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Author contributions
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Introduction With 15 million units transfused per year, red blood cell (RBC) transfusion is the most common procedure performed in United States hospitals (Pfuntner et al., 2013). Exposure to numerous foreign non-ABO RBC antigens during transfusion can induce production of BQ-788 sodium salt manufacturer to “minor” blood group antigens. As a result, approximately 3% of hospitalized patients (Fluit et al., 1990) and as many as 30–50% of patients with sickle cell anemia (Vichinsky et al., 1990), develop alloantibodies against these RBC antigens. Subsequent exposure to the offending RBC antigen may result in potentially fatal hemolytic transfusion reactions, which is a leading cause of transfusion-associated mortality (FDA, 2014). Although such reactions can be avoided by providing antigen-negative RBC units, patients with rare or multiple RBC alloantibodies may experience clinically significant delays in transfusion, as laboratories search for rare compatible blood products (Ryder et al., 2014). To mitigate these adverse effects of alloimmunization, there is heightened interest in identifying genetic and environmental factors that influence the likelihood of alloantibody formation. Multiple factors place patients at higher risk of alloimmunization. These include genetic predisposition (e.g., HLA type), transfusion burden, and co-incident inflammation (Higgins and Sloan, 2008; Telen et al., 2015; Tatari-Calderone et al., 2009). Recipient inflammatory status significantly influences the frequency of alloimmunization; an increased incidence of alloimmunization was reported in patients with inflammatory bowel disease, autoimmune disease, febrile transfusion reactions, and sickle cell-mediated acute chest syndrome (Ramsey and Smietana, 1995; Fasano et al., 2015; Papay et al., 2012; Yazer et al., 2009). In murine RBC transfusion models, exposure to pro-inflammatory pathogen-associated molecular patterns (PAMPs) promotes RBC alloimmune responses (Bao et al., 2009; Hendrickson et al., 2006; Hendrickson et al., 2007; Elayeb et al., 2016). This suggests that RBC alloimmunization, like most adaptive immune responses, depends on innate immune cell activation to initiate antigen presentation and the requisite T cell priming signals (Calabro et al., 2016; Krishnaswamy et al., 2013). However, the specific innate immune stimuli provided by transfused RBCs that regulate a T cell-dependent alloantibody response are not yet known. Recently, the species mismatch of CD47 on transfused xenogeneic sheep RBCs was shown to induce significant inflammation in murine recipients (Yi et al., 2015). However, in allogeneic RBC murine models, such a mismatch does not exist and CD47 expression does not significantly decrease on stored murine RBCs (Gilson et al., 2009). Nonetheless, other alloantigen-independent properties of donor RBCs, including length of refrigerated storage, can influence post-transfusion inflammation and immunogenicity (Ryder et al., 2014; Desai et al., 2015). During prolonged storage, RBCs undergo morphologic transformations, metabolic changes and oxidative damage, leading to increased post-transfusion clearance and decreased RBC viability (Bennett-Guerrero et al., 2007; Tinmouth et al., 2006). However, the cellular and molecular mechanisms, including the recognition of stored RBC factors, underlying these observations are poorly understood. To examine the effect of RBC storage on inflammatory responses and alloimmunization, we previously generated a mouse model of RBC storage that approximates human RBC storage (Gilson et al., 2009). Our studies demonstrated that transfusion of stored RBCs expressing a chimeric protein containing hen egg lysozyme, ovalbumin, and the human Duffy antigen (HOD), leads to accelerated RBC clearance, increased inflammatory cytokine production, and enhanced alloantibody production, compared to freshly collected RBCs (Hod et al., 2010; Hendrickson et al., 2010). Transfusion of stored RBCs also resulted in elevated levels of tissue iron in the spleen and liver and production of circulating non-transferrin bound iron (NTBI) in plasma. The inflammatory response was attributed to phagocytosis of intact damaged stored RBCs (i.e., extravascular hemolysis) (Hod et al., 2010). Although storage of mouse and human RBCs may have differential effects, a prospective study with human volunteers also demonstrated that transfusion of stored RBCs led to elevated NTBI and extravascular hemolysis (Hod et al., 2011). However, the mechanism by which innate immune cells sensed iron or other RBC products was not investigated.