Lactate-Driven HMGB1 Modification: Mechanisms and Implications in Polymicrobial Sepsis

Study Background and Research Question

Sepsis is a severe systemic condition characterized by dysregulated inflammation and organ dysfunction, with high morbidity and mortality worldwide. While serum lactate has long been recognized as a prognostic biomarker in sepsis, its potential causal roles in modulating the inflammatory environment remained poorly understood. High levels of both lactate and high mobility group box-1 (HMGB1) protein are associated with worse outcomes in sepsis, but whether lactate directly influences HMGB1 release from macrophages had not been established (paper).

Key Innovation from the Reference Study

The reference study provides the first direct evidence that extracellular lactate actively promotes post-translational modification (PTM) of HMGB1 within macrophages, namely lactylation and acetylation, leading to enhanced exosomal release of HMGB1 during polymicrobial sepsis. This mechanistic link implicates lactate not merely as a metabolic byproduct or biomarker, but as an upstream regulator of proinflammatory signaling. The study further uncovers the involvement of specific molecular pathways, including monocarboxylate transporters (MCTs), the p300/CBP acetyltransferase system, Hippo/YAP-mediated signaling, and G protein-coupled receptor 81 (GPR81), in mediating these PTMs and HMGB1 secretion (paper).

Methods and Experimental Design Insights

The investigators employed a combination of in vivo and in vitro approaches to dissect the relationship between lactate levels, HMGB1 modifications, and exosomal secretion. Key experimental models and techniques included:

• Animal Models: C57BL/6J wild-type and macrophage-specific YAP knockout mice (generated using the Cre/LoxP system) enabled assessment of Hippo/YAP pathway involvement in vivo.
• Sepsis Induction: A polymicrobial sepsis model was established by cecal ligation and puncture (CLP), a well-validated approach to mimic clinical sepsis.
• Cellular Studies: Mouse bone marrow-derived macrophages (BMDMs) and human THP-1 macrophage-like cells were used to study extracellular lactate uptake, HMGB1 PTMs, and exosome generation.
• Biochemical Analyses: HMGB1 lactylation and acetylation status were assessed using immunoprecipitation and western blotting, while exosome-associated HMGB1 was quantified via ELISA and nanoparticle tracking.
• Pharmacological Modulation: Inhibitors of lactate production, MCTs, p300/CBP, and GPR81, as well as genetic ablation of YAP, were used to dissect specific signaling steps (paper).
• Endothelial Permeability Assays: To assess the functional consequences of HMGB1-laden exosomes, endothelial monolayer permeability was measured after exposure.

Core Findings and Why They Matter

• Lactate Uptake and HMGB1 Modification: Macrophages internalize extracellular lactate via MCTs, resulting in increased HMGB1 lactylation through a p300/CBP-dependent mechanism. Simultaneously, lactate promotes HMGB1 acetylation—mediated by Hippo/YAP signaling and β-arrestin2-facilitated nuclear recruitment of p300/CBP via GPR81 activation (paper).
• Exosomal Release of HMGB1: Both lactylated and acetylated HMGB1 are preferentially packaged into exosomes and released from macrophages. Circulating exosomal HMGB1 levels correlate positively with serum lactate in septic mice, indicating a mechanistic connection in vivo.
• Impact on Endothelial Barrier Function: Exosomal HMGB1 markedly increases endothelial cell permeability, supporting its role as a mediator of vascular leakage and organ dysfunction in sepsis.
• Therapeutic Implications: Pharmacological inhibition of lactate production or GPR81 signaling significantly reduces exosomal HMGB1 release and improves survival in septic mice. This positions lactate/lactate receptor signaling as a promising therapeutic axis for intervention in sepsis (paper).

Protocol Parameters

• CLP-induced sepsis in mice | cecal ligation and puncture | model for systemic inflammation and multi-organ dysfunction | recapitulates clinical sepsis pathophysiology | paper
• Lactate supplementation | 10–20 mM | in vitro macrophage assays | mimics elevated lactate conditions seen in severe sepsis | paper
• p300/CBP inhibition | C646 at 10 μM | tests acetylation/lactylation dependency | validates epigenetic regulator role | paper
• GPR81 antagonist | 3-OBA at 2 mM | blocks lactate signaling | distinguishes receptor-mediated from metabolic effects | paper
• Exosome isolation | ultracentrifugation (100,000g, 70 min) | serum/cell supernatant | standard for extracellular vesicle purification | paper
• Endothelial permeability assay | FITC-dextran flux | evaluates barrier integrity after exosome treatment | functionally links HMGB1 exosomes to vascular leakage | paper
• THP-1 cell inflammasome activation | 1 μg/mL Nigericin, 1 mM ATP | positive control for IL-1β release in inflammasome studies | workflow_recommendation

Comparison with Existing Internal Articles

Recent internal articles have highlighted the strategic importance of NLRP3 inflammasome inhibitors, such as NBC19, in dissecting the mechanisms of inflammatory cytokine release and innate immune responses (internal analysis). While the reference paper focuses specifically on lactate-driven HMGB1 release via exosomal pathways, several internal reviews (e.g., Strategic Innovation in NLRP3 Inflammasome Research) discuss the broader relevance of targeting upstream inflammatory signals, including those modulated by metabolic stress and DAMP release. These resources converge on the view that small molecule inhibitors—such as NBC19—offer powerful tools for functional dissection of inflammasome and cytokine pathways relevant to both sepsis and sterile inflammation.

Limitations and Transferability

Although the study offers robust mechanistic data in mouse models and macrophage cell lines, several limitations should be noted. First, the precise contribution of HMGB1 exosomal release to human sepsis pathophysiology remains to be validated in clinical cohorts. Second, while pharmacological inhibitors of lactate and GPR81 signaling improved outcomes in mice, the translational potential and safety of such strategies in humans require further investigation. Finally, the interplay between lactate, inflammasome activation, and other DAMPs—such as IL-1β—warrants deeper exploration, especially in the context of complex inflammatory milieus (paper).

Research Support Resources

For researchers aiming to extend these findings or interrogate related inflammasome pathways, selective small molecule tools are essential. The NLRP3 inflammasome inhibitor NBC19 (SKU BA6129) from APExBIO offers potent, nanomolar-level suppression of IL-1β release in THP1 cell models upon Nigericin or ATP stimulation (source: product_spec). NBC19 thus provides a practical resource for delineating the crosstalk between metabolic modulators (such as lactate) and inflammasome-driven cytokine output in inflammation research. For detailed workflow recommendations and comparative insights, see “Redefining Translational Inflammation Research: NBC19 and Lactate-Driven HMGB1 Release.”