Search

Acute graft-versus-host disease (GvHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT), a potentially curative treatment for leukemia. Endoplasmic reticulum (ER) stress occurs when the protein folding capacity of the ER is oversaturated. How ER stress modulates tissue homeostasis in the context of alloimmunity is not well understood. We show that ER stress contributes to intestinal tissue injury during GvHD and can be targeted pharmacologically. We observed high levels of ER stress upon GvHD onset in a murine allo- HCT model and in human biopsies. These levels correlated with GvHD severity, underscoring a novel therapeutic potential. Elevated ER stress resulted in increased cell death of intestinal organoids. In a conditional knockout model, deletion of the ER stress regulator transcription factor Xbp1 in intestinal epithelial cells induced a general ER stress signaling disruption and aggravated GvHD lethality. This phenotype was mediated by changes in the production of antimicrobial peptides and the microbiome composition as well as activation of pro-apoptotic signaling. Inhibition of inositol-requiring enzyme 1α (IRE1α), the most conserved signaling branch in ER stress, reduced GvHD development in mice. IRE1α blockade by the small molecule inhibitor 4m8c improved intestinal cell viability, without impairing hematopoietic regeneration and T-cell activity against tumor cells. Our findings in patient samples and mice indicate that excessive ER stress propagates tissue injury during GvHD. Reducing ER stress could improve the outcome of patients suffering from GvHD.

Background Contact sensitizers may interfere with correct protein folding. Generation of un-/misfolded proteins can activate the IRE-1 or PERK signaling pathways initiating the unfolded protein response (UPR) and thereby determine inflammatory immune responses. We have analyzed the effect of sensitizers with different potencies on the induction of UPR activation/inhibition and the subsequent generation of a pro-inflammatory micromilieu in vitro as well as the effect of UPR modulation on the inflammatory response in the murine contact hypersensitivity (CHS) in vivo. Methods Semi-quantitative and quantitative PCR, fluorescence microscopy, ELISA, NF-κB activation and translocation assays, DC/keratinocyte co-culture assay, FACS, and in vivo CHS experiments were performed. Results Sensitizers and irritants activate IRE-1 and PERK in murine and human keratinocytes. Synergistic effects occur after combination of different weak sensitizers / addition of irritants. Moreover, tolerogenic dinitrothiocyanobenzene can be converted into a strong sensitizer by pre-activation of the UPR. Blocking UPR signaling results in decreased NF-κB activation and cytokine production in keratinocytes and in activation marker downregulation in a HaCaT/THP-1 co-culture. Interestingly, not only systemic but also topical application of UPR inhibitors abrogates CHS responses in vivo. Conclusion These observations highlight an important role of the UPR in determination of the inflammatory response in vitro and in vivo further underlining the importance of tissue stress and damage responses in the development of ACD and provide mechanistically based concepts as a basis for the development of new therapeutic approaches to treat allergic contact dermatitis.

Luteolin belongs to the group of flavonoids and can be found in flowers, herbs, vegetables and spices. It plays an important role in defending plants, for example against UV radiation by partially absorbing UVA and UVB radiation. Thus, luteolin can also decrease adverse photobiological effects in the skin by acting as a first line of defense. Furthermore, anti-oxidative and anti-inflammatory activities of luteolin were described on keratinocytes and fibroblasts as well as on several immune cells (e.g., macrophages, mast cell, neutrophils, dendritic cells and T cells). Luteolin can suppress proinflammatory mediators (e.g., IL-1β, IL-6, IL-8, IL-17, IL-22, TNF-α and COX-2) and regulate various signaling pathway (e.g., the NF-κB, JAK-STAT as well as TLR signaling pathway). In this way, luteolin modulates many inflammatory processes of the skin. The present review summarizes the recent in vitro and in vivo research on luteolin in the field of skin aging and skin cancer, wound healing as well as inflammatory skin diseases, including psoriasis, contact dermatitis and atopic dermatitis. In conclusion, luteolin might be a promising molecule for the development of topic formulations and systemic agents against inflammatory skin diseases.

Acute graft-versus-host disease causes significant mortality in patients undergoing allogeneic hematopoietic cell transplantation. Immunosuppressive treatment for graft-versus-host disease can impair the beneficial graft-versus-leukemia effect and facilitate malignancy relapse. Therefore, novel approaches that protect and regenerate injured tissues without impeding the donor immune system are needed. Bile acids regulate multiple cellular processes and are in close contact with the intestinal epithelium, a major target of acute graft-versus-host disease. Here, we found that the bile acid pool is reduced following graft-versus-host disease induction in a preclinical model. We evaluated the efficacy of bile acids to protect the intestinal epithelium without reducing anti-tumor immunity. We observed that application of bile acids decreased cytokine-induced cell death in intestinal organoids and cell lines. Systemic prophylactic administration of tauroursodeoxycholic acid, the most potent compound in our in vitro studies, reduced graft-versus-host disease severity in three different murine transplantation models. This effect was mediated by decreased activity of the antigen presentation machinery and subsequent prevention of apoptosis of the intestinal epithelium. Moreover, bile acid administration did not alter the bacterial composition in the intestine suggesting that its effects are cell-specific and independent of the microbiome. Treatment of human and murine leukemic cell lines with tauroursodeoxycholic acid did not interfere with the expression of antigen presentation-related molecules. Systemic T cell expansion and especially their cytotoxic capacity against leukemic cells remained intact. This study establishes a role for bile acids in the prevention of acute graft-versus-host disease without impairing the graft-versus-leukemia effect. In particular, we provide a scientific rationale for the systematic use of tauroursodeoxycholic acid in patients undergoing allogeneic hematopoietic cell transplantation.

Wahrend sich bei der Entschlusselung der Pathogenese der Kontaktallergie in den letzten Jahren das Augenmerk verstarkt auf die Mechanismen der angeborenen Immunantwort gerichtet hat, fehlt es nach wie vor an validen Therapiemoglichkeiten. Dieser Beitrag soll die Hintergrunde der Kontaktallergieentstehung sowie mogliche Risikofaktoren beleuchten und potenzielle neue Therapiemoglichkeiten aufzeigen. Es erfolgt die Beschreibung der allergischen Kontaktdermatitis (engl. „allergic contact dermatitis“ [ACD]) sowie der Sensibilisierungs- und Auslosephase, potenzieller Risikofaktoren sowie der Therapieoptionen unter Einbeziehung (aktueller) PubMed-gelisteter Literatur. Die Entzundung spielt bei der ACD eine zentrale Rolle. Das angeborene Immunsystem reagiert auf Kontaktallergene wie auf eine Infektion. Die Aufklarung der Mechanismen ermoglicht zukunftig eine gezielte therapeutische Intervention. Auch wenn weiterhin Forschungsbedarf besteht, sind viele Teile der Kontaktallergiepathogenese inzwischen besser verstanden. Insbesondere scheint die essenzielle Rolle der angeborenen Immunantwort nicht nur fur die Sensibilisierungs-, sondern auch fur die Auslosephase etabliert. Wichtig und spannend bleibt die Umsetzung der heutigen Erkenntnisse in neue Therapieansatze und deren Anwendungserprobung.

Although manifold beneficial effects of plant compounds for the treatment of skin disorders are known, cutaneous exposure to plants can also result in various types of incompatibility reactions such as contact dermatitis. In this mini-review, we briefly describe the different clinical forms of contact dermatitis (photoinduced, irritative, and allergic form) and highlight recent publications in the field of contact dermatitis. Major topics are recent recommendations regarding testing for plant contact dermatitis, advances in understanding the immunological mechanisms of plant contact dermatitis, and case reports for plant contact dermatitis. Unfortunately, most people still associate the terms “healthy and safe to use” with plant compounds due to their natural origin, leading to an increased utilization, be it for home-made remedies or as cosmetics. Therefore, it is on the one hand important to raise awareness in a broad audience that plants may cause contact dermatitis and on the other hand to indicate to clinicians that plants should be included in a patch test if a history of plant exposure exists.

Allergies are highly prevalent hypersensitivity responses to usually harmless substances. They are mediated by the immune system which causes pathologic responses such as type I (rhinoconjunctivitis, allergic asthma, atopy) or type IV hypersensitivity (allergic contact dermatitis). The different types of allergy are mediated by effector and memory T cells and, in the case of type I hypersensitivity, B cells. A prerequisite for the activation of these cells of the adaptive immune system is the activation of the innate immune system. The resulting inflammation is essential not only for the initiation but also for the elicitation and maintenance of allergies. Great progress has been made in the elucidation of the cellular and molecular pathomechanisms underlying allergen-induced inflammation. It is now recognized that the innate immune system in concert with tissue stress and damage responses orchestrates inflammation. This should enable the development of novel mechanism-based anti-inflammatory treatment strategies as well as of animal-free in vitro assays for the identification and potency classification of contact allergens.

Background Low molecular weight chemicals or metal ions can cause allergic contact dermatitis, an inflammatory skin disease. Mice lacking Toll-like receptors 2 and 4 (TLR2/4 mice) are resistant to contact hypersensitivity. In the Western population obesity is increasing, which is known to have a pro-inflammatory impact. Objectives The aim of this study was to investigate the impact of high fat diet on the sensitization and elicitation of CHS. We hypothesized that a pro-inflammatory micro-milieu can be caused by an increase in adipose tissue, which might be sufficient to break the resistance of TLR2/4 mice. Methods Four weeks prior to sensitization widltype (wt) or TLR2/4 mice were fed normal chow, control diet or high fat diet. The effects on CHS and inflammation were analysed measuring the ear swelling response, using flow cytometry and ELISA. Results The reaction of wt mice to TNCB was increased by high fat diet. While normal-chow fed TLR2/4 mice were still resistant to CHS, high fat diet and, unexpectedly, control diet feeding broke the resistance of TLR2/4 mice to TNCB. Conclusions These experiments suggest that the increased fat content or the different fatty acid composition of the diets increases inflammation and, therefore, the likelihood of developing CHS. This article is protected by copyright. All rights reserved.

Allergies are highly prevalent hypersensitivity responses to usually harmless substances. They are mediated by the immune system which causes pathologic responses such as type I (rhinoconjunctivitis, allergic asthma, atopy) or type IV hypersensitivity (allergic contact dermatitis). The different types of allergy are mediated by effector and memory T cells and, in the case of type I hypersensitivity, B cells. A prerequisite for the activation of these cells of the adaptive immune system is the activation of the innate immune system. The resulting inflammation is essential not only for the initiation but also for the elicitation and maintenance of allergies. Great progress has been made in the elucidation of the cellular and molecular pathomechanisms underlying allergen-induced inflammation. It is now recognized that the innate immune system in concert with tissue stress and damage responses orchestrates inflammation. This should enable the development of novel mechanism-based anti-inflammatory treatment strategies as well as of animal-free in vitro assays for the identification and potency classification of contact allergens.

Introduction: Hyaluronan (HA) is a major component of the skin that exerts a variety of biological functions. Inter-α-trypsin inhibitor heavy chain (ITIH) proteins comprise a family of hyaladherins of which ITIH5 has recently been described in skin, where it plays a functional role in skin morphology and inflammatory skin diseases including allergic contact dermatitis (ACD). Objective: The current study focused on the ITIH5-HA interaction and its potential clinical and functional impact in extracellular matrix (ECM) stabilization. Methods: Studying the molecular effects of ITIH5 in skin, we established skin models comprising murine skin cells of Itih5 knockout mice and corresponding wild-type controls. In addition, human dermal fibroblasts with an ITIH5 knockdown as well as a murine recombinant Itih5 protein were established to examine the interaction between ITIH5 and HA using in vitro adhesion and HA degradation assays. To understand more precisely the role of ITIH5 in inflammatory skin diseases such as ACD, we generated ITIH5 knockout cells of the KeratinoSens® cell line. Results: Using murine skin models, ITIH5 knockdown fibroblasts, and a reactive oxygen species (ROS)-mediated HA degradation assay, we proved that ITIH5 binds to HA, thereby acting as a stabilizer of HA. Moreover, microarray profiling revealed the impact of ITIH5 on biological processes such as skin development and ECM homeostasis. Performing the in vitro KeratinoSens skin sensitization assay, we detected that ITIH5 decreases the sensitizing potential of moderate and strong contact sensitizers. Conclusion: Taken together, our experiments revealed that ITIH5 forms complexes with HA, thereby on the one hand stabilizing HA and facilitating the formation of ECM structures and on the other hand modulating inflammatory responses.

Drug hypersensitivity involves the activation of T cells in an HLA allele–restricted manner. Because the majority of individuals who carry HLA risk alleles do not develop hypersensitivity, other parameters must control development of the drug-specific T cell response. Thus, we have used a T cell–priming assay and nitroso sulfamethoxazole (SMX-NO) as a model Ag to investigate the activation of specific TCR Vβ subtypes, the impact of programmed death -1 (PD-1), CTL-associated protein 4 (CTLA4), and T cell Ig and mucin domain protein-3 (TIM-3) coinhibitory signaling on activation of naive and memory T cells, and the ability of regulatory T cells (Tregs) to prevent responses. An expansion of the TCR repertoire was observed for nine Vβ subtypes, whereas spectratyping revealed that SMX-NO–specific T cell responses are controlled by public TCRs present in all individuals alongside private TCR repertoires specific to each individual. We proceeded to evaluate the extent to which the activation of these TCR Vβ–restricted Ag-specific T cell responses is governed by regulatory signals. Blockade of PD-L1/CTLA4 signaling dampened activation of SMX-NO–specific naive and memory T cells, whereas blockade of TIM-3 produced no effect. Programmed death-1, CTLA4, and TIM-3 displayed discrete expression profiles during drug-induced T cell activation, and expression of each receptor was enhanced on dividing T cells. Because these receptors are also expressed on Tregs, Treg-mediated suppression of SMX-NO–induced T cell activation was investigated. Tregs significantly dampened the priming of T cells. In conclusion, our findings demonstrate that distinct TCR Vβ subtypes, dysregulation of coinhibitory signaling pathways, and dysfunctional Tregs may influence predisposition to hypersensitivity.

To develop an in vitro assay that recapitulates the key event of Allergic Contact Dermatitis (ACD), that is the priming of human effector T cells by hapten-presenting dendritic cells (DCs), and then allows for the sensitive detection of chemical allergens, represents a highly desirable goal but also a major challenge. Substantial progress has been made in recent years in the development of T cell-based assays, also referred to as human T Cell Priming Assays (hTCPA). hTCPA classically consists of an autologous mixed DC-lymphocyte reaction (MDLR) using test chemical-loaded DCs, as stimulatory cells, and normal human naive lymphocytes, as responding cells. Combining diverse strategies which (i) boosted the stimulatory capacities of DCs, (ii) bypassed the cellular or molecular brakes that mitigate T cell activation, or (iii) improved the solubilisation of test chemicals in culture media, the most newly-refined hTCPA protocols showed good results to predict the allergenic potential of chemicals. Although current protocols still require further investment to improve usability and to demonstrate their capacity to establish the sensitizing potency of chemicals, there is no doubt that the hTCPA will be of great help in the forthcoming years to support the immunotoxicologic assessment of new or challenging chemicals.

Background Missense mutations in keratin 5 and 14 genes cause the severe skin fragility disorder epidermolysis bullosa simplex (EBS) by collapsing of the keratin cytoskeleton into cytoplasmic protein aggregates. Despite intense efforts, no molecular therapies are available, mostly due to the complex phenotype of EBS, comprising cell fragility, diminished adhesion, skin inflammation and itch. Methods We extensively characterized KRT5 and KRT14 mutant keratinocytes from patients with severe generalized EBS following exposure to the chemical chaperone 4-phenylbutyrate (4-PBA). Findings 4-PBA diminished keratin aggregates within EBS cells and ameliorated their inflammatory phenotype. Chemoproteomics of 4-PBA-treated and untreated EBS cells revealed reduced IL1β expression- but also showed activation of Wnt/β-catenin and NF-kB pathways. The abundance of extracellular matrix and cytoskeletal proteins was significantly altered, coinciding with diminished keratinocyte adhesion and migration in a 4-PBA dose-dependent manner. Interpretation Together, our study reveals a complex interplay of benefits and disadvantages that challenge the use of 4-PBA in skin fragility disorders.

Kindler syndrome, a distinct type of epidermolysis bullosa, is a rare disorder caused by mutations in FERMT1, encoding kindlin-1. Most FERMT1 mutations lead to premature termination codons and absence of kindlin-1. Here we investigated the molecular and cellular consequences of a naturally occurring FERMT1 mutation, c.299_301del resulting in a single amino acid deletion, p.R100del. The mutation led to a 50% reduction of FERMT1 mRNA and 90% reduction of kindlin-1 protein in keratinocytes derived from the patient, as compared with control cells. The misfolded p.R100del kindlin-1 mutant was lysosomally degraded and launched a homeostatic unfolded protein response. Sodium-phenylbutyrate significantly increased kindlin-1 mRNA and protein levels and the area of mutant cells, acting as a chemical chaperone and probably also as a histone deacetylase inhibitor. In a recombinant system, low levels of wild-type or p.R100del mutant kindlin-1 were sufficient to improve the cellular phenotype in respect of spreading and proliferation as compared with kindlin-1 negative keratinocytes. The study of this hypomorphic mutation provides evidence that low amounts of kindlin-1 are sufficient to improve the epidermal architecture and Kindler syndrome cellular phenotype and proposes a personalized chaperone therapy for the patient.

The mechanisms underlying thymoma-associated immunodeficiency are largely unknown, and the significance of increased blood γδ Τ cells often remains elusive. In this study we address these questions based on an index patient with thymoma, chronic visceral leishmaniasis, myasthenia gravis, and a marked increase of rare γδ T cell subsets in the peripheral blood. This patient showed cutaneous anergy, even though he had normal numbers of peripheral blood total lymphocytes as well as CD4+ and CD8+ T cells. Despite his chronic infection, analyses of immunophenotypes and spectratyping of his lymphocytes revealed an unusual accumulation of naive γδ and αβ T cells, suggesting a generalized T cell activation defect. Functional studies in vitro demonstrated substantially diminished IL-2 and IFN-γ production following TCR stimulation of his “untouched” naive CD4+ T cells. Biochemical analysis revealed that his γδ and αβ T cells carried an altered TCR complex with reduced amounts of the ζ-chain (CD247). No mutations were found in the CD247 gene that encodes the homodimeric ζ protein. The diminished presence of CD247 and increased numbers of γδ T cells were also observed in thymocyte populations obtained from three other thymoma patients. Thus, our findings describe a novel type of a clinically relevant acquired T cell immunodeficiency in thymoma patients that is distinct from Good’s syndrome. Its characteristics are an accumulation of CD247-deficient, hyporresponsive naive γδ and αβ T cells and an increased susceptibility to infections.

Contact sensitization is the initial process involved in the development of an allergic reaction to xenobiotic environmental substances. Here, we briefly describe the differences between irritant and allergic contact dermatitis. Then, we highlight the essential steps involved in the development of an ACD reaction, i.e., the protein binding of haptens, genetic factors influencing the penetration of sensitizers into the skin, the different mechanisms driving the initial development of an inflammatory cytokine micromilieu enabling the full maturation of dendritic cells, the role of pre- and pro-haptens, antigen presentation and T cell activation via MHC and CD1 molecules, dendritic cell (DC) migration, and potential LC contribution as well as the different T cell subsets involved in ACD. In addition, we discuss the latest publications regarding factors that might influence the sensitizing potential such as repeated sensitizer application, penetration enhancers, humidity of the skin, microbiota, Tregs, and phthalates. Last but not least, we briefly touch upon novel targets for drug development that might serve as treatment options for ACD.

Squamous cell carcinoma (SCC) is one of the most common skin cancers and causes significant morbidity. Although the expression of the epithelial adhesion molecule collagen XVII (ColXVII) has been linked to SCC invasion, only little is known about its mechanistic contribution. Here, we demonstrate that ColXVII expression is essential for SCC cell proliferation and motility. Moreover, it revealed that particularly the post-translational modification of ColXVII by ectodomain shedding is the major driver of SCC progression, because ectodomain-selective immunostaining was mainly localized at the invasive front of human cutaneous SCCs, and exclusive expression of a non-sheddable ColXVII mutant in SCC-25 cells inhibits their matrix-independent growth and invasiveness. This cell surface proteolysis, which is strongly elevated during SCC invasion and metastasis, releases soluble ectodomains and membrane-anchored endodomains. Both released ColXVII domains play distinct roles in tumor progression: the endodomain induces proliferation and survival, whereas the ectodomain accelerates invasiveness. Furthermore, specific blockage of shedding by monoclonal ColXVII antibodies repressed matrix-independent growth and invasion of SCC cells in organotypic co-cultures. Thus, selective inhibition of ColXVII shedding may offer a promising therapeutic strategy to prevent SCC progression.

The induction of allergic contact dermatitis by chemicals depends on their ability to activate a chemical-specific T cell response. This key event that concludes the skin sensitization process is monitored in an in vitro assay, the human T cell priming assay (hTCPA). Naive human T cells are incubated with autologous dendritic cells pulsed with the chemical of interest. After restimulation with the same chemical, the induction of an antigen-specific T cell response can be assessed by the detection of T cell proliferation, cytokine production or surface marker expression. The hTCPA still needs optimization but may nevertheless be a valuable in vitro assay within an integrated testing strategy aiming at replacement of animal testing for contact allergen identification.

Allergic contact dermatitis (ACD) is induced by low-molecular weight electrophilic chemicals and metal ions. Chemical contact sensitizers trigger reactive oxygen species production and provoke electrophilic stress, leading to the accumulation of the transcription factor nuclear-related factor 2 (Nrf2) in innate immune cell types. The objective of this work was to identify the role of Nrf2 in the regulation of ACD. We used the local lymph node assay (LLNA) and the mouse ear swelling test (MEST) to study the role of Nrf2 in both the sensitization and elicitation phase in nrf2 knockout (nrf2(-/-)) and wild-type (nrf2(+/+)) mice. Five chemicals were used: two compounds known to react with cysteine residues, 2,4-dinitrochlorobenzene (DNCB) and cinnamaldehyde (CinA); one sensitizer known to exhibit mixed reactivity to cysteine and lysine residues, isophorone diisocyanate; and one reacting specifically with lysine residues, trimellitic anhydride and croton oil, a well-known irritant. In the MEST assay, DNCB (1 and 2%) induced a significant increase in ear thickness in nrf2(-/-) compared with nrf2(+/+) mice, suggesting a role for Nrf2 in the control of the inflammatory process. When DNCB was used at 0.25 and 0.5% or when mice were treated with CinA, inflammation was found only in nrf2(-/-) mice. In the LLNA, all chemical sensitizers induced an increase of lymphocyte proliferation in nrf2(-/-) compared with nrf2(+/+) mice for the same chemical concentration. These results reveal an important role for Nrf2 in controlling ACD and lymphocyte proliferation in response to sensitizers.

Kindler syndrome (KS), a rare, autosomal recessive disorder comprises mechanical skin fragility and photosensitivity, which manifest early in life. The progression of the disorder is irreversible and results in tissue damage in form of cutaneous and mucosal atrophy and scarring and epithelial cancers. Here, we unravel molecular mechanisms of increased UV-B sensitivity of keratinocytes derived from KS patients. We show that the pro-inflammatory cytokines, IL-1s, IL-6 and TNF-α, are upregulated in KS skin and in UV-B irradiated KS keratinocytes. These cytokines are dependent on p38 activation, which is increased in the absence of kindlin-1 and induced by higher ROS levels. Other dysregulated cytokines and growth factors were identified in this study and might be involved in paracrine interactions contributing to KS pathology. We show a direct relationship between kindlin-1 abundance and UV-B induced apoptosis in keratinocytes, whereas kindlin-2 overexpression has no compensatory effect. Importantly, low levels of kindlin-1 are sufficient to relieve or rescue this feature. Reduction of pro-inflammatory cytokines and of UV-B induced apoptosis is a valid therapeutic goal to influence long term complications of KS. Here, we demonstrate that antioxidants and the plant flavonoid luteolin represent feasible topical therapeutic approaches decreasing UV-B induced apoptosis in two-dimensional and organotypic KS cultures. We provide evidence for potential new therapeutic approaches to mitigate the progressive course of KS, for which no cure is available to date. Furthermore, we established organotypic KS models, a valuable in vitro tool for research with a morphology similar to the skin of patients in situ.

Kindlins are a novel family of intracellular adaptor proteins in integrin-containing focal adhesions. Kindlin-1 and -2 are expressed in the skin, but whether and how they cooperate in adult epithelial cells have remained elusive. We uncovered the overlapping roles of kindlin-1 and -2 in maintaining epithelial integrity and show that the phenotype of kindlin-1-deficient cells can be modulated by regulating kindlin-2 gene expression and vice versa. The experimental evidence is provided by use of human keratinocyte cell lines that express both kindlins, just kindlin-1 or kindlin-2, or none of them. Double deficiency of kindlin-1 and -2 had significant negative effects on focal adhesion formation and actin cytoskeleton organization, cell adhesion, survival, directional migration, and activation of β 1 integrin, whereas deficiency of one kindlin only showed variable perturbation of these functions. Cell motility and formation of cell-cell contacts were particularly affected by lack of kindlin-2. These results predict that kindlin-1 and -2 can functionally compensate for each other, at least in part. The high physiologic and pathologic significance of the compensation was emphasized by the discovery of environmental regulation of kindlin-2 expression. UV-B irradiation induced loss of kindlin-2 in keratinocytes. This first example of environmental regulation of kindlin expression has implications for phenotype modulation in Kindler syndrome, a skin disorder caused by kindlin-1 deficiency.

Chemicals can elicit T-cell-mediated diseases such as allergic contact dermatitis and adverse drug reactions. Therefore, testing of chemicals, drugs and protein allergens for hazard identification and risk assessment is essential in regulatory toxicology. The seventh amendment of the EU Cosmetics Directive now prohibits the testing of cosmetic ingredients in mice, guinea pigs and other animal species to assess their sensitizing potential. In addition, the EU Chemicals Directive REACh requires the retesting of more than 30,000 chemicals for different toxicological endpoints, including sensitization, requiring vast numbers of animals. Therefore, alternative methods are urgently needed to eventually replace animal testing. Here, we summarize the outcome of an expert meeting in Rome on 7 November 2009 on the development of T-cell-based in vitro assays as tools in immunotoxicology to identify hazardous chemicals and drugs. In addition, we provide an overview of the development of the field over the last two decades.

Modification of proteins by reactive small chemicals is a key step in the activation of chemical-specific T cells in allergic contact dermatitis (ACD). However, an integrated approach to characterize both the precise nature of chemically modified proteins and the chemical-specific T cells is currently lacking. Here, we analyze the molecular conditions for adduct formation of the strong human contact sensitizer 2,4-dinitrochlorobenzene (DNCB) and its water-soluble form, 2,4-dinitrobenzenesulfonic acid (DNBS), with both an all amino acid-containing model peptide (± Cys) and the protein human serum albumin (HSA). Mass spectrometric detection and quantification revealed thiol-dependent peptide adduct formation at all pH values found in human skin layers. Highest modification rates were obtained with DNBS. Accordingly, DNBS- but not DNCB-modified human immature dendritic cells (iDC) induced in vitro primary human T-cell responses as did 2,4,6-trinitrobenzenesulfonic acid-modified iDC as measured by dinitrophenyl (DNP)- and trinitrophenyl (TNP)-specific T-cell proliferation and interferon gamma (IFN-γ) production in CD4(+) and CD8(+) T-cell subsets. Moreover, DNP-modified HSA protein effectively induced primary T-cell responses when processed by iDC. Thus, an integrated approach that combines efficient skin-related in chemico coupling analyses with an in vitro T-cell priming assay can be used to predict in vivo reactions of chemical contact allergens with extracellular and cellular proteins. This strategy supports the development of chemical-specific in vitro assays that are urgently required in predictive hazard identification and risk assessment of allergenic and nonallergenic chemicals.

Drug hypersensitivity involves the activation of T-cells in an HLA allele-restricted manner. Since the majority of individuals who carry HLA risk alleles do not develop hypersensitivity, other parameters must control development of the drug-specific T-cell response. Thus, we have utilized a T-cell priming assay and nitroso sulfamethoxazole (SMX-NO) as a model antigen to investigate (1) the activation of specific T-cell receptor (TCR)Vβ subtypes, (2) the impact of PD-1, CTLA4 and TIM-3 co-inhibitory signalling on activation of naïve and memory T-cells and (3) the ability of Tregs to prevent responses. An expansion of the TCR repertoire was observed for nine different Vβ subtypes, while spectratyping revealed that SMX-NO-specific T-cell responses are controlled by public TCRs present in all individuals alongside private TCR repertoires specific to each individual. We proceeded to evaluate the extent to which the activation of these TCR Vβ-restricted antigen-specific T-cell responses is governed by regulatory signals. Blockade of PDL-1/CTLA4 signalling dampened activation of SMX-NO-specific naïve and memory T-cells, while blockade of TIM-3 produced no effect. PD-1, CTLA4, and TIM-3 displayed discrete expression profiles during drug-induced T-cell activation and expression of each receptor was enhanced on dividing T-cells. As these receptors are also expressed on Tregs, Treg-mediated suppression of SMX-NO-induced T-cell activation was investigated. Tregs significantly dampened the priming of T-cells. In conclusion, our findings demonstrate that distinct TCR Vβ subtypes, dysregulation of co-inhibitory signalling pathways and dysfunctional Tregs may influence predisposition to hypersensitivity.

Allergic contact dermatitis is induced by chemicals or metal ions. A hallmark of this T cell mediated skin disease is the activation of the innate immune system by contact allergens. This immune response results in inflammation and is a prerequisite for the activation of the adaptive immune system with tissue-specific migration of effector and regulatory T cells. Recent studies have begun to address in detail the innate immune cells as well as the innate receptors on these cells and the associated signaling pathways which lead to skin inflammation. We review here recent findings regarding innate and adaptive immune responses and immune regulation of contact dermatitis and other skin diseases as well as recent developments towards an in vitro assessment of the allergenic potential of chemicals. The elucidation of the innate inflammatory pathways, cellular components and mediators will help to identify new drug targets for more efficient treatment of allergic contact dermatitis and hopefully also for its prevention.

Allergic contact dermatitis is a T cell-mediated skin disease. Many hundreds of organic chemicals and some metal ions are contact sensitizers. They induce an innate inflammatory immune response in the skin that results in the priming of contact sensitizer-specific T cells by dendritic cells in the draining lymph nodes. The factors that determine the strength of this T cell response and thereby define the potency of a contact sensitizer are largely unknown. This chapter highlights different variables such as precursor frequency of antigen-specific T cells, possible bystander activation, and T cell receptor diversity or avidity of the TCR/peptide-MHC interactions, which might impact the quality and strength of T cell responses to contact sensitizers. In addition, different methods available to determine both the frequency of antigen-specific T cells and T cell receptor repertoires are discussed. Identification of the factors determining potency may allow for the development of suitable in vitro assays for potency assessment of contact sensitizers.

Allergic contact dermatitis is a T cell-mediated skin disease. Many hundreds of organic chemicals and some metal ions are contact sensitizers. They induce an innate inflammatory immune response in the skin that results in the priming of contact sensitizer-specific T cells by dendritic cells in the draining lymph nodes. The factors that determine the strength of this T cell response and thereby define the potency of a contact sensitizer are largely unknown. This chapter highlights different variables such as precursor frequency of antigen-specific T cells, possible bystander activation, and T cell receptor diversity or avidity of the TCR/peptide-MHC interactions, which might impact the quality and strength of T cell responses to contact sensitizers. In addition, different methods available to determine both the frequency of antigen-specific T cells and T cell receptor repertoires are discussed. Identification of the factors determining potency may allow for the development of suitable in vitro assays for potency assessment of contact sensitizers.

We previously demonstrated an age-related decrease in receptor for activated C-kinase (RACK-1) expression and functional deficit in Langerhans cells’ responsiveness. This defect specifically involves the translocation of protein kinase C (PKC)-β. The purpose of this study was to investigate the role of RACK-1 and PKC-β in chemical allergen-induced CD86 expression and IL-8 release in the human promyelocytic cell line THP-1 and primary human dendritic cells (DC). Dinitrochlorobenzene, p-phenylenediamine and diethyl maleate were used as contact allergens. The selective cell-permeable inhibitor of PKC-β and the broad PKC inhibitor GF109203X completely prevented chemical allergen- or lipopolysaccharide (LPS)-induced CD86 expression and significantly modulated IL-8 release (50 % reduction). The selective cell-permeable inhibitor of PKC-e (also known to bind to RACK-1) failed to modulate allergen- or LPS-induced CD86 expression or allergen-induced IL-8 release, while modulating LPS-induced IL-8 release. The use of a RACK-1 pseudosubstrate, which directly activates PKC-β, resulted in dose-related increase in CD86 expression and IL-8 release. Similar results were obtained with human DC, confirming the relevance of results obtained in THP-1 cells. Overall, our findings demonstrate the role of PKC-β and RACK-1 in allergen-induced CD86 expression and IL-8 production, supporting a central role of PKC-β in the initiation of chemical allergen-induced DC activation.

061 Premature aging in Cockayne syndrome due to a failure in ribosomal biogenesis? M Alupei, P esser, K Scharffetter-Kochanek and S Iben 1 University of Ulm, Ulm, Germany and 2 University of freiburg, Freiburg, Germany Cockayne syndrome (CS) is a progeroid disease characterized by growth failure, neurological degeneration, loss of subcutaneous fat and cachexia. The mutations of this polygenic disease inactivate a subbranch of the DNA-repair pathway nucleotide excision repair (NER), thus Cockayne syndrome is regarded as a DNA-repair disease. As the total loss of DNA repair by the NER in xeroderma pigmentosum does not lead to childhood degeneration, alternative functions of the CS proteins might account for the disease. One common alternative function of the Cockayne syndrome proteins is the key step in ribosomal biogenesis, transcription by RNA polymerase I in the nucleolus. We investigated the cellular consequences of a failure of RNA polymerase I transcription in CS-cells and discovered a circulus vitiosus repressing ribosomal biogenesis. A disturbed RNA polymerase I transcription gives rise to ribosomes with a low translational fidelity that produce misfolded proteins. Misfolded proteins provoke endoplasmic reticulum stress and an unfolded protein response that represses RNA polymerase I transcription. The oxidative hypersensitivity of Cockayne syndrome cells might be due to oxidation of misfolded proteins. The elevated apoptosis rate of CS cells after oxidative challenge can be overcome by pharmacological chaperones that dampen endoplasmic reticulum stress and the unfolded protein response. Pharmacological chaperones are restoring proper RNA polymerase I transcription by de-repression thus breaking the circulus vitiosus. This might be a treatment opportunity for children suffering from premature aging. Moreover, loss of proteostasis due to a reduced translational fidelity of ribosomes might be a mechanism contributing to aging and aging associated diseases in humans. 062 Stress perception impacts on clinical signs of skin ageing and modifies the epigenome R Pattinson, C Bundy, L Cordingley, S Kyle, W Yeung, G Beauchef, A Nkengne, A O’Callaghan, CE Griffiths and RE Watson 1 University of Manchester, Manchester, United Kingdom, 2 Laboratoire CLARINS, Pontoise, France and 3 Fios Genomics Ltd., Edinburgh, United Kingdom In this study we tested the commonly held assumption that stress affects the appearance of skin through the premature occurrence of clinical features of skin ageing (lines, wrinkles and dark under-eye circles). We recruited 84 healthy adult female volunteers (age range, 24 e 40 years; mean 30.7 4.7 years); each volunteer completed a comprehensive battery of psychological and sleep questionnaires (State-Trait Anxiety Inventory, Perceived Stress Score, Hospital Anxiety and Depression Scale, Pittsburgh sleep quality index [PSQI], Ford insomnia response to stress test [FIRST] and consensus sleep diary), clinical photography (VISIA-CR ) and provided a sample of whole blood for epigenetic analysis. Image analysis of clinical photographs allowed the objective quantification of wrinkles around the eye (eye bag and crow’s feet), the nasolabial fold and deep mid-forehead frown lines. Initial analysis correlated perceived stress with worse sleep patterns; those volunteers who perceived stress more keenly had significantly disturbed sleep as measured by PSQI and FIRST with reduced sleep efficiency (r1⁄4-0.22; P

Human skin is continuously exposed to solar radiation, which can result in photoaging, a process involving both dermal and, to a lesser extent, epidermal structures. Previously, we have shown that the flavonoid luteolin protects the epidermis from ultraviolet (UV)-induced damage by a combination of UV-absorbing, antioxidant, and antiinflammatory properties. The aim of the present study was to determine direct and indirect effects of luteolin on dermal fibroblasts as major targets of photoaging. Stimulation of fibroblasts with UVA light or the proinflammatory cytokine interleukin-20 (IL-20) is associated with wrinkled skin, increased IL-6 secretion, matrix metalloproteinase (MMP-1) expression, and hyaluronidase activity. All of these targets were inhibited by luteolin via interference with the p38 mitogen-activated protein kinase (MAPK) pathway. Next, we assessed the role of conditioned supernatants from keratinocytes irradiated with solar-simulated radiation (SSR) on nonirradiated dermal fibroblasts. In keratinocytes, luteolin inhibited SSR-induced production of IL-20, also via interference with the p38 MAPK pathway. Similarly, keratinocyte supernatant-induced IL-6 and MMP-1 expression in fibroblasts was reduced by pretreatment of keratinocytes with luteolin. Finally, these results were confirmed ex vivo on skin explants treated with luteolin before UV irradiation. Our results suggest that SSR-mediated production of soluble factors in keratinocytes is modulated by luteolin and may attenuate photoaging in dermal fibroblasts.

BackgroundAllergic contact dermatitis (ACD) represents a severe health problem with increasing worldwide prevalence. It is a T cell-mediated skin disease induced by protein-reactive organic and inorganic chemicals. A key feature of contact allergens is their ability to trigger an innate immune response that leads to skin inflammation. Previous evidence from the mouse contact hypersensitivity (CHS) model suggests a role for endogenous activators of innate immune signaling. Here, we analyzed the role of contact sensitizer induced ROS production and concomitant changes in hyaluronic acid metabolism on CHS responses.Methodology/principal findingsWe analyzed in vitro and in vivo ROS production using fluorescent ROS detection reagents. HA fragmentation was determined by gel electrophoresis. The influence of blocking ROS production and HA degradation by antioxidants, hyaluronidase-inhibitor or p38 MAPK inhibitor was analyzed in the murine CHS model. Here, we demonstrate that organic contact sensitizers induce production of reactive oxygen species (ROS) and a concomitant breakdown of the extracellular matrix (ECM) component hyaluronic acid (HA) to pro-inflammatory low molecular weight fragments in the skin. Importantly, inhibition of either ROS-mediated or enzymatic HA breakdown prevents sensitization as well as elicitation of CHS.Conclusions/significanceThese data identify an indirect mechanism of contact sensitizer induced innate inflammatory signaling involving the breakdown of the ECM and generation of endogenous danger signals. Our findings suggest a beneficial role for anti-oxidants and hyaluronidase inhibitors in prevention and treatment of ACD.

Many small protein reactive organic and inorganic chemicals can cause allergic contact dermatitis, a T cell mediated inflammatory skin disease. In vitro alternatives to animal testing are needed for the identification of chemicals that pose such risks to human health. We here publish the standard operation procedure for a human T cell priming assay developed primarily for the identification of contact allergens within the integrated EU project Sens-it-iv. This multiparametric flow cytometry based assay identifies chemical specific T cells based on their frequency and antigen-specific production of the cytokines IFN-γ and TNF-α at the single cell level. Using sorted naive T cells and monocyte-derived dendritic cells pulsed with the test chemical or with chemical-protein conjugates, the successful priming of an antigen-specific T cell response is controlled after antigen-specific restimulation by cytokine readout. As the most specific response of the immune system to contact allergens the analysis of the chemical-specific T cell response may be a useful in vitro assay for hazard identification in immunotoxicology. This assay may be a valuable, highly specific element of an integrated testing strategy for the identification of chemicals and drugs that cause T cell mediated respiratory or gastrointestinal tract hypersensitivities or adverse drug reactions.

Ultraviolet (UV) radiation induces DNA damage, oxidative stress, and inflammatory processes in human keratinocytes resulting in skin inflammation, photoaging, and photocarcinogenesis. The flavonoid luteolin is one of the most potent antioxidative plant polyphenols. We investigated the UV protective and antioxidant properties of luteolin in human keratinocytes in vitro, ex vivo, and in vivo. Spectrophotometric measurements revealed extinction maxima of luteolin in the UVB and UVA range. UV transmission below 370 nm was

Zeina El Ali 1, Cedric Gerbeix 2, Philippe Esser 3, Pauline Robert 4, Jean-Jacques Legrand 2, Stefan Martin 3, Marc Pallardy 1 and Saadia Kerdine-Romer 1 1 University Paris Sud Faculte de Pharmacie, Châtenay-Malabry, France ; 2 CiToxLAB France, Evreux ; 3 Allergy Research Group, Department of Dermatology, Freiburg, Germany ; 4 University Paris Sud Plateforme Animalerie et Exploration Fonctionnelle ou IPSIT, Faculte de Pharmacie, Châtenay-Malabry, France