Shadmehr Demehri

Skin-derived TSLP triggers progression from epidermal- barrier defects to asthma

A skin-derived cytokine with high systemic availability provides a mechanistic explanation for atopic march and highlights a potential therapeutic target for preventing the development of asthma among people with atopic dermatitis.

Notch-Deficient Skin Induces a Lethal Systemic B-Lymphoproliferative Disorder by Secreting TSLP, a Sentinel for Epidermal Integrity

Epidermal keratinocytes form a highly organized stratified epithelium and sustain a competent barrier function together with dermal and hematopoietic cells. The Notch signaling pathway is a critical regulator of epidermal integrity. Here, we show that keratinocyte-specific deletion of total Notch signaling triggered a severe systemic B-lymphoproliferative disorder, causing death. RBP-j is the DNA binding partner of Notch, but both RBP-j–dependent and independent Notch signaling were necessary for proper epidermal differentiation and lipid deposition. Loss of both pathways caused a persistent defect in skin differentiation/barrier formation. In response, high levels of thymic stromal lymphopoietin (TSLP) were released into systemic circulation by Notch-deficient keratinocytes that failed to differentiate, starting in utero. Exposure to high TSLP levels during neonatal hematopoiesis resulted in drastic expansion of peripheral pre- and immature B-lymphocytes, causing B-lymphoproliferative disorder associated with major organ infiltration and subsequent death, a previously unappreciated systemic effect of TSLP. These observations demonstrate that local skin perturbations can drive a lethal systemic disease and have important implications for a wide range of humoral and autoimmune diseases with skin manifestations.

Atopic dermatitis-like disease and associated lethal myeloproliferative disorder arise from loss of Notch signaling in the murine skin

Background The Notch pathway is essential for proper epidermal differentiation during embryonic skin development. Moreover, skin specific loss of Notch signaling in the embryo results in skin barrier defects accompanied by a B-lymphoproliferative disease. However, much less is known about the consequences of loss of Notch signaling after birth. Methodology and Principal Findings To study the function of Notch signaling in the skin of adult mice, we made use of a series of conditional gene targeted mice that allow inactivation of several components of the Notch signaling pathway specifically in the skin. We demonstrate that skin-specific inactivation of Notch1 and Notch2 simultaneously, or RBP-J, induces the development of a severe form of atopic dermatitis (AD), characterized by acanthosis, spongiosis and hyperkeratosis, as well as a massive dermal infiltration of eosinophils and mast cells. Likewise, patients suffering from AD, but not psoriasis or lichen planus, have a marked reduction of Notch receptor expression in the skin. Loss of Notch in keratinocytes induces the production of thymic stromal lymphopoietin (TSLP), a cytokine deeply implicated in the pathogenesis of AD. The AD-like associated inflammation is accompanied by a myeloproliferative disorder (MPD) characterized by an increase in immature myeloid populations in the bone marrow and spleen. Transplantation studies revealed that the MPD is cell non-autonomous and caused by dramatic microenvironmental alterations. Genetic studies demontrated that G-CSF mediates the MPD as well as changes in the bone marrow microenvironment leading to osteopenia. Significance Our data demonstrate a critical role for Notch in repressing TSLP production in keratinocytes, thereby maintaining integrity of the skin and the hematopoietic system.

Senescent stromal-derived osteopontin promotes preneoplastic cell growth.

Alterations in the tissue microenvironment collaborate with cell autonomous genetic changes to contribute to neoplastic progression. The importance of the microenvironment in neoplastic progression is underscored by studies showing that fibroblasts isolated from a tumor stimulate the growth of preneoplastic and neoplastic cells in xenograft models. Similarly, senescent fibroblasts promote preneoplastic cell growth in vitro and in vivo. Because senescent cells accumulate with age, their presence is hypothesized to facilitate preneoplastic cell growth and tumor formation in older individuals. To identify senescent stromal factors directly responsible for stimulating preneoplastic cell growth, we carried out whole-genome transcriptional profiling and compared senescent fibroblasts with their younger counterparts. We identified osteopontin (OPN) as one of the most highly elevated transcripts in senescent fibroblasts. Importantly, reduction of OPN protein levels by RNA interference did not affect senescence induction in fibroblasts; however, it dramatically reduced the growth-promoting activities of senescent fibroblasts in vitro and in vivo, showing that OPN is necessary for paracrine stimulation of preneoplastic cell growth. In addition, we found that recombinant OPN was sufficient to stimulate preneoplastic cell growth. Finally, we show that OPN is expressed in senescent stroma within preneoplastic lesions that arise following 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate treatment of mice, suggesting that stromal-derived OPN-mediated signaling events affect neoplastic progression.

Elevated Epidermal Thymic Stromal Lymphopoietin Levels Establish an Antitumor Environment in the Skin

Thymic Stromal Lymphopoietin (TSLP), a cytokine implicated in induction of T helper 2 (Th2)-mediated allergic inflammation, has recently been shown to stimulate solid tumor growth and metastasis. Conversely, studying mice with clonal loss of Notch signaling in their skin revealed that high levels of TSLP released by barrier-defective skin caused a severe inflammation, resulting in gradual elimination of Notch-deficient epidermal clones and resistance to skin tumorigenesis. We found CD4(+) T cells to be both required and sufficient to mediate these effects of TSLP. Importantly, TSLP overexpression in wild-type skin also caused resistance to tumorigenesis, confirming that TSLP functions as a tumor suppressor in the skin.

Bi-compartmental communication contributes to the opposite proliferative behavior of Notch1-deficient hair follicle and epidermal keratinocytes.

Notch1-deficient epidermal keratinocytes become progressively hyperplastic and eventually produce tumors. By contrast, Notch1-deficient hair matrix keratinocytes have lower mitotic rates, resulting in smaller follicles with fewer cells. In addition, the ratio of melanocytes to keratinocytes is greatly reduced in hair follicles. Investigation into the underlying mechanism for these phenotypes revealed significant changes in the Kit, Tgfβ and insulin-like growth factor (IGF) signaling pathways, which have not been previously shown to be downstream of Notch signaling. The level of Kitl (Scf) mRNA produced by Notch1-deficient follicular keratinocytes was reduced when compared with wild type, resulting in a decline in melanocyte population. Tgfβ ligands were elevated in Notch1-deficient keratinocytes, which correlated with elevated expression of several targets, including the diffusible IGF antagonist Igfbp3 in the dermal papilla. Diffusible stromal targets remained elevated in the absence of epithelial Tgfβ receptors, consistent with paracrine Tgfβ signaling. Overexpression of Igf1 in the keratinocyte reversed the phenotype, as expected if Notch1 loss altered the IGF/insulin-like growth factor binding protein (IGFBP) balance. Conversely, epidermal keratinocytes contained less stromal Igfbp4 and might thus be primed to experience an increase in IGF signaling as animals age. These results suggest that Notch1 participates in a bi-compartmental signaling network that controls homeostasis, follicular proliferation rates and melanocyte population within the skin.

Notch signaling in bulge stem cells is not required for selection of hair follicle fate

Notch signaling plays an important role in hair follicle maintenance, and it has been suggested that Notch is also required for follicular fate selection by adult hair follicle stem cells in the bulge. Here we demonstrate that, on the contrary, Notch signaling in bi-potential bulge stem cells or their uncommitted descendents acts to suppress the epidermal fate choice, thus ensuring follicular fate selection. To examine the role of Notch signaling in adult hair follicle stem cells, we used a Krt1-15-CrePR1 transgenic mouse line to delete Rbpj or all Notch proteins specifically in the bulge stem cells. We conclusively determined that in the absence of Notch signaling, bulge stem cell descendents retain their capacity to execute the follicular differentiation program but fail to maintain it owing to their genetic deficiency. The defect in terminal differentiation caused the diversion of Notch-deficient hair follicles to epidermal cysts, and the presence of wild-type cells could not prevent this conversion. Importantly, our analysis revealed that a functional Notch signaling pathway was required to block bulge stem cells from migrating into, and assuming the fate of, interfollicular epidermis. Taken together, our findings yield detailed insight into the function of Notch signaling in hair follicle stem cells and reveal the mechanism of the replacement of Notch-deficient adult hair follicles by epidermal cysts.

Chronic itch development in sensory neurons requires BRAF signaling pathways

Chronic itch, or pruritus, is associated with a wide range of skin abnormalities. The mechanisms responsible for chronic itch induction and persistence remain unclear. We developed a mouse model in which a constitutively active form of the serine/threonine kinase BRAF was expressed in neurons gated by the sodium channel Nav1.8 (BRAF(Nav1.8) mice). We found that constitutive BRAF pathway activation in BRAF(Nav1.8) mice results in ectopic and enhanced expression of a cohort of itch-sensing genes, including gastrin-releasing peptide (GRP) and MAS-related GPCR member A3 (MRGPRA3), in nociceptors expressing transient receptor potential vanilloid 1 (TRPV1). BRAF(Nav1.8) mice showed de novo neuronal responsiveness to pruritogens, enhanced pruriceptor excitability, and heightened evoked and spontaneous scratching behavior. GRP receptor expression was increased in the spinal cord, indicating augmented coding capacity for itch subsequent to amplified pruriceptive inputs. Enhanced GRP expression and sustained ERK phosphorylation were observed in sensory neurons of mice with allergic contact dermatitis– or dry skin–elicited itch; however, spinal ERK activation was not required for maintaining central sensitization of itch. Inhibition of either BRAF or GRP signaling attenuated itch sensation in chronic itch mouse models. These data uncover RAF/MEK/ERK signaling as a key regulator that confers a subset of nociceptors with pruriceptive properties to initiate and maintain long-lasting itch sensation.

Desmoglein 4 is regulated by transcription factors implicated in hair shaft differentiation

The hair fiber is made of specialized keratinocytes, known as trichocytes, that primarily express hair keratins, which are cemented by a multitude of keratin-associated proteins (KAPs). The hair keratins form the intermediate filament cytoskeleton of the trichocytes, which are linked to abundant cell-cell adhesion junctions, called desmosomes. Desmoglein 4 (DSG4) is the major desmosomal cadherin expressed in the hair shaft cortex where the hair keratins are highly expressed. In humans, mutations affecting either the hair keratins or DSG4 lead to beaded hair phenotypes with features of monilethrix. In this work, we postulated that the regulatory pathways governing the expression of hair shaft components, such as hair keratins and DSG4, are shared. Therefore, we studied the transcriptional regulation of DSG4 by transcription factors/pathways that are known regulators of hair keratin or KAP expression. We show that HOXC13, LEF1 and FOXN1 repress DSG4 transcription and provide in vitro and in vivo evidence correlating the Notch pathway with the activation and/or maintenance of DSG4 expression in the hair follicle.

The absence of a microbiota enhances TSLP expression in mice with defective skin barrier but does not affect the severity of their allergic inflammation

Evidence is accumulating to suggest that our indigenous microbial communities (microbiota) may play a role in modulating allergic and immune disorders of the skin (Gallo and Nakatsuji, 2011; Macia et al., 2012). To examine the link between the microbiota and atopic dermatitis, we examined a mouse model of defective cutaneous barrier function with an atopic dermatitis-like disease due to loss of Notch signaling. Comparisons of conventionally-raised (CONV-R) and germ-free (GF) mice revealed a similar degree of allergic skin inflammation, systemic atopy, and airway hypersensitivity. GF mutant animals expressed significantly higher levels of thymic stromal lymphopoietin (TSLP), a major proinflammatory cytokine released by skin with defective barrier function, resulting in a more severe B-lymphoproliferative disorder that persisted into adulthood. These findings suggest a role for the microbiota in ameliorating stress signals released by keratinocytes in response to perturbation in cutaneous barrier function.