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Natural killer T (NKT) cells accelerate Shiga toxin type 2 (Stx2) pathology in mice.

Obata F, Subrahmanyam PB, Vozenilek AE, Hippler LM, Jeffers T, Tongsuk M, Tiper I, Saha P, Jandhyala DM, Kolling GL, Latinovic O, Webb TJ - Front Microbiol (2015)

Bottom Line: NKT cell-associated cytokines such as IL-2, IL-4, IFN-γ, and IL-17 were detected in kidney lysates of Stx2-injected WT mice with the peak around 36 h after Stx2 injection.In CD1KO, there was a delay in the kinetics, and increases in these cytokines were observed 60 h post Stx2 injection.We found that murine glomerular endothelial cells and podocytes express functional CD1d molecules and can present exogenous antigen to NKT cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Maryland School of Medicine Baltimore, MD, USA ; Department of Molecular Pathology, University of Yamanashi Graduate School of Medicine Chuo, Japan.

ABSTRACT
Shiga toxin-producing Escherichia coli (STEC) is a leading cause of childhood renal disease Hemolytic Uremic Syndrome (HUS). The involvement of renal cytokines and chemokines is suspected to play a critical role in disease progression. In current article, we tested the hypothesis that NKT cells are involved in Stx2-induced pathology in vivo. To address this hypothesis we compared Stx2 toxicity in WT and CD1 knockout (KO) mice. In CD1KO mice, which lack natural killer T (NKT) cells, Stx2-induced pathologies such as weight loss, renal failure, and death were delayed. In WT mice, Stx2-specific selective increase in urinary albumin occurs in later time points, and this was also delayed in NKT cell deficient mice. NKT cell-associated cytokines such as IL-2, IL-4, IFN-γ, and IL-17 were detected in kidney lysates of Stx2-injected WT mice with the peak around 36 h after Stx2 injection. In CD1KO, there was a delay in the kinetics, and increases in these cytokines were observed 60 h post Stx2 injection. These data suggest that NKT cells accelerate Stx2-induced pathology in mouse kidneys. To determine the mechanism by which NKT cells promote Stx2-associated disease, in vitro studies were performed using murine renal cells. We found that murine glomerular endothelial cells and podocytes express functional CD1d molecules and can present exogenous antigen to NKT cells. Moreover, we observed the direct interaction between Stx2 and the receptor Gb3 on the surface of mouse renal cells by 3D STORM-TIRF which provides single molecule imaging. Collectively, these data suggest that Stx2 binds to Gb3 on renal cells and leads to aberrant CD1d-mediated NKT cell activation. Therefore, strategies targeting NKT cells could have a significant impact on Stx2-associated renal pathology in STEC disease.

No MeSH data available.


Related in: MedlinePlus

Stx2 interacts with murine glomerular renal cells in vitro. (A) Murine podocytes or (B) Vero cells were incubated with 20 nM Stx2 for 0 (no toxin), 5 and 15 min at 37°C. Stx2 was labeled with monoclonal antibody 11E10 followed by anti-mouse IgG-AlexaFluor 647 (pseudo colored red) and Gb3 was labeled with monoclonal antibody 38.13 followed by anti-rat IgM-AlexaFluor 488 (green). Nuclei were stained with DAPI (blue). Samples were observed with confocal microscopy. Representative cells that are chosen from each time point group are shown. Bars indicate 10 μm. Arrowheads point Stx2-AlexaFluor 647 positive cells. (C) Murine podocytes were incubated with 20 nM Stx2 for 0 (no toxin), 5 and 15 min at 37°C and labeled as above. Samples were visualized with 3D STORM-TIRF microscopy and analyzed using Nikon Elements software. Whole cell view presents representative cells from each time point. Bars are 10 μm. Yellow spot view presents highly magnified and three dimensionally shown yellow cluster (red = Stx2-Alexa647, green = Gb3-Alexa488 and yellow = red and green overlapping pixels). Bars are 100 pixels (px). Yellow cluster depth view depicts the intra or extracellular depth of the yellow cluster. The color-coded depth scale at the left differentiate the distance (nm) from the focus plane (0 nm, green). Blue represents intracellular (up to 500 nm) whereas red represents extracellular (up to -500 nm). Contact count given in the graphs presents amount and depth distribution of green and red contact of a whole cell. The color-coded depth scale at the bottom indicates intracellular (blue) to extracellular (red).
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Figure 3: Stx2 interacts with murine glomerular renal cells in vitro. (A) Murine podocytes or (B) Vero cells were incubated with 20 nM Stx2 for 0 (no toxin), 5 and 15 min at 37°C. Stx2 was labeled with monoclonal antibody 11E10 followed by anti-mouse IgG-AlexaFluor 647 (pseudo colored red) and Gb3 was labeled with monoclonal antibody 38.13 followed by anti-rat IgM-AlexaFluor 488 (green). Nuclei were stained with DAPI (blue). Samples were observed with confocal microscopy. Representative cells that are chosen from each time point group are shown. Bars indicate 10 μm. Arrowheads point Stx2-AlexaFluor 647 positive cells. (C) Murine podocytes were incubated with 20 nM Stx2 for 0 (no toxin), 5 and 15 min at 37°C and labeled as above. Samples were visualized with 3D STORM-TIRF microscopy and analyzed using Nikon Elements software. Whole cell view presents representative cells from each time point. Bars are 10 μm. Yellow spot view presents highly magnified and three dimensionally shown yellow cluster (red = Stx2-Alexa647, green = Gb3-Alexa488 and yellow = red and green overlapping pixels). Bars are 100 pixels (px). Yellow cluster depth view depicts the intra or extracellular depth of the yellow cluster. The color-coded depth scale at the left differentiate the distance (nm) from the focus plane (0 nm, green). Blue represents intracellular (up to 500 nm) whereas red represents extracellular (up to -500 nm). Contact count given in the graphs presents amount and depth distribution of green and red contact of a whole cell. The color-coded depth scale at the bottom indicates intracellular (blue) to extracellular (red).

Mentions: In kidney, the first cell types that NKT cells as well as Stx2 may encounter are glomerular cells, such as endothelial cells and podocytes. We tested the possibility of Stx2 directly interacting with murine glomerular cells by using sensitive fluorescence imaging methods. Murine glomerular podocytes and Vero cells were incubated with Stx2 and the localization of Stx2 (red) and the receptor Gb3 (green) was observed by confocal microscopy. Without Stx2, podocytes had none to minimum level of red, whereas Gb3 was clearly positive (Figure 3A, 0 min) and the result was similar in Vero cells (Figure 3B, 0 min). In all NKT cell hypbridomas tested, Gb3 immunofluorescence was undetectable (data not shown). Stx2 incubation of 5 and 15 min resulted in cell surface Stx2 localization in both podocytes and Vero cells (arrowheads, Figures 3A,B, 5 and 15 min). To observe the precise contact of Stx2 and Gb3 molecules at the plasma membrane, 3D STORM-TIRF was utilized. In 3D STORM-TIRF observation, occurrences of Stx2 (red) and Gb3 (green) contact can be visualized as yellow clusters (Figure 3C, whole cell view). The occurrence of yellow clusters in Vero cells, podocytes and endothelial cells are shown in Table 1. Because the whole cell view looks at all signals from the top of z-sections and STORM detects individual fluorescence as single molecule, a close up of a yellow cluster shows more precise three dimensional positioning of Stx2 (red) and Gb3 (green) (Figure 3C, yellow cluster view). The 360° of a yellow cluster view is presented as a movie (Supplemental Figure 1). In the yellow cluster depth view, the colors are assigned according to the depth of the molecules from outside of the cell as Red (distance -500 nm from plasma membrane) to inside of the cells as blue (distance 500 nm from plasma membrane) (Figure 3C, yellow cluster depth view, 360° appearance in Supplemental Figure 2). For example, 0 min sample in which Stx2 is not added, a yellow cluster that occurs occasionally as background is shown in the depth view that colored as green to yellow (membrane to extracellular). In the samples of 5 and 15 min, representative yellow clusters are blue to green (intracellular to membrane) in the depth view. To show the distribution of yellow clusters along with the depth scale, the yellow clusters from each z-section of an entire cell were counted, and shown in contact count graphs (Figure 3C, contact count). The graph plots depth information in x-axis, and cluster counts in y-axis. The results depicts the distribution of Stx2/Gb3 double positive clusters that the longer Stx2 was incubated with the cells, the more cluster counts were obtained and the clusters internalized deeper in the cells. Murine glomerular endothelial cells and Vero cells showed similar results (Supplemental Figure 3). Negative controls such as fluorescence-conjugated secondary antibodies are omitted, had only residual signals of AlexaFluor 647 or 488, and the yellow cluster observation was none to minimum (Supplemental Figure 4).


Natural killer T (NKT) cells accelerate Shiga toxin type 2 (Stx2) pathology in mice.

Obata F, Subrahmanyam PB, Vozenilek AE, Hippler LM, Jeffers T, Tongsuk M, Tiper I, Saha P, Jandhyala DM, Kolling GL, Latinovic O, Webb TJ - Front Microbiol (2015)

Stx2 interacts with murine glomerular renal cells in vitro. (A) Murine podocytes or (B) Vero cells were incubated with 20 nM Stx2 for 0 (no toxin), 5 and 15 min at 37°C. Stx2 was labeled with monoclonal antibody 11E10 followed by anti-mouse IgG-AlexaFluor 647 (pseudo colored red) and Gb3 was labeled with monoclonal antibody 38.13 followed by anti-rat IgM-AlexaFluor 488 (green). Nuclei were stained with DAPI (blue). Samples were observed with confocal microscopy. Representative cells that are chosen from each time point group are shown. Bars indicate 10 μm. Arrowheads point Stx2-AlexaFluor 647 positive cells. (C) Murine podocytes were incubated with 20 nM Stx2 for 0 (no toxin), 5 and 15 min at 37°C and labeled as above. Samples were visualized with 3D STORM-TIRF microscopy and analyzed using Nikon Elements software. Whole cell view presents representative cells from each time point. Bars are 10 μm. Yellow spot view presents highly magnified and three dimensionally shown yellow cluster (red = Stx2-Alexa647, green = Gb3-Alexa488 and yellow = red and green overlapping pixels). Bars are 100 pixels (px). Yellow cluster depth view depicts the intra or extracellular depth of the yellow cluster. The color-coded depth scale at the left differentiate the distance (nm) from the focus plane (0 nm, green). Blue represents intracellular (up to 500 nm) whereas red represents extracellular (up to -500 nm). Contact count given in the graphs presents amount and depth distribution of green and red contact of a whole cell. The color-coded depth scale at the bottom indicates intracellular (blue) to extracellular (red).
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Related In: Results  -  Collection

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Figure 3: Stx2 interacts with murine glomerular renal cells in vitro. (A) Murine podocytes or (B) Vero cells were incubated with 20 nM Stx2 for 0 (no toxin), 5 and 15 min at 37°C. Stx2 was labeled with monoclonal antibody 11E10 followed by anti-mouse IgG-AlexaFluor 647 (pseudo colored red) and Gb3 was labeled with monoclonal antibody 38.13 followed by anti-rat IgM-AlexaFluor 488 (green). Nuclei were stained with DAPI (blue). Samples were observed with confocal microscopy. Representative cells that are chosen from each time point group are shown. Bars indicate 10 μm. Arrowheads point Stx2-AlexaFluor 647 positive cells. (C) Murine podocytes were incubated with 20 nM Stx2 for 0 (no toxin), 5 and 15 min at 37°C and labeled as above. Samples were visualized with 3D STORM-TIRF microscopy and analyzed using Nikon Elements software. Whole cell view presents representative cells from each time point. Bars are 10 μm. Yellow spot view presents highly magnified and three dimensionally shown yellow cluster (red = Stx2-Alexa647, green = Gb3-Alexa488 and yellow = red and green overlapping pixels). Bars are 100 pixels (px). Yellow cluster depth view depicts the intra or extracellular depth of the yellow cluster. The color-coded depth scale at the left differentiate the distance (nm) from the focus plane (0 nm, green). Blue represents intracellular (up to 500 nm) whereas red represents extracellular (up to -500 nm). Contact count given in the graphs presents amount and depth distribution of green and red contact of a whole cell. The color-coded depth scale at the bottom indicates intracellular (blue) to extracellular (red).
Mentions: In kidney, the first cell types that NKT cells as well as Stx2 may encounter are glomerular cells, such as endothelial cells and podocytes. We tested the possibility of Stx2 directly interacting with murine glomerular cells by using sensitive fluorescence imaging methods. Murine glomerular podocytes and Vero cells were incubated with Stx2 and the localization of Stx2 (red) and the receptor Gb3 (green) was observed by confocal microscopy. Without Stx2, podocytes had none to minimum level of red, whereas Gb3 was clearly positive (Figure 3A, 0 min) and the result was similar in Vero cells (Figure 3B, 0 min). In all NKT cell hypbridomas tested, Gb3 immunofluorescence was undetectable (data not shown). Stx2 incubation of 5 and 15 min resulted in cell surface Stx2 localization in both podocytes and Vero cells (arrowheads, Figures 3A,B, 5 and 15 min). To observe the precise contact of Stx2 and Gb3 molecules at the plasma membrane, 3D STORM-TIRF was utilized. In 3D STORM-TIRF observation, occurrences of Stx2 (red) and Gb3 (green) contact can be visualized as yellow clusters (Figure 3C, whole cell view). The occurrence of yellow clusters in Vero cells, podocytes and endothelial cells are shown in Table 1. Because the whole cell view looks at all signals from the top of z-sections and STORM detects individual fluorescence as single molecule, a close up of a yellow cluster shows more precise three dimensional positioning of Stx2 (red) and Gb3 (green) (Figure 3C, yellow cluster view). The 360° of a yellow cluster view is presented as a movie (Supplemental Figure 1). In the yellow cluster depth view, the colors are assigned according to the depth of the molecules from outside of the cell as Red (distance -500 nm from plasma membrane) to inside of the cells as blue (distance 500 nm from plasma membrane) (Figure 3C, yellow cluster depth view, 360° appearance in Supplemental Figure 2). For example, 0 min sample in which Stx2 is not added, a yellow cluster that occurs occasionally as background is shown in the depth view that colored as green to yellow (membrane to extracellular). In the samples of 5 and 15 min, representative yellow clusters are blue to green (intracellular to membrane) in the depth view. To show the distribution of yellow clusters along with the depth scale, the yellow clusters from each z-section of an entire cell were counted, and shown in contact count graphs (Figure 3C, contact count). The graph plots depth information in x-axis, and cluster counts in y-axis. The results depicts the distribution of Stx2/Gb3 double positive clusters that the longer Stx2 was incubated with the cells, the more cluster counts were obtained and the clusters internalized deeper in the cells. Murine glomerular endothelial cells and Vero cells showed similar results (Supplemental Figure 3). Negative controls such as fluorescence-conjugated secondary antibodies are omitted, had only residual signals of AlexaFluor 647 or 488, and the yellow cluster observation was none to minimum (Supplemental Figure 4).

Bottom Line: NKT cell-associated cytokines such as IL-2, IL-4, IFN-γ, and IL-17 were detected in kidney lysates of Stx2-injected WT mice with the peak around 36 h after Stx2 injection.In CD1KO, there was a delay in the kinetics, and increases in these cytokines were observed 60 h post Stx2 injection.We found that murine glomerular endothelial cells and podocytes express functional CD1d molecules and can present exogenous antigen to NKT cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Maryland School of Medicine Baltimore, MD, USA ; Department of Molecular Pathology, University of Yamanashi Graduate School of Medicine Chuo, Japan.

ABSTRACT
Shiga toxin-producing Escherichia coli (STEC) is a leading cause of childhood renal disease Hemolytic Uremic Syndrome (HUS). The involvement of renal cytokines and chemokines is suspected to play a critical role in disease progression. In current article, we tested the hypothesis that NKT cells are involved in Stx2-induced pathology in vivo. To address this hypothesis we compared Stx2 toxicity in WT and CD1 knockout (KO) mice. In CD1KO mice, which lack natural killer T (NKT) cells, Stx2-induced pathologies such as weight loss, renal failure, and death were delayed. In WT mice, Stx2-specific selective increase in urinary albumin occurs in later time points, and this was also delayed in NKT cell deficient mice. NKT cell-associated cytokines such as IL-2, IL-4, IFN-γ, and IL-17 were detected in kidney lysates of Stx2-injected WT mice with the peak around 36 h after Stx2 injection. In CD1KO, there was a delay in the kinetics, and increases in these cytokines were observed 60 h post Stx2 injection. These data suggest that NKT cells accelerate Stx2-induced pathology in mouse kidneys. To determine the mechanism by which NKT cells promote Stx2-associated disease, in vitro studies were performed using murine renal cells. We found that murine glomerular endothelial cells and podocytes express functional CD1d molecules and can present exogenous antigen to NKT cells. Moreover, we observed the direct interaction between Stx2 and the receptor Gb3 on the surface of mouse renal cells by 3D STORM-TIRF which provides single molecule imaging. Collectively, these data suggest that Stx2 binds to Gb3 on renal cells and leads to aberrant CD1d-mediated NKT cell activation. Therefore, strategies targeting NKT cells could have a significant impact on Stx2-associated renal pathology in STEC disease.

No MeSH data available.


Related in: MedlinePlus