Endogenous and exogenous pathways maintain the reductive capacity of the phagosome

Abstract

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. In particular, known inhibitors of the NADPH-dependent selenoprotein, thioredoxin reductase, were shown to inhibit phagosomal disulfide reduction and phagosomal proteolysis. This was supported by the observation that conditional deletion of the selenocysteine tRNA in macrophages decreased phagosomal disulfide reduction capacity. In addition, pharmacologic inhibition of the pentose phosphate pathway decreased rates of disulfide reduction and proteolysis in the phagosome, implicating NADPH as a source of phagosomal reductive energy. Finally, by analyzing the effect of extracellular redox couples, such as cysteine:cystine on thiol-dependent phagosomal processes, we demonstrated that the extracellular space can additionally supply the phagosome with reductive energy. Collectively, these data demonstrate that defined cytosolic reductive pathways act in concert with the uptake of cysteine from the extracellular space to support thiol-dependent chemistries in the phagosome.

Editorial: Decoding the macrophage phagosomal environment: the hunt for key reductases is on

“…As antigen processing in macrophages, as well as dendritic cells, can be influenced by levels of phagosomal proteolysis and disulfide reduction [2,10,11], it can easily be affected by modulation of the sources of reducing compounds. Certainly, the article published in this issue by Balce et al [8] is not the final conclusion to the story, as the authors have not directly implicated TR in supporting the reductive capacity of the phagosome, a finding that needs to be solidified. Nevertheless, as this study [8] was successful in providing novel information regarding NADPH and selenoprotein involvement in phagosomal thiol-dependent functions, it represents an excellent starting platform to discern how the reductive capacity of the phagosome is maintained.…”

“…Certainly, the article published in this issue by Balce et al [8] is not the final conclusion to the story, as the authors have not directly implicated TR in supporting the reductive capacity of the phagosome, a finding that needs to be solidified. Nevertheless, as this study [8] was successful in providing novel information regarding NADPH and selenoprotein involvement in phagosomal thiol-dependent functions, it represents an excellent starting platform to discern how the reductive capacity of the phagosome is maintained. For example, among the many remaining questions to address, further studies will need to delineate the molecular mechanisms by which the reductive energy from NADPH is conveyed through the phagosomal membrane.…”

“…The current study [8] represents an important finding that directly impacts the processing of protein antigens by macrophages and the subsequent presentation on MHC molecules to T cells. As antigen processing in macrophages, as well as dendritic cells, can be influenced by levels of phagosomal proteolysis and disulfide reduction [2,10,11], it can easily be affected by modulation of the sources of reducing compounds.…”

“…Accordingly, it is crucial to decipher the mechanisms responsible for maintaining the reductive capacity of the phagosome in macrophages that could regulate efficient antigen processing in these organelles. In the study published in this issue of JLB, Balce et al [8] describe a collection of intriguing experiments designed to explore these regulatory pathways. To identify the redox systems involved, the authors used a bioactive, chemical-based library-screening technique to assay phagosomal disulfide reduction.…”

“…Their findings, which were later confirmed in the study using specific inhibitors, pointed toward a TR-related enzyme that was needed for the disulfide reduction within phagosomes. Balce et al [8] used NADPH and selenoproteindepletion studies, as well as macrophages from NOX2-deficient mice, to eliminate ROS generation that would typically decrease disulfide reduction. This work excluded the effects of ROS on disulfide reduction within phagosomes and thus, demonstrated a role for NADPH as a possible source of phagosomal-reducing equivalents, requiring at least 1 selenoprotein.…”