Basic science S35 stiffness-mediated induction of aSMA protein expression, FAK phosphorylation, and MLCK and ET-1 gene expression.In addition, CARD-024 partially stimulated members of the COX-2/IL-1b inflammatory pathway.

colitis (UC) patients and 10 healthy controls.Phenotypic analysis was performed by eightcolor flow cytometry and by Reverse-Transcription Polymerase Chain Reaction (Q-RT-PCR).Lymphocytes were stimulated with PMA and ionomycin before intracellular staining (IL17, IFNγ and TNFα).To study the effect of NKG2D stimulation, fresh PBLs or LPLs from CD patients were stimulated with P815 cell line transfected or not with NKG2D ligands (MICA/ B, ULPB 1/2/3), coated with an agonist anti-CD3 antibody (TCR stimulation) or control isotype followed by cytokine staining as above.To assess the Th1 and Th17 cytokine polarization of CD4+NKG2D+ and CD4+NKG2D-T cells, LPMCs were cultured with anti-CD3/anti-CD28 activation beads with different cocktails of cytokines (IL12 for Th1 and IL1β and IL23 Th17 polarization).Results: LP CD4+NKG2D+ T cells were highly positive for IL17 intracellular staining (34.5±25.3%)and expressed significantly higher levels of IL17 than their LP CD4+NKG2D-counterparts (8.8±6%, p=0.001).NKG2D expression on PB CD4+ T cells correlated significantly with IL17 production (p=0.01).In Q-RT-PCR, messenger RNAs of IL-17 and RORC were expressed preferentially in LP CD4+NKG2D+ T cells compared to the CD4+NKG2D-subset (p=0.02).CCR6 and IL23R were significantly more expressed on LP and PB CD4+NKG2D+ T cells as compared to CD4+NKG2D-T cells (p<0.05).A high proportion of IL17 producing T cells co-expressed both NKG2D and CD161, as compared to cells not producing IL17 (52.5±23.7% vs. 4.5±3.2%,p=0.003).Targeting NKG2D by its ligands expressed on P815 cell lines in co-stimulation with the TCR significantly increased the production of IL17 by CD4+ T cells (4.5%±2.1)compared to the stimulation through the TCR alone (1.3%±1.1,p<0.05).LP CD4+NKG2D+ population secreted two times more IL17 in the presence of IL1β and/or IL23 as compared to the IL15 condition, whereas the CD4+NKG2D-showed almost no stimulation under these conditions.Conclusion: The subset of CD4+ T cells expressing NKG2D represents a major source of IL17 in CD, and has typical features of Th17 cells.Interactions between NKG2D and its ligands may strongly influence IL17 production in CD.NKG2D pathway could represent a promising therapeutic target in CD.

Introduction Crohn9s Disease (CD) is thought to arise both from defects in the gut mucosal barrier and from a dysregulated Th1/ Th17 immune response to commensal gut flora. CARD15 polymorphisms confer susceptibility to terminal ileal CD. Nod2 responds to muramyl dipeptide (MDP) leading to NF-κB activation and release of pro-inflammatory cytokines. CD-associated Nod2 mutations are predominantly loss-of-function, and it is uncertain how this predisposes to a pro-inflammatory disease. Published data have revealed Nod2 cross-talks with toll-like receptors. This study investigatesv the role of microRNAs in regulating Nod2 signalling and Nod2/ TLR cross-talk. MicroRNAs are short non-coding RNAs that prevent the translation of mRNA into protein and can negatively regulate innate immunity. Loss of these negative regulators might lower the threshold to the development of a pro-inflammatory state in mucosal tissue. Aims (1) Identify microRNAs differentially expressed in human dendritic cells (DCs) following Nod2 and combined Nod2/ TLR, stimulation. (2) Determine the microRNA targets. (3) Determine the functional consequences of miRNA expression. Methods Monocyte-derived DCs, expressing either WT or 1007fsinsC CARD15, were stimulated with MDP, Pam3CSK4 (TLR2 ligand), or Flagellin (TLR5 ligand) before lysis and extraction of RNA. MiRNA microarray analysis was conducted using Illumina miRNA microarrays. MiRNA expression was validated with quantitative PCR. Potential miRNA targets were identified using available algorithms and from published data. MiRNA mimic and antimir transfection were used to confirm targets by western blot, elisa and 39UTR lucferase assay. Further targets and functional effects were determined by dual-colour Affymetrix cDNA array following miRNA mimic transfection into DCs. Functional consequence of loss of miRNA expression determined by intracellular bacterial killing assay, confocal microscopy, FACS analysis and T-cell proliferation assay. Results Nod2 stimulation is required for expression of two miRNA clusters, in combination with either TLR2 or TLR5 triggering. MiRNA up-regulation is dependent on RIPK2 expression. DCs from patients homozygous for 1007fsinsC CARD15 fail to up-regulate these miRNA clusters. MiRNA cluster over-expression down-regulates the IL12-p40 subunit in human DCs at both mRNA and protein level. This subunit is a component of both IL-12 and IL-23 cytokines. IL-12p40 down-regulation can be ‘rescued’ in FS1007insC DCs by miRNA mimic expression. Conclusion DCs from patients with CD-associated mutations of Nod2 fail to up-regulate two miRNA clusters. These clusters regulate a critical component of the Th1/ Th17 immune response. These findings may help link CARD15 polymorphisms and the pathogenesis of CD.

Introduction NOD2 is an intracellular pattern recognition receptor that induces autophagy in human dendritic cells (DCs) in a manner important for bacterial handling and antigen presentation. Variants of NOD2 associated with terminal ileal Crohn9s disease (CD) fail to induce autophagy normally on Nod2 stimulation and in turn display delayed bacterial handling and defective antigen presentation. We have used phosphoproteomics to map the Nod2 signalling cascade in human monocyte-derived DCs in order to identify proteins involved in Nod2 mediated autophagy and inflammatory pathway induction in these cells. The aim of this study is to determine the molecular basis of Nod2 signalling and its effect on autophagy induction and DC maturation. Methods Primary human monocytes expressing wild type (WT) NOD2 were used to generate monocyte derived DCs and the effect of stimulation of Nod2 with its ligand muramyl dipeptide (MDP) assessed using phosphoproteomics. Here phosphorylated proteins were extracted from cell lysates pre and post MDP triggering, off-gel fractionated and subject to mass spectrometry analysis by LC-MS/MS and Q-TOF. The function of differentially phosphorylated proteins on DC maturation, autophagy induction and bacterial handling was then investigated following knockdown with siRNAs. Results The Nod2 signalling cascade is diverse. One protein identified, SHP-1, is a phosphatase known to control activation thresholds in immune cells. Another is HMGB1, an alarmin that can interact with beclin to facilitate autophagy. Nod2 also differentially phosphorylates L-plastin which is known to be involved in bacterial trafficking and engulfment. Conclusion Phosphoproteomics casts light on how Nod2 signals, how this PRR might control DC maturation and inflammatory cytokine production, interact with the autophagy pathway and handle bacteria in DCs.

IL-8 production was determined by ELISA assay.Cell death was assessed by measuring DNA fragmentation.Total RNA was isolated and IL-8, CXCL1, CXCL2, CCL2, ICAM, VCAM mRNA expression was determined by real time RT-PCR.NF-kB activation was determined by luciferase assay.Results: Exposure of LX2 cells to palmitate significantly induces IL-8 production and gene expression, as well as TLR4, CXCL1, CXCL2, CCL2, ICAM, VCAM mRNA expression.Palmitate induced IL-8 production can not be blocked by pan-caspase inhibitor.However, palmitate induced MIP-2 (rodent homolog of human IL-8) production was completely abrogated in HSCs from TLR4-mutant (C3H/HeJ) mice, but not from wildtype (C3H/HeOuJ) mice.Palmitate also induces NF-kB activation in a dose-dependent manner.Conclusions: Our data demonstrate that palmitate directly stimulates IL-8 production in HSCs via TLR4 signaling pathway, suggesting that HSCs play an important role in FFAs induced liver inflammation and injury.

ABSTRACT Platelets can engulf human immunodeficiency virus type 1 (HIV-1), and a significant amount of HIV-1 in the blood of infected individuals is associated with these cells. However, it is unclear how platelets capture HIV-1 and whether platelet-associated virus remains infectious. DC-SIGN and other lectins contribute to capture of HIV-1 by dendritic cells (DCs) and facilitate HIV-1 spread in DC/T-cell cocultures. Here, we show that platelets express both the C-type lectin-like receptor 2 (CLEC-2) and low levels of DC-SIGN. CLEC-2 bound to HIV-1, irrespective of the presence of the viral envelope protein, and facilitated HIV-1 capture by platelets. However, a substantial fraction of the HIV-1 binding activity of platelets was dependent on DC-SIGN. A combination of DC-SIGN and CLEC-2 inhibitors strongly reduced HIV-1 association with platelets, indicating that these lectins are required for efficient HIV-1 binding to platelets. Captured HIV-1 was maintained in an infectious state over several days, suggesting that HIV-1 can escape degradation by platelets and might use these cells to promote its spread. Our results identify CLEC-2 as a novel HIV-1 attachment factor and provide evidence that platelets capture and transfer infectious HIV-1 via DC-SIGN and CLEC-2, thereby possibly facilitating HIV-1 dissemination in infected patients.

Background. After allogeneic hematopoietic stem cell transplantation, donor T cells interact with an antigen-presenting cell environment that is distorted in number, level of activation, and origin. The role of antigen presentation in the development of chronic graft-versus host disease (cGVHD) is unknown. Methods. The number and origin of peripheral blood immature myeloid (CD19− CD1c+) and plasmacytoid (BDCA-2+) dendritic cells (DCs) was determined in 30 patients at more than 100 days after allogeneic hematopoietic stem cell transplantation. Results. Patients with cGVHD had significantly higher plasmacytoid DC numbers than individuals without this complication (9.1±2.0×106/L versus 3.8±0.6×106/L, P =0.025). Chimerism studies demonstrated that DCs in patients with cGVHD were exclusively of donor origin, whereas persistence of host DCs was observed in some control patients. Conclusions. The antigen-presenting cell environment in patients with cGVHD, as represented by immature blood DCs, is of donor origin but distorted in terms of subset distribution.