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. 2018 Dec 14;9:2962. doi: 10.3389/fimmu.2018.02962

Figure 2.

Figure 2

Translational interference by intracellular pathogens alters NF-κB signaling dynamics. Both the fungal pathogen, C. neoformans (A–C), and bacterial pathogen L. pneumophila (D–E), alter NF-κB signaling by inducing translational interference in host cells. In C. neoformans infected cells, these effects are influenced by microbial burden. (A) Changes in burden can be tracked in live host macrophages. RAW 264.7 murine macrophages were stained with the membrane dye CellTracker™ Red CMTPX dye (Red) and infected with GFP-expressing C. neoformans (Green) then imaged by live cell fluorescence microscopy. The number of intracellular C. neoformans in each cell is marked in white. Burden can increase or decrease due to C. neoformans replication and non-lytic extrusion (NLE), respectively. (B) RAW264.7 cells expressing p65-EGFP were infected with C. neoformans and imaged by live cell fluorescence microscopy in the presence of LPS. For the two infected cells, white and red dashed lines indicate cell and nuclear boundary, respectively. Intracellular C. neoformans are marked with arrows. (C) Quantification of p65-EGFP nuc:cyto ratio in 4 representative non-infected and infected cells (containing ≥3 yeast per cell). Scale bars represents 20 μm. (D,E) Epithelial cells exhibit a biphasic NF-κB response to L. pneumophila. (D) During the first phase, flagellin from extracellular L. pneumophila stimulates transient TLR5:MyD88-dependent nuclear localization of p65. (E) In contrast, the second phase is flagellin, TLR5, and MyD88-independent and requires the L. pneumophila Dot/lcm secretion system. Delivery of effectors into host cells induces translational interference, the partial inhibition of new protein synthesis. This results in a net decrease in the levels of IκBα (and A20) proteins, labile negative regulators of NF-κB signaling. The resulting stable accumulation of p65 proteins in the nucleus promotes increased expression of a subset of pro-inflammatory cytokines, including GM-CSF and IL-23, encoded by the Csf2 and Il23a genes, respectively. The images and data depicted in (B,C) were originally published in Hayes et al. (22), reproduced with permission. © The American Society for Biochemistry and Molecular Biology.