Pro-Inflammatory Effects of Platelet Transfusions in Newborn Mice with and without Underlying Inflammation

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<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<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.

Platelets in the neonate: Not just a small adult

“…From an immunologic perspective, the available data suggest that adult platelets, which express more P‐selectin upon activation and more effectively release their granule content, could be more proinflammatory than neonatal platelets (Figure 1). In support of a potential proinflammatory effect of platelet transfusions in neonates, we recently observed that transfusing healthy C57BL/6J (wild type [WT]) mice on postnatal day 10 (P10) with washed adult platelets resulted in significant increases in interleukin (IL)‐6 and granulocyte colony‐stimulating factor levels, among other cytokines, that were most pronounced 2 and 4 hours after transfusion, respectively 57 . Since platelet transfusions are frequently given to neonates with underlying inflammatory conditions, such as sepsis, we also examined the effects of transfusing adult WT platelets into WT P10 pups following a dose of lipopolysaccharide (LPS).…”

“…Interestingly, transfused mice also had higher levels of the anti-inflammatory cytokine IL-10, but when the correlation between the two was examined, at any level of IL-10, IL-6 levels were 2.3-fold higher in transfused compared to nontransfused littermates, suggesting that platelet transfusions prolonged and amplified the LPSinduced inflammation in newborn mice. 57 Recent work presented ). This resulted in greater monocyte migration toward CCL2 (the CCR2 ligand) in a transwell chamber after treatment with adult, but not neonatal, platelets.…”

“…In these experiments, 18 hours after LPS injection, the mice that had received a platelet transfusion had significantly higher IL‐6 levels than LPS‐injected, nontransfused littermates, which had nearly completely normalized their IL‐6. Interestingly, transfused mice also had higher levels of the anti‐inflammatory cytokine IL‐10, but when the correlation between the two was examined, at any level of IL‐10, IL‐6 levels were 2.3‐fold higher in transfused compared to nontransfused littermates, suggesting that platelet transfusions prolonged and amplified the LPS‐induced inflammation in newborn mice 57 . Recent work presented at the 2021 American Society of Hematology meeting by Maurya et al 58 also investigated the effect of adult versus neonatal platelets on monocyte inflammation and trafficking patterns.…”

“…In support of a potential proinflammatory effect of platelet transfusions in neonates, we recently observed that transfusing healthy C57BL/6J (wild type [WT]) mice on postnatal day 10 (P10) with washed adult platelets resulted in significant increases in interleukin (IL)‐6 and granulocyte colony‐stimulating factor levels, among other cytokines, that were most pronounced 2 and 4 hours after transfusion, respectively. 57 Since platelet transfusions are frequently given to neonates with underlying inflammatory conditions, such as sepsis, we also examined the effects of transfusing adult WT platelets into WT P10 pups following a dose of lipopolysaccharide (LPS). In these experiments, 18 hours after LPS injection, the mice that had received a platelet transfusion had significantly higher IL‐6 levels than LPS‐injected, nontransfused littermates, which had nearly completely normalized their IL‐6.…”

“…Interestingly, transfused mice also had higher levels of the anti‐inflammatory cytokine IL‐10, but when the correlation between the two was examined, at any level of IL‐10, IL‐6 levels were 2.3‐fold higher in transfused compared to nontransfused littermates, suggesting that platelet transfusions prolonged and amplified the LPS‐induced inflammation in newborn mice. 57 Recent work presented at the 2021 American Society of Hematology meeting by Maurya et al 58 also investigated the effect of adult versus neonatal platelets on monocyte inflammation and trafficking patterns. Using an in vitro coculture model of murine adult bone marrow monocytes and either neonatal or adult platelets, they found that monocyte inflammatory mRNAs (nitric oxide synthase 2, chemokine [C‐X‐C motif] ligand 1, chemokine [C‐C motif] ligand 2 [CCL2]) were increased in response to platelets regardless of developmental origin, but only coculture with adult platelets increased monocyte trafficking mRNA (C‐C chemokine receptor type 2 [CCR2]).…”