Fig. 2
Iron does not alter polarization. LPS promotes a pro-inflammatory gene expression profile, with increased expression of CD86 (a) and reduced expression of anti-inflammatory genes ARG1 (b), YM1 (c), and CD206 (d). FeSO4 addition had no effect on gene expression. All groups were compared using two-way ANOVA with Tukey post-test. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. All graphs represent n = 5. Flow cytometry showed similar results, wherein LPS did not significantly alter CD86 expression (e) alone, but did significantly reduce the expression of anti-inflammatory marker CD206 (f). LPS in combination with FeSO4 resulted in a significant increase in CD86 expression; there was no combination effect on CD206 expression. Overlays illustrate fluorescence distribution among individual microglia after 24-h exposure to experimental conditions. All groups were compared using two-way ANOVA with Tukey post-hoc test. *p < 0.05; **p < 0.01; ***p < 0.001 represent significant comparisons with Control. #p < 0.05 represent significant comparisons with FeSO4. N = 4 for each marker. Immunocytochemistry demonstrated elevated expression of CD86 among microglia with LPS and reduced CD206 expression (g). Iron did not influence CD86 or CD206 protein expression. Representative sample of one trial presented. N = 3/group. Size bars represent 50 μm. Finally, Proteome profiler analysis at 24 h after stimulation illustrated a significant pro-inflammatory response to LPS with elevations in IL-1α, IL-1β, IP-10, RANTES, and TNFα (h). Data is shown as protein expression normalized to protein loading control. Iron did not affect this response. All groups were compared using two-way ANOVA with Tukey post-test. FeSO4 vs. #p < 0.05, Control vs. **p < 0.01, ***p < 0.001, N = 3. Bars represent mean ± SEM