Down-regulation of platelet surface CD47 expression in Escherichia coli O157:H7 infection-induced thrombocytopenia

Abstract

Background: Platelet depletion is a key feature of hemolytic uremic syndrome (HUS) caused by Shiga toxin-producing Escherichia coli (STEC) infection. The mechanism underlying STEC-induced platelet depletion, however, is not completely understood.

Methodology/principal findings: Here we demonstrated for the first time that platelet surface expression of CD47 was significantly decreased in C57BL6 mice treated with concentrated culture filtrates (CCF) from STEC O157:H7. STEC O157:H7 CCF treatment also led to a sharp drop of platelet counts. The reduction of cell surface CD47 was specific for platelets but not for neutrophil, monocytes and red blood cells. Down-regulation of platelet surface CD47 was also observed in isolated human platelets treated with O157:H7 CCF. Platelet surface CD47 reduction by O157:H7 CCF could be blocked by anti-TLR4 antibody but not anti-CD62 antibody. Down-regulation of platelet surface CD47 was positively correlated with platelet activation and phagocytosis by human monocyte-derived macrophages. Furthermore, the enhanced phagocytosis process of O157:H7 CCF-treated platelets was abolished by addition of soluble CD47 recombinants.

Conclusions/significance: Our results suggest that platelet CD47 down-regulation may be a novel mechanism underneath STEC-induced platelet depletion, and that the interactions between CD47 and its receptor, signal regulatory protein alpha (SIRPalpha), play an essential role in modulating platelet homeostasis.

Figure 1. Platelet depletion in C57BL6 mice administered with concentrated culture filtrates (CCF) of STEC O157:H7 (strain 99G144).

Note that platelet number was rapidly decreased in mice treated with O157:H7 (strain 99G144) CCF (n = 8) compared to that in mice treated with saline (n = 6) or CCF from O157:H19 (strain 99A041) (n = 6). *, p<0.05, **, p<0.01.

Figure 2. Reduction of platelet surface CD47 expression in mice treated with CCF from STEC O157:H7 (strain 99G144).

A, CD47 expression on platelet surfaces measured by flow cytometry. Cells derived from CD47^−/− mice served as a negative control in CD47 immunofluorescence labeling and measurement by flow cytometry. B, CD47 expression levels on the surfaces of platelets (PLT), neutrophils (PMN), monocytes (MO), and red blood cells (RBC), respectively. Shiga toxin-negative strain 99A041 (O157:H19) was used as a control for STEC O157:H7 (strain 99G144). Data were presented as mean±SD of three independent experiments. *, p<0.05.

Figure 3. Reduction of CD47 surface expression level on isolated human platelets by STEC O157:H7 (strain 99G144) CCF.

A, strain 99G144 CCF but not O157:H19 CCF decreases CD47 surface expression. B, Reduction of CD47 expression levels by strain 99G144 CCF in the presence of various antibodies at concentration of 25 µg/ml each. Data were presented as mean±SD of three independent experiments. *, p<0.05.

Figure 4. Down-regulation of platelet surface CD47 expression level positively correlates with activation of platelets.

The adhesion of platelets to immobilized fibrinogen (FBG) (A) and SIRPα extracellular domain recombinant (SIRPα-GST) (B) was measured by an in vitro binding assay. BSA and GST were used as the controls for FBG and SIRPα-GST in binding assays, respectively. Data were presented as mean±SD of three independent experiments. *, p<0.05.

Figure 5. Enhanced phagocytosis of O157:H7 CCF-treated platelets by human monocytic-derived macrophages.

Percentage of phagocytosis was calculated as the fraction of macrophages with ingested platelets of the total number of macrophages analyzed in 6–8 random selected fields per slide. Soluble CD47 extracellular domain recombinant and GST only were added at 40 µg/ml each. Data were presented as mean±SD of three independent experiments. *, p<0.05.