Autophagy preserves hematopoietic stem cells by restraining MTORC1-mediated cellular anabolism.

Adult stem cells are long-lived and quiescent with unique metabolic requirements. Macroautophagy/autophagy is a fundamental survival mechanism that allows cells to adapt to metabolic changes by degrading and recycling intracellular components. Here we address why autophagy depletion leads to a drastic loss of the stem cell compartment. Using inducible deletion of autophagy specifically in adult hematopoietic stem cells (HSCs) and in mice chimeric for autophagy-deficient and normal HSCs, we demonstrate that the stem cell loss is cell-intrinsic. Mechanistically, autophagy-deficient HSCs showed higher expression of several amino acid transporters (AAT) when compared to autophagy-competent cells, resulting in increased amino acid (AA) uptake. This was followed by sustained MTOR (mechanistic target of rapamycin) activation, with enlarged cell size, glucose uptake and translation, which is detrimental to the quiescent HSCs. MTOR inhibition by rapamycin treatment in vivo was able to rescue autophagy-deficient HSC loss and bone marrow failure and resulted in better reconstitution after transplantation. Our results suggest that targeting MTOR may improve aged stem cell function, promote reprogramming and stem cell transplantation.List of abbreviations: 5FU: fluoracil; AA: amino acids; AKT/PKB: thymoma viral proto-oncogene 1; ATF4: activating transcription factor 4; BafA: bafilomycin A1; BM: bone marrow; EIF2: eukaryotic initiation factor 2; EIF4EBP1/4EBP1: eukaryotic translation initiation factor 4E binding protein 1; KIT/CD117/c-Kit: KIT proto-oncogene receptor tyrosine kinase; HSCs: hematopoietic stem cells; HSPCs: hematopoietic stem and progenitor cells; Kyn: kynurenine; LSK: lineage- (Lin-), LY6A/Sca-1+, KIT/c-Kit/CD117+; LY6A/Sca-1: lymphocyte antigen 6 family member A; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; MTORC2: MTOR complex 2; OPP: O-propargyl-puromycin; PI3K: phosphoinositide 3-kinase; poly(I:C): polyinosinic:polycytidylic acid; RPS6/S6: ribosomal protein S6; tam: tamoxifen; TCA: tricarboxylic acid; TFEB: transcription factor EB; PTPRC/CD45: Protein Tyrosine Phosphatase Receptor Type C, CD45 antigen.

Transcriptomes and metabolism define mouse and human MAIT cell populations.

Mucosal-associated invariant T (MAIT) cells are a subset of T lymphocytes that respond to microbial metabolites. We defined MAIT cell populations in different organs and characterized the developmental pathway of mouse and human MAIT cells in the thymus using single-cell RNA sequencing and phenotypic and metabolic analyses. We showed that the predominant mouse subset, which produced IL-17 (MAIT17), and the subset that produced IFN-γ (MAIT1) had not only greatly different transcriptomes but also different metabolic states. MAIT17 cells in different organs exhibited increased lipid uptake, lipid storage, and mitochondrial potential compared with MAIT1 cells. All these properties were similar in the thymus and likely acquired there. Human MAIT cells in lung and blood were more homogeneous but still differed between tissues. Human MAIT cells had increased fatty acid uptake and lipid storage in blood and lung, similar to human CD8 T resident memory cells, but unlike mouse MAIT17 cells, they lacked increased mitochondrial potential. Although mouse and human MAIT cell transcriptomes showed similarities for immature cells in the thymus, they diverged more strikingly in the periphery. Analysis of pet store mice demonstrated decreased lung MAIT17 cells in these so-called "dirty" mice, indicative of an environmental influence on MAIT cell subsets and function.

Divergent metabolic programmes control two populations of MAIT cells that protect the lung.

Although mucosal-associated invariant T (MAIT) cells provide rapid, innate-like responses, they are not pre-set, and memory-like responses have been described for MAIT cells following infections. The importance of metabolism for controlling these responses, however, is unknown. Here, following pulmonary immunization with a Salmonella vaccine strain, mouse MAIT cells expanded as separate CD127-Klrg1+ and CD127+Klrg1- antigen-adapted populations that differed in terms of their transcriptome, function and localization in lung tissue. These populations remained altered from steady state for months as stable, separate MAIT cell lineages with enhanced effector programmes and divergent metabolism. CD127+ MAIT cells engaged in an energetic, mitochondrial metabolic programme, which was critical for their maintenance and IL-17A synthesis. This programme was supported by high fatty acid uptake and mitochondrial oxidation and relied on highly polarized mitochondria and autophagy. After vaccination, CD127+ MAIT cells protected mice against Streptococcus pneumoniae infection. In contrast, Klrg1+ MAIT cells had dormant but ready-to-respond mitochondria and depended instead on Hif1a-driven glycolysis to survive and produce IFN-γ. They responded antigen independently and participated in protection from influenza virus. These metabolic dependencies may enable tuning of memory-like MAIT cell responses for vaccination and immunotherapies.

Thymus-Derived CD4+CD8+ Cells Reside in Mediastinal Adipose Tissue and the Aortic Arch.

Double-positive CD4+CD8αß+ (DP) cells are thought to reside as T cell progenitors exclusively within the thymus. We recently discovered an unexpected CD4+ and CD8αß+ immune cell population in healthy and atherosclerotic mice by single-cell RNA sequencing. Transcriptomically, these cells resembled thymic DPs. Flow cytometry and three-dimensional whole-mount imaging confirmed DPs in thymus, mediastinal adipose tissue, and aortic adventitia, but nowhere else. Deep transcriptional profiling revealed differences between DP cells isolated from the three locations. All DPs were dependent on RAG2 expression and the presence of the thymus. Mediastinal adipose tissue DPs resided in close vicinity to invariant NKT cells, which they could activate in vitro. Thymus transplantation failed to reconstitute extrathymic DPs, and frequencies of extrathymic DPs were unaltered by pharmacologic inhibition of S1P1, suggesting that their migration may be locally confined. Our results define two new, transcriptionally distinct subsets of extrathymic DPs that may play a role in aortic vascular homeostasis.

Metabolic activation and colitis pathogenesis is prevented by lymphotoxin ß receptor expression in neutrophils.

Inflammatory bowel disease is characterized by an exacerbated intestinal immune response, but the critical mechanisms regulating immune activation remain incompletely understood. We previously reported that the TNF-superfamily molecule TNFSF14 (LIGHT) is required for preventing severe disease in mouse models of colitis. In addition, deletion of lymphotoxin beta receptor (LTßR), which binds LIGHT, also led to aggravated colitis pathogenesis. Here, we aimed to determine the cell type(s) requiring LTßR and the mechanism critical for exacerbation of colitis. Specific deletion of LTßR in neutrophils (LTßRΔN), but not in several other cell types, was sufficient to induce aggravated colitis and colonic neutrophil accumulation. Mechanistically, RNA-Seq analysis revealed LIGHT-induced suppression of cellular metabolism, and mitochondrial function, that was dependent on LTßR. Functional studies confirmed increased mitochondrial mass and activity, associated with excessive mitochondrial ROS production and elevated glycolysis at steady-state and during colitis. Targeting these metabolic changes rescued exacerbated disease severity. Our results demonstrate that LIGHT signals to LTßR on neutrophils to suppress metabolic activation and thereby prevents exacerbated immune pathogenesis during colitis.

Metabolic Triggers of Invariant Natural Killer T-Cell Activation during Sterile Autoinflammatory Disease.

Ample evidence exists for activation of invariant natural killer T (iNKT) cells in a sterile manner by endogenous ligands or microbial antigens from the commensal flora, indicating that iNKT cells are not truly self-tolerant. Their controlled autoreactivity state is disturbed in many types of sterile inflammatory disease, resulting in their central role in modulating autoimmune responses. This review focuses on sterile iNKT-cell responses that are initiated by metabolic triggers, such as obesity-associated inflammation and fatty liver disease, as a manifestation of metabolic disease and dyslipidemia, as well as ischemia reperfusion injuries and sickle cell disease, characterized by acute lack of oxygen and oxidative stress response on reperfusion. In the intestine, inflammation and iNKT-cell response type are shaped by the microbiome as an extended "self". Disease- and organ-specific differences in iNKT-cell response type are summarized and help to define common pathways that shape iNKT-cell responses in the absence of exogenous antigen.

Polyamines Control eIF5A Hypusination, TFEB Translation, and Autophagy to Reverse B Cell Senescence.

Failure to make adaptive immune responses is a hallmark of aging. Reduced B cell function leads to poor vaccination efficacy and a high prevalence of infections in the elderly. Here we show that reduced autophagy is a central molecular mechanism underlying immune senescence. Autophagy levels are specifically reduced in mature lymphocytes, leading to compromised memory B cell responses in old individuals. Spermidine, an endogenous polyamine metabolite, induces autophagy in vivo and rejuvenates memory B cell responses. Mechanistically, spermidine post-translationally modifies the translation factor eIF5A, which is essential for the synthesis of the autophagy transcription factor TFEB. Spermidine is depleted in the elderly, leading to reduced TFEB expression and autophagy. Spermidine supplementation restored this pathway and improved the responses of old human B cells. Taken together, our results reveal an unexpected autophagy regulatory mechanism mediated by eIF5A at the translational level, which can be harnessed to reverse immune senescence in humans.

The Tumor Necrosis Factor Superfamily Members TNFSF14 (LIGHT), Lymphotoxin ß and Lymphotoxin ß Receptor Interact to Regulate Intestinal Inflammation.

Over 1.5 million individuals in the United States are afflicted with inflammatory bowel disease (IBD). While the progression of IBD is multifactorial, chronic, unresolved inflammation certainly plays a key role. Additionally, while multiple immune mediators have been shown to affect pathogenesis, a comprehensive understanding of disease progression is lacking. Previous work has demonstrated that a member of the TNF superfamily, TNFSF14 (LIGHT), which is pro-inflammatory in several contexts, surprisingly plays an important role in protection from inflammation in mouse models of colitis, with LIGHT deficient mice having more severe disease pathogenesis. However, LIGHT is a single member of a complex signaling network. It signals through multiple receptors, including herpes virus entry mediator (HVEM) and lymphotoxin beta receptor (LTßR); these two receptors in turn can bind to other ligands. It remains unknown which receptors and competing ligands can mediate or counteract the outcome of LIGHT-signaling during colitis. Here we demonstrate that LIGHT signaling through LTßR, rather than HVEM, plays a critical role in the progression of DSS-induced colitis, as LTßR deficient mice exhibit a more severe disease phenotype. Further, mice deficient in LTαß do not exhibit differential colitis progression compared to WT mice. However, deletion of both LIGHT and LTαß, but not deletion of both LTαß and LTßR, resulted in a reversal of the adverse effects associated with the loss of LIGHT. In sum, the LIGHT/LTαß/LTßR signaling network contributes to DSS colitis, but there may be additional receptors or indirect effects, and therefore, the relationships between these receptors and ligands remains enigmatic.

B1a B cells require autophagy for metabolic homeostasis and self-renewal.

Specific metabolic programs are activated by immune cells to fulfill their functional roles, which include adaptations to their microenvironment. B1 B cells are tissue-resident, innate-like B cells. They have many distinct properties, such as the capacity to self-renew and the ability to rapidly respond to a limited repertoire of epitopes. The metabolic pathways that support these functions are unknown. We show that B1 B cells are bioenergetically more active than B2 B cells, with higher rates of glycolysis and oxidative phosphorylation, and depend on glycolysis. They acquire exogenous fatty acids and store lipids in droplet form. Autophagy is differentially activated in B1a B cells, and deletion of the autophagy gene Atg7 leads to a selective loss of B1a B cells caused by a failure of self-renewal. Autophagy-deficient B1a B cells down-regulate critical metabolic genes and accumulate dysfunctional mitochondria. B1 B cells, therefore, have evolved a distinct metabolism adapted to their residence and specific functional properties.

Autophagy dictates metabolism and differentiation of inflammatory immune cells.

The role of macroautophagy/autophagy, a conserved lysosomal degradation pathway, during cellular differentiation has been well studied over the last decade. In particular, evidence for its role during immune cell differentiation is growing. Despite the description of a variety of dramatic immune phenotypes in tissue-specific autophagy knockout models, the underlying mechanisms are still under debate. One of the proposed mechanisms is the impact of autophagy on the altered metabolic states during immune cell differentiation. This concept is strengthened through novel molecular insights into how AMPK and MTOR signaling cascades affect both autophagy and metabolism. In this review, we discuss direct and indirect evidence linking autophagy, metabolic pathways and immune cell differentiation including T, B, and innate lymphocytes as well as in myeloid cells that are direct mediators of inflammation. Herein, we propose a model for autophagy-driven immunometabolism controlling immune cell differentiation.

Autophagy-Dependent Generation of Free Fatty Acids Is Critical for Normal Neutrophil Differentiation.

Neutrophils are critical and short-lived mediators of innate immunity that require constant replenishment. Their differentiation in the bone marrow requires extensive cytoplasmic and nuclear remodeling, but the processes governing these energy-consuming changes are unknown. While previous studies show that autophagy is required for differentiation of other blood cell lineages, its function during granulopoiesis has remained elusive. Here, we have shown that metabolism and autophagy are developmentally programmed and essential for neutrophil differentiation in vivo. Atg7-deficient neutrophil precursors had increased glycolytic activity but impaired mitochondrial respiration, decreased ATP production, and accumulated lipid droplets. Inhibiting autophagy-mediated lipid degradation or fatty acid oxidation alone was sufficient to cause defective differentiation, while administration of fatty acids or pyruvate for mitochondrial respiration rescued differentiation in autophagy-deficient neutrophil precursors. Together, we show that autophagy-mediated lipolysis provides free fatty acids to support a mitochondrial respiration pathway essential to neutrophil differentiation.

Mechanistic roles of autophagy in hematopoietic differentiation.

Autophagy is increasingly recognized for its active role in development and differentiation. In particular, its role in the differentiation of hematopoietic cells has been extensively studied, likely because blood cells are accessible, easy to identify and purify, and their progenitor tree is well defined. This review aims to discuss the mechanisms by which autophagy impacts on differentiation, using hematopoietic cell types as examples. Autophagy's roles include the remodeling during terminal differentiation, the maintenance of a long-lived cell type, and the regulation of the balance between self-renewal and quiescence in stem-like cells. We discuss and compare the mechanistic roles of autophagy, such as prevention of apoptosis, supply of energy metabolites and metabolic adaption, and selective degradation of organelles and of regulatory factors.

The autophagy gene Atg16l1 differentially regulates Treg and TH2 cells to control intestinal inflammation.

A polymorphism in the autophagy gene Atg16l1 is associated with susceptibility to inflammatory bowel disease (IBD); however, it remains unclear how autophagy contributes to intestinal immune homeostasis. Here, we demonstrate that autophagy is essential for maintenance of balanced CD4(+) T cell responses in the intestine. Selective deletion of Atg16l1 in T cells in mice resulted in spontaneous intestinal inflammation that was characterized by aberrant type 2 responses to dietary and microbiota antigens, and by a loss of Foxp3(+) Treg cells. Specific ablation of Atg16l1 in Foxp3(+) Treg cells in mice demonstrated that autophagy directly promotes their survival and metabolic adaptation in the intestine. Moreover, we also identify an unexpected role for autophagy in directly limiting mucosal TH2 cell expansion. These findings provide new insights into the reciprocal control of distinct intestinal TH cell responses by autophagy, with important implications for understanding and treatment of chronic inflammatory disorders.

Autophagy limits proliferation and glycolytic metabolism in acute myeloid leukemia.

Decreased autophagy contributes to malignancies, however it is unclear how autophagy impacts on tumour growth. Acute myeloid leukemia (AML) is an ideal model to address this as (i) patient samples are easily accessible, (ii) the hematopoietic stem and progenitor population (HSPC) where transformation occurs is well characterized, and (iii) loss of the key autophagy gene Atg7 in hematopoietic stem and progenitor cells (HSPCs) leads to a lethal pre-leukemic phenotype in mice. Here we demonstrate that loss of Atg5 results in an identical HSPC phenotype as loss of Atg7, confirming a general role for autophagy in HSPC regulation. Compared to more committed/mature hematopoietic cells, healthy human and mouse HSCs displayed enhanced basal autophagic flux, limiting mitochondrial damage and reactive oxygen species in this long-lived population. Taken together, with our previous findings these data are compatible with autophagy limiting leukemic transformation. In line with this, autophagy gene losses are found within chromosomal regions that are commonly deleted in human AML. Moreover, human AML blasts showed reduced expression of autophagy genes, and displayed decreased autophagic flux with accumulation of unhealthy mitochondria indicating that deficient autophagy may be beneficial to human AML. Crucially, heterozygous loss of autophagy in an MLL-ENL model of AML led to increased proliferation in vitro, a glycolytic shift, and more aggressive leukemias in vivo. With autophagy gene losses also identified in multiple other malignancies, these findings point to low autophagy providing a general advantage for tumour growth.