Culture medium for this brief time span (Fig. S2D). Considering the fact that we cultivated cell lines in common culture medium during infection, we in addition excluded the possibility that host cell RNA is stained by non-incorporated EU released from L. monocytogenes. In the data we conclude that during infection, significant amounts of bacRNA are transferred into the cytosol of cells where it could be detected by cytosolic immune receptors. THP-1 cells infected with L. monocytogenes lacking the secA2 secretion program, which was not too long ago identified to become accountable for secretion of bacterial RNA of L. monocytogenes, have been not labeled for EU-containing RNA (Fig. S3), suggesting that translocation of bacterial RNA is rather mediated by active secretion than by lysis of bacteria [53]. Variety I IFN induction by L. monocytogenes in epithelial cells and hepatocytes is triggered by recognition of bacterial RNA. Operate by Ishii et al. [54] suggested that cytosolic recognition of DNA represents an ubiquitous pathway expressed inside a wide array of cell lines and cell sorts. Much more current studies exhibited that in human cell lines this refers only to the recognition of AT-rich [23,55] sequences. Lengthy AT-rich DNA sequences (poly(dA-dT)) had been identified to become the template of RNA polymerase III (pol III) top to synthesis of triphosphorylated RNA, the ligand of RIG-I [23,24]. Nevertheless, non-AT rich dsDNA molecules within the size range of poly(dA-dT) nevertheless induced a kind I IFN response in human monocyte-derived dendritic cells (MoDC) in which RIG-I is silenced [23]. This points to two distinct DNA recognition pathways in immune cells: pol III/RIG-I dependent recognition of AT-rich DNA and STING dependent (polIII/RIG-I independent) type I IFN induction by extended random DNA. This redundancy in DNA sensing mechanisms complicates the delineation of pattern recognition receptors involved in L. monocytogenes sensing.3-AP Recent information suggested that the STING dependentPLOS One | www.plosone.orgRIG-I Detects RNA of Listeria in Non-Immune CellsFigure 2. RNA of L. monocytogenes has access to the cytosol in the host cell during infection. THP-1, A549 and HepG2 had been infected with FITC-tagged and EU-labeled wt and hly- L.Streptomycin sulfate monocytogenes for the indicated duration.PMID:24406011 Cells had been then fixed, stained with Alexa594-azide and counterstained with DAPI. Left column, wt L. monocytogenes infection 1 hr. Middle column, wt L. monocytogenes infection 4 hrs. Suitable column, hly- L. monocytogenes infection 4 hrs. A: THP-1 cells. B: A549 cells. C: HepG2 cells. As determined by counting of single bacteria in cells (50 cells per slidePLOS A single | www.plosone.orgRIG-I Detects RNA of Listeria in Non-Immune Cellswere counted) the average bacterial load was 9(wt) and four(hly-) bacteria per cell for THP-1 cells, 6(wt) and four(hly-) bacteria per cell for A549 cells and six(wt) and 5(hly-) bacteria per cell for HepG2 cells, one particular representative experiment out of two is shown. Whole L. monocytogenes are labeled green with FITC and RNA is visible as red fluorescence (Alexa594), nuclei are stained by DAPI (blue). doi:ten.1371/journal.pone.0062872.gpathway functions primarily in immune cells, whereas tumor cell lines like HEK293 cells only responded to poly(dA-dT) within a pol III/RIG-I dependent fashion [23]. Indeed, we also observed that although THP-1 cells responded to all kinds of transfected DNA such as poly(dA-dT), plasmid DNA (pDNA), genomic bacterial DNA and an 84mer double stranded DNA oligonucleotide (dsODN), the lung epithelial cell line.
