Published papers

Atopic dermatitis (AD) is a multifactorial disease with underlying barrier disruption and altered microbial flora, resulting in dry skin and eczematous inflammation with persistent pruritis. Mouse models have been heavily used to investigate AD pathophysiology. Among various AD mouse models, AD-like inflammation induced by topical calcipotriol, a vitamin D3 analog referred to as MC903 in experimental settings, is a versatile model that can be applied to any strain of mice, which can be used for immunologic and morphologic studies. Herein, we provide basic protocols for the topical application of MC903 and approaches to assess phenotypes. After inducing AD-like inflammation, the skin is harvested for flow cytometry analysis, as well as for histologic and immunofluorescence microscopy analyses. The combination of these approaches enables accurate characterization of the degree of inflammation, type of inflammatory infiltrate, and localization of immune infiltrates. Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Application of MC903 and gross phenotype assessment. Basic Protocol 2: Processing skin for flow cytometry analysis. Support Protocol: Skin immune cell surface staining and flow cytometry analysis. Basic Protocol 3: Harvesting skin for histologic analysis. Basic Protocol 4: Immunofluorescence staining to identify immune cell infiltrates.

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are life-threatening mucocutaneous adverse drug reactions characterized by massive epidermal detachment. Cytotoxic T cells and associated effector molecules are known to drive SJS/TEN pathophysiology, but the contribution of innate immune responses is not well understood. We describe a mechanism by which neutrophils triggered inflammation during early phases of SJS/TEN. Skin-infiltrating CD8+ T cells produced lipocalin-2 in a drug-specific manner, which triggered the formation of neutrophil extracellular traps (NETs) in early lesional skin. Neutrophils undergoing NETosis released LL-37, an antimicrobial peptide, which induced formyl peptide receptor 1 (FPR1) expression by keratinocytes. FPR1 expression caused keratinocytes to be vulnerable to necroptosis that caused further release of LL-37 by necroptotic keratinocytes and induced FPR1 expression on surrounding keratinocytes, which likely amplified the necroptotic response. The NETs-necroptosis axis was not observed in less severe cutaneous adverse drug reactions, autoimmune diseases, or neutrophil-associated disorders, suggesting that this was a process specific to SJS/TEN. Initiation and progression of SJS/TEN keratinocyte necroptosis appear to involve a cascade of events mediated by innate and adaptive immune responses, and understanding these responses may contribute to the identification of diagnostic markers or therapeutic targets for these adverse drug reactions.

The human skin microbiome is intricately intertwined with host immunity. While studies have elucidated microbial influences on immunity, understanding how immune alterations modulate this equilibrium remains limited. We investigated the dual impact of immune deficiency and hematopoietic stem cell transplantation (HSCT) on the skin microbiome in 24 patients with dedicator of cytokinesis 8 (DOCK8) deficiency, a rare inborn error of immunity. Analyzing 590 metagenomic and 534 16S rDNA sequencing samples across eight skin sites, we observed disrupted microbiota pre-HSCT (median eukaryotic viruses 67.6% vs. 0.04% in controls), with extremely diverse human papillomaviruses (HPVs) and polyomaviruses—including oncogenic viruses. Specific bacterial species markedly changed, including decreased Staphylococcus aureus post-HSCT. DNA eukaryotic viruses dramatically decreased (79.7% ± 28.3% to 4.9% ± 8.6%; p < 0.01) 12 months post-HSCT. Recovered microbial communities remained relatively stable through 1-year follow-up with clearance of oncogenic HPV and no convergence with transplant donors. These results highlight the immune system's critical role in restoring microbial balance and skin health.

Stevens-Johnson Syndrome/Toxic Epidermal Necrolysis (SJS/TEN) is a predominantly drug-induced disease, with a mortality rate of 15–20%, that engages the expertise of multiple disciplines: dermatology, allergy, immunology, clinical pharmacology, burn surgery, ophthalmology, urogynecology, and psychiatry. SJS/TEN has an incidence of 1–5/million persons per year in the United States, with even higher rates globally. One of the challenges of SJS/TEN has been developing the research infrastructure and coordination to answer questions capable of transforming clinical care and leading to improved patient outcomes. SJS/TEN 2021, the third research meeting of its kind, was held as a virtual meeting on August 28–29, 2021. The meeting brought together 428 international scientists, in addition to a community of 140 SJS/TEN survivors and family members. The goal of the meeting was to brainstorm strategies to support the continued growth of an international SJS/TEN research network, bridging science and the community. The community workshop section of the meeting focused on eight primary themes: mental health, eye care, SJS/TEN in children, non-drug induced SJS/TEN, long-term health complications, new advances in mechanisms and basic science, managing long-term scarring, considerations for skin of color, and COVID-19 vaccines. The meeting featured several important updates and identified areas of unmet research and clinical need that will be highlighted in this white paper.

Abstract – Chronic itch can arise from a variety of etiologies, ranging from dermatological conditions like eczema and psoriasis to systemic diseases such as liver disease and kidney failure. However, it remains unclear whether there are common molecular features associated with chronic itch, and whether these features are selective for chronic itch compared to chronic pain. To identify potential genes or molecular characteristics that are specifically associated with chronic itch, we examined transcriptomic data from sensory neurons collected from 3 mouse models of chronic itch and a monkey model of contact dermatitis. We compared these data to transcriptomic data from 3 mouse models of pain and clinical data from patients with neuropathic pain. Our analyses revealed that the upregulation of Nppb expression in sensory neurons is consistently associated with models of itch, but not with models of pain. Further, our cellular characterization showed that the increased expression of Nppb arises from increased cell-autonomous expression rather than the recruitment of Nppb expression in other classes of sensory neurons. Given that Nppb is a well-established itch neurotransmitter, our findings suggest that the increased expression of Nppb in sensory neurons may contribute to chronic itch. In addition, based on our results, we propose that Nppb could serve as a conserved biomarker for chronic itch.

Commensals shape host physiology through molecular crosstalk with host receptors. Identifying specific microbial factors that causally influence host immunity is key to understanding homeostasis at the host-microbe interface and advancing microbial-based therapeutics. Here, we identified trehalose monocorynomycolate (TMCM) from Corynebacterium mastitidis as a potent stimulator of interleukin 17 (IL-17) production by Vγ4 γδ T cells at the ocular surface. Mechanistically, TMCM-induced IL-17 responses depended on IL-1R and γδ T cell receptor (TCR) signaling, with TCR engagement further enhancing IL-1R1 expression on γδ T cells. Synthetic TMCM alone recapitulated the effect of Corynebacterium mastitidis in eliciting protective γδ T cell immunity at the ocular surface to prevent bacterial infection. Moreover, TMCM also promoted protective immunity in downstream eye-draining tissues and skin. These findings establish TMCM as a broadly applicable mediator of commensal-driven immune defense and highlight its therapeutic potential to strengthen IL-17-mediated protection at barrier sites.

Hair follicles (HFs) function as hubs for stem cells, immune cells, and commensal microbes, which must be tightly regulated during homeostasis and transient inflammation. Here we found that transmembrane endopeptidase ADAM10 expression in upper HFs was crucial for regulating the skin microbiota and protecting HFs and their stem cell niche from inflammatory destruction. Ablation of the ADAM10-Notch signaling axis impaired the innate epithelial barrier and enabled Corynebacterium species to predominate the microbiome. Dysbiosis triggered group 2 innate lymphoid cell-mediated inflammation in an interleukin-7 (IL-7) receptor-, S1P receptor 1-, and CCR6-dependent manner, leading to pyroptotic cell death of HFs and irreversible alopecia. Double-stranded RNA-induced ablation models indicated that the ADAM10-Notch signaling axis bolsters epithelial innate immunity by promoting β-defensin-6 expression downstream of type I interferon responses. Thus, ADAM10-Notch signaling axis-mediated regulation of host-microbial symbiosis crucially protects HFs from inflammatory destruction, which has implications for strategies to sustain tissue integrity during chronic inflammation.

The IL-2 receptor α chain (IL-2Rα/CD25) is constitutively expressed on double-negative (DN2/DN3 thymocytes and regulatory T cells (Tregs) but induced by IL-2 on T and natural killer (NK) cells, with Il2ra expression regulated by a STAT5-dependent super-enhancer. We investigated CD25 regulation and function using a series of mice with deletions spanning STAT5-binding elements. Deleting the upstream super-enhancer region mainly affected constitutive CD25 expression on DN2/DN3 thymocytes and Tregs, with these mice developing autoimmune alopecia, whereas deleting an intronic region decreased IL-2–induced CD25 on peripheral T and NK cells. Thus, distinct super-enhancer elements preferentially control constitutive versus inducible expression in a cell type–specific manner. The mediator-1 coactivator colocalized with specific STAT5-binding sites. Moreover, both upstream and intronic regions had extensive chromatin interactions, and deletion of either region altered the super-enhancer structure in mature T cells. These results demonstrate differential functions for distinct super-enhancer elements, thereby indicating previously unknown ways to manipulate CD25 expression in a cell type–specific fashion.

Skin is our body's outermost physical barrier and an immunological interface enriched with various immune and non-immune cells. However, efficient generation of single-cell suspensions for flow cytometry analysis can be challenging. Here, we provide protocols to obtain epidermal and whole skin cell suspensions as well as gating strategies to identify mouse keratinocytes and skin immune cell subsets via flow cytometry. For complete details on the use and execution of this protocol, please refer to Sakamoto et al. (2021).