A Bumpy Ride of Mycobacterial Phagosome Maturation: Roleplay of Coronin1 Through Cofilin1 and cAMP

Phagosome-lysosome fusion in innate immune cells like macrophages and neutrophils marshal an essential role in eliminating intracellular microorganisms. In microbe-challenged macrophages, phagosome-lysosome fusion occurs 4 to 6 h after the phagocytic uptake of the microbe. However, live pathogenic mycobacteria hinder the transfer of phagosomes to lysosomes, up to 20 h post-phagocytic uptake. This period is required to evade pro-inflammatory response and upregulate the acid-stress tolerant proteins. The exact sequence of events through which mycobacteria retards phagolysosome formation remains an enigma. The macrophage coat protein Coronin1(Cor1) is recruited and retained by mycobacteria on the phagosome membrane to retard its maturation by hindering the access of phagosome maturation factors. Mycobacteria-infected macrophages exhibit an increased cAMP level, and based on receptor stimulus, Cor1 expressing cells show a higher level of cAMP than non-Cor1 expressing cells. Here we have shown that infection of bone marrow-derived macrophages with H37Rv causes a Cor1 dependent rise of intracellular cAMP levels at the vicinity of the phagosomes. This increased cAMP fuels cytoskeletal protein Cofilin1 to depolymerize F-actin around the mycobacteria-containing phagosome. Owing to reduced F-actin levels, the movement of the phagosome toward the lysosomes is hindered, thus contributing to the retarded phagosome maturation process. Additionally, Cor1 mediated upregulation of Cofilin1 also contributes to the prevention of phagosomal acidification, which further aids in the retardation of phagosome maturation. Overall, our study provides first-hand information on Cor1 mediated retardation of phagosome maturation, which can be utilized in developing novel peptidomimetics as part of host-directed therapeutics against tuberculosis.

Phagocytic Activities of Reactive Microglia and Astrocytes Associated with Prion Diseases Are Dysregulated in Opposite Directions

Phagocytosis is one of the most important physiological functions of the glia directed at maintaining a healthy, homeostatic environment in the brain. Under a homeostatic environment, the phagocytic activities of astrocytes and microglia are tightly coordinated in time and space. In neurodegenerative diseases, both microglia and astrocytes contribute to neuroinflammation and disease pathogenesis, however, whether their phagocytic activities are up- or downregulated in reactive states is not known. To address this question, this current study isolated microglia and astrocytes from C57BL/6J mice infected with prions and tested their phagocytic activities in live-cell imaging assays that used synaptosomes and myelin debris as substrates. The phagocytic uptake by the reactive microglia was found to be significantly upregulated, whereas that of the reactive astrocytes was strongly downregulated. The up- and downregulation of phagocytosis by the two cell types were observed irrespective of whether disease-associated synaptosomes, normal synaptosomes, or myelin debris were used in the assays, indicating that dysregulations are dictated by cell reactive states, not substrates. Analysis of gene expression confirmed dysregulation of phagocytic functions in both cell types. Immunostaining of animal brains infected with prions revealed that at the terminal stage of disease, neuronal cell bodies were subject to engulfment by reactive microglia. This study suggests that imbalance in the phagocytic activities of the reactive microglia and astrocytes, which are dysregulated in opposite directions, is likely to lead to excessive microglia-mediated neuronal death on the one hand, and the inability of astrocytes to clear cell debris on the other hand, contributing to the neurotoxic effects of glia as a whole.

Phagocytic Uptake of Particles by Immune cells Under Flow Conditions

Particles injected intravenously are thought to be cleared by macrophages residing in the liver and spleen, but they also encounter circulating immune cells. It remains to be determined if the circulating cells can take up particles while flowing in the blood. Here, we use an in vitro peristaltic pump setup that mimics pulsatile blood flow to establish if immune cells take up particles under constant fluidic flow. Our results show that the immune cells do phagocytose under flow conditions, and the uptake depends on the cell type, particle size, and flow rate. We demonstrate that cell lines representing myeloid cells, and primary neutrophils and monocytes are similar or better at taking up sub-micrometer-sized particles under flow compared to static conditions. Experiments with whole blood show that even under the crowding effects of red blood cells, neutrophils and monocytes take up particles while flowing. These data suggest that therapeutics may be delivered to circulating immune cells using particulate delivery systems.

Loss of NPC1 enhances phagocytic uptake and impairs lipid trafficking in microglia

Niemann-Pick type C disease is a rare neurodegenerative disorder mainly caused by mutations in NPC1, resulting in abnormal late endosomal/lysosomal lipid storage. Although microgliosis is a prominent pathological feature, direct consequences of NPC1 loss on microglial function remain not fully characterized. We discovered pathological proteomic signatures and phenotypes in NPC1-deficient murine models and demonstrate a cell autonomous function of NPC1 in microglia. Loss of NPC1 triggers enhanced phagocytic uptake and impaired myelin turnover in microglia that precede neuronal death. Npc1−/− microglia feature a striking accumulation of multivesicular bodies and impaired trafficking of lipids to lysosomes while lysosomal degradation function remains preserved. Molecular and functional defects were also detected in blood-derived macrophages of NPC patients that provide a potential tool for monitoring disease. Our study underscores an essential cell autonomous role for NPC1 in immune cells and implies microglial therapeutic potential.

Use of Flow Cytometry to Evaluate Phagocytosis of Staphylococcus aureus by Human Neutrophils

Neutrophils play a key role in the human immune response to Staphylococcus aureus infections. These professional phagocytes rapidly migrate to the site of infection to engulf bacteria and destroy them via specialized intracellular killing mechanisms. Here we describe a robust and relatively high-throughput flow cytometry assay to quantify phagocytosis of S. aureus by human neutrophils. We show that effective phagocytic uptake of S. aureus is greatly enhanced by opsonization, i.e. the tagging of microbial surfaces with plasma-derived host proteins like antibodies and complement. Our rapid assay to monitor phagocytosis can be used to study neutrophil deficiencies and bacterial evasion, but also provides a powerful tool to assess the opsonic capacity of antibodies, either in the context of natural immune responses or immune therapies.

Monitoring phagocytic uptake of amyloid β into glial cell lysosomes in real time

Glial cell phagocytosis of pH-dependent amyloid-β, AβpH, in live and fixed cultures, brain tissue sections, retina, cortex and in live animals useful for studying function in health and disease.

Correction to: Assays of Eosinophil Apoptosis and Phagocytic Uptake

Chapter 10 was previously published non-open access. It has now been changed to open access under a CC BY 4.0 license and the copyright holder has been updated to ‘The Author(s)’. The book has been updated with these changes.