cell maintenance. COL17A1 proteolysis stimulated hair-follicle stem-cell aging, characterized by the loss of stemness signatures and hair-follicle miniaturization associated with androgenic alopecia. In the present study, we demonstrate that HAP stem cells co-express nestin and COL17A1 in vitro and in vivo. The expression of HAP stem cell markers (nestin and SSEA1) increased after HAP stem-cell colonies were formed, then decreased after differentiation to epidermal keratinocytes. In contrast COL17A1 increased after differentiation to epidermal keratinocytes. These results suggest that COL17A1 is important in differentiation of HAP stem cells.

The Diagnosis and Blistering Mechanisms of Mucous Membrane Pemphigoid.

Mucous membrane pemphigoid (MMP) is a mucous membrane-dominated autoimmune subepithelial blistering disease that is caused by autoantibodies against various autoantigens in basement membrane zone (BMZ) proteins, including collagen XVII (COL17). Clinicians face diagnostic problems in detecting circulating antibodies and targeted antigens in MMP. The diagnostic difficulties are mainly attributed to the low titers of MMP autoantibodies in sera and to heterogeneous autoantigens. Additionally, no unanimous diagnostic criteria have been drawn for MMP, which can result in delayed diagnoses or misdiagnoses. This review aims to integrate and present currently available data to clarify diagnostic strategies and to present diagnostic criteria for MMP. The ultimate blistering mechanism in MMP has not been elucidated, and such mechanism is especially obscure in COL17-type MMP. In bullous pemphigoid (BP), which is the most common autoimmune subepidermal blistering disease, some patients show oral lesion as well as predominant skin lesions. However, there is no fundamental explanation for the onset of oral lesions in BP. This article summarizes innovative research perspectives on the pathogenesis of oral lesions in pemphigoid. Finally, we propose a potential pathogenesis for COL17-type MMP.

Type VII and XVII Collagen mRNA Expressions in Regenerated Epidermal Laminae in Chronic Equine Laminitis.

To confirm ability forming the basement membrane of the regenerated laminar epidermis (rLE) in chronic laminitis, expression of type VII and type XVII collagen mRNAs in the rLE was studied applying sequences of two type of murine collagens. On northern blot analysis, complement DNA (cDNA) probes adjusted from the murine type VII and type XVII collagen could hybridize with the equine mRNAs, and each signal was detected as single-bands at approximately 9.5 kb and 5.6 kb, respectively. Contrasting with the expression level of equine glyceraldehyde-3-phosphate dehydrogenease mRNA, the band of type VII collagen mRNA in laminitis was stronger than normal, but the type XVII collagen mRNA in laminitis was less than normal. By in situ hybridization, positive signals in response to the murine type VII and type XVII collagen mRNA probes could be detected in the equine laminitic rLE region. From these results, it is concluded that the keratinocytes constructing the rLE in chronic stage of laminitis can express type VII and type XVII collagen mRNAs and these expression patterns were different from the normal.

Tissue Destruction in Bullous Pemphigoid Can Be Complement Independent and May Be Mitigated by C5aR2.

Bullous pemphigoid (BP), the most frequent autoimmune bullous disorder, is a paradigmatic autoantibody-mediated disease associated with autoantibodies against BP180 (type XVII collagen, Col17). Several animal models have been developed that reflect important clinical and immunological features of human BP. Complement activation has been described as a prerequisite for blister formation, however, the recent finding that skin lesions can be induced by anti-Col17 F(ab')2 fragments indicates complement-independent mechanisms to contribute to blister formation in BP. Here, C5-/- mice injected with anti-Col17 IgG showed a reduction of skin lesions by about 50% associated with significantly less skin-infiltrating neutrophils compared to wild-type mice. Reduction of skin lesions and neutrophil infiltration was seen independently of the employed anti-Col17 IgG dose. Further, C5ar1-/- mice were protected from disease development, whereas the extent of skin lesions was increased in C5ar2-/- animals. Pharmacological inhibition of C5a receptor 1 (C5aR1) by PMX53 led to reduced disease activity when applied in a prophylactic setting. In contrast, PMX-53 treatment had no effect when first skin lesions had already developed. While C5aR1 was critically involved in neutrophil migration in vitro, its role for Col17-anti-Col17 IgG immune complex-mediated release of reactive oxygen species from neutrophils was less pronounced. Our data demonstrate that complement-dependent and -independent mechanisms coexist in anti-Col17-autoantibody-mediated tissue destruction. C5aR1 and C5aR2 seem to play opposing roles in this process with C5aR1 exerting its primary effect in recruiting inflammatory cells to the skin during the early phase of the disease. Further studies are required to fully understand the role of C5aR2 in autoantibody-mediated skin inflammation.

Epidermolysis bullosa: Molecular pathology of connective tissue components in the cutaneous basement membrane zone.

Epidermolysis bullosa (EB), a group of heritable skin fragility disorders, is characterized by blistering, erosions and chronic ulcers in the skin and mucous membranes. In some forms, the blistering phenotype is associated with extensive mutilating scarring and development of aggressive squamous cell carcinomas. The skin findings can be associated with extracutaneous manifestations in the ocular as well as gastrointestinal and vesico-urinary tracts. The phenotypic heterogeneity reflects the presence of mutations in as many as 20 different genes expressed in the cutaneous basement membrane zone, and the types and combinations of the mutations and their consequences at the mRNA and protein levels contribute to the spectrum of severity encountered in different subtypes of EB. This overview highlights the molecular genetics of EB based on mutations in the genes encoding type VII and XVII collagens as well as laminin-332. The mutations identified in these protein components of the extracellular matrix attest to their critical importance in providing stability to the cutaneous basement membrane zone, with implications for heritable and acquired diseases.