Nucleotide-Derived Bacterial Alarmones Attenuation of the Induction of Type-I Interferon Responses in a Murine Macrophage Reporter Cell Line

Description

The stringent response is a well-studied phenomenon in many bacterial systems and regulates resource-consuming activities such as transcription, translation, and replication. The stringent response is a well-conserved signaling framework, as are the nucleotide-derived signaling mediators, collectively referred to as (p)ppGpp or as alarmones. There is a wealth of research evaluating nucleotide-derived alarmone signaling in bacterial models, however, their potential to modulate innate immune signaling has not yet been evaluated. Several common pathogen-synthesized molecules, such as lipopolysaccharide (LPS) and cyclic-di-AMP (c-di-AMP), act as pathogen-associated molecular patterns (PAMPs), which are common patterns that alert the innate immune system of bacterial infection. The goal of this work is to elucidate the impact of (p)ppGpp on innate immune signaling. To explore this, RAW-Dual cells were incubated with guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), two well-studied nucleotide-derived alarmones found in many different pathogenic bacteria, as well as with GTP. Both ppGpp and pppGpp were able to significantly reduce the expression of secreted luciferase in RAW-Dual cells in a dose-dependent manner, indicating a reduction of the interferon-stimulated regulatory elements (ISREs). Neither alarmone impacted secreted embryonic alkaline phosphatase (SEAP) secretion, which reports for NF-kB activation. This is the first work to suggest that nucleotide-derived alarmones produced by bacteria may impact an arm of innate immunity responsible for type-I interferon secretion.

Steps to reproduce

Methods Cell culture The RAW-Dual cell line (InvivoGen), a dual reporter mouse macrophage line, was cultured according to manufacturer specifications and with institutional biosafety approval (24-010). To evaluate immune responses, cells were seeded in a 96-well plate at 2.0 x 105 cells per well and incubated with alarmone (ppGpp or pppGpp, Jena Bioscience), c-di-AMP (InvivoGen), LPS O111:B4 (Sigma), GTP (Biobasic), and/or Dexamethasone (Sigma) for 23h at 37℃ and 5% CO2 in DMEM media without selective antibiotics. After incubation, the luciferase and SEAP assays were completed according to manufacturer instructions (InvivoGen). Analysis Data were normalized within experiment to the mean of 3-5 media-only control replicates (single incubations) or to the mean of 1-3 control replicates of LPS- or c-di-AMP-treated positive controls (co-incubations), then pooled from 12 independent experiments. Significant differences across means of individual treatment groups were evaluated compared using the Kruskal-Wallis test, and means between individual groups were compared using Dunn’s multiple comparison test. Three outliers were identified and removed using the Grubb’s test. Differences between treatments were analyzed by comparing the area under the curve (AUC) over 0-1000uM concentrations of all treatments, and differences between the AUC between treatments were analyzed by Brown-Forsythe ANOVA. All graphical figures and statistical analyses were conducted within GraphPad Prism version 10.2.3.

Institutions

• Old Dominion University

Categories

Funders

• Discretionary account

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