Single-cell transcriptomics analysis of bullous pemphigoid unveils immune-stromal crosstalk in type 2 inflammatory disease.

Bullous pemphigoid (BP) is a type 2 inflammation- and immunity-driven skin disease, yet a comprehensive understanding of the immune landscape, particularly immune-stromal crosstalk in BP, remains elusive. Herein, using single-cell RNA sequencing (scRNA-seq) and in vitro functional analyzes, we pinpoint Th2 cells, dendritic cells (DCs), and fibroblasts as crucial cell populations. The IL13-IL13RA1 ligand-receptor pair is identified as the most significant mediator of immune-stromal crosstalk in BP. Notably, fibroblasts and DCs expressing IL13RA1 respond to IL13-secreting Th2 cells, thereby amplifying Th2 cell-mediated cascade responses, which occurs through the specific upregulation of PLA2G2A in fibroblasts and CCL17 in myeloid cells, creating a positive feedback loop integral to immune-stromal crosstalk. Furthermore, PLA2G2A and CCL17 contribute to an increased titer of pathogenic anti-BP180-NC16A autoantibodies in BP patients. Our work provides a comprehensive insight into BP pathogenesis and shows a mechanism governing immune-stromal interactions, providing potential avenues for future therapeutic research.

Collagen XVII promotes dormancy of colorectal cancer cells by activating mTORC2 signaling.

Tumor dormancy is the underpinning for cancer relapse and chemoresistance, leading to massive cancer-related death in colorectal cancer (CRC). However, our comprehension of the mechanisms dictating tumor dormancy and strategies for eliminating dormant tumor cells remains restricted. In this study, we identified that collagen XVII (COL17A1), a hemidesmosomal transmembrane protein, can promote the dormancy of CRC cells. The upregulation of COL17A1 was observed to prolong quiescence periods and diminish drug susceptibility of CRC cells. Mechanistically, COL17A1 acts as a scaffold, enhancing the crosstalk between mTORC2 and Akt, thereby instigating the mTORC2-mediated dormant signaling. Notably, the activation of mTORC2 is contingent upon the intracellular domain of COL17A1, regardless of its ectodomain shedding. Our findings underscore a pivotal role of the COL17A1-mTORC2 axis in CRC dormancy, suggesting that mTORC2-specific inhibitors may hold therapeutic prospects for the eradication of dormant tumor cells.

[A case of Neonatal generalized atrophic benign epidermolysis bullosa due to variants of COL17A1 gene].

OBJECTIVE: To diagnose and explore the genetic etiology of a neonate with Hereditary epidermolysis bullosa. METHODS: A neonate who was admitted to Suqian Hospital Affiliated to Xuzhou Medical University on July 10, 2021 was selected as the study subject. Peripheral blood samples were collected from the child and his parents for the extraction of genomic DNA. And target gene capture and next-generation sequencing were carried out. Candidate variants were verified by Sanger sequencing and pathogenicity analysis. RESULTS: The child was found to harbor compound heterozygous variants of the COL17A1 gene, namely c.997C>T (p.Q333X) and c.3481dupT (p.Y1161fs*2), which were respectively inherited from his father and mother. Both variants were predicted to be pathogenic. CONCLUSION: The child was diagnosed with Generalized atrophic benign epidermolysis bullosa due to the compound heterozygous variants of the COL17A1 gene.

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.

Heterozygous COL17A1 variants are a frequent cause of amelogenesis imperfecta.

BACKGROUND: Collagen XVII is most typically associated with human disease when biallelic COL17A1 variants (>230) cause junctional epidermolysis bullosa (JEB), a rare, genetically heterogeneous, mucocutaneous blistering disease with amelogenesis imperfecta (AI), a developmental enamel defect. Despite recognition that heterozygous carriers in JEB families can have AI, and that heterozygous COL17A1 variants also cause dominant corneal epithelial recurrent erosion dystrophy (ERED), the importance of heterozygous COL17A1 variants causing dominant non-syndromic AI is not widely recognised. METHODS: Probands from an AI cohort were screened by single molecule molecular inversion probes or targeted hybridisation capture (both a custom panel and whole exome sequencing) for COL17A1 variants. Patient phenotypes were assessed by clinical examination and analyses of affected teeth. RESULTS: Nineteen unrelated probands with isolated AI (no co-segregating features) had 17 heterozygous, potentially pathogenic COL17A1 variants, including missense, premature termination codons, frameshift and splice site variants in both the endo-domains and the ecto-domains of the protein. The AI phenotype was consistent with enamel of near normal thickness and variable focal hypoplasia with surface irregularities including pitting. CONCLUSION: These results indicate that COL17A1 variants are a frequent cause of dominantly inherited non-syndromic AI. Comparison of variants implicated in AI and JEB identifies similarities in type and distribution, with five identified in both conditions, one of which may also cause ERED. Increased availability of genetic testing means that more individuals will receive reports of heterozygous COL17A1 variants. We propose that patients with isolated AI or ERED, due to COL17A1 variants, should be considered as potential carriers for JEB and counselled accordingly, reflecting the importance of multidisciplinary care.

Genotype-Phenotype Correlation in Junctional Epidermolysis Bullosa: Signposts to Severity.

Junctional epidermolysis bullosa (JEB) is a rare autosomal recessive genodermatosis with a broad spectrum of phenotypes. Current genotype-phenotype paradigms are insufficient to accurately predict JEB subtype and characteristics from genotype, particularly for splice site variants, which account for over a fifth of disease-causing variants in JEB. This study evaluated the genetic and clinical findings from a JEB cohort, investigating genotype-phenotype correlations through bioinformatic analyses and comparison with previously reported variants. Eighteen unique variants in LAMB3, LAMA3, LAMC2, or COL17A1 were identified from 17 individuals. Seven had severe JEB, 9 had intermediate JEB, and 1 had laryngo-onycho-cutaneous syndrome. Seven variants were previously unreported. Deep phenotyping was completed for all intermediate JEB cases and demonstrated substantial variation between individuals. Splice site variants underwent analysis with SpliceAI, a state-of-the-art artificial intelligence tool, to predict resultant transcripts. Predicted functional effects included exon skipping and cryptic splice site activation, which provided potential explanations for disease severity and in most cases correlated with laminin-332 immunofluorescence. RT-PCR was performed for 1 case to investigate resultant transcripts produced from the splice site variant. This study expands the JEB genomic and phenotypic landscape. Artificial intelligence tools show potential for predicting the functional effects of splice site variants and may identify candidates for confirmatory laboratory investigation. Investigation of RNA transcripts will help to further elucidate genotype-phenotype correlations for novel variants.

Independent COL17A1 Variants in Cats with Junctional Epidermolysis Bullosa.

Epidermolysis bullosa (EB), characterized by defective adhesion of the epidermis to the dermis, is a heterogeneous disease with many subtypes in human patients and domestic animals. We investigated two unrelated cats with recurring erosions and ulcers on ear pinnae, oral mucosa, and paw pads that were suggestive of EB. Histopathology confirmed the diagnosis of EB in both cats. Case 1 was severe and had to be euthanized at 5 months of age. Case 2 had a milder course and was alive at 11 years of age at the time of writing. Whole genome sequencing of both affected cats revealed independent homozygous variants in COL17A1 encoding the collagen type XVII alpha 1 chain. Loss of function variants in COL17A1 lead to junctional epidermolysis bullosa (JEB) in human patients. The identified splice site variant in case 1, c.3019+1del, was predicted to lead to a complete deficiency in collagen type XVII. Case 2 had a splice region variant, c.769+5G>A. Assessment of the functional impact of this variant on the transcript level demonstrated partial aberrant splicing with residual expression of wildtype transcript. Thus, the molecular analyses provided a plausible explanation of the difference in clinical severity between the two cases and allowed the refinement of the diagnosis in the affected cats to JEB. This study highlights the complexity of EB in animals and contributes to a better understanding of the genotype-phenotype correlation in COL17A1-related JEB.

Functional analysis of Collagen 17a1: A genetic modifier of junctional epidermolysis bullosa in mice.

Previous work strongly implicated Collagen 17a1 (Col17a1) as a potent genetic modifier of junctional epidermolysis bullosa (JEB) caused by a hypomorphic mutation (Lamc2jeb) in mice. The importance of the noncollagenous domain (NC4) of COLXVII was suggested by use of a congenic reduction approach that restricted the modifier effect to 2-3 neighboring amino acid changes in that domain. The current study utilizes TALEN and CRISPR/Cas9 induced amino acid replacements and in-frame indels nested to NC4 to further investigate the role of this and adjoining COLXVII domains both as modifiers and primary risk effectors. We confirm the importance of COLXVI AA 1275 S/G and 1277 N/S substitutions and utilize small nested indels to show that subtle changes in this microdomain attenuate JEB. We further show that large in-frame indels removing up to 1482 bp and 169 AA of NC6 through NC1 domains are surprisingly disease free on their own but can be very potent modifiers of Lamc2jeb/jeb JEB. Together these studies exploiting gene editing to functionally dissect the Col17a1 modifier demonstrate the importance of epistatic interactions between a primary disease-causing mutation in one gene and innocuous 'healthy' alleles in other genes.

Detection of a natural antibody targeting the shed ectodomain of BP180 in mice.

BACKGROUND: Pemphigoid diseases are characterized by subepidermal blister formation accompanied by autoantibodies targeting skin component molecules, such as BP180. It is suggested that an epitope-phenotype correlation exists among autoantibodies recognizing BP180. However, it is unclear which regions of BP180 are likely targets for autoantibodies. OBJECTIVE: To elucidate the portions of BP180 where antibodies tend to react under the breakdown of immune tolerance. METHODS: We immunized mice with full-length mouse BP180 (mBP180) to produce anti-mBP180 antibodies. Using the immunized mice, hybridoma cells were established to produce anti-mBP180 antibodies. We analyzed the characteristics of the anti-mBP180 antibodies that were produced in terms of epitopes, immunoglobulin subclasses, and somatic hypermutations. RESULTS: Hybridoma cells derived from immunized mice with full-length mBP180 produced antibodies targeting the intracellular domain (IC) and the shed ectodomain (EC) of mBP180. Using the domain-deleted mBP180 recombinant protein, we revealed that monoclonal anti-mBP180 EC antibodies react to neoepitopes on the 13th collagenous region of cleaved mBP180, which corresponds to the epitopes of linear IgA bullous dermatosis antibodies in human BP180. Furthermore, the subclasses of these antibodies could be distinguished by epitope: The subclass of the anti-mBP180 IC monoclonal antibodies was IgG, whereas that of the anti-mBP180 EC antibodies was IgM. Of note, a clone of these IgM mBP180 EC antibodies was a germline antibody without somatic hypermutation, which is also known as a natural antibody. CONCLUSION: These data suggest that mice potentially have natural antibodies targeting the neoepitopes of cleaved mBP180 EC.

Absence of NC14A Domain of COLXVII/BP180 in Mice Results in IL-17‒Associated Skin Inflammation.

The deletion of exon 18 from Col17a1 in transgenic ΔNC14A mice results in the absence of the NC14A domain. NC14A corresponds to the human NC16A domain, the immunodominant epitope in bullous pemphigoid. Before the age of 1 year, 84% of ΔNC14A mice have developed severe itch and skin erosion. Further characterization of mice with mutated CoLXVII (Bp180) revealed acanthosis; subepidermal blistering; and inflammatory cell infiltrates, especially neutrophils, eosinophils, and mast cells in the lesional skin. Direct immunofluorescence analysis detected linear complement C3, IgG, and/or IgA deposition in the dermo‒epidermal junction of symptomatic ΔNC14A mice. Elevated gene expression of IL-17‒associated cytokines was detected in the lesional skin. An increased proportion of dendritic cells, myeloid-derived suppressor cells, and NK cells and a decrease of T cells were found in both the spleen and lymph nodes of symptomatic ΔNC14A mice. The proportions of B cells and regulatory T cells were increased in lymph nodes. An 8-week treatment with an anti‒IL-17A decreased the expression of Il6, Il23a, and Cxcl1 in the nonlesional skin. Our results suggest that the absence of the NC14A domain of CoLXVII in mice causes an autoimmune response against the cutaneous basement membrane and manifests as an IL-17‒associated inflammation in the skin.

Paired nicking-mediated COL17A1 reframing for junctional epidermolysis bullosa.

Junctional epidermolysis bullosa (JEB) is a debilitating hereditary skin disorder caused by mutations in genes encoding laminin-332, type XVII collagen (C17), and integrin-α6ß4, which maintain stability between the dermis and epidermis. We designed patient-specific Cas9-nuclease- and -nickase-based targeting strategies for reframing a common homozygous deletion in exon 52 of COL17A1 associated with a lack of full-length C17 expression. Subsequent characterization of protein restoration, indel composition, and divergence of DNA and mRNA outcomes after treatment revealed auspicious efficiency, safety, and precision profiles for paired nicking-based COL17A1 editing. Almost 46% of treated primary JEB keratinocytes expressed reframed C17. Reframed COL17A1 transcripts predominantly featured 25- and 37-nt deletions, accounting for >42% of all edits and encoding C17 protein variants that localized accurately to the cell membrane. Furthermore, corrected cells showed accurate shedding of the extracellular 120-kDa C17 domain and improved adhesion capabilities to laminin-332 compared with untreated JEB cells. Three-dimensional (3D) skin equivalents demonstrated accurate and continuous deposition of C17 within the basal membrane zone between epidermis and dermis. Our findings constitute, for the first time, gene-editing-based correction of a COL17A1 mutation and demonstrate the superiority of proximal paired nicking strategies based on Cas9 D10A nickase over wild-type Cas9-based strategies for gene reframing in a clinical context.

Collagen XVII deficiency alters epidermal patterning.

Vertebrates exhibit patterned epidermis, exemplified by scales/interscales in mice tails and grooves/ridges on the human skin surface (microtopography). Although the role of spatiotemporal regulation of stem cells (SCs) has been implicated in this process, the mechanism underlying the development of such epidermal patterns is poorly understood. Here, we show that collagen XVII (COL17), a niche for epidermal SCs, helps stabilize epidermal patterns. Gene knockout and rescue experiments revealed that COL17 maintains the width of the murine tail scale epidermis independently of epidermal cell polarity. Skin regeneration after wounding was associated with slender scale epidermis, which was alleviated by overexpression of human COL17. COL17-negative skin in human junctional epidermolysis bullosa showed a distinct epidermal pattern from COL17-positive skin that resulted from revertant mosaicism. These results demonstrate that COL17 contributes to defining mouse tail scale shapes and human skin microtopography. Our study sheds light on the role of the SC niche in tissue pattern formation.

Collagen XVII inhibits breast cancer cell proliferation and growth through deactivation of the AKT/mTOR signaling pathway.

Collagen XVII (COL17), a cell-matrix adhesion protein, has been found to be suppressed in breast cancer. Our previous data demonstrated a preventive role of COL17 in breast cancer invasiveness. The present study used the stable COL17-overexpressing MCF7 and MDA-MB-231 cells to reveal an anti-proliferative effect of COL17 on breast cancer cell through mTOR deactivation. Cell proliferation was negatively correlated with the expression level of COL17 in a concentration-dependent manner in both conventional and three-dimensional (3D) culture systems. The correlation was confirmed by decreased expression of the proliferative marker Ki67 in COL17-expressing cells. In addition, overexpression of COL17 reduced the clonogenicity and growth of the cells. We demonstrated that COL17 affects the AKT/mTOR signaling pathway by deactivation of AKT, mTOR and downstream effectors, particularly 4EBP1. Moreover, mice xenografted with high COL17-expressing cells exhibited delayed tumor progression and prolonged survival time. The high expression of COL17A1 gene encoding COL17 is associated with low-proliferation tumors, extended tumor-free period, and overall survival of breast cancer patients. In conclusion, our results revealed the novel function of COL17 using in vitro and in vivo models and elucidated the related pathway in breast cancer cell growth and proliferation.

The unique molecular targets associated antioxidant and antifibrotic activity of curcumin in in vitro model of acute lung injury: A proteomic approach.

Bleomycin (BLM) injury is associated with the severity of acute lung injury (ALI) leading to fibrosis, a high-morbidity, and high-mortality respiratory disease of unknown etiology. BLM-induced ALI is marked by the activation of a potent fibrogenic cytokine transcription growth factor beta-1 (TGFß-1), which is considered a critical cytokine in the progression of alveolar injury. Previously, our work demonstrated that a diet-derived compound curcumin (diferuloylmethane), represents its antioxidative and antifibrotic application in TGF-ß1-mediated BLM-induced alveolar basal epithelial cells. However, curcumin-specific protein targets, as well as its mechanism using mass spectrometry-based proteomic approach, remain elusive. To elucidate the underlying mechanism, a quantitative proteomics approach and bioinformatics analysis were employed to identify the protein targets of curcumin in BLM or TGF-ß1-treated cells. With subsequent in vitro experiments, curcumin-related pathways and cellular processes were predicted and validated. The current study discusses two separate proteomics experiments using BLM and TGF-ß1-treated cells with the proteomics approach, various unique target proteins were identified, and proteomic analysis revealed that curcumin reversed the expressions of unique proteins like DNA topoisomerase 2-alpha (TOP2A), kinesin-like protein (KIF11), centromere protein F (CENPF), and so on BLM or TGF-ß1 injury. For the first time, the current study reveals that curcumin restores TGF-ß1 induced peroxisomes like PEX-13, PEX-14, PEX-19, and ACOX1. This was verified by subsequent in vitro assays. This study generated molecular evidence to deepen our understanding of the therapeutic role of curcumin at the proteomic level and may be useful to identify molecular targets for future drug discovery.

Human gastrointestinal epithelia of the esophagus, stomach, and duodenum resolved at single-cell resolution.

The upper gastrointestinal tract, consisting of the esophagus, stomach, and duodenum, controls food transport, digestion, nutrient uptake, and hormone production. By single-cell analysis of healthy epithelia of these human organs, we molecularly define their distinct cell types. We identify a quiescent COL17A1high KRT15high stem/progenitor cell population in the most basal cell layer of the esophagus and detect substantial gene expression differences between identical cell types of the human and mouse stomach. Selective expression of BEST4, CFTR, guanylin, and uroguanylin identifies a rare duodenal cell type, referred to as BCHE cell, which likely mediates high-volume fluid secretion because of continual activation of the CFTR channel by guanylin/uroguanylin-mediated autocrine signaling. Serotonin-producing enterochromaffin cells in the antral stomach significantly differ in gene expression from duodenal enterochromaffin cells. We, furthermore, discover that the histamine-producing enterochromaffin-like cells in the oxyntic stomach express the luteinizing hormone, yet another member of the enteroendocrine hormone family.

Cell Fitness: More Than Push-Ups.

Cell competition (CC) is a feature that allows tumor cells to outcompete and eliminate adjacent cells that are deemed less fit. Studies of CC, first described in Drosophila melanogaster, reveal a diversity of underlying mechanisms. In this review, we will discuss three recent studies that expand our understanding of the molecular features governing CC. In particular, we will focus on a molecular fitness fingerprint, oncogenic pathways, and the importance of cell junction stability. A fitness fingerprint, mediated by flower (hFWE) protein isoforms, dictates that cells expressing the flower-win isoforms will outcompete adjacent flower-loss-expressing cells. The impact of the flower protein isoforms is seen in cancer progression and may have diagnostic potential. The yes-associated protein (YAP) and TAZ transcription factors, central mediators of the oncogenic Hippo pathway, elevate peritumoral fitness thereby protecting against tumor progression and provide a suppressive barrier. Similarly, COL17A1 is a key component in hemidesmosome stability, and its expression in epidermal stem cells contributes to fitness competition and aging characteristics. The contributions of these pathways to disease development and progression will help define how CC is hijacked to favor cancer growth. Understanding these features will also help frame the diagnostic and therapeutic possibilities that may place CC in the crosshairs of cancer therapeutics.