Individuals with JAK1 variants are affected by syndromic features encompassing autoimmunity, atopy, colitis, and dermatitis.

Inborn errors of immunity lead to autoimmunity, inflammation, allergy, infection, and/or malignancy. Disease-causing JAK1 gain-of-function (GoF) mutations are considered exceedingly rare and have been identified in only four families. Here, we use forward and reverse genetics to identify 59 individuals harboring one of four heterozygous JAK1 variants. In vitro and ex vivo analysis of these variants revealed hyperactive baseline and cytokine-induced STAT phosphorylation and interferon-stimulated gene (ISG) levels compared with wild-type JAK1. A systematic review of electronic health records from the BioME Biobank revealed increased likelihood of clinical presentation with autoimmunity, atopy, colitis, and/or dermatitis in JAK1 variant-positive individuals. Finally, treatment of one affected patient with severe atopic dermatitis using the JAK1/JAK2-selective inhibitor, baricitinib, resulted in clinically significant improvement. These findings suggest that individually rare JAK1 GoF variants may underlie an emerging syndrome with more common presentations of autoimmune and inflammatory disease (JAACD syndrome). More broadly, individuals who present with such conditions may benefit from genetic testing for the presence of JAK1 GoF variants.

Comparative analyses of Netherton syndrome patients and Spink5 conditional knock-out mice uncover disease-relevant pathways.

Netherton syndrome (NS) is a rare skin disease caused by loss-of-function mutations in the serine peptidase inhibitor Kazal type 5 (SPINK5) gene. Disease severity and the lack of efficacious treatments call for a better understanding of NS mechanisms. Here we describe a novel and viable, Spink5 conditional knock-out (cKO) mouse model, allowing to study NS progression. By combining transcriptomics and proteomics, we determine a disease molecular profile common to mouse models and NS patients. Spink5 cKO mice and NS patients share skin barrier and inflammation signatures defined by up-regulation and increased activity of proteases, IL-17, IL-36, and IL-20 family cytokine signaling. Systemic inflammation in Spink5 cKO mice correlates with disease severity and is associated with thymic atrophy and enlargement of lymph nodes and spleen. This systemic inflammation phenotype is marked by neutrophils and IL-17/IL-22 signaling, does not involve primary T cell immunodeficiency and is independent of bacterial infection. By comparing skin transcriptomes and proteomes, we uncover several putative substrates of tissue kallikrein-related proteases (KLKs), demonstrating that KLKs can proteolytically regulate IL-36 pro-inflammatory cytokines. Our study thus provides a conserved molecular framework for NS and reveals a KLK/IL-36 signaling axis, adding new insights into the disease mechanisms and therapeutic targets.

Pachyonychia Congenita: A Research Agenda Leading to New Therapeutic Approaches.

Pachyonychia congenita (PC) is a dominantly inherited genetic disorder of cornification. PC stands out among other genodermatoses because despite its rarity, it has been the focus of a very large number of pioneering translational research efforts over the past 2 decades, mostly driven by a patient support organization, the Pachyonychia Congenita Project. These efforts have laid the ground for innovative strategies that may broadly impact approaches to the management of other inherited cutaneous and noncutaneous diseases. This article outlines current avenues of research in PC, expected outcomes, and potential hurdles.

Highly efficient ex-vivo correction of COL7A1 through RNP-based CRISPR/Cas9 and Homology-Directed Repair to treat recessive dystrophic epidermolysis bullosa.

Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a rare and severe genetic skin disease responsible for blistering of the skin and mucosa after minor trauma. RDEB is caused by a wide variety of mutations in COL7A1 encoding C7, the major component of anchoring fibrils (AFs) which form key attachment structures for dermal-epidermal adherence. Here, we achieved highly efficient COL7A1 editing in primary RDEB keratinocytes (RDEB-K) and fibroblasts (RDEB-F) from two patients homozygous for the c.6508C>T (p.Gln2170*) mutation through CRISPR/Cas9-mediated Homology-Directed Repair. Three gRNAs targeting the c.6508C>T mutation or harboring sequences, were delivered together with hfCas9 as a ribonucleoprotein complex (RNP). Among them, one achieved 73% cleavage activity in primary RDEB-K and RDEB-F. Then, we treated RDEB-K and RDEB-F with this specific RNP and the corresponding donor template delivered as ssODN and achieved up to 58% of genetic correction as well as C7 rescue. Finally, grafting of corrected 3D skin onto nude mice induced re-expression and normal localization of C7 as well as AFs formation at the DEJ at five and ten weeks post-grafting. With this promising non-viral approach, we achieved therapeutically relevant specific gene editing which could be applicable to all mutations in exon 80 of COL7A1 in primary RDEB cells.

Large-scale functional inference for skin-expressing lncRNAs using expression and sequence information.

Long noncoding RNAs (lncRNAs) regulate the expression of protein-coding genes and have been shown to play important roles in inflammatory skin diseases. However, we still have limited understanding of the functional impact of lncRNAs in skin, partly due to their tissue specificity and lower expression levels compared with protein-coding genes. We compiled a comprehensive list of 18,517 lncRNAs from different sources and studied their expression profiles in 834 RNA-Seq samples from multiple inflammatory skin conditions and cytokine-stimulated keratinocytes. Applying a balanced random forest to predict involvement in biological functions, we achieved a median AUROC of 0.79 in 10-fold cross-validation, identifying significant DNA binding domains (DBDs) for 39 lncRNAs. G18244, a skin-expressing lncRNA predicted for IL-4/IL-13 signaling in keratinocytes, was highly correlated in expression with F13A1, a protein-coding gene involved in macrophage regulation, and we further identified a significant DBD in F13A1 for G18244. Reflecting clinical implications, AC090198.1 (predicted for IL-17 pathway) and AC005332.6 (predicted for IFN-γ pathway) had significant negative correlation with the SCORAD metric for atopic dermatitis. We also utilized single-cell RNA and spatial sequencing data to validate cell type specificity. Our research demonstrates lncRNAs have important immunological roles and can help prioritize their impact on inflammatory skin diseases.

Successful treatment of JAK1-associated inflammatory disease.

BACKGROUND: Gain-of-function variants of JAK1 drive a rare immune dysregulation syndrome associated with atopic dermatitis, allergy, and eosinophilia. OBJECTIVES: This study sought to describe the clinical and immunological characteristics associated with a new gain-of-function variant of JAK1 and report the therapeutic efficacy of Janus kinase (JAK) inhibition. METHODS: The investigators identified a family affected by JAK1-associated autoinflammatory disease and performed clinical assessment and immunological monitoring on 9 patients. JAK1 signaling was studied by flow and mass cytometry in patients' cells at basal state or after immune stimulation. A molecular disease signature in the blood was studied at the transcriptomic level. Patients were treated with 1 of 2 JAK inhibitors: either baricitinib or upadacitinib. Clinical, cellular, and molecular response were evaluated over a 2-year period. RESULTS: Affected individuals displayed a syndromic disease with prominent allergy including atopic dermatitis, ichthyosis, arthralgia, chronic diarrhea, disseminated calcifying fibrous tumors, and elevated whole blood histamine levels. A variant of JAK1 localized in the pseudokinase domain was identified in all 9 affected, tested patients. Hyper-phosphorylation of STAT3 was found in 5 of 6 patients tested. Treatment of patients' cells with baricitinib controlled most of the atypical hyper-phosphorylation of STAT3. Administration of baricitinib to patients led to rapid improvement of the disease in all adults and was associated with reduction of systemic inflammation. CONCLUSIONS: Patients with this new JAK1 gain-of-function pathogenic variant displayed very high levels of blood histamine and showed a variable combination of atopy with articular and gastrointestinal manifestations as well as calcifying fibrous tumors. The disease, which appears to be linked to STAT3 hyperactivation, was well controlled under treatment by JAK inhibitors in adult patients.

Generation and characterization of induced pluripotent stem cell lines from two patients with recessive dystrophic epidermolysis Bullosa.

Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a rare and severe genetic disease responsible for blistering of the skin and mucosa caused by a wide variety of mutations in COL7A1 encoding type VII collagen. We have generated Induced Pluripotent Stem Cells (iPSCs) from two RDEB patients' fibroblasts harboring homozygous recurrent mutations in COL7A1. Their pluripotent state was confirmed by gene and protein expression of stem cell markers OCT4, SOX2, TRA1/60 and SSEA4. Embryoid body formation followed by immunostaining and TaqMan scorecard analysis confirmed the capacity of RDEB iPSCs to differentiate into cell types from the three germ layers in vitro.

Type I IFNs link skin-associated dysbiotic commensal bacteria to pathogenic inflammation and angiogenesis in rosacea.

Rosacea is a common chronic inflammatory skin disease with a fluctuating course of excessive inflammation and apparent neovascularization. Microbial dysbiosis with a high density of Bacillus oleronius and increased activity of kallikrein 5, which cleaves cathelicidin antimicrobial peptide, are key pathogenic triggers in rosacea. However, how these events are linked to the disease remains unknown. Here, we show that type I IFNs produced by plasmacytoid DCs represent the pivotal link between dysbiosis, the aberrant immune response, and neovascularization. Compared with other commensal bacteria, B. oleronius is highly susceptible and preferentially killed by cathelicidin antimicrobial peptides, leading to enhanced generation of complexes with bacterial DNA. These bacterial DNA complexes but not DNA complexes derived from host cells are required for cathelicidin-induced activation of plasmacytoid DCs and type I IFN production. Moreover, kallikrein 5 cleaves cathelicidin into peptides with heightened DNA binding and type I IFN-inducing capacities. In turn, excessive type I IFN expression drives neoangiogenesis via IL-22 induction and upregulation of the IL-22 receptor on endothelial cells. These findings unravel a potentially novel pathomechanism that directly links hallmarks of rosacea to the killing of dysbiotic commensal bacteria with induction of a pathogenic type I IFN-driven and IL-22-mediated angiogenesis.

AXIN2 germline testing in a French cohort validates pathogenic variants as a rare cause of predisposition to colorectal polyposis and cancer.

Only a few patients with germline AXIN2 variants and colorectal adenomatous polyposis or cancer have been described, raising questions about the actual contribution of this gene to colorectal cancer (CRC) susceptibility. To assess the clinical relevance for AXIN2 testing in patients suspected of genetic predisposition to CRC, we collected clinical and molecular data from the French Oncogenetics laboratories analyzing AXIN2 in this context. Between 2004 and June 2020, 10 different pathogenic/likely pathogenic AXIN2 variants were identified in 11 unrelated individuals. Eight variants were from a consecutive series of 3322 patients, which represents a frequency of 0.24%. However, loss-of-function AXIN2 variants were strongly associated with genetic predisposition to CRC as compared with controls (odds ratio: 11.89, 95% confidence interval: 5.103-28.93). Most of the variants were predicted to produce an AXIN2 protein devoid of the SMAD3-binding and DIX domains, but preserving the ß-catenin-binding domain. Ninety-one percent of the AXIN2 variant carriers who underwent colonoscopy had adenomatous polyposis. Forty percent of the variant carriers developed colorectal or/and other digestive cancer. Multiple tooth agenesis was present in at least 60% of them. Our report provides further evidence for a role of AXIN2 in CRC susceptibility, arguing for AXIN2 testing in patients with colorectal adenomatous polyposis or cancer.

EGFR Signaling Is Overactive in Pachyonychia Congenita: Effective Treatment with Oral Erlotinib.

Pachyonychia congenita (PC) is a rare keratinizing disorder characterized by painful palmoplantar keratoderma for which there is no standard current treatment. PC is caused by dominant mutations in keratin (K) K6A, K6B, K6C, K16, or K17 genes involved in stress, wound healing, and epidermal barrier formation. Mechanisms leading to pain and painful palmoplantar keratoderma in PC remain elusive. In this study, we show overexpression of EGFR ligands epiregulin and TGF-α as well as HER1‒EGFR and HER2 in the upper spinous layers of PC lesions. EGFR activation was confirmed by upregulated MAPK/ERK and mTOR signaling. Abnormal late terminal keratinization was associated with elevated TGM1 activity. In addition, the calcium ion permeable channel TRPV3 was significantly increased in PC-lesional skin, suggesting a predominant role of the TRPV3/EGFR signaling complex in PC. We hypothesized that this complex contributes to promoting TGM1 activity and induces the expression and shedding of EGFR ligands. To counteract this biological cascade, we treated three patients with PC with oral erlotinib for 6‒8 months. The treatment was well-tolerated and led to an early, drastic, and sustained reduction of neuropathic pain with a major improvement of QOL. Our study provides evidence that targeted pharmacological inhibition of EGFR is an effective strategy in PC.

Phage Display Selected Cyclic Peptide Inhibitors of Kallikrein-Related Peptidases 5 and 7 and Their In Vivo Delivery to the Skin.

Kallikrein-related peptidases 5 (KLK5) and 7 (KLK7) are serine proteases with homeostatic functions in the epidermis that play a critical role in Netherton syndrome (NS), a rare yet life-threatening genetic disorder that currently lacks specific treatment. Previous research suggests that controlling KLKs could lead to the development of NS therapies, but existing synthetic inhibitors have limitations. Herein, we used phage display to screen libraries comprising more than 100 billion different cyclic peptides and found selective, high-affinity inhibitors of KLK5 (Ki = 2.2 ± 0.1 nM) and KLK7 (Ki = 16 ± 4 nM). By eliminating protease-prone sites and conjugating the inhibitors to an albumin-binding peptide, we enhanced the inhibitor stability and prolonged the elimination half-life to around 5 h in mice. In tissue sections taken from mice, a fluorescently labeled peptide was detected in the epidermis, suggesting that the inhibitors can reach the KLKs upon systemic delivery and should be suited to control deregulated protease activity in NS.

Human OTULIN haploinsufficiency impairs cell-intrinsic immunity to staphylococcal α-toxin.

The molecular basis of interindividual clinical variability upon infection with Staphylococcus aureus is unclear. We describe patients with haploinsufficiency for the linear deubiquitinase OTULIN, encoded by a gene on chromosome 5p. Patients suffer from episodes of life-threatening necrosis, typically triggered by S. aureus infection. The disorder is phenocopied in patients with the 5p- (Cri-du-Chat) chromosomal deletion syndrome. OTULIN haploinsufficiency causes an accumulation of linear ubiquitin in dermal fibroblasts, but tumor necrosis factor receptor-mediated nuclear factor κB signaling remains intact. Blood leukocyte subsets are unaffected. The OTULIN-dependent accumulation of caveolin-1 in dermal fibroblasts, but not leukocytes, facilitates the cytotoxic damage inflicted by the staphylococcal virulence factor α-toxin. Naturally elicited antibodies against α-toxin contribute to incomplete clinical penetrance. Human OTULIN haploinsufficiency underlies life-threatening staphylococcal disease by disrupting cell-intrinsic immunity to α-toxin in nonleukocytic cells.

Biological treatments for pediatric Netherton syndrome.

Netherton syndrome (NS) is a rare and potentially life-threatening genetic skin disease responsible for skin inflammation and scaling, hair abnormalities and severe allergic manifestations. NS is caused by loss-of-function variants in Serine Peptidase Inhibitor Kazal-Type 5 (SPINK5), encoding the serine protease inhibitor LEKTI. NS patients have a profound skin barrier defect caused by unopposed kallikrein-related proteases activity (KLKs). They develop severe skin inflammation with eczematous-like lesions and high serum IgE levels. Multiomics studies have revealed that the IL-17/IL-36 pathway is the most predominant upregulated pathway in NS. It is associated with a Th2 signature with complement activation in the ichthyosis linearis circumflexa subtype, and with interferon and Th9 activation in the scaly erythrodermic form. Several case reports proved the efficacy of different biotherapies targeting IL-17A, IL-12/IL-23, IL-4R and IL-13R, TNF-a and IL-1ß in pediatric NS patients. Intravenous immunoglobulins (IVIG) have also shown efficacy. These studies showed no severe side effects. At present, IL-17 blockade seems to be the most efficient treatment, but case reports remain limited with small numbers of patients and no placebo-control. Additional pathways must also be explored, and more efficient strategies could be used to block IL-17 and IL-23 pathways. In the future, the combination of specific strategies aiming at repairing the initial skin barrier defect could potentiate the efficacy of biologics. The current reports suggest that biological therapy is safe and often effective at pediatric age. However, controlled clinical trials that include a larger number of patients need to be conducted to reach more reliable conclusions.

Drug Repurposing Reveals mTOR Inhibition as a Promising Strategy for Epidermolysis Bullosa Simplex.

Drug repurposing has the potential to discover new treatments for diseases with high unmet medical needs. Lee et al. (2021) combined transcriptomics and computational analysis of drug-target databases to identify novel therapies for epidermolysis bullosa simplex. Differential gene expression analysis of blister epidermis identified the phosphoinositide 3-kinase/protein kinase B/mTOR signaling pathway as central. A pilot study using a topical mTOR inhibitor showed marked improvement.

Netherton syndrome subtypes share IL-17/IL-36 signature with distinct IFN-α and allergic responses.

BACKGROUND: Netherton syndrome (NS) is a rare recessive skin disorder caused by loss-of-function mutations in SPINK5 encoding the protease inhibitor LEKTI (lymphoepithelial Kazal-type-related inhibitor). NS patients experience severe skin barrier defects, display inflammatory skin lesions, and have superficial scaling with atopic manifestations. They present with typical ichthyosis linearis circumflexa (NS-ILC) or scaly erythroderma (NS-SE). OBJECTIVE: We used a combination of several molecular profiling methods to comprehensively characterize the skin, immune cells, and allergic phenotypes of NS-ILC and NS-SE patients. METHODS: We studied a cohort of 13 patients comprising 9 NS-ILC and 4 NS-SE. RESULTS: Integrated multiomics revealed abnormal epidermal proliferation and differentiation and IL-17/IL-36 signatures in lesion skin and in blood in both NS endotypes. Although the molecular profiles of NS-ILC and NS-SE lesion skin were very similar, nonlesion skin of each disease subtype displayed distinctive molecular features. Nonlesion and lesion NS-SE epidermis showed activation of the type I IFN signaling pathway, while lesion NS-ILC skin differed from nonlesion NS-ILC skin by increased complement activation and neutrophil infiltration. Serum cytokine profiling and immunophenotyping of circulating lymphocytes showed a TH2-driven allergic response in NS-ILC, whereas NS-SE patients displayed mainly a TH9 axis with increased CCL22/MDC and CCL17/TARC serum levels. CONCLUSIONS: This study confirms IL-17/IL-36 as the predominant signaling axes in both NS endotypes and unveils molecular features distinguishing NS-ILC and NS-SE. These results identify new therapeutic targets and could pave the way for precision medicine of NS.

Clinical trial of ABCB5+ mesenchymal stem cells for recessive dystrophic epidermolysis bullosa.

BACKGROUNDRecessive dystrophic epidermolysis bullosa (RDEB) is a rare, devastating, and life-threatening inherited skin fragility disorder that comes about due to a lack of functional type VII collagen, for which no effective therapy exists. ABCB5+ dermal mesenchymal stem cells (ABCB5+ MSCs) possess immunomodulatory, inflammation-dampening, and tissue-healing capacities. In a Col7a1-/- mouse model of RDEB, treatment with ABCB5+ MSCs markedly extended the animals' lifespans.METHODSIn this international, multicentric, single-arm, phase I/IIa clinical trial, 16 patients (aged 4-36 years) enrolled into 4 age cohorts received 3 i.v. infusions of 2 × 106 ABCB5+ MSCs/kg on days 0, 17, and 35. Patients were followed up for 12 weeks regarding efficacy and 12 months regarding safety.RESULTSAt 12 weeks, statistically significant median (IQR) reductions in the Epidermolysis Bullosa Disease Activity and Scarring Index activity (EBDASI activity) score of 13.0% (2.9%-30%; P = 0.049) and the Instrument for Scoring Clinical Outcome of Research for Epidermolysis Bullosa clinician (iscorEB­c) score of 18.2% (1.9%-39.8%; P = 0.037) were observed. Reductions in itch and pain numerical rating scale scores were greatest on day 35, amounting to 37.5% (0.0%-42.9%; P = 0.033) and 25.0% (-8.4% to 46.4%; P = 0.168), respectively. Three adverse events were considered related to the cell product: 1 mild lymphadenopathy and 2 hypersensitivity reactions. The latter 2 were serious but resolved without sequelae shortly after withdrawal of treatment.CONCLUSIONThis trial demonstrates good tolerability, manageable safety, and potential efficacy of i.v. ABCB5+ MSCs as a readily available disease-modifying therapy for RDEB and provides a rationale for further clinical evaluation.TRIAL REGISTRATIONClinicaltrials.gov NCT03529877; EudraCT 2018-001009-98.FUNDINGThe trial was sponsored by RHEACELL GmbH & Co. KG. Contributions by NYF and MHF to this work were supported by the NIH/National Eye Institute (NEI) grants RO1EY025794 and R24EY028767.

A Potent and Selective Kallikrein-5 Inhibitor Delivers High Pharmacological Activity in Skin from Patients with Netherton Syndrome.

Regulation of proteolytic activity in the skin plays a pivotal role in epidermal homeostasis. This is best exemplified in Netherton syndrome, a severe genetic skin condition caused by loss-of-function mutations in the gene serine protease inhibitor Kazal-type 5 encoding lympho-epithelial Kazal-type-related inhibitor, a serine protease inhibitor that regulates kallikrein (KLK)-related peptidase 5, 7, and 14 activities. KLK5 plays a central role in stratum corneum shedding and inflammatory cell signaling, activates KLK7 and KLK14, and is therefore an optimal therapeutic target. We aimed to identify a potent and selective small-molecule inhibitor of KLK5 amenable to epidermal delivery. GSK951 was identified using a structure-based design strategy and showed a half maximal inhibitory concentration of 250 pM for KLK5 and greater than 100-fold selectivity over KLK7 and KLK14. Cocrystal structure analysis identified the critical catalytic site interactions to a surrogate for KLK5. Topical application of GSK951-containing cream inhibited KLK5 activity in TgKLK5 mouse skin, reduced transepidermal water loss, and decreased proinflammatory cytokine expression. GSK951 achieved high concentrations in healthy human epidermis following topical application in a cream formulation. Finally, KLK5 protease activity was increased in stratum corneum of patients with Netherton syndrome and significantly inhibited by GSK951. These findings unveil a KLK5-specific small-molecule inhibitor with a high therapeutic potential for patients with Netherton syndrome.

Diagnosis of Epidermolysis Bullosa Acquisita: Multicentre Comparison of Different Assays for Serum Anti-type VII Collagen Reactivity.

Epidermolysis bullosa acquisita is a pemphigoid disease characterized by autoantibodies against type VII collagen. This study compared the sensitivity and specificity of 6 diagnostic assays: type VII collagen non-collagenous domains enzyme-linked immunoassay (NC1/2 ELISA) (MBL, Nagoya, Japan); type VII collagen NC1 ELISA (Euroimmun, Lübeck, Germany); indirect immunofluorescence (IF) microscopy test based on the expression of recombinant NC1 in a human cell line (NC1 BIOCHIP®; Euroimmun); full-length recombinant type VII collagen ELISA; immunoblotting with full-length type VII collagen in the extract of human dermis; and immunoblotting with recombinant NC1. Immunoblotting with recombinant NC1 showed a sensitivity of 93.1% and specificity of 100%, follow-ed by NC1 BIOCHIP® (sensitivity, 89.1%; specificity, 100%), immunoblotting with human dermis (sensitivity, 87.1%; specificity 100%), NC1-ELISA (sensitivity 82.2%; specificity 98.6%), NC1/NC2 ELISA (sensitivity 88.1%; specificity 93.3%), and full-length type VII collagen ELISA (sensitivity 80.2%; specificity 93.8%).