Altered type 1 interferon responses in alloimmunized and nonalloimmunized patients with sickle cell disease

Abstract 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.

the development of strategies to prevent alloimmunization. However, mechanisms underlying the increased frequency of alloimmunization in patients with SCD are poorly understood.
Prior studies have also implicated IFNα/β in the pathogenesis of autoimmune diseases [14,15]. Patients with systemic lupus erythematosus (SLE) have a high frequency of alloimmunization and express a type 1 interferon (IFNα/β) gene signature defined as the production of IFNα/β and numerous ISGs [16,17]. Although a role for IFNα/β in RBC alloimmunization has not been investigated in any patient population, IFNα/β promotes RBC alloimmunization in a lupus mouse model [10].
Recent studies indicate that patients with SCD also express an IFNα/β gene signature. Hounkpe et al. performed a meta-analysis of gene expression studies and identified a cluster of ISGs enriched in patients with SCD [18]. In addition, Hermand et al. recently reported that serum IFNα and ISGs produced by neutrophils are elevated in children with SCD, compared to healthy blood donors [19]. Meinderts et al. also found associations between single-nucleotide polymorphisms in IFNα/β-related genes and RBC alloimmunization in patients with SCD [20].
Here, we examine the IFNα/β gene signature in whole blood and peripheral blood mononuclear cells (PBMCs) of adults with SCD and race-matched controls. In addition, we test the hypothesis that IFNα/β contributes to human RBC alloimmunization by examining ISG expression in alloimmunized and nonalloimmunized patients with SCD.

Patients and control subjects
Sixteen patients with sickle cell hemoglobinopathy and five race-   Table S1. Thermo Fisher Connect software was used to determine the expression of target genes relative to GAPDH.

Statistics
Statistical analyses were performed using GraphPad Prism software.
Statistical significance of demographic data between two groups was determined using an unpaired t-test, and statistical significance between three groups was determined using a one-way ANOVA with a Tukey's posttest. All non-demographic data were nonparametric.
Statistical significance between two groups was determined using a

Participant demographics
To examine IFNα/β activity in SCD, patients with SCD (SS patients) and healthy race-matched controls (AA) were enrolled. The study included 21 participants: nine SS patients with a history of RBC alloimmunization, seven SS patients without a history of alloimmunization, and five controls (Table 1). A "nonalloimmunized patient" had at least one alloantibody screen at least 15 days after any RBC transfusion, with no antibodies detected at that screen or any other RBC antibody screen. There were no significant differences between the ages of the three groups or the number of transfusions between alloimmunized and nonalloimmunized SS patients. Anti-E, anti-K, and warm autoantibodies were the common antibodies identified. Six of the nine alloimmunized patients had one to two historical alloantibodies ( Figure S1).

Increased ISG expression in whole blood of SS patients
As IFNα/β are transient in blood, we examined the expression of a well-characterized ISG, myxovirus resistance protein 1 (MxA), in leukocytes of SS patients and controls by whole blood immunoassay. Although not statistically significant, SS patients had elevated levels of MxA compared to controls. There was no significant difference in MxA between patients with and without alloantibodies ( Figure 1).  [21]. By quantitative PCR, IFIT3, ISG-15, and IFI27 were significantly elevated in blood leukocytes of SS patients compared to controls, and there were nonsignificant trends toward increased expression of MxA, IFI44, and IFI44L in SS patients (Figure 2A). The IFNα/β gene score, which is a measurement of the bioactivity of IFNα/β, was calculated by summing the differences in expression, between SS patients and controls, of all tested ISGs for each subject (Figure 2B) [22]. There was a significant increase in IFNα/β scores in SS patients compared to controls. However, the IFNα/β scores did not differ between the two SS patient groups ( Figure 2C,D).

Elevated ISGs in IFNβ-stimulated PBMCs of SS patients
As baseline IFNα/β gene scores are elevated in blood leukocytes of SS patients, we considered that PBMCs from SS patients may have altered sensitivity to IFNα/β. Thus, PBMCs were unstimulated or stimulated

Increased CD86 on lymphocytes of alloimmunized patients with SCD
We then sought to determine the effect of elevated IFNα/β activity in SS patients on immune cell activation. IFNα/β induces maturation of antigen presenting cells (APCs). Increased expression of the costimulatory protein CD86 on APCs, including monocytes and B cells, is a marker of APC maturation [12]. Thus, we examined the expression of CD86 on APCs from SS patients and controls by flow cytometry using the gating strategy in Figure S4

Resistance of SCD monocytes to IFNβ stimulation of whole blood
In addition to CD86, we examined the expression of ISGs on monocytes and lymphocytes by flow cytometry. Whole blood was unstimulated or stimulated with IFNβ prior to measurement of ISG expression, Siglec-1 and CD38, on CD64 + CD14 + monocytes ( Figure 5A). The baseline expression of these ISGs was not different between SS patients and controls. However, following IFNβ stimulation of whole blood from controls, Siglec-1 and CD38 were significantly upregulated on monocytes. However, in contrast to IFNβ stimulation of isolated PBMCs,

IFNβ stimulation of whole blood from SS patients failed to upregulate
ISGs on monocytes ( Figure 5B

Increase in immunomodulatory cytokines in patients with SCD
The ability of IFNβ to upregulate ISGs in isolated PBMCs but not in whole blood leukocytes indicates that other components of SS whole blood regulate IFNβ responses. Thus, we examined plasma cytokines of SS patients and controls with an ELISA-based multiplex assay. IP-10, also known as CXCL10, is an ISG that recruits granulocytes to sites of inflammation. IL-10 has anti-inflammatory properties, while IL-8, IL-6, and TNFα are NFκb-regulated cytokines that antagonize IFNα/β activation [24][25][26]. We observed a significant increase in IP-10, IL-6, and IL-10 in SS patients compared to controls and a nonsignificant trend toward an increase in TNFα and IL-8. Alloimmunized SS patients had significantly higher IL-6, IL-10, and IP-10 than controls, and nonsignificant increases in IL-10, TNFα, and IL-8 compared to nonalloimmunized patients (Figure 7). In sum, SS patients had elevated levels of multiple cytokines that have the potential to inhibit IFNβ stimulation in whole blood.

DISCUSSION
Patients with SCD have the highest frequency of RBC alloimmunization compared to any other disease population, including those with similar transfusion burdens [17,27]. Defining the cause of this disparity is inte-gral to preventing alloimmunization and improving transfusion safety.
We previously reported that IFNα/β promotes alloimmunization in mouse models [8][9][10][11]. Here, we report that a cohort of adult patients with SCD expresses an IFNα/β gene signature. Although there were trends toward increased ISGs in alloimmunized patients, there were no significant differences between alloimmunized and nonalloimmunized patients with SCD. We also report that isolated PBMCs from patients with SCD may be more sensitive to IFNβ stimulation, while leukocytes in whole blood of patients with SCD are resistant to IFNβ stimulation.
Hermand et al. reported that neutrophils of pediatric patients with SCD express elevated levels of ISGs, compared to adult blood donors [19]. Here, we found elevated ISGs and IFNα/β gene scores in whole blood, but not in unstimulated PBMCs. These results support the prior finding that SCD neutrophils, which are increased in patients with SCD [30], contribute to the IFNα/β gene signature. Both studies highlight variability in IFNα/β activity in SCD, as Hermand et al. reported that serum IFNα was elevated in half of patients, and we found that IFNα/β scores of blood leukocytes were increased in approximately half of patients. The variability may be related to varying clinical presentations. However, the prior study did not find any association of serum IFNα levels with clinical outcomes, and concluded that examining a link between IFNα/β activity and alloimmunization in patients with SCD was needed [19].
A prior study reported that elevations of non-IFNα/β cytokines are not correlated with RBC alloimmunization frequency [31]. Here, there were no significant differences in ISGs of unstimulated or IFNβ-stimulated samples between alloimmunized and nonalloimmu- in SCD plasma, alternate chronic inflammatory pathways may limit IFNα/β responses and inhibit ISG expression in whole blood. IL-8, previously shown to be elevated in patients with SCD [29], is a chemokine induced following viral infection that inhibits IFNα/β antiviral functions [26]. Upon macrophage stimulation, IFNα/β induces IL-10, which feedbacks to suppress further cytokine production and responses [35]. Finally, TNFα inhibits IFNα/β activity in patients with SLE, while IFNα/β inhibits TNFα production in multiple models [24]. Collectively, the antagonistic effects of elevated IL-8, IL-10, and TNFα may inhibit

ACKNOWLEDGMENTS
This work was supported by grants from the National Blood Foundation (R13672), the American Society of Hematology (5283), and the NIH/NHLBI (5 K08 HL141446) to David R. Gibb, and the NIH/NHLBI (R01 HL132951) to Jeanne E. Hendrickson.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interest relevant to the manuscript.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.