Phagocytosis and phagosome maturation are regulated by calcium in J774 macrophages interacting with unopsonized prey

Phagocytosis by neutrophils, macrophages, and other professional phagocytes requires rapid remodeling of actin. Early phagosomes are surrounded by a rim of F-actin that is disassembled during phagosomoal maturation. Breakdown of periphagosomal F-actin and phagolysosome fusion are calcium dependent processes in neutrophils interacting with serum-opsonized prey, but appears to be calcium independent in macrophages interacting with serum- or IgG-opsonized prey. In the present study, we found that calcium was necessary for phagocytosis, breakdown of periphagosomal F-actin, and phagosomal maturation in J774 macrophages interacting with unopsonized prey. We also observed that lipophosphoglycan (LPG) from Leishmania donovani promastigotes required calcium to exert its inhibitory effect on macrophage phagocytosis and periphagosomal F-actin breakdown. We conclude that calcium is essential for phagocytosis, depolymerization of periphagosomal F-actin, and phagosomal maturation in J774 macrophages interacting with unopsonized prey, as well as for proper functioning of LPG.     

Compromised phagosome maturation underlies RPE pathology in cell culture and whole animal models of Smith-Lemli-Opitz Syndrome

Treatment of rats with the cholesterol pathway inhibitor AY9944 produces an animal model of Smith-Lemli-Opitz syndrome (SLOS), an autosomal recessive disease caused by defective cholesterol synthesis. This SLOS rat model undergoes progressive and irreversible degeneration of the neural retina, with associated pathological features of the retinal pigmented epithelium (RPE). Here, we provide further insights into the mechanism involved in the RPE pathology. In the SLOS rat model, markedly increased RPE apical autofluorescence is observed, compared to untreated animals, which correlates with increased levels of A2E and other bisretinoids. Utilizing cultured human induced pluripotent stem cell (iPSC)- derived SLOS RPE cells, we found significantly elevated steady-state levels of 7-dehydrocholesterol (7DHC) and decreased cholesterol levels (key biochemical hallmarks of SLOS). Western blot analysis revealed altered levels of the macroautophagy/autophagy markers MAP1LC3B-II and SQSTM1/p62, and build-up of ubiquitinated proteins. Accumulation of immature autophagosomes was accompanied by inefficient degradation of phagocytized, exogenously supplied retinal rod outer segments (as evidenced by persistence of the C-terminal 1D4 epitope of RHO [rhodopsin]) in SLOS RPE compared to iPSC-derived normal human control. SLOS RPE cells exhibited lysosomal pH levels and CTSD activity within normal physiological limits, thus discounting the involvement of perturbed lysosomal function. Furthermore, 1D4-positive phagosomes that accumulated in the RPE in both pharmacological and genetic rodent models of SLOS failed to fuse with lysosomes. Taken together, these observations suggest that defective phagosome maturation underlies the observed RPE pathology. The potential relevance of these findings to SLOS and the requirement of cholesterol for phagosome maturation are discussed.     

Role of Immune Serum in the Killing of Helicobacter pylori by Macrophages

Background: Helicobacter pylori infection can lead to the development of gastritis, peptic ulcers and gastric cancer, which makes this bacterium an important concern for human health. Despite evoking a strong immune response in the host, H. pylori persists, requiring complex antibiotic therapy for eradication. Here we have studied the impact of a patient’s immune serum on H. pylori in relation to macrophage uptake, phagosome maturation, and bacterial killing. Materials and Methods: Primary human macrophages were infected in vitro with both immune serum‐treated and control H. pylori. The ability of primary human macrophages to kill H. pylori was characterized at various time points after infection. H. pylori phagosome maturation was analyzed by confocal immune fluorescence microscopy using markers specific for H. pylori, early endosomes (EEA1), late endosomes (CD63) and lysosomes (LAMP‐1). Results: Immune serum enhanced H. pylori uptake into macrophages when compared to control bacteria. However, a sufficient inoculum remained for recovery of viable H. pylori from macrophages, at 8 hours after infection, for both the serum‐treated and control groups. Both serum‐treated and control H. pylori phagosomes acquired EEA1 (15 minutes), CD63 and LAMP‐1 (30 minutes). These markers were then retained for the rest of an 8 hour time course. Conclusions: While immune sera appeared to have a slight positive effect on bacterial uptake, both serum‐treated and control H. pylori were not eliminated by macrophages. Furthermore, the same disruptions to phagosome maturation were observed for both serum‐treated and control H. pylori. We conclude that to eliminate H. pylori, a strategy is required to restore the normal process of phagosome maturation and enable effective macrophage killing of H. pylori, following a host immune response.     

Spatiotemporal Changes of the Phagosomal Proteome in Dendritic Cells in Response to LPS Stimulation*

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Highlights

• •Characterization of the phagosomal proteome comparing resting and LPS-treated BMDCs.
• •Label-free quantification determined 2843 phagosomal proteins.
• •Reduced recruitment of hydrolases and V-ATPase to phagosomes of LPS-treated cells.
• •Increased recruitment of antigen cross-presentation molecules to these phagosomes.

     

Autophagosome–lysosome fusion in neurons requires INPP5E,a protein associated with Joubert syndrome

Autophagy is a multistep membrane traffic pathway. In contrast to autophagosome formation, the mechanisms underlying autophagosome–lysosome fusion remain largely unknown. Here, we describe a novel autophagy regulator, inositol polyphosphate‐5‐phosphatase E (INPP5E), involved in autophagosome–lysosome fusion process. In neuronal cells, INPP5E knockdown strongly inhibited autophagy by impairing the fusion step. A fraction of INPP5E is localized to lysosomes, and its membrane anchoring and enzymatic activity are necessary for autophagy. INPP5E decreases lysosomal phosphatidylinositol 3,5‐bisphosphate (PI(3,5)P₂), one of the substrates of the phosphatase, that counteracts cortactin‐mediated actin filament stabilization on lysosomes. Lysosomes require actin filaments on their surface for fusing with autophagosomes. INPP5E is one of the genes responsible for Joubert syndrome, a rare brain abnormality, and mutations found in patients with this disease caused defects in autophagy. Taken together, our data reveal a novel role of phosphoinositide on lysosomes and an association between autophagy and neuronal disease.     

Vacuolar ATPase in Phagosome-Lysosome Fusion

The vacuolar H⁺-ATPase (v-ATPase) complex is instrumental in establishing and maintaining acidification of some cellular compartments, thereby ensuring their functionality. Recently it has been proposed that the transmembrane V₀ sector of v-ATPase and its a-subunits promote membrane fusion in the endocytic and exocytic pathways independent of their acidification functions. Here, we tested if such a proton-pumping independent role of v-ATPase also applies to phagosome-lysosome fusion. Surprisingly, endo(lyso)somes in mouse embryonic fibroblasts lacking the V₀ a3 subunit of the v-ATPase acidified normally, and endosome and lysosome marker proteins were recruited to phagosomes with similar kinetics in the presence or absence of the a3 subunit. Further experiments used macrophages with a knockdown of v-ATPase accessory protein 2 (ATP6AP2) expression, resulting in a strongly reduced level of the V₀ sector of the v-ATPase. However, acidification appeared undisturbed, and fusion between latex bead-containing phagosomes and lysosomes, as analyzed by electron microscopy, was even slightly enhanced, as was killing of non-pathogenic bacteria by V₀ mutant macrophages. Pharmacologically neutralized lysosome pH did not affect maturation of phagosomes in mouse embryonic cells or macrophages. Finally, locking the two large parts of the v-ATPase complex together by the drug saliphenylhalamide A did not inhibit in vitro and in cellulo fusion of phagosomes with lysosomes. Hence, our data do not suggest a fusion-promoting role of the v-ATPase in the formation of phagolysosomes.     

Death commitment point is advanced by axotomy in sympathetic neurons

Axotomized neurons have several characteristics that are different from intact neurons. Here we show that, unlike established cultures, the axotomized sympathetic neurons deprived of NGF become committed to die before caspase activation, since the same proportion of NGF-deprived neurons are rescued by NGF regardless of whether caspases are inhibited by the pan-caspase inhibitor Boc-Asp(O-methyl)-CH(2)F (BAF). Despite prolonged Akt and ERK signaling induced by NGF after BAF treatment has prevented death, the neurons fail to increase protein synthesis, recover ATP levels, or grow. Within 3 d, all the mitochondria disappear without apparent removal of any other organelles or loss of membrane integrity. Although NGF does rescue intact BAF-treated 6-d cultures after NGF deprivation, rescue by NGF fails when these neurons are axotomized before NGF deprivation and BAF treatment. Moreover, cytosolic cytochrome c rapidly kills axotomized neurons. We propose that axotomy induces signals that make sympathetic neurons competent to die prematurely. NGF cannot repair these NGF-deprived, BAF-treated neurons because receptor signaling (which is normal) is uncoupled from protein renewal, and the mitochondria (which are damaged) go on to be eliminated. Hence, the order of steps underlying neuronal death commitment is mutable and open to regulation.     

Targeting of aequorin for calcium monitoring in intracellular compartments

We have recently developed a new method for monitoring Ca²⁺ concentrations in defined cell compartments. The cDNA encoding the Ca²⁺-sensitive photoprotein aequorin has been modified in order to include specific targeting sequences and expressed in eukaryotic cells; the recombinant protein, specifically located inside the cells, has allowed the direct study of mitochondrial and nuclear Ca²⁺ concentrations in living cells. The principles, and the application, of this new methodology are discussed in this article.     

Altered secretion of kidney lysosomal enzymes in the mouse pigment mutants ruby-eye,ruby-eye-2-J,and maroon

Melanosomes and lysosomes share structural and biosynthetic properties. Three mouse pigment mutants, ruby-eye, ruby-eye-2-J, and maroon, have abnormally high concentrations of kidney lysosomal enzymes. Concentrations of kidney nonlysosomal enzymes and of liver and serum lysosomal enzymes are normal. By light microscopy the mutants have normal kidney lysosome morphology. It does not appear that the mutant genes cause an increased rate of production of lysosomes since the increased kidney -glucuronidase concentration is not accompanied by a corresponding increase in rate of synthesis. The common defect in all mutants is a decreased rate of secretion of lysosomal enzymes from kidney into urine. Eight mouse pigment mutants are now known which affect both melanosome and lysosome function. They should serve as useful models for the study of the biogenesis, structure, and processing of these and other subcellular organelles.This work was supported in part by United States Public Health Service Research Grant GM-19521 and by National Science Foundation Grant PCM77-24804. E. K. N. was supported in part by United States Public Health Service Grant GM07093-03. F. W. was a high school student in the summer program supported by National Science Foundation Grant SP177-26980.