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Cross-interference of RLR and TLR signaling pathways modulates antibacterial T cell responses Hideo Negishi, Hideyuki Yanai, Akira Nakajima, Ryuji Koshiba, Koji Atarashi, Atsushi Matsuda, Kosuke Matsuki, Shoji Miki, Takahiro Doi, Alan Aderem, Junko Nishio, Stephen T Smale, Kenya Honda & Tadatsugu Taniguchi Nature Immunology volume 13 7 July 2012 p659 Pathogen Pattern Recognition Receptors (PPRR) Bind Pathogen Associated Molecular Pattern (PAMP) molecules. 1. MBP and C-RP are soluble PPRR 2. TLR and LPS-BP are membrane-bound PPRR 3. NLR (Nod-like receptor) and RLH (Rig-like Helicase) are cytoplasmic PPRR Viral infection (VSV) increases susceptibility to bacterial infection (LM). Negishi Hideo et al. (2012) Figure Suppl 6a Negishi Hideo et al. (2012) Figure Suppl 6b Different cytokine gene profiles induced (expressed) by RLRs and TLRs. Stimulation with CpG (bacterial-DNA) Negishi Hideo et al. (2012) Figure 1a Stimulation with B-DNA (virus infection) Imiquimod (R-837) stimulates TLR-7 Quantitative RT-PCR. Negishi Hideo et al. (2012) Figure 1b ELISA Negishi Hideo et al. (2012) Figure Suppl. 1d Listeria monocytogenes Klebsiella pneumoniae Escherichia coli Salmonella typhimurium Vesicular Stomatitis Virus Newcastle Disease Virus Negishi Hideo et al. (2012) Figure 1c Selective suppression of TLRinduced Il12b by RLR signaling. Negishi Hideo et al. (2012) Figure 1d Caption of Figure 1: “Data are representative of three experiments (mean and s.d. of triplicates in b–d).” “… simultaneous stimulation of RLRs and TLRs also resulted in considerable suppression of the expression of Il12b mRNA (Supplementary Fig. 2b).” Negishi Hideo et al. (2012) Figure Supp. 2b Virus-induced interferon exerts anti-viral activity by inhibiting protein synthesis in neighboring cells. Thus, it is plausible that the decrease in Il12 upon virus infection (RLR stimulation) resulted from the generalized inhibition of protein synthesis by interferon. Virus Interferon RLR Inhibition of protein synthesis Interferon receptor INFaR1-deficient cells Negishi Hideo et al. (2012) Figure Suppl 2d WT cells Negishi Hideo et al. (2012) Figure 1d INFaR deficient Negishi Hideo et al. (2012) Figure Suppl. 2d WT cells Cycloheximide treatment Negishi Hideo et al. (2012) Figure Suppl 2e Suppressive effect of IRF3 on the Il12b promotor. IRF-RE Negishi Hideo et al. (2012) Figure 2a Chip assay (Chromatin immunoprecipitation) [poly(I:C) stimulation; Il12b promotor] Negishi Hideo et al. (2012) Figure 2b Negishi Hideo et al. (2012) Figure 2c Negishi Hideo (2012) Figure suppl. 3a Chip assay (Chromatin immunoprecipitation) [poly(I:C) stimulation; Infb1 promotor] Negishi Hideo et al. (2012) Figure Suppl. 3b ChIP assay. [Il12b promotor in WT and IRF3-/- cells; Poly (I:C) stimulation] Negishi Hideo et al. (2012) Figure 3a Negishi Hideo et al. (2012) Figure 3b Microarray. Induction of gene expression by VSV infection of WT and IRF3 -/- macrophages. Negishi Hideo et al. (2012) Figure Suppl 3f Negishi Hideo et al. (2012) Figure 3c Negishi Hideo et al. (2012) Figure 3d Negishi Hideo et al. (2012) Figure 3e Binding of Transcription Factors to Il12b promotor by LPS AFTER stimulation with poly(I:C) Negishi Hideo et al. (2012) Figure 4a Negishi Hideo et al. (2012) Figure 4b Negishi Hideo et al. (2012) Figure 4c Dominant effect of IRF3 over IRF5 on the Il12b promotor. Activation of IRF-3 with different TLR- and RLR-inducers. (Native PAGE) (SDS PAGE) Western blot followed by immunostaining. Negishi Hideo et al. (2012) Figure 5a Plasmid coding for IRF-3-5D IRF-3 Plasmid coding for IL-12b promotor 5’ of Luciferase gene Luciferase gene Promotor Luciferase Reporter Assay Plasmid coding for Infb promotor 5’ of Luciferase gene Transient luciferase reporter system for Il12b and INF-b promotor activation. Negishi Hideo et al. (2012) Figure Suppl 4b Transient luciferase reporter system: two ISREs for IRF5 in the Il12b promotor. Negishi Hideo et al. (2012) Figure Suppl 4d Active form of IRF3 that spontaneously undergoes dimerization Constitutively active IRF-5 Negishi Hideo et al. (2012) Figure 5b Negishi Hideo et al. (2012) Figure 5c (Dual transfection!!) Negishi Hideo et al. (2012) Figure Suppl. 4e. Polarization of CD4+ cells by innate signaling. Negishi Hideo et al. (2012) Figure 6a Eli-spot: Cytokine-producing Th cells. Negishi Hideo et al. (2012) Figure 6b Negishi Hideo et al. (2012) Figure Suppl. 5b Negishi Hideo et al. (2012) Figure 6c Negishi Hideo et al. (2012) Figure Suppl. 5e Negishi Hideo et al. (2012) Figure 6d Attenuation of antibacterial immune responses by viral infection. Negishi Hideo et al. (2012) Figure 7a Negishi Hideo et al. (2012) Figure Suppl 6a Negishi Hideo et al. (2012) Figure Suppl 6b Negishi Hideo et al. (2012) Figure 7b Negishi Hideo et al. (2012) Figure 7c “VSV + LM had nearly 10,000 fold more bacterial mRNA than LM alone” Negishi Hideo et al. (2012) Figure 7d Splenic CD11b+ cells. IL-12 mRNA measured by qRTPCR and expressed relative to mRNA of GAPDH gene Negishi Hideo et al. (2012) Figure 7e Negishi Hideo et al. (2012) Figure 7f Negishi Hideo et al. (2012) Figure 7g Negishi Hideo et al. (2012) Figure 7h Negishi Hideo et al. (2012) Figure 7i