Interaction of Deubiquitinase 2A-DUB/MYSM1 with DNA Repair and Replication Factors.

The deubiquitination of histone H2A on lysine 119 by 2A-DUB/MYSM1, BAP1, USP16, and other enzymes is required for key cellular processes, including transcriptional activation, apoptosis, and cell cycle control, during normal hematopoiesis and tissue development, and in tumor cells. Based on our finding that MYSM1 colocalizes with γH2AX foci in human peripheral blood mononuclear cells, leukemia cells, and melanoma cells upon induction of DNA double-strand breaks with topoisomerase inhibitor etoposide, we applied a mass spectrometry-based proteomics approach to identify novel 2A-DUB/MYSM1 interaction partners in DNA-damage responses. Differential display of MYSM1 binding proteins significantly enriched after exposure of 293T cells to etoposide revealed an interacting network of proteins involved in DNA damage and replication, including factors associated with poor melanoma outcome. In the context of increased DNA-damage in a variety of cell types in Mysm1-deficient mice, in bone marrow cells upon aging and in UV-exposed Mysm1-deficient skin, our current mass spectrometry data provide additional evidence for an interaction between MYSM1 and key DNA replication and repair factors, and indicate a potential function of 2A-DUB/MYSM1 in DNA repair processes.

A Face-Aging Smoking Prevention/Cessation Intervention for Nursery School Students in Germany: An Appearance-Focused Interventional Study.

The Education Against Tobacco (EAT) network delivers smoking prevention advice in secondary schools, typically using the mirroring approach (i.e., a "selfie" altered with a face-aging app and shared with a class). In November 2017, however, the German assembly of EAT opted to expand its remit to include nursing students. To assess the transferability of the existing approach, we implemented it with the self-developed face-aging app "Smokerface" (=mixed - methods approach) in six nursing schools. Anonymous questionnaires were used to assess the perceptions of 197 students (age 18⁻40 years; 83.8% female; 26.4% smokers; 23.3% daily smokers) collecting qualitative and quantitative data for our cross-sectional study. Most students perceived the intervention to be fun (73.3%), but a minority disagreed that their own animated selfie (25.9%) or the reaction of their peers (29.5%) had motivated them to stop smoking. The impact on motivation not to smoke was considerably lower than experienced with seventh graders (63.2% vs. 42.0%; notably, more smokers also disagreed (45.1%) than agreed (23.5%) with this statement. Agreement rates on the motivation not to smoke item were higher in females than in males and in year 2⁻3 than in year 1 students. Potential improvements included greater focus on pathology (29%) and discussing external factors (26%). Overall, the intervention seemed to be appealing for nursing students.

A Face-Aging App for Smoking Cessation in a Waiting Room Setting: Pilot Study in an HIV Outpatient Clinic.

BACKGROUND: There is strong evidence for the effectiveness of addressing tobacco use in health care settings. However, few smokers receive cessation advice when visiting a hospital. Implementing smoking cessation technology in outpatient waiting rooms could be an effective strategy for change, with the potential to expose almost all patients visiting a health care provider without preluding physician action needed. OBJECTIVE: The objective of this study was to develop an intervention for smoking cessation that would make use of the time patients spend in a waiting room by passively exposing them to a face-aging, public morphing, tablet-based app, to pilot the intervention in a waiting room of an HIV outpatient clinic, and to measure the perceptions of this intervention among smoking and nonsmoking HIV patients. METHODS: We developed a kiosk version of our 3-dimensional face-aging app Smokerface, which shows the user how their face would look with or without cigarette smoking 1 to 15 years in the future. We placed a tablet with the app running on a table in the middle of the waiting room of our HIV outpatient clinic, connected to a large monitor attached to the opposite wall. A researcher noted all the patients who were using the waiting room. If a patient did not initiate app use within 30 seconds of waiting time, the researcher encouraged him or her to do so. Those using the app were asked to complete a questionnaire. RESULTS: During a 19-day period, 464 patients visited the waiting room, of whom 187 (40.3%) tried the app and 179 (38.6%) completed the questionnaire. Of those who completed the questionnaire, 139 of 176 (79.0%) were men and 84 of 179 (46.9%) were smokers. Of the smokers, 55 of 81 (68%) said the intervention motivated them to quit (men: 45, 68%; women: 10, 67%); 41 (51%) said that it motivated them to discuss quitting with their doctor (men: 32, 49%; women: 9, 60%); and 72 (91%) perceived the intervention as fun (men: 57, 90%; women: 15, 94%). Of the nonsmokers, 92 (98%) said that it motivated them never to take up smoking (men: 72, 99%; women: 20, 95%). Among all patients, 102 (22.0%) watched another patient try the app without trying it themselves; thus, a total of 289 (62.3%) of the 464 patients were exposed to the intervention (average waiting time 21 minutes). CONCLUSIONS: A face-aging app implemented in a waiting room provides a novel opportunity to motivate patients visiting a health care provider to quit smoking, to address quitting at their subsequent appointment and thereby encourage physician-delivered smoking cessation, or not to take up smoking.

Facial-Aging Mobile Apps for Smoking Prevention in Secondary Schools in Brazil: Appearance-Focused Interventional Study.

BACKGROUND: Most smokers start smoking during their early adolescence, often with the idea that smoking is glamorous. Interventions that harness the broad availability of mobile phones as well as adolescents' interest in their appearance may be a novel way to improve school-based prevention. A recent study conducted in Germany showed promising results. However, the transfer to other cultural contexts, effects on different genders, and implementability remains unknown. OBJECTIVE: In this observational study, we aimed to test the perception and implementability of facial-aging apps to prevent smoking in secondary schools in Brazil in accordance with the theory of planned behavior and with respect to different genders. METHODS: We used a free facial-aging mobile phone app ("Smokerface") in three Brazilian secondary schools via a novel method called mirroring. The students' altered three-dimensional selfies on mobile phones or tablets and images were "mirrored" via a projector in front of their whole grade. Using an anonymous questionnaire, we then measured on a 5-point Likert scale the perceptions of the intervention among 306 Brazilian secondary school students of both genders in the seventh grade (average age 12.97 years). A second questionnaire captured perceptions of medical students who conducted the intervention and its conduction per protocol. RESULTS: The majority of students perceived the intervention as fun (304/306, 99.3%), claimed the intervention motivated them not to smoke (289/306, 94.4%), and stated that they learned new benefits of not smoking (300/306, 98.0%). Only a minority of students disagreed or fully disagreed that they learned new benefits of nonsmoking (4/306, 1.3%) or that they themselves were motivated not to smoke (5/306, 1.6%). All of the protocol was delivered by volunteer medical students. CONCLUSIONS: Our data indicate the potential for facial-aging interventions to reduce smoking prevalence in Brazilian secondary schools in accordance with the theory of planned behavior. Volunteer medical students enjoyed the intervention and are capable of complete implementation per protocol.

Targeted Tumor Therapy Remixed-An Update on the Use of Small-Molecule Drugs in Combination Therapies.

Over the last decade, the treatment of tumor patients has been revolutionized by the highly successful introduction of novel targeted therapies, in particular small-molecule kinase inhibitors and monoclonal antibodies, as well as by immunotherapies. Depending on the mutational status, BRAF and MEK inhibitor combinations or immune checkpoint inhibitors are current first-line treatments for metastatic melanoma. However, despite great improvements of survival rates limitations due to tumor heterogeneity, primary and acquired therapy resistance, immune evasion, and economical considerations will need to be overcome. Accordingly, ongoing clinical trials explore the individualized use of small-molecule drugs in new targeted therapy combinations based on patient parameters and tumor biopsies. With focus on melanoma therapy this review aims at providing a comprehensive overview of such novel alternative and combinational therapy strategies currently emerging from basic research. The molecular principles and drug classes that may hold promise for improved tumor therapy combination regimens including kinase inhibition, induction of apoptosis, DNA-damage response inhibition, epigenetic reprogramming, telomerase inhibition, redox modulation, metabolic reprogramming, proteasome inhibition, cancer stem cell transdifferentiation, immune cell signaling modulation, and others, are explained in brief. In addition, relevant targeted therapy combinations in current clinical trials and individualized treatment strategies are highlighted.

2A-DUB/Mysm1 Regulates Epidermal Development in Part by Suppressing p53-Mediated Programs.

Development and homeostasis of the epidermis are governed by a complex network of sequence-specific transcription factors and epigenetic modifiers cooperatively regulating the subtle balance of progenitor cell self-renewal and terminal differentiation. To investigate the role of histone H2A deubiquitinase 2A-DUB/Mysm1 in the skin, we systematically analyzed expression, developmental functions, and potential interactions of this epigenetic regulator using Mysm1-deficient mice and skin-derived epidermal cells. Morphologically, skin of newborn and young adult Mysm1-deficient mice was atrophic with reduced thickness and cellularity of epidermis, dermis, and subcutis, in context with altered barrier function. Skin atrophy correlated with reduced proliferation rates in Mysm1-/- epidermis and hair follicles, and increased apoptosis compared with wild-type controls, along with increases in DNA-damage marker γH2AX. In accordance with diminished α6-Integrinhigh+CD34⁺ epidermal stem cells, reduced colony formation of Mysm1-/- epidermal progenitors was detectable in vitro. On the molecular level, we identified p53 as potential mediator of the defective Mysm1-deficient epidermal compartment, resulting in increased pro-apoptotic and anti-proliferative gene expression. In Mysm1-/-p53-/- double-deficient mice, significant recovery of skin atrophy was observed. Functional properties of Mysm1-/- developing epidermis were assessed by quantifying the transepidermal water loss. In summary, this investigation uncovers a role for 2A-DUB/Mysm1 in suppression of p53-mediated inhibitory programs during epidermal development.

A skin cancer prevention photoageing intervention for secondary schools in Brazil delivered by medical students: protocol for a randomised controlled trial.

INTRODUCTION: The incidence of melanoma is increasing faster than any other major cancer both in Brazil and worldwide. The Southeast of Brazil has especially high incidences of melanoma, and early detection is low. Exposure to UV radiation represents a primary risk factor for developing melanoma. Increasing attractiveness is a major motivation for adolescents for tanning. A medical student-delivered intervention that harnesses the broad availability of mobile phones as well as adolescents' interest in their appearance may represent a novel method to improve skin cancer prevention. METHODS AND ANALYSIS: We developed a free mobile app (Sunface), which will be implemented in at least 30 secondary school classes, each with 21 students (at least 30 classes with 21 students for control) in February 2018 in Southeast Brazil via a novel method called mirroring. In a 45 min classroom seminar, the students' altered three-dimensional selfies on tablets are 'mirrored' via a projector in front of their entire class, showing the effects of unprotected UV exposure on their future faces. External block randomisation via computer is performed on the class level with a 1:1 allocation. Sociodemographic data, as well as skin type, ancestry, UV protection behaviour and its predictors are measured via a paper-pencil questionnaire before as well as at 3 and 6 months postintervention. The primary end point is the group difference in the 30-day prevalence of daily sunscreen use at a 6-month follow-up. Secondary end points include (1) the difference in daily sunscreen use at a 3-month follow-up, (2) if a self-skin examination in accordance with the ABCDE rule was performed within the 6-month follow-up and (3) the number of tanning sessions. ETHICS AND DISSEMINATION: Ethical approval was obtained from the ethics committee of the University of Itauna. Results will be disseminated at conferences and in peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT03178240; Pre-results.

A Skin Cancer Prevention Facial-Aging Mobile App for Secondary Schools in Brazil: Appearance-Focused Interventional Study.

BACKGROUND: The incidence of melanoma is increasing faster than any other major cancer both in Brazil and worldwide. Southeast Brazil has especially high incidences of melanoma, and early detection is low. Exposure to ultraviolet (UV) radiation is a primary risk factor for developing melanoma. Increasing attractiveness is a major motivation among adolescents for tanning. A medical student-delivered intervention that takes advantage of the broad availability of mobile phones and adolescents' interest in their appearance indicated effectiveness in a recent study from Germany. However, the effect in a high-UV index country with a high melanoma prevalence and the capability of medical students to implement such an intervention remain unknown. OBJECTIVE: In this pilot study, our objective was to investigate the preliminary success and implementability of a photoaging intervention to prevent skin cancer in Brazilian adolescents. METHODS: We implemented a free photoaging mobile phone app (Sunface) in 15 secondary school classes in southeast Brazil. Medical students "mirrored" the pupils' altered 3-dimensional (3D) selfies reacting to touch on tablets via a projector in front of their whole grade accompanied by a brief discussion of means of UV protection. An anonymous questionnaire capturing sociodemographic data and risk factors for melanoma measured the perceptions of the intervention on 5-point Likert scales among 356 pupils of both sexes (13-19 years old; median age 16 years) in grades 8 to 12 of 2 secondary schools in Brazil. RESULTS: We measured more than 90% agreement in both items that measured motivation to reduce UV exposure and only 5.6% disagreement: 322 (90.5%) agreed or strongly agreed that their 3D selfie motivated them to avoid using a tanning bed, and 321 (90.2%) that it motivated them to improve their sun protection; 20 pupils (5.6%) disagreed with both items. The perceived effect on motivation was higher in female pupils in both tanning bed avoidance (n=198, 92.6% agreement in females vs n=123, 87.2% agreement in males) and increased use of sun protection (n=197, 92.1% agreement in females vs n=123, 87.2% agreement in males) and independent of age or skin type. All medical students involved filled in a process evaluation revealing that they all perceived the intervention as effective and unproblematic, and that all pupils tried the app in their presence. CONCLUSIONS: The photoaging intervention was effective in changing behavioral predictors for UV protection in Brazilian adolescents. The predictors measured indicated an even higher prospective effectiveness in southeast Brazil than in Germany (>90% agreement in Brazil vs >60% agreement in Germany to both items that measured motivation to reduce UV exposure) in accordance with the theory of planned behavior. Medical students are capable of complete implementation. A randomized controlled trial measuring prospective effects in Brazil is planned as a result of this study.

Loss of p53 compensates osteopenia in murine Mysm1 deficiency.

Histone modifications critically contribute to the epigenetic orchestration of bone homeostasis-in part, by modifying the access of transcription factors to specific genes involved in the osteogenic differentiation process of bone marrow mesenchymal stem cells (MSCs) and osteoblasts. Based on our previous finding that histone H2A deubiquitinase 2A-DUB/Mysm1 interacts with the p53 axis in hematopoiesis and tissue development, we analyzed the molecular basis of the skeletal phenotype of Mysm1-deficient mice and dissected the underlying p53-dependent and -independent mechanisms. Visible morphologic, skeletal deformations of young Mysm1-deficient mice-including a kinked and truncated tail and shortened long bones-were associated with osteopenia of long bones. On the cellular level, Mysm1-deficient primary osteoblasts displayed reduced potential to differentiate into mature osteoblasts, as indicated by decreased expression of osteogenic markers. Reduced osteogenic differentiation capacity of Mysm1-deficient osteoblasts was accompanied by an impaired induction of osteogenic transcription factor Runx2. Osteogenic differentiation of Mysm1-/- MSCs, however, was not compromised in vitro. In line with defective hematopoietic development of Mysm1-deficient mice, Mysm1-/- osteoclasts had reduced resorption activity and were more prone to apoptosis in TUNEL assays. Skeletal alterations and osteopenia of Mysm1-deficient mice were phenotypically completely rescued by simultaneous ablation of p53 in p53-/-Mysm1-/- double-deficient mice-although p53 deficiency did not restore Runx2 expression in Mysm1-/- osteoblasts on the molecular level but, instead, enhanced proliferation and osteogenic differentiation of MSCs. In summary, our results demonstrate novel roles for Mysm1 in osteoblast differentiation and osteoclast formation, resulting in osteopenia in Mysm1-deficient mice that could be abrogated by the loss of p53 from increased osteogenic differentiation of Mysm1-/-p53-/- MSCs.-Haffner-Luntzer, M., Kovtun, A., Fischer, V., Prystaz, K., Hainzl, A., Kroeger, C. M., Krikki, I., Brinker, T. J., Ignatius, A., Gatzka, M. Loss of p53 compensates osteopenia in murine Mysm1 deficiency.

MYSM1/2A-DUB is an epigenetic regulator in human melanoma and contributes to tumor cell growth.

Histone modifying enzymes, such as histone deacetylases (HDACs) and polycomb repressive complex (PRC) components, have been implicated in regulating tumor growth, epithelial-mesenchymal transition, tumor stem cell maintenance, or repression of tumor suppressor genes - and may be promising targets for combination therapies of melanoma and other cancers. According to recent findings, the histone H2A deubiquitinase 2A-DUB/Mysm1 interacts with the p53-axis in hematopoiesis and tissue differentiation in mice, in part by modulating DNA-damage responses in stem cell and progenitor compartments. Based on the identification of alterations in skin pigmentation and melanocyte specification in Mysm1-deficient mice, we hypothesized that MYSM1 may be involved in melanoma formation. In human melanoma samples, expression of MYSM1 was increased compared with normal skin melanocytes and nevi and co-localized with melanocyte markers such as Melan-A and c-KIT. Similarly, in melanoma cell lines A375 and SK-MEL-28 and in murine skin, expression of the deubiquitinase was detectable at the mRNA and protein level that was inducible by growth factor signals and UVB exposure, respectively. Upon stable silencing of MYSM1 in A375 and SK-MEL-28 melanoma cells by lentivirally-mediated shRNA expression, survival and proliferation were significantly reduced in five MYSM1 shRNA cell lines analyzed compared with control cells. In addition, MYSM1-silenced melanoma cells proliferated less well in softagar assays. In context with our finding that MYSM1 bound to the c-MET promoter region in close vicinity to PAX3 in melanoma cells, our data indicate that MYSM1 is an epigenetic regulator of melanoma growth and potentially promising new target for tumor therapy.

A Dermatologist's Ammunition in the War Against Smoking: A Photoaging App.

This viewpoint reviews the perspectives for dermatology as a specialty to go beyond the substantial impact of smoking on skin disease and leverage the impact of skin changes on a person's self-concept and behavior in the design of effective interventions for smoking prevention and cessation.

Photoaging Mobile Apps in School-Based Melanoma Prevention: Pilot Study.

BACKGROUND: Around 90% of melanomas are caused by exposure to ultraviolet (UV) radiation and are therefore eminently preventable. Tanning behavior is mostly initiated in early adolescence, often with the belief that it increases attractiveness; the problems related to malignant melanoma and other skin cancers are too far in the future to fathom. Given the substantial amount of time children and adolescents spend in schools, as well as with their mobile phones, addressing melanoma prevention via both of these ways is crucial. However, no school-based intervention using mobile apps has been evaluated to date. We recently released a photoaging mobile app, in which a selfie is altered to predict future appearance dependent on UV protection behavior and skin type. OBJECTIVE: In this pilot study, we aimed to use mobile phone technology to improve school-based melanoma prevention and measure its preliminary success in different subgroups of students with regard to their UV protection behavior, Fitzpatrick skin type and age. METHODS: We implemented a free photoaging mobile phone app (Sunface) in 2 German secondary schools via a method called mirroring. We "mirrored" the students' altered 3-dimensional (3D) selfies reacting to touch on mobile phones or tablets via a projector in front of their whole grade. Using an anonymous questionnaire capturing sociodemographic data as well as risk factors for melanoma we then measured their perceptions of the intervention on a 5-point Likert scale among 205 students of both sexes aged 13-19 years (median 15 years). RESULTS: We measured more than 60% agreement in both items that measured motivation to reduce UV exposure and only 12.5% disagreement: 126 (63.0%) agreed or strongly agreed that their 3D selfie motivated them to avoid using a tanning bed, and 124 (61.7%) to increase use of sun protection. However, only 25 (12.5%) disagreed with both items. The perceived effect on motivation was increased in participants with Fitzpatrick skin types 1-2 in both tanning bed avoidance (n=74, 71.8% agreement in skin types 1-2 vs n=50, 53.8% agreement in skin types 3-6) and increased use of sun protection (n=70, 68.0% agreement in skin types 1-2 vs n=52, 55.3% agreement in skin types 3-6), and also positively correlated with higher age. CONCLUSIONS: We present a novel way of integrating photoaging in school-based melanoma prevention that affects the students' peer group, considers the predictors of UV exposure in accordance with the theory of planned behavior, and is particularly effective in changing behavioral predictors in fair-skinned adolescents (Fitzpatrick skin types 1-2). Further research is required to evaluate the intervention's prospective effects on adolescents of various cultural backgrounds.

Skin under Tnf influence: how regulatory T cells work against macrophages in psoriasis.

Tumour necrosis factor (TNF)-α and interleukin (IL)-17 are key cytokines driving psoriasis and other inflammatory autoimmune diseases, and thus represent effective targets for anti-psoriatic therapy. In a recent issue of The Journal of Pathology, Leite Dantas et al explore a mouse model of TNF-mediated psoriasiform dermatitis and arthritis with doxycyclin-inducible general overexpression of human TNF (ihTNFtg) mice for the contributions of macrophages and T cells in skin inflammation - with some unexpected and interesting findings. Although T cells are commonly known as major proinflammatory players in psoriasis, in the ihTNFtg mouse model macrophages were the predominant cells causing inflammation, and T cells, represented by Foxp3+ regulatory T cells, mainly formed the opposition to keep inflammation in check. In addition to offering a new perspective on potential alternative initiation mechanisms in psoriatic skin inflammation, this constellation illustrates how cellular networks in inflammatory conditions evolve according to the prevailing cytokine, and may help to explain individual responses to either anti-TNF-α or anti-IL-17 therapy regimens in psoriasis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Senescent fibroblast-derived Chemerin promotes squamous cell carcinoma migration.

Aging is associated with a rising incidence of cutaneous squamous cell carcinoma (cSCC), an aggressive skin cancer with the potential for local invasion and metastasis. Acquisition of a senescence-associated secretory phenotype (SASP) in dermal fibroblasts has been postulated to promote skin cancer progression in elderly individuals. The underlying molecular mechanisms are largely unexplored. We show that Chemerin, a previously unreported SASP factor released from senescent human dermal fibroblasts, promotes cSCC cell migration, a key feature driving tumor progression. Whereas the Chemerin abundance is downregulated in malignant cSCC cells, increased Chemerin transcripts and protein concentrations are detected in replicative senescent fibroblasts in vitro and in the fibroblast of skin sections from old donors, indicating that a Chemerin gradient is built up in the dermis of elderly. Using Transwell® migration assays, we show that Chemerin enhances the chemotaxis of different cSCC cell lines. Notably, the Chemerin receptor CCRL2 is remarkably upregulated in cSCC cell lines and human patient biopsies. Silencing Chemerin in senescent fibroblasts or the CCRL2 and GPR1 receptors in the SCL-1 cSCC cell line abrogates the Chemerin-mediated chemotaxis. Chemerin triggers the MAPK cascade via JNK and ERK1 activation, whereby the inhibition impairs the SASP- or Chemerin-mediated cSCC cell migration.Taken together, we uncover a key role for Chemerin, as a major factor in the secretome of senescent fibroblasts, promoting cSCC cell migration and possibly progression, relaying its signals through CCRL2 and GPR1 receptors with subsequent MAPK activation. These findings might have implications for targeted therapeutic interventions in elderly patients.

T-cell plasticity in inflammatory skin diseases--the good, the bad, and the chameleons.

According to the concept of T-cell plasticity, peripheral T cells - once differentiated into a specific T cell subset - may, in response to new environmental cues or signaling alterations, adopt the phenotype of a different helper cell subset with regard to cytokine production and regulatory functions. In a variety of T cell-mediated inflammatory disorders, such as rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, and psoriasis, T-cell plasticity - in particular the conversion of regulatory T cells (Tregs) to IL-17-producing inflammatory cells - has recently been described as key pathomechanism contributing to the aggravation of inflammatory symptoms and disease chronification. Apart from psoriasis, the phenomenon of immune cell plasticity may also have a role in other inflammatory conditions of the skin showing a T cell component and/or an IL-17-mediated pathology, such as lichen planus, lupus erythematosus, blistering diseases, allergic disorders, and others. This review summarizes the basic molecular mechanisms regulating T-cell fate decisions and plasticity in inflamed skin and in other lymphoid organs. Moreover, it explores the effect of established targeted therapies as well as alternative concepts with a focus on how to prevent the unwanted conversion of "helpful" T cells and other beneficial immune cells to pathological inflammatory vermin.

Reduction of CD18 promotes expansion of inflammatory γδ T cells collaborating with CD4+ T cells in chronic murine psoriasiform dermatitis.

IL-17 is a critical factor in the pathogenesis of psoriasis and other inflammatory diseases. The impact of γδ T cells, accounting for an important source of IL-17 in acute murine IL-23- and imiquimod-induced skin inflammation, in human psoriasis is still unclear. Using the polygenic CD18(hypo) PL/J psoriasis mouse model spontaneously developing chronic psoriasiform dermatitis due to reduced CD18/ß2 integrin expression to 2-16% of wild-type levels, we investigated in this study the influence of adhesion molecule expression on generation of inflammatory γδ T cells and analyzed the occurrence of IL-17-producing γδ and CD4(+) T cells at different disease stages. Severity of CD18(hypo) PL/J psoriasiform dermatitis correlated with a loss of skin-resident Vγ5(+) T cells and concurrent skin infiltration with IL-17(+), IL-22(+), and TNF-α(+) γδTCR(low) cells preceded by increases in Vγ4(+) T cells in local lymph nodes. In vitro, reduced CD18 levels promoted expansion of inflammatory memory-type γδ T cells in response to IL-7. Similar to IL-17 or IL-23/p19 depletion, injection of diseased CD18(hypo) PL/J mice with anti-γδTCR Abs significantly reduced skin inflammation and largely eliminated pathological γδ and CD4(+) T cells. Moreover, CD18(hypo) γδ T cells induced allogeneic CD4(+) T cell responses more potently than CD18(wt) counterparts and, upon adoptive transfer, triggered psoriasiform dermatitis in susceptible hosts. These results demonstrate a novel function of reduced CD18 levels in generation of pathological γδ T cells that was confirmed by detection of increases in CD18(low) γδ T cells in psoriasis patients and may also have implications for other inflammatory diseases.

Reduced CD18 levels drive regulatory T cell conversion into Th17 cells in the CD18hypo PL/J mouse model of psoriasis.

Defective development and function of CD4(+)CD25(high+)Foxp3(+) regulatory T cells (Tregs) contribute to the pathogenesis of psoriasis and other autoimmune diseases. Little is known about the influence of adhesions molecules on the differentiation of Foxp3(+) Tregs into proinflammatory Th17 cells occurring in lesional skin and blood of psoriasis patients. In the CD18(hypo) PL/J mouse model of psoriasis, reduced expression of CD18/ß2 integrin to 2-16% of wild-type levels is associated with progressive loss of Tregs, impaired cell-cell contact between Tregs and dendritic cells (DCs), as well as Treg dysfunction as reported earlier. In the present investigation, Tregs derived from CD18(hypo) PL/J mice were analyzed for their propensity to differentiate into IL-17-producing Th17 cells in vivo and in in vitro Treg-DC cocultures. Adoptively transferred CD18(hypo) PL/J Tregs were more inclined toward conversion into IL-17-producing Th17 cells in vivo in an inflammatory as well as noninflammatory environment compared with CD18(wt) PL/J Tregs. Addition of neutralizing Ab against CD18 to Treg-DC cocultures in vitro promoted conversion of CD18(wt) PL/J Tregs to Th17 cells in a dose-dependent manner similar to conversion rates of CD18(hypo) PL/J Tregs. Reduced thymic output of naturally occurring Tregs and peripheral conversion of Tregs into Th17 cells therefore both contribute to the loss of Tregs and the psoriasiform dermatitis observed in CD18(hypo) PL/J mice. Our data overall indicate that CD18 expression levels impact Treg development as well as Treg plasticity and that differentiation of Tregs into IL-17-producing Th17 cells is distinctly facilitated by a subtotal deficiency of CD18.

Chromatin condensation via the condensin II complex is required for peripheral T-cell quiescence.

Naive T cells encountering their cognate antigen become activated and acquire the ability to proliferate in response to cytokines. Stat5 is an essential component in this response. We demonstrate that Stat5 cannot access DNA in naive T cells and acquires this ability only after T-cell receptor (TCR) engagement. The transition is not associated with changes in DNA methylation or global histone modification but rather chromatin decondensation. Condensation occurs during thymocyte development and proper condensation is dependent on kleisin-ß of the condensin II complex. Our findings suggest that this unique chromatin condensation, which can affect interpretations of chromatin accessibility assays, is required for proper T-cell development and maintenance of the quiescent state. This mechanism ensures that cytokine driven proliferation can only occur in the context of TCR stimulation.

Altered thymic selection and increased autoimmunity caused by ectopic expression of DRAK2 during T cell development.

Negative regulation of TCR signaling is an important mechanism enforcing immunological self-tolerance to prevent inappropriate activation of T cells and thus the development of autoimmune diseases. The lymphoid-restricted serine/threonine kinase death-associated protein-related apoptotic kinase-2 (DRAK2) raises the TCR activation threshold by targeting TCR-induced calcium mobilization in thymocytes and peripheral T cells and regulates positive thymic selection and peripheral T cell activation. Despite a hypersensitivity of peripheral drak2-deficient T cells, drak2-deficient mice are enigmatically resistant to induced autoimmunity in the model experimental autoimmune encephalomyelitis. To further evaluate the differential role of DRAK2 in central vs peripheral tolerance and to assess its impact on the development of autoimmune diseases, we have generated a transgenic (Tg) mouse strain ectopically expressing DRAK2 via the lck proximal promoter (1017-DRAK2 Tg mice). This transgene led to highest expression levels in double-positive thymocytes that are normally devoid of DRAK2. 1017-DRAK2 Tg mice displayed a reduction of single-positive CD4(+) and CD8(+) thymocytes in context with diminished negative selection in male HY TCR x 1017-DRAK2 Tg mice as well as peripheral T cell hypersensitivity, enhanced susceptibility to experimental autoimmune encephalomyelitis, and spontaneous autoimmunity. These findings suggest that alteration in thymocyte signaling thresholds impacts the sensitivity of peripheral T cell pools.

Enhanced T cell apoptosis within Drak2-deficient mice promotes resistance to autoimmunity.

Clonal expansion of T cells is vital to adaptive immunity, yet this process must be tightly controlled to prevent autoimmune disease. The serine/threonine kinase death-associated protein kinase-related apoptosis-inducing kinase 2 (DRAK2) is a negative regulator of TCR signaling and sets the threshold for the activation of naive and memory T cells and selected thymocytes. Despite enhanced T cell activation, Drak2(-/-) mice are resistant to experimental autoimmune encephalomyelitis, an autoimmune demyelinating disease that resembles multiple sclerosis. However, the basis for this autoimmune resistance is currently unknown. In this study, we show that, in the absence of DRAK2 signaling, T cells require greater tonic signaling for maintenance during clonal expansion. Following stimulation, Drak2(-/-) T cells were more sensitive to an intrinsic form of apoptosis that was prevented by CD28 ligation, homeostatic cytokines, or enforced Bcl-x(L) expression. T cell-specific Bcl-x(L) expression also restored the susceptibility of Drak2(-/-) mice to experimental autoimmune encephalomyelitis and enhanced thymic positive selection. These findings demonstrate that DRAK2 is selectively important for T cell survival and highlight the potential that DRAK2 blockade may lead to permanent autoimmune T cell destruction via intrinsic apoptosis pathways.