Protein S Enhances the Phagocytosis of Phosphatidylserine-Positive Erythrocyte: Implications for the Erythrocytes Turnover

Introduction: Protein S (ProS) promotes the clearance of apoptotic cells (efferocytosis) exposing phosphatidylserine (PtdSer), by activating MerTK on macrophages (Anderson et al., Nat. Immunol. 2003). Klei et al (2020) showed that senescent erythrocytes transform into erythrocyte ghosts (eryghosts i.e erythrocytes lacking their hemoglobin content) before being engulfed by splenic red pulp macrophages. Eryghosts are observed in the splenic blood of healthy individuals but are virtually absent in the peripheral blood, while eryghosts with extensive PtdSer exposure were observed in the peripheral blood of patients with hemolytic anemia (Özpolat et al., 2022).

We hypothesized that ProS may play a role in erythrocytes turn-over enhancing the phagocytosis of PtSer-exposing eryghosts by RPM.

Materials and Methods: Erythrocytes were treated with ionomycin to induce PdSer exposure (iono-RBC), or transformed into eryghosts by osmotic shock, and characterized by imaging flow cytometry. PtdSer exposure was quantified by lactadherin staining. CFSE-labelled Iono-RBC or eryghosts were added to THP-1-derived macrophages in the presence or absence of ProS (50 or 100 nM) and of a MerTK inhibitor (UNC2025, 5µM). The percentage of fluorescent macrophages, thus having phagocytosed fluorescent iono-RBC or eryghosts, was quantified by flow cytometry. ProS effect on eryghosts phagocytosis was assessed using monocyte-derived macrophages (MDM) in a second model. PBMC were isolated from buffy coat and CD14-positive monocytes were selected. For MDM differentiation, purified cells were cultured with M-CSF for 5 days and IL-10 was added for 2 more days. Results are expressed as mean ± standard error of mean.

Results: A high proportion of iono-RBC and eryghosts exposed PdSer (76.4 ± 21.7%, n=41 and 53,8 ± 23,6% n=10, respectively). When incubated with iono-RBC, the percentage of fluorescent THP-1 macrophages significantly increased from 19.5 ± 0.6% to 26.8 ± 0.9% (p<0.05) and 31.9 ± 3.1% (p<0.01) with 50 and 100 nM of ProS respectively (n=3), and decreased from 33.4 ± 2.6% to 9.7 ± 1.0% (p<0.01) when the MerTK inhibitor was added (n=4). Imaging flow cytometry confirmed the ghost aspect in the brightfield, and eryghosts were positive for glycophorin A staining, confirming their erythroid nature. ProS enhanced the phagocytosis of eryghosts by THP-1 macrophages even more than that of iono-RBC, by an 11.8 ± 1.2-fold and a 1.9 ± 0.12- fold, respectively (n = 4, p<0.001). The prophagocytic effect of ProS on eryghosts was also confirmed using monocyte-derived macrophages. ProS significantly enhanced eryghosts phagocytosis from 11.2± 5.5% to 50.8 ± 9.3% (n=6, p<0.001).

Conclusion: As for nucleated cells, ProS enhances the phagocytosis of PdSer-exposing RBC by a MerTK-dependent mechanism. This discovery has implications in physiology, as ProS could be involved in erythrocyte turnover by enhancing the phagocytosis of eryghosts resulting from senescent erythrocyte hemolysis in the spleen. In hemolytic anemias such as sickle cell disease (SCD), circulating eryghosts have been described (Özpolat et al., 2022), probably due to hemolysis and functional hyposplenism. ProS levels are frequently moderately decreased in SCD patients, while ProS is detected on the surface of SCD patient's erythrocytes (Whelihan et al, 2016). ProS may be trapped on the surface of circulating eryghosts, worsening coagulation's ongoing activation. Hence, ProS may be an interesting target for future treatments in hemolytic anemias such as SCD.