Martin Olivier

Disruption of the trypanothione reductase gene of Leishmania decreases its ability to survive oxidative stress in macrophages

Parasitic protozoa belonging to the order Kinetoplastida contain trypanothione as their major thiol. Trypanothione reductase (TR), the enzyme responsible for maintaining trypanothione in its reduced form, is thought to be central to the redox defence systems of trypanosomatids. To investigate further the physiological role of TR in Leishmania, we attempted to create TR‐knockout mutants by gene disruption in L.donovani and L.major strains using the selectable markers neomycin and hygromycin phosphotransferases. TR is likely to be an important gene for parasite survival since all our attempts to obtain a TR null mutant in L.donovani failed. Instead, we obtained mutants with a partial trisomy for the TR locus where, despite the successful disruption of two TR alleles by gene targeting, a third TR copy was generated as a result of genomic rearrangements involving the translocation of a TR‐containing region to a larger chromosome. Mutants of L.donovani and L.major possessing only one wild‐type TR allele express less TR mRNA and have lower TR activity compared with wild‐type cells carrying two copies of the TR gene. Significantly, these mutants show attenuated infectivity with a markedly decreased capacity to survive intracellularly within macrophages, provided that the latter are producing reactive oxygen intermediates.

Leishmania‐induced increases in activation of macrophage SHP‐1 tyrosine phosphatase are associated with impaired IFN‐γ‐triggered JAK2 activation

Leishmania‐induced macrophage (Mϕ) dysfunctions have been correlated with altered signaling events. Recent findings from our laboratory suggest that modulation of host protein tyrosine phosphatase (PTP) following Leishmania infection could lead to these Mϕ defects. To address this issue, Mϕ PTP activity and IFN‐γ‐inducible signaling events were evaluated in Leishmania donovani (Ld)‐infected cells. We observed that Ld promastigotes can rapidly trigger host PTP activity simultaneously with dephosphorylation of Mϕ protein tyrosyl residues and inhibition of protein tyrosine kinase (PTK). Our results further revealed that Mϕ SHP‐1 PTP was rapidly activated by the infection. This Ld‐evoked signaling alteration was reflected by absence of IFN‐γ‐induced intracellular phosphorylation. IFN‐γ‐inducible JAK2 PTK phosphorylation was also markedly diminished in Ld‐infected cells. We also observed that co‐immunoprecipitation of JAK2 with SHP‐1 was considerably higher in infected as compared to uninfected cells. Altogether, these results suggest that SHP‐1‐mediated JAK2 dephosphorylation triggered by Leishmania is partly responsible for abnormal Mϕ IFN‐γ signaling and represent an important mechanism supporting persistent parasitic infection.

Episomal and stable expression of the luciferase reporter gene for quantifying Leishmania spp. infections in macrophages and in animal models.

We have expressed the reporter firefly luciferase gene (LUC) in Leishmania donovani and Leishmania major either as part of episomal vectors or integrated into the parasite genome under the control of their respective ribosomal promoter regions. An excellent linear correlation between parasite number and luciferase activity was observed with all the transfectants. LUC-expressing recombinant parasites were useful to monitor Leishmania spp. infections in macrophages or in animal models. For prolonged growth in absence of drug selection, such as within animal models, quantitation of parasites is more reliable when the reporter gene LUC is stably integrated in the parasite genome. These recombinant strains should be useful tools to monitor Leishmania growth under a number of conditions.

Enteropathogenic Escherichia coli mediates antiphagocytosis through the inhibition of PI 3‐kinase‐dependent pathways

The extracellular pathogen enteropathogenic Escherichia coli (EPEC) uses a type III secretion system to inhibit its uptake by macrophages. We show that EPEC antiphagocytosis is independent of the translocated intimin receptor Tir and occurs by preventing F‐actin polymerization required for bacterial uptake. EPEC–macrophage contact triggered activation of phosphatidylinositol (PI) 3‐kinase, which was subsequently inhibited in a type III secretion‐dependent manner. Inhibition of PI 3‐kinase significantly reduced uptake of a secretion‐deficient mutant, without affecting antiphagocytosis by the wild type, suggesting that EPEC blocks a PI 3‐kinase‐dependent phagocytic pathway. EPEC specifically inhibited Fcγ receptor‐ but not CR3‐receptor mediated phagocytosis of opsonized zymosan. We showed that EPEC inhibits PI 3‐kinase activity rather than its recruitment to the site of bacterial contact. Phago cytosis of a secretion mutant correlated with the association of PI 3‐kinase with tyrosine‐phosphorylated proteins, which wild‐type EPEC prevented. These results show that EPEC blocks its uptake by inhibiting a PI 3‐kinase‐mediated pathway, and translocates effectors other than Tir to interfere with actin‐driven host cell processes. This constitutes a novel mechanism of phagocytosis avoidance by an extracellular pathogen.

Mannosylated Lipoarabinomannan Antagonizes Mycobacterium tuberculosis-Induced Macrophage Apoptosis by Altering Ca+2-Dependent Cell Signaling

Mycobacterium tuberculosis-induced macrophage apoptosis can be inhibited by mannosylated lipoarabinomannan (ManLAM), although it induces tumor necrosis factor (TNF)-alpha and NO production, which participate in apoptosis induction. ManLAM also modulates Ca(+2)-dependent intracellular events, and Ca(+2) participates in apoptosis in different systems. Ca(+2) was assessed for involvement in M. tuberculosis-induced macrophage apoptosis and for modulation by ManLAM. The role of Ca(+2) was supported by the blockade of apoptosis by cAMP inhibitors and the Ca(+2) chelator, BAPTA/AM. These agents also inhibited caspase-1 activation and cAMP-responsive element-binding protein translocation without affecting TNF-alpha production. Infection of macrophages with M. tuberculosis induced an influx of Ca(+2) that was prevented by ManLAM. Similarly, M. tuberculosis infection-altered mitochondrial permeability transition was prevented by ManLAM and BAPTA/AM. Finally, ManLAM and BAPTA/AM reversed the effects of M. tuberculosis on p53 and Bcl-2 expression. ManLAM counteracts the alterations of calcium-dependent intracellular events that occur during M. tuberculosis-induced macrophage apoptosis.

Hemozoin-Inducible Proinflammatory Events In Vivo: Potential Role in Malaria Infection

During malaria infection, high levels of proinflammatory molecules (e.g., cytokines, chemokines) correlate with disease severity. Even if their role as activators of the host immune response has been studied, the direct contribution of hemozoin (HZ), a parasite metabolite, to such a strong induction is not fully understood. Previous in vitro studies demonstrated that both Plasmodium falciparum HZ and synthetic HZ (sHZ), β-hematin, induce macrophage/monocyte chemokine and proinflammatory cytokine secretion. In the present study, we investigated the proinflammatory properties of sHZ in vivo. To this end, increasing doses of sHZ were injected either i.v. or into an air pouch generated on the dorsum of BALB/c mice over a 24-h period. Our results showed that sHZ is a strong modulator of leukocyte recruitment and more specifically of neutrophil and monocyte populations. In addition, evaluation of chemokine and cytokine mRNA and protein expression revealed that sHZ induces the expression of chemokines, macrophage-inflammatory protein (MIP)-1α/CCL3, MIP-1β/CCL4, MIP-2/CXCL2, and monocyte chemoattractant protein-1/CCL2; chemokine receptors, CCR1, CCR2, CCR5, CXCR2, and CXCR4; cytokines, IL-1β and IL-6; and myeloid-related proteins, S100A8, S100A9, and S100A8/A9, in the air pouch exudates. Of interest, chemokine and cytokine mRNA up-regulation were also detected in the liver of i.v. sHZ-injected mice. In conclusion, our study demonstrates that sHZ is a potent proinflammatory agent in vivo, which could contribute to the immunopathology related to malaria.

Hemozoin Increases IFN-γ-Inducible Macrophage Nitric Oxide Generation Through Extracellular Signal-Regulated Kinase- and NF-κB-Dependent Pathways

NO overproduction has been suggested to contribute to the immunopathology related to malaria infection. Even though a role for some parasite molecules (e.g., GPI) in NO induction has been proposed, the direct contribution of hemozoin (HZ), another parasite metabolite, remains to be established. Therefore, we were interested to determine whether Plasmodium falciparum (Pf) HZ and synthetic HZ, β-hematin, alone or in combination with IFN-γ, were able to induce macrophage (Mφ) NO synthesis. We observed that neither Pf HZ nor synthetic HZ led to NO generation in B10R murine Mφ; however, they significantly increased IFN-γ-mediated inducible NO synthase (iNOS) mRNA and protein expression, and NO production. Next, by investigating the transductional mechanisms involved in this cellular regulation, we established that HZ induces extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase phosphorylation as well as NF-κB binding to the iNOS promoter, and enhances the IFN-γ-dependent activation of both second messengers. Of interest, cell pretreatment with specific inhibitors against either NF-κB or the ERK1/2 pathway blocked the HZ + IFN-γ-inducible NF-κB activity and significantly reduced the HZ-dependent increase on IFN-γ-mediated iNOS and NO induction. Even though selective inhibition of the Janus kinase 2/STAT1α pathway suppressed NO synthesis in response to HZ + IFN-γ, HZ alone did not activate this signaling pathway and did not have an up-regulating effect on the IFN-γ-induced Janus kinase 2/STAT1α phosphorylation and STAT1α binding to the iNOS promoter. In conclusion, our results suggest that HZ exerts a potent synergistic effect on the IFN-γ-inducible NO generation in Mφ via ERK- and NF-κB-dependent pathways.

Leishmania- Induced Cellular Recruitment during the Early Inflammatory Response: Modulation of Proinflammatory Mediators

This study investigated whether Leishmania species, the etiologic agent of cutaneous (Leishmania major) and visceral (Leishmania donovani) leishmaniasis, could differentially elicit early inflammatory events in vivo correlating with the subsequent development of their reciprocal pathogenesis. By use of the murine air pouch system, injection of Leishmania led to a rapid and transient accumulation of a mixed population of leukocytes, and L. major recruited 31-fold more leukocytes than did controls, compared with 7-fold more leukocytes for L. donovani. L. major promastigotes were better than L. donovani promastigotes at inducing proinflammatory cytokine secretion and chemokine gene expression in pouch exudates. L. major infection elicited significantly increased chemokine receptor gene expression, compared with L. donovani infection. Collectively, the data reveal that L. major is a strong inducer of the early inflammatory response, compared with L. donovani, and suggest that such an immunologic event potentially could restrain this parasite to the inoculation site, favoring the development of local swelling and cutaneous lesions.

Kinetic Study of Host Defense and Inflammatory Response to Aspergillus fumigatus in Steroid-Induced Immunosuppressed Mice

The sequential pathogenesis of pulmonary aspergillosis was studied and the role of inflammatory cytokines in host response to Aspergillus fumigatus was characterized in immunocompetent and immunosuppressed mice. Two distinct phases were observed in immunocompetent mice: First, an intense clearance of A. fumigatus occurred, possibly through alveolar macrophages and recruited neutrophils (PMNL), accompanied by rapid release of tumor necrosis factor-alpha, interleukin (IL)-6, and IL-1beta, and second, cellular and fungal debris were cleaned by recruited monocytes, cytokine production rapidly decreased, and pneumonia self-healed. In contrast, cortisone-treated animals had, first, an altered clearance of conidia and delayed cytokine production and inflammatory cell recruitment; second, an invasive process in lungs, recruitment of PMNL, and release of IL-6 and IL-1beta; and third, widespread tissue necrosis, sustained release of IL-6 and IL-1beta, further increases in PMNL trafficking but no monocyte recruitment, respiratory failure, and 100% mortality within 5 days. These insights may be useful in the development of new treatment strategies for pulmonary aspergillosis.

Signalling events involved in interferon-γ-inducible macrophage nitric oxide generation

Nitric oxide (NO) produced by macrophages (Mφ) in response to interferon‐γ (IFN‐γ) plays a pivotal role in the control of intracellular pathogens. Current knowledge of the specific biochemical cascades involved in this IFN‐γ‐inducible Mφ function is still limited. In the present study, we evaluated the participation of various second messengers – Janus kinase 2 (JAK2), signal transducer and activator of transcription (STAT) 1α, MAP kinase kinase (MEK1/2), extracellular signal‐regulated kinases 1 and 2 (Erk1/Erk2) and nuclear factor kappa B (NF‐κB) – in the regulation of NO production by IFN‐γ‐stimulated J774 murine Mφ. The use of specific signalling inhibitors permitted us to establish that JAK2/STAT1α‐ and Erk1/Erk2‐dependent pathways are the main players in IFN‐γ‐inducible Mφ NO generation. To determine whether the inhibitory effect was taking place at the pre‐ and/or post‐transcriptional level, we evaluated the effect of each antagonist on inducible nitric oxide synthase (iNOS) gene and protein expression, and on the capacity of IFN‐γ to induce JAK2, Erk1/Erk2 and STAT1α phosphorylation. All downregulatory effects occurred at the pretranscriptional level, except for NF‐κB, which seems to exert its role in NO production through an iNOS‐independent event. In addition, electrophoretic mobility shift assay (EMSA) analysis revealed that STAT1α is essential for IFN‐γ‐inducible iNOS expression and NO production, whereas the contribution of NF‐κB to this cellular regulation seems to be minimal. Moreover, our data suggest that Erk1/Erk2 are responsible for STAT1α Ser727 residue phosphorylation in IFN‐γ‐stimulated Mφ, thus contributing to the full activation of STAT1α. Taken together, our results indicate that JAK2, MEK1/2, Erk1/Erk2 and STAT1α are key players in the IFN‐γ‐inducible generation of NO by Mφ.