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Vitiligo is an autoimmune disease that results from the loss of melanocytes, associated with skin infiltration of CD8+ effector memory T cells with a Tc1 skewed immune response. NKG2D is an activating receptor found on immune cells, in particular natural killer and activated CD8+ T cells, that are able to produce a high amount of IFN-γ. Here we found that NKG2D expression was increased in vitiligo skin CD8+ effector memory T cells and was promoted by IL-15. Phenotypic and functional analyses showed that NKG2D+ CD8+ skin effector memory T cells displayed an activated phenotype and produced elevated levels of both IFN-γ and tumor necrosis factor-α. Additional experiments revealed that vitiligo skin dendritic cells expressed the NKG2D ligands MICA-MICB, and in vitro experiments showed that these ligands could be induced on dendritic cells by IFN-α. Cultures of IFN-α–stimulated dendritic cells with skin NKG2D+ CD8+ T cells potentiated the production of type 1 cytokines, which was next inhibited by blocking the NKG2D/MICA-MICB interaction. These data show that NKG2D is a potential marker of pathogenic skin CD8+ effector memory T cells during vitiligo. Therefore, targeting NKG2D could be an attractive strategy in vitiligo, a disease for which there is a strong need of innovative treatments.

Loss of melanocytes is the pathological hallmark of vitiligo, a chronic inflammatory skin depigmenting disorder induced by exaggerated immune response, including autoreactive CD8 T cells producing high levels of type 1 cytokines. However, the interplay between this inflammatory response and melanocyte disappearance remains to be fully characterized. Here, we demonstrate that vitiligo skin contains a significant proportion of suprabasal melanocytes, associated with disruption of E-cadherin expression, a major protein involved in melanocyte adhesion. This phenomenon is also observed in lesional psoriatic skin. Importantly, apoptotic melanocytes were mainly observed once cells were detached from the basal layer of the epidermis, suggesting that additional mechanism(s) could be involved in melanocyte loss. The type 1 cytokines IFN-γ and TNF-α induce melanocyte detachment through E-cadherin disruption and the release of its soluble form, partly due to MMP-9. The levels of MMP-9 are increased in the skin and sera of patients with vitiligo, and MMP-9 is produced by keratinocytes in response to IFN-γ and TNF-α. Inhibition of MMP-9 or the JAK/STAT signaling pathway prevents melanocyte detachment in vitro and in vivo. Therefore, stabilization of melanocytes in the basal layer of the epidermis by preventing E-cadherin disruption appears promising for the prevention of depigmentation occurring in vitiligo and during chronic skin inflammation.

Vitiligo is a chronic autoimmune depigmenting skin disorder that results from a loss of melanocytes. Multiple combinatorial factors have been involved in disease development, with a prominent role of the immune system, in particular T cells. After repigmentation, vitiligo frequently recurs in the same area, suggesting that vitiligo could involve the presence of resident memory T cells (TRM). We sought to perform a thorough characterization of the phenotype and function of skin memory T cells in vitiligo. We show that stable and active vitiligo perilesional skin is enriched with a population of CD8 TRM expressing both CD69 and CD103 compared with psoriasis and control unaffected skin. CD8 TRM expressing CD103 are mainly localized in the epidermis. Expression of CXCR3 is observed on most CD8 TRM in vitiligo, including the population of melanocyte-specific CD8 T cells. CD8 TRM displayed increased production of IFN-γ and tumor necrosis factor-α with moderate cytotoxic activity. Our study highlights the presence of functional CD8 TRM in both stable and active vitiligo, reinforcing the concept of vitiligo as an immune memory skin disease. The CD8 TRM that remain in stable disease could play a role during disease flares, emphasizing the interest in targeting this cell subset in vitiligo.

Background The use of anti-programmed cell death (PD)-1 therapies in metastatic tumors is associated with cutaneous side effects including vitiligo-like lesions. Objective We sought to characterize clinically and biologically vitiligo-like lesions occurring in patients receiving anti-PD-1 therapies by studying a case series of 8 patients with metastatic tumors and 30 control subjects with vitiligo. Methods Eight patients receiving anti-PD-1 therapies with features of vitiligo-like lesions seen in our department were recruited. Clinical features and photographs were analyzed. For some patients, skin and blood samples were obtained. Results were compared with the vitiligo group. Results All patients developed lesions localized on photoexposed areas with a specific depigmentation pattern consisting of multiple flecked lesions without Koebner phenomenon. In contrast to vitiligo, patients receiving anti-PD-1 therapies who developed vitiligo-like lesions did not report any personal or family histories of vitiligo, thyroiditis, or other autoimmune disorders. Analysis of blood and skin samples revealed increased C-X-C motif ligand 10 levels in serum of patients developing vitiligo-like lesions, associated with skin infiltration of CD8 T-cells expressing C-X-C motif receptor 3 and producing elevated levels of interferon-γ and tumor necrosis factor-alfa. Limitations This cross-sectional study concerned a single center. Conclusions Clinical and biological patterns of vitiligo-like lesions occurring in patients receiving anti-PD-1 therapies differ from vitiligo, suggesting a different mechanism.

Folliculitis decalvans (FD) is a chronic inflammatory disease leading to scarring alopecia with poorly defined pathogenesis. The aim of this study was to investigate the expression of markers associated with the activation of innate immune signals, such as inflammasome (NALP1 and NALP3), interleukin (IL)-1β and IL-8 and type I interferon (MxA). A retrospective monocentric study was conducted and included 17 patients with FD with available biopsies. Disease activity (stable vs. active) was defined clinically and histologically. Immunostaining was performed using antibodies directed against NALP1, NALP3, IL-1β, IL-8, and MxA on FD skin biopsies. Results were compared with normal controls and lichen planopilaris. Eleven patients had active disease and 6 had stable disease. NALP1, NALP3, and IL-1β expression were significantly increased in hair follicles in FD compared with controls and lichen planopilaris. This study highlights the predominant immune signal associated with inflammasome activation in FD, suggesting the use of IL-1β blockade in FD.

SummaryBackground Plasmacytoid dendritic cells (pDCs) are a subset of dendritic cells specialized in the production of type I interferon (IFN-α/β) and involved in various cutaneous inflammatory and autoimmune disorders, such as cutaneous lupus erythematosus (CLE) and vitiligo. Heat shock proteins (HSPs) are molecular chaperones essential for maintaining cellular functions, but they can act as a danger signal during inflammation. Objectives To decipher the role of HSP70 in the production of IFN-α by pDCs in CLE and vitiligo. Methods Expression of HSP70 and CD123+ pDCs was analysed by immunohistochemistry or immunofluorescence in CLE and vitiligo skin samples. Flow cytometry was performed to analyse expression of HSP70 receptors, activation markers on pDCs and DNA uptake by pDCs in the presence of HSP70. The impact of HSP70 on DNA-induced IFN-α secretion by pDCs was evaluated by enzyme-linked immunosorbent assay (ELISA). The effect of IFN-α on chemokine (C-X-C motif) ligand 9 (CXCL9)/10 gene and protein expression by keratinocytes was determined by real-time polymerase chain reaction and ELISA. Results Infiltration of pDCs in CLE and progressive vitiligo was primarily located in the epidermis, close to keratinocytes expressing HSP70. In vitro experiments revealed that the pDCs expressing HSP70 receptor Lox-1 (lectin-like oxidized low-density lipoprotein-receptor-1) were able to aggregate HSP70. Exogenous HSP70 induced activation of pDCs and increased the uptake of exogenous DNA. Furthermore, HSP70 potentiated DNA-induced IFN-α production by pDCs. Finally, IFN-α induced expression of CXCL9 and CXCL10 by keratinocytes. Conclusions These data demonstrate that interaction between HSP70 and pDCs in CLE and vitiligo is a prerequisite for the enhancement of IFN-α production, and could be an interesting target.

Anti-tumor necrosis factor alpha (TNFα) therapies are used with success in many chronic inflammatory diseases. A wide range of adverse events has been reported in patients receiving these agents1, including lichenoid eruptions, lupus-like syndrome1, 2 and psoriasiform eruptions3. Lupus erythematosus panniculitis (LEP) is a rare variant of cutaneous lupus erythematosus (CLE), which occurs approximately in 1%-3% of CLE. Here we report a case of a patient with rheumatoid arthritis (RA) treated with anti-TNFα that developed skin lesions clinically and pathologically compatible with LEP. This article is protected by copyright. All rights reserved.

Pentahydroxyflavone dihydrate, quercetin (QU) is one of common flavonols biosynthesized by plants and has been suggested to modulate inflammatory responses in various models. In the present study, we investigated in vivo effects of oral or intra-cutaneous QU in chronic rat adjuvant-induced arthritis (AA). Growth delay and arthritic scores were evaluated daily after AA induction in Lewis rats. Oral administration of QU (5 x 160 mg/kg) to arthritic rats resulted in a clear decrease of clinical signs compared to untreated controls. Intra-cutaneous injections of lower doses (5 x 60 mg/kg) of QU gave similar anti-arthritic effects, while 5 x 30 mg/kg concentrations were inefficient in this respect. Finally, injection of relatively low QU doses (5 x 30 mg/kg) prior to AA induction significantly reduced arthritis signs. As QU was suggested to inhibit macrophage-derived cytokines and nitric oxide (NO), we then analyzed macrophage response ex vivo. Anti-arthritic effects of QU correlated with significant decrease of inflammatory mediators produced by peritoneal macrophages, ex vivo and in vitro. These data indicate that QU is a potential anti-inflammatory therapeutic and preventive agent targeting the inflammatory response of macrophages.

In addition to parasite spread, the severity of disease observed in cases of human African trypanosomiasis (HAT), or sleeping sickness, is associated with increased levels of inflammatory mediators, including tumor necrosis factor (TNF)-α and nitric oxide derivatives. In the present study, quercetin (3,3′,4′,5,7-pentahydroxyflavone), a potent immunomodulating flavonoid, was shown to directly induce the death of Trypanosoma brucei gambiense , the causative agent of HAT, without affecting normal human cell viability. Quercetin directly promoted T. b. gambiense death by apoptosis as shown by Annexin V binding. In addition to microbicidal activity, quercetin induced dose-dependent decreases in the levels of TNF-α and nitric oxide produced by activated human macrophages. These results highlight the potential use of quercetin as an antimicrobial and anti-inflammatory agent for the treatment of African trypanomiasis.

Introduction Le vitiligo est une dermatose depigmentante multifactorielle, caracterisee par la disparition progressive des melanocytes. Cette pathologie reste a ce jour orpheline de traitement efficace. Le role de l’auto-immunite dans la perte des melanocytes a ete bien decrit dans la litterature, impliquant a la fois des lymphocytes T (LT) CD4+ et CD8+. Toutefois, le phenotype et la fonction de ces cellules restent a ce jour controverses en raison de l’absence d’une analyse approfondie sur les modeles humains et animaux reproduisant clairement la complexite de la maladie. Ainsi, l’objectif de notre travail est d’etudier le role des lymphocytes T effecteurs memoires (LTEM) dans la depigmentation au cours du vitiligo. Materiel et methodes L’analyse multiparametrique du profil phenotypique et fonctionnel des populations lymphocytaires TEM CD4+ et CD8+ circulantes et cutanees de patients atteints de vitiligo et de sujets sains a ete realisee par cytometrie en flux. Les effets des cytokines produites par ces populations lymphocytaires ont ensuite ete evalues in vitro sur des cultures primaires de melanocytes et des epidermes reconstruits pigmentes par PCR quantitative et par Elisa. Resultats Nos resultats montrent une diminution de la frequence des LTEM circulants CD4+ et CD8+ exprimant le CXCR3 chez les patients atteints du vitiligo par rapport aux sujets sains, suggerant un recrutement de ces populations du compartiment sanguin vers la peau, ce que nous avons confirme au niveau cutane. Par ailleurs, un pourcentage important de ces lymphocytes TEM presente un phenotype resident memoire, exprimant les marqueurs CD103 et CD69. Au niveau cutane, ces LTEM produisent des niveaux eleves d’interferon (IFN)-g et de « tumor necrosis factor » (TNF)-a, deux cytokines inflammatoires connues pour leur implication dans plusieurs pathologies auto-immunes. L’etude des effets de ces deux cytokines in vitro a revele un effet synergique dans l’inhibition de la fonction et l’adhesion du melanocyte, reproduisant les observations realisees chez les patients. Par ailleurs, l’IFNg et le TNFa induisent la production de chimiokines et cytokines pro-inflammatoires a la fois par les keratinocytes et les melanocytes, permettant ainsi le maintien de la reponse inflammatoire a l’origine de la depigmentation. Conclusion Nos resultats apportent une meilleure comprehension des mecanismes physiopathologiques impliques dans la pathogenie du vitiligo et permettent ainsi d’etablir un lien direct entre immunite, facteurs solubles inflammatoires et perte des melanocytes afin de definir de nouvelles cibles therapeutiques prometteuses.

A novel series of isoindolo[2,1-a]quinoxaline and indolo[1,2-a]quinoxaline derivatives was synthesized and evaluated in vitro against various human cancer cell lines for antiproliferative activity. These new compounds displayed activity against leukemia and breast cancer cell lines in the 3- to 18-µM concentration range.

Background In addition to helminthic infections, elevated serum IgE levels were observed in many protozoal infections, while their contribution during immune response to these pathogens remained unclear. As IgE/antigen immune complexes (IgE-IC) bind to human cells through FceRI or FceRII/CD23 surface molecules, the present study aimed to identify which functional receptor may be involved in IgE-IC interaction with human macrophages, the major effector cell during parasite infection. Methodology/Principal Findings Human monocyte-derived macrophages were infected with Toxoplasma gondii before being incubated with IgE-IC. IgE receptors were then identified using appropriate blocking antibodies. The activation of cells and parasiticidal activity were evaluated by mediator quantification and direct counting of infected macrophages. RNAs were extracted and cell supernatants were also collected for their content in tumor necrosis factor (TNF)-α, interleukin-10 (IL-10) and nitrites. Sera from symptomatic infected patients were also tested for their content of IgE, IL-10 and nitrites, and compared to values found in healthy donors. Results showed that IgE-IC induced intracellular elimination of parasites by human macrophages. IgE-mediated effect was FceRI-independent, but required cross-linking of surface FceRII/CD23, cell activation and the generation of nitric oxide (NO). Although TNF-α was shown to be produced during cell activation, this cytokine had minor contribution in this phenomenon while endogenous and exogenous IL-10 down-regulated parasite killing. Inverse relationship was found between IL-10 and NO expression by infected human macrophages at both mRNA and mediator levels. The relationship between these in vitro data and in vivo levels of various factors in T. gondii infected patients supports the involvement of CD23 antigen and IL-10 expression in disease control. Conclusion Thus, IgE may be considered as immune mediator during antiprotozoal activity of human macrophages through its ability to trigger CD23 signaling. Increased cell activation by IgE-IC may also account for chronic inflammatory diseases observed in some patients.

Attenuation of protein kinases by selective inhibitors is an extremely active field of activity in anticancer drug development. Therefore, Akt, a serine/threonine protein kinase, also known as protein kinase B (PKB), represents an attractive potential target for therapeutic intervention. Recent efforts in the development and biological evaluation of small molecule inhibitors of Akt have led to the identification of novel inhibitors with various heterocycle scaffolds. Based on previous results obtained on the antiproliferative activities of new pyrrolo[1,2-a]quinoxalines, a novel series was designed and synthesized from various substituted phenyl-1H-pyrrole-2-carboxylic acid alkyl esters via a multistep heterocyclization process. These new compounds were tested for their in vitro ability to inhibit the proliferation of the human leukemic cell lines K562, U937, and HL60, and the breast cancer cell line MCF7. The first biological evaluation of our new substituted pyrrolo[1,2-a]quinoxalines showed antiproliferative activity against the tested cell lines. From a general SAR point of view, these preliminary biological results highlight the importance of substitution at the C-4 position of the pyrroloquinoxaline scaffold by a benzylpiperidinyl fluorobenzimidazole group, and also the need for a functionalization on the pyrrole ring.

Akt kinases are attractive targets for small molecule drug discovery because of their key role in tumor cell survival/proliferation and their overexpression/activation in many human cancers. Recent efforts in the development and biological evaluation of small molecule inhibitors of Akt have led to the identification of novel Akt kinase inhibitors, based on a quinoxaline or pyrazinone scaffold. A series of new substituted pyrrolo[1,2-a]quinoxaline derivatives, structural analogues of these active quinoxaline or pyrazinone pharmacophores, was synthesized from various substituted 2-nitroanilines or 1,2-phenylenediamine via multistep heterocyclization process. These new compounds were tested for their in vitro ability to inhibit the proliferation of the human leukemic cell lines K562, U937 and HL60, and the breast cancer cell line MCF7. Three of these human cell lines (K562, U937 and MCF7) exhibited an active phosphorylated Akt form. The most promising active pyrroloquinoxalines were found to be 1a that inhibited K562 cell line proliferation with an IC(50) of 4.5 microM, and 1h that inhibited U937 and MCF7 cell lines with IC(50) of 5 and 8 microM, respectively. These two candidates exhibited more potent activities than the reference inhibitor A6730.

A series of new 4-(E)-alkenylpyrrolo[1,2-a]quinoxaline derivatives, structural analogues of alkaloid chimanine B, was synthesized in good yields using efficient palladium(0)-catalyzed Suzuki-Miyaura cross-coupling reactions. These new compounds were tested for in vitro antiparasitic activity upon Leishmania amazonensis and Leishmania infantum strains. Biological results showed activity against the promastigote forms of L. amazonensis and L. infantum with IC50 ranging from 0.5 to 7 microM. From a Structure-Activity Relationships point of view, these pharmacological results mainly enlightened the importance of the 4-lateral C6, C7 or C8 alpha-unsaturated trans-alkenyl chain of unsubstituted pyrrolo[1,2-a]quinoxaline moiety.

Introduction Le lupus erythemateux (LE) est une maladie auto-immune frequente, evoluant par poussees, parfois declenchees ou aggravees par l’exposition solaire. Les mecanismes physiopathologiques impliques dans l’activation des cellules dendritiques plasmacytoides (pDC), a l’origine du declenchement des lesions cutanees restent mal connus. Nous avons montre que l’exposition des keratinocytes aux ultraviolets entraine une production importante de la proteine de choc thermique HSP72. Nous avons emis l’hypothese que cette proteine chaperon moleculaire HSP72 pouvait participer au declenchement des lesions cutanees lupiques suite a l’exposition solaire. Materiel et methodes Vingt-quatre patients suivis en dermatologie au centre hospitalier de Bordeaux ont ete inclus. Pour chaque patient, une biopsie de lesion cutanee lupique en zone photo-exposee a ete analysee en immunohistochimie afin d’evaluer l’expression de HSP72 et la presence de pDC. Ces analyses ont ete comparees a des biopsies temoins et psoriasiques. L’analyse de l’expression des recepteurs d’HSP72 a ete evaluee. Par la suite, des cultures in vitro de pDC en presence ou non d’HSP72 et de fragments d’ADN ont ete realisees afin d’evaluer l’expression des marqueurs d’activation des pDC et leur capacite de production d’IFN-alpha. Resultats Les immuno-marquages sur peau lupique montrent une forte expression d’HSP72 au niveau epidermique comparativement aux tissus temoins et psoriasiques. Par ailleurs contrairement au psoriasis, les pDC infiltrant les lesions lupiques sont preferentiellement localisees au niveau de la jonction dermo-epidermique au contact des keratinocytes exprimant HSP72. Les pDC expriment le recepteur d’HSP72 : LOX-1. Par ailleurs, la presence d’HSP72 augmente l’expression des marqueurs d’activation a la surface des pDC ainsi que leur capacite de fixer de l’ADN. Enfin les analyses fonctionnelles montrent que HSP72 potentialise la secretion d’IFN-alpha des pDC en presence d’ADN stimulant le TLR9. Conclusion Ces resultats suggerent pour la premiere fois chez l’homme la possible implication d’HSP72 dans l’activation des pDC a l’origine de l’initiation des lesions cutanees lupiques declenchee par l’exposition solaire. La meilleure comprehension des mecanismes physiopathologiques impliques dans l’initiation du LEC ouvre de nouvelles voies de recherche therapeutique.

Le lupus erythemateux (LE) est une maladie auto-immune, evoluant par poussees, parfois declenchees ou aggravees par l’exposition solaire. Les mecanismes impliques dans l’activation des cellules dendritiques plasmacytoides (pCD), a l’origine du declenchement des lesions cutanees, restent mal connus. Nous avons montre que les keratinocytes irradies par un rayonnement UV secretent des quantites importantes d’une proteine de choc thermique HSP72, decrites notamment pour ses proprietes pro-inflammatoires. Nous avons alors emis l’hypothese que cette molecule pouvait participer au declenchement de l’atteinte cutanee lupique suite a l’exposition solaire. Une expression d’HSP72 est retrouvee augmentee dans les atteintes cutanees lupiques de facon comparative aux temoins. L’infiltration des pCD au cours du LE predomine au niveau de l’interface dermo-epidermique au contact des keratinocytes exprimant HSP72. Par la suite, nous montrons que les pCD exprimant certains recepteurs de HSP72, comme LOX-1, sont capables de fixer HSP72. Il en resulte une activation des pCD, potentialisant leur fonction dans l’emergence des lesions cutanees lupiques. Ainsi, notre etude apporte une meilleure comprehension des mecanismes physiopathologiques impliques dans l’initiation du LE et suggere de nouvelles voies de recherche therapeutique.

An original series of 4-substituted pyrrolo[1,2-a]quinoxaline derivatives, new structural analogues of Galipea species quinoline alkaloids, was synthesized from various substituted 2-nitroanilines via multistep heterocyclizations and tested for in vitro antiparasitic activity upon Leishmania amazonensis and Leishmania infantum strains. Structure-activity relationships enlighten the importance of the 4-substituted alkenyl side chain on the pyrrolo[1,2-a]quinoxaline moiety to modulate the antileishmanial activity.

The synthesis of new 7-hydroxy-2-substituted-methyl-5 H -oxazolo[3,2- a] pyrimidin-5-ones derivatives, designed as structural bicyclic analogues of the iron chelator deferiprone, is described. They were tested for their ability to inhibit proliferation in human Bcr-Abl + leukemia cells.

Background Dietary flavonols may play an important role in the adjunct therapy of chronic inflammation. The availability of therapeutic formulations of pentahydroxyflavone glycoside, rutoside (RU), led us to investigate the ability of this molecule to modulate the release of various proinflammatory mediators from human activated macrophages in vitro and to ameliorate arthritic markers in a rat model. Methods RU was added simultaneously to human macrophages during their activation. Cells were then analyzed for inflammation-related gene expression using a specific array, and cell supernatants were collected to measure inflammatory mediators. RU was also injected into adjuvant-induced arthritic rats, and disease progression and body weight were evaluated until 50 days after injection. Sera and peritoneal macrophages were also collected to quantify the RU effect on various inflammatory markers. Results RU inhibited inflammation-related gene expression in activated human macrophages and the release of nitric oxide, tumor necrosis factor-alpha, interleukin (IL)-1, and IL-6 from these cells. In a rat model, RU inhibited clinical signs of chronic arthritis, correlating with decreased levels of inflammatory cytokines detected in rat sera and macrophage supernatants. Conclusion Thus, RU may have clinical value in reducing inflammatory manifestations in human arthritis and other inflammatory diseases.

It is often postulated that trans-3,4',5-trihydroxystilbene (resveratrol, RES) exhibits cell growth regulatory and chemopreventive activities. However, mechanisms by which this polyphenol inhibits tumor cell growth, and its therapeutic potential are poorly understood. Using various human leukemia cells, we have first defined the anti-tumoral doses of this compound. RES inhibited the proliferation and induced the apoptosis of all tested lymphoid and myeloid leukemia cells with IC(50) = 5-43 microM. Prior to apoptosis, RES-induced caspase activity in a dose-dependent manner and cell cycle arrest in G(2)/M-phase, correlating with a significant accumulation of cyclins A and B. Leukemia cell death with RES required both caspase-dependent and -independent proteases, as it was significantly inhibited by simultaneous addition of Z-VAD-FMK and leupeptin to these cultures. While RES did not affect non-activated normal lymphocytes, this agent decreased the growth and induced the apoptosis of cycling normal human peripheral blood lymphocytes at lower concentrations (IC(50)