All authors read and approved the final manuscript
All authors read and approved the final manuscript. Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Footnotes Funding. cell treatment with LABs beneficially modulated the mRNA levels of C-X-C motif chemokine 10 (CXCL-10), 2-5A oligoadenylate synthetase 1 (OSA1), myxovirus resistance protein (MxA), TLR3, and retinoic acid inducible gene-I (RIG-I), and TLR negative regulators. In addition, LABs increased IFN-, IFN-, and interleukin-10 (IL-10) and decreased tumor necrosis factor- (TNF-) and IL-1 protein/mRNA levels in THP-1 cells. LABs also protected the cells by maintaining tight-junction proteins (zonula occludens-1 and occludin). The beneficial effects of these LABs were mediated via modulation of the interferon regulatory factor 3 (IRF3) and nuclear factor-kappa B (NF-B) pathways. Overall, the results of this study indicate that immunobiotics have potent antiviral and anti-inflammatory activities that may use as antiviral substitutes for human and animal applications. model to study innate anti-viral immune response of LAB strains against rotavirus (RV) and PolyI:C (21C23). Human intestinal epithelial (HT-29) cells potently respond to PolyI:C by up-regulating immune gene proteins related to the TLR signaling pathway (10), and a study using porcine jejunal cells (IPEC-J2) showed treatment with GG reduced inflammatory response and RV infection (22). Also, PolyI:C increased the mRNA level of inflammatory cytokines (IL-6, IL-8, MCP-1) and interferon (IFN- and IFN-) in porcine IECs (PIE cells) (17, 23), whereas PIE cells treated with immunobiotic MEP221106 up-regulated IFN- and IFN- and down-regulated IL-6 and MCP-1 in response to the TLR3 agonist PolyI:C (16). These studies also suggest that IECs are the useful model to select and study of probiotic PI-1840 bacteria against viral or PolyI:C induced immune response DU1, DU1, and DU2 used in this study were previously isolated from Korean fermented foods and maintained in MRS (deMan-Rogosa-Sharp) medium at ?70C. LABs were grown at 37C for 19 h, centrifuged, washed with distilled phosphate buffered saline (PBS), and re-suspended in Roswell Park Memorial Institute 1640 medium (RPMI 1640, Gyeongsagbuk-do, South Korea) at desired concentrations, then stored at ?4C until required. The cytotoxicity of these strains on human cell line was determined previously using a cell viability assay kit (EZ-CYTOX, DOGEN Bio Co. Ltd) (24). Cell Culture The human colon and monocytic cells (HCT116 cells and THP-1) were used in this study that were obtained from the Korean Cell Line Bank (Seoul). HCT116 cells were cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin (P/S) at 37C, under 5% CO2. The medium was changed at 1-day interval for 5C6 days. Cells from passages 20C40 were used in the present study. In addition, THP-1 Bmpr2 cells were also cultured in RPMI-1640 medium containing FBS (1%), P/S (1%), and mercaptoethanol (0.05 mM) at 37C, under 5% CO2 for 5C6 days. To induce differentiation, cells were incubated with PMA (phorbol-12-myristate-13-acetate) medium for 48 h and then in fresh RPMI medium for 24 h. PMA medium was prepared by adding PMA (50 ng/ml) to RPMI medium. Analysis of Antiviral Activity of LABs PI-1840 in HCT116 Cells HCT116 cells (3 104 cells/ml) were placed in collagen coated plates (SPL Life Sciences Co. Ltd, Gyeonggi-do, Korea), and incubated at 37C, under 5% CO2 for 5C6 days. Cultured cells were then incubated with LAB strains (5 107 cells/ml) for 48 h PI-1840 and post-incubated with PolyI:C (10 g/ml) for 3 or 12 h. HCT116 cells stimulated with PolyI:C and medium alone were used as positive and negative controls, respectively. The RNA was extracted from treated cells and the PI-1840 expressions of type I interferon (IFN-, IFN-), antiviral proteins [(MxA, OAS1, C-X-C motif chemokine 10 (CXCL-10)], signaling receptors (RIG-I, TLR3), inflammatory cytokines/chemokine (IL-6, IL-8, MCP1, IL-1), and TLR negative regulators such as A20, toll-interacting protein (Tollip), single Ig interleukin 1 Crelated receptor (SIGIRR) and IL-1 receptor-associated kinase-M (IRAK-M) were analyzed by qRT-PCR. RNA Extraction and PI-1840 Quantitative Polymerase Chain Reaction (qPCR) Total RNA was isolated from cells by adding TRIzol reagent (Invitrogen), and used to synthesize cDNA using a Thermal cycler (BioRad, Hercules, CA, USA). qPCR was performed with a 7300 real-time PCR system (Roche Applied Science, Indianapolis, IN,.