Freund’s adjuvants have been used in vaccine and autoimmune settings, and their effects can be overlapping or unique to each. While both incomplete Freund’s adjuvants (IFA) and complete Freund’s adjuvants (CFA) influence antibody and T cell responses, the robust T helper 1 cytokines induced by the mycobacterial components make CFA the powerful immunostimulating adjuvant. In these studies, the adjuvant effects are investigated in a select population of cells, and the changes, if any, with the metabolic alterations in the systemic compartment are unclear. We investigated whether the effects of IFA and CFA can be influenced by the metabolic shifts in mice immunized with saline, IFA, or CFA using Mycobacterium tuberculosis var. bovis Bacillus Calmette–Guérin (BCG) as a positive control. After seven days of immunization, we analyzed the serum metabolite profiles using liquid chromatography coupled with high-resolution mass spectrometry and multivariate statistical analysis to identify metabolic features between the groups. The data revealed that, in the scores space, the CFA and BCG groups were more closely aligned compared to the saline group, while the IFA group displayed an intermediate profile. Furthermore, comparisons between the CFA and BCG groups showed more significant perturbations in lipid and amino acid metabolism, particularly involving glycerophospholipids, cysteine, and aromatic amino acids. In contrast, comparisons between the BCG and IFA groups indicated a more pronounced disruption in central energy metabolism pathways, such as the citric acid cycle and pyruvate metabolism. Together, the data suggest that the serum metabolite profiles in response to IFA and CFA might play a role in modulating the immune responses.

Background/Objectives: Freund’s adjuvants induce different immunomodulatory effects, but their underlying molecular mechanisms are unclear. In this study, we investigated whether the immune-stimulating effects of the complete Freund’s adjuvant (CFA) involve the mechanisms of trained immunity (TI). Methods: We examined bone marrow cells (BMCs) isolated from CFA-immunized A/J mice to address this question. Incomplete Freund’s adjuvant (IFA) and Mycobacterium tuberculosis var. bovis Bacillus Calmette-Guérin (BCG) served as negative and positive controls, respectively. We evaluated cytokine profiles, metabolic, and epigenetic changes. Results: First, BMCs from all groups except saline showed varied levels of IL-1β, IL-6, and TNF-α. But expression of CCL5 and CXCL10 was significantly elevated only in the CFA and BCG groups. Transcriptionally, significant elevations were noted for TNF-α and IL-1β in the CFA and BCG groups, whereas CXCL10, IL-6, and IL-10 were upregulated in the CFA and BCG groups, respectively. Second, while BMCs from the BCG group expressed the markers of both the M1 and M2 macrophages, no clear trends were noted in the CFA and IFA groups. Third, cell lysates from the CFA group revealed metabolic reprogramming in the BMCs. Specifically, we observed an increased level of lactate, indicative of aerobic glycolysis, which is implicated in TI, and this was also detected in the IFA group. Fourth, epigenetic analysis revealed histone enrichment in the promoter region of TNF-α, in the CFA group, but to a lesser degree than the BCG group. However, no epigenetic changes were observed in the IFA group. Conclusions: Our data provide new insights into the mechanisms of Freund’s adjuvants and the immunomodulatory effects of CFA could involve the features of TI.