Innate non immune and immune cells including macrophages and

Innate non-immune and immune cells, including macrophages and dendritic cells, utilize pattern recognition receptors (PRRs) to detect PAMPs (Takeuchi and Akira, 2010). In addition, they can recognize damage-associated molecular patterns (DAMPs), which derive from endogenous molecules modified or released following cellular stress or damage (Krishnaswamy et al., 2013). Many PRRs exist, including Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), each sensing discrete types of stimuli. For example, TLR4 recognizes the Gram-negative bacterial LY2228820 component lipopolysaccharide (LPS) whereas NLR containing pyrin domain 3 (NLRP3) is activated by chemically diverse PAMPs and DAMPs including protein aggregates, insoluble crystals, and bacterial pore-forming toxins. Thus, identifying PRRs that promote storage-induced alloimmunization can inform the identity of potential RBC DAMPs. Many NLRs, including NLRP3, form a multi-molecular complex, termed an ‘inflammasome’, that results in NLR oligomerization, caspase-1 activation and enzymatic release of IL-1β and IL-18, which contribute to T cell activity (Latz et al., 2013) (Liu et al., 2013). NLRP3 is activated by a wide array of DAMPs, including extracellular ATP (Mariathasan et al., 2006), which is released from stored RBCs (Dern et al., 1967). Hemozoin, a byproduct of heme catabolism in malarial infection was also shown to activate NLRP3 (Shio et al., 2009). More recently, Dutra et al. demonstrated that inflammation caused by iron-containing heme is critically regulated by NLRP3 and downstream inflammasome components (Dutra et al., 2014). Therefore, we hypothesized that activation of NLRP3 or other NLR inflammasomes by RBC-associated DAMPs promotes the alloimmune response to sterile RBC transfusion. Using genetically modified mice lacking either Nlrp3 or multiple downstream common inflammasome response elements, we tested whether the NLRP3 inflammasome was a sensor of stored RBCs. Our data unambiguously excludes a critical role for the NLRP3 inflammasome or any caspase-1 or -11 dependent inflammasome in alloimmunization to stored HOD RBCs.
Materials & Methods
Results
Discussion Identifying patients with an elevated risk of forming alloantibodies to non-ABO RBC antigens could lead to directed preventative measures, including antigen matching, or therapeutic interventions that mitigate the alloimmune response (Hendrickson et al., 2014). In light of the clinical significance of RBC alloimmunization, progress in this area could substantially decrease associated adverse events, including hemolytic transfusion reactions and general morbidity and mortality (Nickel et al., 2016). However, to develop criteria for biomarkers or cytokine profiles that have predictive value, mechanisms underlying the alloantibody-forming adaptive immune responses must be determined. Using the mouse transfusion model of HOD RBCs, we and others have shown that refrigerated RBC storage increases the inflammatory (Hod et al., 2010) and alloantibody (Hendrickson et al., 2010) responses of recipients. In parallel, a clinical study has shown that the age of transfused RBCs is associated with alloimmunization in patients with sickle cell disease (Desai et al., 2015). The pronounced increase in alloantibodies in our studies occurred following 7–14days of murine RBC storage. Significant differences have been noted in the risk of alloantibody formation between 7 and 35days of human RBC storage (Desai et al., 2015). In addition, a recent study by Veale et al. found that the susceptibility of stored human RBCs to in vitro erythrophagocytosis increased significantly with storage duration (Veale et al., 2014). We conclude that DAMPs are formed and contained within intact murine RBCs during the second week of storage, and hypothesize that DAMPs may also be formed in human RBCs the week prior to outdate. This finding is supported by a prior study demonstrating that intact HOD RBCs, but not RBC lysate or RBC ghosts generated from stored cells, induce inflammatory cytokine production following transfusion (Hod et al., 2010). Further studies addressing metabolic changes within intact stored RBCs could potentially identify immunogenic DAMPs.