CD14-Dependent Monocyte Isolation Enhances Phagocytosis of Listeria monocytogenes by Proinflammatory, GM-CSF-Derived Macrophages

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.

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“…61,62 Apoptotic and viable promastigotes were purified from stationary phase promastigotes by magnetic separation, as described previously. 1 …”

Section: Methodsmentioning

confidence: 99%

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

et al. 2015

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“…Bone marrow-derived macrophages (BMDM) were generated from bone marrow cells in M-CSF-containing L929-conditioned medium as described 10, 14 , or by incubation with mouse recombinant M-CSF or GM-CSF (50ng/ml each) 15 . Experiments were carried out for 24 h in 6-well trays (1.5 × 10 6 cells/ml; Costar, Cambridge, MA) in the absence or presence of eRNA (1, 10 or 25 µg/ml), as indicated.…”

Section: Methodsmentioning

confidence: 99%

Role of Extracellular RNA in Atherosclerotic Plaque Formation in Mice

et al. 2014

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Background Atherosclerosis and vascular remodeling after injury are driven by inflammation and mononuclear cell infiltration. Extracellular RNA (eRNA) has recently been implicated to become enriched at sites of tissue damage, and to act as a pro-inflammatory mediator. We here addressed the role of eRNA in high-fat-diet (HFD)-induced atherosclerosis and neointima formation after injury in atherosclerosis-prone mice. Methods and Results The presence of eRNA was revealed in atherosclerotic lesions from HFD-fed low density lipoprotein receptor-deficient (Ldlr−/−) mice in a time-progressive fashion. RNase activity in plasma increased within the first 2 weeks (44 ± 9 vs. 70 ± 7 mU/mg protein; p=0.0012), followed by a decrease to levels below baseline after 4 weeks of HFD (44 ± 9 vs. 12 ± 2 mU/mg protein; p<0.0001). Exposure of bone marrow-derived macrophages to eRNA resulted in a concentration-dependent upregulation of the pro-inflammatory mediators tumor necrosis factor-α, arginase-2, Interleukin (IL)-1β, IL-6 and Interferon-γ. In a model of accelerated atherosclerosis after arterial injury in apolipoprotein E-deficient (apoE−/−) mice, treatment with RNase1 diminished the increased plasma level of eRNA, evidenced after injury. Likewise, RNase1 administration reduced neointima formation compared to vehicle-treated apoE−/− controls (25.0 ± 6.2 vs. 46.9 ± 6.9 × 103 µm2, p=0.0339), and was associated with a significant decrease in plaque macrophage content. Functionally, RNase1 treatment impaired monocyte arrest on activated smooth muscle cells under flow conditions in vitro, and inhibited leukocyte recruitment to injured carotid arteries in vivo. Conclusions As eRNA is associated with atherosclerotic lesions and contributes to inflammation-dependent plaque progression in atherosclerosis-prone mice, its targeting with RNase1 may serve as a new treatment option against atherosclerosis.

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“…Primary human macrophages were generated essentially as described previously (40). Briefly, lymphocytes were isolated from fresh buffy coats by density gradient centrifugation using lymphocyte separation medium (LSM 1077; PAA Laboratories).…”

Section: Cfu MLmentioning

confidence: 99%

Expression of Fluorescent Proteins in Bifidobacteria for Analysis of Host-Microbe Interactions

Grimm

Gleinser

Neu

et al. 2014

Appl Environ Microbiol

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Bifidobacteria are an important component of the human gastrointestinal microbiota and are frequently used as probiotics. The genetic inaccessibility and lack of molecular tools commonly used in other bacteria have hampered a detailed analysis of the genetic determinants of bifidobacteria involved in their adaptation to, colonization of, and interaction with the host. In the present study, a range of molecular tools were developed that will allow the closing of some of the gaps in functional analysis of bifidobacteria. A number of promoters were tested for transcriptional activity in Bifidobacterium bifidum S17 using pMDY23, a previously published promoter probe vector. The promoter of the gap gene (P gap ) of B. bifidum S17 yielded the highest promoter activity among the promoters tested. Thus, this promoter and the pMDY23 backbone were used to construct a range of vectors for expression of different fluorescent proteins (FPs). Successful expression of cyan fluorescent protein (CFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), and mCherry could be shown for three strains representing three different Bifidobacterium spp. The red fluorescent B. bifidum S17/pVG-mCherry was further used to demonstrate application of fluorescent bifidobacteria for adhesion assays and detection in primary human macrophages cultured in vitro. Furthermore, pMGCmCherry was cloned by combining a chloramphenicol resistance marker and expression of the FP mCherry under the control of P gap . The chloramphenicol resistance marker of pMGC-mCherry was successfully used to determine gastrointestinal transit time of B. bifidum S17. Moreover, B. bifidum S17/pMGC-mCherry could be detected in fecal samples of mice after oral administration.

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CD14-Dependent Monocyte Isolation Enhances Phagocytosis of Listeria monocytogenes by Proinflammatory, GM-CSF-Derived Macrophages

Cited by 34 publications

References 45 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

Role of Extracellular RNA in Atherosclerotic Plaque Formation in Mice

Expression of Fluorescent Proteins in Bifidobacteria for Analysis of Host-Microbe Interactions

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