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 due to similarities with benign dermatoses such as atopic dermatitis (AD). Furthermore, the delineation from so-called "parapsoriasis en plaque," a disease that can appear either in a small- or large-plaque form, is still controversial. OBJECTIVE: 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: 6 out 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. By contrast, 6 out 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+ IL13-expressing "Th2A" cells or strong type 17 inflammation, respectively. CONCLUSION: These data position polyclonal small- and large-plaque dermatitis lesions as a separate disease entity, that characteristically harbors a so far undescribed ILC population. We thus propose the new term "polyclonal parapsoriasis en plaque" to this kind of lesions, as they can be clearly differentiated from early and advanced-stage MF, psoriasis and AD on several cellular and molecular levels.

Single-cell RNA sequencing comparison of CD4+, CD8+ and TCR-γδ+ cutaneous T-cell lymphomas reveals subset-specific molecular phenotypes.

BACKGROUND: Malignant clones of primary cutaneous T-cell lymphomas (CTCL) can show a CD4, CD8 or TCR-γδ phenotype, but their individual impact on tumor biology and skin lesion formation remains ill-defined. OBJECTIVES: To perform a comprehensive molecular characterization of CD4+ vs. CD8+ and TCR-γ/δ+ CTCL lesions. METHODS: We performed scRNA-seq of 18 CTCL skin biopsies to compare classic CD4+ advanced-stage mycosis fungoides (MF) with TCR-γ/δ+MF and primary cutaneous CD8+ aggressive epidermotropic cytotoxic T-cell lymphoma (Berti's lymphoma). RESULTS: Malignant clones of TCR-γ/δ+MF and Berti's lymphoma showed similar clustering patterns distinct from CD4+MF, along with increased expression of cytotoxic markers such as NKG7, CTSW, GZMA, and GZMM. Only advanced-stage CD4+MF clones expressed central memory T-cell markers (SELL, CCR7, LEF1), alongside B1/B2 blood involvement, whereas TCR-γ/δ+MF and Berti's lymphoma harbored a more tissue-resident phenotype (CD69, CXCR4, NR4A1) without detectable cells in the blood. CD4+MF and TCR-γ/δ+MF skin lesions harbored strong type 2 immune activation across myeloid cells, while Berti's lymphoma was more skewed towards type 1 immune responses. Both CD4+MF and TCR-γ/δ+MF lesions showed upregulation of keratinocyte hyperactivation markers such as S100As and KRT16 genes. This increase was entirely absent in Berti's lymphoma, possibly reflecting an aberrant keratinocyte response to invading tumor cells, that could contribute to the formation of the typical ulcero-necrotic lesions within this entity. CONCLUSIONS: Our scRNAseq profiling study reveals specific molecular patterns associated with distinct CTCL subtypes.

Dupilumab-associated head and neck dermatitis shows a pronounced type 22 immune signature mediated by oligoclonally expanded T cells.

Dupilumab, an IL4R-blocking antibody, has shown clinical efficacy for atopic dermatitis (AD) treatment. In addition to conjunctivitis/blepharitis, the de novo appearance of head/neck dermatitis is now recognized as a distinct side effect, occurring in up to 10% of patients. Histopathological features distinct from AD suggest a drug effect, but exact underlying mechanisms remain unknown. We profiled punch biopsies from dupilumab-associated head and neck dermatitis (DAHND) by using single-cell RNA sequencing and compared data with untreated AD and healthy control skin. We show that dupilumab treatment was accompanied by normalization of IL-4/IL-13 downstream activity markers such as CCL13, CCL17, CCL18 and CCL26. By contrast, we found strong increases in type 22-associated markers (IL22, AHR) especially in oligoclonally expanded T cells, accompanied by enhanced keratinocyte activation and IL-22 receptor upregulation. Taken together, we demonstrate that dupilumab effectively dampens conventional type 2 inflammation in DAHND lesions, with concomitant hyperactivation of IL22-associated responses.

BPA, BPAF and TMBPF Alter Adipogenesis and Fat Accumulation in Human Mesenchymal Stem Cells, with Implications for Obesity.

Bisphenol A (BPA) is an endocrine-disrupting chemical used in the production of plastics, and is linked to developmental, reproductive, and metabolic disorders including obesity. Manufacturers have begun using 'BPA-free' alternatives instead of BPA in many consumer products. However, these alternatives have had much less testing and oversight, yet they are already being mass-produced and used across industries from plastics to food-contact coatings. Here, we used human female adipose-derived stem cells (hASCs), a type of adult mesenchymal stem cell, to compare the effects of BPA and BPA alternatives on adipogenesis or fat cell development in vitro. We focused on two commonly used BPA replacements, bisphenol AF (BPAF) and tetramethyl bisphenol F (TMBPF; monomer of the new valPure V70 food-contact coating). Human ASCs were differentiated into adipocytes using chemically defined media in the presence of control differentiation media with and without 17ß-estradiol (E2; 10 µM), or with increasing doses of BPA (0, 0.1 and 1 µM), BPAF (0, 0.1, 1 and 10 nM), or TMBPF (0, 0.01 and 0.1 µM). After differentiation, the cells were stained and imaged to visualize and quantify the accumulation of lipid vacuoles and number of developing fat cells. Treated cells were also examined for cell viability and apoptosis (programmed cell death) using the respective cellular assays. Similar to E2, BPA at 0.1 µM and BPAF at 0.1 nM, significantly increased adipogenesis and lipid production by 20% compared to control differentiated cells (based on total lipid vacuole number to cell number ratios), whereas higher levels of BPA and BPAF significantly decreased adipogenesis (p < 0.005). All tested doses of TMBPF significantly reduced adipogenesis and lipid production by 30-40%, likely at least partially through toxic effects on stem cells, as viable cell numbers decreased and apoptosis levels increased throughout differentiation. These findings indicate that low, environmentally-relevant doses of BPA, BPAF, and TMBPF have significant effects on fat cell development and lipid accumulation, with TMBPF having non-estrogenic, anti-adipogenic effects. These and other recent results may provide a potential cellular mechanism between exposure to bisphenols and human obesity, and underscore the likely impact of these chemicals on fat development in vivo.

Data demonstrating distinct embryonic developmental defects induced by bisphenol a alternatives.

Embryos of Xenopus laevis (African clawed frog) were exposed to the widespread environmental plasticizers bisphenol AF (BPAF; 0.003-3 µM), bisphenol A (BPA; 1-50 µM), or 17ß-estradiol (E2; 10 µM) from just after fertilization through 96 hours of development. The potencies and cellular and morphological effects were compared across chemical treatments and controls. The embryos were staged, counted and imaged, and time-lapse movies collected, on an inverted stereomicroscope and camera. The data show there were both shared and unique effects of BPAF, BPA, and E2, on early cleavage divisions and development of the spinal cord, head, and gut, with BPAF having the greatest potency and toxicity (1000 times more potent than BPA). Specifically, cleavage divisions, within 1-6 hours of exposure had severe irregularities including asymmetrical division, slowed mitosis and cytokinesis, cellular dissociation, and fewer numbers of cells per embryo. By 48 hours of exposure the embryos had curved body axis defects, neural tube defects including curved, incomplete, or two neural tubes, ventral and gut blisters, and overall extreme abnormalities. By 96 hours of exposure estradiol caused tail flexures/bent spines, severe pigmentation reduction, long loosely coiled gut, and a ventral blister in 100% of embryos. BPA caused truncated body axis defects, tail flexures, and head and eye malformations in over 60% of embryos. BPAF, at the lowest doses tested, caused craniofacial defects, shorter tails, ventral blisters, edema and peritoneal effusion in over 75% of the surviving embryos. For a complete description, interpretation of the data and a discussion refer to the article in press Arancio et al., 2018.

Bisphenol A, Bisphenol AF, di-n-butyl phthalate, and 17ß-estradiol have shared and unique dose-dependent effects on early embryo cleavage divisions and development in Xenopus laevis.

Bisphenol A (BPA), Bisphenol AF (BPAF), and di-n-butyl phthalate (DBP) are widespread compounds used in the production of plastics. We used Xenopus laevis to compare their effects on early embryo cell division and development. Directly after in vitro fertilizations, embryos were exposed to BPA, BPAF, DBP, or 17ß-estradiol (E2) for up to 96 h. BPA (1-50 µM) and BPAF (0.003-25 µM) caused disrupted cleavage divisions, slowed cytokinesis, and cellular dissociation within 1-6 h. Flexures of the spinal cord, shorter body axis/tail, craniofacial malformations, and significant mortality occurred with environmentally relevant doses of BPAF (LC50 = 0.013 µM). DBP (10-200 µM) showed similar effects, but with severe ventral edema. There were both shared and unique effects of all compounds, with BPAF having the greatest potency and toxicity (BPAF > BPA > estradiol > DBP). These findings underscore the pleiotropic effects of widespread toxicants on early development and highlight the need for better toxicological characterization.