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Abstract
Anemia commonly occurs in systemic lupus erythematosus (SLE), a disease characterized by innate immune activation by nucleic acids. Overactivation of cytoplasmic sensors by self-DNA or RNA can cause erythroid cell death while sparing other hematopoietic cell lineages. However, little is currently known about the impact of nucleic acid sensing innate receptors on the bone marrow (BM) erythropoietic niche.
The ssRNA endosomal receptors TLR7 and human TLR8 both recognize ssRNA. TLR7 overexpression causes a lupus syndrome that includes the development of mild to moderate anemia (Hb >10) and thrombocytopenia and is associated with spleen histiocytosis, autoimmune hemolysis, erythrophagocytosis and compensatory stress erythropoiesis in the spleen. A recent study demonstrated that patients with TLR8 gain of function present with immunodeficiency, inflammation and bone marrow failure. However, the role of TLR8 in SLE has been difficult to study in mice because it has a 5 amino acid deletion that attenuates its RNA binding capacity.
To address the role of TLR8 in SLE, we overexpressed human TLR8 in a lupus mouse model (huTLR8tg.Sle1.Yaa) using a BAC transgene. 50% of homozygous huTLR8tg.Sle1.Yaa mice developed severe anemia (Hb < 9) resulting in early mortality starting at 3–4.5 months of age. This phenotype required both the Sle1 and Yaa loci that promote the formation of high titer anti-chromatin and anti-RNA antibodies and onset of nephritis at > 6 months of age. There was no difference in autoantibody titers between Sle1.Yaa wt and huTLR8tg mice and early death in the transgenic mice was not due to premature onset of renal disease. All mice had normal RBC indices prior to 10 weeks of age. Anemia was associated with an increase in bone marrow (BM) trabecular bone and a decrease in erythromyeloblastic islands (EMBI) in the BM with compensatory stress erythropoiesis leading to reticulocytosis and vast splenomegaly. RBC half- life decreased in severely anemic mice as a pre-terminal event and was due to hemophagocytosis by 3 subsets of phagocytic red pulp macrophages.
Flow cytometry of BMs showed a block in CFU-E to proerythroblast differentiation that was confirmed by single cell RNASeq of bone marrow EMBIs. The erythroblast cluster proximal to the block had a signature of mitochondrial stress and decreased proliferation. We found 6 closely related clusters of EMBI central macrophages in the BM, most of which displayed an inflammatory and Type 1 IFN signature. One cluster (M2) expressed all the classical central macrophage phenotypic markers was characterized in transgenic mice by downregulation of multiple phagocytic receptors and a 5-fold decrease of VCAM1 expression. Loss of VCAM1 and downregulation of Mertk and CD169 in BM central macrophages was confirmed by flow cytometry. By contrast spleen central macrophages from transgenic mice retained VCAM1 and CD169 expression (figures 1–3).
Together, these results suggest that erythropoiesis in the BM of huTLR8tg SLE-prone mice fails due to a block in differentiation from CFU-E to proerythroblasts in the BM and is associated with an inflammatory phenotype specifically in BM erythroblastic island central macrophages and down regulation of adhesion and phagocytic receptors. These data implicate the endosomal RNA sensor TLR8 as an additional innate receptor whose overactivation causes acquired failure of erythropoiesis via myeloid cell dysregulation. Compensatory stress erythropoiesis in the spleens is associated with expansion of several subsets of macrophages with phagocytic properties and fatal anemia is associated with a decrease in red blood cell half-life, suggesting that excessive RBC phagocytosis, coupled with insufficient erythroblast progenitors, eventually exceeds the capability of stress erythropoiesis to replace the RBC mass.
Premature death in huTLR8tg Sle1.Yaa mice is due to severe anemia that is due to loss of RBC precursors at the proerythroblast stage as shown by flow cytometry of bone marrow
Single cell analysis of isolated BM EMBIs shows a decrease in central macrophage adhesion molecules, confirmed by flow cytometry
Decrease in RBC half-life in anemic mice (A) is acquired when RBC from young mice are transferred to anemic mice (B). Hemophagocytic macrophages (C) from anemic mice comprise 3 subsets including iHPCs and red pulp macrophages (D).
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