The Nrf2 Activator CDDO-Imidazole Suppresses Inflammation-Induced Red Blood Cell Alloimmunization

Abstract

Experimental Objective: During red blood cell (RBC) transfusion, inflammation promotes the production of anti-RBC alloantibodies that can cause significant hemolytic events. Avoiding RBC antigen exposure is the only strategy to prevent RBC alloimmunization in transfusion recipients. Identifying mechanisms that inhibit alloimmunization may lead to novel prophylactic interventions. One potential regulatory mechanism is the activation of the transcription factor nuclear factor erythroid-derived 2-like 2 (Nrf2), a master regulator of antioxidant pathways. Pharmacologic Nrf2 activators induce antioxidant production and improve the sequelae of inflammatory diseases. Thus, we tested the hypothesis that a Nrf2 activator, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]-imidazole (CDDO-Im), regulates inflammation-induced RBC alloimmunization.

Methods: WT and Nrf2-deficient mice were treated with inflammatory stimuli and CDDO-Im prior to transfusion with RBCs expressing the KEL antigen (KEL+ RBCs). Anti-KEL IgM and IgG were measured in the serum of transfused mice. Nrf2-activated gene expression and interferon activity were measured in mice and human macrophages pre-treated with CDDO-Im and interferon stimuli.

Results: Here, we report that CDDO-Im induces Nrf2-activated gene expression and inhibits type 1 interferon activity, which promotes RBC alloimmunization in transfusion models. In mice transfused with KEL+ RBCs, pre-treatment with CDDO-Im inhibited inflammation-induced anti-KEL antibody production and increased the post-transfusion recovery of KEL+ RBCs in a Nrf2-dependent manner. CDDO-Im also inhibited RBC alloimmunization in mice with pre-existing inflammation.

Conclusions: These results indicate that the activation of the Nrf2 antioxidant pathway regulates RBC alloimmunization to the KEL antigen in a pre-clinical model. If these findings translate to other models and human studies, Nrf2 activators may represent a potential prophylactic intervention to inhibit alloimmunization.

Keywords: Nrf2; RBC alloimmunization; inflammation; transfusion; type 1 interferons.

Figure 1

CDDO-Im induces Nrf2-activated gene expression in mice. WT mice were treated (i.p.) with 5–10 mg/kg CDDO-Im and/or 100 µg poly(I:C) 6 h and 3 h prior to analysis, respectively. (A) HMOX1 and (B) NQO1 expression by blood leukocytes measured by RT-qPCR. * p < 0.05, ** p < 0.01, *** p < 0.001 by one-way ANOVA with Tukey’s post-test; 5 mice per group; representative of 3 independent experiments.

Figure 2

CDDO-Im inhibits inflammation-induced RBC alloimmunization. WT mice were treated with or without poly(I:C) and/or CDDO-Im 3 and 6 h before transfusion, respectively, with KEL + RBCs. (A) An experimental timeline showing the schedule of CDDO-Im and poly(I:C) treatments, transfusions, and serum collections. (B) Serum anti-KEL IgM levels measured 5 days after transfusion. (C) Serum anti-KEL IgG levels measured 7 and 14 days after transfusion. (D) The post-transfusion recovery of KEL+ RBCs in peripheral blood 1–3 days after a second transfusion, measured by flow cytometry. The data shown are from one experiment, representative of three independent experiments with five mice per group. * p < 0.05 by the Kruskal–Wallis test with Dunn’s post-test for comparison of poly(I:C) vs. poly(I:C) + CDDO-Im groups. (B,C) * p < 0.05 by the Kruskal–Wallis test with Dunn’s post-test.

Figure 3

Nrf2 activation inhibits inflammation-induced RBC alloimmunization. WT and Nrf2^-/- mice were treated with poly(I:C), 3 h before transfusion with KEL+ RBCs. Mice were treated with or without CDDO-Im 6 h before transfusion. (A) Serum anti-KEL IgM levels measured 5 days after transfusion. (B) Serum anti-KEL IgG levels collected 7 and 14 days after transfusion. (C) Post-transfusion recovery of KEL+ RBCs in peripheral blood 1–3 days after a second transfusion, measured by flow cytometry. Data are from one experiment, representative of 3 independent experiments with 5 mice per group. (A,B) ** p < 0.01 by Kruskal–Wallis test with Dunn’s post-test. ns, non-significant. (C) * p < 0.05, ** p < 0.01 by Kruskal–Wallis test with Dunn’s post-test for comparison of WT + CDDO-Im vs. Nrf2^-/- + CDDO-Im groups.

Figure 4

CDDO-Im regulates IFNα/β and NFκB-induced cytokine production in mice. WT mice were treated (i.p.) with 5–10 mg/kg CDDO-Im and/or 100 µg poly(I:C) 6 h and 3 h prior to analysis, respectively. (A) IFNα, IFNβ, and (B) NF-κB-induced cytokines CXCL1, TNF, MCP-1, and IL-6 levels in serum measured by multiplex bead assay. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 by one-way ANOVA with Tukey’s post-test; 5 mice per group; representative of 3 independent experiments; n.d. = not detectable.

Figure 5

CDDO-Im induces Nrf2-activated gene expression in human macrophages. Human monocyte-derived macrophages were treated with CDDO-Im for 18 h. (A) AKR1C1, (B) HMOX1, and (C) NQO1 expression in macrophages treated with either 0, 0.2, 0.4, or 0.8 µM CDDO-Im, measured by RT-qPCR. (D) Representative intracellular flow cytometric analysis of Hmox1 expression by CD64+ macrophages treated with or without 0.8 µM CDDO-Im, gated on live cells (E) Cumulative data of Hmox1 expression by macrophages from 5 independent experiments; ** p < 0.01 by Student’s t-test. (A–C) Each circle represents expression from anindependent experiment, n = 5. * p < 0.05, ** p < 0.01 by one-way ANOVA with Tukey’s post-test.

Figure 6

CDDO-Im inhibits IFNα/β activity in human macrophages. Human monocyte-derived macrophages were treated with CDDO-Im for 18 h followed by 1 µg/mL poly(I:C) treatment for 3 (D–F) or 24 h (A–C). (A) Representative flow cytometric analysis of CD38 and Siglec-1 expression on CD64+ macrophages treated with or without poly(I:C) and 0.8 µM CDDO-Im. (B,C) Cumulative data of (B) CD38 and (C) Siglec-1 expression by macrophages from (A), n = 5. (D–F) Fold expression of ISGs (D) MXA, (E) IP-10, and (F) ISG15 in macrophages treated with poly(I:C) and either 0.2, 0.4, or 0.8 µM CDDO-Im, relative to untreated cells (None), measured by RT-qPCR. Each circle represents an independent experiment (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 by one-way ANOVA with Tukey’s post-test. ns, not significant.

Figure 7

CDDO-Im regulates RBC alloimmunization in mice with pre-existing inflammation. WT mice were treated with or without poly(I:C) and/or CDDO-Im 6 h and 3 h before transfusion, respectively (Poly(I:C) → CDDO-Im), with KEL+ RBCs. (A) A timeline of treatments, transfusions, and serum collections. (B) Serum cytokines at the time of transfusion. (C) Serum anti-KEL IgM levels measured 5 days after transfusion. (D) Serum anti-KEL IgG levels measured 7 and 14 days after transfusion. (E) The post-transfusion recovery of KEL+ RBCs in peripheral blood 1–3 days after the second transfusion, measured by flow cytometry. The data shown are from one experiment, representative of three independent experiments with five mice per group. (C,D) * p < 0.05, ** p < 0.01 by the Kruskal–Wallis test with Dunn’s post-test. (E) * p < 0.05 by the Kruskal–Wallis test with Dunn’s post-test for a comparison of poly(I:C) vs. Poly(I:C) → CDDO-Im groups.

Figure 7

CDDO-Im regulates RBC alloimmunization in mice with pre-existing inflammation. WT mice were treated with or without poly(I:C) and/or CDDO-Im 6 h and 3 h before transfusion, respectively (Poly(I:C) → CDDO-Im), with KEL+ RBCs. (A) A timeline of treatments, transfusions, and serum collections. (B) Serum cytokines at the time of transfusion. (C) Serum anti-KEL IgM levels measured 5 days after transfusion. (D) Serum anti-KEL IgG levels measured 7 and 14 days after transfusion. (E) The post-transfusion recovery of KEL+ RBCs in peripheral blood 1–3 days after the second transfusion, measured by flow cytometry. The data shown are from one experiment, representative of three independent experiments with five mice per group. (C,D) * p < 0.05, ** p < 0.01 by the Kruskal–Wallis test with Dunn’s post-test. (E) * p < 0.05 by the Kruskal–Wallis test with Dunn’s post-test for a comparison of poly(I:C) vs. Poly(I:C) → CDDO-Im groups.