Single-cell analyses reveal an attenuated NF-κB response in theSalmonella-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.

Escalating Threat Levels of Bacterial Infection Can Be Discriminated by Distinct MAPK and NF-κB Signaling Dynamics in Single Host Cells

“…Expanding upon a recent study of the TLR response to lipid A (Gottschalk et al, 2016), our work confirms that engagement of MAPK signaling is a universal signature of an escalation in the threat to the host cell, be it high doses of PAMPs, infection, or bacterial replication. Identifying the role of MAPKs, and perhaps more importantly, the MAPK-NF-kB combination, in discrimination of the infection threat level, also adds a critical new dimension to the recent finding that NF-kB activation dynamics are altered in Salmonella-infected fibroblasts (Ramos-Marquè s et al, 2017). Furthermore, JNK signaling was a signature both of cells with replicating S. typhimurium and those that can secrete effectors.…”

“…Thus, while an immune cell could co-opt signaling pathways beyond the TLRs to transmit contextual information about a given bacterial stimulus, there is increasing evidence that a cell could also use the dynamics of TLR signaling proteins for this purpose. For example, S. typhimurium infection of fibroblasts has been shown to reduce NF-kB dynamics in infected cells via the secretion of effectors (Ramos-Marquè s et al, 2017), while Cryptococcus neoformans infection of macrophages was found to increase NF-kB signaling by modulating the negative feedback response (Hayes et al, 2016).…”

RNA-Seq unveils new attributes of the heterogeneous Salmonella-host cell communication

“…Similar observations have recently been found in S. Typhimurium-infected fibroblasts, in which infected and uninfected cell subpopulations were sorted and examined separately for analysis of the NF-kB defense response. 25 Third, and most importantly, the authors were able to correlate the bimodal induction of type I INF response genes in infected macrophages with differences in the activity of PhoP-PhoQ regulatory system in intracellular bacteria. This latter finding was possible by FACS sorting of the infected macrophages populations using a fluorescent reporter to an IFNstimulated response element (ISRE).…”

Single-cell analysis: Understanding infected cell heterogeneity

“…Electron or classical fluorescence microscopy require the use of fixed samples that preclude an observation in a dynamic mode. This recent study 5 analyses at the single cell level the dynamics of the NF-kB pathway during an infection. Using a software that automatically defines cell segmentation based on the Hoechst nucleus staining while tracking the p65-GFP signal, they were able to calculate the nuclear to cytoplasmic ratio of the GFP intensity, which was then exploited as a read-out of NF-kB activation.…”

“…Over the last number of years, the number of publications reporting such an approach has increased with applications in a wide variety of fields including bacterial replication, 1,2 bacterial cell morphology, 3 and deformation of red blood cells. 4 In this issue of Virulence Ramos-Marques et al 5 combine up-to-date technologies and single cell analysis to obtain an insight of how bacterial pathogens interact with host cells.…”