Dataset Information

Phosphoproteome analysis of bone marrow-derived dendritic cells infected by Francisella tularensis

ABSTRACT: Francisella tularensis is a highly infective Gram-negative bacterium, also known as the causative agent of tularemia. The disease is characterized by the delayed onset of the adaptive immune response which provides time for the bacterial replication. As an intracellular pathogen, Francisella proliferates mainly in the cytosol of host phagocytes, which are linkers between the innate and the adaptive immunity. In particular, dendritic cells (DCs) are known to be the most effective antigen-presenting cells and thus they are crucial for the induction of the adaptive response. However, Francisella is able to invade and to proliferate inside DCs while avoiding their effective activation and maturation. The goal of this study was to map phosphoproteome changes in DCs infected by Francisella in order to reveal host signaling pathways involved in Francisella-DC interaction. The focus was laid on early intervals (<1 h p.i.) because of the ability of Francisella to escape in this time period from the phagosome to the cytosol where it establishes its replicative niche. For comparison, attenuated and in vivo-protective Francisella strain was used in parallel in experiments. DC phosphoproteome was analyzed by the means of shotgun LC-MS-based proteomics and relative changes were quantified by the stable isotope labeling method specially developed for primary bone marrow-derived DCs.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Francisella Tularensis Subsp. Holarctica Fsc200 Mus Musculus (mouse)

TISSUE(S): Dendritic Cell, Cell Culture, Bone Marrow

DISEASE(S): Tularemia

SUBMITTER: Ivo Fabrik

LAB HEAD: Jiri Stulik

PROVIDER: PXD005747 | Pride | 2017-10-26

REPOSITORIES: Pride

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Publications

The Early Dendritic Cell Signaling Induced by Virulent Francisella tularensis Strain Occurs in Phases and Involves the Activation of Extracellular Signal-Regulated Kinases (ERKs) and p38 In the Later Stage.

Fabrik Ivo I Link Marek M Putzova Daniela D Plzakova Lenka L Lubovska Zuzana Z Philimonenko Vlada V Pavkova Ivona I Rehulka Pavel P Krocova Zuzana Z Hozak Pavel P Santic Marina M Stulik Jiri J

Molecular & cellular proteomics : MCP 20171018 1

Dendritic cells (DCs) infected by Francisella tularensis are poorly activated and do not undergo classical maturation process. Although reasons of such unresponsiveness are not fully understood, their impact on the priming of immunity is well appreciated. Previous attempts to explain the behavior of Francisella-infected DCs were hypothesis-driven and focused on events at later stages of infection. Here, we took an alternative unbiased approach by applying methods of global phosphop ...[more]

PMID: 29046388

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Project description:Dendritic cells (DCs) are key initiators of the adaptive immunity, and upon recognition of pathogens are able to skew T cell differentiation to elicit appropriate responses. DCs possess this extraordinary capacity to discern external signals using receptors that recognize pathogen-associated molecular patterns. These can be glycan-binding receptors that recognize carbohydrate structures on pathogens or pathogen-associated patterns that additionally bind receptors, such as Toll-like receptors (TLRs). This study explores the early signaling events in DCs upon binding of α2-3 sialic acid (α2-3sia) that are recognized by Immune inhibitory Sialic acid binding immunoglobulin type lectins. α2-3sias are commonly found on bacteria, e.g. Group B Streptococcus, but can also be expressed by tumor cells. We investigated whether α2-3sia conjugated to a dendrimeric core alters DC signaling properties. Through phosphoproteomic analysis, we found differential signaling profiles in DCs after α2-3sia binding alone or in combination with LPS/TLR4 co-stimulation. α2-3sia was able to modulate the TLR4 signaling cascade, resulting in 109 altered phosphoproteins. These phosphoproteins were annotated to seven biological processes, including the regulation of the IL-12 cytokine pathway. Secretion of IL-10, the inhibitory regulator of IL-12 production, by DCs was found upregulated after overnight stimulation with the α2-3sia dendrimer. Analysis of kinase activity revealed altered signatures in the JAK-STAT signaling pathway. PhosphoSTAT3 (Ser727) and phosphoSTAT5A (Ser780), involved in the regulation of the IL-12 pathway, were both downregulated. Flow cytometric quantification indeed revealed de- phosphorylation over time upon stimulation with α2-3sia, but no α2-6sia. Inhibition of both STAT3 and -5A in moDCs resulted in a similar cytokine secretion profile as α-3sia triggered DCs. Conclusively, this study revealed a specific alteration of the JAK-STAT pathway in DCs upon simultaneous α2-3sia and LPS stimulation, altering the IL10:IL-12 cytokine secretion profile associated with reduction of inflammation. Targeted control of the STAT phosphorylation status is therefore an interesting lead for the abrogation of immune escape that bacteria or tumors impose on the host.

Dataset Information

Phosphoproteome analysis of bone marrow-derived dendritic cells infected by Francisella tularensis

Publications

The Early Dendritic Cell Signaling Induced by Virulent <i>Francisella tularensis</i> Strain Occurs in Phases and Involves the Activation of Extracellular Signal-Regulated Kinases (ERKs) and p38 In the Later Stage.

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