HAMLET a human milk protein‐lipid complex induces a pro‐inflammatory phenotype of myeloid cells

HAMLET is a protein‐lipid complex with a specific and broad bactericidal and tumoricidal activity, that lacks cytotoxic activity against healthy cells. In this study, we show that HAMLET also has general immune‐stimulatory effects on primary human monocyte‐derived dendritic cells and macrophages (Mo‐DC and Mo‐M) and murine RAW264.7 macrophages. HAMLET, but not its components alpha‐lactalbumin or oleic acid, induces mature CD14(low/–)CD83(+) Mo‐DC and M1‐like CD14(+)CD86(++) Mo‐M surface phenotypes. Concomitantly, inflammatory mediators, including IL‐2, IL‐6, IL‐10, IL‐12 and MIP‐1α, were released in the supernatant of HAMLET‐stimulated cells, indicating a mainly pro‐inflammatory phenotype. The HAMLET‐induced phenotype was mediated by calcium, NFκB and p38 MAPK signaling in Mo‐DCs and calcium, NFκB and ERK signaling in Mo‐M as inhibitors of these pathways almost completely blocked the induction of mature Mo‐DCs and M1‐like Mo‐M. Compared to unstimulated Mo‐DCs, HAMLET‐stimulated Mo‐DCs were more potent in inducing T cell proliferation and HAMLET‐stimulated macrophages were more efficient in phagocytosis of Streptococcus pneumoniae in vitro. This indicates a functionally activated phenotype of HAMLET‐stimulated DCs and macrophages. Combined, we propose that HAMLET has a two‐fold anti‐bacterial activity; one inducing direct cytotoxic activity, the other indirectly mediating elimination of bacteria by activation of immune cells of the myeloid lineage.