Single-cell sequencing delineates T-cell clonality and pathogenesis of the parapsoriasis disease group.

BACKGROUND: Mycosis fungoides (MF), the most common cutaneous T-cell lymphoma, is often underdiagnosed in early stages because of similarities with benign dermatoses such as atopic dermatitis (AD). Furthermore, the delineation from what is called "parapsoriasis en plaque", a disease that can appear either in a small- or large-plaque form, is still controversial. OBJECTIVE: We sought to characterize the parapsoriasis disease spectrum. METHODS: We performed single-cell RNA sequencing of skin biopsies from patients within the parapsoriasis-to-early-stage MF spectrum, stratified for small and large plaques, and compared them to AD, psoriasis, and healthy control skin. RESULTS: Six of 8 large-plaque lesions harbored either an expanded alpha/beta or gamma/delta T-cell clone with downregulation of CD7 expression, consistent with a diagnosis of early-stage MF. In contrast, 6 of 7 small-plaque lesions were polyclonal in nature, thereby lacking a lymphomatous phenotype, and also revealed a less inflammatory microenvironment than early-stage MF or AD. Of note, polyclonal small- and large-plaque lesions characteristically harbored a population of NPY+ innate lymphoid cells and displayed a stromal signature of complement upregulation and antimicrobial hyperresponsiveness in fibroblasts and sweat gland cells, respectively. These conditions were clearly distinct from AD or psoriasis, which uniquely harbored CD3+CRTH2+ IL-13 expressing "TH2A" cells, or strong type 17 inflammation, respectively. CONCLUSION: These data position polyclonal small- and large-plaque parapsoriasis lesions as a separate disease entity that characteristically harbors a so far undescribed innate lymphoid cell population. We thus propose a new term, "polyclonal parapsoriasis en plaque", for this kind of lesion because they can be clearly differentiated from early- and advanced-stage MF, psoriasis, and AD on several cellular and molecular levels.

Single-cell RNA sequencing defines disease-specific differences between chronic nodular prurigo and atopic dermatitis.

BACKGROUND: Chronic nodular prurigo (CNPG) is an inflammatory skin disease that is maintained by a chronic itch-scratch cycle likely rooted in neuroimmunological dysregulation. This condition may be associated with atopy in some patients, and there are now promising therapeutic results from blocking type 2 cytokines such as IL-4, IL-13, and IL-31. OBJECTIVES: This study aimed to improve the understanding of pathomechanisms underlying CNPG as well as molecular relationships between CNPG and atopic dermatitis (AD). METHODS: We profiled skin lesions from patients with CNPG in comparison with AD and healthy control individuals using single-cell RNA sequencing combined with T-cell receptor sequencing. RESULTS: We found type 2 immune skewing in both CNPG and AD, as evidenced by CD4+ helper T cells expressing IL13. However, only AD harbored an additional, oligoclonally expanded CD8A+IL9R+IL13+ cytotoxic T-cell population, and immune activation pathways were highly upregulated in AD, but less so in CNPG. Conversely, CNPG showed signatures of extracellular matrix organization, collagen synthesis, and fibrosis, including a unique population of CXCL14-IL24+ secretory papillary fibroblasts. Besides known itch mediators such as IL31 and oncostatin M, we also detected increased levels of neuromedin B in fibroblasts of CNPG lesions compared with AD and HC, with neuromedin B receptors detectable on some nerve endings. CONCLUSIONS: These data show that CNPG does not harbor the strong disease-specific immune activation pathways that are typically found in AD but is rather characterized by upregulated stromal remodeling mechanisms that might have a direct impact on itch fibers.

Single-cell analysis reveals innate lymphoid cell lineage infidelity in atopic dermatitis.

BACKGROUND: Although ample knowledge exists about phenotype and function of cutaneous T lymphocytes, much less is known about the lymphocytic components of the skin's innate immune system. OBJECTIVE: To better understand the biologic role of cutaneous innate lymphoid cells (ILCs), we investigated their phenotypic and molecular features under physiologic (normal human skin [NHS]) and pathologic (lesional skin of patients with atopic dermatitis [AD]) conditions. METHODS: Skin punch biopsies and reduction sheets as well as blood specimens were obtained from either patients with AD or healthy individuals. Cell and/or tissue samples were analyzed by flow cytometry, immunohistochemistry, and single-cell RNA sequencing or subjected to in vitro/ex vivo culture. RESULTS: Notwithstanding substantial quantitative differences between NHS and AD skin, we found that the vast majority of cutaneous ILCs belong to the CRTH2+ subset and reside in the upper skin layers. Single-cell RNA sequencing of cutaneous ILC-enriched cell samples confirmed the predominance of biologically heterogeneous group 2 ILCs and, for the first time, demonstrated considerable ILC lineage infidelity (coexpression of genes typical of either type 2 [GATA3 and IL13] or type 3/17 [RORC, IL22, and IL26] immunity within individual cells) in lesional AD skin, and to a much lesser extent, in NHS. Similar events were demonstrated in ILCs from skin explant cultures and in vitro expanded ILCs from the peripheral blood. CONCLUSION: These findings support the concept that instead of being a stable entity with well-defined components, the skin immune system consists of a network of highly flexible cellular players that are capable of adjusting their function to the needs and challenges of the environment.

Single-cell RNA sequencing reveals markers of disease progression in primary cutaneous T-cell lymphoma.

BACKGROUND: In early-stage mycosis fungoides (MF), the most common primary cutaneous T-cell lymphoma, limited skin involvement with patches and plaques is associated with a favorable prognosis. Nevertheless, approximately 20-30% of cases progress to tumors or erythroderma, resulting in poor outcome. At present, factors contributing to this switch from indolent to aggressive disease are only insufficiently understood. METHODS: In patients with advanced-stage MF, we compared patches with longstanding history to newly developed plaques and tumors by using single-cell RNA sequencing, and compared results with early-stage MF as well as nonlesional MF and healthy control skin. RESULTS: Despite considerable inter-individual variability, lesion progression was uniformly associated with downregulation of the tissue residency markers CXCR4 and CD69, the heat shock protein HSPA1A, the tumor suppressors and immunoregulatory mediators ZFP36 and TXNIP, and the interleukin 7 receptor (IL7R) within the malignant clone, but not in benign T cells. This phenomenon was not only found in conventional TCR-αß MF, but also in a case of TCR-γδ MF, suggesting a common mechanism across MF subtypes. Conversely, malignant cells in clinically unaffected skin from MF patients showed upregulation of these markers. CONCLUSIONS: Our data reveal a specific panel of biomarkers that might be used for monitoring MF disease progression. Altered expression of these genes may underlie the switch in clinical phenotype observed in advanced-stage MF.

Single-cell transcriptomics combined with interstitial fluid proteomics defines cell type-specific immune regulation in atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is the most common chronic inflammatory skin disease, but its complex pathogenesis is only insufficiently understood, resulting in still limited treatment options. OBJECTIVE: We sought to characterize AD on both transcriptomic and proteomic levels in humans. METHODS: We used skin suction blistering, a painless and nonscarring procedure that can simultaneously sample skin cells and interstitial fluid. We then compared results with conventional biopsies. RESULTS: Suction blistering captured epidermal and most immune cells equally well as biopsies, except for mast cells and nonmigratory CD163+ macrophages that were only present in biopsy isolates. Using single-cell RNA sequencing, we found comparable transcriptional profiles of key inflammatory pathways between blister and biopsy AD, but suction blistering was superior in cell-specific resolution for high-abundance transcripts (KRT1/KRT10, KRT16/KRT6A, S100A8/S100A9), which showed some background signals in biopsy isolates. Compared with healthy controls, we found characteristic upregulation of AD-typical cytokines such as IL13 and IL22 in Th2 and Th22 cells, respectively, but we also discovered these mediators in proliferating T cells and natural killer T cells, that also expressed the antimicrobial cytokine IL26. Overall, not T cells, but myeloid cells were most strongly enriched in AD, and we found dendritic cell (CLEC7A, amphiregulin/AREG, EREG) and macrophage products (CCL13) among the top upregulated proteins in AD blister fluid proteomic analyses. CONCLUSION: These data show that by using cutting-edge technology, suction blistering offers several advantages over conventional biopsies, including better transcriptomic resolution of skin cells, combined with proteomic information from interstitial fluid, unraveling novel inflammatory players that shape the cellular and proteomic microenvironment of AD.

Evidence that a neutrophil-keratinocyte crosstalk is an early target of IL-17A inhibition in psoriasis.

The response of psoriasis to antibodies targeting the interleukin (IL)-23/IL-17A pathway suggests a prominent role of T-helper type-17 (Th17) cells in this disease. We examined the clinical and immunological response patterns of 100 subjects with moderate-to-severe psoriasis receiving 3 different intravenous dosing regimens of the anti-IL-17A antibody secukinumab (1 × 3 mg/kg or 1 × 10 mg/kg on Day 1, or 3 × 10 mg/kg on Days 1, 15 and 29) or placebo in a phase 2 trial. Baseline biopsies revealed typical features of active psoriasis, including epidermal accumulation of neutrophils and formation of microabscesses in >60% of cases. Neutrophils were the numerically largest fraction of infiltrating cells containing IL-17 and may store the cytokine preformed, as IL-17A mRNA was not detectable in neutrophils isolated from active plaques. Significant clinical responses to secukinumab were observed 2 weeks after a single infusion, associated with extensive clearance of cutaneous neutrophils parallel to the normalization of keratinocyte abnormalities and reduction of IL-17-inducible neutrophil chemoattractants (e.g. CXCL1, CXCL8); effects on numbers of T cells and CD11c-positive dendritic cells were more delayed. Histological and immunological improvements were generally dose dependent and not observed in the placebo group. In the lowest-dose group, a recurrence of neutrophils was seen in some subjects at Week 12; these subjects relapsed faster than those without microabscesses. Our findings are indicative of a neutrophil-keratinocyte axis in psoriasis that may involve neutrophil-derived IL-17 and is an early target of IL-17A-directed therapies such as secukinumab.

Temporal association between COVID-19 vaccination and Raynaud's phenomenon: A case series.

COVID-19 vaccine-related adverse events are mostly minor to moderate, and serious events are rare. Single cases of Raynaud's phenomenon (RP) in temporal proximity to COVID-19 vaccination have been reported. Demographic data, medical history, and detailed information regarding vaccination status and RP characteristics were obtained from patients with confirmed RP after COVID-19 vaccination. Fifteen participants reported the initial manifestation of RP, which occurred in 40% after the first, in 33% after the second, and in 27% after the third vaccination. RP development and occurrence of episodes were not linked to any specific vaccine type. New onset of disease was observed in 40% of the vaccinees after BNT162b2, in 33% after mRNA-1273, and in 27% after ChAdOx1 vaccination. Three out of four participants with preexisting RP prior to COVID-19 vaccination reported aggravation in frequency and intensity after immunization. Although COVID-19 vaccination is pivotal in controlling the pandemic, the observed temporal association between vaccine administration and RP occurrence warrants global activities to support pharmacovigilance for the detection of adverse reactions, one of which may include RP.

Comprehensive Analysis of Nasal Polyps Reveals a More Pronounced Type 2 Transcriptomic Profile of Epithelial Cells and Mast Cells in Aspirin-Exacerbated Respiratory Disease.

Chronic rhinosinusitis with nasal polyps is affecting up to 3% of Western populations. About 10% of patients with nasal polyps also suffer from asthma and intolerance to aspirin, a syndrome called aspirin-exacerbated respiratory disease. Although eosinophilic inflammation is predominant in polyps of both diseases, phenotypic differences in the tissue-derived microenvironment, elucidating disease-specific characteristics, have not yet been identified. We sought to obtain detailed information about phenotypic and transcriptional differences in epithelial and immune cells in polyps of aspirin-tolerant and intolerant patients. Cytokine profiles in nasal secretions and serum of patients suffering from aspirin-exacerbated respiratory disease (n = 10) or chronic rhinosinusitis with nasal polyps (n = 9) were assessed using a multiplex mesoscale discovery assay. After enrichment for immune cell subsets by flow cytometry, we performed transcriptomic profiling by employing single-cell RNA sequencing. Aspirin-intolerant patients displayed significantly elevated IL-5 and CCL17 levels in nasal secretions corresponding to a more pronounced eosinophilic type 2 inflammation. Transcriptomic profiling revealed that epithelial and mast cells not only complement one another in terms of gene expression associated with the 15-lipoxygenase pathway but also show a clear type 2-associated inflammatory phenotype as identified by the upregulation of POSTN, CCL26, and IL13 in patients with aspirin-exacerbated respiratory disease. Interestingly, we also observed cellular stress responses indicated by an increase of MTRNR2L12, MTRNR2L8, and NEAT1 across all immune cell subsets in this disease entity. In conclusion, our findings support the hypothesis that epithelial and mast cells act in concert as potential drivers of the pathogenesis of the aspirin-exacerbated respiratory disease.

Single-Cell RNA Sequencing Reveals Tissue Compartment-Specific Plasticity of Mycosis Fungoides Tumor Cells.

Mycosis fungoides (MF) is the most common primary cutaneous T-cell lymphoma. While initially restricted to the skin, malignant cells can appear in blood, bone marrow and secondary lymphoid organs in later disease stages. However, only little is known about phenotypic and functional properties of malignant T cells in relationship to tissue environments over the course of disease progression. We thus profiled the tumor micromilieu in skin, blood and lymph node in a patient with advanced MF using single-cell RNA sequencing combined with V-D-J T-cell receptor sequencing. In skin, we identified clonally expanded T-cells with characteristic features of tissue-resident memory T-cells (TRM, CD69+CD27-NR4A1+RGS1+AHR+ ). In blood and lymph node, the malignant clones displayed a transcriptional program reminiscent of a more central memory-like phenotype (KLF2+TCF7+S1PR1+SELL+CCR7+ ), while retaining tissue-homing receptors (CLA, CCR10). The skin tumor microenvironment contained potentially tumor-permissive myeloid cells producing regulatory (IDO1) and Th2-associated mediators (CCL13, CCL17, CCL22). Given their expression of PVR, TNFRSF14 and CD80/CD86, they might be under direct control by TIGIT+CTLA4+CSF2+TNFSF14+ tumor cells. In sum, this study highlights the adaptive phenotypic and functional plasticity of MF tumor cell clones. Thus, the TRM-like phenotype enables long-term skin residence of MF cells. Their switch to a TCM-like phenotype with persistent skin homing molecule expression in the circulation might explain the multi-focal nature of MF.

Persistence of mature dendritic cells, TH2A, and Tc2 cells characterize clinically resolved atopic dermatitis under IL-4Rα blockade.

Therapeutic options for autoimmune diseases typically consist of broad and targeted immunosuppressive agents. However, sustained clinical benefit is rarely achieved, as the disease phenotype usually returns after cessation of treatment. To better understand tissue-resident immune memory in human disease, we investigated patients with atopic dermatitis (AD) who underwent short-term or long-term treatment with the IL-4Rα blocker dupilumab. Using multi-omics profiling with single-cell RNA sequencing and multiplex proteomics, we found significant decreases in overall skin immune cell counts and normalization of transcriptomic dysregulation in keratinocytes consistent with clearance of disease. However, we identified specific immune cell populations that persisted for up to a year after clinical remission while being absent from healthy controls. These populations included LAMP3 + CCL22+ mature dendritic cells, CRTH2 + CD161 + T helper ("TH2A") cells, and CRTAM + cytotoxic T cells, which expressed high levels of CCL17 (dendritic cells) and IL13 (T cells). TH2A cells showed a characteristic cytokine receptor constellation with IL17RB, IL1RL1 (ST2), and CRLF2 expression, suggesting that these cells are key responders to the AD-typical epidermal alarmins IL-25, IL-33, and TSLP, respectively. We thus identified disease-linked immune cell populations in resolved AD indicative of a persisting disease memory, facilitating a rapid response system of epidermal-dermal cross-talk between keratinocytes, dendritic cells, and T cells. This observation may help to explain the disease recurrence upon termination of immunosuppressive treatments in AD, and it identifies potential disease memory-linked cell types that may be targeted to achieve a more sustained therapeutic response.

Euphorbia myrsinites Sap-Induced Phytodermatitis: A Prototype of Irritant Contact Dermatitis?

BACKGROUND: A hallmark of Euphorbia myrsinites (EM), a member of the widespread perennial Euphorbia species, is the extrusion of a poisonous, latex-like sap irritant to the skin and eye after contact. The exact mechanisms underlying these effects have not been unraveled so far. OBJECTIVES: The aims of the study were to allocate EM sap-induced phytodermatitis to irritant or allergic contact dermatitis (ACD) and to investigate mechanism(s) causing keratinocyte damage. METHODS: Cutaneous effects of EM sap on healthy human skin were investigated by clinical scoring and reflectance confocal microscopy analyses and compared with ACD. In addition, the effects of sap exposure to keratinocytes were analyzed in vitro using histological analyses and flow cytometry. CONCLUSIONS: We report on 2 cases of EM sap-induced phytodermatitis. Patch testing with fresh EM sap induced dermatitis in 100% of the tested sites with a clinical course following a decrescendo pattern. Compared with ACD, the lesional phenotype was more severe and epidermal disruption was more pronounced. Exposure of human skin tissues and cultivated keratinocytes to EM sap in vitro resulted in a dose-dependent increase in keratinocyte apoptosis. The reported findings support the primarily toxic irritant nature of EM sap-induced phytodermatitis. The contribution of ingenol mebutate to (nontoxic) proinflammatory effects remains to be elucidated.

Epigenomics and Single-Cell Sequencing Define a Developmental Hierarchy in Langerhans Cell Histiocytosis.

Langerhans cell histiocytosis (LCH) is a rare neoplasm predominantly affecting children. It occupies a hybrid position between cancers and inflammatory diseases, which makes it an attractive model for studying cancer development. To explore the molecular mechanisms underlying the pathophysiology of LCH and its characteristic clinical heterogeneity, we investigated the transcriptomic and epigenomic diversity in primary LCH lesions. Using single-cell RNA sequencing, we identified multiple recurrent types of LCH cells within these biopsies, including putative LCH progenitor cells and several subsets of differentiated LCH cells. We confirmed the presence of proliferative LCH cells in all analyzed biopsies using IHC, and we defined an epigenomic and gene-regulatory basis of the different LCH-cell subsets by chromatin-accessibility profiling. In summary, our single-cell analysis of LCH uncovered an unexpected degree of cellular, transcriptomic, and epigenomic heterogeneity among LCH cells, indicative of complex developmental hierarchies in LCH lesions. SIGNIFICANCE: This study sketches a molecular portrait of LCH lesions by combining single-cell transcriptomics with epigenome profiling. We uncovered extensive cellular heterogeneity, explained in part by an intrinsic developmental hierarchy of LCH cells. Our findings provide new insights and hypotheses for advancing LCH research and a starting point for personalizing therapy.See related commentary by Gruber et al., p. 1343.This article is highlighted in the In This Issue feature, p. 1325.

Human and Mouse Hematopoietic Stem Cells Are a Depot for Dormant Mycobacterium tuberculosis.

An estimated third of the world's population is latently infected with Mycobacterium tuberculosis (Mtb), with no clinical signs of tuberculosis (TB), but lifelong risk of reactivation to active disease. The niches of persisting bacteria during latent TB infection remain unclear. We detect Mtb DNA in peripheral blood selectively in long-term repopulating pluripotent hematopoietic stem cells (LT-pHSCs) as well as in mesenchymal stem cells from latently infected human donors. In mice infected with low numbers of Mtb, that do not develop active disease we, again, find LT-pHSCs selectively infected with Mtb. In human and mouse LT-pHSCs Mtb are stressed or dormant, non-replicating bacteria. Intratracheal injection of Mtb-infected human and mouse LT-pHSCs into immune-deficient mice resuscitates Mtb to replicating bacteria within the lung, accompanied by signs of active infection. We conclude that LT-pHSCs, together with MSCs of Mtb-infected humans and mice serve as a hitherto unappreciated quiescent cellular depot for Mtb during latent TB infection.

In Situ Mapping of Innate Lymphoid Cells in Human Skin: Evidence for Remarkable Differences between Normal and Inflamed Skin.

Although innate lymphoid cells (ILCs) have recently been identified also in skin, their role in this organ remains poorly understood. In this study, we aimed at developing a technique to assess ILCs in situ and to determine their topographical distribution in human skin. We collected lesional skin biopsies from patients with atopic dermatitis and psoriasis (both n = 13) and normal human skin from healthy controls. After establishing immunofluorescence ILC in situ stainings, we developed an analysis approach (gating combined with manual validation) to reliably identify ILCs. Topographical mapping was obtained by automated calculations of the distances between ILCs and different cellular/structural elements of the skin. Whereas normal human skin harbored a very scarce ILC population (mostly ILC1s and AHR+ILC3s), atopic dermatitis and psoriasis skin was infiltrated by clearly visible ILC subsets. We observed atopic dermatitis skin to contain not only ILC2s but also a prominent AHR+ILC3 population. Conversely, we encountered almost equal proportions of ILC1s and RORC+ILC3s in psoriasis skin. Distance calculations revealed ILCs to reside near the epidermis and in close proximity to T lymphocytes. ILC mapping in situ will provide valuable information about their likely communication partners in normal and diseased skin and forms the basis for the appropriate mechanistic studies.