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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

Delay in Stx2-induced pathology in NKT cell deficient mice. (A) Percent survival of WT (n = 5) and CD1KO (n = 5) after Stx2 injection is plotted. Log-rank (Mantel-Cox) test, p = 0.0042. (B) Percent weight change of WT (n = 5) and CD1KO (n = 5) after Stx2 injection is plotted. Error bars are standard deviation. Two-Way ANOVA followed by Bonferroni test, *p < 0.05. (C) BUN value of each mouse are plotted from WT (n = 5 per time point) and CD1KO (n = 3 per time point). One-Way ANOVA followed by Tukey test, *p < 0.05 WT compared to 0 h, #p < 0.05 CD1KO compared to 0 h. (D) Urinary albumin of WT (n = 2–9 per time point) and CD1KO (n = 4–8 per time point) are plotted for each urine sample obtained at indicated time points. One-Way ANOVA followed by Tukey test, *p < 0.05 WT compared to 0 h, #p < 0.05 CD1KO compared to 0 h. (E) Averages of plasma concentration of WT (n = 5 per time point) and CD1KO (n = 3 per time point) from matching samples to (C) are shown. Error bars are standard deviation. One-Way ANOVA followed by Tukey test and no significance was detected. (F) Averages of urinary protein concentration of WT (n = 2 to9 per time point) and CD1KO (n = 4 to 8 per time point) from matching samples to (D) are shown. One-Way ANOVA followed by Tukey test, *p < 0.05 WT compared to 0 h, #p < 0.05 CD1KO compared to 0 h.
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Figure 1: Delay in Stx2-induced pathology in NKT cell deficient mice. (A) Percent survival of WT (n = 5) and CD1KO (n = 5) after Stx2 injection is plotted. Log-rank (Mantel-Cox) test, p = 0.0042. (B) Percent weight change of WT (n = 5) and CD1KO (n = 5) after Stx2 injection is plotted. Error bars are standard deviation. Two-Way ANOVA followed by Bonferroni test, *p < 0.05. (C) BUN value of each mouse are plotted from WT (n = 5 per time point) and CD1KO (n = 3 per time point). One-Way ANOVA followed by Tukey test, *p < 0.05 WT compared to 0 h, #p < 0.05 CD1KO compared to 0 h. (D) Urinary albumin of WT (n = 2–9 per time point) and CD1KO (n = 4–8 per time point) are plotted for each urine sample obtained at indicated time points. One-Way ANOVA followed by Tukey test, *p < 0.05 WT compared to 0 h, #p < 0.05 CD1KO compared to 0 h. (E) Averages of plasma concentration of WT (n = 5 per time point) and CD1KO (n = 3 per time point) from matching samples to (C) are shown. Error bars are standard deviation. One-Way ANOVA followed by Tukey test and no significance was detected. (F) Averages of urinary protein concentration of WT (n = 2 to9 per time point) and CD1KO (n = 4 to 8 per time point) from matching samples to (D) are shown. One-Way ANOVA followed by Tukey test, *p < 0.05 WT compared to 0 h, #p < 0.05 CD1KO compared to 0 h.

Mentions: Male C57BL/6 (wild type, WT) mice weighing 22 to 24 g were purchased from Charles River Laboratories (Wilmington, MA, USA). Male CD1.1−/− (CD1KO) mice of the matched age were maintained in Webb lab (Carnaud et al., 1999; Park et al., 1999; Hua et al., 2011). Mice were housed in a 12 h/12 h light and dark cycle and given access to food and water ad libitum. Mice were injected intraperitoneally 250 ng/kg Stx2 (two times the 50% lethal dose). At this dose of Stx2, 50% of WT mice die at approximately 72 h, and 100% die by 96 h (Figure 1). This dose was chosen based on previous studies with our mouse model of HUS (Keepers et al., 2006). Weight and survival were monitored every 12 h using five mice per strain. In the different set of experiment, at selected time points after injection, three mice per time point were euthanized by CO2 inhalation and kidneys were removed. Kidneys collected at 0 h (prior to Stx2 injection) were used for controls. Kidneys were processed for enzyme-linked immunosorbent assay (ELISA) as described below. Mouse blood was withdrawn into a 0.5 M of sodium ethylenediaminetetraacetate (Na2EDTA) wetted needle and syringe from their heart at each time point to isolate plasma for blood urea nitrogen (BUN) assay. Blood from 0 h (prior to Stx2 injection) animals were used as controls. All animal procedures were performed in accordance with University of Maryland School of Medicine Animal Care and Use Committee policies (animal use protocol number 0811012).


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)

Delay in Stx2-induced pathology in NKT cell deficient mice. (A) Percent survival of WT (n = 5) and CD1KO (n = 5) after Stx2 injection is plotted. Log-rank (Mantel-Cox) test, p = 0.0042. (B) Percent weight change of WT (n = 5) and CD1KO (n = 5) after Stx2 injection is plotted. Error bars are standard deviation. Two-Way ANOVA followed by Bonferroni test, *p < 0.05. (C) BUN value of each mouse are plotted from WT (n = 5 per time point) and CD1KO (n = 3 per time point). One-Way ANOVA followed by Tukey test, *p < 0.05 WT compared to 0 h, #p < 0.05 CD1KO compared to 0 h. (D) Urinary albumin of WT (n = 2–9 per time point) and CD1KO (n = 4–8 per time point) are plotted for each urine sample obtained at indicated time points. One-Way ANOVA followed by Tukey test, *p < 0.05 WT compared to 0 h, #p < 0.05 CD1KO compared to 0 h. (E) Averages of plasma concentration of WT (n = 5 per time point) and CD1KO (n = 3 per time point) from matching samples to (C) are shown. Error bars are standard deviation. One-Way ANOVA followed by Tukey test and no significance was detected. (F) Averages of urinary protein concentration of WT (n = 2 to9 per time point) and CD1KO (n = 4 to 8 per time point) from matching samples to (D) are shown. One-Way ANOVA followed by Tukey test, *p < 0.05 WT compared to 0 h, #p < 0.05 CD1KO compared to 0 h.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4389548&req=5

Figure 1: Delay in Stx2-induced pathology in NKT cell deficient mice. (A) Percent survival of WT (n = 5) and CD1KO (n = 5) after Stx2 injection is plotted. Log-rank (Mantel-Cox) test, p = 0.0042. (B) Percent weight change of WT (n = 5) and CD1KO (n = 5) after Stx2 injection is plotted. Error bars are standard deviation. Two-Way ANOVA followed by Bonferroni test, *p < 0.05. (C) BUN value of each mouse are plotted from WT (n = 5 per time point) and CD1KO (n = 3 per time point). One-Way ANOVA followed by Tukey test, *p < 0.05 WT compared to 0 h, #p < 0.05 CD1KO compared to 0 h. (D) Urinary albumin of WT (n = 2–9 per time point) and CD1KO (n = 4–8 per time point) are plotted for each urine sample obtained at indicated time points. One-Way ANOVA followed by Tukey test, *p < 0.05 WT compared to 0 h, #p < 0.05 CD1KO compared to 0 h. (E) Averages of plasma concentration of WT (n = 5 per time point) and CD1KO (n = 3 per time point) from matching samples to (C) are shown. Error bars are standard deviation. One-Way ANOVA followed by Tukey test and no significance was detected. (F) Averages of urinary protein concentration of WT (n = 2 to9 per time point) and CD1KO (n = 4 to 8 per time point) from matching samples to (D) are shown. One-Way ANOVA followed by Tukey test, *p < 0.05 WT compared to 0 h, #p < 0.05 CD1KO compared to 0 h.
Mentions: Male C57BL/6 (wild type, WT) mice weighing 22 to 24 g were purchased from Charles River Laboratories (Wilmington, MA, USA). Male CD1.1−/− (CD1KO) mice of the matched age were maintained in Webb lab (Carnaud et al., 1999; Park et al., 1999; Hua et al., 2011). Mice were housed in a 12 h/12 h light and dark cycle and given access to food and water ad libitum. Mice were injected intraperitoneally 250 ng/kg Stx2 (two times the 50% lethal dose). At this dose of Stx2, 50% of WT mice die at approximately 72 h, and 100% die by 96 h (Figure 1). This dose was chosen based on previous studies with our mouse model of HUS (Keepers et al., 2006). Weight and survival were monitored every 12 h using five mice per strain. In the different set of experiment, at selected time points after injection, three mice per time point were euthanized by CO2 inhalation and kidneys were removed. Kidneys collected at 0 h (prior to Stx2 injection) were used for controls. Kidneys were processed for enzyme-linked immunosorbent assay (ELISA) as described below. Mouse blood was withdrawn into a 0.5 M of sodium ethylenediaminetetraacetate (Na2EDTA) wetted needle and syringe from their heart at each time point to isolate plasma for blood urea nitrogen (BUN) assay. Blood from 0 h (prior to Stx2 injection) animals were used as controls. All animal procedures were performed in accordance with University of Maryland School of Medicine Animal Care and Use Committee policies (animal use protocol number 0811012).

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