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Introduction: Vaccinating patients with immune-mediated inflammatory diseases (IMIDs) poses two key challenges: Impaired vaccine responses due to immunosuppression and the risk of disease flares triggered by adjuvant-induced innate immune activation. Many adjuvants act via pattern recognition receptors (PRRs), including Toll-like receptor 4 (TLR4). In rheumatoid arthritis (RA), endogenous ligands such as S100A8/A9 chronically activate TLR4, leading to persistent monocyte/macrophage (Mφ) priming. Thus, innate immune cells in RA operate in a pre-activated state, which may alter their response to vaccines. Emerging evidence suggests that metabolic reprogramming critically shapes Mφ inflammatory responses.The aim of this study is to elucidate the metabolic and functional mechanisms underlying sustained TLR4-induced macrophage reprogramming and to determine how this altered state shapes responses to vaccines with distinct adjuvant profiles.

Methods: HoxB8-derived Mφ were exposed to prolonged TLR4 stimulation to model chronic PRR activation. Metabolic profiling was performed tandem mass spectrometry (HILIC-MS/MS). Immune responses upon TLR4 restimulation were assessed by TNFα ELISA and qPCR. Mitochondrial function was analyzed using flow cytometry. Metabolic pathways were targeted using inhibitors of pyruvate dehydrogenase kinase 1 (PDHK1) and glutaminase 1 (GLS1). Nuclear factor erythroid 2-related factor 2 (Nrf2) activity was assessed by Western blot and inhibited using ML385. Three licensed respiratory syncytial virus (RSV) vaccines with distinct adjuvant mechanisms were tested under basal and pre-activated conditions.

Results: Sustained TLR4 activation induced stable metabolic and functional reprogramming of Mφ, characterized by Krebs cycle disruption, reduced ATP production, mitochondrial dysfunction, and altered urea cycle metabolism. Nrf2 emerged as a central regulator, with glutamine metabolism being essential for establishing this state. Pharmacological inhibition of PDHK1 and GLS1 reversed key metabolic alterations. In translational experiments, RSV vaccines induced distinct activation profiles depending on their adjuvant composition, leading to differential responses upon subsequent inflammatory stimulation.

Conclusion: Chronic TLR4 stimulation reprograms Mφ metabolism and function, altering responses to subsequent PRR activation. These findings indicate that vaccine-induced immune responses may differ in RA due to innate immune pre-activation, underscoring the need to consider disease-specific immune states in vaccine design.

References

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