Measuring the Killing of Intracellular Pathogens: <i>Leishmania</i>

Stenger, Steffen; Zandbergen, Ger van

doi:10.1002/0471142735.im1423s93

Cited by 11 publications

(10 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To investigate the consequences of T-cell proliferation on parasite infection rate and parasite load we used transgenic Lm parasites, of which the viable parasites express the dsRed protein. 32 This offered us the ability to use flow cytometry to analyze parasite survival by means of infection rate (dsRed C hMDM) and parasite load (mean fluorescent intensity, MFI). Next, the hMDM were infected with log.ph.…”

Section: Apoptotic-likementioning

confidence: 99%

Apoptotic-likeLeishmaniaexploit the host´s autophagy machinery to reduce T-cell-mediated parasite elimination

et al. 2015

View full text Add to dashboard Cite

Apoptosis is a well-defined cellular process in which a cell dies, characterized by cell shrinkage and DNA fragmentation. In parasites like Leishmania, the process of apoptosis-like cell death has been described. Moreover upon infection, the apoptotic-like population is essential for disease development, in part by silencing host phagocytes. Nevertheless, the exact mechanism of how apoptosis in unicellular organisms may support infectivity remains unclear. Therefore we investigated the fate of apoptotic-like Leishmania parasites in human host macrophages. Our data showed-in contrast to viable parasites-that apoptotic-like parasites enter an LC3 C , autophagy-like compartment. The compartment was found to consist of a single lipid bilayer, typical for LC3-associated phagocytosis (LAP). As LAP can provoke anti-inflammatory responses and autophagy modulates antigen presentation, we analyzed how the presence of apoptotic-like parasites affected the adaptive immune response. Macrophages infected with viable Leishmania induced proliferation of CD4 C T-cells, leading to a reduced intracellular parasite survival. Remarkably, the presence of apoptotic-like parasites in the inoculum significantly reduced T-cell proliferation. Chemical induction of autophagy in human monocyte-derived macrophage (hMDM), infected with viable parasites only, had an even stronger proliferationreducing effect, indicating that host cell autophagy and not parasite viability limits the T-cell response and enhances parasite survival. Concluding, our data suggest that apoptotic-like Leishmania hijack the host cells´autophagy machinery to reduce T-cell proliferation. Furthermore, the overall population survival is guaranteed, explaining the benefit of apoptosis-like cell death in a single-celled parasite and defining the host autophagy pathway as a potential therapeutic target in treating Leishmaniasis.

show abstract

Section: Apoptotic-likementioning

confidence: 99%

Apoptotic-likeLeishmaniaexploit the host´s autophagy machinery to reduce T-cell-mediated parasite elimination

et al. 2015

View full text Add to dashboard Cite

show abstract

“…As expected the infection rate was increased after 24 h to 45% in control cells, but dramatically reduced in C2 toxin-treated cells to 8% ( Figure 6B). This cannot be explained solely by depolymerization of the actin cytoskeleton in the macrophages, and rather might involve cytotoxic effects on the internalized promastigotes, which lose their EGFP fluorescence when undergoing cell death (Stenger and van Zandbergen, 2010). Intriguingly, the infection rate was only slightly decreased in C2-streptavidintreated cells as compared with control cells.…”

Section: The C2-streptavidin Transporter Does Not Compromise Phagocytmentioning

confidence: 90%

“…To this end, cells were stained with anti-HLA-DR antibody labeled with PerCP (BD Biosciences, San Jose, USA) in FACS-buffer consisting of PBS, 1% FCS, 1% BSA and 1% human plasma. Subsequently, infection rates were quantified in MHC-II positive MF using FACS analysis after 3 and 24 h of co-incubation with L. major promastigotes as described very recently (Stenger and van Zandbergen, 2010).…”

Section: Infection Of Human Primary Macrophages (Mf) With Leishmania mentioning

confidence: 99%

The C2-streptavidin delivery system promotes the uptake of biotinylated molecules in macrophages and T-leukemia cells

Fahrer¹,

Rieger²,

Zandbergen

et al. 2010

Biological Chemistry

View full text Add to dashboard Cite

Macrophages are tightly associated with inflammatory diseases as well as carcinogenesis, and therefore represent promising targets for drug delivery and gene transfer. We have recently established a novel protein delivery system based on the binary C2 toxin of Clostridium botulinum and streptavidin, allowing the uptake of exogenous biotinylated molecules into mammalian cells. Here, we applied this C2-streptavidin delivery system to macrophages and other leukocytes. First, the effect of wild-type C2 toxin on different leukocyte cell lines was tested, indicating no differences in sensitivity. Next, the uptake and stability of the engineered C2-streptavidin was analyzed in macrophages and Jurkat T-cells, showing internalization into the cytosol of both cell types with similar kinetics. The transporter did not exhibit any cytotoxic effect and did not interfere with phagocytosis in primary human macrophages. The C2-streptavidin system promoted specific uptake of biotinylated fluorophores into the cytosol of macrophages as revealed by confocal microscopy. In addition, flow cytometry analysis showed a significantly enhanced uptake of biotinylated fluorescent tracers in Jurkat leukemia cells mediated by the C2-streptavidin transporter. Our results demonstrate that C2-streptavidin is a functional delivery system for transport of biotinylated molecules into macrophages and other leukocytes without compromising cell viability and intrinsic functions such as phagocytosis.

show abstract

“…Evaluation of parasitic load can be determined by staining macrophages with Giemsa and counting parasites and cell nuclei under the microscope then estimating the infection index (Lezama-Dávila et al, 2014). The generation of transgenic parasites expressing fluorescent proteins (GFP, RFP, and DsRed) enables the use of flow cytometry to estimate parasitic loads more broadly and rapidly as the analysis is based on thousands of cells (Kram et al, 2008; Stenger and Zandbergen, 2011). Further, an indirect evidence of infection could be estimated using the mean fluorescence intensity of the infected population.…”

Section: Introductionmentioning

confidence: 99%

Uncovering Leishmania–macrophage interplay using imaging flow cytometry

Terrazas

Oghumu

Martínez-Saucedo

et al. 2015

Journal of Immunological Methods

View full text Add to dashboard Cite

Host–pathogen interaction is an area of considerable interest. Intracellular parasites such as Leishmania reside inside phagocytes such as macrophages, dendritic cells and neutrophils. Macrophages can be activated by cytokines such as IFN-γ and Toll like receptor (TLR) agonists resulting in enhanced microbicidal activity. Leishmania parasites hijack the microbicidal function of macrophages, mainly by interfering with intracellular signaling initiated by IFN-γ and TLR ligands. Here we used transgenic Leishmania donovani parasites expressing the red fluorescent protein DsRed2 and imaging-flow cytometry technology to evaluate parasitic loads inside the macrophage in vitro. Further, this methodology enables us to visualize impairment in NFκB translocation to the nucleus in L. donovani infected macrophages. Additionally we show that uninfected bystander macrophages have a similar impairment in NFκB translocation as in L. donovani infected macrophages in response to the TLR4 agonist LPS. This evidence suggests a possible immunosuppressive role for infected macrophages in regulating the activation of uninfected bystander macrophages.

show abstract

Measuring the Killing of Intracellular Pathogens: Leishmania

Cited by 11 publications

References 23 publications

Apoptotic-likeLeishmaniaexploit the host´s autophagy machinery to reduce T-cell-mediated parasite elimination

Apoptotic-likeLeishmaniaexploit the host´s autophagy machinery to reduce T-cell-mediated parasite elimination

The C2-streptavidin delivery system promotes the uptake of biotinylated molecules in macrophages and T-leukemia cells

Uncovering Leishmania–macrophage interplay using imaging flow cytometry

Contact Info

Product

Resources

About