Role of bulge epidermal stem cells and TSLP signaling in psoriasis
2019; Springer Nature; Volume: 11; Issue: 11 Linguagem: Inglês
10.15252/emmm.201910697
ISSN1757-4684
AutoresNuria Gago‐López, Liliana F. Mellor, Diego Megı́as, Guillermo Martín‐Serrano, Ander Izeta, Francisco Jiménez, Erwin F. Wagner,
Tópico(s)Dermatology and Skin Diseases
ResumoArticle26 September 2019Open Access Source DataTransparent process Role of bulge epidermal stem cells and TSLP signaling in psoriasis Nuria Gago-Lopez Genes, Development and Disease Group, Cancer Cell Biology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Liliana F Mellor Genes, Development and Disease Group, Cancer Cell Biology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Diego Megías Confocal Unit at Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Guillermo Martín-Serrano Bioinformatics Unit at Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Ander Izeta Tissue Engineering Group, Biodonostia Health Research Institute, San Sebastian, Spain Search for more papers by this author Francisco Jimenez Grupo de Patología Médica, Mediteknia Dermatologic Clinic, Universidad Fernando Pessoa Canarias, Universidad Las Palmas Gran Canaria, Las Palmas de Gran Canaria, Spain Search for more papers by this author Erwin F Wagner Corresponding Author [email protected] orcid.org/0000-0001-7872-0196 Genes, Development and Disease Group, Cancer Cell Biology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Department of Dermatology and Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria Search for more papers by this author Nuria Gago-Lopez Genes, Development and Disease Group, Cancer Cell Biology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Liliana F Mellor Genes, Development and Disease Group, Cancer Cell Biology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Diego Megías Confocal Unit at Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Guillermo Martín-Serrano Bioinformatics Unit at Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Ander Izeta Tissue Engineering Group, Biodonostia Health Research Institute, San Sebastian, Spain Search for more papers by this author Francisco Jimenez Grupo de Patología Médica, Mediteknia Dermatologic Clinic, Universidad Fernando Pessoa Canarias, Universidad Las Palmas Gran Canaria, Las Palmas de Gran Canaria, Spain Search for more papers by this author Erwin F Wagner Corresponding Author [email protected] orcid.org/0000-0001-7872-0196 Genes, Development and Disease Group, Cancer Cell Biology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Department of Dermatology and Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria Search for more papers by this author Author Information Nuria Gago-Lopez1, Liliana F Mellor1, Diego Megías2, Guillermo Martín-Serrano3, Ander Izeta4, Francisco Jimenez5 and Erwin F Wagner *,1,6 1Genes, Development and Disease Group, Cancer Cell Biology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain 2Confocal Unit at Spanish National Cancer Research Centre (CNIO), Madrid, Spain 3Bioinformatics Unit at Spanish National Cancer Research Centre (CNIO), Madrid, Spain 4Tissue Engineering Group, Biodonostia Health Research Institute, San Sebastian, Spain 5Grupo de Patología Médica, Mediteknia Dermatologic Clinic, Universidad Fernando Pessoa Canarias, Universidad Las Palmas Gran Canaria, Las Palmas de Gran Canaria, Spain 6Department of Dermatology and Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria *Corresponding author. Tel: +43 1 40400 78760; E-mail: [email protected] EMBO Mol Med (2019)11:e10697https://doi.org/10.15252/emmm.201910697 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Psoriasis is a common inflammatory skin disease involving a cross-talk between epidermal and immune cells. The role of specific epidermal stem cell populations, including hair follicle stem cells (HF-SCs) in psoriasis is not well defined. Here, we show reduced expression of c-JUN and JUNB in bulge HF-SCs in patients with scalp psoriasis. Using lineage tracing in mouse models of skin inflammation with inducible deletion of c-Jun and JunB, we found that mutant bulge HF-SCs initiate epidermal hyperplasia and skin inflammation. Mechanistically, thymic stromal lymphopoietin (TSLP) was identified in mutant cells as a paracrine factor stimulating proliferation of neighboring non-mutant epidermal cells, while mutant inter-follicular epidermal (IFE) cells are lost over time. Blocking TSLP in psoriasis-like mice reduced skin inflammation and decreased epidermal proliferation, VEGFα expression, and STAT5 activation. These findings unravel distinct roles of HF-SCs and IFE cells in inflammatory skin disease and provide novel mechanistic insights into epidermal cell interactions in inflammation. Synopsis Lineage tracing in models of skin inflammation reveals that thymic stromal lymphopoietin (TSLP) activates pro-inflammatory cues in mutant hair follicle stem cells (HF-SCs) and keratinocytes (KCs) (1). Primed non-mutant HF-SCs also produce TSLP, thus contributing to the disease (2). Scalp psoriasis was associated with a reduction of c-JUN/JUNB in bulge hair follicle stem cells (HF-SCs) in psoriatic patients. Specific deletion of c-Jun/JunB in bulge HF-SCs (mutantGFP HF-SCs) was sufficient for the development of psoriasis-like disease in mice. Keratinocytes derived from mutantGFPHF-SCs survived, while keratinocytes from mutantGFP inter-follicular epidermal cells (IFE) were lost during psoriasis progression. TSLP was an important mediator for epidermal hyper-proliferation and pro-inflammatory signaling in keratinocytes. Neutralization with anti-TSLP reduced epidermal cell proliferation and psoriasis-like progression in mice. TSLP was highly expressed in the epidermis and hair follicles of scalp psoriasis. Introduction The epidermis is a stratified epithelium undergoing constant renewal due to the presence of stem cells (Alberts et al, 2002). It is composed of the inter-follicular epidermis (IFE), which consists of layers of keratinocytes, hair follicles, and associated sebaceous and sweat glands (Fuchs, 2008). Within the IFE, epidermal stem cells are localized in the basal cell layer. Upon commitment to terminal differentiation, basal stem cells asymmetrically divide giving rise to post-mitotic suprabasal layers establishing an epidermal barrier (Fuchs, 2008). In addition to the IFE, distinct regions in the hair follicle contain hair follicle stem cells (HF-SCs), such as the bulge, isthmus and junctional zone of hair follicle, and sebaceous glands (Liu et al, 2003; Horsley et al, 2006; Jensen et al, 2009). Differential expression of specific markers characterizes the different stem cell subpopulations, allowing the study of their specific contributions to different aspects of cell behavior. For instance, in mice, HF-SCs in the bulge region express CD34, while both HF-SCs and IFE basal keratinocytes express integrin-α6 (CD49f; Liu et al, 2003; Trempus et al, 2003; Horsley et al, 2006, 2008; Jaks et al, 2008; Jensen et al, 2009; Snippert et al, 2010). These different stem cell populations from IFE and hair follicles also have distinct roles during skin remodeling, wound healing, and homeostasis. For example, during homeostasis, committed progenitor cells (transit-amplifying keratinocytes) of the IFE maintain the tissue renewal, while HF-SCs remain largely quiescent until the onset of HF growth (Ito et al, 2005; Blanpain & Simons, 2013). During wound healing and acute inflammation, both IFE epidermal stem cells and K15+ bulge HF-SCs contribute to early wound re-epithelialization by increasing their proliferation rates, thereby expanding the pool of progenitor cells that migrate out of the follicles toward the wound (Ito et al, 2005; Levy et al, 2005; Arwert et al, 2012; Mascre et al, 2012; Blanpain & Simons, 2013). Furthermore, it is reported that basal and squamous skin cell carcinomas arise from K15+ bulge HF-SCs (Trempus et al, 2007; Lapouge et al, 2011; Schober & Fuchs, 2011; Wang et al, 2011). However, little is known about the specific contribution of K15+ bulge HF-SCs and IFE stem cells to the development of chronic inflammatory skin diseases such as psoriasis. Psoriasis is a common heterogeneous inflammatory disease of unknown etiology, with an estimated worldwide prevalence of 3% in the population (Wagner et al, 2010). The vast majority of patients develop a chronic "plaque-type" psoriasis referred to as psoriasis vulgaris. The most common sites of psoriasis are the elbows, knees, forearms, shins, retro-auricular regions, and scalp (Wolf-Henning Boehncke, 2015). Scalp psoriasis is the most common type of psoriasis, representing 50% of the cases of psoriatic patients (Icen et al, 2009). Approximately 75–90% of patients with plaque-type psoriasis in other areas also develop psoriasis in the scalp (Ortonne et al, 2009). Psoriatic plaques are characterized and perpetuated by hyper-proliferation of keratinocytes, impaired differentiation and permanent infiltration of neutrophils and T cells (Wagner et al, 2010). Many studies have focused on immunological aspects of the disease; however, treatments against immunological targets are not completely effective, and recurrence and chronicity prevail. In an epidermal context, it has been demonstrated that human transit-amplifying keratinocytes in psoriatic plaques exhibit increased proliferation and a reduced apoptotic rate, fostering epidermal hyper-proliferation and altered differentiation in situ and in vitro (McKay & Leigh, 1995; Truzzi et al, 2011). However, it remains unclear which epidermal lineages give rise to psoriasis, since a longitudinal analysis of the behavior of psoriatic keratinocytes in human patients is not achievable. Genetically engineered mouse models (GEMMs) of psoriasis have been generated to mimic various aspects of the disease (Wagner et al, 2010). We have previously generated an inducible epidermal-specific double knockout mouse model by epidermal deletion of c-Jun and JunB using the K5 promoter (referred as DKO*). This model develops a psoriasis-like disease within 2 weeks after induction, exhibiting several psoriatic hallmarks including hyper- and parakeratosis, inflammatory infiltrate, elevated levels of cytokines/chemokines, increased subepidermal vascularization, and some co-morbidities like bone loss and arthritic joints (Zenz et al, 2005). Our goal in this study is to elucidate the specific contribution of different epidermal stem cell populations during the development of psoriasis-like disease, by applying a novel strategy to simultaneously track the IFE and K15+ bulge HF-SCs stem cell lineages in the DKO*-psoriasis-like mouse model. This model allows us to permanently label different cell populations involved in the development of psoriasis-like disease in vivo, and to investigate the specific role of each population during disease initiation and development. Using this new mouse model, we found that a small fraction of K15+ bulge HF-SCs can initiate a psoriasis-like disease. Mutant bulge HF-SCs are able to survive, whereas IFE cells disappear during disease progression. Furthermore, mutant bulge HF-SCs and basal keratinocytes secrete thymic stromal lymphopoietin (TSLP), which induces proliferation of neighboring non-mutant keratinocytes. These results emphasize the heterogeneity of epidermal stem cells and their role in psoriasis-like development, and define a mechanistic basis for epidermal hyperplasia through TSLP paracrine signaling. Results c-JUN and JUNB levels are reduced in bulge hair follicle stem cells (HF-SCs) from scalp psoriasis patients Previous results have reported alterations in the expression levels of c-JUN and JUNB in the inter-follicular epidermis (IFE) of psoriatic plaques from human patients (Zenz et al, 2005); however, it is not known whether the expression levels of c-JUN and JUNB are affected in epidermal stem cells. While there are no specific markers to identify IFE stem cells, keratin 15 (K15)-positive cells represent the vast majority of HF-SCs located in the bulge of hair follicles (Liu et al, 2003; Purba et al, 2014). Another marker that identifies putative bulge HF-SCs in human HFs is CD200 (Purba et al, 2014). We analyzed the expression of c-JUN and JUNB in the bulge region of HFs from scalp psoriatic patients, a common form of human psoriasis (Figs 1 and EV1A). K15 was highly expressed in hair follicles, and low expression was also found in some basal keratinocytes of the scalp epidermis (Fig 1A and B). K15+ basal cells located in the outer root sheath (ORS) of the bulge region showed significantly reduced expression of c-JUN and JUNB in lesional hair follicles in anagen phase (38 and 12%, respectively), while c-JUN and JUNB were expressed in 70 and 50%, respectively, in non-lesional hair follicles and healthy scalp (Fig 1A–J). The reduction was confirmed in putative bulge HF-SCs identified by the surface marker CD200 (Fig EV1A). Figure 1. Scalp psoriasis exhibits reduced expression of c-JUN and JUNB in hair follicle stem cells (HF-SCs) A–D. Representative composite immunofluorescence images of whole hair follicle units from non-lesional and lesional scalp psoriasis patients. K15 (green), c-JUN (red in A, C) and JUNB (red in B, D) and DAPI (blue). E–H. Confocal images of the bulge region of human psoriatic hair follicles from non-lesional and lesional regions of the scalp. K15 (green), c-JUN (red in E, G) and JUNB (red in F, H) and DAPI (blue). I, J. Percentage of HF-SCs (K15+) that express c-JUN (I) and JUNB (J) in the bulge region of human psoriatic hair follicles from non-lesional and lesional scalp in comparison with healthy scalp. n = 2–6 hair follicles in anagen per group from five psoriatic patients and two healthy patients. Data represent mean ± SD. Statistical significance *P < 0.05, **P < 0.01, ***P < 0.001 (Student's two-tailed t-test relative to controls). See Appendix Table S2 for exact P-values. Source data are available online for this figure. Source Data for Figure 1 [emmm201910697-sup-0006-SDataFig1.xlsx] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Characterization of c-JUN/JUNB in distinct hair follicle stem cell populations from scalp psoriasis patients (related to Fig 1) A. Confocal images of the bulge region of human psoriatic hair follicles from non-lesional and lesional regions of the scalp. CD200 (green), c-JUN (red), DAPI (blue). B, C. Representative composite immunofluorescence images of whole hair follicle units by confocal from non-lesional and lesional scalp psoriasis patients (B) and magnification images of specific regions from the HFs (C). c-JUN (green), JUNB (gray), GATA-6 (red), and DAPI (blue). Yellow arrows represent the outer root sheath basal layer in the bulge (R1, R3) and proximal bulb (Pb, R2, R4). Yellow asterisks represent overexpression of GATA-6 in suprabasal layers of the outer root sheath in lesional bulge region. Download figure Download PowerPoint Only bulge HF-SCs exhibited reduced c-JUN/JUNB expression, while basal and suprabasal ORS layers in the sub-bulge and proximal bulb regions maintained the expression of c-JUN/JUNB (Fig EV1B and C). In addition, other regulators of HF-SC renewal from other regions in the hair follicle, such as GATA-6, expressed in progenitor matrix cells (bulb) in mice were analyzed (Wang et al, 2017). Interestingly, GATA-6 was up-regulated in suprabasal ORS layers in the bulge region from psoriatic lesional scalps, while no differences were observed in GATA-6-positive cells from the proximal bulb (Fig EV1C). These findings show that scalp psoriasis is associated with a reduction of c-JUN and JUNB in bulge HF-SCs along with GATA-6 increased expression. Epidermal stem cell lineage tracing in the DKO* psoriasis-like mouse model The differential expression of c-JUN and JUNB in CD200+/K15+ bulge HF-SCs in human samples of psoriatic plaques directed us to explore the potential causal contribution of epidermal stem cell populations to the initiation and development of psoriasis. To this end, we applied mouse genetics to ablate the expression of c-Jun and JunB, first globally in all epidermal stem cell populations present in both the basal layer of the IFE and hair follicle units during homeostasis (Byrne et al, 1994). We crossed the well-established DKO* psoriasis-like mouse model (Zenz et al, 2005) with the mT/mG fluorescent reporter mouse. The psoriasis-like phenotype in DKO*-mT/mG mice was induced by the genetic deletion of c-Jun and JunB floxed alleles in K5+ basal keratinocytes and K5+ epidermal stem cells after tamoxifen administration. This treatment also induced an irreversible replacement of the constitutive Tomato expression (red) by the expression of GFP only in K5+ basal keratinocytes and K5+ epidermal stem cells (Fig 2A), allowing tracing of epidermal stem cells and their progeny (transient-amplifying and differentiated keratinocytes) during psoriasis progression. Mice expressing Cre recombinase under the control of the K5 promoter with wild-type or heterozygous c-Jun and JunB were used as controls (Co-mT/mG). Figure 2. Mutant and non-mutant inter-follicular keratinocytes have different proliferation and apoptotic rates during psoriasis-like disease progression A. Schematic representation of the JunB and c-Jun double knockout mouse model (DKO*) with lineage tracing used to investigate the psoriasis-like disease development in mice. Briefly, DKO* psoriasis-like mouse model was generated by the cross of mice carrying the floxed JunB allele (JunBf/f) and floxed c-Jun allele (c-Junf/f) with the transgenic mice expressing the Cre recombinase–estrogen receptor fusion under the control of the basal keratinocyte-specific K5 promoter (K5-Cre-ERT) to obtain JunBf/f c-Junf/f K5-Cre-ERT mice. To trace epidermal-specific deletion of JunBf/f and c-Junf/f in DKO* mice, global double-fluorescent Cre reporter mouse Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J (referred as mT/mG) was crossed with DKO* mice (referred as DKO*-mT/mG). B. Experimental timeline to induce psoriasis-like disease in 8-week-old mice with four consecutive doses of tamoxifen injections (2 mg). Upon induction, non-mutant keratinocytes express Tomato, while mutant keratinocytes express GFP. GFP expression was analyzed at 0, 5, 7, 15, and 30 days post-induction. C. Composite immunofluorescence images showing GFP expression of whole ear sections from DKO*-mT/mG mice at day 5, 7, 15, and 30. Increased GFP expression is shown at day 7 followed by progressive decrease of GFP+ keratinocytes (white dotted line represents basal layer, and red dotted line represents outermost skin layer). n = 3 per time point. D. Quantification analysis of GFP expression (top) and epidermal thickness (bottom) of DKO* mice at different time points during psoriasis-like disease progression. n = 6 per time point. Data represent mean ± SD. Statistical significance **P < 0.01, ***P < 0.001 (Student's two-tailed t-test relative to control group). See Appendix Table S2 for exact P-values. E. Quantification analysis of cleaved caspase-3 (cCas3, top) and Ki67 (bottom) in ear skin of DKO* mice at different time points during psoriasis-like disease progression. n = 3 per time point. Data represent mean ± SD. Statistical significance *P < 0.05, ***P < 0.001 (Student's two-tailed t-test). See Appendix Table S2 for exact P-values. F. Intravital confocal time-lapse imaging of non-mutant Tomato keratinocytes and mutant GFP keratinocytes of DKO*-mT/mG at day 7 after first tamoxifen injection. Dotted circles emphasize areas in which mutant GFP+ keratinocytes are replaced by non-mutant Tomato+ keratinocytes. G. Percentage of GFP+ area eliminated during intravital confocal time-lapse imaging at day 5, 7, and 15 in DKO*-mT/mG mice (Time-lapse 1:8 h). Positive value: Area of GFP increased. Negative value: Area of GFP reduced. H. Fluorescence imaging of DKO*-mT/mG ear skin at day 30 after first tamoxifen injection shows that remaining mutant GFP+ keratinocytes reside along the hair follicles. White dotted line separates epidermis and dermis. White arrows represent GFP+ hair follicles. I. Intravital confocal time-lapse imaging of DKO*-mT/mG at day 15 after first tamoxifen injection. Mutant GFP+ keratinocytes (white arrows) are maintained around hair follicles. White arrows represent GFP+ hair follicles. Source data are available online for this figure. Source Data for Figure 2 [emmm201910697-sup-0007-SDataFig2.xlsx] Download figure Download PowerPoint We first analyzed the labeling efficiency and epidermal specificity of GFP in Co-mT/mG ear skin 15 days after tamoxifen injection. Without tamoxifen, 95 ± 5% of epidermal cells expressed Tomato and only few keratinocytes expressed GFP (Fig EV2A). Following tamoxifen treatment, Tomato expression decreased and GFP expression increased by ~ 60–70% in K5+ epidermal cells of IFE, hair follicles, and sebaceous glands (Fig EV2B, upper panels). DKO*-mT/mG mice treated with tamoxifen developed a psoriasis-like phenotype 2 weeks after induction, mainly in the ears, head, paws, and tail, as previously reported (Zenz et al, 2005). We next analyzed the expression of GFP in this psoriasis-like DKO*-mT/mG ear skin. Interestingly, although hyperplasia was present throughout the epidermis, only small patches of hyperplastic keratinocytes expressed GFP in IFE, hair follicles, and sebaceous glands, showing a mosaic pattern of mutantGFP-positive keratinocytes among a majority of non-mutantTom-positive keratinocytes (Fig EV2B, lower panels). We confirmed that the deletion of c-Jun and JunB occurred only in mutantGFP epidermal cells using fluorescence microscopy (Fig EV2C). Therefore, our tracing system successfully allows studying the dynamics of mutantGFP epidermal stem cells and their progenitor cells, and non-mutantTom epidermal stem cells and their progenitor cells in this psoriasis-like mouse model. Click here to expand this figure. Figure EV2. Validation of the lineage tracing system in Co-mT/mG and DKO*-mT/mG mice (related to Fig 2) A. Whole mount of ear skin from co-mT/mG mice before (VEHICLE) and after tamoxifen at day 15. White arrows represent isolated spots of GFP in vehicle skin. B. Immunofluorescence images of Co-mT/mG ear skin sections and DKO*-mT/mG at day 15 after tamoxifen treatment, co-stained with K5 (white). Yellow dotted line separates epidermis and dermis. C. Immunofluorescence images of c-Jun and JunB staining in DKO*-mT/mG at day 15 after tamoxifen treatment showing GFP+ keratinocytes are negative for c-Jun and JunB expression. GFP (green), Tomato (red), JunB and c-Jun (white), DAPI (blue). Yellow dotted line separates epidermis and dermis. D. Representative images of whole mounts of ear skin from DKO*-mT/mG at day 0 after tamoxifen treatment in two views, from the epidermis and from the dermis, to distinguish the expression of GFP+ epidermal cell into the IFE and HFs. Download figure Download PowerPoint Mutant and non-mutant IFE keratinocytes exhibit different proliferation and apoptosis rates during psoriasis-like development To further understand the mosaic deletion pattern in the DKO*-mT/mG psoriasis-like mouse model, we traced GFP expression after tamoxifen injection during the initiation (day 0–5), mid-term (day 7–9), and late-term (day 15–30) stages of the disease (Fig 2B). Initial epidermal cells labeled for GFP+ at day 0 after the last injection with tamoxifen are observed in isolated groups of cells in IFE and hair follicles (Figs 2C and EV2D). Composite images of whole ear sections showed that mutantGFP keratinocytes are increased at 5–7 days followed by a drastic reduction at days 15–30, whereas immune cell infiltration and epidermal thickening sharply increased over time (Fig 2C and D; Appendix Fig S1A–C). We then analyzed apoptosis and proliferation by cleaved caspase-3 (cCas3) and Ki67 expression, respectively. Apoptosis was observed in mutantGFP and non-mutantTom keratinocytes from the IFE of DKO*-mT/mG mice during psoriasis-like progression, although at day 7 after induction, the apoptosis rate was higher in mutantGFP keratinocytes (38%), when compared to non-mutantTom keratinocytes (10%) (Fig 2E, upper panel). Interestingly, 78% of non-mutantTom keratinocytes expressed Ki67 vs. 40% of mutantGFP keratinocytes, suggesting a proliferative advantage of non-mutantTom keratinocytes (Fig 2E, lower panel). The rapid loss of mutantGFP keratinocytes in the ear skin of DKO*-mT/mG mice was also confirmed by intravital confocal imaging analyses at day 7 after induction (Fig 2F and G, and Movie EV1). Overall, these findings suggest that the psoriasis-like phenotype in the IFE of DKO*-mT/mG mice is characterized by different proliferation and apoptotic rates of distinct mutant and non-mutant populations during psoriasis-like progression. The global proliferation/apoptosis imbalance led to almost complete depletion of mutantGFP basal and suprabasal keratinocytes in the IFE at the latest stage (day 15–30). This drastic loss could imply that the psoriasis-like phenotype should also decrease over time. However, the psoriasis-like phenotype is sustained until the late term of disease progression (D30) (Fig 2D). Interestingly, remaining mutantGFP keratinocytes at the late term of disease progression were located in the outer root sheath (ORS) in both hair follicles and in adjacent IFE keratinocytes (Fig 2H and I). In fact, same dynamic pattern of GFP/Tomato cells was observed in the IFE of tail and back skin during psoriasis development in DKO* mice (Appendix Fig S2). Interestingly, mutant GFP+ hair follicles from tail psoriatic skin underwent anagen induction suggesting the activation of these mutantGFP HF-SCs in psoriasis-like disease. Our next goal was to further investigate the contribution of these HF-associated mutantGFP keratinocytes during psoriasis-like progression. Deletion of c-Jun/JunB in bulge HF-SCs is sufficient for the development of inflammatory skin disease Bulge HF-SCs are an important stem cell population for wound healing and for the initiation and maintenance of skin carcinomas, and are characterized by the expression of K15 and the surface marker CD34 in mice. We hypothesized that the remaining mutantGFP keratinocytes observed in the ORS of the hair follicles and in adjacent IFE regions are mainly derived from mutant HF-SCs. To test this hypothesis, we first conducted FACS analyses to determine the proportion of the different HF-SC subpopulations expressing CD34, CD49f, and Sca-1 markers (Jensen et al, 2010) in the ear skin of the Co-mT/mG and DKO*-mT/mG mice during the psoriasis-like progression (Appendix Fig S3A). As expected, the number of mutantGFP basal IFE cells decreased during disease progression, whereas non-mutantTom basal and suprabasal IFE cells increased over time (Appendix Fig S3B and C). With regard to the hair follicles, the number of mutantGFP HF-SCs from bulge and junctional zone was increased twofold during disease progression, whereas the number of non-mutantTom HF-SCs decreased over time (Appendix Fig S3D–F). To confirm that CD34+ mutantGFP cells belong to bulge HF-SCs and not to the IFE epidermis, we conducted histological section and ear whole-mount confocal analyses and observed co-expression of GFP+ and CD34+ cells only in the bulge region of hair follicles (Fig EV3A and Appendix Fig S4A). Interestingly, both bulge HF-SC populations (mutantGFP and non-mutantTom) had reduced mRNA levels of the quiescence transcription factors Foxc1 and Nfatc1 (Horsley et al, 2008; Wang et al, 2016; Fig EV3B and C), suggesting an activation of bulge HF-SCs during psoriasis progression. In fact, FACS-isolated CD34+ mutantGFP bulge HF-SCs displayed an increased clonogenic capacity in vitro, compared to non-mutantTom bulge HF-SC counterparts and control bulge HF-SCs (Fig EV3D and Appendix Fig S4B). Overall, all these findings suggest that bulge HF-SCs are activated and exit quiescence in the psoriasis-like disease. Click here to expand this figure. Figure EV3. MutantGFP bulge HF-SCs are active and initiate psoriasis-like development (related to Fig 3) A. Immunofluorescence images of the ears of control (Co) and DKO* mice showing co-expression of mutantGFP with CD34 (red arrows) in HF-SCs. n = 3. Scale bar = 50 μm. B, C. Gene expression of quiescence transcription factors, Foxc1a and Nfatc1 show that HF-SCs exit the quiescent stage during psoriasis-like development in DKO* mice. n = 3 mice per group. Data represent mean ± SD. Statistical significance ***P < 0.001 (Student's two-tailed t-test relative to control group). See Appendix Table S2 for exact P-values. D. Colony formation in vitro of bulge hair follicle stem cells (HF-SCs, CD34+ CD49fhigh) vs. basal keratinocytes (b-KCs, CD49fhigh) from control and DKO*-mT/mG ears shows that mutantGFP HF-SCs have significant increased colony formation when compared to non-mutantTom HF-SCs or control HF-SCs. n = 3 mice per group. Data represent mean ± SD. Statistical significance **P < 0.01 (Student's two-tailed t-test relative to control groups). See Appendix Table S2 for exact P-values. E. Representative images of whole mo
Referência(s)