Distinct roles of SOX9 in self-renewal of progenitors and mesenchymal transition of the endothelium.

Regenerative capabilities of the endothelium rely on vessel-resident progenitors termed endothelial colony forming cells (ECFCs). This study aimed to investigate if these progenitors are impacted by conditions (i.e., obesity or atherosclerosis) characterized by increased serum levels of oxidized low-density lipoprotein (oxLDL), a known inducer of Endothelial-to-Mesenchymal Transition (EndMT). Our investigation focused on understanding the effects of EndMT on the self-renewal capabilities of progenitors and the associated molecular alterations. In the presence of oxLDL, ECFCs displayed classical features of EndMT, through reduced endothelial gene and protein expression, function as well as increased mesenchymal genes, contractility, and motility. Additionally, ECFCs displayed a dramatic loss in self-renewal capacity in the presence of oxLDL. RNA-sequencing analysis of ECFCs exposed to oxLDL validated gene expression changes suggesting EndMT and identified SOX9 as one of the highly differentially expressed genes. ATAC sequencing analysis identified SOX9 binding sites associated with regions of dynamic chromosome accessibility resulting from oxLDL exposure, further pointing to its importance. EndMT phenotype and gene expression changes induced by oxLDL in vitro or high fat diet (HFD) in vivo were reversed by the silencing of SOX9 in ECFCs or the endothelial-specific conditional knockout of Sox9 in murine models. Overall, our findings support that EndMT affects vessel-resident endothelial progenitor's self-renewal. SOX9 activation is an early transcriptional event that drives the mesenchymal transition of endothelial progenitor cells. The identification of the molecular network driving EndMT in vessel-resident endothelial progenitors presents a new avenue in understanding and preventing a range of condition where this process is involved.

Bimodal behaviour of interfollicular epidermal progenitors regulated by hair follicle position and cycling.

Interfollicular epidermal (IFE) homeostasis is a major physiological process allowing maintenance of the skin barrier function. Despite progress in our understanding of stem cell populations in different hair follicle compartments, cellular mechanisms of IFE maintenance, in particular, whether a hierarchy of progenitors exists within this compartment, have remained controversial. We here used multicolour lineage tracing with Brainbow transgenic labels activated in the epidermis to track individual keratinocyte clones. Two modes of clonal progression could be observed in the adult murine dorsal skin. Clones attached to hair follicles showed rapid increase in size during the growth phase of the hair cycle. On the other hand, clones distant from hair follicles were slow cycling, but could be mobilized by a proliferative stimulus. Reinforced by mathematical modelling, these data support a model where progenitor cycling characteristics are differentially regulated in areas surrounding or away from growing hair follicles. Thus, while IFE progenitors follow a non-hierarchical mode of development, spatiotemporal control by their environment can change their potentialities, with far-reaching implications for epidermal homeostasis, wound repair and cancer development.

Igf1r signalling acts on the anagen-to-catagen transition in the hair cycle.

Insulin-like growth factor 1 (Igf1) is important for skin development and homoeostasis. However, overexpression and inactivation studies have produced variable findings regarding its role in hair follicle (HF) biology. Here, we studied a conditional and inducible knockout of the Igf1 receptor (Igf1r) in keratin 15-expressing bulge cells. Deletion of Igf1r after the development of the skin appendages in K15-Igf1rKO mice showed no abnormalities in epidermal homoeostasis. Numbers of bulge cells were lower in K15-Igf1rKO mice than in controls, without consequences on wound healing, at least in young mice. K15-Igf1rKO HFs entered anagen phase earlier than controls and showed a delay in the anagen/catagen switch. The expression of Bmp-4 mRNA was inhibited in HFs from K15-Igf1rKO . MED1 transcription was impaired in the epidermis of K15-Igf1rKO mice. These findings suggest that Igf1r controls the hair cycle, partly through Bmp-4 activation.

Concise review: understanding clonal dynamics in homeostasis and injury through multicolor lineage tracing.

Lineage tracing is an essential tool to study stem cell fate. Although traditional lineage tracing techniques have considerably advanced our understanding of stem cell behavior, they pose significant limitations for identification and longitudinal tracking of the progeny of individual stem cells, to compare their behaviors. This is of importance given the well-established heterogeneity among stem cells both in terms of potentialities and proliferative capacities. The recent development of multicolor genetic reporters addressable to specific cell populations largely overcomes these issues. These new "rainbow" technologies provide increased resolution in clonal identification and offer the possibility to study the relative distribution, contacts, tiled arrangement, and competitive interactions among cells or groups of cells of the same type.

Biphasic recruitment of microchimeric fetal mesenchymal cells in fibrosis following acute kidney injury.

Fetal microchimeric cells (FMCs) enter the maternal circulation and persist in tissue for decades. They have capacity to home to injured maternal tissue and differentiate along that tissue's lineage. This raises the question of the origin(s) of cells transferred to the mother during pregnancy. FMCs with a mesenchymal phenotype have been documented in several studies, which makes mesenchymal stem cells an attractive explanation for their broad plasticity. Here we assessed the recruitment and mesenchymal lineage contribution of FMCs in response to acute kidney fibrosis induced by aristolochic acid injection. Serial in vivo bioluminescence imaging revealed a biphasic recruitment of active collagen-producing FMCs during the repair process of injured kidney in post-partum wild-type mothers that had delivered transgenic pups expressing luciferase under the collagen type I-promoter. The presence of FMCs long-term post injury (day 60) was associated with profibrotic molecules (TGF-ß/CTGF), serum urea levels, and collagen deposition. Immunostaining confirmed FMCs at short term (day 15) using post-partum wild-type mothers that had delivered green fluorescent protein-positive pups and suggested a mainly hematopoietic phenotype. We conclude that there is biphasic recruitment to, and activity of, FMCs at the injury site. Moreover, we identified five types of FMC, implicating them all in the reparative process at different stages of induced renal interstitial fibrosis.

Nck adaptors are positive regulators of the size and sensitivity of the T-cell repertoire.

The size and sensitivity of the T-cell repertoire governs the effectiveness of immune responses against invading pathogens. Both are modulated by T-cell receptor (TCR) activity through molecular mechanisms, which remain unclear. Here, we provide genetic evidence that the SH2/SH3 domain containing proteins Nck lower the threshold of T-cell responsiveness. The hallmarks of Nck deletion were T-cell lymphopenia and hyporeactivity to TCR-mediated stimulation. In the absence of the Nck adaptors, peripheral T cells expressing a TCR with low avidity for self-antigens were strongly reduced, whereas an overall impairment of T-cell activation by weak antigenic stimulation was observed. Mechanistically, Nck deletion resulted in a significant decrease in calcium mobilization and ERK phosphorylation upon TCR engagement. Taken together, our findings unveil a crucial role for the Nck adaptors in shaping the T-cell repertoire to ensure maximal antigenic coverage and optimal T cell excitability.

Epidermal mutation accumulation in photodamaged skin is associated with skin cancer burden and can be targeted through ablative therapy.

The main carcinogen for keratinocyte skin cancers (KCs) such as basal and squamous cell carcinomas is ultraviolet (UV) radiation. There is growing evidence that accumulation of mutations and clonal expansion play a key role in KC development. The relationship between UV exposure, epidermal mutation load, and KCs remains unclear. Here, we examined the mutation load in both murine (n = 23) and human (n = 37) epidermal samples. Epidermal mutations accumulated in a UV dose-dependent manner, and this mutation load correlated with the KC burden. Epidermal ablation (either mechanical or laser induced), followed by spontaneous healing from underlying epithelial adnexae reduced the mutation load markedly in both mouse (n = 8) and human (n = 6) clinical trials. In a model of UV-induced basal cell carcinoma, epidermal ablation reduced incident lesions by >80% (n = 5). Overall, our findings suggest that mutation burden is strongly associated with KC burden and represents a target to prevent subsequent KCs.

Fine tuning of the threshold of T cell selection by the Nck adapters.

Thymic selection shapes the T cell repertoire to ensure maximal antigenic coverage against pathogens while preventing autoimmunity. Recognition of self-peptides in the context of peptide-MHC complexes by the TCR is central to this process, which remains partially understood at the molecular level. In this study we provide genetic evidence that the Nck adapter proteins are essential for thymic selection. In vivo Nck deletion resulted in a reduction of the thymic cellularity, defective positive selection of low-avidity T cells, and impaired deletion of thymocytes engaged by low-potency stimuli. Nck-deficient thymocytes were characterized by reduced ERK activation, particularly pronounced in mature single positive thymocytes. Taken together, our findings identify a crucial role for the Nck adapters in enhancing TCR signal strength, thereby fine-tuning the threshold of thymocyte selection and shaping the preimmune T cell repertoire.

Abrogation of ICOS/ICOS ligand costimulation in NOD mice results in autoimmune deviation toward the neuromuscular system.

NOD mice spontaneously develop insulin-dependent diabetes around 10-40 wk of age. Numerous immune gene variants contribute to the autoimmune process. However, genes that direct the autoimmune response toward ß cells remain ill defined. In this study, we provide evidence that the Icos and Icosl genes contribute to the diabetes process. Protection from diabetes in ICOS(-/-) and ICOSL(-/-) NOD mice was unexpectedly associated with the development of an autoimmune disorder of the neuro-muscular system, characterized by myositis, sensory ganglionitis and, to a reduced extent, inflammatory infiltrates in the CNS. This syndrome was reproduced upon adoptive transfer of CD4(+) and CD8(+) T cells from diseased donors to naïve NOD.scid recipients. Our data further show that protection from diabetes results from defective activation of autoimmune diabetogenic effector T cells in ICOS(-/-) NOD mice, whereas acceleration of diabetes in BDC2.5 ICOS(-/-) NOD mice is induced by a dominant defect in Treg. Taken together, our findings indicate that costimulation signals play a key role in regulating immune tolerance in peripheral tissues and that the ICOS/ICOSL costimulatory pathway influences the balance between Treg and diabetogenic effector T cells.

Specific maternal microchimeric T cells targeting fetal antigens in ß cells predispose to auto-immune diabetes in the child.

OBJECTIVE: During pregnancy there is an exchange of cells between the fetus and the mother including T lymphocytes that can persist after delivery. Previous studies have described an increased numbers of maternal cells in children with juvenile diabetes as compared to their unaffected siblings. Our objective was to assess the possibility for these chimeric T cells to trigger an anti-beta cell response. RESEARCH DESIGN AND METHODS: We mated OT2 transgenic female mice having T cells specifically targeting ovalbumin to RIP-OVA males expressing ovalbumin in pancreatic ß cells. This allowed us to examine RIP-OVA progeny from OT2 mothers to assess the consequences of maternal T cells acquired during gestation or lactation. We quantitatively analyzed the pancreas of RIP-OVA mice from OT2 mothers for islet infiltration and compared them to RIP-OVA mice not exposed to OT2 mothers or to wild-type mice from OT2 mothers. RESULTS: RIP-OVA mice from OT2 mothers had significantly more peri-insulitis (p=0.0083) compared to wild-type littermates. Similarly RIP-OVA mice from OT2 mothers had more peri-insulitis as compared to RIP-OVA mice from RIP-OVA mothers (p=0.0073). Presence and specific anti-ovalbumin activity of maternal OT2 cells in the offsprings' peripheral lymphoid tissues was found in a separate group of mice. In animals presenting islet inflammation, CD3+ infiltrating cells in the pancreas were however derived from the offspring and not from OT2 mothers. In accordance, OT2 and RIP-OVA double transgenic mice with high levels of auto-reactive T cells had more peri-insulitis and sometimes intense insulitis when they were from OT2 mothers as compared to RIP-OVA mothers (p=0.046). CONCLUSIONS: In highly specific fetal/maternal combinations, maternal T cells with activity against the offspring pancreatic beta cells, presumably chimeric in fetal organs, initiate islet inflammation and may therefore predispose to auto-immune diabetes.

Whole-mount staining coupled to a UV-inducible basal cell carcinoma murine model.

The origin of basal cell carcinomas (BCC) remains elusive. This protocol combines the use of an ultraviolet (UV)-inducible BCC murine model (K14CrexPtchwt/lox) and an optimized protocol for florescent whole-mount cytokeratin 17 immunostaining to address the spatiotemporal dynamics of BCC formation. The high-resolution three-dimensional confocal images are an excellent tool to visualize and quantify the distribution of skin cancers at a given time point. For complete details on the use and execution of this protocol, please refer to Roy et al. (2020).

Subtype-Specific Analyses Reveal Infiltrative Basal Cell Carcinomas Are Highly Interactive with their Environment.

Little is known regarding the molecular differences between basal cell carcinoma (BCC) subtypes, despite clearly distinct phenotypes and clinical outcomes. In particular, infiltrative BCCs have poorer clinical outcomes in terms of response to therapy and propensity for dissemination. In this project, we aimed to use exome sequencing and RNA sequencing to identify somatic mutations and molecular pathways leading to infiltrative BCCs. Using whole-exome sequencing of 36 BCC samples (eight infiltrative) combined with previously reported exome data (58 samples), we determine that infiltrative BCCs do not contain a distinct somatic variant profile and carry classical UV-induced mutational signatures. RNA sequencing on both datasets revealed key differentially expressed genes, such as POSTN and WISP1, suggesting increased integrin and Wnt signaling. Immunostaining for periostin and WISP1 clearly distinguished infiltrative BCCs, and nuclear ß-catenin staining patterns further validated the resulting increase in Wnt signaling in infiltrative BCCs. Of significant interest, in BCCs with mixed morphology, infiltrative areas expressed WISP1, whereas nodular areas did not, supporting a continuum between subtypes. In conclusion, infiltrative BCCs do not differ in their genomic alteration in terms of initiating mutations. They display a specific type of interaction with the extracellular matrix environment regulating Wnt signaling.

Sox9 and Rbpj differentially regulate endothelial to mesenchymal transition and wound scarring in murine endovascular progenitors.

Endothelial to mesenchymal transition (EndMT) is a leading cause of fibrosis and disease, however its mechanism has yet to be elucidated. The endothelium possesses a profound regenerative capacity to adapt and reorganize that is attributed to a population of vessel-resident endovascular progenitors (EVP) governing an endothelial hierarchy. Here, using fate analysis, we show that two transcription factors SOX9 and RBPJ specifically affect the murine EVP numbers and regulate lineage specification. Conditional knock-out of Sox9 from the vasculature (Sox9fl/fl/Cdh5-CreER RosaYFP) depletes EVP while enhancing Rbpj expression and canonical Notch signalling. Additionally, skin wound analysis from Sox9 conditional knock-out mice demonstrates a significant reduction in pathological EndMT resulting in reduced scar area. The converse is observed with Rbpj conditionally knocked-out from the murine vasculature (Rbpjfl/fl/Cdh5-CreER RosaYFP) or inhibition of Notch signaling in human endothelial colony forming cells, resulting in enhanced Sox9 and EndMT related gene (Snail, Slug, Twist1, Twist2, TGF-ß) expression. Similarly, increased endothelial hedgehog signaling (Ptch1fl/fl/Cdh5-CreER RosaYFP), that upregulates the expression of Sox9 in cells undergoing pathological EndMT, also results in excess fibrosis. Endothelial cells transitioning to a mesenchymal fate express increased Sox9, reduced Rbpj and enhanced EndMT. Importantly, using topical administration of siRNA against Sox9 on skin wounds can substantially reduce scar area by blocking pathological EndMT. Overall, here we report distinct fates of EVPs according to the relative expression of Rbpj or Notch signalling and Sox9, highlighting their potential plasticity and opening exciting avenues for more effective therapies in fibrotic diseases.

Ectopic expression of SOX18 in Basal cell carcinoma negatively regulates tumour progression.

BACKGROUND: Basal Cell Carcinoma is the most common tumour and yet much remains to be determined regarding the molecular mechanisms that leads to its development. Hedgehog signal activation is sufficient for BCC induction, but the molecular mediators of BCC growth are not well understood. SoxF transcription factor Sox18 has been identified in human BCC, but its role in growth of the tumour is as yet unknown. OBJECTIVE: To determine if Sox18 is involved in the regulation of Basal Cell Carcinoma growth. METHODS: We analysed the function of Sox18 by combining a dominant negative Sox18 mouse model, Sox18+/OP with murine BCC RESULTS: We determine that Sox18 is ectopically expressed in the epidermal cells of a murine model of Basal Cell Carcinoma. We then show that dominant negative mutation of Sox18 increases the severity of murine Basal Cell Carcinoma. Finally, decreased Hey1 in Sox18+/OP BCC suggests Sox18 may negatively regulate BCC progression via Notch signaling. CONCLUSIONS: These data suggest that Sox18 is a hedgehog regulated mediator of tumour suppression within Basal Cell Carcinoma epidermis.

Regional Variation in Epidermal Susceptibility to UV-Induced Carcinogenesis Reflects Proliferative Activity of Epidermal Progenitors.

To better understand the influence of ultraviolet (UV) irradiation on the initial steps of skin carcinogenesis, we examine patches of labeled keratinocytes as a proxy for clones in the interfollicular epidermis (IFE) and measure their size variation upon UVB irradiation. Multicolor lineage tracing reveals that in chronically irradiated skin, patches near hair follicles (HFs) increase in size, whereas those far from follicles do not change. This is explained by proliferation of basal epidermal cells within 60 µm of HF openings. Upon interruption of UVB, patch size near HFs regresses significantly. These anatomical differences in proliferative behavior have significant consequences for the cell of origin of basal cell carcinomas (BCCs). Indeed, a UV-inducible murine BCC model shows that BCC patches are more frequent, larger, and more invasive near HFs. These findings have major implications for the prevention of field cancerization in the epidermis.

Whole-Mount Immunofluorescent Staining Coupled to Multicolor Lineage Tracing Model for Analyzing the Spatiotemporal Organization of Epidermal Stem Cells.

An overall three-dimensional picture of the distribution of epidermal cells at a given time point is of importance to better characterize epidermal progenitors. We introduce a whole-mount immunofluorescent staining coupled to a multicolor lineage tracing model for analyzing the spatiotemporal organization of epidermal stem cells. Laser scanning confocal microscopy with multiple labelling allows for robust imaging of dorsal skin with excellent resolution.

Immunosuppression Agent Cyclosporine Reduces Self-Renewal and Vessel Regeneration Potentiation of Human Endothelial Colony Forming Cells.

Endothelial colony forming cells (ECFC) and mesenchymal stem cells (MSC) combined have great potential to be used for cell therapy of ischemic vascular diseases. However, to improve allogeneic stem cell engraftment the use of immunosuppression, such as cyclosporine has been suggested. Our aim was to assess the impact of cyclosporine on hind limb revascularisation upon MSC and ECFC combination therapy. Balb/c immunocompetent mice subjected to hind limb ischemia (right femoral artery ligation) were given both human ECFC and MSC (weekly intramuscular injections) with or without cyclosporine (daily injection). Surprisingly, mice receiving cyclosporine had a significant decrease in reperfusion based on laser Doppler imaging compared to vehicle controls and had poorer limb survival. In vitro, the downstream calcineurin target NFATC4 was highly expressed in the self-renewing fraction of ECFCs. ECFCs cultured with cyclosporine had reduced colony formation capacity and tube formation in Matrigel. Lastly, ECFC displayed increased proliferation and loss of capacity for long term culture when in the presence of cyclosporine clearly showing a loss of quiescence and progenitor function. Our findings demonstrate the deleterious impact of cyclosporine on ECFC function, with significant impact on ECFC-based allogeneic cellular therapy. Stem Cells Translational Medicine 2019;8:162&7.