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

Virulence. 2017 Aug 18;8(6):719-740. doi: 10.1080/21505594.2016.1229727. Epub 2016 Aug 30.


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 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 (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 as IL1B, NFKBIA, TNFAIP3, and TRAF1 in uninfected (ST-) fibroblasts. These observations proved that S. Typhimurium dampens the NF-κB response in the infected fibroblast. Higher expression of SOCS3, encoding a "suppressor of cytokine signaling," was also observed in the ST+ population. Intracellular S. 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 of SOCS3 and all NF-κB 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 ST+ and ST- populations are analyzed separately. This tune-down in a central host defense might be instrumental for S. Typhimurium to establish intracellular persistent infections.

Keywords: NF-κB response; Salmonella; attenuation; cell sorting; fibroblast; intracellular; live cell imaging; single-cell analysis.

MeSH terms

  • Cell Line
  • Fibroblasts / drug effects
  • Fibroblasts / metabolism
  • Fibroblasts / microbiology*
  • Humans
  • Interleukin-1beta / genetics
  • Microfluidics
  • NF-KappaB Inhibitor alpha / genetics
  • NF-kappa B / genetics
  • NF-kappa B / metabolism*
  • Salmonella typhimurium / drug effects
  • Salmonella typhimurium / metabolism
  • Salmonella typhimurium / pathogenicity*
  • Signal Transduction
  • Single-Cell Analysis
  • Suppressor of Cytokine Signaling 3 Protein / genetics
  • TNF Receptor-Associated Factor 1 / genetics
  • Time-Lapse Imaging
  • Tumor Necrosis Factor alpha-Induced Protein 3 / genetics
  • Tumor Necrosis Factor-alpha / pharmacology


  • IL1B protein, human
  • Interleukin-1beta
  • NF-kappa B
  • NFKBIA protein, human
  • SOCS3 protein, human
  • Suppressor of Cytokine Signaling 3 Protein
  • TNF Receptor-Associated Factor 1
  • Tumor Necrosis Factor-alpha
  • NF-KappaB Inhibitor alpha
  • TNFAIP3 protein, human
  • Tumor Necrosis Factor alpha-Induced Protein 3