Transfusion of Adult, but Not Neonatal, Platelets Promotes Monocyte Trafficking in Neonatal Mice

Abstract

Background: Thrombocytopenia is common in preterm neonates. Platelet transfusions are sometimes given to thrombocytopenic neonates with the hope of reducing the bleeding risk, however, there are little clinical data to support this practice, and platelet transfusions may increase the bleeding risk or lead to adverse complications. Our group previously reported that fetal platelets expressed lower levels of immune-related mRNA compared with adult platelets. In this study, we focused on the effects of adult versus neonatal platelets on monocyte immune functions that may have an impact on neonatal immune function and transfusion complications.

Methods: Using RNA sequencing of postnatal day 7 and adult platelets, we determined age-dependent platelet gene expression. Platelets and naive bone marrow-isolated monocytes were cocultured and monocyte phenotypes determined by RNA sequencing and flow cytometry. An in vivo model of platelet transfusion in neonatal thrombocytopenic mice was used in which platelet-deficient TPOR (thrombopoietin receptor) mutant mice were transfused with adult or postnatal day 7 platelets and monocyte phenotypes and trafficking were determined.

Results: Adult and neonatal platelets had differential immune molecule expression, including Selp. Monocytes incubated with adult or neonatal mouse platelets had similar inflammatory (Ly6C^hi) but different trafficking phenotypes, as defined by CCR2 and CCR5 mRNA and surface expression. Blocking P-sel (P-selectin) interactions with its PSGL-1 (P-sel glycoprotein ligand-1) receptor on monocytes limited the adult platelet-induced monocyte trafficking phenotype, as well as adult platelet-induced monocyte migration in vitro. Similar results were seen in vivo, when thrombocytopenic neonatal mice were transfused with adult or postnatal day 7 platelets; adult platelets increased monocyte CCR2 and CCR5, as well as monocyte chemokine migration, whereas postnatal day 7 platelets did not.

Conclusions: These data provide comparative insights into adult and neonatal platelet transfusion-regulated monocyte functions. The transfusion of adult platelets to neonatal mice was associated with an acute inflammatory and trafficking monocyte phenotype that was platelet P-sel dependent and may have an impact on complications associated with neonatal platelet transfusions.

Keywords: P-selectin; blood platelets; mice; monocytes; platelet transfusion.

Figure 1.

Adult and neonatal mouse platelets have different immune molecule expression. A-D) P7 and adult platelet mRNA were isolated for RNA-seq. A) Heat Map and B) PCI plot of platelet mRNA expression demonstrated differential mRNA expression. C-D). Pathway analysis. Adult platelets were enriched for immune related transcripts compared to P7 platelets. E-G) Validation of RNA-seq. E) P7 and adult mouse plasma β2M were measured by ELISA. Adult plasma had more β2M compared to P7 plasma. F) Adult and P7 platelets were stimulated with thrombin and surface P-selectin determined by flow cytometry. Adult platelets had more P-selectin compared to P7 platelets. G) Human cord blood plasma had less β2M compared to adult blood plasma as determined by ELISA (mean ± SEM, Student t-test).

Figure 2.

Adult and P7 Platelets Induce Different Monocyte Phenotypes. A-D) Mouse BMM were co-incubated with adult or P7 mouse platelets for 4 hrs and mRNA expression was assessed by RNA-seq. A) Heat Map, and B) PCI of monocyte mRNA indicated that platelets induced monocyte gene expression in an age dependent manner. C-D) Top 30 monocyte gene expression pathways C) upregulated and D) downregulated by adult vs P7 platelets. E) Confirmation of RNA-seq. Monocytes were co-incubated with adult or P7 platelets and 4 hrs later gene expression was determined by qRT-PCR. Adult platelets increased Ccr2, Ccr5, and Ccl2 more than P7 platelets (mean ± SEM, 1-way ANOVA Kruskal-Wallis test with Dunn’s multiple comparison ).

Figure 3:

Adult platelets induce a trafficking monocyte phenotype in vitro. Primary mouse BMM were co-incubated with adult or P7 platelets and 4 hrs later monocyte CD41, Ly6C, CCR2 and CCR5 expression were determined. A) Adult platelets formed significantly more PMAs compared to P7 platelets, but B) induced similar inflammatory monocyte phenotypes. C-D). Adult, but not P7, platelets induced a trafficking monocyte phenotype. Adult platelets increased C) CCR2 and D) CCR5 expression compared to P7 platelets (mean ± SEM, 1-way ANOVA with Bonferroni correction).

Figure 4.

Adult, but not P7 platelet transfusions, induced a trafficking monocyte response in vivo. P14 TPOR^−/− mice were transfused with PBS, adult platelets, or P7 platelets and 4 hrs later blood was collected. A) Circulating PMAs were quantified by flow cytometry and adult platelet transfusions formed more PMAs in vivo. B) Adult and P7 platelets both induced an inflammatory monocyte phenotype (Ly6C^hi). C-D) Adult, but not P7, platelet transfusions induced a trafficking monocyte phenotype including increased C) CCR2 and D) CCR5 expression (mean ± SEM, 1-way ANOVA with Bonferroni correction).

Figure 5:

P-selectin-PSGL1 interactions mediate adult platelet induced trafficking monocyte phenotype. Adult or P7 platelets were co-incubated with monocytes with IgG or anti-PSGL1 Ab. 4 hrs later PMAs, Ly6C, CCR2 and CCR5 expression were determined. A) Adult platelet associated PMAs were reduced and B) Ly6C was unchanged by anti-PSGL1 Ab. C-D) Adult platelets increased CCR2 and CCR5 compared to P7 platelets, which was decreased by anti-PSGL1 antibody. E-H) Monocyte trafficking in vitro. Adult or P7 platelets and monocytes were co-cultured in the top of a transwell chamber with control IgG or anti-PSGL1. CCL2 or CCL5/RANTES were placed in the bottom chamber and monocyte migration determined. E-F) Adult platelets induced monocyte trafficking to CCL2 which was decreased by anti-PSGL1 Ab. E) Representative Image and F) Quantification. G-H) Adult platelets induced monocyte trafficking to CCL5, which was reduced by anti-PSGL1 Ab. G) Representative Image and H) Quantification (mean ± SEM, 1-way ANOVA with Bonferroni correction). Scale bar =200 μm.

Figure 6:

P-selectin-PSGL1 mediated monocyte trafficking in vivo. P14 TPOR^−/− mice were transfused with adult or P7 platelets in the presence IgG or anti-PSGL-1 Ab. 4hrs later A) CD41, B) Ly6C, C) CCR2 and D) CCR5 expression were determined. Adult platelet induced changes were inhibited by anti-PSGL-1 Ab. E-F) Mice were also transfused and treated with the CCR2 ligand CCL2 via an IP injection 4 hrs after platelet transfusions. 24 hrs later the number of migrated E) monocytes and F) neutrophils was determined by peritoneal lavage and flow cytometry. Adult platelet transfusions greatly increased monocyte and neutrophil peritoneal migration, which was blunted by anti-(mean ± SEM, 1-way ANOVA with Bonferroni correction).