Indoleamine 2,3-dioxygenase (IDO), an enzyme involved in the catabolism of tryptophan, is expressed in certain cells and tissues, particularly in antigen-presenting cells of lymphoid organs and in the placenta. It was shown that IDO prevents rejection of the fetus during pregnancy, probably by inhibiting alloreactive T cells, and it was suggested that IDO-expression in antigen-presenting cells may control autoreactive immune responses. Degradation of tryptophan, an essential amino acid required for cell proliferation, was reported to be the mechanism of IDO-induced T cell suppression. Because we wanted to study the action of IDO-expressing dendritic cells (DCs) on allogeneic T cells, the human IDO gene was inserted into an adenoviral vector and expressed in DCs. Transgenic DCs decreased the concentration of tryptophan, increased the concentration of kynurenine, the main tryptophan metabolite, and suppressed allogeneic T cell proliferation in vitro. Kynurenine, 3-hydroxykynurenine, and 3-hydroxyanthranilic acid, but no other IDO-induced tryptophan metabolites, suppressed the T cell response, the suppressive effects being additive. T cells, once stopped in their proliferation, could not be restimulated. Inhibition of proliferation was likely due to T cell death because suppressive tryptophan catabolites exerted a cytotoxic action on CD3+ cells. This action preferentially affected activated T cells and increased gradually with exposure time. In addition to T cells, B and natural killer (NK) cells were also killed, whereas DCs were not affected. Our findings shed light on suppressive mechanisms mediated by DCs and provide an explanation for important biological processes in which IDO activity apparently is increased, such as protection of the fetus from rejection during pregnancy and possibly T cell death in HIV-infected patients.

BackgroundCirculating tumour cells (CTCs) have shown prognostic relevance in metastatic breast, prostate, colon and pancreatic cancer. For further development of CTCs as a biomarker, we compared the performance of different protocols for CTC detection in murine breast cancer xenograft models (MDA-MB-231, MDA-MB-468 and KPL-4). Blood samples were taken from tumour bearing animals (20 to 200 mm2) and analysed for CTCs using 1. an epithelial marker based enrichment method (AdnaTest), 2. an antibody independent technique, targeting human gene transcripts (qualitative PCR), and 3. an antibody-independent approach, targeting human DNA-sequences (quantitative PCR). Further, gene expression changes associated with epithelial-to-mesenchymal transition (EMT) were determined with an EMT-specific PCR assay.MethodsWe used the commercially available Adna Test, RT-PCR on human housekeeping genes and a PCR on AluJ sequences to detect CTCs in xenografts models. Phenotypic changes in CTCs were tested with the commercially available “Human Epithelial to Mesenchymal Transition RT-Profiler PCR Array”.ResultsAlthough the AdnaTest detects as few as 1 tumour cell in 1 ml of mouse blood spiking experiments, no CTCs were detectable with this approach in vivo despite visible metastasis formation. The presence of CTCs could, however, be demonstrated by PCR targeting human transcripts or DNA-sequences - without epithelial pre-enrichment. The failure of CTC detection by the AdnaTest resulted from downregulation of EpCAM, whereas mesenchymal markers like Twist and EGFR were upregulated on CTCs. Such a change in the expression profile during metastatic spread of tumour cells has already been reported and was linked to a biological program termed epithelial-mesenchymal transition (EMT).ConclusionsThe use of EpCAM-based enrichment techniques leads to the failure to detect CTC populations that have undergone EMT. Our findings may explain clinical results where low CTC numbers have been reported even in patients with late metastatic cancers. These results are a starting point for the identification of new markers for detection or capture of CTCs, including the mesenchymal-like subpopulations.

Due to the steadily increasing incidence of atopic dermatitis (AD), especially in children, there is a high medical need for new therapies and improved animal models. In mice, trimellitic anhydride (TMA) is routinely used to trigger T-cell-dependent contact hypersensitivity (CHS) reactions. In this study, we compared the standard acute TMA-induced CHS in Balb/c mice with subacute and chronic models of TMA-induced ear inflammation. Compared to the acute model, the chronic CHS model more closely reflects characteristics of AD, such as typical morphological changes of the inflamed skin, strong infiltration with T cells, major histocompatibility complex II-positive cells, eosinophils, and mast cells, a T-helper cell-type (Th) 2 cytokine profile and a strong increase of serum IgE levels. Moreover, a strong lymph node involvement with T-helper cell dominance and a mixed Th1/Th2 T-cell differentiation and activation pattern was demonstrated. Importantly, as demonstrated by successful therapy with prednisolone, the chronic TMA-induced CHS model, in contrast to acute and subacute models, made prolonged therapeutic treatment of a pre-established skin inflammation possible. Altogether, we present an improved model of mouse T-cell-dependent skin inflammation for AD. We hope this model will enhance the predictive value of animal models for therapeutic treatment of atopic eczema.

The complex tissue remodeling process of menstruation is experienced by humans and some primates, whereas most placental mammals, including mice, go through an estrous cycle. How menstruation and the underlying mechanisms evolved is still unknown. Here we demonstrate that the process of menstruation is not just species-specific but also depends on factors which can be induced experimentally. In intact female mice endogenous progesterone levels were raised by the induction of pseudopregnancy. Following an intrauterine oil injection, the decidualization of the endometrium was reliably induced as a prerequisite for menstruation. The natural drop of endogenous progesterone led to spontaneous breakdown of endometrial tissue within an average of 3 days post induction of decidualization. Interestingly, morphological changes such as breakdown and repair of the endometrial layer occurred in parallel in the same uterine horn. Most importantly, endometrial breakdown was accompanied by vaginally visible (overt) bleeding and flushing out of shed tissue comparable to human menstruation. Real-time PCR data clearly showed temporal changes in the expression of multiple factors participating in inflammation, angiogenesis, tissue modulation, proliferation, and apoptosis, as has been described for human menstruating endometrium. In conclusion, human menstruation can be mimicked in terms of extravaginally visible bleeding, tissue remodeling, and gene regulation in naturally non-menstruating species such as intact female mice without the need for an exogenous hormone supply.

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Mitogen-activated protein kinase-activated protein kinase 2 (MK2) is a downstream molecule of p38, involved in the production of TNF-alpha, a key cytokine, and an established drug target for many inflammatory diseases. We investigated the role of MK2 in skin inflammation to determine its drug target potential. MK2 deficiency significantly decreased plasma TNF-alpha levels after systemic endotoxin application. Deficient mice showed decreased skin edema formation in chronic 2-O-tetradecanoylphorbol-13-acetate (TPA)-induced irritative dermatitis and in subacute 2,4-dinitrofluorobenzene (DNFB)-induced contact hypersensitivity. Surprisingly, MK2 deficiency did not inhibit edema formation in subacute 2,4-dinitrochlorobenzene (DNCB)-induced contact allergy and even increased TNF-alpha and IL-1beta levels as well as granulocyte infiltration in diseased ears. Ear inflammation in this model, however, was inhibited by TNF-alpha neutralization as it was in the subacute DNFB model. MK2 deficiency also did not show anti-inflammatory effects in acute DNFB-induced contact hypersensitivity, whereas the p38 inhibitor, SB203580, ameliorated skin inflammation supporting a pathophysiological role of p38. When evaluating possible mechanisms, we found that TNF-alpha production in MK2-deficient spleen cells was strongly diminished after TLR stimulation but less affected after T-cell receptor stimulation. Our data suggest that MK2, in contrast to its downstream effector molecule, TNF-alpha, has a rather elusive role in T-cell-dependent cutaneous inflammation.

Despite recent developments, there is a high medical need for new treatment options for chronic inflammatory dermatoses like allergic contact dermatitis (ACD) and psoriasis. Particularly, more predictive skin inflammation models are required to facilitate the process of drug discovery. Murine contact hypersensitivity (CHS) models adequately reflect ACD and are also used to characterize therapeutic approaches for psoriasis. Using the hapten 2,4-dinitrofluorobenzene (DNFB), we established new subacute and subchronic DNFB-induced CHS models in C57BL/6 mice, which more closely reflect the characteristics of chronic T-cell-dependent inflammatory dermatoses as pronounced keratinocyte proliferation, strong hypervascularization, immune cell infiltration and overexpression of T cell and inflammatory cytokines. For the subacute DNFB model, we demonstrated anti-inflammatory activity of the glucocorticoid, prednisolone, as well as of neutralization of TNFα, IL-12/IL-23 or IL-18. In the subchronic DNFB-induced CHS model, deficiency for MyD88 and IL-12/IL-35 p35 chain but not IL-12/IL-23 p40 chain led to decreased skin inflammation. Furthermore, as exemplified by the dose-dependently effective therapeutic prednisolone treatment, the subchronic model allows the continuous therapy of a pre-established stable contact dermatitis. Altogether, prolonged DNFB-induced mouse CHS models closely reflect ACD sensitive to glucocorticoids as standard therapy, reveal a more chronic skin inflammation and are responsive to cytokine antagonization.

T-cell-mediated processes play an essential role in the pathogenesis of several inflammatory skin diseases such as atopic dermatitis, allergic contact dermatitis and psoriasis. The aim of this study was to investigate the role of the IL-2-inducible tyrosine kinase (Itk), an enzyme acting downstream of the T-cell receptor (TCR), in T-cell-dependent skin inflammation using three approaches. Itk knockout mice display significantly reduced inflammatory symptoms in mouse models of acute and subacute contact hypersensitivity (CHS) reactions. Systemic administration of a novel small molecule Itk inhibitor, Compound 44, created by chemical optimization of an initial high-throughput screening hit, inhibited Itk's activity with an IC50 in the nanomolar range. Compound 44 substantially reduced proinflammatory immune responses in vitro and in vivo after systemic administration in two acute CHS models. In addition, our data reveal that human Itk, comparable to its murine homologue, is expressed mainly in T cells and is increased in lesional skin from patients with atopic dermatitis and allergic contact dermatitis. Finally, silencing of Itk by RNA interference in primary human T cells efficiently blocks TCR-induced lymphokine secretion. In conclusion, Itk represents an interesting new target for the therapy of T-cell-mediated inflammatory skin diseases.