Lactate Induces Alternative Polarization (M2) of Macrophages Under Lipopolysaccharide Stimulation In Vitro Through G-Protein Coupled Receptor 81

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Lactate Induces Alternative Polarization (M2) of Macrophages Under Lipopolysaccharide Stimulation In Vitro Through G-Protein Coupled Receptor 81

Introduction

Sepsis, a life-threatening condition characterized by systemic inflammation and subsequent immunosuppression, remains a critical challenge in critical care medicine. Hyperlactatemia, a hallmark of severe sepsis, correlates with poor clinical outcomes and mortality. Emerging evidence suggests lactate not only serves as a metabolic byproduct but also modulates immune responses. This study investigates the role of lactate in regulating macrophage polarization under lipopolysaccharide (LPS) stimulation and explores the involvement of G-protein coupled receptor 81 (GPR81) in this process.

Experimental Design and Methodology

Cell Culture and Treatment Groups

The study utilized RAW 264.7 mouse macrophages divided into three primary experimental groups:

  1. Control 1 Group: Untreated macrophages (no LPS or lactate).
  2. LPS Group: Macrophages stimulated with 100 ng/mL LPS for 24 hours.
  3. LPS + Lactate Group: Macrophages treated with LPS (100 ng/mL) and 10 mmol/L lactate. Lactate exposure lasted 15 minutes, followed by a 4-hour incubation post-washout.

For GPR81 knockdown experiments, two additional groups were established:

  • GPR81(–) Group: Macrophages transfected with GPR81-targeting siRNA using Lipofectamine.
  • Control 2 Group: Macrophages treated with scrambled siRNA.

Assays and Measurements

  • Cytokine and Chemokine Analysis: Supernatants were analyzed via ELISA for TNF-α, IL-6, IL-10, CCL-3, CCL-20, CCL-22, and CCL-24 levels.
  • Protein Expression: Western blotting quantified GPR81 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels.
  • siRNA Transfection: Silencer Select siRNA targeting GPR81 was administered twice, 24 hours apart, to ensure effective knockdown.

Key Findings

Lactate Modulates Macrophage Polarization Under LPS Stimulation

  1. Pro-inflammatory Cytokine Suppression:

    • LPS stimulation significantly elevated TNF-α (P < 0.001) and IL-6 (P < 0.01) levels compared to Control 1.
    • Co-treatment with lactate reduced TNF-α by 45% and IL-6 by 38% (P < 0.05 for both), indicating suppression of M1 polarization.

  2. Chemokine Profile Shifts:

    • LPS increased M1-associated chemokines CCL-3 (P < 0.01) and CCL-20 (P < 0.05). Lactate reduced their expression by 32% and 28%, respectively.
    • Conversely, lactate elevated M2-associated chemokines CCL-22 (1.7-fold increase, P < 0.05) and CCL-24 (2.1-fold increase, P < 0.01).

  3. GPR81 Upregulation:

    • Western blotting revealed a 2.5-fold increase in GPR81 expression in the LPS + Lactate group compared to LPS alone (P < 0.001).

GPR81 Mediates Lactate’s Immunomodulatory Effects

  1. Reversal of Lactate Effects with GPR81 Knockdown:

    • In GPR81(–) macrophages, lactate failed to suppress TNF-α and IL-6, with levels comparable to LPS-only treatment.
    • CCL-3 and CCL-20 suppression by lactate was abolished (P < 0.05 vs. Control 2), while CCL-22 and CCL-24 induction decreased by 41% and 53%, respectively (P < 0.01).

  2. Mechanistic Insights:

    • GPR81 activation by lactate attenuated LPS-induced NF-κB signaling, reducing pro-inflammatory cytokine release.
    • Enhanced CCL-22 and CCL-24 secretion suggests lactate promotes M2 polarization via GPR81-dependent pathways.

Discussion

Lactate and Sepsis Immunopathology

Sepsis progression involves an initial hyperinflammatory phase followed by immunosuppression, where macrophage polarization plays a pivotal role. This study demonstrates lactate’s dual role: suppressing M1-associated inflammation while promoting M2 polarization, which aligns with the immunosuppressive phase of sepsis. The finding that lactate elevates IL-10 (a key anti-inflammatory cytokine) further supports its role in immune tolerance.

GPR81 as a Therapeutic Target

GPR81’s involvement in lactate-mediated immunomodulation highlights its potential as a therapeutic target. Prior studies in immune hepatitis models showed GPR81 activation reduces liver injury by dampening macrophage activation. In sepsis, targeting GPR81 could mitigate excessive inflammation early or enhance immunosuppression in later stages, depending on clinical context.

Clinical Implications and Limitations

  1. Contradictory Cytokine Observations:

    • While lactate reduced TNF-α and IL-6 in vitro, septic patients often exhibit elevated plasma levels of these cytokines. This discrepancy may arise from the complexity of in vivo microenvironments, where multiple cell types and signaling pathways interact.

  2. Translational Challenges:

    • The 10 mmol/L lactate concentration used here exceeds physiological levels (normal: 1–2 mmol/L), raising questions about clinical relevance. However, tissue-specific lactate accumulation (e.g., in hypoxic regions) may mirror these concentrations locally.

  3. M2 Polarization and Immunosuppression:

    • Increased CCL-22 and CCL-24 correlates with impaired pathogen clearance and secondary infections in sepsis. Thus, lactate’s M2-polarizing effects might exacerbate immunosuppression, necessitating balanced therapeutic strategies.

Conclusion

This study establishes lactate as a critical regulator of macrophage polarization via GPR81 under LPS stimulation. By suppressing M1-associated cytokines (TNF-α, IL-6) and chemokines (CCL-3, CCL-20) while enhancing M2 markers (CCL-22, CCL-24), lactate drives immunosuppressive reprogramming in macrophages. GPR81 knockdown abolishes these effects, confirming its central role. These findings provide a mechanistic foundation for targeting lactate-GPR81 signaling in sepsis, though further in vivo validation is essential to address translational gaps.

doi.org/10.1097/CM9.0000000000000955

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