Searching for a role of oxidative stress marker 4-hydroxynonenal in in vitro erythrophagocytosis

Background: 4-hydroxynonenal (4-HNE) is a by-product derived from lipoperoxidation of polyunsaturated fatty acids in cell membranes that acts as signalling molecule in physiological conditions but, it is also involved in the pathogenesis of several inflammatory-based diseases, in which oxidative stress plays an important role. Due to its endogenous nature, and its possible physiological role, 4-HNE was studied for a correlation between 4-HNE conjugate red blood cell (RBCs) membrane proteins and in vitro erythrophagocytosis. Objectives of the present study are: 1) to evaluate the possible presence of 4-HNE protein conjugates in surface of human healthy RBCs at different cell age; 2) to understand the relationship between 4-HNE and cell senescence 3) to demonstrate its possible role as a marker for physiological erythrophagocytosis. Methods: Separation of human whole blood from healthy donors by Percoll gradient allowed to collect RBC fractions of different densities which correlate with the time of permanence in circulation (from youngest to senescent cells). RBC fractions were tested for CD47 and 4-HNE protein conjugates by FACS to confirm the different cell age and to evaluate the presence of oxidative stress, respectively. Western Blot (WB) analysis with specific anti-4-HNE antibodies was necessary to understand which and how many RBC surface proteins are the most affected by 4-HNE binding. Erythrophagocytosis was performed feeding cultured primary human monocytes with the different RBC fractions and luminescence analysis was applied to quantify the amount of haemin in monocytes as measure for occurred erythrophagocytosis. Results: whole blood RBCs were separated in young, old, and very old cells characterized by increasing density and decreasing cell size and CD47 expression. Old and very old RBCs represented the minority of whole cell population (10% and 2%, respectively) and can be considered senescent considering the low CD47 expression compared to the young cells fraction. 4-HNE binds preferentially proteins of senescent RBCs. 4-HNE conjugates levels showed a significant difference between old RBC fractions and young RBCs both in the surface antigens and in the total membrane protein (p=0,04 and p=0,01, respectively). Of note, among cytoskeleton proteins, the spectrins showed a significant difference of bound 4-HNE conjugates between the oldest cells and the young ones reaching a 3-fold higher value in the oldest. Also, the erythrophagocytosis in vitro reveals the preferential removal of the oldest and senescent RBCs, the mostly modified by 4-HNE. Conclusion: A significant accumulation of 4-HNE protein conjugates on RBCs surface and in membrane was revealed during cell aging. The kinetics of accumulation differed a bit between cell surface antigens and membrane proteins, and involving the oldest fraction of the RBCs. Considering the senescent RBCs as a model for RBC elimination, presented results pave the way for pathomechanistic studies of diseases in which the removal of RBCs is involved, such as anaemia.