Influence of red blood cells inducing hypoxic stress on lipid droplet formation in human monocytes.

Abstract

Introduction: Red blood cells (RBCs) are now recognized as active immunomodulatory cells capable of influencing innate immune responses. Monocytes, as central effectors of innate immunity, accumulate intracellular lipid droplets (LDs) upon activation — organelles established as inducible inflammatory platforms. The functional communication between hemoglobin-deficient erythrocytes (RBC⁻/Hb⁻) and monocyte lipid metabolism remains poorly characterized. This study aimed to assess ex vivo the impact of autologous hemoglobin-deficient RBCs on monocyte lipid metabolism, including total cellular cholesterol (tccCHOL), intracellular triglycerides (iTAG), and lipid droplet accumulation. Materials and Methods: Peripheral venous blood was collected from hemoglobindeficient patients. Monocytes were isolated by density gradient centrifugation and purified by selective plastic adherence. Autologous RBCs were recovered from the same gradient and resuspended in complete DMEM. Four experimental conditions were established. tccCHOL and iTAG were quantified by enzymatic colorimetric methods and normalized to total proteins. Lipid droplets were visualized by Oil Red O staining and quantified by corrected total cell fluorescence (CTCF) using Fiji/ImageJ and CellProfiler. Results: Total cellular cholesterol remained stable across all four conditions, with no statistically significant difference between groups. In contrast, intracellular triglyceride levels showed a markedly different profile. Lipid droplet accumulation, assessed by CTCF (Oil Red O), increased significantly in response to LPS and/or Hb stimulation, with a maximal effect observed under dual LPS+Hb+ stimulation. Conclusion: This study demonstrates that Hb-deficient RBCs modulate monocyte lipid metabolism by promoting lipid droplet accumulation, particularly under dual LPS+Hb+ stimulation, positioning these organelles as key players in innate inflammation in the context of hemoglobin deficiency.