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Effect of stimulation with lipopolysaccharide (LPS) at 0,30, and 60 minutes on gene expression in immortalized mouse macrophages (IMM).

ABSTRACT: The interactions between RNA and protein within cellular signaling pathways, known as the interactome, have modulatory effects on RNA binding proteins (RBP’s) effector functions, which contribute to post-transcriptional control of inflammatory cytokine release in the innate immune response. Post-translational modifications (PTMs) have a regulatory effect on a protein’s effector function. Thus, investigating the effect of RNA-protein interactions can answer questions central to our understanding of cellular signaling. Global RNA sequencing was used to investigate the differentially expressed (DE) genes from baseline following induction with LPS. Shotgun proteomics along with SILAC labeling was used to quantify relative abundance and differential protein expression in response to LPS. To assess the proteins interacting with RNA after LPS induction, total RNA associated protein purification was utilized. Using the tandem MS approach and methodology mentioned above, we were able to identify a list of candidate RNA binding proteins. Global RNA sequencing provided a baseline transcriptomic profile as well as quantification of DE genes across a stimulation time course (0, 30, 60 minutes). We then handpicked potential RBPs of immunological interest (Il1a, MARCKS, and Acod1). Interestingly, Il1a, MARCKS, and Acod1 were all highly DE compared to baseline in the RNA Seq results and displayed a tendency to disassociate from RNA as the LPS time course pursued. Il1a, MARCKS, and Acod1 can all directly regulate pro-inflammatory gene expression in macrophages. These RBPs could offer a new regulatory mechanism to tune their effector function, which could lead to new therapeutic innovations for disease pathologies these proteins are implicated in. This research was supported by the Intramural Research Program of NIAID, NIH.

ORGANISM(S): Mus musculus

PROVIDER: GSE248765 | GEO | 2024/03/04

REPOSITORIES: GEO

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Project description:The interactions between RNA and protein within cellular signaling pathways, known as the interactome, have modulatory effects on RNA binding proteins (RBP’s) effector functions, which contribute to post-transcriptional control of inflammatory cytokine release in the innate immune response. Post-translational modifications (PTMs) have a regulatory effect on a protein’s effector function. Thus, investigating the effect of RNA-protein interactions can answer questions central to our understanding of cellular signaling. Global RNA sequencing was used to investigate the differentially expressed (DE) genes from baseline following induction with LPS. Shotgun proteomics along with SILAC labeling was used to quantify relative abundance and differential protein expression in response to LPS. To assess the proteins interacting with RNA after LPS induction, total RNA associated protein purification was utilized. Using the tandem MS approach and methodology mentioned above, we were able to identify a list of candidate RNA binding proteins. Global RNA sequencing provided a baseline transcriptomic profile as well as quantification of DE genes across a stimulation time course (0, 30, 60 minutes). To investigate the discordance between the transcriptome and proteome, we performed miRNA sequencing at 0,30,60, and 120 minutes of LPS stimulation to assess expression changes in LPS induced miRNA’s which may regulate the transcriptome. We then handpicked potential RBPs of immunological interest (Il1a, MARCKS, and Acod1). Interestingly, Il1a, MARCKS, and Acod1 were all highly DE compared to baseline in the RNA Seq results and displayed a tendency to disassociate from RNA as the LPS time course pursued. Il1a, MARCKS, and Acod1 can all directly regulate pro-inflammatory gene expression in macrophages. These RBPs could offer a new regulatory mechanism to tune their effector function, which could lead to new therapeutic innovations for disease pathologies these proteins are implicated in. This research was supported by the Intramural Research Program of NIAID, NIH.

Project description:Background: Environmental lipopolysaccharide (LPS) and microbial component-enriched organic dusts cause significant lung diseases. These environmental exposures induce the recruitment and activation of distinct lung monocyte/macrophage subpopulations involved in disease pathogenesis. Given monocyte/macrophage activation is tightly linked to metabolism, the objective of these studies was to determine the role of the immunometabolic regulator, aconitate decarboxylase 1 (ACOD1), in environmental exposure-induced lung inflammation. Methods: Wild-type (WT) mice were intratracheally instilled (I.T.) with 10 ug LPS or saline. Whole lungs were profiled using bulk RNA sequencing or sorted to isolate monocyte/macrophage subpopulations. Sorted subpopulations were then characterized transcriptomically using a NanoString innate immunity multiplex array 48 hours post-exposure. Next, WT and Acod1-/- mice were instilled with LPS, 25% organic dust extract (ODE), or saline with serum, bronchoalveolar lavage fluid (BALF), and lung tissues collected. BALF metabolites of the tricarboxylic acid (TCA) cycle were quantified by mass-spectrometry. Cytokines/chemokines and tissue remodeling mediators were quantitated by ELISA. Lung immune cells were characterized by flow cytometry. Whole body plethysmography was performed 3 hours post-LPS with WT and Acod1-/- mice. Results: By bulk sequencing, Acod1 was one of the most significantly upregulated genes following LPS (vs. saline) exposure of murine whole lungs. Transcriptomic profiling of sorted lung monocyte/macrophage subpopulations corroborated Acod1 significance. Acod1-/- mice treated with LPS (vs. WT) demonstrated decreased BALF levels of itaconate, TCA cycle reprogramming, decreased BALF neutrophils, increased lung CD4+ T cells, decreased BALF and lung levels of TNF-a, and decreased BALF CXCL1. In comparison, Acod1-/- mice treated with ODE (vs. WT) demonstrated decreased serum pentraxin-2, BALF levels of itaconate, lung total cell, neutrophil, monocyte, and B cell infiltrates with decreased BALF levels of TNF-a, IL-6, and decreased lung CXCL1. Mediators of tissue remodeling (TIMP1, MMP8, MMP9) were also decreased in the LPS-exposed Acod1-/- mice, with MMP-9 also decreased in the ODE-exposed Acod1-/- mice. Lung function assessments demonstrated a blunted response to LPS-induced airway hyper-responsiveness in Acod1-/- mice. Conclusion: Acod1 is robustly upregulated in the lungs following LPS-exposure and encodes a key immunometabolic regulator. ACOD1 mediates the pro-inflammatory response to acute inhaled, environmental LPS and organic dust exposure-induced lung inflammation.

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Effect of stimulation with lipopolysaccharide (LPS) at 0,30, and 60 minutes on gene expression in immortalized mouse macrophages (IMM).

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