Effect of Melittin on Metabolomic Profile and Cytokine Production in PMA-Differentiated THP-1 Cells

Abstract

Melittin, the major active peptide of honeybee venom (BV), has potential for use in adjuvant immunotherapy. The immune system response to different stimuli depends on the secretion of different metabolites from macrophages. One potent stimulus is lipopolysaccharide (LPS), a component isolated from gram-negative bacteria, which induces the secretion of pro-inflammatory cytokines in macrophage cell cultures. This secretion is amplified when LPS is combined with melittin. In the present study, pure melittin was isolated from whole BV by flash chromatography to obtain pure melittin. The ability of melittin to enhance the release of tumour necrosis factor-α (TNF-α), Interleukin (IL-1β, IL-6, and IL-10) cytokines from a macrophage cell line (THP-1) was then assessed. The response to melittin and LPS, applied alone or in combination, was characterised by metabolic profiling, and the metabolomics results were used to evaluate the potential of melittin as an immune adjuvant therapy. The addition of melittin enhanced the release of inflammatory cytokines induced by LPS. Effective chromatographic separation of metabolites was obtained by liquid chromatography-mass spectrometry (LC-MS) using a ZIC-pHILIC column and an ACE C4 column. The levels of 108 polar and non-polar metabolites were significantly changed (p ˂ 0.05) following cell activation by the combination of LPS and melittin when compared to untreated control cells. Overall, the findings of this study suggested that melittin might have a potential application as a vaccine adjuvant.

Metabolomic Profiling of the Immune Stimulatory Effect of Eicosenoids on PMA-Differentiated THP-1 Cells

“…Several studies have shown that metabolic reprogramming of the cell regulates macrophage activation. In the present study, the findings for LPS activation of THP-1 macrophages regarding specific pathway and biomarker metabolites are consistent with those of previous reports [17,37]. In general, pro-inflammatory stimuli cause the macrophages to undergo a metabolic switch from oxidative phosphorylation (OXPHOS) to glycolysis [38].…”

“…Multivariate and univariate statistical analysis were used to visualise and examine the metabolite effects on the following treatment combinations: T1 (9 µg/mL (Z)-11-eicosenol + 0.5 µg/mL LPS), T2 (150 µg/mL eicosenoate + 0.5 µg/mL LPS) and T3 (40 µg/mL eicosenoic acid + 0.5 µg/mL LPS). The effect of LPS alone was also evaluated to confirm the previous findings [17] and to investigate new pathways that might be involved in immune stimulation by eicosenoids. LPS synergism was clearly evident from the cytokine assessment, particularly with IL-1β and IL-10.…”

“…THP-1 cells differentiated with PMA were cultured in 6-well plates at a seeding density of 4.5 × 10 5 cells/mL (n = 6) for 48 h, after which the medium was removed by aspiration and replaced prior to incubation with LPS (0.5 µg/mL) either alone or together with (Z)-11-eicosenol (9 µg/mL), eicosenoate (150 µg/mL) and eicosenoic acid (40 µg/mL) for another 24 h. Extraction of the metabolites was carried out as described previously [17]. Seven different analytical standard solutions were prepared by adding each metabolite standard (10 µg/mL final concentration) containing glycine-13 C 2 [25].…”

“…LC-MS was carried out using an Exactive Orbitrap (Thermo Fisher Scientific, Bremen, Germany); the conditions used were as described in our previous paper [17].…”

“…Data extraction was performed using the MZMatch software (SourceForge, La Jolla, CA, USA) and the metabolite peaks were filtered, compared and characterised using the Microsoft Excel IDEOM [26] as described in our previous paper [17]. The metabolite data were also subjected to multivariate analysis by fitting PCA-X and OPLS-DA using the SIMCA-P software v.14.0 (Umetrics, Umea, Sweden).…”