Exploring the proteomic landscape of THP-1 monocytes through two-challenge LPS induction.

Mass Spectrometry-Based Multiomic Profiling Defines Proteome, Lipidome, and Metabolome Remodeling in IFN-γ and LPS-Stimulated BV-2 Microglial Cells

Microglial inflammatory activation is accompanied by extensive molecular remodeling, yet proteomic, lipidomic, and metabolomic responses are often analyzed independently. Here, we applied an integrated mass spectrometry-based multiomic workflow to characterize proteomic, lipidomic, and polar metabolomic remodeling from matched BV-2 biological samples following stimulation with interferon-γ and lipopolysaccharide (IFN-γ and LPS). Inflammatory activation was confirmed by increased nitrite accumulation, elevated TNF-α and IL-6 secretion, and treatment-associated morphological changes. Discovery proteomics quantified 8,676 proteins and identified 562 significantly altered proteins, including 344 increased and 218 decreased proteins. Increased proteins were enriched for interferon-responsive, innate immune, inflammatory effector, and antigen-associated pathways, whereas decreased proteins were associated with cellular organization, protein biogenesis, vesicular trafficking, and metabolic regulation. Targeted lipidomics identified 237 significantly altered lipid features out of 356 measured lipids, including increased triacylglycerols and diacylglycerols and broad remodeling of glycerophospholipids, lysophospholipids, and sphingolipid-related species. Targeted polar metabolomics identified 75 significantly altered metabolites out of 98 measured metabolites, including changes in nucleotide/NAD-related metabolism, amino acid metabolism, methylation-associated metabolites, acylcarnitine abundance, phospholipid precursors, polyamine metabolism, arginine/nitric oxide-associated metabolism, and redox-associated metabolites. Process-level integration of significant features revealed coordinated remodeling of inflammatory protein programs with lipid storage, membrane remodeling, nucleotide metabolism, amino acid availability, phospholipid precursor abundance, nitric oxide-associated metabolism, and redox/osmolyte pathways. These findings demonstrate that IFN-γ and LPS-induced activation of BV-2 cells involves integrated immune, lipid, and metabolic adaptation rather than isolated induction of canonical inflammatory mediators. This integrated multiomic framework provides a resource for investigating how lipid and metabolic remodeling regulate microglial inflammatory states. Graphical Abstract

Proteomic analysis of migrasomes derived from IL-6 amplifier-induced human breast cancer cells.

It is indicated that IL-6 amplifier activation is associated with coordinated changes in migrasome proteins, including those involved in inflammatory signaling and trafficking, including those involved in inflammatory signaling and trafficking.

TSPO Regulates TLR4-Mediated Inflammation Through Calcium Homeostasis and Immunometabolic Adaptation

Bacterial infection triggered excessive inflammatory responses, yet the mechanisms linking inflammatory activation to immunometabolic adaptation remained incompletely understood. The mitochondrial translocator protein (TSPO) has been implicated in inflammatory activation and cellular metabolism. This study aimed to investigate the role of TSPO in inflammation mediated by Toll-like receptor 4 (TLR4). Herein, we integrated transcriptomic data from the human peripheral blood dataset GSE72829, and single-cell transcriptomic profiles from the CELLxGENE platform with cellular mechanistic experiments in BV2 microglia and RAW264.7 macrophages. Transcriptomic analyses revealed that TSPO expression was markedly upregulated in patients with bacterial infection (n = 52) and exhibited diagnostic potential to distinguish bacterial infection from healthy controls (HCs, n = 16) and viral infection (n = 92). TSPO-correlated genes were enriched in Toll-like receptor (TLR) signaling, inflammatory response, and immunometabolic pathways. Mechanistically, TSPO interacted with TLR4 and selectively modulated TLR4-driven inflammatory activation. TSPO deficiency augmented lipopolysaccharide (LPS) induced tumor necrosis factor‑α (TNF-α) and interleukin‑6 (IL-6) secretion, accompanied by disrupted Ca2+ homeostasis, impaired cholesterol balance, and compensatory metabolic remodeling characterized by elevated L-lactate and sustained Adenosine triphosphate (ATP) levels. Collectively, these findings identified TSPO as an immunometabolic regulator bridging TLR4 signaling and metabolic adaptation during inflammatory activation. Besides, TSPO represented a promising biomarker and therapeutic target to limit exaggerated inflammatory responses.

Transcriptomic profiling reveals transcriptomic remodeling linked to inflammatory activation and cell cycle-related gene expression in the early hours of macrophage activation

The integrated stress response kinase GCN2 prevents ZAKα-Dependent inflammatory hyperactivation in macrophages.

Translational control is redefined as a central checkpoint in macrophage activation, revealing how GCN2 mitigates ribosomal stress to prevent inflammatory hyperactivation, with potential therapeutic implications for TNFα-driven inflammatory diseases.

Nuclear Localization of IRG1 Modulates COX2 Expression and Prostaglandin Biosynthesis in Primary Human Macrophages 2308479

Immunometabolism within macrophages represents a dynamic process modulated by environmental factors and pathological conditions. Immune Responsive Gene 1 (IRG1) catalyzes the conversion of cis-aconitate, an intermediate in the tricarboxylic acid (TCA) cycle, to itaconate, and is robustly upregulated in macrophages following toll-like receptor engagement or type II interferon stimulation. The upregulation of IRG1 results in reduced macrophage cytokine production and influences polarization. To date, IRG1 localization has primarily been associated with mitochondria. In this study, we present evidence that IRG1 is also present in the nucleus of macrophages, where it associates with several promoters, including one controlling expression of PTGS2 (COX2), the rate-limiting enzyme in prostaglandin synthesis. In these studies, we employed a multi-omic strategy incorporating ChIP-seq, RNA-seq, global proteomics, and metabolomics. To establish the biological significance of this novel IRG1 function, we generated CRISPR knockouts in human monocyte-derived macrophages and utilized a DSS-induced colitis model to assess inflammation in vivo. Through a multi-omic approach encompassing ChIP-seq, RNA-seq, global differential proteomics, and metabolomics, we have elucidated a nuclear function for IRG1 in primary human macrophages influencing prostaglandin biosynthesis. Additionally, investigations using a CRISPR-generated catalytically inactive IRG1 point mutant in THP1 cells demonstrated significant binding of IRG1 to the PTGS2 promoter, resulting in increased COX2 transcript and protein expression independent of itaconate production. Collectively, these results reveal a novel, nuclear role for IRG1 in macrophages that provides a deeper understanding of how IRG1 may regulate macrophage function in the context of inflammation. Eli Lilly and Company Cellular Adhesion, Migration, and Inflammation (CAM)