Albert Descoteaux

Protein Kinase C-ζ Regulates Transcription of the Matrix Metalloproteinase-9 Gene Induced by IL-1 and TNF-α in Glioma Cells via NF-κB

The regulation of matrix metalloproteinase-9 (MMP-9) expression in glioma cells is one of the key processes in tumor invasion through the brain extracellular matrix. Although some studies have demonstrated the implication of classic protein kinase C (PKC) isoforms in the regulation of MMP-9 production by phorbol esters or lipopolysaccharide, the involvement of specific PKC isoforms in the signaling pathways leading to MMP-9 expression by inflammatory cytokines remains unclear. Here we report that the atypical PKC-ζ isoform participates in the induction of MMP-9 expression by interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) in rat C6 glioma cells. Indeed, zymography and semi-quantitative reverse transcriptase-PCR analysis showed that pretreatment of C6 cells with PKC-ζ pseudosubstrate abolished MMP-9 activity and gene expression induced by IL-1 or TNF-α. Accordingly, IL-1 and TNF-α were able to induce PKC-ζ activity, as demonstrated by in vitro kinase assay using immunoprecipitated PKC-ζ. Furthermore, stable C6 clones overexpressing PKC-ζ, but not PKC-ε, displayed an up-regulation of MMP-9 constitutive expression as well as an increase ofmmp-9 promoter activity. These processes were inhibited by an NF-κB-blocking peptide and completely prevented by NF-κB-binding site mutation in the mmp-9 promoter. Taken together, these results indicate that PKC-ζ plays a key role in the regulation of MMP-9 expression in C6 glioma cells through NF-κB.

Impaired recruitment of the small GTPase rab7 correlates with the inhibition of phagosome maturation by Leishmania donovani promastigotes

We have shown recently that one of the survival strategies used by Leishmania donovani promastigotes during the establishment of infection in macrophages consists in inhibiting phagosome–endosome fusion. This inhibition requires the expression of lipophosphoglycan (LPG), the predominant surface glycoconjugate of promastigotes, as parasites expressing truncated forms of LPG reside in phagosomes that fuse extensively with endocytic organelles. In the present study, we developed a single‐organelle fluorescence analysis approach to study and analyse the intracellular trafficking of ‘fusogenic’ and ‘low‐fusogenic’ phagosomes induced by an LPG repeating unit‐defective mutant (lpg2 KO) or by wild‐type L. donovani promastigotes respectively. The results obtained indicate that phagosomes containing mutant parasites fuse extensively with endocytic organelles and transform into phagolysosomes by losing the early endosome markers EEA1 and transferrin receptor, and acquiring the late endocytic and lysosomal markers rab7 and LAMP1. In contrast, a majority of ‘low‐fusogenic’ phagosomes containing wild‐type L. donovani promastigotes do not acquire rab7, wheres they acquire LAMP1 with slower kinetics. These results suggest that L. donovani parasites use LPG to restrict phagosome–endosome fusion at the onset of infection in order to prevent phagosome maturation. This is likely to permit the transformation of hydrolase‐sensitive promastigotes into hydrolase‐resistant amastigotes within a hospitable vacuole not displaying the harsh environment of phagolysosomes.

Leishmania donovani promastigotes evade the activation of mitogen-activated protein kinases p38, c-Jun N-terminal kinase, and extracellular signal-regulated kinase-1/2 during infection of naive macrophages.

The protozoan parasite Leishmania fails to activate naive macrophages for proinflammatory cytokines production, and selectively impairs signal transduction pathways in infected macrophages. Because mitogen‐activated protein kinases (MAPK)‐ and NF‐κB‐dependent signaling pathways regulate proinflammatory cytokines release, we investigated their activation in mouse bone marrow‐derived macrophages (BMM) exposed to Leishmania donovani promastigotes. In naive BMM, the parasite failed to induce the phosphorylation of p38 MAPK, c‐Jun N‐terminal kinase (JNK), and extracellular signal‐regulated kinase (ERK)1/2, as well as the degradation of IκB‐α. The use of L. donovani mutants defective in the biosynthesis of lipophosphoglycan revealed that evasion of ERK1/2 activation requires surface expression of the repeating unit moiety of this virulence determinant. In IFN‐γ‐primed BMM, L. donovani promastigotes strongly induced the phosphorylation of p38 MAPK and ERK1/2, and the use of selective inhibitors for ERK (PD98059) and p38 MAPK (SB203580) revealed that both kinases are required for L. donovani‐induced TNF‐α but not NO2– release. Collectively, these data suggest that both p38 MAPK and ERK1/2 pathways participate in some Leishmania‐induced responses in IFN‐γ‐primed BMM. The ability of L. donovani promastigotes to avoid MAPK and NF‐κB activation in naive macrophages may be part of the strategy evolved by this parasite to evade innate immune responses.

Leishmania promastigotes require lipophosphoglycan to actively modulate the fusion properties of phagosomes at an early step of phagocytosis.

The lipophosphoglycan (LPG) of Leishmania promastigotes plays key roles in parasite survival in both insect and mammalian hosts. Evidence suggests that LPG decreases phagosome fusion properties at the onset of infection in macrophages. The mechanisms of action of this molecule are, however, poorly understood. In the present study, we used a panoply of Leishmania mutants displaying modified LPG structures to determine more precisely how LPG modulates phagosome–endosome fusion. Using an in vivo fusion assay measuring, at the electron microscope, the transfer of solute materials from endosomes to phagosomes, we provided further evidence that the repeating Gal(β1,4)Man(α1‐PO4) units of LPG are responsible for the alteration in phagosome fusion. The inhibitory effect of LPG on phagosome fusion was shown to be more potent towards late endocytic organelles and lysosomes than early endosomes, explaining how Leishmania promastigotes can avoid degradation in hydrolase‐enriched compartments. The involvement of other repeating unit‐containing molecules, including the secreted acid phosphatase, in the inhibition process was ruled out, as an LPG‐defective mutant (lpg1−) which secretes repeating unit‐containing glycoconjugates was present in highly fusogenic phagosomes. In L. major, oligosaccharide side‐chains of LPG did not contribute to the inhibition process, as Spock, an L. major mutant lacking LPG side‐chains, blocked fusion to the same extent as wild‐type parasites. Finally, dead parasites internalized from the culture medium were not as efficient as live parasites in altering phagosome–endosome fusion, despite the presence of LPG. However, the killing of parasites with vital dyes after their sequestration in phagosomes had no effect on the fusion properties of this organelle. Collectively, these results suggest that living promastigotes displaying full‐length cell surface LPG can actively influence macrophages at an early stage of phagocytosis to generate phagosomes with poor fusogenic properties.

Leishmania donovani lipophosphoglycan causes periphagosomal actin accumulation: correlation with impaired translocation of PKCα and defective phagosome maturation

Lipophosphoglycan (LPG) is the major surface glycoconjugate of Leishmania donovani promastigotes. The repeating disaccharide–phosphate units of LPG are crucial for promastigote survival inside macrophages and establishment of infection. LPG has a number of effects on the host cell, including inhibition of PKC activity, inhibition of nitric oxide production and altered expression of cytokines. LPG also inhibits phagosomal maturation, a process requiring depolymerization of periphagosomal F‐actin. In the present study, we have characterized the dynamics of F‐actin during the phagocytosis of L. donovani promastigotes in J774 macrophages. We observed that F‐actin accumulated progressively around phagosomes containing wild‐type L. donovani promastigotes during the first hour of phagocytosis. Using LPG‐defective mutants and yeast particles coated with purified LPG, we obtained evidence that this effect could be attributed to the repeating units of LPG. LPG also disturbed cortical actin turnover during phagocytosis. The LPG‐dependent accumulation of periphagosomal F‐actin correlated with an impaired recruitment of the lysosomal marker LAMP1 and PKCα to the phagosome. Accumulation of periphagosomal F‐actin during phagocytosis of L. donovani promastigotes may contribute to the inhibition of phagosomal maturation by physically preventing vesicular trafficking to and from the phagosome.

Functional aspects of the Leishmania donovani lipophosphoglycan during macrophage infection.

The most abundant surface glycoconjugate of the Leishmania promastigotes is lipophosphoglycan, a glycosylphosphatidyl-inositol-anchored polymer of the repeating disaccharide-phosphate Gal(beta1,4)Manalpha1-PO4 unit. This complex molecule possesses properties that contribute to the ability of Leishmania to modulate macrophage signaling pathways during the initiation of infection.

Role of protein kinase C α for uptake of unopsonized prey and phagosomal maturation in macrophages

Protein kinase C alpha (PKC alpha) participates in F-actin remodeling during phagocytosis and phagosomal maturation in macrophages. Leishmania donovani promastigotes, which inhibit phagosomal maturation, cause accumulation of periphagosomal F-actin instead of the disassembly observed around other prey [Cell. Microbiol. 7 (2001) 439]. This accumulation is induced by promastigote lipophosphoglycan (LPG), which has several effects on macrophages including inhibition of PKC alpha. To investigate a possible connection between PKC alpha and LPGs effects on actin dynamics, we utilized RAW264.7 macrophages overexpressing dominant-negative PCK alpha (DN PKC alpha). We found increased cortical F-actin and decreased phagocytic capacity, as well as defective periphagosomal F-actin breakdown and inhibited phagosomal maturation in the DN PKC alpha-overexpressing cells, effects similar to those seen in controls subjected to LPG-coated prey. The results indicate that PKC alpha is involved in F-actin turnover in macrophages and that PKC alpha-dependent breakdown of periphagosomal F-actin is required for phagosomal maturation, and endorse the hypothesis that intracellular survival of L. donovani involves inhibition of PKC alpha by LPG.

Contribution of Electron and Confocal Microscopy in the Study of Leishmania –Macrophage Interactions

Promastigotes of the protozoan parasite genus Leishmania are inoculated into a mammalian host when an infected sand fly takes a bloodmeal. Following their opsonization by complement, promastigotes are phagocytosed by macrophages. There, promastigotes differentiate into amastigotes, the form of the parasite that replicates in the phagolysosomal compartments of host macrophages. Although the mechanisms by which promastigotes survive the microbicidal consequence of phagocytosis remain, for the most part, to be elucidated, evidence indicates that glycoconjugates play a role in this process. One such glycoconjugate is lipophosphoglycan, an abundant promastigote surface glycolipid. Using quantitative electron and confocal laser scanning microscopy approaches, evidence was provided that L. donovani promastigotes inhibit phagolysosome biogenesis in a lipophosphoglycan-dependent manner. This inhibition correlates with an accumulation of periphagosomal F-actin, which may potentially form a physical barrier that prevents L. donovani promastigote-containing phagosomes from interacting with endocytic vacuoles. Inhibition of phagosome maturation may constitute a strategy to provide an environment propitious to the promastigote-to-amastigote differentiation.

Modulation of lipopolysaccharide‐induced NF‐IL6 activation by protein kinase C‐α in a mouse macrophage cell line

We have previously shown that overexpression of a dominant‐negative (DN) mutant of protein kinase C‐α (PKC‐α) in RAW 264.7 macrophages inhibited lypopolysaccharide (LPS)‐induced IL‐1α, inducible nitric oxide synthase and cyclooxygenase‐2 expression. This inhibition was not related to defective NF‐κB nuclear translocation, suggesting that PKC‐α might be involved in the modulation of other LPS‐inducible transcription factors. In the present study, we have investigated the impact of PKC‐α on the activation of AP‐1 and NF‐IL6 in LPS‐treated RAW 264.7 macrophages. Electrophoretic mobility shift assays and luciferase reporter constructs revealed that LPS‐induced AP‐1 transcriptional activity was normal in DN PKC‐α‐overexpressing RAW 264.7 cells. In contrast, LPS‐induced DNA‐binding and transcriptional activities of NF‐IL6 were inhibited in DN PKC‐α‐overexpressing RAW 264.7 cells and correlated with an impairment of NF‐IL6 nuclear translocation. Conversely, overexpression of either wild‐type PKC‐α or a constitutively active PKC‐α mutant significantly enhanced LPS‐stimulated NF‐IL6‐dependent promoter activity. Finally, LPS‐induced expression of two genes regulated by NF‐IL6, namely IL‐1β and granulocyte colony‐stimulating factor, was impaired in DN PKC‐α‐overexpressing RAW 264.7 cells. Taken together, these results suggest that regulation of NF‐IL6 activity constitutes one of the mechanisms by which PKC‐α modulates LPS‐induced gene expression in the mouse macrophage cell line RAW 264.7.

Large-Scale Phagosome Preparation

Phagocytosis is the process by which cells engulf and destroy large particles such as pathogens or apoptotic cells. In this way, macrophages play a pivotal role in the resolution of microbial infections. However, many microorganisms have evolved efficient strategies to preempt the weaponry of macrophages. A better understanding of the components engaged in the phagosome formation and maturation is necessary to devise novel approaches aimed at counteracting these microbial strategies. Recently, large-scale approaches have been used to improve our understanding of phagosome functional properties by the identification of hundreds of proteins and by studying each of them. Presently, purification of pathogen-containing phagosomes presents several technical challenges, whereas the use of latex beads to isolate phagosomes presents many advantages because this system can mimic host-pathogen interactions during phagocytosis. This system thus remains the best approach to advance our knowledge of phagosome biology, notably when used in conjunction with functional approaches. In this chapter, we outline an approach for the isolation of large-scale phagosome preparations with high degrees of purity.