Inflammatory signaling selectively disrupts GATA factor-regulated networks governing erythroid differentiation and hemoglobin synthesis Free

Chronic inflammation, often associated with aging, instigates anemia of inflammation (AI) that afflicts hundreds of thousands in the USA (Ganz, N Engl J Med. 2019). Human erythroid cells express cytokine/chemokine receptor genes, yet erythroid-intrinsic actions of many receptor agonists are unstudied, even though they correlate with AI. Using primary human hematopoietic stem and progenitor cells (HSPCs) cultured ex vivo and flow cytometric assays, we discovered an amalgam (TNFα, IFNγ, and IL-6) of inflammatory mediators implicated in AI synergistically abrogated erythropoiesis. To dissect the mechanism, we treated human HSPCs with or without the amalgam and harvested cells for single-cell RNA-sequencing (scRNA-seq) on days 9 and 13. Cell types were annotated using Azimuth human bone marrow as a reference (Oetjen et al., JCI Insight. 2018; Granja et al. Nat Biotechnol. 2019). There were no major cellularity changes between vehicle- and inflammation-treated day 9 cells. On day 13, >80% of vehicle-treated cells were late erythroid with few non-erythroid cells. The majority of late erythroid cells were lost, whereas early erythroid and select myeloid cells were retained in inflammation-treated cells. These results, which were confirmed by flow cytometric analysis and Giemsa staining, suggest that erythroblasts are hypersensitive to inflammation.

We performed differential gene expression (DEG) analysis in early and late erythroid cells using pseudobulk limma-voom workflow. Inflammation highly induced TNFα, IFNγ, and IL-6 targets established in non-erythroid systems in early and late populations. We asked if inflammation impacts transcription factors and cytokine receptors vital for erythroid survival and differentiation. Inflammation induced GATA2 in early (logFC=1.26, p-adj=5.17e^-4) and late erythroid cells (logFC=0.94, p-adj=4.68e^-3) and slightly reduced KLF1, LDB1, and EPOR; GATA1, ZFPM1, LMO2, NFE2, TAL1, and KIT were unaffected. Pseudotime analysis revealed GATA2 decreased over pseudotime and increased with inflammation. KLF1, LDB1 and EPOR decreased with inflammation, and GATA1 and KIT were constant. Inflammation increased GATA2 protein 2.0-fold (p=0.0001) without affecting GATA1. TNFα, but not IFNγ or IL-6, increased GATA2 (1.9-fold, p=0.0033), while GATA1 was unaltered. To test the hypothesis that GATA and inflammatory mechanisms intersect, we quantified expression of established targets. For the top 100 GATA1-regulated genes from G1E-ER-GATA1 rescue (Tanimura et al., EMBO Rep. 2016), inflammation mimicked GATA1 activation of select targets (e.g., ALAS2, SLC4A1, and HBE1) and antagonized GATA1 function to regulate another cohort, e.g., HBB and VIM implicated in terminal differentiation. GATA1 regulates 145 SLC genes in G1E-ER-GATA1 cells that mediate small molecule transport (Zwifelhofer et al., PLoS Genet. 2020). Inflammation promoted or antagonized GATA1 regulation of SLCs, including Zn²⁺ transporters SLC30A1 and SLC39A8 and adenosine transporters SLC29A1 and A2 with roles in erythroid survival and differentiation. Inflammation upregulated most GATA2-induced genes (GATA2 rescue in GATA2^+/- HUDEP2), consistent with elevated GATA2 mRNA and protein. As inflammation antagonized GATA2 regulation of SLC4A1, PRG2, and CD69, inflammation promoted or inhibited GATA factor function in a context-dependent mechanism.

DEG and pseudotime analysis revealed decreased HBB and increased HBG1 and HBG2 upon inflammation in late erythroid cells. Interrogation of established γ-globin regulators revealed BCL11A downregulation by inflammation in early (logFC=-1.17, p-adj=3.45e^-3) and late erythroid cells (logFC=-0.92, p-adj=2.53e^-3). Amalgam or IFNγ decreased BCL11A protein and increased γ-globin. BCL11A expression is activated by intronic enhancers that are gene editing targets for sickle cell disease and β-thalassemia (Canver et al., Nature. 2015). ATAC-seq revealed inflammation abrogated accessibility at all enhancers. Using gene editing strategies and multiomics, we are dissecting the underlying mechanisms.

In summary, our multiomic analyses with normal human erythroid progenitor cells unveiled mechanistic intersections between GATA factor and inflammation networks governing erythropoiesis. The study provide mechanistic insights into the control of erythropoiesis under normal and pathological conditions and translational opportunities vis-à-vis anemia of inflammation.