Lactate–HMGB1 Signaling in Polymicrobial Sepsis

Sepsis research has long treated circulating lactate as an indicator of impaired perfusion, metabolic stress, or disease severity. The study by Yang and colleagues, published in Cell Death & Differentiation, expands this view by showing that lactate can directly influence inflammatory protein trafficking. In the reference study, lactate promoted both lactylation and acetylation of high mobility group box 1 (HMGB1) in macrophages and enhanced its release in exosomes during polymicrobial sepsis.

Study Background and Research Question

HMGB1 is normally a nuclear chromatin-associated protein, but activated macrophages can release it as a late inflammatory mediator. Once outside the cell, HMGB1 can amplify innate immune signaling and contribute to vascular dysfunction. Clinical observations have linked high circulating HMGB1 and lactate concentrations with severe sepsis, but a correlation does not establish whether lactate participates mechanistically in HMGB1 release.

The central question was therefore whether extracellular or glycolysis-associated lactate could alter HMGB1 biology in macrophages. The authors specifically examined three connected processes: whether lactate changes HMGB1 post-translational modification, how lactate-associated signaling controls those modifications, and whether modified HMGB1 is released through extracellular vesicles that affect endothelial barrier function.

This question is important because HMGB1 release is not simply a consequence of cell lysis. Nuclear export, modification of lysine residues, and vesicular trafficking can determine when and how HMGB1 enters the extracellular environment. A metabolic intermediate that regulates these steps would provide a mechanistic link between altered metabolism and inflammatory tissue injury.

Key Innovation from the Reference Study

The major innovation is the identification of lactate as a signaling and substrate-related regulator of HMGB1 release. Earlier work had established histone lactylation and had associated aerobic glycolysis with HMGB1 acetylation, but the direct contribution of lactate to HMGB1 modification during sepsis was unresolved. The authors show that macrophages take up extracellular lactate through monocarboxylate transporters and use lactate-associated signaling to promote HMGB1 lactylation through a p300/CBP-dependent mechanism.

The study also separates two related but mechanistically distinct routes to HMGB1 modification. Lactate stimulated HMGB1 acetylation through G protein-coupled receptor 81 (GPR81). One branch involved Hippo/YAP signaling and suppression of the deacetylase SIRT1. Another involved β-arrestin2-dependent recruitment of the acetyltransferases p300 and CBP to the nucleus. Together, these findings suggest that lactate can affect HMGB1 through both intracellular metabolic handling and receptor-mediated signal transduction.

A further advance is the connection between modification and secretion route. The lactylated and acetylated HMGB1 accumulated in macrophage-derived exosomes rather than being considered only as freely soluble protein. These exosomes increased endothelial permeability in functional assays. In vivo experiments then connected reduced lactate production or inhibition of GPR81-associated signaling with lower circulating exosomal HMGB1 and improved outcomes in polymicrobial sepsis. Thus, the proposed pathway is not merely a biochemical observation; it links metabolism, nuclear protein regulation, extracellular vesicle biology, and vascular dysfunction.

Methods and Experimental Design Insights

The experimental design combined cell culture, mouse genetics, biochemical analysis, extracellular vesicle studies, and a polymicrobial sepsis model. This layered strategy is useful because each experimental system addresses a different level of causality. Macrophage experiments test direct effects of lactate, genetic manipulation examines pathway dependence, and animal experiments determine whether the mechanism is relevant to systemic disease.

Macrophages were exposed to lactate and analyzed for HMGB1 modification, intracellular localization, and release. The investigators examined transporter-dependent lactate uptake, p300/CBP activity, GPR81 signaling, SIRT1 regulation, and the role of β-arrestin2. Macrophage-specific YAP deletion generated with the Lyz2-Cre system provided a genetic test of the Hippo/YAP branch. In parallel, serum exosomes from septic animals were evaluated for HMGB1 content and related to lactate-associated changes in vivo.

The study also used endothelial permeability assays to test biological activity of macrophage-derived exosomal material. This is a significant design feature: measuring exosomal HMGB1 alone would establish cargo association, whereas a permeability assay asks whether that cargo can produce a vascular phenotype. Pharmacological reduction of lactate production and interference with GPR81-mediated signaling further tested whether the pathway could be modified after sepsis induction.

Protocol Parameters

• Sepsis comparison: Use matched sham and polymicrobial sepsis groups when evaluating lactate, serum exosomes, and HMGB1; these study-oriented comparisons are more informative than measuring either analyte in isolation.
• Macrophage perturbation: Compare lactate exposure with transporter or GPR81 pathway manipulation, while including controls for cell viability and nonspecific stress.
• Modification analysis: Preserve nuclear and cytosolic fractions rapidly and analyze total HMGB1 together with lactylation and acetylation signals; workflow recommendations should not be interpreted as exact replication parameters from the reference study.
• Exosome assessment: Pair HMGB1 cargo measurements with an endothelial permeability readout to distinguish vesicle abundance from functional activity.
• Pathway validation: Combine pharmacological interventions with genetic tests such as macrophage-specific YAP deletion where available, because either approach alone may have off-target or compensatory effects.

Core Findings and Why They Matter

The first major finding was that lactate increased HMGB1 lactylation in macrophages. The p300/CBP dependence supports a model in which lactate availability influences lysine lactylation through an enzymatic writer system. This observation extends lactylation from a chromatin-centered concept to a nonhistone inflammatory protein with a defined secretion consequence.

Second, lactate increased HMGB1 acetylation through complementary signaling mechanisms. Suppression of SIRT1 would be expected to reduce deacetylation, while p300/CBP recruitment would favor acetyl group addition. Because HMGB1 localization is influenced by modifications near its nuclear localization sequences, these changes can facilitate movement from the nucleus to the cytoplasm and subsequent release.

Third, the modified HMGB1 was associated with exosome secretion and endothelial barrier disruption. This result provides a plausible route by which macrophage metabolic state could influence distant tissues. Exosomal transport may protect inflammatory cargo from immediate degradation and deliver it to vascular targets, although the relative contribution of exosomal versus soluble HMGB1 remains an important question.

Finally, interventions that reduced lactate generation or blocked GPR81-associated signaling lowered circulating exosomal HMGB1 and improved survival in polymicrobial sepsis. These data support lactate-associated signaling as a potential therapeutic entry point, but they do not imply that lactate is uniformly harmful. Lactate also participates in normal energy exchange and tissue repair, so pathway-selective intervention will likely be more appropriate than indiscriminate depletion.

Comparison with Existing Internal Articles

The internal article Protease and Phosphatase Inhibitor Cocktail: Workflow Mastery addresses practical control of protein degradation and dephosphorylation during extraction. It complements the reference study at the sample-handling level: accurate assessment of HMGB1 abundance, SIRT1, YAP, and signaling proteins requires rapid lysis and preservation of labile protein states. However, a preservation workflow does not itself demonstrate lactate-dependent lactylation or acetylation.

Similarly, Optimizing Protein Extraction: EDTA Free Protease and Phosphatase Inhibitor Cocktail focuses on maintaining protein integrity and phosphorylation fidelity across extraction workflows. Its relevance here is methodological rather than evidentiary. The Yang et al. study supplies the biological mechanism; extraction guidance can help researchers reproduce reliable immunoblotting or immunoprecipitation measurements without confusing improved sample preservation with proof of pathway activation.

Limitations and Transferability

The findings are compelling but should be interpreted within the boundaries of the experimental systems. Mouse polymicrobial sepsis models reproduce selected features of human sepsis but do not capture the full heterogeneity of infection source, comorbidity, treatment history, and immune status in patients. Macrophage cultures also simplify the cellular environment and may expose cells to lactate concentrations or exposure durations that do not precisely match individual human disease states.

Mechanistic attribution is strengthened by transporter, receptor, pharmacological, and YAP genetic experiments, yet each intervention has limitations. GPR81 manipulation may affect several downstream responses, Lyz2-Cre-directed deletion is not restricted to one macrophage subtype, and pharmacological lactate reduction can change systemic metabolism beyond the proposed pathway. The study also supports an association between exosomal HMGB1 and endothelial permeability, but exosome preparations can contain heterogeneous vesicle populations and co-isolated soluble proteins. Additional work is needed to establish the relevant HMGB1 modification sites, quantify their stoichiometry, and determine whether the same modified species dominate in human septic plasma.

Why this cross-domain matters, maturity, and limitations

The work bridges immunometabolism and protein biochemistry: a metabolite-associated signal is translated into a specific post-translational and extracellular trafficking program. That bridge is mature enough to guide mechanistic experiments, but not yet sufficient to justify a clinical lactate-targeting strategy. For transferability, future studies should verify the pathway in human macrophages and patient-derived exosomes, define which intervention window is beneficial, and distinguish effects on HMGB1 modification from general changes in inflammation or hemodynamics.

Research Support Resources

For comparable macrophage, tissue, or cell-lysate workflows, researchers can use the Protease and Phosphatase Inhibitor Cocktail (EDTA Free, 100X in ddH2O) (SKU K4006) during extraction or lysis. This EDTA free protease inhibitor cocktail contains inhibitors directed against aminopeptidases, cysteine proteases, and serine proteases, together with phosphatase inhibitors relevant to serine/threonine and tyrosine phosphatases. As a protein extraction protease inhibitor and phosphatase inhibitor for cell lysate, it can support preservation of HMGB1 and signaling proteins; it does not replace validation of lactylation or acetylation assays. The product information reports a concentrated 100X formulation for dilution and storage at −20°C.