Amanda J. Stranks

The autophagy protein Atg7 is essential for hematopoietic stem cell maintenance

Adult mouse LSK cells unable to undergo autophagy contain fewer HSCs, accumulate mitochondria, and fail to reconstitute lethally irradiated mice.

Autophagy is activated in systemic lupus erythematosus and required for plasmablast development

Background Autophagy has emerged as a critical homeostatic mechanism in T lymphocytes, influencing proliferation and differentiation. Autophagy in B cells has been less studied, but genetic deficiency causes impairment of early and late developmental stages Objectives To explore the role of autophagy in the pathogenesis of human and murine lupus, a disease in which B cells are critical effectors of pathology. Methods Autophagy was assessed using multiple techniques in NZB/W and control mice, and in patients with systemic lupus erythematosus (SLE) compared to healthy controls. We evaluated the phenotype of the B cell compartment in Vav-Atg7−/− mice in vivo, and examined human and murine plasmablast formation following inhibition of autophagy. Results We found activation of autophagy in early developmental and transitional stages of B cell development in a lupus mouse model even before disease onset, and which progressively increased with age. In human disease, again autophagy was activated compared with healthy controls, principally in naïve B cells. B cells isolated from Vav-Atg7F/F mice failed to effectively differentiate into plasma cells following stimulation in vitro. Similarly, human B cells stimulated in the presence of autophagy inhibition did not differentiate into plasmablasts. Conclusions Our data suggest activation of autophagy is a mechanism for survival of autoreactive B cells, and also demonstrate that it is required for plasmablast differentiation, processes that induce significant cellular stress. The implication of autophagy in two major pathogenic pathways in SLE suggests the potential to use inhibition of autophagy as a novel treatment target in this frequently severe autoimmune disease.

Activation of Autophagy by α-Herpesviruses in Myeloid Cells Is Mediated by Cytoplasmic Viral DNA through a Mechanism Dependent on Stimulator of IFN Genes

Autophagy has been established as a player in host defense against viruses. The mechanisms by which the host induces autophagy during infection are diverse. In the case of HSV type 1 (HSV-1), dsRNA-dependent protein kinase is essential for induction of autophagy in fibroblasts through phosphorylation of eukaryotic initiation factor 2α (eIF2α). HSV-1 counteracts autophagy via ICP34.5, which dephosphorylates eIF2α and inhibits Beclin 1. Investigation of autophagy during HSV-1 infection has largely been conducted in permissive cells, but recent work suggests the existence of a eIF2α-independent autophagy-inducing pathway in nonpermissive cells. To clarify and further characterize the existence of a novel autophagy-inducing pathway in nonpermissive cells, we examined different HSV and cellular components in murine myeloid cells for their role in autophagy. We demonstrate that HSV-1–induced autophagy does not correlate with phosphorylation of eIF2α, is independent of functional dsRNA-dependent protein kinase, and is not antagonized by ICP34.5. Autophagy was activated independent of viral gene expression, but required viral entry. Importantly, we found that the presence of genomic DNA in the virion was essential for induction of autophagy and, conversely, that transfection of HSV-derived DNA induced microtubule-associated protein 1 L chain II formation, a marker of autophagy. This occurred through a mechanism dependent on stimulator of IFN genes, an essential component for the IFN response to intracellular DNA. Finally, we observed that HSV-1 DNA was present in the cytosol devoid of capsid material following HSV-1 infection of dendritic cells. Thus, our data suggest that HSV-1 genomic DNA induces autophagy in nonpermissive cells in a stimulator of IFN gene-dependent manner.

Essential role for autophagy during invariant NKT cell development

Significance Autophagy is an evolutionarily conserved catabolic process essential to maintaining cellular homeostasis through the breakdown and recycling of damaged organelles and long-lived proteins. We report that autophagy plays an essential cell-intrinsic role in maintaining the survival of a subset of innate-like cells known as invariant natural killer T (iNKT) cells. Autophagy deficiency prevents transition to a quiescent state after population expansion of thymic iNKT cells. Hence, autophagy-deficient iNKT cells accumulate mitochondria and oxygen radicals and subsequently die of apoptosis. Autophagy is an evolutionarily conserved cellular homeostatic pathway essential for development, immunity, and cell death. Although autophagy modulates MHC antigen presentation, it remains unclear whether autophagy defects impact on CD1d lipid loading and presentation to invariant natural killer T (iNKT) cells and on iNKT cell differentiation in the thymus. Furthermore, it remains unclear whether iNKT and conventional T cells have similar autophagy requirements for differentiation, survival, and/or activation. We report that, in mice with a conditional deletion of the essential autophagy gene Atg7 in the T-cell compartment (CD4 Cre-Atg7−/−), thymic iNKT cell development—unlike conventional T-cell development—is blocked at an early stage and mature iNKT cells are absent in peripheral lymphoid organs. The defect is not due to altered loading of intracellular iNKT cell agonists; rather, it is T-cell–intrinsic, resulting in enhanced susceptibility of iNKT cells to apoptosis. We show that autophagy increases during iNKT cell thymic differentiation and that it developmentally regulates mitochondrial content through mitophagy in the thymus of mice and humans. Autophagy defects result in the intracellular accumulation of mitochondrial superoxide species and subsequent apoptotic cell death. Although autophagy-deficient conventional T cells develop normally, they show impaired peripheral survival, particularly memory CD8+ T cells. Because iNKT cells, unlike conventional T cells, differentiate into memory cells while in the thymus, our results highlight a unique autophagy-dependent metabolic regulation of adaptive and innate T cells, which is required for transition to a quiescent state after population expansion.

Autophagy limits proliferation and glycolytic metabolism in acute myeloid leukemia.

Decreased autophagy contributes to malignancies; however, it is unclear how autophagy has an impact on tumor 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 cells (HSPC) where transformation occurs is well characterized and (iii) loss of the key autophagy gene Atg7 in 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 with more committed/mature hematopoietic cells, healthy human and mouse HSPCs 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 tumor growth.

A humanized mouse model of anaphylactic peanut allergy

Background: Food allergy is a growing health problem with very limited treatment options. Investigation of the immunologic pathways underlying allergic sensitization to foods in humans has been greatly constrained by the limited availability of intestinal tissue and gut‐resident immune cells. Although mouse models have offered insights into pathways of food sensitization, differences between rodent and human immune physiology limit the extension of these findings to our understanding of human disease. Objective: We sought to develop a strategy for the generation of mice with humanized adaptive immune systems, complete with tissue engraftment by human mast cells that are competent to mount specific IgE‐mediated responses and drive systemic anaphylaxis on ingestion challenge. Methods: Nonobese diabetic severe combined immunodeficient mice lacking the cytokine receptor common gamma chain (&ggr;c−/−) and carrying a human stem cell factor transgene were engrafted with human hematopoietic stem cells. The impact of peanut (PN) feeding and IgE neutralization on the development of immune responses, mast cell homeostasis, and anaphylactic food allergy was assessed in these animals. Results: Humanized nonobese diabetic severe combined immunodeficient common gamma chain–deficient stem cell factor (huNSG) mice exhibited robust engraftment with functional human T and B lymphocytes and human mast cells were found in significant numbers in their tissues, including the intestinal mucosa. Following gavage feeding with PN, they mounted specific antibody responses, including PN‐specific IgE. When enterally challenged with PN, they exhibited mast‐cell–mediated systemic anaphylaxis, as indicated by hypothermia and increases in plasma tryptase levels. Anti‐IgE (omalizumab) treatment ablated this anaphylactic response. Conclusions: huNSG mice provide a novel tool for studying food allergy and IgE‐mediated anaphylaxis.

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

Summary 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.

Allergen-specific IgG antibody signaling through FcγRIIb promotes food tolerance

Background: Patients with food allergy produce high‐titer IgE antibodies that bind to mast cells through Fc&egr;RI and trigger immediate hypersensitivity reactions on antigen encounter. Food‐specific IgG antibodies arise in the setting of naturally resolving food allergy and accompany the acquisition of food allergen unresponsiveness in oral immunotherapy. Objective: In this study we sought to delineate the effects of IgG and its inhibitory Fc receptor, Fc&ggr;RIIb, on both de novo allergen sensitization in naive animals and on established immune responses in the setting of pre‐existing food allergy. Methods: Allergen‐specific IgG was administered to mice undergoing sensitization and desensitization to the model food allergen ovalbumin. Cellular and molecular mechanisms were interrogated by using mast cell– and Fc&ggr;RIIb‐deficient mice. The requirement for Fc&ggr;RII in IgG‐mediated inhibition of human mast cells was investigated by using a neutralizing antibody. Results: Administration of specific IgG to food allergy–prone IL4raF709 mice during initial food exposure prevented the development of IgE antibodies, TH2 responses, and anaphylactic responses on challenge. When given as an adjunct to oral desensitization in mice with established IgE‐mediated hypersensitivity, IgG facilitated tolerance restoration, favoring expansion of forkhead box protein 3–positive regulatory T cells along with suppression of existing TH2 and IgE responses. IgG and Fc&ggr;RIIb suppress adaptive allergic responses through effects on mast cell function. Conclusion: These findings suggest that allergen‐specific IgG antibodies can act to induce and sustain immunologic tolerance to foods.

IgE promotes type 2 innate lymphoid cells in murine food allergy.

Mast cells serve an important sentinel function at mucosal barriers and have been implicated as key early inducers of type 2 immune responses in food allergy. The generation of Th2 and IgE following food allergen ingestion is inhibited in the absence of mast cells. Group 2 innate lymphoid cells are also thought to play an important early role in nascent allergic responses.

Tissue-Specific Expression of the Low-Affinity IgG Receptor, FcγRIIb, on Human Mast Cells

Immediate hypersensitivity reactions are induced by the interaction of allergens with specific IgE antibodies bound via FcεRI to mast cells and basophils. While these specific IgE antibodies are needed to trigger such reactions, not all individuals harboring IgE exhibit symptoms of allergy. The lack of responsiveness seen in some subjects correlates with the presence of IgG antibodies of the same specificity. In cell culture studies and in vivo animal models of food allergy and anaphylaxis such IgG antibodies have been shown to exert suppression via FcγRIIb. However, the reported absence of this inhibitory receptor on primary mast cells derived from human skin has raised questions about the role of IgG-mediated inhibition of immediate hypersensitivity in human subjects. Here, we tested the hypothesis that mast cell FcγRIIb expression might be tissue specific. Utilizing a combination of flow cytometry, quantitative PCR, and immunofluorescence staining of mast cells derived from the tissues of humanized mice, human skin, or in fixed paraffin-embedded sections of human tissues, we confirm that FcγRIIb is absent from dermal mast cells but is expressed by mast cells throughout the gastrointestinal tract. IgE-induced systemic anaphylaxis in humanized mice is strongly inhibited by antigen-specific IgG. These findings support the concept that IgG, signaling via FcγRIIb, plays a physiological role in suppressing hypersensitivity reactions.