Red Blood Cells As a Novel Mediator of Chronic Inflammation in Diet-Induced Obesity: Implications for Atherosclerosis

Red blood cell (RBC) extravasation contributes to the growth of atherosclerotic plaque, and is associated with plaque rupture. High Fat Diet (HFD) fuels systemic inflammation by promoting monocyte activation and their recruitment/transendothelial migration. RBC bind chemokines such as monocyte chemotactic protein-1 (MCP-1) via Duffy Antigen Receptor for Chemokines (DARC). While it was previously reported that HFD may impact RBC cholesterol content, very little is known about the impact – if any – of HFD on RBC function(s). Hence, we studied whether HFD affects biochemical and functional properties of RBC in ways potentially relevant to the progression of atherosclerosis.

Wild type (WT) C56BL6/J mice were fed either chow (10% total fat) or HFD (60% total fat) for ³12 weeks. MCP-1 levels were measured in plasma before and after the release of DARC-bound MCP-1 by heparin. Released MCP-1 was 1.5 fold higher in HFD RBC (12 weeks on diet, n=3) compared to chow RBC. Levels of intracellular reactive oxygen species (ROS) were measured by DCFH fluorescence using flow cytometry, and were increased in HFD RBC (17 weeks on diet, n=3) by ∼1.2 fold compared to chow RBC (p<0.05). We also observed a 1.5 fold increase in external phosphatidylserine (PS) in HFD RBC (12 weeks on diet, n=3) as revealed by Annexin V binding (flow cytometry). In vitro erythrophagocytosis was assayed by incubating thioglycollate-induced peritoneal macrophages from WT mice with calcein-AM labeled RBC obtained from mice on HFD (12 weeks on diet, n=3) or chow. A ∼1.5 fold (p<0.05) increase in phagocytosis was observed for HFD RBC compared to chow RBC. When HFD RBC served as a source of chemoattractant(s), we observed a ∼1.4 fold (p<0.05) increase (compared to chow RBC) in the number of monocytes undergoing transendothelial migration in a Boyden chamber assay. Experiments performed on RBC from LDLr −/− mice fed either HFD or chow for 12 weeks (n=3), displayed the fold changes analogous to those observed in RBC from WT mice with respect to ROS levels, transendothelial monocyte migration, and PS externalization but a significant increase in RBC phagocytosis of ∼1.7 fold (p<0.05). The increased PS exposure was associated with an increase in splenic uptake of RBC: calcein-AM labeled packed RBC (chow or HFD) were injected retroorbitally into WT mice maintained on chow and after 24 hrs, peripheral blood was removed by cardiac perfusion, spleens harvested, embedded in OCT media, and 10 mm sections were analyzed using Image J software. Macrophages located predominantly in splenic sinuses engulfed ∼3 fold more HFD RBC (12 weeks on diet, n=3) compared to chow RBC (p<0.05). Microarray analysis was then performed on peritoneal macrophages derived from HFD mice that were incubated with either HFD RBC or chow RBC for 4 hrs (results validated by qRT-PCR). Interestingly, several chemokines such as Il-1b, Ccl3, and Cxcl2 were upregulated in HFD macrophages that engulfed HFD RBC compared to those that engulfed chow RBC (3 fold, 3 fold, and 12 fold, respectively). RBC deformability, a crucial characteristic enabling RBC to traverse through narrow capillaries/splenic sinuses, was measured using ektacytometry. Deformability expressed as elongation index was decreased in HFD RBC compared to chow RBC, especially at intermediate shear stress (2 – 20 Pa). Finally, when freshly harvested aortic segments were incubated with packed RBC (HFD or chow), washed, and exposed to pre-labeled fluorescent WT murine macrophages, there was an enhancement of macrophage adhesion to the luminal endothelium when HFD RBC were used (∼3 fold, p<0.05).

We here show that HFD elicits an increase in surface levels of MCP-1 in RBC, which is associated with an enhanced macrophage adhesion to the intact vascular endothelium and increased transendothelial migration in vitro. Further, HFD increases ROS levels and PS externalization in RBC, and HFD RBC exhibit an enhanced splenic uptake in vivo. These effects of HFD on circulating RBC are likely to promote chronic inflammation, rendering RBC a heretofore unknown contributor to local as well as systemic changes that lead to atherosclerosis.

No relevant conflicts of interest to declare.