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Abstract

Cell Biology, Immunology and Biochemistry (basic and preclinical research)

Plenary Oral Abstract Session

PL3-SN-27 - Antioxidant Responses Regulate RBC Alloimmunization

Sunday, October 20, 2024
9:30 AM - 11:00 AM  
CE: Zero
Background/Case Studies: Most transfusion recipients do not form anti-RBC alloantibodies. Identifying mechanisms that inhibit alloimmunization may lead to prophylactic interventions. One potential regulatory mechanism is oxidative stress-induced activation of the transcription factor, nuclear factor erythroid-derived 2-like 2 (Nrf2), a master regulatory of antioxidant pathways. During erythrophagocytosis, oxidized heme activates Nrf2 which induces antioxidant enzymes, including heme oxygenase 1 (HMOX1), and promotes iron recycling. It also suppresses production of inflammatory cytokines including type 1 interferons (IFNα/β), which have been implicated in promoting RBC alloimmunization in pre-clinical models. Pharmacologic Nrf2 activators improve sequelae of sickle cell disease in pre-clinical models, but their role in transfusion has not been examined. Thus, we tested the hypothesis that Nrf2 activation regulates RBC alloimmunization.

Study

Design/Methods: Wildtype (WT) and Nrf2-deficient (Nrf2-/-) mice were treated with or without the Nrf2 activator, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im), and the IFNα/β stimulus, poly(I:C), prior to transfusion with murine RBCs expressing the KEL antigen. Anti-KEL IgG antibodies and post-transfusion recovery of KEL RBCs were measured by flow cytometry. Expression of Nrf2- and IFNα/β-stimulated genes (NSGs, ISGs) by blood leukocytes were measured by qPCR. To examine Nrf2 regulation of IFNα/β responses in human cells, monocyte-derived macrophages were co-cultured with opsonized or non-opsonized RBCs or treated with CDDO-Im prior to poly(I:C) treatment. NSGs and ISGs were measured by qPCR and flow cytometry.

Results/Findings: In human macrophage cultures, erythrophagocytosis of opsonized RBCs (Ops) and treatment with CDDO-Im promoted expression of NSGs (Fold relative HMOX1 gene expression compared to untreated: CDDO-Im = 16.1 ± 3.4 SD, **p < 0.001; Ops RBCs = 10.3 ± 1.8, *p < 0.05; Non-ops RBCs = 1.1 ± 0.5. not significant) and inhibited expression of poly(I:C)-induced ISGs. CDDO-Im treatment of WT mice induced the expression of NSGs and inhibited the production of ISGs by blood leukocytes (Mean IRF7 (ISG) gene expression: +poly(I:C) = 9273 ± 1584 SD; +poly(I:C) and CDDO-Im = 1355.6 ± 928, **p < 0.01). Following transfusion with KEL RBCs, CDDO-Im treatment inhibited poly(I:C)-induced anti-KEL IgG production and promoted RBC post-transfusion recovery in WT mice but failed to do so in Nrf2-/‑ mice (Figure).

Conclusions:

Results indicate that activation of the Nrf2 antioxidant pathway regulates RBC alloimmunization to the KEL antigen in a pre-clinical model. Although it is not yet clear whether Nrf2 regulates RBC alloimmunization by inhibiting IFNα/β, Nrf2 activation inhibited IFNα/β responses in mice and human cells. If findings translate to other models and human studies, Nrf2 activators may represent a potential prophylactic intervention to inhibit alloimmunization.