(P18-010-25) Naringenin Suppresses M1 Macrophage Polarization but Does Not Promote M2 Macrophage Polarization

Objectives: M1 macrophages are known as pro-inflammatory while M2 macrophages play a crucial role in anti-inflammation and tissue repair. The balance between M1 and M2 is important to maintain cellular homeostasis and immuno-defense systems. Naringenin (NAR), a natural flavonoid in citrus fruits and apiaceous vegetables, is known to exert anti-inflammatory effects. However, its role in macrophage polarization is not fully understood. Here, we investigated whether NAR modulates macrophage polarization in Raw264.7 cells.

Methods: Raw264.7 cells were treated with NAR for 2 hr prior to exposure to lipopolysacharride (LPS) and interferon γ (IFNγ) for M1 macrophage differentiation, or IL-4 for M2 macrophage differentiation. After 22 hr, culture media was collected for nitric oxide (NO) quantification and the cells were used for Western blot or qPCR analysis. Data were was analyzed by One-way ANOVA with Duncan’s post-test.

Results: In M1 macrophages, NO production was increased by LPS/IFNγ from 1.7 μM to 7.3 μM while NAR dose-dependently suppressed the induction of NO production (6.0, 5.8, 4.7 μM of NO in 25, 50, 100 μM of NAR-treated cells, respectively, P < 0.05). M1 macrophage markers (IL-1β, IL-6, TNFα, iNOS, CD86) were induced by LPS/IFNγ, but were suppressed by 50 and 100 μM of NAR at the RNA level (P < 0.05). M2 macrophage markers (Arg-1, CD206, IL-10, IL-13) were markedly decreased in M1 macrophages (P < 0.05), however NAR attenuated the reduction of Arg-1 at 100 μM (P < 0.05) but not others, indicating that NAR may partially repolarize M1 macrophages toward M2. On the other hand, NAR suppressed M2 marker genes (Arg1, CD206, IL-13) in M2 macrophages, however there was no effect on protein level of CD206 and pSTAT6 (IL4 downstream signal transducer), suggesting that NAR did not promote M2 macrophage polarization. NAR did not improve M1 macrophage phenotypes (i.e., induction of iNOS, phosphorylation of p65, NLRP3 inflammasome activation), however NAR markedly suppressed the nuclear localization of p65 induced by LPS/IFNγ (P < 0.05).

Conclusions: In conclusion, NAR suppressed M1 polarization through inhibition of p65 nuclear translocation and repolarized M1 partly toward M2 macrophage. To understand the mechanism of NAR in this context, further studies are needed.

Funding Sources: 21st Century Endowed Chair in Human Environmental Sciences, University of Arkansas