Single-cell analyses reveal an attenuated NF-κB response in the salmonella-infected fibroblast

Abstract

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-kB 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 to Salmonella enterica serovar Typhimurium. Fibroblasts were used due to the capacity of S. 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 (STC) cells translocate NF-κB to the nucleus at higher rate than the uninfected (ST-) cells. Surprisingly, in non-flow (static) culture conditions, STfibroblasts exhibited higher NF-κB nuclear translocation than the STC population, with these latter cells turning refractory to external stimuli such as TNF-a or a second infection. Sorting of STC and STcell populations confirmed enhanced expression of NF-kB target genes such as IL1B, NFKBIA, TNFAIP3, and TRAF1 in uninfected (ST-) fibroblasts. These observations proved that S. Typhimurium dampens the NF-kB response in the infected fibroblast. Higher expression of SOCS3, encoding a “suppressor of cytokine signaling,” was also observed in the STC population. Intracellular S. Typhimurium subverts NF-kB activity using protein effectors translocated by the secretion systems encoded by pathogenicity islands 1 (T1) and 2 (T2). T1 is required for regulating expression of SOCS3 and all NF-kB target genes analyzed whereas T2 displayed no role in the control of SOCS3 and IL1B expression. Collectively, these data demonstrate that S. Typhimurium attenuates NF-κB signaling in fibroblasts, an effect only perceptible when STC and STpopulations are analyzed separately. This tune-down in a central host defense might be instrumental for S. Typhimurium to establish intracellular.