Microenvironmental conditions and serum availability alter primary human macrophage NF-κB inflammatory response and...

Area of Science:

• Immunology
• Cell Biology

Background:

• Macrophages are crucial for innate immunity and widely used in vitro to study immune signaling.
• Optimal in vitro culturing methods for macrophages lack consensus, raising questions about culture condition impacts on experimental outcomes.

Purpose of the Study:

• To compare the effects of different culture media compositions on Toll-like receptor 4 (TLR4)-mediated inflammatory activity in primary human monocyte-derived macrophages (hMDM).
• To investigate how culture conditions influence macrophage morphology, phagocytosis, and transcriptional profiles.

Main Methods:

• Primary hMDM were cultured in media with or without fetal bovine serum (FBS), including DMEM, RPMI, and Macrophage-Serum Free Medium (M-SFM).
• Lipopolysaccharide (LPS)-mediated responses were assessed via NF-κB activation, cytokine/chemokine secretion, and phagocytosis assays.
• Morphological and transcriptional analyses were performed on unstimulated hMDM.

Main Results:

• M-SFM cultures showed muted LPS-induced NF-κB activation, cytokine/chemokine secretion, and reduced phagocytosis compared to DMEM and RPMI.
• FBS supplementation enhanced cytokine secretion but also increased baseline secretion, indicating a pro-inflammatory phenotype.
• FBS-free and M-SFM conditions induced distinct transcriptional profiles, affecting cell cycle and lipid homeostasis pathways.

Conclusions:

• DMEM and RPMI media yield comparable morphologic and functional outcomes in hMDM, with minor differences.
• M-SFM significantly alters macrophage inflammatory responses and morphology, leading to a muted inflammatory phenotype.
• The in vitro microenvironment critically influences human macrophage inflammatory outcomes, underscoring its importance in experimental design.