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.

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 FcεRI 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, FcγRIIb, 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 FcγRIIb-deficient mice. The requirement for FcγRII 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 FcγRIIb 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.

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 (γc-/-) 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 Controls Acquisition of Aging Features in Macrophages.

Macrophages provide a bridge linking innate and adaptive immunity. An increased frequency of macrophages and other myeloid cells paired with excessive cytokine production is commonly seen in the aging immune system, known as 'inflamm-aging'. It is presently unclear how healthy macrophages are maintained throughout life and what connects inflammation with myeloid dysfunction during aging. Autophagy, an intracellular degradation mechanism, has known links with aging and lifespan extension. Here, we show for the first time that autophagy regulates the acquisition of major aging features in macrophages. In the absence of the essential autophagy gene Atg7, macrophage populations are increased and key functions such as phagocytosis and nitrite burst are reduced, while the inflammatory cytokine response is significantly increased - a phenotype also observed in aged macrophages. Furthermore, reduced autophagy decreases surface antigen expression and skews macrophage metabolism toward glycolysis. We show that macrophages from aged mice exhibit significantly reduced autophagic flux compared to young mice. These data demonstrate that autophagy plays a critical role in the maintenance of macrophage homeostasis and function, regulating inflammation and metabolism and thereby preventing immunosenescence. Thus, autophagy modulation may prevent excess inflammation and preserve macrophage function during aging, improving immune responses and reducing the morbidity and mortality associated with inflamm-aging.

Essential role for autophagy during invariant NKT cell development.

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.

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.

The autophagy protein Atg7 is essential for hematopoietic stem cell maintenance.

The role of autophagy, a lysosomal degradation pathway which prevents cellular damage, in the maintenance of adult mouse hematopoietic stem cells (HSCs) remains unknown. Although normal HSCs sustain life-long hematopoiesis, malignant transformation of HSCs leads to leukemia. Therefore, mechanisms protecting HSCs from cellular damage are essential to prevent hematopoietic malignancies. In this study, we crippled autophagy in HSCs by conditionally deleting the essential autophagy gene Atg7 in the hematopoietic system. This resulted in the loss of normal HSC functions, a severe myeloproliferation, and death of the mice within weeks. The hematopoietic stem and progenitor cell compartment displayed an accumulation of mitochondria and reactive oxygen species, as well as increased proliferation and DNA damage. HSCs within the Lin(-)Sca-1(+)c-Kit(+) (LSK) compartment were significantly reduced. Although the overall LSK compartment was expanded, Atg7-deficient LSK cells failed to reconstitute the hematopoietic system of lethally irradiated mice. Consistent with loss of HSC functions, the production of both lymphoid and myeloid progenitors was impaired in the absence of Atg7. Collectively, these data show that Atg7 is an essential regulator of adult HSC maintenance.