Pro-Inflammatory Effects of Platelet Transfusions in Newborn Mice with and without Underlying Inflammation
Authors: Davenport Patricia E , Nolton Emily , Feldman Henry , Liu Zhi-Jian , Sola-Visner Martha 
Published: November 2021

Abstract
Platelet transfusions (PTx) are frequently given to thrombocytopenic preterm neonates at higher platelet count (PC) thresholds than those used in adults, in an attempt to reduce their bleeding risk. However, in the largest randomized trial of neonatal PTx thresholds, infants transfused at a higher PC threshold had a significantly higher mortality and/or major bleeding compared to infants transfused at a lower threshold. Since platelets carry multiple cytokines and chemokines, and since activated adult platelets may have a higher ability to interact with immune cells than neonatal platelets (due to their higher P-selectin expression levels), we hypothesized that the deleterious effects of PTx in neonates would be related to pro-inflammatory effects. We further hypothesized that the effects of PTx on the systemic inflammatory response would be different in thrombocytopenic neonates with non-inflammatory conditions (e.g., intrauterine growth restriction, drugs, genetic syndromes) compared to neonates with underlying inflammation (e.g., sepsis, necrotizing enterocolitis). To test the effects of PTx in the absence of inflammation, we transfused healthy post-natal day 10 (P10) C57BL/6 pups with washed platelets (5x10 7/g, isolated from adult C57BL/6J mice or eGFP+ mice) or with Tyrode's buffer (TY control). Blood was collected via terminal bleed 2h, 4h, and 6h after transfusion, and plasma was separated for quantification of 31 pro- and anti-inflammatory cytokines by multiplex (n=5-10 mice per group/timepoint). Two hours after PTx, the transfused mice exhibited significantly higher levels of G-CSF, IL-1, IL-1, IL-6, IL-17, KC (CXCL1) and MCP-1 compared to controls, with the most striking increases observed in IL-6 (928±19 vs. 135±36 pg/dL, p&lt;0.001) and KC (1201±239 vs 371±77 pg/dL, p=0.001). At 4h post-transfusion, the levels of most cytokines were decreasing, with the exception of G-CSF (1940±276 vs. 825±126 pg/dL, p=0.003), MCP-1 (185±39 vs. 58±14 pg/dL, p=0.003), and IL-17 (2.12±1 vs. 0.66±0.3 pg/dL, p=0.002), which peaked at four hours. All cytokines were decreasing by 6h. Next, to model neonates with inflammatory conditions, we injected P10 pups with lipopolysaccharide (LPS) IP at a sub-lethal dose (1µg/g), which induced mild weight loss, thrombocytopenia (~ 50% drop in PC), and leukopenia followed by leukocytosis. Two hours after LPS injection, pups were transfused with washed platelets from adult C57BL/6 mice or TY (as above). Blood was obtained by terminal phlebotomy 4h, 8h or 18h post LPS injection and plasma was separated and stored for cytokine quantification by multiplex. 4h after LPS, PTx pups had significantly higher levels of leukemia inhibitory factor (LIF, a member of the IL-6 family) compared to TY controls (35±6 pg/mL vs. 17±3.9 pg/mL, p&lt;0.01). At both 4 and 8h, IL-6 and G-CSF levels were extremely high and at or above the upper limit of the standard curve in both groups. By 18h post-LPS, the majority of cytokines had decreased to near-normal levels in TY control pups, while IL-6, IL-5, KC (CXCL1) and IL-10 remained significantly elevated in PTx mice (IL-6: 601±114 vs. 187±38 pg/mL, p=0.0007; IL-5: 659±257 vs. 486±191 pg/mL, p=0.01; KC: 4569±1370 vs. 2686±827 pg/mL, p=0.04; IL-10: 729±283 vs. 330±131 pg/mL, p=0.009). Since IL-10 is an anti-inflammatory cytokine, we also evaluated the relation of IL-6 to IL-10 in PTx vs. TY control mice. This analysis showed that IL-6 levels were 2.3 times higher for any given IL-10 level in pups who received PTx compared to controls. In conclusion, our findings suggest that platelet transfusions induce an inflammatory response in newborn mice without underlying inflammation, characterized mostly by elevations in IL-6, G-CSF and KC. In newborn pups with underlying sub-lethal inflammation, platelet transfusions seem to prolong the inflammatory response. These observations may provide an explanation for the increased morbidity and mortality in human neonates receiving liberal PTx. Studies to identify the mechanisms through which platelets induce these responses are ongoing.

Disclosures
No relevant conflicts of interest to declare.