Immune cells kill microbes by engulfing them in a membrane-enclosed compartment, the phagosome. Phagocytosis is initiated when foreign particles bind to receptors on the membrane of phagocytes. The best-studied phagocytic receptors, those for Igs (FcγR) and for complement proteins (CR), activate PLC and PLD, resulting in the intracellular production of the Ca²⁺-mobilizing second messengers InsP3 and S1P, respectively. The ensuing release of Ca²⁺ from the ER activates SOCE channels in the plasma and/or phagosomal membrane, leading to sustained or oscillatory elevations in cytosolic Ca²⁺ concentration. Cytosolic Ca²⁺ elevations are required for efficient ingestion of foreign particles by some, but not all, phagocytic receptors and stringently control the subsequent steps involved in the maturation of phagosomes. Ca²⁺ is required for the solubilization of the actin meshwork that surrounds nascent phagosomes, for the fusion of phagosomes with granules containing lytic enzymes, and for the assembly and activation of the superoxide-generating NADPH oxidase complex. Furthermore, Ca²⁺ entry only occurs at physiological voltages and therefore, requires the activity of proton channels that counteract the depolarizing action of the phagocytic oxidase. The molecules that mediate Ca²⁺ ion flux across the phagosomal membrane are still unknown but likely include the ubiquitous SOCE channels and possibly other types of Ca²⁺ channels such as LGCC and VGCC. Understanding the molecular basis of the Ca²⁺ signals that control phagocytosis might provide new, therapeutic tools against pathogens that subvert phagocytic killing.

Macrophages 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.

Statins are 3-hydroxyl-3-methyglutaryl-CoA reductase inhibitors that are commonly used to inhibit cholesterol biosynthesis. Emerging data have suggested that they also have “pleotropic effects,” including modulating actin cytoskeleton reorganization. Here, we report an effect of simvastatin on the trafficking of aquaporin-2 (AQP2). Specifically, simvastatin induced the membrane accumulation of AQP2 in cell cultures and kidneys in situ. The effect of simvastatin was independent of protein kinase A activation and phosphorylation at AQP2-Ser²⁵⁶, a critical event involved in vasopressin (VP)-regulated AQP2 trafficking. Further investigation showed that simvastatin inhibited endocytosis in parallel with downregulation of RhoA activity. Overexpression of active RhoA attenuated simvastatin's effect, suggesting the involvement of this small GTPase in simvastatin-mediated AQP2 trafficking. Finally, the effect of simvastatin on urinary concentration was investigated in VP-deficient Brattleboro rats. Simvastatin acutely (3–6 h) increased urinary concentration and decreased urine output in these animals. In summary, simvastatin regulates AQP2 trafficking in vitro and urinary concentration in vivo via events involving downregulation of Rho GTPase activity and inhibition of endocytosis. Our study provides an alternative mechanism to regulate AQP2 trafficking, bypassing the VP-vasopressin receptor signaling pathway.

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.Stim1ablation 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.