Immune Mechanisms in Inflammatory Anemia.

Maintaining the correct number of healthy red blood cells (RBCs) is critical for proper oxygenation of tissues throughout the body. Therefore, RBC homeostasis is a tightly controlled balance between RBC production and RBC clearance, through the processes of erythropoiesis and macrophage hemophagocytosis, respectively. However, during the inflammation associated with infectious, autoimmune, or inflammatory diseases this homeostatic process is often dysregulated, leading to acute or chronic anemia. In each disease setting, multiple mechanisms typically contribute to the development of inflammatory anemia, impinging on both sides of the RBC production and RBC clearance equation. These mechanisms include both direct and indirect effects of inflammatory cytokines and innate sensing. Here, we focus on common innate and adaptive immune mechanisms that contribute to inflammatory anemias using examples from several diseases, including hemophagocytic lymphohistiocytosis/macrophage activation syndrome, severe malarial anemia during Plasmodium infection, and systemic lupus erythematosus, among others.

Iron dysregulation and inflammatory stress erythropoiesis associates with long-term outcome of COVID-19.

Persistent symptoms following SARS-CoV-2 infection are increasingly reported, although the drivers of post-acute sequelae (PASC) of COVID-19 are unclear. Here we assessed 214 individuals infected with SARS-CoV-2, with varying disease severity, for one year from COVID-19 symptom onset to determine the early correlates of PASC. A multivariate signature detected beyond two weeks of disease, encompassing unresolving inflammation, anemia, low serum iron, altered iron-homeostasis gene expression and emerging stress erythropoiesis; differentiated those who reported PASC months later, irrespective of COVID-19 severity. A whole-blood heme-metabolism signature, enriched in hospitalized patients at month 1-3 post onset, coincided with pronounced iron-deficient reticulocytosis. Lymphopenia and low numbers of dendritic cells persisted in those with PASC, and single-cell analysis reported iron maldistribution, suggesting monocyte iron loading and increased iron demand in proliferating lymphocytes. Thus, defects in iron homeostasis, dysregulated erythropoiesis and immune dysfunction due to COVID-19 possibly contribute to inefficient oxygen transport, inflammatory disequilibrium and persisting symptomatology, and may be therapeutically tractable.

Erythroid mitochondrial retention triggers myeloid-dependent type I interferon in human SLE.

Emerging evidence supports that mitochondrial dysfunction contributes to systemic lupus erythematosus (SLE) pathogenesis. Here we show that programmed mitochondrial removal, a hallmark of mammalian erythropoiesis, is defective in SLE. Specifically, we demonstrate that during human erythroid cell maturation, a hypoxia-inducible factor (HIF)-mediated metabolic switch is responsible for the activation of the ubiquitin-proteasome system (UPS), which precedes and is necessary for the autophagic removal of mitochondria. A defect in this pathway leads to accumulation of red blood cells (RBCs) carrying mitochondria (Mito+ RBCs) in SLE patients and in correlation with disease activity. Antibody-mediated internalization of Mito+ RBCs induces type I interferon (IFN) production through activation of cGAS in macrophages. Accordingly, SLE patients carrying both Mito+ RBCs and opsonizing antibodies display the highest levels of blood IFN-stimulated gene (ISG) signatures, a distinctive feature of SLE.

A role of PIEZO1 in iron metabolism in mice and humans.

Iron overload causes progressive organ damage and is associated with arthritis, liver damage, and heart failure. Elevated iron levels are present in 1%-5% of individuals; however, iron overload is undermonitored and underdiagnosed. Genetic factors affecting iron homeostasis are emerging. Individuals with hereditary xerocytosis, a rare disorder with gain-of-function (GOF) mutations in mechanosensitive PIEZO1 ion channel, develop age-onset iron overload. We show that constitutive or macrophage expression of a GOF Piezo1 allele in mice disrupts levels of the iron regulator hepcidin and causes iron overload. We further show that PIEZO1 is a key regulator of macrophage phagocytic activity and subsequent erythrocyte turnover. Strikingly, we find that E756del, a mild GOF PIEZO1 allele present in one-third of individuals of African descent, is strongly associated with increased plasma iron. Our study links macrophage mechanotransduction to iron metabolism and identifies a genetic risk factor for increased iron levels in African Americans.

Identification of RIOK2 as a master regulator of human blood cell development.

Anemia is a major comorbidity in aging, chronic kidney and inflammatory diseases, and hematologic malignancies. However, the transcriptomic networks governing hematopoietic differentiation in blood cell development remain incompletely defined. Here we report that the atypical kinase RIOK2 (right open reading frame kinase 2) is a master transcription factor (TF) that not only drives erythroid differentiation, but also simultaneously suppresses megakaryopoiesis and myelopoiesis in primary human stem and progenitor cells. Our study reveals the previously uncharacterized winged helix-turn-helix DNA-binding domain and two transactivation domains of RIOK2 that are critical to regulate key hematopoietic TFs GATA1, GATA2, SPI1, RUNX3 and KLF1. This establishes RIOK2 as an integral component of the transcriptional regulatory network governing human hematopoietic differentiation. Importantly, RIOK2 mRNA expression significantly correlates with these TFs and other hematopoietic genes in myelodysplastic syndromes, acute myeloid leukemia and chronic kidney disease. Further investigation of RIOK2-mediated transcriptional pathways should yield therapeutic approaches to correct defective hematopoiesis in hematologic disorders.

Blockade of IL-22 signaling reverses erythroid dysfunction in stress-induced anemias.

Patients with myelodysplastic syndromes (MDSs) display severe anemia but the mechanisms underlying this phenotype are incompletely understood. Right open-reading-frame kinase 2 (RIOK2) encodes a protein kinase located at 5q15, a region frequently lost in patients with MDS del(5q). Here we show that hematopoietic cell-specific haploinsufficient deletion of Riok2 (Riok2f/+Vav1cre) led to reduced erythroid precursor frequency leading to anemia. Proteomic analysis of Riok2f/+Vav1cre erythroid precursors suggested immune system activation, and transcriptomic analysis revealed an increase in p53-dependent interleukin (IL)-22 in Riok2f/+Vav1cre CD4+ T cells (TH22). Further, we discovered that the IL-22 receptor, IL-22RA1, was unexpectedly present on erythroid precursors. Blockade of IL-22 signaling alleviated anemia not only in Riok2f/+Vav1cre mice but also in wild-type mice. Serum concentrations of IL-22 were increased in the subset of patients with del(5q) MDS as well as patients with anemia secondary to chronic kidney disease. This work reveals a possible therapeutic opportunity for reversing many stress-induced anemias by targeting IL-22 signaling.

Interplay between liver and blood stages of Plasmodium infection dictates malaria severity via γδ T cells and IL-17-promoted stress erythropoiesis.

Plasmodium replicates within the liver prior to reaching the bloodstream and infecting red blood cells. Because clinical manifestations of malaria only arise during the blood stage of infection, a perception exists that liver infection does not impact disease pathology. By developing a murine model where the liver and blood stages of infection are uncoupled, we showed that the integration of signals from both stages dictated mortality outcomes. This dichotomy relied on liver stage-dependent activation of Vγ4+ γδ T cells. Subsequent blood stage parasite loads dictated their cytokine profiles, where low parasite loads preferentially expanded IL-17-producing γδ T cells. IL-17 drove extra-medullary erythropoiesis and concomitant reticulocytosis, which protected mice from lethal experimental cerebral malaria (ECM). Adoptive transfer of erythroid precursors could rescue mice from ECM. Modeling of γδ T cell dynamics suggests that this protective mechanism may be key for the establishment of naturally acquired malaria immunity among frequently exposed individuals.

A Red Carpet for Iron Metabolism.

200 billion red blood cells (RBCs) are produced every day, requiring more than 2 × 1015 iron atoms every second to maintain adequate erythropoiesis. These numbers translate into 20 mL of blood being produced each day, containing 6 g of hemoglobin and 20 mg of iron. These impressive numbers illustrate why the making and breaking of RBCs is at the heart of iron physiology, providing an ideal context to discuss recent progress in understanding the systemic and cellular mechanisms that underlie the regulation of iron homeostasis and its disorders.

Functional Selectivity in Cytokine Signaling Revealed Through a Pathogenic EPO Mutation.

Cytokines are classically thought to stimulate downstream signaling pathways through monotonic activation of receptors. We describe a severe anemia resulting from a homozygous mutation (R150Q) in the cytokine erythropoietin (EPO). Surprisingly, the EPO R150Q mutant shows only a mild reduction in affinity for its receptor but has altered binding kinetics. The EPO mutant is less effective at stimulating erythroid cell proliferation and differentiation, even at maximally potent concentrations. While the EPO mutant can stimulate effectors such as STAT5 to a similar extent as the wild-type ligand, there is reduced JAK2-mediated phosphorylation of select downstream targets. This impairment in downstream signaling mechanistically arises from altered receptor dimerization dynamics due to extracellular binding changes. These results demonstrate how variation in a single cytokine can lead to biased downstream signaling and can thereby cause human disease. Moreover, we have defined a distinct treatable form of anemia through mutation identification and functional studies.

Inactive Parp2 causes Tp53-dependent lethal anemia by blocking replication-associated nick ligation in erythroblasts.

Poly (ADP-ribose) polymerase (PARP) 1 and 2 enzymatic inhibitors (PARPi) are promising cancer treatments. But recently, their use has been hindered by unexplained severe anemia and treatment-related leukemia. In addition to enzymatic inhibition, PARPi also trap PARP1 and 2 at DNA lesions. Here we report that, unlike Parp2-/- mice, which develop normally, mice expressing catalytically inactive Parp2 (E534A and Parp2EA/EA) succumb to Tp53- and Chk2-dependent erythropoietic failure in utero, mirroring Lig1-/- mice. While DNA damage mainly activates PARP1, we demonstrate that DNA replication activates PARP2 robustly. PARP2 is selectively recruited and activated by 5'-phosphorylated nicks (5'p-nicks), including those between Okazaki fragments, resolved by ligase 1 (Lig1) and Lig3. Inactive PARP2, but not its active form or absence, impedes Lig1- and Lig3-mediated ligation, causing dose-dependent replication fork collapse, which is detrimental to erythroblasts with ultra-fast forks. This PARylation-dependent structural function of PARP2 at 5'p-nicks explains the detrimental effects of PARP2 inactivation on erythropoiesis, shedding light on PARPi-induced anemia and the selection for TP53/CHK2 loss.

Systematic Functional Dissection of Common Genetic Variation Affecting Red Blood Cell Traits.

Genome-wide association studies (GWAS) have successfully identified thousands of associations between common genetic variants and human disease phenotypes, but the majority of these variants are non-coding, often requiring genetic fine-mapping, epigenomic profiling, and individual reporter assays to delineate potential causal variants. We employ a massively parallel reporter assay (MPRA) to simultaneously screen 2,756 variants in strong linkage disequilibrium with 75 sentinel variants associated with red blood cell traits. We show that this assay identifies elements with endogenous erythroid regulatory activity. Across 23 sentinel variants, we conservatively identified 32 MPRA functional variants (MFVs). We used targeted genome editing to demonstrate endogenous enhancer activity across 3 MFVs that predominantly affect the transcription of SMIM1, RBM38, and CD164. Functional follow-up of RBM38 delineates a key role for this gene in the alternative splicing program occurring during terminal erythropoiesis. Finally, we provide evidence for how common GWAS-nominated variants can disrupt cell-type-specific transcriptional regulatory pathways.

Infection perturbs Bach2- and Bach1-dependent erythroid lineage 'choice' to cause anemia.

Elucidation of how the differentiation of hematopoietic stem and progenitor cells (HSPCs) is reconfigured in response to the environment is critical for understanding the biology and disorder of hematopoiesis. Here we found that the transcription factors (TFs) Bach2 and Bach1 promoted erythropoiesis by regulating heme metabolism in committed erythroid cells to sustain erythroblast maturation and by reinforcing erythroid commitment at the erythro-myeloid bifurcation step. Bach TFs repressed expression of the gene encoding the transcription factor C/EBPß, as well as that of its target genes encoding molecules important for myelopoiesis and inflammation; they achieved the latter by binding to their regulatory regions also bound by C/EBPß. Lipopolysaccharide diminished the expression of Bach TFs in progenitor cells and promoted myeloid differentiation. Overexpression of Bach2 in HSPCs promoted erythroid development and inhibited myelopoiesis. Knockdown of BACH1 or BACH2 in human CD34+ HSPCs impaired erythroid differentiation in vitro. Thus, Bach TFs accelerate erythroid commitment by suppressing the myeloid program at steady state. Anemia of inflammation and myelodysplastic syndrome might involve reduced activity of Bach TFs.

Mitochondrial ClpX Activates a Key Enzyme for Heme Biosynthesis and Erythropoiesis.

The mitochondrion maintains and regulates its proteome with chaperones primarily inherited from its bacterial endosymbiont ancestor. Among these chaperones is the AAA+ unfoldase ClpX, an important regulator of prokaryotic physiology with poorly defined function in the eukaryotic mitochondrion. We observed phenotypic similarity in S. cerevisiae genetic interaction data between mitochondrial ClpX (mtClpX) and genes contributing to heme biosynthesis, an essential mitochondrial function. Metabolomic analysis revealed that 5-aminolevulinic acid (ALA), the first heme precursor, is 5-fold reduced in yeast lacking mtClpX activity and that total heme is reduced by half. mtClpX directly stimulates ALA synthase in vitro by catalyzing incorporation of its cofactor, pyridoxal phosphate. This activity is conserved in mammalian homologs; additionally, mtClpX depletion impairs vertebrate erythropoiesis, which requires massive upregulation of heme biosynthesis to supply hemoglobin. mtClpX, therefore, is a widely conserved stimulator of an essential biosynthetic pathway and uses a previously unrecognized mechanism for AAA+ unfoldases.

Resilient anatomy and local plasticity of naive and stress haematopoiesis.

The bone marrow adjusts blood cell production to meet physiological demands in response to insults. The spatial organization of normal and stress responses are unknown owing to the lack of methods to visualize most steps of blood production. Here we develop strategies to image multipotent haematopoiesis, erythropoiesis and lymphopoiesis in mice. We combine these with imaging of myelopoiesis1 to define the anatomy of normal and stress haematopoiesis. In the steady state, across the skeleton, single stem cells and multipotent progenitors distribute through the marrow enriched near megakaryocytes. Lineage-committed progenitors are recruited to blood vessels, where they contribute to lineage-specific microanatomical structures composed of progenitors and immature cells, which function as the production sites for each major blood lineage. This overall anatomy is resilient to insults, as it was maintained after haemorrhage, systemic bacterial infection and granulocyte colony-stimulating factor (G-CSF) treatment, and during ageing. Production sites enable haematopoietic plasticity as they differentially and selectively modulate their numbers and output in response to insults. We found that stress responses are variable across the skeleton: the tibia and the sternum respond in opposite ways to G-CSF, and the skull does not increase erythropoiesis after haemorrhage. Our studies enable in situ analyses of haematopoiesis, define the anatomy of normal and stress responses, identify discrete microanatomical production sites that confer plasticity to haematopoiesis, and uncover unprecedented heterogeneity of stress responses across the skeleton.

Co-transcriptional splicing regulates 3' end cleavage during mammalian erythropoiesis.

Pre-mRNA processing steps are tightly coordinated with transcription in many organisms. To determine how co-transcriptional splicing is integrated with transcription elongation and 3' end formation in mammalian cells, we performed long-read sequencing of individual nascent RNAs and precision run-on sequencing (PRO-seq) during mouse erythropoiesis. Splicing was not accompanied by transcriptional pausing and was detected when RNA polymerase II (Pol II) was within 75-300 nucleotides of 3' splice sites (3'SSs), often during transcription of the downstream exon. Interestingly, several hundred introns displayed abundant splicing intermediates, suggesting that splicing delays can take place between the two catalytic steps. Overall, splicing efficiencies were correlated among introns within the same transcript, and intron retention was associated with inefficient 3' end cleavage. Remarkably, a thalassemia patient-derived mutation introducing a cryptic 3'SS improved both splicing and 3' end cleavage of individual ß-globin transcripts, demonstrating functional coupling between the two co-transcriptional processes as a determinant of productive gene output.

Activation of γ-globin expression by hypoxia-inducible factor 1α.

Around birth, globin expression in human red blood cells (RBCs) shifts from γ-globin to ß-globin, which results in fetal haemoglobin (HbF, α2γ2) being gradually replaced by adult haemoglobin (HbA, α2ß2)1. This process has motivated the development of innovative approaches to treat sickle cell disease and ß-thalassaemia by increasing HbF levels in postnatal RBCs2. Here we provide therapeutically relevant insights into globin gene switching obtained through a CRISPR-Cas9 screen for ubiquitin-proteasome components that regulate HbF expression. In RBC precursors, depletion of the von Hippel-Lindau (VHL) E3 ubiquitin ligase stabilized its ubiquitination target, hypoxia-inducible factor 1α (HIF1α)3,4, to induce γ-globin gene transcription. Mechanistically, HIF1α-HIF1ß heterodimers bound cognate DNA elements in BGLT3, a long noncoding RNA gene located 2.7 kb downstream of the tandem γ-globin genes HBG1 and HBG2. This was followed by the recruitment of transcriptional activators, chromatin opening and increased long-range interactions between the γ-globin genes and their upstream enhancer. Similar induction of HbF occurred with hypoxia or with inhibition of prolyl hydroxylase domain enzymes that target HIF1α for ubiquitination by the VHL E3 ubiquitin ligase. Our findings link globin gene regulation with canonical hypoxia adaptation, provide a mechanism for HbF induction during stress erythropoiesis and suggest a new therapeutic approach for ß-haemoglobinopathies.

MicroRNA-451 Escapes Global MicroRNA Crisis by Clustered Neighboring MicroRNA-144 During Erythropoiesis.

In a recent issue of Molecular Cell, Kretov et al. (2020) demonstrate that microRNA-144 targets Dicer in a negative feedback loop, affecting global canonical microRNA expression in erythrocytes. MicroRNA-451 is refractory to the loss of Dicer because of its Ago2-dependent processing.

Absolute Quantification of Transcription Factors Reveals Principles of Gene Regulation in Erythropoiesis.

Dynamic cellular processes such as differentiation are driven by changes in the abundances of transcription factors (TFs). However, despite years of studies, our knowledge about the protein copy number of TFs in the nucleus is limited. Here, by determining the absolute abundances of 103 TFs and co-factors during the course of human erythropoiesis, we provide a dynamic and quantitative scale for TFs in the nucleus. Furthermore, we establish the first gene regulatory network of cell fate commitment that integrates temporal protein stoichiometry data with mRNA measurements. The model revealed quantitative imbalances in TFs' cross-antagonistic relationships that underlie lineage determination. Finally, we made the surprising discovery that, in the nucleus, co-repressors are dramatically more abundant than co-activators at the protein level, but not at the RNA level, with profound implications for understanding transcriptional regulation. These analyses provide a unique quantitative framework to understand transcriptional regulation of cell differentiation in a dynamic context.

Ago2-Dependent Processing Allows miR-451 to Evade the Global MicroRNA Turnover Elicited during Erythropoiesis.

MicroRNAs (miRNAs) are sequentially processed by two RNase III enzymes, Drosha and Dicer. miR-451 is the only known miRNA whose processing bypasses Dicer and instead relies on the slicer activity of Argonaute-2 (Ago2). miR-451 is highly conserved in vertebrates and regulates erythrocyte maturation, where it becomes the most abundant miRNA. However, the basis for the non-canonical biogenesis of miR-451 is unclear. Here, we show that Ago2 is less efficient than Dicer in processing pre-miRNAs, but this deficit is overcome when miR-144 represses Dicer in a negative-feedback loop during erythropoiesis. Loss of miR-144-mediated Dicer repression in zebrafish embryos and human cells leads to increased canonical miRNA production and impaired miR-451 maturation. Overexpression of Ago2 rescues some of the defects of miR-451 processing. Thus, the evolution of Ago2-dependent processing allows miR-451 to circumvent the global repression of canonical miRNAs elicited, in part, by the miR-144 targeting of Dicer during erythropoiesis.

The HIF signaling pathway in osteoblasts directly modulates erythropoiesis through the production of EPO.

Osteoblasts are an important component of the hematopoietic microenvironment in bone. However, the mechanisms by which osteoblasts control hematopoiesis remain unknown. We show that augmented HIF signaling in osteoprogenitors results in HSC niche expansion associated with selective expansion of the erythroid lineage. Increased red blood cell production occurred in an EPO-dependent manner with increased EPO expression in bone and suppressed EPO expression in the kidney. In contrast, inactivation of HIF in osteoprogenitors reduced EPO expression in bone. Importantly, augmented HIF activity in osteoprogenitors protected mice from stress-induced anemia. Pharmacologic or genetic inhibition of prolyl hydroxylases1/2/3 in osteoprogenitors elevated EPO expression in bone and increased hematocrit. These data reveal an unexpected role for osteoblasts in the production of EPO and modulation of erythropoiesis. Furthermore, these studies demonstrate a molecular role for osteoblastic PHD/VHL/HIF signaling that can be targeted to elevate both HSCs and erythroid progenitors in the local hematopoietic microenvironment.