Red blood cell (RBC) transfusion exposes recipients to hundreds of unmatched minor RBC antigens. This exposure can lead to production of alloantibodies that promote clinically significant hemolytic events. Multiple studies have reported an increased frequency of RBC alloimmunization in patients with autoimmunity. However, cellular and molecular mechanisms that underlie autoimmunity-induced alloimmunization have not been reported. Patients with systemic lupus erythematosus (SLE) have a high frequency of alloimmunization and express a type 1 interferon (IFNα/β) gene signature. Thus, we utilized the pristane-induced lupus mouse model to test the hypothesis that inflammation in lupus promotes RBC alloimmunization, and to examine the potential role of IFNα/β. Intraperitoneal injection of pristane, a hydrocarbon oil, led to autoantibody production, glomerulonephritis, and pulmonary hemorrhage in wild type (WT) mice. Pristane treatment significantly induced serum IFNα and expression of multiple interferon-stimulated genes (ISGs) in peripheral blood and peritoneal fluid cells, including inflammatory macrophages. Following transfusion with allogeneic RBCs expressing the KEL glycoprotein, pristane-treated WT mice produced significantly elevated levels of anti-KEL IgM and anti-KEL IgG, compared to untreated mice. Pristane induced comparable levels of inflammatory cells and cytokines in mice lacking the IFNα/β receptor (IFNAR1 −/−) or the IFNα/β-inducing transcriptions factors (IRF3/7 −/−), compared to WT mice. However, pristane-treated IFNAR1 −/− and IRF3/7 −/− mice failed to produce ISGs and produced significantly lower levels of transfusion-induced anti-KEL IgG, compared to WT mice. Thus, pristane induction of a lupus-like phenotype promoted alloimmunization to the KEL RBC antigen in an IFNα/β-dependent manner. To our knowledge, this is the first examination of molecular mechanisms contributing to RBC alloimmunization in a model of autoimmunity. These results warrant further investigation of the role of IFNα/β in alloimmunization to other RBC antigens and the contribution of the IFNα/β gene signature to the elevated frequency of alloimmunization in patients with SLE.
Patients with sickle cell disease (SCD) have a high prevalence of RBC alloimmunization.However, underlying mechanisms are poorly understood. Given that proinflammatory type 1 interferons (IFNα/β) and interferon stimulated genes (ISGs) promote alloimmunization in mice, we hypothesized that IFNα/β may contribute to the increased frequency of alloimmunization in patients with SCD. To investigate this, expression of ISGs in blood leukocytes and peripheral blood mononuclear cells (PBMCs) of previously transfused SCD patients with or without alloimmunization and race-matched healthy controls were quantified, and IFNα/β gene scores were calculated. IFNα/β gene scores of SCD leukocytes and plasma cytokines were elevated, compared to controls (gene score, p < 0.01). Upon stimulation with IFNβ, isolated PBMCs from patients with SCD had elevated ISGs and IFNα/β gene scores (p < 0.05), compared to stimulated PBMCs from controls. However, IFNβ-stimulated and unstimulated ISG expression did not significantly differ between alloimmunized and non-alloimmunized patients. These findings indicate that patients with SCD express an IFNα/β gene signature, and larger studies are needed to fully determine its role in alloimmunization. Further, illustration of altered IFNα/β responses in SCD has potential implications for IFNα/β-mediated viral immunity, responses to IFNα/β-based therapies, and other sequelae of SCD.
Graphical AbstractHypothesis: Baseline type I interferon activity may contribute to variable COVID-19 progression in SCD. (Top) At early stages of SARS-CoV-2 infection, high baseline IFNα/β activity may contribute to the anti-viral response in patients with SCD. Recognition of damage-associated molecular patterns by pattern recognition receptors (PRRs) induces IFNα/β production. Heme released from hemolyzed sickle cells binds Toll-like receptor 4 (TLR4), which may induce IFNα/β in vascular endothelial cells. IFNα/β bind to the IFNα/β receptor (IFNAR) in neutrophils and other cells types, leading to production of MxA and other interferon-stimulated genes (ISGs). ISGs can directly inhibit viral replication and promote B cell production of neutralizing antibodies. The IFNα/β response is one of multiple responses, including production of IL-6, TNFα, and IL-1b, by innate and adaptive immune cells that have the potential to limit COVID-19 progression. (Bottom) In contrast, reduced or absent IFNα/β activity may increase susceptibility to viral infection, leading to airway epithelial cell death and COVID-19. Dashed lines indicate potentially connected pathways, while solid lines are supported by prior studies.
Background RBC alloimmunization is a clinically significant issue in transfusion medicine; patients with sickle cell disease have an increased risk of alloantibody production (30-50% of SS patients) compared to that of other hospitalized patients (3-10%). However, mechanisms underlying the increased frequency of alloimmunization in sickle cell patients are poorly understood. In previous studies, inflammation in the recipient has been shown to promote alloimmunization. Transfusion models, type 1 interferons (IFNα/β) and Interferon Stimulated Genes (ISGs) have been shown to promote alloimmunization in mice. Other studies have shown that patients with inflammatory autoimmune diseases express an IFNα/β signature which may contribute to the increased frequency of alloimmunization in these populations. One recent study reported significantly elevated ISGs in neutrophils as well as evidence that IFNα is upregulated in SS patients compared to controls. Given the chronic inflammatory state in SS patients, we sought to determine the role of PBMCs and whether they also expressed an IFN gene signature that contributes to the increased frequency of alloimmunization. Methods The expression of the ISG, myxovirus resistance protein 1 (MxA), was measured in the blood of SS patients with more patients with SS disease (SS, n=13) and race matched healthy controls (ββ, n=3) by whole blood immunoassay (ELISA). qPCR was performed on 5 previously established ISGs to determine an IFN score, a measure of overall gene expression, from whole blood and IFNβ stimulated PBMCs of SS patients (SS, n=15) and healthy race matched controls (ββ, n=5). A LEGENDplex™ Human Anti-Virus Response Panel assay was used to determine the expression of various type 1 IFNs, cytokines and ISGs in patients with SS disease (SS, n=15) and healthy race matched controls (ββ, n=5). Results SS patients had significantly elevated levels of MxA (mean ± standard error of the mean, SS MxA = 12.27 ng/mL ± 15.68) compared to control patients without SS (ββ MxA = 1.52 ng/mL± 0.26, p< 0.05) (Figure 1 A) . The Legendplex showed a significant increase in IL-6, IL-10 and the ISG, IP-10. (SS IP-10= 147.81 pg/mL ± 49.24) (ββ IP-10 = 68.85 pg/mL ±10.70, p<0.01) (Figure 1 B) Analyzing the 5 ISGs, we saw a trend towards a higher IFN score in patients with SS disease than healthy controls in whole blood; this difference was significant in PBMCs stimulated with IFNB (IFN Score SS = 20.76 ± 17.18 ,ββ = 0.00 ± 3.71 , p<0.01) (Figure 1 C). Discussion SCD is a complex disease with many environmental and genetic factors that play roles in the severity of the disease. Any number of these factors may influence the high rates of alloimmunization found in sickle cell patients. We found increased cytokines, ISGs and IFN scores in SS patients compared to healthy controls. These findings suggest the presence of an IFNα/β gene signature in patients with sickle cell disease. Due to the relatively small sample size, we are unable to determine a correlation between alloantibodies and MxA levels or high IFN scores with this cohort. Further studies will allow us to determine if the increased interferon gene signature plays a role in the increased alloimmunization burden that these patients experience. Disclosures No relevant conflicts of interest to declare.
Red blood cell (RBC) transfusion exposes recipients to hundreds of unmatched minor RBC antigens. This exposure can lead to production of alloantibodies that can cause potentially fatal hemolytic events. Prior studies have reported that inflammatory states, including autoimmune disease, in the transfusion recipient can promote alloimmunization. Recent studies reported that pro-inflammatory type 1 interferons (IFNα/β) regulated RBC alloimmunization in multiple mouse transfusion models. Interestingly, patients with systemic lupus erythematosus (SLE) have an increased frequency of alloimmunization and express an IFNα/β gene signature. Thus, we tested the hypothesis that IFNα/β activation in lupus promotes RBC alloimmunization by utilizing the pristane-induced lupus mouse model. Pristane significantly induced serum IFNα and expression of multiple interferon-stimulated genes (ISG) in peritoneal fluid cells, including inflammatory macrophages. Following transfusion with allogeneic RBCs expressing the KEL antigen, pristane-treated WT mice produced significantly elevated levels of anti-KEL IgM and anti-KEL IgG, compared to untreated mice. Pristane-treated mice lacking the IFNα/β receptor (IFNAR1−/−) or IFNα/β-inducing transcription factors (IRF3/7−/−) mice failed to produce ISGs and produced significantly lower levels of anti-KEL IgG compared to WT mice. In conclusion, pristane induction of a lupus-like phenotype promoted alloimmunization to the KEL RBC antigen in an IFNα/β-dependent manner. These results warrant further investigation of the role of IFNα/β in alloimmunization to other RBC antigens and the contribution of IFNα/β responses to the increased frequency of alloimmunization in patients with SLE.
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