Autophagy gene-dependent intracellular immunity triggered by interferon-γ.

Interferon-γ (IFNγ) is a critical mediator of cell-intrinsic immunity to intracellular pathogens. Understanding the complex cellular mechanisms supporting robust interferon-γ-induced host defenses could aid in developing new therapeutics to treat infections. Here, we examined the impact of autophagy genes in the interferon-γ-induced host response.

Approaches to Measuring Reductive and Oxidative Events in Phagosomes.

The phagosome is a redox-active organelle. Numerous reductive and oxidative systems play both direct and indirect roles in phagosomal function. With the advent of newer methodologies to study these redox events in live cells, the details of how redox conditions change within the maturing phagosome, how they are regulated, and how they influence other phagosomal functions can be investigated.

A pan-influenza antibody inhibiting neuraminidase via receptor mimicry.

Rapidly evolving influenza A viruses (IAVs) and influenza B viruses (IBVs) are major causes of recurrent lower respiratory tract infections. Current influenza vaccines elicit antibodies predominantly to the highly variable head region of haemagglutinin and their effectiveness is limited by viral drift and suboptimal immune responses. Here we describe a neuraminidase-targeting monoclonal antibody, FNI9, that potently inhibits the enzymatic activity of all group 1 and group 2 IAVs, as well as Victoria/2/87-like, Yamagata/16/88-like and ancestral IBVs.

Macrophages disseminate pathogen associated molecular patterns through the direct extracellular release of the soluble content of their phagolysosomes.

Recognition of pathogen-or-damage-associated molecular patterns is critical to inflammation. However, most pathogen-or-damage-associated molecular patterns exist within intact microbes/cells and are typically part of non-diffusible, stable macromolecules that are not optimally immunostimulatory or available for immune detection. Partial digestion of microbes/cells following phagocytosis potentially generates new diffusible pathogen-or-damage-associated molecular patterns, however, our current understanding of phagosomal biology would have these molecules sequestered and destroyed within phagolysosomes.

A non-immunological role for γ-interferon-inducible lysosomal thiol reductase (GILT) in osteoclastic bone resorption.

The extracellular bone resorbing lacuna of the osteoclast shares many characteristics with the degradative lysosome of antigen-presenting cells. γ-Interferon-inducible lysosomal thiol reductase (GILT) enhances antigen processing within lysosomes through direct reduction of antigen disulfides and maintenance of cysteine protease activity. In this study, we found the osteoclastogenic cytokine RANKL drove expression of GILT in osteoclast precursors in a STAT1-dependent manner, resulting in high levels of GILT in mature osteoclasts, which could be further augmented by γ-interferon.

UFMylation inhibits the proinflammatory capacity of interferon-γ-activated macrophages.

Macrophages activated with interferon-γ (IFN-γ) in combination with other proinflammatory stimuli, such as lipopolysaccharide or tumor necrosis factor-α (TNF-α), respond with transcriptional and cellular changes that enhance clearance of intracellular pathogens at the risk of damaging tissues. IFN-γ effects must therefore be carefully balanced with inhibitory mechanisms to prevent immunopathology. We performed a genome-wide CRISPR knockout screen in a macrophage cell line to identify negative regulators of IFN-γ responses.

Intercellular Mitochondria Transfer to Macrophages Regulates White Adipose Tissue Homeostasis and Is Impaired in Obesity.

Recent studies suggest that mitochondria can be transferred between cells to support the survival of metabolically compromised cells. However, whether intercellular mitochondria transfer occurs in white adipose tissue (WAT) or regulates metabolic homeostasis in vivo remains unknown. We found that macrophages acquire mitochondria from neighboring adipocytes in vivo and that this process defines a transcriptionally distinct macrophage subpopulation.

TFEB Transcriptional Responses Reveal Negative Feedback by BHLHE40 and BHLHE41.

Transcription factor EB (TFEB) activates lysosomal biogenesis genes in response to environmental cues. Given implications of impaired TFEB signaling and lysosomal dysfunction in metabolic, neurological, and infectious diseases, we aim to systematically identify TFEB-directed circuits by examining transcriptional responses to TFEB subcellular localization and stimulation. We reveal that steady-state nuclear TFEB is sufficient to activate transcription of lysosomal, autophagy, and innate immunity genes, whereas other targets require higher thresholds of stimulation.

Select autophagy genes maintain quiescence of tissue-resident macrophages and increase susceptibility to Listeria monocytogenes.

Innate and adaptive immune responses that prime myeloid cells, such as macrophages, protect against pathogens. However, if left uncontrolled, these responses may lead to detrimental inflammation. Macrophages, particularly those resident in tissues, must therefore remain quiescent between infections despite chronic stimulation by commensal microorganisms.

Autophagy genes in myeloid cells counteract IFNγ-induced TNF-mediated cell death and fatal TNF-induced shock.

Host inflammatory responses must be tightly regulated to ensure effective immunity while limiting tissue injury. IFN gamma (IFNγ) primes macrophages to mount robust inflammatory responses. However, IFNγ also induces cell death, and the pathways that regulate IFNγ-induced cell death are incompletely understood.

Identification of Antinorovirus Genes in Human Cells Using Genome-Wide CRISPR Activation Screening.

Noroviruses (NoVs) are a leading cause of gastroenteritis worldwide, yet host factors that restrict NoV replication are not well understood. Here, we use a CRISPR activation genome-wide screening to identify host genes that can inhibit murine norovirus (MNoV) replication in human cells. Our screens identified with high confidence 49 genes that can inhibit MNoV infection when overexpressed.

Author Correction: Smac mimetics and oncolytic viruses synergize in driving anticancer T-cell responses through complementary mechanisms.

The originally published version of this article contained an error in the spelling of the author Pankaj Tailor, which was incorrectly given as Pankaj Taylor. This has now been corrected in both the PDF and HTML versions of the article.

Tropism for tuft cells determines immune promotion of norovirus pathogenesis.

Complex interactions between host immunity and the microbiome regulate norovirus infection. However, the mechanism of host immune promotion of enteric virus infection remains obscure. The cellular tropism of noroviruses is also unknown.

Smac mimetics and oncolytic viruses synergize in driving anticancer T-cell responses through complementary mechanisms.

Second mitochondrial activator of caspase (Smac)-mimetic compounds and oncolytic viruses were developed to kill cancer cells directly. However, Smac-mimetic compound and oncolytic virus therapies also modulate host immune responses in ways we hypothesized would complement one another in promoting anticancer T-cell immunity. We show that Smac-mimetic compound and oncolytic virus therapies synergize in driving CD8 T-cell responses toward tumors through distinct activities.

A role for cathepsin Z in neuroinflammation provides mechanistic support for an epigenetic risk factor in multiple sclerosis.

Background: Hypomethylation of the cathepsin Z locus has been proposed as an epigenetic risk factor for multiple sclerosis (MS). Cathepsin Z is a unique lysosomal cysteine cathepsin expressed primarily by antigen presenting cells. While cathepsin Z expression has been associated with neuroinflammatory disorders, a role for cathepsin Z in mediating neuroinflammation has not been previously established.

Fluorometric Approaches to Measuring Reductive and Oxidative Events in Phagosomes.

The phagosome is a redox-active organelle. Numerous reductive and oxidative systems play both direct and indirect roles in phagosomal function. With the advent of newer methodologies to study these redox events in live cells, the details of how redox conditions change within the maturing phagosome, how they are regulated, and how they influence other phagosomal functions can be investigated.

Ligation of FcγR Alters Phagosomal Processing of Protein via Augmentation of NADPH Oxidase Activity.

Proteolysis and the reduction of disulfides, both major components of protein degradation, are profoundly influenced by phagosomal redox conditions in macrophages. We evaluated the activation of phagocytic receptors that are known to influence activation of the phagocyte NADPH oxidase (NOX2), and its effect on phagosomal protein degradation. Population-based and single phagosome analyses of phagosomal chemistries in murine macrophages revealed that activation of NOX2 via the Fcγ receptor (FcγR) during phagocytosis decreased rates of proteolysis and disulfide reduction.

Endogenous and exogenous pathways maintain the reductive capacity of the phagosome.

Although endosomes, lysosomes, and phagosomes require a reductive environment for the optimal activity of disulfide reductases and other thiol-dependent enzymes, how these reductive environments are established and maintained remain unknown. Our goal in this study was to begin to elucidate the redox control systems responsible for maintaining redox-sensitive enzymatic activities in the phagolysosome of murine macrophages. Through the use of specific inhibitors and genetic knockdown of known redox enzymes, we identified redox pathways that influence phagosomal disulfide reduction.

Infection of porcine bone marrow-derived macrophages by porcine respiratory and reproductive syndrome virus impairs phagosomal maturation.

Porcine reproductive and respiratory syndrome virus (PRRSV), a positive-sense, ssRNA virus of the genus Arterivirus, is a devastating disease of swine worldwide. Key early targets of PRRSV infection in pigs include professional phagocytes in the lung, such as alveolar and interstitial macrophages and dendritic cells, the dysfunction of which is believed to be responsible for much of the associated mortality. In order to study the effect of virus infection on phagocyte function, the development of a robust, reproducible model would be advantageous.

γ-Interferon-inducible lysosomal thiol reductase (GILT) maintains phagosomal proteolysis in alternatively activated macrophages.

Although it is known that lysosomal cysteine cathepsins require a reducing environment for optimal activity, it is not firmly established how these enzymes are maintained in their reduced-active state in the acidic and occasionally oxidative environment within phagosomes and lysosomes. γ-Interferon-inducible lysosomal thiol reductase (GILT) has been the only enzyme described in the endosomes, lysosomes, and phagosomes with the potential to catalyze the reduction of cysteine cathepsins. Our goal in the current study was to assess the effect of GILT on major phagosomal functions with an emphasis on proteolytic efficiency in murine bone marrow-derived macrophages.

NADPH oxidase modifies patterns of MHC class II-restricted epitopic repertoires through redox control of antigen processing.

The chemistries within phagosomes of APCs mediate microbial destruction as well as generate peptides for presentation on MHC class II. The antimicrobial effector NADPH oxidase (NOX2), which generates superoxide within maturing phagosomes, has also been shown to regulate activities of cysteine cathepsins through modulation of the lumenal redox potential. Using real-time analyses of lumenal microenvironmental parameters, in conjunction with hydrolysis pattern assessment of phagocytosed proteins, we demonstrated that NOX2 activity not only affects levels of phagosomal proteolysis as previously shown, but also the pattern of proteolytic digestion.

Redox-sensitive probes for the measurement of redox chemistries within phagosomes of macrophages and dendritic cells.

There is currently much interest in factors that affect redox chemistries within phagosomes of macrophages and dendritic cells. In addition to the antimicrobial role of reactive oxygen species generation within phagosomes, accumulating evidence suggests that phagosomal redox chemistries influence other phagosomal functions such as macromolecular degradation and antigen processing. Whilst the redox chemistries within many sub-cellular compartments are being heavily scrutinized with the increasing use of fluorescent probe technologies, there is a paucity of tools to assess redox conditions within phagosomes.

Phagosomal proteolysis in dendritic cells is modulated by NADPH oxidase in a pH-independent manner.

The level of proteolysis within phagosomes of dendritic cells (DCs) is thought to be tightly regulated, as it directly impacts the cell's efficiency to process antigen. Activity of the antimicrobial effector NADPH oxidase (NOX2) has been shown to reduce levels of proteolysis within phagosomes of both macrophages and DCs. However, the proposed mechanisms underlying these observations in these two myeloid cell lineages are dissimilar.