David J. Kusner

Cutting Edge: Mycobacterium tuberculosis Blocks Ca2+ Signaling and Phagosome Maturation in Human Macrophages Via Specific Inhibition of Sphingosine Kinase

One-third of the world’s population is infected with Mycobacterium tuberculosis (Mtb), and three million people die of tuberculosis each year. Following its ingestion by macrophages (MPs), Mtb inhibits the maturation of its phagosome, preventing progression to a bactericidal phagolysosome. Phagocytosis of Mtb is uncoupled from the elevation in MP cytosolic Ca2+ that normally accompanies microbial ingestion, resulting in inhibition of phagosome-lysosome fusion and increased intracellular viability. This study demonstrates that the mechanism responsible for this failure of Ca2+-dependent phagosome maturation involves mycobacterial inhibition of MP sphingosine kinase. Thus, inhibition of sphingosine kinase directly contributes to survival of Mtb within human MPs and represents a novel molecular mechanism of pathogenesis.

ATP-Induced Killing of Virulent Mycobacterium tuberculosis Within Human Macrophages Requires Phospholipase D

The global dissemination of antibiotic-resistant Mycobacterium tuberculosis has underscored the urgent need to understand the molecular mechanisms of immunity to this pathogen. Use of biological immunomodulatory compounds to enhance antituberculous therapy has been hampered by the limited efficacy of these agents toward infected human macrophages and lack of information regarding their mechanisms of activity. We tested the hypotheses that extracellular ATP (ATPe) promotes killing of virulent M. tuberculosis within human macrophages, and that activation of a specific macrophage enzyme, phospholipase D (PLD), functions in this response. ATPe treatment of infected monocyte-derived macrophages resulted in 3.5-log reduction in the viability of three different virulent strains of M. tuberculosis. Stimulation of macrophage P2X7 purinergic receptors was necessary, but not sufficient, for maximal killing by primary macrophages or human THP-1 promonocytes differentiated to a macrophage phenotype. Induction of tuberculocidal activity by ATPe was accompanied by marked stimulation of PLD activity, and two mechanistically distinct inhibitors of PLD produced dose-dependent reductions in ATPe-induced killing of intracellular bacilli. Purified PLD restored control levels of mycobacterial killing to inhibitor-treated cells, and potentiated ATPe-dependent tuberculocidal activity in control macrophages. These results demonstrate that ATPe promotes killing of virulent M. tuberculosis within infected human macrophages and strongly suggest that activation of PLD plays a key role in this process.

Mycobacterium tuberculosis Phagosomes Exhibit Altered Calmodulin-Dependent Signal Transduction: Contribution to Inhibition of Phagosome-Lysosome Fusion and Intracellular Survival in Human Macrophages

Mycobacterium tuberculosis successfully parasitizes macrophages by disrupting the maturation of its phagosome, creating an intracellular compartment with endosomal rather than lysosomal characteristics. We have recently demonstrated that live M. tuberculosis infect human macrophages in the absence of an increase in cytosolic Ca2+ ([Ca2+]c), which correlates with inhibition of phagosome-lysosome fusion and intracellular viability. In contrast, killed M. tuberculosis induces an elevation in [Ca2+]c that is coupled to phagosome-lysosome fusion. We tested the hypothesis that defective activation of the Ca2+-dependent effector proteins calmodulin (CaM) and CaM-dependent protein kinase II (CaMKII) contributes to the intracellular pathogenesis of tuberculosis. Phagosomes containing live M. tuberculosis exhibited decreased levels of CaM and the activated form of CaMKII compared with phagosomes encompassing killed tubercle bacilli. Furthermore, ionophore-induced elevations in [Ca2+]c resulted in recruitment of CaM and activation of CaMKII on phagosomes containing live M. tuberculosis. Specific inhibitors of CaM or CaMKII blocked Ca2+ ionophore-induced phagosomal maturation and enhanced the bacilli’s intracellular viability. These results demonstrate a novel role for CaM and CaMKII in the regulation of phagosome-lysosome fusion and suggest that defective activation of these Ca2+-activated signaling components contributes to the successful parasitism of human macrophages by M. tuberculosis.

ATP stimulates human macrophages to kill intracellular virulent Mycobacterium tuberculosis via calcium-dependent phagosome-lysosome fusion.

Advances in therapy for tuberculosis will require greater understanding of the molecular mechanisms of pathogenesis and the human immune response in this disease. Exposure of Mycobacterium tuberculosis-infected human macrophages to extracellular ATP (ATPe) results in bacterial killing, but the molecular mechanisms remain incompletely characterized. In this study, we demonstrate that ATPe-induced bactericidal activity toward virulent M. tuberculosis requires an increase in cytosolic Ca2+ in infected macrophages. Based on our previous work with primary infection of human macrophages, we hypothesized that the Ca2+ dependence of ATP-induced killing of intracellular M. tuberculosis was linked to promotion of phagosome-lysosome fusion. Using confocal laser-scanning microscopy, we demonstrate that ATPe induces fusion of the M. tuberculosis-containing phagosome with lysosomes, defined by accumulation of three lysosomal proteins and an acidophilic dye. Stimulation of phagosome-lysosome fusion by ATPe exhibited distinct requirements for both Ca2+ and phospholipase D and was highly correlated with killing of intracellular bacilli. Thus, key signal transduction pathways are conserved between two distinct models of human macrophage antituberculous activity: primary infection of naive macrophages and physiologic stimulation of macrophages stably infected with M. tuberculosis.

Sphingosine Kinase 1 (SK1) Is Recruited to Nascent Phagosomes in Human Macrophages: Inhibition of SK1 Translocation by Mycobacterium tuberculosis

Mycobacterium tuberculosis (M.tb) is a leading cause of global infectious mortality. The pathogenesis of tuberculosis involves inhibition of phagosome maturation, leading to survival of M.tb within human macrophages. A key determinant is M.tb-induced inhibition of macrophage sphingosine kinase (SK) activity, which normally induces Ca2+ signaling and phagosome maturation. Our objective was to determine the spatial localization of SK during phagocytosis and its inhibition by M.tb. Stimulation of SK activity by killed M.tb, live Staphylococcus aureus, or latex beads was associated with translocation of cytosolic SK1 to the phagosome membrane. In contrast, SK1 did not associate with phagosomes containing live M.tb. To characterize the mechanism of phagosomal translocation, live cell confocal microscopy was used to compare the localization of wild-type SK1, catalytically inactive SK1G82D, and a phosphorylation-defective mutant that does not undergo plasma membrane translocation (SK1S225A). The magnitude and kinetics of translocation of SK1G82D and SK1S225A to latex bead phagosomes were indistinguishable from those of wild-type SK1, indicating that novel determinants regulate the association of SK1 with nascent phagosomes. These data are consistent with a model in which M.tb inhibits both the activation and phagosomal translocation of SK1 to block the localized Ca2+ transients required for phagosome maturation.

Phospholipases D1 and D2 Coordinately Regulate Macrophage Phagocytosis

Phagocytosis is a fundamental feature of the innate immune system, required for antimicrobial defense, resolution of inflammation, and tissue remodeling. Furthermore, phagocytosis is coupled to a diverse range of cytotoxic effector mechanisms, including the respiratory burst, secretion of inflammatory mediators and Ag presentation. Phospholipase D (PLD) has been linked to the regulation of phagocytosis and subsequent effector responses, but the identity of the PLD isoform(s) involved and the molecular mechanisms of activation are unknown. We used primary human macrophages and human THP-1 promonocytes to characterize the role of PLD in phagocytosis. Macrophages, THP-1 cells, and other human myelomonocytic cells expressed both PLD1 and PLD2 proteins. Phagocytosis of complement-opsonized zymosan was associated with stimulation of the activity of both PLD1 and PLD2, as demonstrated by a novel immunoprecipitation-in vitro PLD assay. Transfection of dominant-negative PLD1 or PLD2 each inhibited the extent of phagocytosis (by 55–65%), and their combined effects were additive (reduction of 91%). PLD1 and PLD2 exhibited distinct localizations in resting macrophages and those undergoing phagocytosis, and only PLD1 localized to the phagosome membrane. The COS-7 monkey fibroblast cell line, which has been used as a heterologous system for the analysis of receptor-mediated phagocytosis, expressed PLD2 but not PLD1. These data support a model in which macrophage phagocytosis is coordinately regulated by both PLD1 and PLD2, with isoform-specific localization. Human myelomonocytic cell lines accurately model PLD-dependent signal transduction events required for phagocytosis, but the heterologous COS cell system does not.

Evolutionary conservation of physical and functional interactions between phospholipase D and actin

Phospholipase D (PLD) enzymes from bacteria to mammals exhibit a highly conserved core structure and catalytic mechanism, but whether protein-protein interactions exhibit similar commonality is unknown. Our objective was to determine whether the physical and functional interactions of mammalian PLDs with actin are evolutionarily conserved among bacterial and plant PLDs. Highly purified bacterial and plant PLDs cosedimented with mammalian skeletal muscle alpha-actin, indicating direct interaction with F-actin. The binding of bacterial PLD to G-actin exhibited two affinity states, with dissociation constants of 1.13 pM and 0.58 microM. The effects of actin on the activities of bacterial and plant PLDs were polymerization dependent; monomeric G-actin inhibited PLD activity, whereas polymerized F-actin augmented PLD activity. Actin modulation of bacterial and plant PLDs demonstrated kinetic characteristics, efficacies, and potencies similar to those of human PLD1. Thus, physical and functional interactions between PLD and actin in PLD family members from bacteria to mammals are highly conserved throughout evolution.

ROR1, an embryonic protein with an emerging role in cancer biology

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a member of the ROR family consisting of ROR1 and ROR2. RORs contain two distinct extracellular cysteine-rich domains and one transmembrane domain. Within the intracellular portion, ROR1 possesses a tyrosine kinase domain, two serine/threonine-rich domains and a proline-rich domain. RORs have been studied in the context of embryonic patterning and neurogenesis through a variety of homologs. These physiologic functions are dichotomous based on the requirement of the kinase domain. A growing literature has established ROR1 as a marker for cancer, such as in CLL and other blood malignancies. In addition, ROR1 is critically involved in progression of a number of blood and solid malignancies. ROR1 has been shown to inhibit apoptosis, potentiate EGFR signaling, and induce epithelial-mesenchymal transition (EMT). Importantly, ROR1 is only detectable in embryonic tissue and generally absent in adult tissue, making the protein an ideal drug target for cancer therapy.

Paracrine WNT5A Signaling Inhibits Expansion of Tumor-Initiating Cells

It is not well understood how paracrine communication between basal and luminal cell populations in the mammary gland affects tumorigenesis. During ErbB2-induced mammary tumorigenesis, enriched mammary stem cells that represent a subpopulation of basal cells exhibit enhanced tumorigenic capacity compared with the corresponding luminal progenitors. Transcript profiling of tumors derived from basal and luminal tumor-initiating cells (TIC) revealed preferential loss of the noncanonical Wnt ligand WNT5A in basal TIC-derived tumors. Heterozygous loss of WNT5A was correlated with shorter survival of breast cancer patients. In a mouse model of ErbB2-induced breast cancer, Wnt5a heterozygosity promoted tumor multiplicity and pulmonary metastasis. As a TGFβ substrate, luminal cell-produced WNT5A induced a feed-forward loop to activate SMAD2 in a RYK and TGFβR1-dependent manner to limit the expansion of basal TIC in a paracrine fashion, a potential explanation for the suppressive effect of WNT5A in mammary tumorigenesis. Our results identify the WNT5A/RYK module as a spatial regulator of the TGFβ-SMAD signaling pathway in the context of mammary gland development and carcinogenesis, offering a new perspective on tumor suppression provided by basal-luminal cross-talk in normal mammary tissue.

Phospholipase D1 Regulates Phagocyte Adhesion

Adhesion is a fundamental cellular response that is essential to the physiologic processes of development, differentiation, proliferation, and motility, as well as to the pathology of inflammation, transformation, and metastasis. Adhesion of phagocytic leukocytes is a critical modulator of antimicrobial and cytotoxic functions, including the respiratory burst, secretion, and apoptosis. Because phospholipase D (PLD) is linked to several signaling pathways implicated in these processes, we tested the hypothesis that PLD regulates phagocyte adhesion. Adhesion of primary human neutrophils and monocyte-derived macrophages to fibronectin was accompanied by marked stimulation of PLD activity. Similarly, adhesion of both human (PLB, THP-1) and murine (RAW) myeloid-macrophage cell lines to fibronectin, fibrinogen, collagen, or plastic resulted in significant activation of PLD. Stimulation of PLD activity was rapid and persisted for at least 90 min. Confocal microscopy indicated that PLD1 exhibited partial colocalization with actin filaments at the adherent interface, in proximity to the focal adhesion protein, paxillin. Reductions in PLD activity by chemical inhibitors or specific short-interfering RNA-induced knockdown of PLD1 resulted in significant inhibition of phagocyte adhesion and was accompanied by reductions in total cellular F-actin. These data support the hypotheses that adhesion stimulates PLD activity, and that PLD1 regulates the initial stages of phagocyte adhesion. Stimulation of PLD activity may promote adhesion-dependent phagocyte effector responses.