Selective Ablation of Ctip2/Bcl11b in Epidermal Keratinocytes Triggers Atopic Dermatitis-Like Skin Inflammatory Responses in Adult Mice

Background: Ctip2 is crucial for epidermal homeostasis and protective barrier formation in developing mouse embryos. Selective ablation of Ctip2 in epidermis leads to increased transepidermal water loss (TEWL), impaired epidermal proliferation, terminal differentiation, as well as altered lipid composition during development. However, little is known about the role of Ctip2 in skin homeostasis in adult mice. Methodology/Principal Findings: To study the role of Ctip2 in adult skin homeostasis, we utilized Ctip2[superscript ep2/2] mouse model in which Ctip2 is selectively deleted in epidermal keratinocytes. Measurement of TEWL, followed by histological, immunohistochemical, and RT[subscript -q]PCR analyses revealed an important role of Ctip2 in barrier maintenance and in regulating adult skin homeostasis. We demonstrated that keratinocytic ablation of Ctip2 leads to atopic dermatitis (AD)-like skin inflammation, characterized by alopecia, pruritus and scaling, as well as extensive infiltration of immune cells including T lymphocytes, mast cells, and eosinophils. We observed increased expression of T-helper 2 (Th2)-type cytokines and chemokines in the mutant skin, as well as systemic immune responses that share similarity with human AD patients. Furthermore, we discovered that thymic stromal lymphopoietin (TSLP) expression was significantly upregulated in the mutant epidermis as early as postnatal day 1 and ChIP assay revealed that TSLP is likely a direct transcriptional target of Ctip2 in epidermal keratinocytes. Conclusions/Significance: Our data demonstrated a cell-autonomous role of Ctip2 in barrier maintenance and epidermal homeostasis in adult mice skin. We discovered a crucial non-cell autonomous role of keratinocytic Ctip2 in suppressing skin inflammatory responses by regulating the expression of Th2-type cytokines. It is likely that the epidermal hyperproliferation in the Ctip2-lacking epidermis may be secondary to the compensatory response of the adult epidermis that is defective in barrier functions. Our results establish an initiating role of epidermal TSLP in AD pathogenesis via a novel repressive regulatory mechanism enforced by Ctip2

BCL11B Regulates Epithelial Proliferation and Asymmetric Development of the Mouse Mandibular Incisor

Mouse incisors grow continuously throughout life with enamel deposition uniquely on the outer, or labial, side of the tooth. Asymmetric enamel deposition is due to the presence of enamel-secreting ameloblasts exclusively within the labial epithelium of the incisor. We have previously shown that mice lacking the transcription factor BCL11B/CTIP2 (BCL11B hereafter) exhibit severely disrupted ameloblast formation in the developing incisor. We now report that BCL11B is a key factor controlling epithelial proliferation and overall developmental asymmetry of the mouse incisor: BCL11B is necessary for proliferation of the labial epithelium and development of the epithelial stem cell niche, which gives rise to ameloblasts; conversely, BCL11B suppresses epithelial proliferation, and development of stem cells and ameloblasts on the inner, or lingual, side of the incisor. This bidirectional action of BCL11B in the incisor epithelia appears responsible for the asymmetry of ameloblast localization in developing incisor. Underlying these spatio-specific functions of BCL11B in incisor development is the regulation of a large gene network comprised of genes encoding several members of the FGF and TGFβ superfamilies, Sprouty proteins, and Sonic hedgehog. Our data integrate BCL11B into these pathways during incisor development and reveal the molecular mechanisms that underlie phenotypes of both Bcl11b−/− and Sprouty mutant mice

Deficiency in the autophagy modulator Dram1 exacerbates pyroptotic cell death of Mycobacteria-infected macrophages

DNA damage regulated autophagy modulator 1 (DRAM1) is a stress-inducible regulator of autophagy and cell death. DRAM1 has been implicated in cancer, myocardial infarction, and infectious diseases, but the molecular and cellular functions of this transmembrane protein remain poorly understood. Previously, we have proposed DRAM1 as a host resistance factor for tuberculosis (TB) and a potential target for host-directed anti-infective therapies. In this study, we generated a zebrafish dram1 mutant and investigated its loss-of-function effects during Mycobacterium marinum (Mm) infection, a widely used model in TB research. In agreement with previous knockdown analysis, dram1 mutation increased the susceptibility of zebrafish larvae to Mm infection. RNA sequencing revealed major effects of Dram1 deficiency on metabolic, immune response, and cell death pathways during Mm infection, and only minor effects on proteinase and metabolic pathways were found under uninfected conditions. Furthermore, unchallenged dram1 mutants did not display overt autophagic defects, but autophagic targeting of Mm was reduced in the absence of Dram1. The phagocytic ability of macrophages in dram1 mutants was unaffected, but acidification of Mm-containing vesicles was strongly reduced, indicating that Dram1 is required for phagosome maturation. By in vivo imaging, we observed that Dram1-deficient macrophages fail to restrict Mm during early stages of infection. The resulting increase in bacterial burden could be reverted by knockdown of inflammatory caspase a (caspa) and gasdermin Eb (gsdmeb), demonstrating pyroptosis as the mechanism underlying premature cell death of Mm-infected macrophages in dram1 mutants. Collectively, these data demonstrate that dissemination of mycobacterial infection in zebrafish larvae is promoted in the absence of Dram1 due to reduced maturation of mycobacteria-containing vesicles, failed intracellular containment, and consequent pyroptotic death of infected macrophages. These results provide new evidence that Dram1 plays a central role in host resistance to intracellular infection, acting at the crossroad of autophagy and cell death

Selective Ablation of Ctip2/Bcl11b in Epidermal Keratinocytes Triggers Atopic Dermatitis-Like Skin Inflammatory Responses in Adult Mice

<div><h3>Background</h3><p>Ctip2 is crucial for epidermal homeostasis and protective barrier formation in developing mouse embryos. Selective ablation of Ctip2 in epidermis leads to increased transepidermal water loss (TEWL), impaired epidermal proliferation, terminal differentiation, as well as altered lipid composition during development. However, little is known about the role of Ctip2 in skin homeostasis in adult mice.</p> <h3>Methodology/Principal Findings</h3><p>To study the role of Ctip2 in adult skin homeostasis, we utilized Ctip2^ep−/− mouse model in which Ctip2 is selectively deleted in epidermal keratinocytes. Measurement of TEWL, followed by histological, immunohistochemical, and RT-qPCR analyses revealed an important role of Ctip2 in barrier maintenance and in regulating adult skin homeostasis. We demonstrated that keratinocytic ablation of Ctip2 leads to atopic dermatitis (AD)-like skin inflammation, characterized by alopecia, pruritus and scaling, as well as extensive infiltration of immune cells including T lymphocytes, mast cells, and eosinophils. We observed increased expression of T-helper 2 (Th2)-type cytokines and chemokines in the mutant skin, as well as systemic immune responses that share similarity with human AD patients. Furthermore, we discovered that thymic stromal lymphopoietin (TSLP) expression was significantly upregulated in the mutant epidermis as early as postnatal day 1 and ChIP assay revealed that TSLP is likely a direct transcriptional target of Ctip2 in epidermal keratinocytes.</p> <h3>Conclusions/Significance</h3><p>Our data demonstrated a cell-autonomous role of Ctip2 in barrier maintenance and epidermal homeostasis in adult mice skin. We discovered a crucial non-cell autonomous role of keratinocytic Ctip2 in suppressing skin inflammatory responses by regulating the expression of Th2-type cytokines. It is likely that the epidermal hyperproliferation in the Ctip2-lacking epidermis may be secondary to the compensatory response of the adult epidermis that is defective in barrier functions. Our results establish an initiating role of epidermal TSLP in AD pathogenesis via a novel repressive regulatory mechanism enforced by Ctip2.</p> </div

Th2-dependent cytokine and chemokine expression levels in WT and Ctip2^ep−/− skin.

<p>(<b>A–F</b>) Quantitative RT-PCR (RT-qPCR) analyses of cytokines and chemokines in the dorsal skin of 1day, 1 week, 2 weeks, 1month, 2 months and 4 months old WT and mutant mice using specific primers as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051262#pone.0051262.s008" target="_blank">Table S1</a>. Relative mRNA expression levels of (<b>A</b>) TSLP, (<b>B</b>) CCL17, (<b>C</b>) IL13, (<b>D</b>) IL4, and (<b>E</b>) IL6 in Ctip2^ep−/− skin was compared to wildtype (WT) skin (set as 1.0). (<b>F</b>) RT-qPCR analyses of TSLP mRNA levels in separated epidermis and dermis from tail skin. All values represent relative transcript level after normalization with HPRT transcripts. (<b>G</b>) Chromatin immunoprecipitation (ChIP) assay was performed on freshly isolated neonatal mouse skin keratinocytes using anti-Ctip2 antibody and results were analyzed by qPCR using primers indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051262#pone.0051262.s008" target="_blank">Table S1</a>. Rat IgG was used as a control. Ctip2 was recruited to the distal promoter regions of TSLP. Statistical analyses were performed by student's unpaired t-test using GraphPad Prism software; * P<0.05, ** P<0.01, *** P<0.001.</p

Characterization of inflammatory cell infiltrates in dorsal skin of WT and in Ctip2^ep−/− adult mice.

<p>(<b>A</b>) Combined eosinophil and mast cell (C.E.M) staining for eosinophils (pink) and mast cells (blue). Black arrows point to eosinophils. Scale bar: 50 µm. (<b>B</b>) Immunohistochemical staining of dorsal skin biopsies from WT and Ctip2^ep−/− mice were performed with specific antibodies against CD3 (red). Yellow arrowhead indicates dermal infiltrates of CD3+ cells. Scale bar: 100 µm. (<b>C</b>) Percent CD3+ T cells at 2 m and 4 m. (<b>D</b>) Immunostaining of CD4+ T cells (red) in WT and mutant mice. Scale bar: 100 µm. (<b>E</b>) Percent CD4+ T cells at 2 m and 4 m. All sections were counterstained with DAPI (blue). Statistical analyses were performed by student's unpaired t-test using GraphPad Prism software; ** P<0.005.</p

Ctip2^ep−/− mice develop a chronic skin lesions and enhanced epidermal proliferation and differentiation.

<p>(<b>A</b>) Gross morphology of 4 months old wildtype (WT) and Ctip2^ep−/− mice. The yellow arrowheads indicate lesion and alopecia of Ctip2^ep−/− mice in the back, face and neck, to be compared with the normal appearance in a wildtype mouse. (<b>B</b>) Measurement of trans-epidermal water loss (TEWL) from dorsal skin of wildtype and Ctip2^ep−/− mice at different time points. Statistical analyses were performed by student's unpaired t-test using GraphPad Prism software; ** P<0.01, *** P<0.001. (<b>C</b>) Hemotoxylin & Eosin stained 5 µm thick paraffin sections from dorsal skin of WT and Ctip2^ep−/− mice at 1 week (1W), 2 weeks (2w), 1month (1 m), 2 months (2 m) and 4 months (4 m). Immunohistochemical staining of dorsal skin biopsies from WT and Ctip2^ep−/− mice was performed with antibodies directed against (<b>D</b>) Ki67, (<b>E</b>) Filaggrin and (<b>F</b>) Loricrin (all in red). All sections were counterstained with DAPI (blue). Scale bar: 100 µm. Epidermis (E) and dermis (D) are indicated.</p