title: 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-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.
authors: a Laboratory of Intracellular Bacterial Pathogens , Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas (CNB-CSIC) , Madrid , Spain.
authors: Estel
authors: E
authors: Ramos-Marquès
authors: b San Raffaele University , Milan , Italy.
authors: Samuel
authors: S
authors: Zambrano
authors: a Laboratory of Intracellular Bacterial Pathogens , Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas (CNB-CSIC) , Madrid , Spain.
authors: Alberto
authors: A
authors: Tiérrez
authors: c Genetics and Cell Biology Division , San Raffaele Scientific Institute , Milan , Italy.
authors: Marco E
authors: ME
authors: Bianchi
authors: c Genetics and Cell Biology Division , San Raffaele Scientific Institute , Milan , Italy.
authors: Alessandra
authors: A
authors: Agresti
authors: a Laboratory of Intracellular Bacterial Pathogens , Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas (CNB-CSIC) , Madrid , Spain.
authors: Francisco
authors: F
authors: García-Del Portillo
mesh_terms: D002460:Cell Line; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism / Q000382:microbiology*; D006801:Humans; D053583:Interleukin-1beta / Q000235:genetics; D044085:Microfluidics; D000072000:NF-KappaB Inhibitor alpha / Q000235:genetics; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D012486:Salmonella typhimurium / Q000187:drug effects / Q000378:metabolism / Q000472:pathogenicity*; D015398:Signal Transduction; D059010:Single-Cell Analysis; D000071223:Suppressor of Cytokine Signaling 3 Protein / Q000235:genetics; D047990:TNF Receptor-Associated Factor 1 / Q000235:genetics; D059008:Time-Lapse Imaging; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3 / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: C508585:IL1B protein, human; D053583:Interleukin-1beta; D016328:NF-kappa B; C000606204:NFKBIA protein, human; C106644:SOCS3 protein, human; D000071223:Suppressor of Cytokine Signaling 3 Protein; D047990:TNF Receptor-Associated Factor 1; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; C076529:TNFAIP3 protein, human; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3
keywords: NF-κB response; Salmonella; attenuation; cell sorting; fibroblast; intracellular; live cell imaging; single-cell analysis