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. 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.