In vitro interactions of Candida parapsilosis wild type and lipase deficient mutants with human monocyte derived dendritic cells

<p>Abstract</p> <p>Background</p> <p><it>Candida parapsilosis </it>typically is a commensal of human skin. However, when host immune defense is compromised or the normal microflora balance is disrupted, <it>C. parapsilosis </it>transforms itself into an opportunistic pathogen. <it>Candida</it>-derived lipase has been identified as potential virulence factor. Even though cellular components of the innate immune response, such as dendritic cells, represent the first line of defense against invading pathogens, little is known about the interaction of these cells with invading <it>C. parapsilosis</it>. Thus, the aim of our study was to assess the function of dendritic cells in fighting <it>C. parapsilosis </it>and to determine the role that <it>C. parapsilosis</it>-derived lipase plays in the interaction with dendritic cells.</p> <p>Results</p> <p>Monocyte-derived immature and mature dendritic cells (iDCs and mDCs, respectively) co-cultured with live wild type or lipase deficient <it>C. parapsilosis </it>strains were studied to determine the phagocytic capacity and killing efficiency of host cells. We determined that both iDCs and mDCs efficiently phagocytosed and killed <it>C. parapsilosis</it>, furthermore our results show that the phagocytic and fungicidal activities of both iDCs and mDCs are more potent for lipase deficient compared to wild type yeast cells. In addition, the lipase deficient <it>C. parapsilosis </it>cells induce higher gene expression and protein secretion of proinflammatory cytokines and chemokines in both DC types relative to the effect of co-culture with wild type yeast cells.</p> <p>Conclusions</p> <p>Our results show that DCs are activated by exposure to <it>C. parapsilosis</it>, as shown by increased phagocytosis, killing and proinflammatory protein secretion. Moreover, these data strongly suggest that <it>C. parapsilosis </it>derived lipase has a protective role during yeast:DC interactions, since lipase production in wt yeast cells decreased the phagocytic capacity and killing efficiency of host cells and downregulated the expression of host effector molecules.</p

A Brain Region-Dependent Alteration in the Expression of Vasopressin, Corticotropin-Releasing Factor, and Their Receptors Might Be in the Background of Kisspeptin-13-Induced Hypothalamic-Pituitary-Adrenal Axis Activation and Anxiety in Rats

Previously, we reported that intracerebroventricularly administered kisspeptin-13 (KP-13) induces anxiety-like behavior and activates the hypothalamic-pituitary-adrenal (HPA) axis in rats. In the present study, we aimed to shed light on the mediation of KP-13′s stress-evoking actions. The relative gene expressions of the corticotropin-releasing factor (Crf, Crfr1, and Crfr2) and arginine vasopressin (Avp, Avpr1a, and Avpr1b) systems were measured in the amygdala and hippocampus of male Wistar rats after icv KP-13 treatment. CRF and AVP protein content were also determined. A different set of animals received CRF or V1 receptor antagonist pretreatment before the KP-13 challenge, after which either an open-field test or plasma corticosterone levels measurement was performed. In the amygdala, KP-13 induced an upregulation of Avp and Avpr1b expression, and a downregulation of Crf. In the hippocampus, the mRNA level of Crf increased and the level of Avpr1a decreased. A significant rise in AVP protein content was also detected in the amygdala. KP-13 also evoked anxiety-like behavior in the open field test, which the V1 receptor blocker antagonized. Both CRF and V1 receptor blockers reduced the KP-13-evoked rise in the plasma corticosterone level. This suggests that KP-13 alters the AVP and CRF signaling and that might be responsible for its effect on the HPA axis and anxiety-like behavior

The expression of inflammatory cytokines, TAM tyrosine kinase receptors and their ligands is upregulated in venous leg ulcer patients: A novel insight into chronic wound immunity

The systemic host defence mechanisms, especially innate immunity, in venous leg ulcer patients are poorly investigated. The aim of the current study was to measure Candida albicans killing activity and gene expressions of pro- and anti-inflammatory cytokines and innate immune response regulators, TAM receptors and ligands of peripheral blood mononuclear cells separated from 69 venous leg ulcer patients and 42 control probands. Leg ulcer patients were stratified into responder and non-responder groups on the basis of wound healing properties. No statistical differences were found in Candida killing among controls, responders and non-responders. Circulating blood mononuclear cells of patients overexpress pro-inflammatory (IL-1α, TNFα, CXCL-8) and anti-inflammatory (IL-10) cytokines as well as TAM receptors (Tyro, Axl, MerTK) and their ligands Gas6 and Protein S compared with those of control individuals. IL-1α is notably overexpressed in venous leg ulcer treatment non-responders; in contrast, Axl gene expression is robustly stronger among responders. These markers may be considered as candidates for the prediction of treatment response among venous leg ulcer patients. © 2015 Medicalhelplines.com Inc and John Wiley & Sons Ltd

Genome Wide Transcriptome Analysis of Dendritic Cells Identifies Genes with Altered Expression in Psoriasis

Activation of dendritic cells by different pathogens induces the secretion of proinflammatory mediators resulting in local inflammation. Importantly, innate immunity must be properly controlled, as its continuous activation leads to the development of chronic inflammatory diseases such as psoriasis. Lipopolysaccharide (LPS) or peptidoglycan (PGN) induced tolerance, a phenomenon of transient unresponsiveness of cells to repeated or prolonged stimulation, proved valuable model for the study of chronic inflammation. Thus, the aim of this study was the identification of the transcriptional diversity of primary human immature dendritic cells (iDCs) upon PGN induced tolerance. Using SAGESeq approach, a tag-based transcriptome sequencing method, we investigated gene expression changes of primary human iDCs upon stimulation or restimulation with Staphylococcus aureus derived PGN, a widely used TLR2 ligand. Based on the expression pattern of the altered genes, we identified non-tolerizeable and tolerizeable genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (Kegg) analysis showed marked enrichment of immune-, cell cycle- and apoptosis related genes. In parallel to the marked induction of proinflammatory mediators, negative feedback regulators of innate immunity, such as TNFAIP3, TNFAIP8, Tyro3 and Mer are markedly downregulated in tolerant cells. We also demonstrate, that the expression pattern of TNFAIP3 and TNFAIP8 is altered in both lesional, and non-lesional skin of psoriatic patients. Finally, we show that pretreatment of immature dendritic cells with anti-TNF-α inhibits the expression of IL-6 and CCL1 in tolerant iDCs and partially releases the suppression of TNFAIP8. Our findings suggest that after PGN stimulation/restimulation the host cell utilizes different mechanisms in order to maintain critical balance between inflammation and tolerance. Importantly, the transcriptome sequencing of stimulated/restimulated iDCs identified numerous genes with altered expression to date not associated with role in chronic inflammation, underlying the relevance of our in vitro model for further characterization of IFNprimed iDCs

Reverse Signaling Contributes to Control of Chronic Inflammation by Anti-TNF Therapeutics

Anti-tumor necrosis factor (TNF) monoclonal antibodies and TNF receptor ectodomain fusion protein are in clinical use to neutralize circulating TNF and ameliorate symptoms of many autoimmune diseases and pathological conditions with chronic inflammation. In this paper we present data to prove that reverse signaling, elicited by agonist molecules interacting with the membrane-bound TNF of myeloid cells, significantly contributes to the therapeutic effect of these anti-TNF medicines. Interaction of agonist monoclonals with cell surface TNF significantly attenuates the expression of pro-inflammatory cytokines and induces changes in the production of extracellular and intracellular signaling molecules. This phenomenon is not dependent on the Fc portion of antibodies as Fab constructs are as efficient as full antibody molecules

A Brain Region-Dependent Alteration in the Expression of Vasopressin, Corticotropin-Releasing Factor, and Their Receptors Might Be in the Background of Kisspeptin-13-Induced Hypothalamic-Pituitary-Adrenal Axis Activation and Anxiety in Rats

Previously, we reported that intracerebroventricularly administered kisspeptin-13 (KP-13) induces anxiety-like behavior and activates the hypothalamic-pituitary-adrenal (HPA) axis in rats. In the present study, we aimed to shed light on the mediation of KP-13′s stress-evoking actions. The relative gene expressions of the corticotropin-releasing factor (Crf, Crfr1, and Crfr2) and arginine vasopressin (Avp, Avpr1a, and Avpr1b) systems were measured in the amygdala and hippocampus of male Wistar rats after icv KP-13 treatment. CRF and AVP protein content were also determined. A different set of animals received CRF or V1 receptor antagonist pretreatment before the KP-13 challenge, after which either an open-field test or plasma corticosterone levels measurement was performed. In the amygdala, KP-13 induced an upregulation of Avp and Avpr1b expression, and a downregulation of Crf. In the hippocampus, the mRNA level of Crf increased and the level of Avpr1a decreased. A significant rise in AVP protein content was also detected in the amygdala. KP-13 also evoked anxiety-like behavior in the open field test, which the V1 receptor blocker antagonized. Both CRF and V1 receptor blockers reduced the KP-13-evoked rise in the plasma corticosterone level. This suggests that KP-13 alters the AVP and CRF signaling and that might be responsible for its effect on the HPA axis and anxiety-like behavior

Targeting TNF-α with infliximab decreases the expression of IL-6 and CCL1 in tolerant iDCs at 27h.

<p>Soluble chimeric monoclonal anti-TNF-α antibody (infliximab) was added to the culture supernatant in a therapeutic concentration (white bars) and the effects on gene and protein expression were monitored by QRT-PCR (A–E) and ELISA (F). Samples without infliximab treatment (gray bars) were used as controls. While infliximab pre-treatment has no effect on the expression of TNF-α (A), TNFAIP3 (B), TNFAIP8 (C), IL-6 (D) and CCL1 (E, F) in naïve and induced iDCs, it partially releases the inhibition of TNFAIP8 (C) and significantly dowregulates the expression of IL-6 (D) and CCL-1 (E, F) in tolerant iDCs. The ratio of each mRNA relative to the 18S rRNA was calculated using the 2^-ΔΔCT method. Data are representative of 3 or more independent experiments and are presented as interquartile range (box) with median (horizontal black bar) and minimum and maximum values. The significance of differences between sets of data was determined by Student’s paired t-test using SPSS Statistics; *<i>p</i><0.05.</p

The expression of cell cycle and apoptosis regulators in induced and tolerant iDCs at 27h.

<p>Based on the SAGE-Seq data, we have chosen 2 cell cycle regulators, cyclin B2 (A) and cyclin D1 (B), and three apoptosis regulators, caspase-8 (C), FADD (D) and TGF-β (E) for the validation by QRT-PCR. All of the selected genes show significantly dowregulated expression in both induced and tolerant iDCs when compared to naive cells. In addition, while still downregulated relative to naive cells, the expression of TGF-β in tolerant iDCs is significantly upregulated as compared to induced iDCs (E). (F) Induced and tolerant iDCs exhibit decreased apoptosis as determined by fluorescence intensity measurements of phosphatidylserine expressed on cell membrane, measured with Annexin V-Alexa Fluor 488 binding. The ratio of each mRNA relative to the 18S rRNA was calculated using the 2^-ΔΔCT method. Data are representative of 3 or more independent experiments and are presented as interquartile range (box) with median (horizontal black bar) and minimum and maximum values except for the phosphatidylserine expression intensity (F). The significance of differences between sets of data was determined by Student’s paired t-test using SPSS Statistics; *<i>p</i><0.05.</p