Aging and chronic inflammation are independent risk factors for the development of atherothrombosis and cardiovascular disease. We hypothesized that aging-associated inflammation promotes the development of platelet hyperreactivity and increases thrombotic risk during aging. Functional platelet studies in aged-frail adults and old mice demonstrated that their platelets are hyperreactive and form larger thrombi. We identified tumor necrosis factor α (TNF-α) as the key aging-associated proinflammatory cytokine responsible for platelet hyperreactivity. We further showed that platelet hyperreactivity is neutralized by abrogating signaling through TNF-α receptors in vivo in a mouse model of aging. Analysis of the bone marrow compartments showed significant platelet-biased hematopoiesis in old mice reflected by increased megakaryocyte-committed progenitor cells, megakaryocyte ploidy status, and thrombocytosis. Single-cell RNA-sequencing analysis of native mouse megakaryocytes showed significant reprogramming of inflammatory, metabolic, and mitochondrial gene pathways in old mice that appeared to play a significant role in determining platelet hyperreactivity. Platelets from old mice (where TNF-α was endogenously increased) and from young mice exposed to exogenous TNF-α exhibited significant mitochondrial changes characterized by elevated mitochondrial mass and increased oxygen consumption during activation. These mitochondrial changes were mitigated upon TNF-α blockade. Similar increases in platelet mitochondrial mass were seen in platelets from patients with myeloproliferative neoplasms, where TNF-α levels are also increased. Furthermore, metabolomics studies of platelets from young and old mice demonstrated age-dependent metabolic profiles that may differentially poise platelets for activation. Altogether, we present previously unrecognized evidence that TNF-α critically regulates megakaryocytes resident in the bone marrow niche and aging-associated platelet hyperreactivity and thrombosis.

SummaryDifferentiation of nitrogen-fixing cells, called heterocysts, by the cyanobacterium Anabaena sp. strain PCC 7120 requires HetR, which is considered the master regulator of heterocyst differentiation. In this study, ectopic expression of hetP from an inducible promoter was shown to partially bypass the need for hetR in heterocyst differentiation. A hetR-deletion strain with hetP expressed ectopically produced cells morphologically similar to heterocysts that produced exopolysaccharide and glycolipids specific to heterocysts, and nitrogenase activity was present under anaerobic conditions, as indicated by an acetylene reduction assay. Five potential transcription start points or transcript processing sites located at positions -727, -545, -208, -177 and -12 bp relative to the putative translational start site were identified in the promoter region of hetP. Relative levels of transcription and their location in filaments resembled that previously reported for HetR protein in various genetic backgrounds, and an inverted repeat sequence in the promoter of hetP was necessary for binding of HetR. Two of the three identified homologues of hetP found in PCC 7120 partially complemented a hetP-null mutant. Taken together, these results suggest that HetP functions directly downstream of HetR in the regulatory network responsible for heterocyst differentiation.

SummaryNitrogen-fixing heterocysts are arranged in a periodic pattern on filaments of the cyanobacterium Anabaena sp. strain PCC 7120 under conditions of limiting combined nitrogen. Patterning requires two inhibitors of heterocyst differentiation, PatS and HetN, which work at different stages of differentiation by laterally suppressing levels of an activator of differentiation, HetR, in cells adjacent to source cells. Here we show that the RGSGR sequence in the 287-amino-acid HetN protein, which is shared by PatS, is critical for patterning. Conservative substitutions in any of the five amino acids lowered the extent to which HetN inhibited differentiation when overproduced and altered the pattern of heterocysts in filaments with an otherwise wild-type genetic background. Conversely, substitution of amino acids comprising the putative catalytic triad of this predicted reductase had no effect on inhibition or patterning. Deletion of putative domains of HetN suggested that the RGSGR motif is the primary component of HetN required for both its inhibitory and patterning activity, and that localization to the cell envelope is not required for patterning of heterocysts. The intercellular signalling proteins PatS and HetN use the same amino acid motif to regulate different stages of heterocyst patterning.

Hematopoietic stem cells (HSCs) are capable of entering the cell cycle to replenish the blood system in response to inflammatory cues; however, excessive proliferation in response to chronic inflammation can lead to either HSC attrition or expansion. The mechanism(s) that limit HSC proliferation and expansion triggered by inflammatory signals are poorly defined. Here, we show that long-term HSCs (HSCLT) rapidly repress protein synthesis and cell cycle genes following treatment with the proinflammatory cytokine interleukin (IL)-1. This gene program is associated with activation of the transcription factor PU.1 and direct PU.1 binding at repressed target genes. Notably, PU.1 is required to repress cell cycle and protein synthesis genes, and IL-1 exposure triggers aberrant protein synthesis and cell cycle activity in PU.1-deficient HSCs. These features are associated with expansion of phenotypic PU.1-deficient HSCs. Thus, we identify a PU.1-dependent mechanism triggered by innate immune stimulation that limits HSC proliferation and pool size. These findings provide insight into how HSCs maintain homeostasis during inflammatory stress.

The early events that drive myeloid oncogenesis are not well understood. Most studies focus on the cell-intrinsic genetic changes and how they impact cell fate decisions. We consider how chronic exposure to the proinflammatory cytokine, interleukin-1β (IL-1β), impacts Cebpa-knockout hematopoietic stem and progenitor cells (HSPCs) in competitive settings. Surprisingly, we found that Cebpa loss did not confer a hematopoietic cell–intrinsic competitive advantage; rather chronic IL-1β exposure engendered potent selection for Cebpa loss. Chronic IL-1β augments myeloid lineage output by activating differentiation and repressing stem cell gene expression programs in a Cebpa-dependent manner. As a result, Cebpa-knockout HSPCs are resistant to the prodifferentiative effects of chronic IL-1β, and competitively expand. We further show that ectopic CEBPA expression reduces the fitness of established human acute myeloid leukemias, coinciding with increased differentiation. These findings have important implications for the earliest events that drive hematologic disorders, suggesting that chronic inflammation could be an important driver of leukemogenesis and a potential target for intervention.

While dietary folate deficiency is associated with increased risk for birth defects and other diseases, evidence suggests that supplementation with folic acid can contribute to predisposition to some diseases, including immune dysfunction and cancer. Herein, we show that diets supplemented with folic acid both below and above the recommended levels led to significantly altered metabolism in multiple tissues in mice. Surprisingly, both low and excessive dietary folate induced similar metabolic changes, which were particularly evident for nucleotide biosynthetic pathways in B-progenitor cells. Diet-induced metabolic changes in these cells partially phenocopied those observed in mice treated with anti-folate drugs, suggesting that both deficiency and excessive levels of dietary folic acid compromise folate-dependent biosynthetic pathways. Both folate deficiency and excessive dietary folate levels compromise hematopoiesis, resulting in defective cell cycle progression, persistent DNA damage, and impaired production of lymphocytes. These defects reduce the reconstitution potential in transplantation settings and increase radiation-induced mortality. We conclude that excessive folic acid supplementation can metabolically mimic dietary folate insufficiency, leading to similar functional impairment of hematopoiesis.

In the filamentous cyanobacterium Anabaena, patS and hetN encode peptide-derived signals with many of the properties of morphogens. These signals regulate the formation of a periodic pattern of heterocysts by lateral inhibition of differentiation. Here we show that intercellular transfer of the patS-and hetN-dependent developmental signals from heterocysts to vegetative cells requires HetC, a predicted ATP-binding cassette transporter (ABC transporter). Relative to the wild type, in a hetC mutant differentiation resulted in a reduced number of heterocysts that were incapable of nitrogen fixation, but deletion of patS or hetN restored heterocyst number and function in a hetC background. These epistasis results suggest that HetC is necessary for conferring self-immunity to the inhibitors on differentiating cells. Nine hours after induction of differentiation, HetC was required for neither induction of transcription of patS nor intercellular transfer of the patS-encoded signal to neighboring cells. Conversely, in strains lacking HetC, the patS-and hetN-encoded signals were not transferred from heterocyst cells to adjacent vegetative cells. The results support a model in which the patS-dependent signal is initially transferred between vegetative cells in a HetCindependent fashion, but some time before morphological differentiation of heterocysts is complete, transfer of both signals transitions to a HetC-dependent process. IMPORTANCEHow chemical cues that regulate pattern formation in multicellular organisms move from one cell to another is a central question in developmental biology. In this study, we show that an ABC transporter, HetC, is necessary for transport of two developmental signals between different types of cells in a filamentous cyanobacterium. ABC transporters are found in organisms as diverse as bacteria and humans and, as the name implies, are often involved in the transport of molecules across a cellular membrane. The activity of HetC was shown to be required for signaling between heterocysts, which supply fixed nitrogen to the organism, and other cells, as well as for conferring immunity to self-signaling on developing heterocysts. Differentiation of heterocysts by filamentous cyanobacteria represents a simple but elegant model of biological patterning. In response to a shortage of combined nitrogen, linear filaments of Anabaena sp. strain PCC 7120 (here, Anabaena) form a periodic pattern of single, terminally differentiated, nitrogen-fixing heterocysts separated by approximately 10 totipotent vegetative cells. The spacing between heterocysts reflects the metabolic interdependence of the two cell types; fixed nitrogen is supplied to vegetative cells from heterocysts, and in return, heterocysts receive a source of carbon and reductant to compensate for their lack of photosystem II and the Calvin cycle (for a review of heterocyst differentiation, see reference 1). As filaments lengthen by growth and division of vegetative cells, subsequent rounds of heterocyst differentiation place new hetero...

Recent reports by Martincorena et al and Yokoyama et al reveal unanticipated dynamics of somatic evolution in the esophageal epithelium, with clonal expansions apparently driven by mutations in Notch1 dominating the epithelium even in middle‐aged individuals, far outpacing the prevalence of these mutations in esophageal cancers. We propose a model whereby the promotion of clonal expansions by mutations such as in Notch1 can limit more malignant somatic evolutionary trajectories until old ages.