D2.138 - The Effect of Human Milk Oligosaccharides 2’FL and 3FL on Macrophage polarization

Macrophages play a central role in both viral infections and allergic diseases. Human milk oligosaccharides (HMOs) may be able to modulate the macrophage phenotype. A macrophage polarization model for M1 and M2a macrophages was set up to study the effects of 2’FL and 3FL on macrophage phenotype. 

Monocytes were purified from healthy donor peripheral blood mononuclear cells (PBMCs), using magnetic bead positive selection. Monocytes were differentiated with 100 ng/mL M-CSF for 6 days to induce differentiation into naïve macrophages (M0). After 6 days, stimuli were added to induce polarization towards M1 (20 ng/mL LPS and/or 100 ng/mL IFNγ) and M2a (20 ng/ml IL4) subtypes. Cells were harvested after 48h and analyzed using flow cytometry (CD14, CD80, CD86, CD206 (mannose receptor), CD209 (DC-SIGN)). Supernatant was collected after 24h and 48h and analyzed with ELISA (TNFα and CCL22). Moreover, the effect of 0.01% and 0.05% 2’FL or 3FL on the polarization was studied.

The combination of LPS+IFNγ significantly induced the expression of CD80 and CD86 (in ±50% of cells, N=7). CD206 and CD209 expression remained low in the CD80⁺CD86⁺ cells, a phenotypic characteristic of M1 macrophages. Preliminary results also indicate an increase in TNFα secretion under LPS+IFNγ conditions (N=3). Naïve macrophages exposed to IL4 exhibited a CD80^-CD86⁺ phenotype, of which ±85% expressed CD209, indicative of M2a macrophage polarization (N=7). The expression of CD206 increased in ±50% of CD80^-CD86⁺ cells. Although there was considerable donor variation in CD206 expression, in general all CD206+ cells were also CD209+. Additionally, CCL22 secretion was increased under IL4 conditions (N=3). Furthermore, 2’FL and 3FL did not affect M1 macrophage phenotype. However, a trend towards increased CD206 MFI, CD209 MFI , %CD206⁺ cells and/or %CD206⁺CD209⁺ M2a macrophages was observed with 0.01% 2’FL or 3FL (all P≤ 0.10, N=3). 

The polarization protocol yielded phenotypically distinct M1 and M2a macrophages. While, 2’FL and 3FL did not significantly affect M1 marker expression, there was a trend towards upregulation of CD206 in M2a macrophages. Therefore, the frequency of M2a macrophages expressing both mannose receptor (CD206) and DC-SIGN (CD209) tended to increase in response to 2’FL and 3FL exposure.