Red Blood Cell-Conditioned Media from Non-Alcoholic Fatty Liver Disease Patients Contain Increased MCP1 and Induce TNF-α Release

Abstract

BACKGROUND: Non-Alcoholic Fatty Liver Disease (NAFLD) constitutes a global pandemic. An intricate network among cytokines and lipids possesses a central role in NAFLD pathogenesis. Red blood cells comprise an important source of both cytokines and signaling lipids and have an important role in the molecular crosstalk during immunometabolic deregulation. However, their role in NAFLD has not been investigated in deep. METHODS: Conditioned media from erythrocytes derived from 10 NAFLD patients (4 men, 6 women, aged 57.875+-15,16) and 10 healthy controls (4 men, 6 women, aged 39.3+-15.55) were produced and used for the analysis of 9 cytokines (IFN-γ, TNF-α, CCL2, CCL5, IL-8, IL-1β, IL-12p40, IL-17, MIP-1β), 2 signaling lipids (Sphingosine 1-phosphate and Lysophosphatidic Acid), and cholesterol. Their effect on the cytokine profile released by RAW 264.7 macrophages was also studied. RESULTS: Erythrocytes from patients with NAFLD augmented the levels of MCP1 in the growth medium in comparison to the erythrocytes derived from healthy controls (37+-40 pg/ml vs 6.51+-5.63). No statistically significant differences were found between patients and healthy controls with regard to S1P, LPA, cholesterol and 8 other cytokines. TNF-a release by RAW 264.7 cells was increased after incubation with patient-derived erythrocyte conditioned medium compared to medium without RAW 264.7 cells from either healthy of NAFLD subjects. CONCLUSIONS: Erythrocytes could contribute to the liver infiltration by monocytes and to the activation of macrophages, partially due to release of CCL2, in the context of NAFLD.

Non-alcoholic steatohepatitis patients exhibit reduced CD47, and increased sphingosine, cholesterol and MCP1 levels in the erythrocyte membranes

“…MCP1 levels in erythrocyte membranes of NASH patients were slightly increased, albeit to marginally non-statistically significant levels. However, increased membrane bound MCP1, in combination to other stimulatory factors, like ATP acting on P2X7 (unpublished results), can promote high MCP1 release from NASH patients’ erythrocytes 14 .…”

“…Interestingly, erythrocyte sphingolipid metabolism can result in the formation of sphingosine 1-phosphate (S1P) 9 , a bioactive lipid mediator, implicated in the pathogenesis of NASH 33 . It has previously been shown that erythrocyte membranes of NASH patients, contain decreased levels of sphingomyelin 34 , and that erythrocyte-conditioned media do not contain elevated S1P 14 . Hence, we wondered, if there is sphingosine accumulation in the erythrocyte membrane in the context of NASH.…”

“…In addition, chemotactic molecules, like MCP1, can participate in efferocytosis, functioning as “find me” signals 23 . Indeed, increased MCP1 release from red blood cells of NASH patients 14 has been reported. In addition to the “find me”, “eat me”, and “do not eat me” signals, phagocytized cells also release “goodbye” signals: metabolites that regulate cellular function 16 .…”

“…Since, cholesterol accumulation contributes to the inflammatory process of NASH, we wondered whether erythrocyte membranes of NASH patients contain increased cholesterol levels. A previous study showed that erythrocytes from NASH patients do not release increased cholesterol to the growth media 14 . Hence, our results here of increased membrane cholesterol could indicate that increased efferocytosis of erythrocytes in the liver of NASH patients could result in augmented cholesterol disposition in Kupffer cells.…”

“…It was recently shown that erythrocytes from NASH patients release the chemokine MCP1, without significant changes in the release of other cytokines and of the signaling lipids, sphingosine 1 phosphate (S1P) and lysophosphatidic acid (LPA). This event was followed by increased TNF-α release from macrophages RAW 264.7 14 .…”