Milk modulates macrophage polarization in vitro

Abstract

Objective: Milk holds an anti-inflammatory response that is particularly important to protecting infants against necrotizing enterocolitis. Milk might also exert anti-inflammatory effects in adulthood, including the oral cavity where macrophages of the oral mucosal control innate immunity defense. It remains unknown, however, whether milk can modulate the local inflammatory response by affecting the polarization of macrophages.

Material and methods: To determine whether pasteurized human milk and pasteurized cow milk can provoke macrophage polarization, murine bone marrow macrophages and RAW264.7 cells were exposed to human saliva or the inflammatory cytokines IL1β and TNFα. Activation of pro-(M1) inflammatory response is indicated by the expression of IL1 and IL8. To determine polarization towards a M2 phenotype, the expression of arginase 1 (ARG1) and chitinase-like 3 (Chil3) was determined by reverse transcriptase PCR and immunoassay. Western blot was done on phosphorylated p38 and JNK.

Results: Aqueous fractions of human milk and cow milk from different donors, respectively, significantly decreased the inflammatory response of primary macrophages and RAW264.7 cells when exposed to saliva or IL1 and TNFα. Similar to IL4, human milk and cow milk caused a robust expression of ARG1 and Chil3 in primary macrophages. The polarization of macrophages by pasteurized milk occurred independent of the phosphorylation of p38 and JNK.

Conclusion: These data suggest that pasteurized milk, independent of the origin, can cause the polarization of macrophages from a pro-inflammatory M1 towards a pro-resolving M2 phenotype. Thus, milk might have a protective role for the oral cavity by modulation of the macrophage-based innate immune system.

Keywords: Inflammation; Macrophages; Milk; Mucositis; Oral; Polarisation.

Fig. 1

Milk suppress saliva-induced inflammation in primary macrophages and RAW264.7 cells. Murine bone marrow-derived macrophages (A, C) and RAW264.7 cells (B, D) were exposed to 5% saliva with and without 5% aqueous fractions of pasteurized human (HM; A, B) and cow milk (CM; C, D). Expression of inflammatory genes is indicated in percentage (%) compared to stimulated controls (100%). Dot-blots represent independent experiments. p-values are based on a Wilcoxon matched-pairs signed rank test.

Fig. 2

Milk suppress IL1β-TNFα-induced inflammation in primary macrophages and RAW264.7 cells. Murine bone marrow-derived macrophages (A, C) and RAW264.7 cells (B, D) were exposed to IL1β-TNFα with and without 5% aqueous fractions of pasteurized human (HM; A, B) and cow milk (CM; C, D). Expression of inflammatory genes is indicated in percentage (%) compared to stimulated controls (100%). Dot-blots represent independent experiments. p-values are based on a Wilcoxon matched-pairs signed rank test.

Fig. 3

2FL does not suppress saliva or IL1β-TNFα-induced inflammation in primary macrophages and RAW264.7 cells. Murine bone marrow-derived macrophages and RAW264.7 cells were exposed to (A) 5% saliva or (B) IL1β-TNFα with and without 1% 2FL. Expression of IL1 is indicated in x-fold change of stimulated controls. Dot-blots represent independent experiments. p-values are based on a Wilcoxon matched-pairs signed rank test.

Fig. 4

Milk but not 2FL modulate saliva-induced phosphorylation of p38 and JNK in RAW264.7 cells. Serum-starved RAW264.7 cells were exposed to (A) 5% aqueous fractions of pasteurized human (HM) and cow milk (CM) or (B) 2FL for 10 min before being exposed to saliva for 25 min. Targets were detected by antibodies raised against phospho-ERK (pERK), phosphor-p38 (pp38) and phospho-JNK (pJNK).

Fig. 5

Milk increases ARG1 and Chil3 in primary macrophages and RAW264.7 cells. Murine bone marrow-derived macrophages (A, C) and RAW264.7 cells (B, D) were exposed to IL4, 5% aqueous fractions of pasteurized human (HM), and cow milk (CM). Expression of M2 marker genes is indicated in x-fold change of unstimulated control. Dot-blots represent independent experiments. p-values are based on a Wilcoxon matched-pairs signed rank test. (E) Increased arginase 1 in RAW264.7 cells was confirmed by Western blot.

Fig. 6

Milk at 10% does not affect the viability of in primary macrophages and RAW264.7 cells. Murine bone marrow-derived macrophages (A, B) and RAW264.7 cells (C, D) were incubated overnight with 1 to 100% pasteurized human (A, C; HM), and cow milk (B, D; CM). MTT conversion into solubilized formazan crystals was determined on a photometer. Mean values from at least four independent experiments are expressed as percentage of optical density in the treatment groups normalized to unstimulated control values.