Therapeutic potential of lipids obtained from γ-irradiated PBMCs in dendritic cell-mediated skin inflammation.

BACKGROUND: Since numerous pathological conditions are evoked by unwanted dendritic cell (DC) activity, therapeutic agents modulating DC functions are of great medical interest. In regenerative medicine, cellular secretomes have gained increasing attention and valuable immunomodulatory properties have been attributed to the secretome of γ-irradiated peripheral blood mononuclear cells (PBMCs). Potential effects of the PBMC secretome (PBMCsec) on key DC functions have not been elucidated so far. METHODS: We used a hapten-mediated murine model of contact hypersensitivity (CH) to study the effects of PBMCsec on DCs in vivo. Effects of PBMCsec on human DCs were investigated in monocyte-derived DCs (MoDC) and ex vivo skin cultures. DCs were phenotypically characterised by transcriptomics analyses and flow cytometry. DC function was evaluated by cytokine secretion, antigen uptake, PBMC proliferation and T-cell priming. FINDINGS: PBMCsec significantly alleviated tissue inflammation and cellular infiltration in hapten-sensitized mice. We found that PBMCsec abrogated differentiation of MoDCs, indicated by lower expression of classical DC markers CD1a, CD11c and MHC class II molecules. Furthermore, PBMCsec reduced DC maturation, antigen uptake, lipopolysaccharides-induced cytokine secretion, and DC-mediated immune cell proliferation. Moreover, MoDCs differentiated with PBMCsec displayed diminished ability to prime naïve CD4+T-cells into TH1 and TH2 cells. Furthermore, PBMCsec modulated the phenotype of DCs present in the skin in situ. Mechanistically, we identified lipids as the main biomolecule accountable for the observed immunomodulatory effects. INTERPRETATION: Together, our data describe DC-modulatory actions of lipids secreted by stressed PBMCs and suggest PBMCsec as a therapeutic option for treatment of DC-mediated inflammatory skin conditions. FUNDING: This research project was supported by the Austrian Research Promotion Agency (Vienna, Austria; grant "APOSEC" 862068; 2015-2019) and the Vienna Business Agency (Vienna, Austria; grant "APOSEC to clinic" 2343727).

Analysis of region specific gene expression patterns in the heart and systemic responses after experimental myocardial ischemia.

AIMS: Ischemic myocardial injury leads to the activation of inflammatory mechanisms and results in ventricular remodeling. Although great efforts have been made to unravel the molecular and cellular processes taking place in the ischemic myocardium, little is known about the effects on the surrounding tissue and other organs. The aim of this study was to determine region specific differences in the myocardium and in distant organs after experimental myocardial infarction by using a bioinformatics approach. METHODS AND RESULTS: A porcine closed chest reperfused acute myocardial infarction model and mRNA microarrays have been used to evaluate gene expression changes. Myocardial infarction changed the expression of 8903 genes in myocardial-, 856 in hepatic- and 338 in splenic tissue. Identification of myocardial region specific differences as well as expression profiling of distant organs revealed clear gene-regulation patterns within the first 24 hours after ischemia. Transcription factor binding site analysis suggested a strong role for Kruppel like factor 4 (Klf4) in the regulation of gene expression following myocardial infarction, and was therefore investigated further by immunohistochemistry. Strong nuclear Klf4 expression with clear region specific differences was detectable in porcine and human heart samples after myocardial infarction. CONCLUSION: Apart from presenting a post myocardial infarction gene expression database and specific response pathways, the key message of this work is that myocardial ischemia does not end at the injured myocardium. The present results have enlarged the spectrum of organs affected, and suggest that a variety of organ systems are involved in the co-ordination of the organism´s response to myocardial infarction.

Expression of Merkelcell polyomavirus (MCPyV) large T-antigen in Merkel cell carcinoma lymph node metastases predicts poor outcome.

BACKGROUND: The aim of this study was to determine the prevalence of MCPyV in Merkel cell carcinoma (MCC) primaries versus lymph node metastasis and to evaluate possible prognostic factors. METHODS: Samples of MCC primaries and lymph node metastases were stained immunohistochemically for the MCPyV large T-antigen and expression was compared to patients´ clinical outcome. RESULTS: 41 MCC patients were included. 33 (61%) out of 54 specimens were MCPyV-positive in the immunohistochemistry. 15 (47%) out of 32 primary tumors were positive compared to 18 (82%) out of 22 lymph node metastases. Eleven patients with positive polyomavirus expression died from the carcinoma compared to 4 patients without virus expression. Cox regression analysis showed worse disease-free survival in patients with MCPyV compared to virus-negative lymph nodes (p = 0.002). CONCLUSIONS: To our knowledge this is the first study to describe a negative prognostic effect of the MCPyV expression in lymph node metastasis in MCC patients.

Ionizing radiation regulates long non-coding RNAs in human peripheral blood mononuclear cells.

Long non-coding RNAs (lncRNAs) are non-protein coding transcripts that modulate mRNA and microRNA (miRNA) expression, thereby controlling multiple cellular processes, including transcriptional regulation of gene expression, cell differentiation and apoptosis. Ionizing radiation (IR), a strong cellular stressor, is known to influence gene expression of irradiated cells, mainly by activation of oxidative processes. Whether and how IR also affects lncRNA expression in human peripheral blood mononuclear cells (PBMCs) is still poorly understood. Exposure of PBMCs to IR dose-dependently activated p53 and its downstream target p21, ultimately leading to cell-cycle arrest and/or apoptosis. Cleavage of caspase-3, a specific process during apoptotic cell death, was detectable at doses as low as 30 Gy. Transcriptome analysis of 60 Gy-irradiated PBMCs revealed a strong time-dependent regulation of a variety of lncRNAs. Among many unknown lncRNAs we also identified a significant upregulation of Trp53cor1, MEG3 and TUG1, which have been shown to be involved in the regulation of cell cycle and apoptotic processes mediated by p53. In addition, we found 177 miRNAs regulated in the same samples, including several miRNAs that are known targets of upregulated lncRNAs. Our data show that IR dose-dependently regulates the expression of a wide spectrum of lncRNAs in PBMCs, suggesting a crucial role for lncRNAs in the complex regulatory machinery activated in response to IR.

Dying blood mononuclear cell secretome exerts antimicrobial activity.

BACKGROUND: Several activities are attributed to antimicrobial peptides (AMPs), including bacterial killing, leucocyte recruitment and angiogenesis. Despite promises of advanced cellular therapies for treatment of diabetic foot ulcer, it is currently accepted that paracrine factors rather than cellular components are causative for the observed effects. Whether AMPs are present in the mononuclear cell (MNC) secretome (MNC-sec) of white blood cells that are beneficial in experimental wound healing is not known. MATERIALS AND METHODS: Antimicrobial activity of the secretomes of nonirradiated (MNC-sec) and γ-irradiated MNCs (MNC-sec rad) was analysed by microdilution assay. AMPs were determined by quantitative real-time PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Whether human MNC-sec rad causes AMP secretion in vivo was examined in an experimental rat model. Image flow cytometry was used to determine the type of cell death induced in MNCs after exposure to γ-radiation. RESULTS: The antimicrobial activity assay revealed a bactericidal activity of MNC-sec rad and to a lesser degree also of MNC-sec. Image flow cytometry showed that γ-irradiation of MNCs induced early apoptosis followed mainly by necroptosis. RT-PCR and ELISA revealed a high abundance of different AMPs in the secretome of MNCs. In addition, human MNC-sec elicited an increase in de novo endogenous AMP production in rats in vivo. CONCLUSION: We provide evidence that the secretome of MNCs has direct and indirect positive effects on the immune defence system, including augmentation of antibacterial properties. Our data further suggest that necroptosis could play a key role for the release of paracrine factors and the therapeutic action of MNC-sec rad.

Development of Blood and Lymphatic Endothelial Cells in Embryonic and Fetal Human Skin.

Blood and lymphatic vessels provide nutrients for the skin and fulfill important homeostatic functions, such as the regulation of immunologic processes. In this study, we investigated the development of blood and lymphatic endothelial cells in prenatal human skin in situ using multicolor immunofluorescence and analyzed angiogenic molecules by protein arrays of lysates and cell culture supernatants. We found that at 8 to 10 weeks of estimated gestational age, CD144(+) vessels predominantly express the venous endothelial cell marker PAL-E, whereas CD144(+)PAL-E(-) vessels compatible with arteries only appear at the end of the first trimester. Lymphatic progenitor cells at 8 weeks of estimated gestational age express CD31, CD144, Prox1, and temporary PAL-E. At that developmental stage not all lymphatic progenitor cells express podoplanin or Lyve-1, which are acquired with advancing gestational age in a stepwise fashion. Already in second-trimester human skin, the phenotype of blood and lymphatic vessels roughly resembles the one in adult skin. The expression pattern of angiogenic molecules in lysates and cell culture supernatants of prenatal skin did not reveal the expected bent to proangiogenic molecules, indicating a complex regulation of angiogenesis during ontogeny. In summary, this study provides enticing new insights into the development and phenotypic characteristics of the vascular system in human prenatal skin.