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

E Ramos-Marquès, S Zambrano, A Tiérrez, ME Bianchi, A Agresti, F García-del Portillo

Research output: Contribution to journalArticle

9 Citations (Scopus)

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. © 2016 Taylor & Francis
Original languageEnglish
Pages (from-to)719-740
Number of pages22
JournalVirulence
Volume8
Issue number6
DOIs
Publication statusPublished - 2017

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Single-Cell Analysis
NF-kappa B
Salmonella
Fibroblasts
Population
TNF Receptor-Associated Factor 1
Infection
Cytokines
Genomic Islands
Salmonella enterica
Microfluidics
Proxy
Genes

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Single-cell analyses reveal an attenuated NF-κB response in the Salmonella-infected fibroblast. / Ramos-Marquès, E; Zambrano, S; Tiérrez, A; Bianchi, ME; Agresti, A; García-del Portillo, F.

In: Virulence, Vol. 8, No. 6, 2017, p. 719-740.

Research output: Contribution to journalArticle

Ramos-Marquès, E ; Zambrano, S ; Tiérrez, A ; Bianchi, ME ; Agresti, A ; García-del Portillo, F. / Single-cell analyses reveal an attenuated NF-κB response in the Salmonella-infected fibroblast. In: Virulence. 2017 ; Vol. 8, No. 6. pp. 719-740.
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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. {\circledC} 2016 Taylor & Francis",
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AB - 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. © 2016 Taylor & Francis

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