A New Murine Model of Primary Autoimmune Hemolytic Anemia (AIHA)

Abstract

Loss of humoral tolerance to red blood cells (RBCs) can lead to autoimmune hemolytic anemia (AIHA), a severe, and sometimes fatal disease. Patients with AIHA present with pallor, fatigue, decreased hematocrit, and splenomegaly. While secondary AIHA is associated with lymphoproliferative disorders, infections, and more recently, as an adverse event secondary to cancer immunotherapy, the etiology of primary AIHA is unknown. Several therapeutic strategies are available; however, there are currently no licensed treatments for AIHA and few therapeutics offer treatment-free durable remission. Moreover, supportive care with RBC transfusions can be challenging as most autoantibodies are directed against ubiquitous RBC antigens; thus, virtually all RBC donor units are incompatible. Given the severity of AIHA and the lack of treatment options, understanding the cellular and molecular mechanisms that facilitate the breakdown in tolerance would provide insight into new therapeutics. Herein, we report a new murine model of primary AIHA that reflects the biology observed in patients with primary AIHA. Production of anti-erythrocyte autoantibodies correlated with sex and age, and led to RBC antigen modulation, complement fixation, and anemia, as determined by decreased hematocrit and hemoglobin values and increased reticulocytes in peripheral blood. Moreover, autoantibody-producing animals developed splenomegaly, with altered splenic architecture characterized by expanded white pulp areas and nearly diminished red pulp areas. Additional analysis suggested that compensatory extramedullary erythropoiesis occurred as there were increased frequencies of RBC progenitors detectable in the spleen. No significant correlations between AIHA onset and inflammatory status or microbiome were observed. To our knowledge, this is the first report of a murine model that replicates observations made in humans with idiopathic AIHA. Thus, this is a tractable murine model of AIHA that can serve as a platform to identify key cellular and molecular pathways that are compromised, thereby leading to autoantibody formation, as well as testing new therapeutics and management strategies.

Keywords: RBC (red-blood-cell); autoimmune disease; autoimmune hemolytic anemia; erythrocyte; idiopathic autoimmune hemolytic anemia; primary autoimmune hemolytic anemia; red blood cell; tolerance.

Figure 1

A subset of HOD+OTII+ mice develop RBC autoantibodies. (A) Graphical illustrations of the HOD RBC (left) and OTII T cells (right). Whole blood and sera were collected from HODxOTII F1 animals and analyzed monthly. HOD-specific autoantibodies were detected in sera by flow crossmatch and mean fluorescence intensity (MFI) of (B) total immunoglobulins (Igs) and (C) IgM and IgG subclasses were calculated. Two standard deviations from mean values of IgM and each IgG subtype of HOD-OTII+ mice were included in the graphs (dotted lines). (D) The frequency of HOD+OTII+ animals that made detectable RBC autoantibodies was determined and broken down by sex. Results are representative of 4 independent cohorts of HODxOTII F1 animals (n = 10 HOD-OTII+ and 10-20 HOD+OTII+ mice per cohort). Each line represents an individual mouse of a particular genotype and autoantibody status: HOD-OTII+ (blue circles), HOD+OTII+ without autoantibodies (red filled circles, dashed lines), and HOD+OTII+ with autoantibodies (black circles).

Figure 2

Effects of autoantibodies production on RBCs. Peripheral RBCs were collected and (A) evaluated for DAT, (B) HOD antigen expression and (C) C3 complement deposition. (D) Hematocrit, (E) hemoglobin and (F) reticulocytes were calculated from whole blood samples. Results are representative of 4 independent cohorts of HODxOTII F1 animals (n = 10 HOD-OTII+ and 10-20 HOD+OTII+ mice per cohort). HOD-OTII+ (blue circles), HOD+OTII+ without autoantibodies (red circles), and HOD+OTII+ with autoantibodies (black circles). Statistical significance was determined by one-way ANOVA with multiple comparisons post-test and p-value > 0.05 n.s., < 0.05 *, < 0.01 **, < 0.001 ***, < 0.0001 ****.

Figure 3

HOD+OTII+ mice have splenomegaly and disrupted splenic architecture. Spleens from aged HODxOTII F1 animals were collected and (A) weights were determined. To visualize splenic architecture, spleens were fixed, sectioned, and stained with H&E. (B) Spleens from HOD-OTII+ and HOD+OTII+ without autoantibodies and (C) HOD+OTII+ mice with autoantibodies are shown at 2x and 4x magnification. (D) In select splenic sections from HOD+OTII+ mice with autoantibodies, megakaryocyte-like cells were identified (boxes on 10x and 20x images) and arrows shown in the 20x image point to hemosiderin macrophages. Images are representative of 4 individual mice of each genotype and condition. Scale bars are indicated on each image. Statistical significance was determined by one-way ANOVA with multiple comparisons post-test and p-value > 0.05 n.s. and < 0.01 **.

Figure 4

Autoantibody-producing HOD+OTII+ mice have extramedullary hematopoiesis. Spleens and bone marrow were collected from HODxOTII F1 animals, processed into a single cell suspension, and stained with antibodies to delineate RBC progenitors and development. (A) Representative images of each RBC developmental stage are shown. Flow plots and gating strategy used to identify each RBC progenitor in the (B) spleen and (C) bone marrow. Images are representative of 3 individual mice of each genotype and condition.

Figure 5

Representing scheme of HOD-OTII, HOD+OTII+ without and with Autoantibodies.