Type: Oral
Session: 101. Red Cells and Erythropoiesis, Excluding Iron: Erythropoiesis in Disease
Hematology Disease Topics & Pathways:
Research, Translational Research, Autoimmune hemolytic anemia, Genetic Disorders, Diseases, Immune Disorders, Metabolism, Biological Processes, Molecular biology
First, glucose uptake is ~2.5-fold higher in primary FL cells from Nan/+ cells than from WT. Pharmacological inhibition of glucose transporters reduces uptake, resulting in decreased mitochondrial activity and survival (90%) in Nan/+ but not WT FL cells. This shows that the elevated Nan/+ energy requirement is specifically due to and thus can be attenuated by glucose inhibition.
Second, to evaluate the contribution of inflammation to the anemia, we treated Nan/+ or WT FL (E13.5) cells with pharmacological STING inhibitors in vitro and find this reduces the number of early-stage erythroblasts and accelerates the transition to late-stage erythroblasts/mature red blood cells. Compared to WT, RNA expression of IFN (4α, β, and γ), ISGs, and inflammatory genes are reduced several fold in Nan/+ FL cells treated with the STING inhibitors, as is repression of key enzymes involved in activation of the cGAS-STING pathway, such as pTBK-1 and p-IRFs (-3,-7).
Third and as a complement, in vivo effects of STING inhibition were assessed by intravenous treatment of adult mice with H151. STING inhibitor significantly elevates anemic hallmarks such as red blood cell count, hemoglobin, and hematocrit in Nan/+ mice. H151 treatment improves ineffective erythropoiesis in the bone marrow (BM) of Nan/+ mice by enhancing the number of Ter119+ positive cells and reducing circulating reticulocytes and spleen weights. IFN (α and β) protein levels in serum are reduced in Nan/+ treated with H151 compared to WT mice. This shows that the Nan/+ phenotype can be alleviated by decreasing inflammatory pathways.
Fourth, to understand the underlying molecular mechanism of this effect we performed single-cell RNA-Seq of BM from Nan/+ or WT mice. Nan/+ cells show starkly different clustered erythroid sub-populations compared to WT cells, with reduced mature erythroid cells and increased immature, defective cells that also exhibit elevated metabolic and inflammatory gene expression levels. Remarkably, H151-treated Nan/+ BM cells reverses this pattern and presents with normal mature erythroid sub-populations and reduced defective erythroid populations similar to WT. Expression of genes affected by the Nan/+ mutation (both downregulated and ectopic) are restored to WT levels upon treatment. STING inhibitor treatment thus explicitly targets the defective erythroid population in the BM of Nan/+ mice, reduces ectopic inflammatory gene expression, and dramatically restores normal erythroid maturation.
Finally, oxidatively stressed mitochondria release mtDNA fragments via the channel formed by VDAC1 oligomers in the cytosol, thus inducing inflammation in Nan/+ FL cells. We tested the effect of the VDAC1 inhibitor, VBIT4. VDAC1 oligomerization and increased mtROS production are inhibited by VBIT-4 in Nan/+ as compared to WT FL cells in vitro. We then tested the effect of VBIT4 in vivo in adult mice and find that treatment significantly improves the anemic hallmarks and ineffective erythropoiesis in the BM of Nan/+ mice, which also show decreased circulating reticulocytes and spleen weights. IFNs, ISGs, and inflammatory genes transcript levels in erythroid cells, and IFNs (α and β) protein levels in serum, are reduced in Nan/+ mice treated with VBIT4, demonstrating that treatment directed at mtDNA release/VDAC activity reduces inflammation in the Nan/+ mouse.
Our results establish that STING and VDAC inhibitors positively impact inflammatory signaling and greatly reduce disease severity in the Nan anemic mouse model, and may be a potential therapeutic approach relevant to the dyserythropoiesis and anemia seen in patients with CDA type IV.
Disclosures: No relevant conflicts of interest to declare.
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