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. Using real-time analysis of lumenal microenvironmental parameters within phagosomes in live bone marrow-derived DCs, we show that the levels of phagosomal proteolysis are diminished in the presence of NOX2 activity, but in contrast to previous reports, the acidification of the phagosome is largely unaffected. As found in macrophages, we show that NOX2 controls phagosomal proteolysis in DCs through redox modulation of local cysteine cathepsins. Aspartic cathepsins were unaffected by redox conditions, indicating that NOX2 skews the relative protease activities in these antigen processing compartments. The ability of DC phagosomes to reduce disulphides was also compromised by NOX2 activity, implicating this oxidase in the control of an additional antigen processing chemistry of DCs.

The phagosomal lumen in macrophages is the site of numerous interacting chemistries that mediate microbial killing, macromolecular degradation, and antigen processing. Using a non-hypothesisbased screen to explore the interconnectivity of phagosomal functions, we found that NADPH oxidase (NOX2) negatively regulates levels of proteolysis within the maturing phagosome of macrophages. Unlike the NOX2 mechanism of proteolytic control reported in dendritic cells, this phenomenon in macrophages is independent of changes to lumenal pH and is also independent of hydrolase delivery to the phagosome. We found that NOX2 mediates the inhibition of phagosomal proteolysis in macrophages through reversible oxidative inactivation of local cysteine cathepsins. We also show that NOX2 activity significantly compromises the phagosome's ability to reduce disulfides. These findings indicate that NOX2 oxidatively inactivates cysteine cathepsins through sustained ablation of the reductive capacity of the phagosomal lumen. This constitutes a unique mechanism of spatiotemporal control of phagosomal chemistries through the modulation of the local redox environment. In addition, this work further implicates the microbicidal effector NOX2 as a global modulator of phagosomal physiologies, particularly of those pertinent to antigen processing.phagocytosis | cathepsin | disulfide reduction | antigen processing | lysosome U nlike many specialized lineages of the mononuclear phagocyte system, tissue macrophages function in a diverse array of homeostatic and immune physiologies. Critical to many of these functions is the phagosome. Over the past decade, proteomic characterization of the phagosome in conjunction with biochemical analysis of phagosomal chemistries in reconstituted systems has given great insight into the function of this organelle (1, 2). More recently, measurement of phagosomal properties in live cells has enabled the in situ dissection of the complex crosstalk between spatiotemporally intimate phagosomal chemistries (3, 4). In particular, cross-talk influencing the control of phagosomal proteolysis has recently received much attention (4). It has become increasingly apparent that a tightly controlled, limited level of proteolysis within the endolysosomal system, as found in dendritic cells (DCs), is essential for efficient antigen processing (5, 6). Macrophages possess a reported 20-to 60-fold higher level of lysosomal proteolysis than DCs, which is implicated in limiting their efficiency as antigen-presenting cells (7-9). Nonetheless, macrophages are capable of productively presenting antigen to T cells and play an important role in the secondary immune response (10). This is presumably aided by the stringent control of the macrophage's lysosomal protease activities in its antigenprocessing compartments.Control of lysosomal proteases such as cathepsins occurs at several regulatory levels, including transcription, trafficking, prodomain removal, regulatory proteins (e.g. cystatins), and vacuolar pH (11). Modification of the redox...

Alternatively activated macrophages, generated in a T-helper 2 environment, have demonstrated roles in wound repair and tissue remodeling in addition to being charged with immune tasks. Because the hydrolytic chemistries of the phagosomal lumen are central to many of these functions, we investigated their modification after alternative activation with IL-4 and IL-13. Most significantly, we found striking up-regulation of the proteolytic levels within the phagosome of IL-4-activated macrophages. Two synergistic mechanisms were determined to underlie this up-regulation. First, IL-4-activated macrophages displayed increased expression of cathepsin S and L, providing greater proteolytic machinery to the phagosome despite unchanged rates of lysosomal contribution. Secondly, decreased phagosomal NADPH oxidase (NOX2) activity, at least partially resulting from decreased expression of the NOX2 subunit gp91 phox , resulted in a more reductive lumenal microenvironment, which in turn, enhanced activities of local cysteine cathepsins.Decreased NOX2 activity additionally increased the phagosome's ability to reduce disulfides, further enhancing the efficiency of the macrophage to degrade proteins containing disulfide bonds. Together, these changes initiated by IL-4 act synergistically to rapidly and dramatically enhance the macrophage's ability to degrade phagocytosed protein, which, we reason, better equips this cell for its roles in wound repair and tissue remodeling. IntroductionThe ability of the macrophage to dramatically remodel its cell biology in response to environmental cues and immune signals permits this ubiquitous cell lineage to function in a wide range of homeostatic and immune physiologies. Classic activation of the macrophage in response to microbial products and T-helper 1 cytokines has been long known to reprogram the macrophage's biology to enhance microbial killing and its ability to present antigen. [1][2][3] Modulation of the macrophage's key organelle, the phagosome, contributes to this functional reprogramming. [4][5][6] During the past 2 decades, alternative activation of macrophages by the T-helper 2 cytokines IL-4 and IL-13 has been shown to result in a unique phenotype that enables the macrophage to perform distinct physiologic functions. In addition to their demonstrated roles in helminthic infection, alternatively activated macrophages (AAMØs) are gaining considerable attention for their roles in tissue remodeling, wound repair, and control of inflammation. 7,8 Concomitant with these roles, several features of AAMØs have been identified that are consistent with the clearance of apoptotic cells and debris, such as up-regulation of certain phagocytic receptors and modification of endolysosomal dynamics. 7,[9][10][11][12] Hitherto changes to the functional chemistries within the phagosome that facilitate the AAMØ's function have not been elucidated.The lumenal chemistries of phagosomes play a central role in many macrophage functions. In addition to microbial killing and antigen processing, the ph...

SummaryIntracellular chloride channel protein 1 (CLIC1) is a 241 amino acid protein of the glutathione S transferase fold family with redox-and pH-dependent membrane association and chloride ion channel activity. Whilst CLIC proteins are evolutionarily conserved in Metazoa, indicating an important role, little is known about their biology. CLIC1 was first cloned on the basis of increased expression in activated macrophages. We therefore examined its subcellular localisation in murine peritoneal macrophages by immunofluorescence confocal microscopy. In resting cells, CLIC1 is observed in punctate cytoplasmic structures that do not colocalise with markers for endosomes or secretory vesicles. However, when these macrophages phagocytose serum-opsonised zymosan, CLIC1 translocates onto the phagosomal membrane. Macrophages from CLIC1 2/2 mice display a defect in phagosome acidification as determined by imaging live cells phagocytosing zymosan tagged with the pH-sensitive fluorophore Oregon Green. This altered phagosomal acidification was not accompanied by a detectable impairment in phagosomal-lysosomal fusion. However, consistent with a defect in acidification, CLIC1 2/2 macrophages also displayed impaired phagosomal proteolytic capacity and reduced reactive oxygen species production. Further, CLIC12/2 mice were protected from development of serum transfer induced K/BxN arthritis. These data all point to an important role for CLIC1 in regulating macrophage function through its ion channel activity and suggest it is a suitable target for the development of anti-inflammatory drugs.

Entamoeba histolytica is an enteric dwelling human protozoan parasite that causes the disease amoebiasis, which is endemic in the developing world. Over the past four decades, considerable effort has been made to understand the parasite and the disease. Improved diagnostics can now differentiate pathogenic E. histolytica from that of the related but nonpathogenic Entamoeba dispar, thus minimizing screening errors. Classically, the triad of Gal-lectin, cysteine proteinases and amoebapores of the parasite were thought to be the major proteins involved in the pathogenesis of amoebiasis. However, other amoebic molecules such as lipophosphopeptidoglycan, perioxiredoxin, arginase, and lysine and glutamic acid-rich proteins are also implicated. Recently, the genome of E. histolytica has been sequenced, which has widened our scope to study additional virulence factors. E. histolytica genome-based approaches have now confirmed the presence of Golgi apparatus-like vesicles and the machinery for glycosylation, thus improving the chances of identifying potential drug targets for chemotherapeutic intervention. Apart from Gal-lectin-based vaccines, promising vaccine targets such as serine-rich E. histolytica protein have yielded encouraging results. Considerable efforts have also been made to skew vaccination responses towards appropriate T-helper cell immunity that could augment the efficacy of vaccine candidates under study. Thus, ongoing efforts mining the information made available with the sequencing of the E. histolytica genome will no doubt identify and characterize other important potential vaccine/drug targets and lead to effective immunologic strategies for the control of amoebiasis.