Experimental Macrophage Polarization: A Two-Pronged Approach To Evaluate Modulation Of Macrophage Function

This study aimed to create an in vitro procedure for testing macrophage function following human-subject serum incubation. The study involved three optimizations: 1. THP-1 monocyte (THP-1) differentiation to monocyte derived macrophages (MDMs) with PMA; 2. LPS polarization of MDMs to M1-macrophages; 3. MDM serum incubation. Phagocytosis was determined via commercially available kit and cytokine concentrations were analyzed from supernatant of select samples. NucBlue staining assessed cell numbers in optimizations 2 and 3. Phagocytosis data were analyzed by one-way ANOVA. Serum incubation time x treatment effects were analyzed by two-way ANOVA. Significance was set to 0.05. Post-hoc analyses used Bonferroni Correction. Insufficient samples prevented statistical analysis of cytokine data. Greater phagocytosis was observed with 48/24-hr incubation/rest than longer incubations (p<0.01). Longer incubations led to lower levels of all cytokines but IL-1B. 0.5ug/mL LPS led to the highest, though not significant, phagocytosis (p=0.386). Higher serum concentrations and longer incubations led to higher and lower phagocytosis respectively (p<0.01, p<0.001), with similar trends in secreted cytokines. The results inform the following procedure: THP-1 differentiation via 48-hr incubation (25nM PMA) plus 24-hr rest, 3.0-hr incubation of MDMs with 35% human serum media, then 24-hr MDM polarization using 0.5ug/mL LPS prior to functional measures.