SummaryMacrophages represent the first line of immune defense against pathogens, and phagosome acidification is a necessary step in pathogen clearance. Here, we identified the bicarbonate transporter SLC4A7, which is strongly induced upon macrophage differentiation, as critical for phagosome acidification. Loss of SLC4A7 reduced acidification of phagocytosed beads or bacteria and impaired the intracellular microbicidal capacity in human macrophage cell lines. The phenotype was rescued by wild-type SLC4A7, but not by SLC4A7 mutants, affecting transport capacity or cell surface localization. Loss of SLC4A7 resulted in increased cytoplasmic acidification during phagocytosis, suggesting that SLC4A7-mediated, bicarbonate-driven maintenance of cytoplasmic pH is necessary for phagosome acidification. Altogether, we identify SLC4A7 and bicarbonate-driven cytoplasmic pH homeostasis as an important element of phagocytosis and the associated microbicidal functions in macrophages.

Antigen cross-presentation by dendritic cells (DC) stimulates cytotoxic T cell activation to promote immunity to intracellular pathogens, viruses and cancer. Phagocytosed antigens generate potent T cell responses, but the signalling and trafficking pathways regulating their cross-presentation are unclear. Here, we show that ablation of the store-operated-Ca2+-entry regulator STIM1 in mouse myeloid cells impairs cross-presentation and DC migration in vivo and in vitro. Stim1 ablation reduces Ca2+ signals, cross-presentation, and chemotaxis in mouse bone-marrow-derived DCs without altering cell differentiation, maturation or phagocytic capacity. Phagosomal pH homoeostasis and ROS production are unaffected by STIM1 deficiency, but phagosomal proteolysis and leucyl aminopeptidase activity, IRAP recruitment, as well as fusion of phagosomes with endosomes and lysosomes are all impaired. These data suggest that STIM1-dependent Ca2+ signalling promotes the delivery of endolysosomal enzymes to phagosomes to enable efficient cross-presentation.

Dendritic cells (DC) have the unique ability to present exogenous antigens via the major histocompatibility complex class I pathway to stimulate naive CD8+ T cells. In DCs with a non-functional mutation in Unc93b1 (3d mutation), endosomal acidification, phagosomal maturation, antigen degradation, antigen export to the cytosol and the function of the store-operated-Ca2+-entry regulator STIM1 are impaired. These defects result in compromised antigen cross-presentation and anti-tumor responses in 3d-mutated mice. Here, we show that UNC93B1 interacts with the calcium sensor STIM1 in the endoplasmic reticulum, a critical step for STIM1 oligomerization and activation. Expression of a constitutively active STIM1 mutant, which no longer binds UNC93B1, restores antigen degradation and cross-presentation in 3d-mutated DCs. Furthermore, ablation of STIM1 in mouse and human cells leads to a decrease in cross-presentation. Our data indicate that the UNC93B1 and STIM1 cooperation is important for calcium flux and antigen cross-presentation in DCs.

Phagocytosis is an essential mechanism through which innate immune cells ingest foreign material that is either destroyed or used to generate and present antigens and initiate adaptive immune responses. While a role for the ER during phagosome biogenesis has been recognized, whether fusion with ER cisternae or vesicular derivatives occurs has been the source of much contention. Membrane contact sites (MCS) are tight appositions between ER membranes and various organelles that coordinate multiple functions including localized signalling, lipid transfer and trafficking. The discovery that MCS form between the ER and phagosomes now begs the question of whether MCS play a role in connecting the ER to phagosomes under different contexts. In this review, we consider the implications of MCS between the ER and phagosomes during cross-presentation and infection with intracellular pathogens. We also discuss the similarities between these contacts and those between the ER and plasma membrane and acidic organelles such as endosomes and lysosomes. This article is part of a Special Issue entitled: Membrane Contact Sites edited by Christian Ungermann and Benoit Kornmann.

Oxysterol binding related proteins 5 and 8 (ORP5 and ORP8) are two close homologs of the larger oxysterol binding protein (OSBP) family of sterol sensors and lipid transfer proteins (LTP). Early studies indicated these transmembrane proteins, anchored to the endoplasmic reticulum (ER), bound and sensed cholesterol and oxysterols. They were identified as important for diverse cellular functions including sterol homeostasis, vesicular trafficking, proliferation and migration. In addition, they were implicated in lipid-related diseases such as atherosclerosis and diabetes, but also cancer, although their mechanisms of action remained poorly understood. Then, alongside the increasing recognition that membrane contact sites (MCS) serve as hubs for non-vesicular lipid transfer, added to their structural similarity to other LTPs, came discoveries showing that ORP5 and 8 were in fact phospholipid transfer proteins that rather sense and exchange phosphatidylserine (PS) for phosphoinositides, including phosphatidylinositol-4-phosphate (PI(4)P) and potentially phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2). Evidence now points to their action at MCS between the ER and various organelles including the plasma membrane, lysosomes, mitochondria, and lipid droplets. Dissecting exactly how this unexpected phospholipid transfer function connects with sterol regulation in health or disease remains a challenge for future studies.