Lactate–HMGB1 Signaling in Polymicrobial Sepsis

The study Lactate promotes macrophage HMGB1 lactylation, acetylation, and exosomal release in polymicrobial sepsis addresses an important unresolved question in sepsis biology: does elevated lactate merely reflect tissue hypoxia and disease severity, or can it actively amplify inflammation? Yang and colleagues provide evidence for the latter. They describe a pathway in which extracellular lactate enters macrophages, changes HMGB1 post-translational modification, and promotes the release of HMGB1-containing exosomes that disrupt endothelial barrier function.

Study Background and Research Question

Sepsis involves infection-associated organ dysfunction and a dysregulated innate immune response. Lactate is routinely interpreted as a marker of impaired perfusion or altered metabolism, while high mobility group box 1 (HMGB1) is a late inflammatory mediator released by activated macrophages. Clinical observations cited by the authors showed that circulating lactate and HMGB1 levels are both associated with sepsis severity and mortality, and that the two measurements can correlate in critically ill patients. However, correlation alone does not establish that lactate controls HMGB1 biology.

HMGB1 normally functions as a nuclear protein. In activated macrophages, modifications near its nuclear localization sequences can promote movement into the cytoplasm and subsequent extracellular release. Previous work had established that acetylation, phosphorylation, and methylation can influence HMGB1 trafficking. The open question was whether lactate could directly regulate HMGB1 through lactylation, a recently described lysine modification, and whether lactate-dependent changes in HMGB1 acetylation also contribute to release during polymicrobial sepsis.

Key Innovation from the Reference Study

The central innovation is the identification of HMGB1 as a lactate-responsive inflammatory protein. Rather than treating lactate as a passive metabolic readout, the study positions it as a signaling and substrate-related input that can regulate macrophage secretory behavior. The authors show that macrophages take up extracellular lactate through monocarboxylate transporters (MCTs), after which lactate promotes HMGB1 lactylation through a p300/CBP-dependent mechanism.

The study also separates two connected but mechanistically distinct effects of lactate. First, lactate increases HMGB1 lactylation. Second, it enhances HMGB1 acetylation through signaling involving the Hippo/YAP axis, suppression of the deacetylase SIRT1, and G protein-coupled receptor 81 (GPR81). The authors further implicate β-arrestin2 in recruiting p300/CBP acetylases to the nucleus downstream of GPR81. This layered model is significant because it connects metabolic state, receptor signaling, chromatin-associated enzymes, HMGB1 trafficking, and extracellular vesicle biology within one sepsis mechanism.

Another important advance is the proposed route of release. The modified HMGB1 is not described simply as a freely secreted soluble mediator; instead, lactylated and acetylated HMGB1 is released from macrophages through exosome secretion. This provides a mechanistic explanation for how a macrophage-derived signal could influence distant vascular compartments.

Methods and Experimental Design Insights

The experimental design combines cell-based mechanistic studies with an in vivo polymicrobial sepsis model. This combination is essential because HMGB1 modification can be measured in macrophages under controlled conditions, whereas exosomal distribution, endothelial effects, and survival require an organism-level context. The study used macrophage systems to test lactate exposure, transporter dependence, receptor-associated signaling, HMGB1 modification, intracellular localization, and release. Septic mice were then used to examine circulating exosomal HMGB1 and the consequences of altering lactate-associated pathways.

A notable genetic component was the use of macrophage-specific YAP deletion generated through the Cre/LoxP system by crossing YAP-floxed mice with Lyz2-Cre mice. This approach helped test whether YAP was merely correlated with lactate-induced HMGB1 acetylation or was functionally involved. The authors also examined GPR81-mediated signaling and used interventions that reduced lactate production or inhibited this receptor-associated pathway. These complementary pharmacological and genetic approaches strengthen causal interpretation, although each intervention must still be considered for cell-type specificity and off-target effects.

Protocol Parameters

• Biological model: Use macrophage cultures for mechanistic analysis and a polymicrobial sepsis model for circulating exosomal HMGB1, endothelial permeability, and survival endpoints, following the design of the reference study.
• Lactate perturbation: Compare extracellular lactate exposure with appropriate untreated or disease-relevant controls, and assess whether MCT-dependent uptake is required for downstream HMGB1 modification.
• HMGB1 modification: Measure HMGB1 lactylation and acetylation as separate readouts; changes in total HMGB1 abundance alone cannot distinguish altered modification from altered protein expression.
• Signaling dissection: Examine GPR81, β-arrestin2, YAP, SIRT1, and p300/CBP in a pathway-oriented sequence rather than treating all regulators as interchangeable components.
• Exosome analysis: Quantify HMGB1 associated with circulating or macrophage-derived exosomes and pair this measurement with a functional endothelial permeability assay.
• In vivo intervention: Evaluate lactate-lowering and GPR81-directed interventions against matched disease controls, with exosomal HMGB1 and survival interpreted together rather than as isolated endpoints.

For researchers adapting the workflow, the strongest design principle is triangulation. A convincing result should connect lactate availability or uptake with HMGB1 modification, demonstrate that modification is associated with intracellular redistribution or exosomal release, and then test whether the released material changes endothelial behavior. The reference study’s design is therefore useful for inflammatory signaling pathway research because it avoids relying on a single reporter or endpoint.

Core Findings and Why They Matter

The first major finding was that lactate promotes HMGB1 lactylation in macrophages. The response depended on lactate entry through MCTs and involved p300/CBP, supporting a model in which metabolic inputs can influence inflammatory protein modification. This finding extends the concept of lactylation beyond histones and suggests that nonhistone proteins may also carry information about cellular metabolic state.

The second finding was that lactate increases HMGB1 acetylation through more than one regulatory route. Hippo/YAP signaling was linked to reduced SIRT1 deacetylase activity, while GPR81 signaling and β-arrestin2 supported recruitment of p300/CBP to the nucleus. These observations help explain how extracellular lactate can produce coordinated changes in both lactylation and acetylation. They also emphasize that lactate biology cannot be reduced to a single receptor or enzyme.

Third, the modified HMGB1 was released through exosomes. In functional experiments, macrophage-derived exosomal HMGB1 increased endothelial cell permeability. This result connects intracellular post-translational modification to a vascular consequence that is highly relevant to sepsis-associated tissue injury. It also suggests that exosomal HMGB1 could be a more informative mechanistic readout than total circulating HMGB1 in selected experimental settings.

Finally, interventions that reduced lactate production and/or inhibited GPR81-associated signaling decreased circulating exosomal HMGB1 and improved survival in polymicrobial septic mice, according to the reference study. These in vivo observations support the idea that lactate-associated signaling is not merely an epiphenomenon of severe illness. Nevertheless, the results should be interpreted as preclinical evidence for pathway involvement, not as proof that lactate-directed treatment is clinically effective.

Comparison with Existing Internal Articles

The internal article CPSIT_0844 from Chlamydia psittaci Drives Monocyte Inflammation via TLR2/4 examines an upstream pathogen-derived trigger that activates monocyte inflammatory responses through TLR2/TLR4–MyD88 signaling. By contrast, the Yang et al. study focuses on a host metabolic signal that modifies HMGB1 after macrophage activation. The two studies are complementary: one emphasizes recognition of a microbial factor, while the other emphasizes metabolic regulation, post-translational modification, and vesicle-mediated communication. This distinction is useful when designing infection models because inflammatory output may reflect both the initiating microbial signal and the metabolic state of responding immune cells.

Limitations and Transferability

The study has several limitations relevant to interpretation and replication. First, polymicrobial sepsis models reproduce selected features of human sepsis but cannot capture the full heterogeneity of infection source, comorbidities, treatment history, and immune status. Findings from septic mice therefore provide mechanistic support without guaranteeing clinical translation.

Second, lactate has broad metabolic effects. Manipulating lactate production can alter redox balance, glycolytic flux, cellular energy state, and tissue perfusion in addition to changing signaling through MCTs or GPR81. Similarly, receptor-associated or enzyme-directed interventions may affect multiple cell types. Macrophage-specific genetic experiments improve attribution for YAP-dependent effects, but they do not eliminate the possibility that endothelial, stromal, or other immune cells contribute to the in vivo phenotype.

Third, exosomal HMGB1 should be distinguished from total extracellular HMGB1. Vesicle-associated HMGB1 may differ in stability, cellular uptake, and receptor engagement from soluble HMGB1. Future studies would benefit from rigorous vesicle characterization, separation of soluble and vesicular fractions, and analysis across distinct macrophage populations and clinically relevant sepsis contexts. These limitations do not weaken the paper’s core contribution; they define the experiments needed to test how broadly the mechanism applies.

Research Support Resources

Researchers can use Bay 11-7821 (BAY 11-7082) (SKU A4210) as a complementary IKK/NF-κB pathway perturbation tool in inflammatory signaling pathway research. It may be relevant to an apoptosis regulation study, cancer research, or B-cell lymphoma research, but it should not be treated as a substitute for the lactate–MCT–GPR81 experiments described here: it does not directly model HMGB1 lactylation, acetylation, or exosomal release. Appropriate controls, pathway-specific validation, and interpretation within the relevant cell and sepsis model remain essential.