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

AbstractThere 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. Hence the systems that control redox homeostasis in these unique environments remain poorly defined. This review highlights current redox-sensitive probes that can measure oxidative or reductive activity in phagosomes and discusses their suitability and limitations of use. Probes that are easily targeted to the phagosome by using established approaches are emphasized

Investigating Thiol-Dependent Processes in the Phagosome of Macrophages

The work presented here evaluates the control of redox sensitive processes involved in phagosomal protein degradation which include the activities of cysteine cathepsins and gamma-interferon-inducible lysosomal thiol reductase (GILT). The catalytic mechanisms of these enzymes are dependent on reduced active site thiol (SH) groups making them susceptible to oxidative inactivation via reactive oxygen species (ROS). First, we evaluate how phagosomal thiol-dependent processes are influenced by the activation status of the macrophage. We show that interleukin-4 (IL-4) activation of murine bone marrow derived macrophages (BMMØs) results in a dramatic increase in phagosomal protein degradation. This was due to a decrease in oxidative inactivation of cysteine cathepsins and phagosomal disulfide reduction as a result of decreased NADPH oxidase (NOX2) -mediated ROS production. In addition, we found that IL-4 activation resulted in increased expression of cysteine cathepsins S and L which also enhanced phagosomal proteolysis in a NOX2-independent manner. Second, the influence of GILT on phagosomal thiol-dependent protease activity was evaluated in BMMØs to address how cysteine cathepsins are maintained in their reduced active state. We show that during NOX2 inhibition, GILT maintains phagosomal proteolysis in the early phagosome. The effect of GILT on proteolysis was enhanced in IL-4-activated BMMØs (with/without NOX2 inhibition). Furthermore, in a reconstituted system, we observed enhanced cathepsin S activity in the presence of recombinant active GILT. Last, we investigated the involvement of cytosolic redox control systems on phagosomal thiol-dependent processes. A novel phagosome-specific disulfide reduction assay was screened against a small molecule bioactives library. Results of the screen indicated the putative involvement of thioredoxin reductase (TrxR) in supporting phagosomal disulfide reduction. Furthermore, nicotinamide adenine dinucleotide phosphate (NADPH) depletion in BMMØs decreased both phagosomal disulfide reduction and proteolysis, implicating NADPH as a putative source of phagosomal reducing equivalents. Finally, through conditional depletion of the selenocysteine tRNA in BMMØs, we provide evidence for the involvement of selenoproteins, a family of redox enzymes which includes TrxR, in phagosomal disulfide reduction. Overall, the work presented in this dissertation contributes to our understanding of how macrophages regulate redox sensitive protein degradation in the phagosome

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

Abstract 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. Methods Experimental autoimmune encephalomyelitis (EAE) was induced in both wildtype mice and mice deficient in cathepsin Z. The effects of cathepsin Z-deficiency on the processing and presentation of the autoantigen myelin oligodendrocyte glycoprotein, and on the production of IL-1β and IL-18 were determined in vitro from cells derived from wildtype and cathepsin Z-deficient mice. The effects of cathepsin Z-deficiency on CD4+ T cell activation, migration, and infiltration to the CNS were determined in vivo. Statistical analyses of parametric data were performed by one-way ANOVA followed by Tukey post-hoc tests, or by an unpaired Student’s t test. EAE clinical scoring was analyzed using the Mann–Whitney U test. Results We showed that mice deficient in cathepsin Z have reduced neuroinflammation and dramatically lowered circulating levels of IL-1β during EAE. Deficiency in cathepsin Z did not impact either the processing or the presentation of MOG, or MOG- specific CD4+ T cell activation and trafficking. Consistently, we found that cathepsin Z-deficiency reduced the efficiency of antigen presenting cells to secrete IL-1β, which in turn reduced the ability of mice to generate Th17 responses—critical steps in the pathogenesis of EAE and MS. Conclusion Together, these data support a novel role for cathepsin Z in the propagation of IL-1β-driven neuroinflammation

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. Furthermore, we identify shared and distinct transcriptional signatures between mTOR inhibition and bacterial autophagy. Using a genome-wide CRISPR library, we find TFEB targets that protect cells from or sensitize cells to lysosomal cell death. BHLHE40 and BHLHE41, genes responsive to high, sustained levels of nuclear TFEB, act in opposition to TFEB upon lysosomal cell death induction. Further investigation identifies genes counter-regulated by TFEB and BHLHE40/41, adding this negative feedback to the current understanding of TFEB regulatory mechanisms. Employing RNA sequencing, genome-wide CRISPR screening, and high-content subcellular imaging, Carey et al. systematically unravel localization- and stimulation-specific transcriptional responses to TFEB, including target gene activation at steady state. The authors further uncover a negative feedback loop by BHLHE40 and BHLHE41 that counteracts a TFEB transcriptional signature induced by lysosomal stress

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. Here, we show the controlled release of partially-digested, soluble material from phagolysosomes of macrophages through transient, iterative fusion-fission events between mature phagolysosomes and the plasma membrane, a process we term eructophagy. Eructophagy is most active in proinflammatory macrophages and further induced by toll like receptor engagement. Eructophagy is mediated by genes encoding proteins required for autophagy and can activate vicinal cells by release of phagolysosomally-processed, partially-digested pathogen associated molecular patterns. We propose that eructophagy allows macrophages to amplify local inflammation through the processing and dissemination of pathogen-or-damage-associated molecular patterns