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Fasting enhances TRAIL-mediated liver natural killer cell activity via HSP70 upregulation.

Dang VT, Tanabe K, Tanaka Y, Tokumoto N, Misumi T, Saeki Y, Fujikuni N, Ohdan H - PLoS ONE (2014)

Bottom Line: Liver natural killer cells also showed high TRAIL-mediated antitumor function in response to 3-day fasting.Treatment of liver lymphocytes with 50 µg/mL of recombinant heat shock protein 70 led to the upregulation of both TRAIL and CD69 in liver natural killer cells (n = 6, p <0.05).These findings indicate that acute fasting enhances TRAIL-mediated liver natural killer cell activity against neoplastic cells through upregulation of heat shock protein 70.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterological and Transplant Surgery, Applied Life Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.

ABSTRACT
Acute starvation, which is frequently observed in clinical practice, sometimes augments the cytolytic activity of natural killer cells against neoplastic cells. In this study, we investigated the molecular mechanisms underlying the enhancement of natural killer cell function by fasting in mice. The total number of liver resident natural killer cells in a unit weight of liver tissue obtained from C57BL/6J mice did not change after a 3-day fast, while the proportions of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)+ and CD69+ natural killer cells were significantly elevated (n = 7, p <0.01), as determined by flow cytometric analysis. Furthermore, we found that TRAIL- natural killer cells that were adoptively transferred into Rag-2-/- γ chain-/- mice could convert into TRAIL+ natural killer cells in fasted mice at a higher proportion than in fed mice. Liver natural killer cells also showed high TRAIL-mediated antitumor function in response to 3-day fasting. Since these fasted mice highly expressed heat shock protein 70 (n = 7, p <0.05) in liver tissues, as determined by western blot, the role of this protein in natural killer cell activation was investigated. Treatment of liver lymphocytes with 50 µg/mL of recombinant heat shock protein 70 led to the upregulation of both TRAIL and CD69 in liver natural killer cells (n = 6, p <0.05). In addition, HSP70 neutralization by intraperitoneally injecting an anti- heat shock protein 70 monoclonal antibody into mice prior to fasting led to the downregulation of TRAIL expression (n = 6, p <0.05). These findings indicate that acute fasting enhances TRAIL-mediated liver natural killer cell activity against neoplastic cells through upregulation of heat shock protein 70.

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Assay analyzing cytotoxic effects of liver lymphocytes obtained from fasted mice on tumor cells.(A) The cytotoxic activity of freshly isolated liver lymphocytes from fed mice (solid lines) and 3-day-fasted mice (dashed lines) against TRAIL-resistant YAC-1 and (B) TRAIL-sensitive Hepa1-6 cells was analyzed using the 51Cr-release assay. The effector to target (E/T) ratios were 40∶1, 20∶1, 10∶1, and 5∶1. The cytotoxicity percentage was calculated as the percentage of specific 51Cr release, as described in the materials and methods section. Data are presented as mean ± standard error of the mean from triplicate samples of 11 repeated assays, each including 1 fed and 1 fasted mouse. (C) Histograms show the phenotype of Hepa1-6 cells that was analyzed using antibodies against mouse Fas, death receptor 5 (DR5), and decoy TRAIL receptor 1 and 2 (DcR1 and DcR2) in solid lines. Negative controls, which were stained with isotype-math antibodies, are indicated using dotted lines. The proportion of Hepa1-6 cells positive for those markers is provided. (D) Liver lymphocytes that were obtained from 3-day-fasted mice were incubated with CMA, anti-TRAIL mAb, anti-FasL mAb, or their combination before incubation with 51Cr-labeled-Hepa1-6 for 4 hours, at a lymphocyte: Hepa1-6 ratio of 40∶1. Bar graph shows the mean cytotoxicity percentage plus standard deviation for each group. Statistical analysis was performed for each ratio using the independent samples T test; *p <0.05.
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pone-0110748-g005: Assay analyzing cytotoxic effects of liver lymphocytes obtained from fasted mice on tumor cells.(A) The cytotoxic activity of freshly isolated liver lymphocytes from fed mice (solid lines) and 3-day-fasted mice (dashed lines) against TRAIL-resistant YAC-1 and (B) TRAIL-sensitive Hepa1-6 cells was analyzed using the 51Cr-release assay. The effector to target (E/T) ratios were 40∶1, 20∶1, 10∶1, and 5∶1. The cytotoxicity percentage was calculated as the percentage of specific 51Cr release, as described in the materials and methods section. Data are presented as mean ± standard error of the mean from triplicate samples of 11 repeated assays, each including 1 fed and 1 fasted mouse. (C) Histograms show the phenotype of Hepa1-6 cells that was analyzed using antibodies against mouse Fas, death receptor 5 (DR5), and decoy TRAIL receptor 1 and 2 (DcR1 and DcR2) in solid lines. Negative controls, which were stained with isotype-math antibodies, are indicated using dotted lines. The proportion of Hepa1-6 cells positive for those markers is provided. (D) Liver lymphocytes that were obtained from 3-day-fasted mice were incubated with CMA, anti-TRAIL mAb, anti-FasL mAb, or their combination before incubation with 51Cr-labeled-Hepa1-6 for 4 hours, at a lymphocyte: Hepa1-6 ratio of 40∶1. Bar graph shows the mean cytotoxicity percentage plus standard deviation for each group. Statistical analysis was performed for each ratio using the independent samples T test; *p <0.05.

Mentions: The cytotoxic potential of NK cells against the cell lines YAC-1 and Hepa1-6, which differ in their sensitivity to TRAIL, was determined using the 51Cr release assay. Liver lymphocytes from fed and 3-day-fasted mice were used as the effectors. There was no difference in the cytotoxicity of the two lymphocyte groups against TRAIL-resistant YAC-1 (Figure 5A). However, liver lymphocytes from fasted mice showed significantly higher cytotoxicity against TRAIL-sensitive Hepa1-6 than liver lymphocytes from fed mice at effector: target ratios of 40∶1, 20∶1, and 10∶1 (Figure 5B). To further investigate whether the upregulated cytotoxicity was mediated via TRAIL, we incubated liver lymphocytes from fasted mice with perforin inhibitor (CMA), anti-TRAIL mAb, anti-FasL mAb, or their combination at an effector: target ratio of 40∶1. Hepa1-6 receptor expression was also examined. Hepa1-6 cells highly expressed both Fas and death receptor 5 (TRAIL receptor 2) (Figure 5C). Lymphocytes treated with CMA, anti-TRAIL mAb, or their combination presented a significantly reduced cytotoxicity in comparison with the untreated group (Figure 5D). In contrast, the group treated with anti-FasL showed no significant difference. These results indicate that liver NK cells from fasted mice presented an increased perforin- and TRAIL-mediated antitumor activity.


Fasting enhances TRAIL-mediated liver natural killer cell activity via HSP70 upregulation.

Dang VT, Tanabe K, Tanaka Y, Tokumoto N, Misumi T, Saeki Y, Fujikuni N, Ohdan H - PLoS ONE (2014)

Assay analyzing cytotoxic effects of liver lymphocytes obtained from fasted mice on tumor cells.(A) The cytotoxic activity of freshly isolated liver lymphocytes from fed mice (solid lines) and 3-day-fasted mice (dashed lines) against TRAIL-resistant YAC-1 and (B) TRAIL-sensitive Hepa1-6 cells was analyzed using the 51Cr-release assay. The effector to target (E/T) ratios were 40∶1, 20∶1, 10∶1, and 5∶1. The cytotoxicity percentage was calculated as the percentage of specific 51Cr release, as described in the materials and methods section. Data are presented as mean ± standard error of the mean from triplicate samples of 11 repeated assays, each including 1 fed and 1 fasted mouse. (C) Histograms show the phenotype of Hepa1-6 cells that was analyzed using antibodies against mouse Fas, death receptor 5 (DR5), and decoy TRAIL receptor 1 and 2 (DcR1 and DcR2) in solid lines. Negative controls, which were stained with isotype-math antibodies, are indicated using dotted lines. The proportion of Hepa1-6 cells positive for those markers is provided. (D) Liver lymphocytes that were obtained from 3-day-fasted mice were incubated with CMA, anti-TRAIL mAb, anti-FasL mAb, or their combination before incubation with 51Cr-labeled-Hepa1-6 for 4 hours, at a lymphocyte: Hepa1-6 ratio of 40∶1. Bar graph shows the mean cytotoxicity percentage plus standard deviation for each group. Statistical analysis was performed for each ratio using the independent samples T test; *p <0.05.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4214715&req=5

pone-0110748-g005: Assay analyzing cytotoxic effects of liver lymphocytes obtained from fasted mice on tumor cells.(A) The cytotoxic activity of freshly isolated liver lymphocytes from fed mice (solid lines) and 3-day-fasted mice (dashed lines) against TRAIL-resistant YAC-1 and (B) TRAIL-sensitive Hepa1-6 cells was analyzed using the 51Cr-release assay. The effector to target (E/T) ratios were 40∶1, 20∶1, 10∶1, and 5∶1. The cytotoxicity percentage was calculated as the percentage of specific 51Cr release, as described in the materials and methods section. Data are presented as mean ± standard error of the mean from triplicate samples of 11 repeated assays, each including 1 fed and 1 fasted mouse. (C) Histograms show the phenotype of Hepa1-6 cells that was analyzed using antibodies against mouse Fas, death receptor 5 (DR5), and decoy TRAIL receptor 1 and 2 (DcR1 and DcR2) in solid lines. Negative controls, which were stained with isotype-math antibodies, are indicated using dotted lines. The proportion of Hepa1-6 cells positive for those markers is provided. (D) Liver lymphocytes that were obtained from 3-day-fasted mice were incubated with CMA, anti-TRAIL mAb, anti-FasL mAb, or their combination before incubation with 51Cr-labeled-Hepa1-6 for 4 hours, at a lymphocyte: Hepa1-6 ratio of 40∶1. Bar graph shows the mean cytotoxicity percentage plus standard deviation for each group. Statistical analysis was performed for each ratio using the independent samples T test; *p <0.05.
Mentions: The cytotoxic potential of NK cells against the cell lines YAC-1 and Hepa1-6, which differ in their sensitivity to TRAIL, was determined using the 51Cr release assay. Liver lymphocytes from fed and 3-day-fasted mice were used as the effectors. There was no difference in the cytotoxicity of the two lymphocyte groups against TRAIL-resistant YAC-1 (Figure 5A). However, liver lymphocytes from fasted mice showed significantly higher cytotoxicity against TRAIL-sensitive Hepa1-6 than liver lymphocytes from fed mice at effector: target ratios of 40∶1, 20∶1, and 10∶1 (Figure 5B). To further investigate whether the upregulated cytotoxicity was mediated via TRAIL, we incubated liver lymphocytes from fasted mice with perforin inhibitor (CMA), anti-TRAIL mAb, anti-FasL mAb, or their combination at an effector: target ratio of 40∶1. Hepa1-6 receptor expression was also examined. Hepa1-6 cells highly expressed both Fas and death receptor 5 (TRAIL receptor 2) (Figure 5C). Lymphocytes treated with CMA, anti-TRAIL mAb, or their combination presented a significantly reduced cytotoxicity in comparison with the untreated group (Figure 5D). In contrast, the group treated with anti-FasL showed no significant difference. These results indicate that liver NK cells from fasted mice presented an increased perforin- and TRAIL-mediated antitumor activity.

Bottom Line: Liver natural killer cells also showed high TRAIL-mediated antitumor function in response to 3-day fasting.Treatment of liver lymphocytes with 50 µg/mL of recombinant heat shock protein 70 led to the upregulation of both TRAIL and CD69 in liver natural killer cells (n = 6, p <0.05).These findings indicate that acute fasting enhances TRAIL-mediated liver natural killer cell activity against neoplastic cells through upregulation of heat shock protein 70.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterological and Transplant Surgery, Applied Life Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.

ABSTRACT
Acute starvation, which is frequently observed in clinical practice, sometimes augments the cytolytic activity of natural killer cells against neoplastic cells. In this study, we investigated the molecular mechanisms underlying the enhancement of natural killer cell function by fasting in mice. The total number of liver resident natural killer cells in a unit weight of liver tissue obtained from C57BL/6J mice did not change after a 3-day fast, while the proportions of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)+ and CD69+ natural killer cells were significantly elevated (n = 7, p <0.01), as determined by flow cytometric analysis. Furthermore, we found that TRAIL- natural killer cells that were adoptively transferred into Rag-2-/- γ chain-/- mice could convert into TRAIL+ natural killer cells in fasted mice at a higher proportion than in fed mice. Liver natural killer cells also showed high TRAIL-mediated antitumor function in response to 3-day fasting. Since these fasted mice highly expressed heat shock protein 70 (n = 7, p <0.05) in liver tissues, as determined by western blot, the role of this protein in natural killer cell activation was investigated. Treatment of liver lymphocytes with 50 µg/mL of recombinant heat shock protein 70 led to the upregulation of both TRAIL and CD69 in liver natural killer cells (n = 6, p <0.05). In addition, HSP70 neutralization by intraperitoneally injecting an anti- heat shock protein 70 monoclonal antibody into mice prior to fasting led to the downregulation of TRAIL expression (n = 6, p <0.05). These findings indicate that acute fasting enhances TRAIL-mediated liver natural killer cell activity against neoplastic cells through upregulation of heat shock protein 70.

Show MeSH
Related in: MedlinePlus