T Cell-Expressed microRNA-155 Reduces Lifespan in a Mouse Model of Age-Related Chronic Inflammation.

Aging-related chronic inflammation is a risk factor for many human disorders through incompletely understood mechanisms. Aged mice deficient in microRNA (miRNA/miR)-146a succumb to life-shortening chronic inflammation. In this study, we report that miR-155 in T cells contributes to shortened lifespan of miR-146a-/- mice. Using single-cell RNA sequencing and flow cytometry, we found that miR-155 promotes the activation of effector T cell populations, including T follicular helper cells, and increases germinal center B cells and autoantibodies in mice aged over 15 months. Mechanistically, aerobic glycolysis genes are elevated in T cells during aging, and upon deletion of miR-146a, in a T cell miR-155-dependent manner. Finally, skewing T cell metabolism toward aerobic glycolysis by deleting mitochondrial pyruvate carrier recapitulates age-dependent T cell phenotypes observed in miR-146a-/- mice, revealing the sufficiency of metabolic reprogramming to influence immune cell functions during aging. Altogether, these data indicate that T cell-specific miRNAs play pivotal roles in regulating lifespan through their influences on inflammaging.

MicroRNAs and acute myeloid leukemia: therapeutic implications and emerging concepts.

Acute myeloid leukemia (AML) is a deadly hematologic malignancy characterized by the uncontrolled growth of immature myeloid cells. Over the past several decades, we have learned a tremendous amount regarding the genetic aberrations that govern disease development in AML. Among these are genes that encode noncoding RNAs, including the microRNA (miRNA) family. miRNAs are evolutionarily conserved small noncoding RNAs that display important physiological effects through their posttranscriptional regulation of messenger RNA targets. Over the past decade, studies have identified miRNAs as playing a role in nearly all aspects of AML disease development, including cellular proliferation, survival, and differentiation. These observations have led to the study of miRNAs as biomarkers of disease, and efforts to therapeutically manipulate miRNAs to improve disease outcome in AML are ongoing. Although much has been learned regarding the importance of miRNAs in AML disease initiation and progression, there are many unanswered questions and emerging facets of miRNA biology that add complexity to their roles in AML. Moving forward, answers to these questions will provide a greater level of understanding of miRNA biology and critical insights into the many translational applications for these small regulatory RNAs in AML.

miR-155 promotes FLT3-ITD-induced myeloproliferative disease through inhibition of the interferon response.

FLT3-ITD+ acute myeloid leukemia (AML) accounts for ∼25% of all AML cases and is a subtype that carries a poor prognosis. microRNA-155 (miR-155) is specifically overexpressed in FLT3-ITD+ AML compared with FLT3 wild-type (FLT3-WT) AML and is critical for the growth of FLT3-ITD+ AML cells in vitro. However, miR-155's role in regulating FLT3-ITD-mediated disease in vivo remains unclear. In this study, we used a genetic mouse model to determine whether miR-155 influences the development of FLT3-ITD-induced myeloproliferative disease. Results indicate that miR-155 promotes FLT3-ITD-induced myeloid expansion in the bone marrow, spleen, and peripheral blood. Mechanistically, miR-155 increases proliferation of the hematopoietic stem and progenitor cell compartments by reducing the growth-inhibitory effects of the interferon (IFN) response, and this involves targeting of Cebpb. Consistent with our observations in mice, primary FLT3-ITD+ AML clinical samples have significantly higher miR-155 levels and a lower IFN response compared with FLT3-WT AML samples. Further, inhibition of miR-155 in FLT3-ITD+ AML cell lines using CRISPR/Cas9, or primary FLT3-ITD+ AML samples using locked nucleic acid antisense inhibitors, results in an elevated IFN response and reduces colony formation. Altogether, our data reveal that miR-155 collaborates with FLT3-ITD to promote myeloid cell expansion in vivo and that this involves a multitarget mechanism that includes repression of IFN signaling.

Antitumor immunity is defective in T cell-specific microRNA-155-deficient mice and is rescued by immune checkpoint blockade.

MicroRNA-155 (miR-155) regulates antitumor immune responses. However, its specific functions within distinct immune cell types have not been delineated in conditional KO mouse models. In this study, we investigated the role of miR-155 specifically within T cells during the immune response to syngeneic tumors. We found that miR-155 expression within T cells is required to limit syngeneic tumor growth and promote IFNγ production by T cells within the tumor microenvironment. Consequently, we found that miR-155 expression by T cells is necessary for proper tumor-associated macrophage expression of IFNγ-inducible genes. We also found that immune checkpoint-blocking (ICB) antibodies against programmed cell death protein 1/programmed death ligand 1 (PD-1/PD-L1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) restored antitumor immunity in miR-155 T cell-conditional KO mice. We noted that these ICB antibodies rescued the levels of IFNγ-expressing T cells, expression of multiple activation and effector genes expressed by tumor-infiltrating CD8+ and CD4+ T cells, and tumor-associated macrophage activation. Moreover, the ICB approach partially restored expression of several derepressed miR-155 targets in tumor-infiltrating, miR-155-deficient CD8+ T cells, suggesting that miR-155 and ICB regulate overlapping pathways to promote antitumor immunity. Taken together, our findings highlight the multifaceted role of miR-155 in T cells, in which it promotes antitumor immunity. These results suggest that the augmentation of miR-155 expression could be used to improve anticancer immunotherapies.

Reductions in the mitochondrial ABC transporter Abcb10 affect the transcriptional profile of heme biosynthesis genes.

ATP-binding cassette subfamily B member 10 (Abcb10) is a mitochondrial ATP-binding cassette (ABC) transporter that complexes with mitoferrin1 and ferrochelatase to enhance heme biosynthesis in developing red blood cells. Reductions in Abcb10 levels have been shown to reduce mitoferrin1 protein levels and iron import into mitochondria, resulting in reduced heme biosynthesis. As an ABC transporter, Abcb10 binds and hydrolyzes ATP, but its transported substrate is unknown. Here, we determined that decreases in Abcb10 did not result in protoporphyrin IX accumulation in morphant-treated zebrafish embryos or in differentiated Abcb10-specific shRNA murine Friend erythroleukemia (MEL) cells in which Abcb10 was specifically silenced with shRNA. We also found that the ATPase activity of Abcb10 is necessary for hemoglobinization in MEL cells, suggesting that the substrate transported by Abcb10 is important in mediating increased heme biosynthesis during erythroid development. Inhibition of 5-aminolevulinic acid dehydratase (EC 4.2.1.24) with succinylacetone resulted in both 5-aminolevulinic acid (ALA) accumulation in control and Abcb10-specific shRNA MEL cells, demonstrating that reductions in Abcb10 do not affect ALA export from mitochondria and indicating that Abcb10 does not transport ALA. Abcb10 silencing resulted in an alteration in the heme biosynthesis transcriptional profile due to repression by the transcriptional regulator Bach1, which could be partially rescued by overexpression of Alas2 or Gata1, providing a mechanistic explanation for why Abcb10 shRNA MEL cells exhibit reduced hemoglobinization. In conclusion, our findings rule out that Abcb10 transports ALA and indicate that Abcb10's ATP-hydrolysis activity is critical for hemoglobinization and that the substrate transported by Abcb10 provides a signal that optimizes hemoglobinization.

miRNA-1 promotes acute myeloid leukemia cell pathogenesis through metabolic regulation.

Acute myeloid leukemia (AML) is a heterogeneous and deadly disease characterized by uncontrolled expansion of malignant blasts. Altered metabolism and dysregulated microRNA (miRNA) expression profiles are both characteristic of AML. However, there is a paucity of studies exploring how changes in the metabolic state of the leukemic cells regulate miRNA expression leading to altered cellular behavior. Here, we blocked pyruvate entry into mitochondria by deleting the Mitochondria Pyruvate Carrier (MPC1) gene in human AML cell lines, which decreased Oxidative Phosphorylation (OXPHOS). This metabolic shift also led to increased expression of miR-1 in the human AML cell lines tested. AML patient sample datasets showed that higher miR-1 expression correlates with reduced survival. Transcriptional and metabolic profiling of miR-1 overexpressing AML cells revealed that miR-1 increased OXPHOS, along with key metabolites that fuel the TCA cycle such as glutamine and fumaric acid. Inhibition of glutaminolysis decreased OXPHOS in miR-1 overexpressing MV4-11 cells, highlighting that miR-1 promotes OXPHOS through glutaminolysis. Finally, overexpression of miR-1 in AML cells exacerbated disease in a mouse xenograft model. Together, our work expands current knowledge within the field by uncovering novel connections between AML cell metabolism and miRNA expression that facilitates disease progression. Further, our work points to miR-1 as a potential new therapeutic target that may be used to disrupt AML cell metabolism and thus pathogenesis in the clinic.

Mitochondrial Pyruvate Carrier 1 Promotes Peripheral T Cell Homeostasis through Metabolic Regulation of Thymic Development.

Metabolic pathways regulate T cell development and function, but many remain understudied. Recently, the mitochondrial pyruvate carrier (MPC) was identified as the transporter that mediates pyruvate entry into mitochondria, promoting pyruvate oxidation. Here we find that deleting Mpc1, an obligate MPC subunit, in the hematopoietic system results in a specific reduction in peripheral αß T cell numbers. MPC1-deficient T cells have defective thymic development at the ß-selection, intermediate single positive (ISP)-to-double-positive (DP), and positive selection steps. We find that early thymocytes deficient in MPC1 display alterations to multiple pathways involved in T cell development. This results in preferred escape of more activated T cells. Finally, mice with hematopoietic deletion of Mpc1 are more susceptible to experimental autoimmune encephalomyelitis. Altogether, our study demonstrates that pyruvate oxidation by T cell precursors is necessary for optimal αß T cell development and that its deficiency results in reduced but activated peripheral T cell populations.

Neonatal NET-inhibitory factor and related peptides inhibit neutrophil extracellular trap formation.

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMNs), extrude molecular lattices of decondensed chromatin studded with histones, granule enzymes, and antimicrobial peptides that are referred to as neutrophil extracellular traps (NETs). NETs capture and contain bacteria, viruses, and other pathogens. Nevertheless, experimental evidence indicates that NETs also cause inflammatory vascular and tissue damage, suggesting that identifying pathways that inhibit NET formation may have therapeutic implications. Here, we determined that neonatal NET-inhibitory factor (nNIF) is an inhibitor of NET formation in umbilical cord blood. In human neonatal and adult neutrophils, nNIF inhibits key terminal events in NET formation, including peptidyl arginine deiminase 4 (PAD4) activity, neutrophil nuclear histone citrullination, and nuclear decondensation. We also identified additional nNIF-related peptides (NRPs) that inhibit NET formation. nNIFs and NRPs blocked NET formation induced by pathogens, microbial toxins, and pharmacologic agonists in vitro and in mouse models of infection and systemic inflammation, and they improved mortality in murine models of systemic inflammation, which are associated with NET-induced collateral tissue injury. The identification of NRPs as neutrophil modulators that selectively interrupt NET generation at critical steps suggests their potential as therapeutic agents. Furthermore, our results indicate that nNIF may be an important regulator of NET formation in fetal and neonatal inflammation.