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The eukaryotic transcriptional regulator Nuclear Factor kappa B (NF-κB) plays a central role in the defense to pathogens. Despite this, few studies have analyzed NF-κB activity in single cells during infection. Here, we investigated at the single cell level how NF-κB nuclear localization – a proxy for NF-κB activity – oscillates in infected and uninfected fibroblasts co-existing in cultures exposed toSalmonella entericaserovar Typhimurium. Fibroblasts were used due to the capacity ofS. Typhimurium to persist in this cell type. Real-time dynamics of NF-κB was examined in microfluidics, which prevents cytokine accumulation. In this condition, infected (ST+) cells translocate NF-κB to the nucleus at higher rate than the uninfected (ST-) cells. Surprisingly, in non-flow (static) culture conditions, ST- fibroblasts exhibited higher NF-κB nuclear translocation than the ST+ population, with these latter cells turning refractory to external stimuli such as TNF-α or a second infection. Sorting of ST+ and ST- cell populations confirmed enhanced expression of NF-κB target genes such asIL1B, NFKBIA, TNFAIP3, andTRAF1in uninfected (ST-) fibroblasts. These observations proved thatS. Typhimurium dampens the NF-κB response in the infected fibroblast. Higher expression ofSOCS3, encoding a “suppressor of cytokine signaling,” was also observed in the ST+ population. IntracellularS. Typhimurium subverts NF-κB activity using protein effectors translocated by the secretion systems encoded by pathogenicity islands 1 (T1) and 2 (T2). T1 is required for regulating expression ofSOCS3and all NF-κB target genes analyzed whereas T2 displayed no role in the control ofSOCS3andIL1Bexpression. Collectively, these data demonstrate thatS. Typhimurium attenuates NF-κB signaling in fibroblasts, an effect only perceptible when ST+ and ST- populations are analyzed separately. This tune-down in a central host defense might be instrumental forS. Typhimurium to establish intracellular persistent infections. [ABSTRACT FROM AUTHOR]