1 The inhibitory effect of PVC-CM on inflammasome activation in human macrophage

1 The inhibitory effect of PVC-CM on inflammasome activation in human macrophage. be responsible for the inhibitory effects on mitochondrial ROS generation PK11007 as well as on inflammasome activation. Conclusions Our results suggest that PVCs may be therapeutically useful for PK11007 the treatment of macrophage- and inflammation-mediated diseases by paracrine action via the secretion of various biological factors. (1). Their assembly is triggered when a member of the NOD-like receptor (NLR) proteins (NLRP1 and NLRP3) and non-NLR receptors, such as absent in melanoma-2 (AIM2), Mmp10 are activated (2). Upon activation, these innate immune sensor proteins oligomerize, leading to the recruitment of an adaptor protein such as apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC), which then binds with pro-caspase-1 to form an inflammasome. Inflammasome assembly promotes pro-caspase-1 PK11007 cleavage to generate the active form, which leads to promoting of caspase-1 (Casp1)-dependent inflammatory cell death, termed pyroptosis, and the secretion of pro-inflammatory cytokines, such as interleukin (IL)-1or IL-18. Accordingly, the aberrant activation of the inflammasomes is a risk factor for the emergence of autoimmune, autoinflammatory, chronic inflammatory, and metabolic diseases (3C6). Mesenchymal stem cells (MSCs) exhibit multipotency with immunomodulatory and anti-inflammatory activity, which make them attractive for treating many diseases (7, 8). MSCs migrate to sites of tissue injury and produce various immunosuppressive factors such as tumor necrosis factor-(TNF-that PVCs effectively attenuate the secretion of pro-inflammatory cytokines in human and murine macrophages by suppressing inflammasome activation in a paracrine fashion. We also demonstrated that the PVC secretome significantly reduces inflammatory activity and endoplasmic reticulum (ER) stress in peritoneal macrophages in a monosodium urate (MSU)-induced peritonitis mouse model. Secretome analysis of PVCs revealed serpin E1 and angiogenin as potential factors for inhibiting mitochondrial reactive oxygen species (ROS) generation and inflammasome activation. Our findings provide a rationale for the possible application of PVCs in the treatment of macrophage- and inflammation-mediated diseases by paracrine action via the secretion of various biological factors. Materials and Methods PVC isolation and cultures Unless otherwise indicated, all materials for PVC culture were purchased from Gibco (Thermo Fisher Scientific Inc., MA, PK11007 USA). The study was approved by the institutional review board of Kangwon National University Hospital (2012-11-003-008). All participants provided written informed consent. As previously described (15), PVCs were isolated from HUC obtained following full-term birth after caesarian section. PVC purification was confirmed by FACS to represent 87%, CD146+ populations and be free of CD45+, CD34+, CD31+ cells. PVCs were maintained with antibody (AF-201-NA/ AF-401-NA, PK11007 R&D systems), anti-caspase-1 p20 antibody (ab207802, Abcam., Cambridge, UK), anti-ASC antibody (sc-22514, Santa Cruz Biotechnology, CA, USA), anti-caspase-1 antibody (sc-622, Santa Cruz Biotechnology), anti-and Caspase-1/ICE, cell culture supernatants from PMA-differentiated THP-1 cells and mouse peritoneal macrophages were measured using the human IL-1and human Caspase-1/ICE Quantikine ELISA Kit (R&D Systems). The ELISA plates were read-out using a microplate reader (Molecular Devices, CA, USA). Caspase-1 activity assay For caspase-1 activity assay, human recombinant caspase-1 (1 unit/rx, #1081, BioVison Inc., CA, USA) was incubated with YVAD-pNA, a substrate of caspase-1, in the present of PVC-CM, Serpin E1, Angiogenin or Z-VAD-FMK (10 activation that can be stimulated by various triggers (Fig. 1A). We first treated LPS-primed human macrophages (PMA-differentiated THP-1 cells) with PVC-CM in the absence and presence of inflammasome activators. As shown in Fig. 1B, PVC-CM alone did not mediate the secretion of the active form of IL-1(p17) in THP-1 cells, whereas ATP, a well-known NLRP3 inflammasome activator, significantly induced IL-1secretion. To elucidate whether PVC-CM acts as an inhibitor for the NLRP3, NLRC4, and AIM2 inflammasomes, we treated inflammasomes triggered in LPS-primed human macrophages with PVC-CM and measured the active forms of Casp1 and IL-1in the cellular supernatants. PVC-CM significantly inhibited Casp1 and IL-1secretion mediated by several.