Natural killer (NK) cells are critical to both innate and adaptive immunity. However, the development and heterogeneity of human NK cells are yet to be fully defined. Using single-cell RNA-sequencing technology, here we identify distinct NK populations in human bone marrow and blood, including one population expressing higher levels of immediate early genes indicative of a homeostatic activation. Functionally matured NK cells with high expression of CX3CR1 , HAVCR2 (TIM-3), and ZEB2 represents terminally differentiated status with the unique transcriptional profile. Transcriptomic and pseudotime analyses identify a transitional population between CD56 bright and CD56 dim NK cells. Finally, a donor with GATA2 T354M mutation exhibits reduced percentage of CD56 bright NK cells with altered transcriptome and elevated cell death. These data expand our understanding of the heterogeneity and development of human NK cells.

Rationale: A hallmark of chronic inflammatory disorders is persistence of pro-inflammatory macrophages in diseased tissues. In atherosclerosis this is associated with dyslipidemia and oxidative stress, but mechanisms linking these phenomena to macrophage activation remain incompletely understood. Objective: To investigate mechanisms linking dyslipidemia, oxidative stress and macrophage activation through modulation of immunometabolism, and to explore therapeutic potential targeting specific metabolic pathways. Methods and Results: Using a combination of biochemical, immunological, and ex vivo cell metabolic studies, we report that CD36 mediates a mitochondrial metabolic switch from oxidative phosphorylation to superoxide production in response to its ligand, oxLDL. Mitochondrial-specific inhibition of superoxide inhibited oxLDL-induced NF-κB activation and inflammatory cytokine generation. RNAseq, flow cytometry, 3H-labeled palmitic acid uptake, lipidomic analysis, confocal and EM imaging, and functional energetics revealed that oxLDL upregulated effectors of long-chain fatty acid (FA) uptake and mitochondrial import, while downregulating FA oxidation and inhibiting ATP5A, an electron transport chain (ETC) component. The combined effect is long-chain FA accumulation, alteration of mitochondrial structure and function, repurposing of the ETC to superoxide production, and NF-κB activation. Apoe null mice challenged with high fat diet showed similar metabolic changes in circulating Ly6C+ monocytes and peritoneal macrophages, along with increased CD36 expression. Moreover, mitochondrial ROS was positively correlated with CD36 expression in aortic lesional macrophages. Conclusions: These findings reveal that oxLDL/CD36 signaling in macrophages links dys-regulated FA metabolism to oxidative stress from the mitochondria, which drives chronic inflammation. Thus, targeting to CD36 and its downstream effectors may serve as potential new strategies against chronic inflammatory diseases such as atherosclerosis.

Acute myelogenous leukemia (AML) is a high-risk hematopoietic malignancy caused by a variety of mutations, including genes encoding the cohesin complex. Recent studies have demonstrated that reduction in cohesin complex levels leads to enhanced self-renewal in hematopoietic stem and progenitors (HSPCs). We sought to delineate the molecular mechanisms by which cohesin mutations promote enhanced HSPC self-renewal since this represents a critical initial step during leukemic transformation. We verified that RNAi against the cohesin subunit Rad21 causes enhanced self-renewal of HSPCs in vitro through derepression of Polycomb Repressive Complex 2 (PRC2) target genes, including Hoxa7 and Hoxa9. Importantly, knockdown of either Hoxa7 or Hoxa9 suppressed self-renewal, implying both are critical downstream effectors of reduced cohesin levels. We further demonstrate that the cohesin and PRC2 complexes interact and are bound in close proximity to Hoxa7 and Hoxa9. Rad21 depletion resulted in decreased levels of H3K27me3 at the Hoxa7 and Hoxa9 promoters, consistent with Rad21 being critical to proper gene silencing by recruiting the PRC2 complex. Our data demonstrates that the cohesin complex regulates PRC2 targeting to silence Hoxa7 and Hoxa9 and negatively regulate self-renewal. Our studies identify a novel epigenetic mechanism underlying leukemogenesis in AML patients with cohesin mutations.