Entative experiment out of two is shown. Entire L. monocytogenes are labeled green with FITC and RNA is visible as red fluorescence (Alexa594), nuclei are stained by DAPI (blue). doi:10.1371/journal.pone.0062872.gpathway functions mostly in immune cells, whereas tumor cell lines like HEK293 cells only responded to poly(dAdT) within a pol III/RIGI dependent fashion [23]. Indeed, we also observed that although THP1 cells responded to all kinds of transfected DNA which includes poly(dAdT), plasmid DNA (pDNA), genomic bacterial DNA and an 84mer double stranded DNA oligonucleotide (dsODN), the lung epithelial cell line A549 only responded to poly(dAdT) and double stranded 59 triphosphate RNA (3PdsRNA) (Fig. 3A). Analogous final results were obtained when we made use of bacRNA or bacDNA derived from Listeria (Fig. 3B). Both transfected bacRNA and bacDNA induced equal amounts of type I IFN in monocytic THP1 cells (Fig. 3B). By contrast, A549, HepG2 (hepatocarcinoma) and Colo205 (colon carinoma) cells had been capable to sense transfected bacRNA but didn’t induce a form I IFN response upon transfection of bacDNA (Fig.1551176-24-9 Data Sheet 3B).1-Bromo-3-fluoro-2-methyl-4-nitrobenzene Chemscene We thereby conclude that type I IFN induction by L. monocytogenes bacDNA is restricted to immune cells with intact STING dependent recognition pathways, that are not functional in human nonimmune cells. By contrast, nonimmune cells are exclusively triggered by bacRNA to induce form I IFN. Subsequently we tested no matter if L. monocytogenes infection of cell lines, unresponsive to bacDNA, can nevertheless induce a kind I IFN response.PMID:33522428 To this finish, we infected the indicated cell lines with wild variety (wt) or LLO deficient (hly) L. monocytogenes in the MOI shown and assessed the form I IFN secretion (Fig. 3C,D, E, F). THP1 cells, which can respond to bacDNA, raised a robust kind I IFN response upon infection with wt L. monocytogenes (Fig. 3C). Strikingly, cell lines derived from nonimmune cells lacking a type I IFN response to bacDNA (A549, HepG2, Colo205) also induced substantial amounts of form I IFN or the type I IFN regulated chemokine CXCL10 (HepG2) when infected with wt L. monocytogenes (Fig. 3D, E, F). In all analyzed cell lines L. monocytogenes induced variety I IFN/CXCL10 inside a LLO dependent manner, strongly suggesting cytosolic recognition of bacRNA (Fig. 3C, D, E, F). Collectively these final results indicate that bacRNA translocated for the cytosol may be the causative agent for L. monocytogenesmediated form I IFN induction in nonimmune cells.siRNAmediated knockdown of MAVS for the duration of infection with L. monocytogenes (Fig. 4C), whilst the variety I IFN response to the RIGI ligand 3PdsRNA was effectively downregulated. The response to DNA was still intact soon after knockdown of MAVS, excluding the involvement of a polIII/RIGI stimulatory effect of this DNA kind in these cells. Currently, STING is the only known adaptor protein upstream of IRF3 in the DNA recognition signaling pathway and has been shown to be important for the type I IFN response induced upon L. monocytogenes infection [16,17,42]. Knockdown of STING strongly inhibited the type I IFN induction response to plasmid DNA(pDNA) in THP1 cells (Fig. 4C). In contrast to MAVS, knockdown of STING substantially lowered type I IFN induction throughout L. monocytogenes infection of THP1 cells. We conclude that induction of sort I IFN through Listeria infection of STING pathway deficient cells is dependent on RIGI, although the RIGI pathway is redundant in immune cells which include monocytes, as they possess a STINGdependent pathway and are for that reason.