Differentiated THP-1-ASC-GFP cells were plated in 24-well plates at 105 cells/well in 200 l of total medium

Differentiated THP-1-ASC-GFP cells were plated in 24-well plates at 105 cells/well in 200 l of total medium. cytometry. MoDC were preincubated with NLRP3-inhibitors KCl (20 and 40 mM) and 10 M MCC950 for 1 h. or A) mock-treated or exposed to LPS/nigericin and B) 20 and 40 mM KCl C) 10 M MCC950. These experiments were carried out in at least 2 individual donors with comparable results.(TIF) ppat.1005871.s004.tif (445K) GUID:?9D031A4F-38E3-4079-9000-E3339AC0DC25 S4 Fig: Expression levels of inflammasome sensors. RT-qPCR analysis of A) B) and mRNA levels in monocytes and MoDCs and after challenge with LPS or IC-HAdV in MoDC. These assays were performed in triplicate using 3 donors with comparable results. C) Immunoblotting demonstrating lentivirus-mediated shRNA knockdown of AIM2 in MoDC. D) Viral DNA is usually readily detected in the cells and remains associated with viral capsid in IC-HAdV-challenged MoDC. MoDC were exposed to IC-HAdV-488 for 3 h and prepared for fluorescence microscopy with DAPI as counterstaining.(TIF) ppat.1005871.s005.tif (361K) GUID:?C2BA1AAA-DCD6-433D-998E-FFDA5585DC0B S5 Fig: Plasmid DNA induces loss of membrane integrity. MoDCs were pre-incubated with 10, 50 or 100 M ODN A151 for 2 h and transfected with plasmid DNA complexed by Lipofectamine PIK3C1 LTX and CCT251545 cell membrane integrity was assessed by PI/circulation cytometry (n = 2).(TIF) ppat.1005871.s006.tif (33K) GUID:?67EA7336-4396-4951-A3CC-D62DBF55B7BA Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract Human adenoviruses (HAdVs) are nonenveloped proteinaceous particles made up of a linear double-stranded DNA genome. HAdVs cause a spectrum of pathologies in all populations regardless of health requirements. Following repeat exposure to multiple HAdV types, we develop strong and long-lived humoral and cellular immune responses that provide life-long protection from de novo infections and prolonged HAdV. How HAdVs, anti-HAdV antibodies and antigen presenting cells (APCs) interact to influence infection is still incompletely understood. In our study, we used physical, pharmacological, biochemical, fluorescence and electron microscopy, molecular and cell biology approaches to dissect the impact of immune-complexed HAdV (IC-HAdV) on human monocyte-derived dendritic cells (MoDCs). We show that IC-HAdV generate stabilized complexes of ~200 nm that are efficiently internalized by, and aggregate in, MoDCs. By comparing IC-HAdV, IC-empty capsid, IC-Ad2ts1 (a HAdV-C2 CCT251545 impaired in endosomal escape due to a mutation that impacts protease encapsidation) and IC-AdL40Q (a HAdV-C5 impaired in endosomal escape due to a mutation in protein VI), we demonstrate that protein VI-dependent endosomal escape is required for the HAdV genome to engage the DNA pattern recognition receptor AIM2 (absent in melanoma 2). AIM2 engagement induces pyroptotic MoDC death via ASC (apoptosis-associated speck protein made up of a caspase activation/recruitment domain name) aggregation, inflammasome formation, caspase 1 activation, and IL-1 and gasdermin D (GSDMD) cleavage. Our study provides mechanistic insight into how humoral immunity initiates an innate immune response to HAdV-C5 in human professional APCs. Author CCT251545 Summary While numerous studies have resolved the response to main virus infections, we know relatively little about the interplay between recurrent and/or persistent infections and the memory humoral immune response on professional antigen-presenting cells. Immune complexed-adenoviruses are present in patients suffering from adenoviremia. In CCT251545 addition to the impact of HAdV infections on healthy and immune suppressed hosts, humoral immunity hampers the use of human adenovirus vectors during gene transfer. Our study shows that anti-adenovirus humoral immunity engages an innate immune response to cause pyroptosis of antigen-presenting cells. The downstream effects of this cells death is unknown and may impact the activation and differentiation of T cells into an inflammatory phenotype that may be associated with the complications during adenovirus disease and adenovirus vector use. Our study generates insight into how humoral immunity designs the response to adenoviruses in healthy and immune-compromised individuals, during human adenovirus-based vaccine use,.