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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-treated murine glomerular renal cells are able to activate NKT cells in vitro. (A) Murine podocytes or (B) murine glomerular endothelial cells were treated with (stx) or without (untx) Stx2, washed and incubated either with or without 100 ng/ml of αGC for 2 h. These antigen loaded cells were then washed and cocultured with DN32.D3, N37-1A12 or N38-3C3 NKT cell hybridomas for 20 h. IL-2 was measured in the supernatant by ELISA as readout for NKT cell activation. Medium alone and empty vector-transfected L-cells (L-vector) were used as negative controls, and CD1d-transfected L-cells (L-CD1d) was used as a positive control. (C) Murine podocytes and murine glomerular endothelial cells were incubated with (bold line) or without (dashed line) Stx2 and stained with PE-conjugated anti-CD1d antibody and cell surface expression of CD1d was determined by flow cytometry. An isotype control Ab-PE served as a negative control (gray). (D) L-vector and L-CD1d were used as negative and positive controls, respectively.
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Figure 4: Stx2-treated murine glomerular renal cells are able to activate NKT cells in vitro. (A) Murine podocytes or (B) murine glomerular endothelial cells were treated with (stx) or without (untx) Stx2, washed and incubated either with or without 100 ng/ml of αGC for 2 h. These antigen loaded cells were then washed and cocultured with DN32.D3, N37-1A12 or N38-3C3 NKT cell hybridomas for 20 h. IL-2 was measured in the supernatant by ELISA as readout for NKT cell activation. Medium alone and empty vector-transfected L-cells (L-vector) were used as negative controls, and CD1d-transfected L-cells (L-CD1d) was used as a positive control. (C) Murine podocytes and murine glomerular endothelial cells were incubated with (bold line) or without (dashed line) Stx2 and stained with PE-conjugated anti-CD1d antibody and cell surface expression of CD1d was determined by flow cytometry. An isotype control Ab-PE served as a negative control (gray). (D) L-vector and L-CD1d were used as negative and positive controls, respectively.

Mentions: CD1d-restricted NKT cells can be activated by recognizing lipid antigen that is presented in the context of CD1d molecules that are expressed on antigen presenting cells. The ability of Stx2-treated murine renal glomerular cells to induce NKT cell activation was measured by ELISA. Podocytes (Figure 4A) or endothelial cells (Figure 4B) were incubated with or without Stx2, and αGC was added as a lipid antigen, then co-cultured with NKT cells. Stx2-treated cells with αGC induced higher levels of IL-2, compared to the no toxin control in the presence of an exogenous antigen, αGC (p < 0.001). Without αGC, both Stx2-treated and non-treated cells did not induce IL-2 production by NKT cells. The positive control, mouse L-cells stably transfected with mCD1d1 (L-CD1d) induced IL-2 production in all NKT cell hybridomas. In order to determine whether an increase in NKT activation in Stx2-treated cells is due to an increase in CD1d surface expression, flow cytometry was performed with podocytes (Figure 4C) or endothelial cells (Figure 4D). Addition of Stx2 did not change the surface expression of CD1d in podocytes or endothelial cells, suggesting an increase in NKT activation in Stx2-treated renal cells is not due to an increased the levels of CD1d.


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-treated murine glomerular renal cells are able to activate NKT cells in vitro. (A) Murine podocytes or (B) murine glomerular endothelial cells were treated with (stx) or without (untx) Stx2, washed and incubated either with or without 100 ng/ml of αGC for 2 h. These antigen loaded cells were then washed and cocultured with DN32.D3, N37-1A12 or N38-3C3 NKT cell hybridomas for 20 h. IL-2 was measured in the supernatant by ELISA as readout for NKT cell activation. Medium alone and empty vector-transfected L-cells (L-vector) were used as negative controls, and CD1d-transfected L-cells (L-CD1d) was used as a positive control. (C) Murine podocytes and murine glomerular endothelial cells were incubated with (bold line) or without (dashed line) Stx2 and stained with PE-conjugated anti-CD1d antibody and cell surface expression of CD1d was determined by flow cytometry. An isotype control Ab-PE served as a negative control (gray). (D) L-vector and L-CD1d were used as negative and positive controls, respectively.
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Figure 4: Stx2-treated murine glomerular renal cells are able to activate NKT cells in vitro. (A) Murine podocytes or (B) murine glomerular endothelial cells were treated with (stx) or without (untx) Stx2, washed and incubated either with or without 100 ng/ml of αGC for 2 h. These antigen loaded cells were then washed and cocultured with DN32.D3, N37-1A12 or N38-3C3 NKT cell hybridomas for 20 h. IL-2 was measured in the supernatant by ELISA as readout for NKT cell activation. Medium alone and empty vector-transfected L-cells (L-vector) were used as negative controls, and CD1d-transfected L-cells (L-CD1d) was used as a positive control. (C) Murine podocytes and murine glomerular endothelial cells were incubated with (bold line) or without (dashed line) Stx2 and stained with PE-conjugated anti-CD1d antibody and cell surface expression of CD1d was determined by flow cytometry. An isotype control Ab-PE served as a negative control (gray). (D) L-vector and L-CD1d were used as negative and positive controls, respectively.
Mentions: CD1d-restricted NKT cells can be activated by recognizing lipid antigen that is presented in the context of CD1d molecules that are expressed on antigen presenting cells. The ability of Stx2-treated murine renal glomerular cells to induce NKT cell activation was measured by ELISA. Podocytes (Figure 4A) or endothelial cells (Figure 4B) were incubated with or without Stx2, and αGC was added as a lipid antigen, then co-cultured with NKT cells. Stx2-treated cells with αGC induced higher levels of IL-2, compared to the no toxin control in the presence of an exogenous antigen, αGC (p < 0.001). Without αGC, both Stx2-treated and non-treated cells did not induce IL-2 production by NKT cells. The positive control, mouse L-cells stably transfected with mCD1d1 (L-CD1d) induced IL-2 production in all NKT cell hybridomas. In order to determine whether an increase in NKT activation in Stx2-treated cells is due to an increase in CD1d surface expression, flow cytometry was performed with podocytes (Figure 4C) or endothelial cells (Figure 4D). Addition of Stx2 did not change the surface expression of CD1d in podocytes or endothelial cells, suggesting an increase in NKT activation in Stx2-treated renal cells is not due to an increased the levels of CD1d.

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