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Histoplasma capsulatum yeast phase-specific protein Yps3p induces Toll-like receptor 2 signaling.

Aravalli RN, Hu S, Woods JP, Lokensgard JR - J Neuroinflammation (2008)

Bottom Line: Our data show that H. capsulatum Yps3p induced TLR2 signaling in wild-type microglia, but not in microglia isolated from TLR2 KO mice, confirming that Yps3p is a ligand for TLR2.Furthermore, Yps3p-induced TLR2 signaling was suppressed by vaccinia virus-encoded TLR inhibitors.This is the first demonstration of a fungal protein serving as a TLR ligand and mediating signaling in primary brain cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Infectious Diseases and Microbiology Translational Research, University of Minnesota Medical School, Minneapolis, Minnesota, USA. arava001@umn.edu

ABSTRACT
Histoplasma capsulatum is a common cause of fungal infection in certain geographic areas, and although most infections are asymptomatic, it is capable of causing histoplasmosis, a disseminated, life-threatening disease, especially in immunocompromised individuals. A deeper understanding of this host-pathogen interaction is needed to develop novel therapeutic strategies to counter lethal infection. Although several lines of evidence suggest that this fungus is neurotropic in HIV patients, little is known about the immunobiology of Histoplasma infection in the central nervous system [CNS]. The goal of the present study was to understand the innate neuroimmune mechanisms that recognize H. capsulatum during the initial stages of infection. Using a 293T stable cell line expressing murine Toll-like receptor 2 [TLR2], we show here that TLR2 recognizes H. capsulatum cell wall protein Yps3p and induces the activation of NF-kappaB. In further experiments, we tested the ability of Yps3p to induce signaling from TLR2 in primary microglial cells, the resident brain macrophages of the CNS. Our data show that H. capsulatum Yps3p induced TLR2 signaling in wild-type microglia, but not in microglia isolated from TLR2 KO mice, confirming that Yps3p is a ligand for TLR2. Furthermore, Yps3p-induced TLR2 signaling was suppressed by vaccinia virus-encoded TLR inhibitors. This is the first demonstration of a fungal protein serving as a TLR ligand and mediating signaling in primary brain cells.

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Activation of NF-κB in primary murine microglia following treatment with Yps3p. Purified microglial cells [1 × 106] from wild-type and TLR2 knockout mice were transiently transfected with pNiFty2-Luc plasmid by electroporation using the mouse macrophage nucleofector kit. The cells were incubated overnight at 37°C and 1.5 μg Yps3p was added to the cell culture medium. The microglia were harvested after 5 h and the amount of luciferase produced as a result of NF-κB activation was quantified using the bright glow substrate. Data are presented as mean ± SD of triplicate samples and are representative of three independent experiments. Statistical analysis was performed by student's t test. **P < 0.01.
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Figure 4: Activation of NF-κB in primary murine microglia following treatment with Yps3p. Purified microglial cells [1 × 106] from wild-type and TLR2 knockout mice were transiently transfected with pNiFty2-Luc plasmid by electroporation using the mouse macrophage nucleofector kit. The cells were incubated overnight at 37°C and 1.5 μg Yps3p was added to the cell culture medium. The microglia were harvested after 5 h and the amount of luciferase produced as a result of NF-κB activation was quantified using the bright glow substrate. Data are presented as mean ± SD of triplicate samples and are representative of three independent experiments. Statistical analysis was performed by student's t test. **P < 0.01.

Mentions: Having determined that H. capsulatum protein Yps3p engages TLR2 signaling pathway and causes NF-κB activation in our 293T-mTLR2 cell line, we went on to determine whether Yps3p protein also induced NF-kB activation in primary brain cells. For this experiment, microglial cells from wild-type C57BL/6 mice as well as TLR2 KO mice were isolated and transfected with pNiFty2-Luc plasmid using nucleofection. After overnight incubation at 37°C, the microglia were exposed to the recombinant fungal protein for 6 h. The cells were then harvested and a luciferase assay was performed. In these experiments, Yps3p-induced TLR2 signaling in wild-type microglial cells resulted in high levels of luciferase expression, demonstrating an increased level of NF-κB activation (Fig. 4). In contrast, a significant reduction in luciferase expression occurred following the identical treatment using TLR2 KO microglia. This result further demonstrates that Yps3p triggered signaling through TLR2.


Histoplasma capsulatum yeast phase-specific protein Yps3p induces Toll-like receptor 2 signaling.

Aravalli RN, Hu S, Woods JP, Lokensgard JR - J Neuroinflammation (2008)

Activation of NF-κB in primary murine microglia following treatment with Yps3p. Purified microglial cells [1 × 106] from wild-type and TLR2 knockout mice were transiently transfected with pNiFty2-Luc plasmid by electroporation using the mouse macrophage nucleofector kit. The cells were incubated overnight at 37°C and 1.5 μg Yps3p was added to the cell culture medium. The microglia were harvested after 5 h and the amount of luciferase produced as a result of NF-κB activation was quantified using the bright glow substrate. Data are presented as mean ± SD of triplicate samples and are representative of three independent experiments. Statistical analysis was performed by student's t test. **P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2474602&req=5

Figure 4: Activation of NF-κB in primary murine microglia following treatment with Yps3p. Purified microglial cells [1 × 106] from wild-type and TLR2 knockout mice were transiently transfected with pNiFty2-Luc plasmid by electroporation using the mouse macrophage nucleofector kit. The cells were incubated overnight at 37°C and 1.5 μg Yps3p was added to the cell culture medium. The microglia were harvested after 5 h and the amount of luciferase produced as a result of NF-κB activation was quantified using the bright glow substrate. Data are presented as mean ± SD of triplicate samples and are representative of three independent experiments. Statistical analysis was performed by student's t test. **P < 0.01.
Mentions: Having determined that H. capsulatum protein Yps3p engages TLR2 signaling pathway and causes NF-κB activation in our 293T-mTLR2 cell line, we went on to determine whether Yps3p protein also induced NF-kB activation in primary brain cells. For this experiment, microglial cells from wild-type C57BL/6 mice as well as TLR2 KO mice were isolated and transfected with pNiFty2-Luc plasmid using nucleofection. After overnight incubation at 37°C, the microglia were exposed to the recombinant fungal protein for 6 h. The cells were then harvested and a luciferase assay was performed. In these experiments, Yps3p-induced TLR2 signaling in wild-type microglial cells resulted in high levels of luciferase expression, demonstrating an increased level of NF-κB activation (Fig. 4). In contrast, a significant reduction in luciferase expression occurred following the identical treatment using TLR2 KO microglia. This result further demonstrates that Yps3p triggered signaling through TLR2.

Bottom Line: Our data show that H. capsulatum Yps3p induced TLR2 signaling in wild-type microglia, but not in microglia isolated from TLR2 KO mice, confirming that Yps3p is a ligand for TLR2.Furthermore, Yps3p-induced TLR2 signaling was suppressed by vaccinia virus-encoded TLR inhibitors.This is the first demonstration of a fungal protein serving as a TLR ligand and mediating signaling in primary brain cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Infectious Diseases and Microbiology Translational Research, University of Minnesota Medical School, Minneapolis, Minnesota, USA. arava001@umn.edu

ABSTRACT
Histoplasma capsulatum is a common cause of fungal infection in certain geographic areas, and although most infections are asymptomatic, it is capable of causing histoplasmosis, a disseminated, life-threatening disease, especially in immunocompromised individuals. A deeper understanding of this host-pathogen interaction is needed to develop novel therapeutic strategies to counter lethal infection. Although several lines of evidence suggest that this fungus is neurotropic in HIV patients, little is known about the immunobiology of Histoplasma infection in the central nervous system [CNS]. The goal of the present study was to understand the innate neuroimmune mechanisms that recognize H. capsulatum during the initial stages of infection. Using a 293T stable cell line expressing murine Toll-like receptor 2 [TLR2], we show here that TLR2 recognizes H. capsulatum cell wall protein Yps3p and induces the activation of NF-kappaB. In further experiments, we tested the ability of Yps3p to induce signaling from TLR2 in primary microglial cells, the resident brain macrophages of the CNS. Our data show that H. capsulatum Yps3p induced TLR2 signaling in wild-type microglia, but not in microglia isolated from TLR2 KO mice, confirming that Yps3p is a ligand for TLR2. Furthermore, Yps3p-induced TLR2 signaling was suppressed by vaccinia virus-encoded TLR inhibitors. This is the first demonstration of a fungal protein serving as a TLR ligand and mediating signaling in primary brain cells.

Show MeSH
Related in: MedlinePlus