Underlying Inflammation Impacts Immune Modulation Following Exposure to Platelet Concentrates and Packed Red Blood Cells

Introduction

Transfusion of platelet concentrates (PC) and packed red blood cells (PRBC) are lifesaving treatments for patients suffering from thrombocytopenia, haematological disorders, severe trauma and cancer. Platelets play a central role in mediating coagulation and wound healing, inflammation and innate immune responses. Red blood cells are crucial for delivery of oxygen to surrounding tissues and translocation of carbon dioxide in the lungs. In Australia, PC and PRBC are routinely stored for up to 5- or 42- days respectively prior to transfusion. Storage related changes of PC and PRBC may contribute to adverse patient outcomes and include increased release of biological mediators (sCD40L, IL-8), partial platelet activation, loss of 2,3-diphosphoglycerate and ATP, change in oxygen dissociation and altered morphology. Despite the therapeutic benefits of PC and PRBC transfusion, clinical studies suggest transfusion-associated modulation of the recipient immune response contributes to adverse outcomes including infection and mortality. Underlying inflammation is an important factor contributing to the development of transfusion-related acute lung injury (TRALI), a pro-inflammatory response, whereas in vitro transfusion models largely report leucocyte suppression following exposure to PC and PRBC. The relationship between the level of underlying inflammation and the impact on cell function post-transfusion remains unclear. We investigated the relationship between inflammation and modulation of dendritic cell (DC) and monocyte phenotype following exposure to PC and PRBC.

Methods

An in-vitro transfusion model was used to investigate changes in DC and monocyte phenotype following exposure to PC-supernatants (PC-SN) or PRBC-supernatants (PRBC-SN) in co-culture with a range of lipopolysaccharide concentrations (LPS; 0 µg/mL (no LPS), 0.25 µg/mL, 0.5 µg/mL, 0.75 µg/mL, 1 µg/mL LPS) to model different levels of underlying inflammation. Based on results of recent randomised controlled trials, we used fresh PC- and PRBC- SN. Freshly collected whole blood was cultured with RPMI media and day 2 (D2) PC-SN or D2 PRBC-SN at doses representing a 1 (10%), or 2-3 (25%) unit transfusion. Golgi-plug (containing Brefeldin-a) was used to inhibit protein release to facilitate intracellular staining of cytokines (IL-6, IL-8, IL-10, IL-12, IL-1α, TNF-α, MIP-1α, MIP-1β, MCP-1, IP-10) from DC and monocytes using via flow cytometry. Changes in monocyte and DC cytokine and chemokine production at different concentrations of LPS were assessed by a repeated measures one-way ANOVA with Tukey's post-test (P<0.05).

Results

Culture with LPS alone resulted in a concentration dependent decrease in the production of most monocyte and DC cytokines. Modelling a low dose (1 unit) transfusion with D2 PC-or PRBC- SN resulted in a reduction in monocyte and DC cytokine production in the presence of 0.25 µg/mL and 0.5 µg/mL LPS. This effect was less evident when higher concentrations of LPS (0.75 µg/mL, 1 µg/mL) were used. When modelling a high dose (2-3 unit) transfusion, exposure to D2 PC-or PRBC- SN resulted in a predominant suppression of monocyte and DC responses at all LPS concentrations tested.

Conclusion

Our data provides evidence that the level of underlying inflammation differentially affects DC and monocyte responses following exposure to PC-SN or PRBC-SN. Dose in combination with level of underlying inflammation significantly impacts monocyte and DC responses which may contribute and increase the risk of transfusion-associated poor patient outcomes including infection.