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Rapamycin ameliorates CCl4-induced liver fibrosis in mice through reciprocal regulation of the Th17/Treg cell balance.

Gu L, Deng WS, Sun XF, Zhou H, Xu Q - Mol Med Rep (2016)

Bottom Line: It was found that rapamycin treatment led to a significant reduction in the number of Th17 cells and in the expression levels of ROR‑γt in the liver tissues.Consequently, rapamycin treatment prevented the development of CCl4-induced hepatic fibrosis, which was shown by its histological appearances.These results suggested that the immunosuppressive effect of rapamycin on liver fibrosis was associated with the suppression of hepatic fibrogenesis and regulation of the Th17/Treg cell balance.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastrointestinal Surgery, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200127, P.R. China.

ABSTRACT
Previous investigations have suggested that the activation of Th17 cells and/or deficiency of regulatory T cells (Tregs) are involved in the pathogenesis of liver fibrosis. The aim of the present study was to investigate the effect of rapamycin on immune responses in a carbon tetrachloride (CCl4)-induced murine liver fibrosis model. Liver fibrosis was induced by intraperitoneal administration with CCl4. Following injection of CCl4, the mice were treated intraperitoneally with rapamycin (1.25 mg/kg/day) for 8 weeks. Hematoxylin and eosin staining and Masson's trichrome staining were used for histological examination. The protein levels of forkhead/winged helix transcription factor P3, retinoic-acid-related orphan receptor (ROR)‑γt in liver tissue were determined by western blotting, the frequency of Th17 and Treg cells in the liver was evaluated by flow cytometry, and a suppression assay was measured by incorporating [3H]‑thymidine. In addition, to explore the effect of Tregs expanded with rapamycin on hepatic stellate cells (HSC), HSCs were co‑cultured with Tregs from rapamycin or phosphate‑buffered saline‑treated mice. It was found that rapamycin treatment led to a significant reduction in the number of Th17 cells and in the expression levels of ROR‑γt in the liver tissues. Simultaneously, the results of the present study showed a significant increase in the frequency of Tregs and a marked enhancement in the expression of forkhead/winged helix transcription factor P3 in the rapamycin‑treated mice. Furthermore, the Tregs in rapamycin‑treated mice had significantly higher suppressive effects, compared with the cells from mice treated with phospphate‑buffered saline. Consequently, rapamycin treatment prevented the development of CCl4-induced hepatic fibrosis, which was shown by its histological appearances. These results suggested that the immunosuppressive effect of rapamycin on liver fibrosis was associated with the suppression of hepatic fibrogenesis and regulation of the Th17/Treg cell balance.

No MeSH data available.


Related in: MedlinePlus

Rapamycin treatment upregulates the suppressive efficacy of Tregs in mice with liver fibrosis. (A) Cells were isolated from the spleen and liver tissues of mice in each group and subjected to intracellular FoxP3 staining. Treg cell frequency was analyzed using flow cytometry and (B) quantified. Data represent one experiment of three with similar results and values are expressed as the mean ± standard error of the mean (n=5). *P<0.01, vs. PBS-treated group. (C) Protein levels of FoxP3 in the liver were determined using western blot analysis and (D) expressed as relative change, compared with the control animals. Data are presented as the mean ± standard error of the mean (n=6). *P<0.01, vs. PBS-treated group. (E) CD4+CD25+ T cells were purified from the spleen tissues of the rapamycin- or PBS-treated mice. The titrated CD4+CD25+ T cells were co-cultured with 2×105 CD4+CD25- T cells, as responder cells, which were obtained from PBS-treated mice in the presence of anti-CD3/CD28 antibody. The co-cultured cells were maintained for 72 h, and 1 µCi [3H]-thymidine was added to the culture 18 h prior to harvesting. The ratios of CD4+CD25− T cells: CD4+CD25+ T cells are shown. Data are presented as the mean ± standard error of the mean (n=3 in each group). *P<0.05, vs. PBS-treated group. Tregs, regulatory T cells; CCl4, carbon tetrachloride; PBS, phosphate-buffered saline; FoxP3, forkhead/winged helix transcription factor P3.
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f3-mmr-14-02-1153: Rapamycin treatment upregulates the suppressive efficacy of Tregs in mice with liver fibrosis. (A) Cells were isolated from the spleen and liver tissues of mice in each group and subjected to intracellular FoxP3 staining. Treg cell frequency was analyzed using flow cytometry and (B) quantified. Data represent one experiment of three with similar results and values are expressed as the mean ± standard error of the mean (n=5). *P<0.01, vs. PBS-treated group. (C) Protein levels of FoxP3 in the liver were determined using western blot analysis and (D) expressed as relative change, compared with the control animals. Data are presented as the mean ± standard error of the mean (n=6). *P<0.01, vs. PBS-treated group. (E) CD4+CD25+ T cells were purified from the spleen tissues of the rapamycin- or PBS-treated mice. The titrated CD4+CD25+ T cells were co-cultured with 2×105 CD4+CD25- T cells, as responder cells, which were obtained from PBS-treated mice in the presence of anti-CD3/CD28 antibody. The co-cultured cells were maintained for 72 h, and 1 µCi [3H]-thymidine was added to the culture 18 h prior to harvesting. The ratios of CD4+CD25− T cells: CD4+CD25+ T cells are shown. Data are presented as the mean ± standard error of the mean (n=3 in each group). *P<0.05, vs. PBS-treated group. Tregs, regulatory T cells; CCl4, carbon tetrachloride; PBS, phosphate-buffered saline; FoxP3, forkhead/winged helix transcription factor P3.

Mentions: The present study subsequently investigated whether the effect of rapamycin was associated with modulation of Treg cell function in CCl4-induced hepatic fibrosis. The percentages of Tregs in the spleen and liver were analyzed using flow cytometry (Fig. 3A). The frequencies of Tregs in the spleen and liver from the rapamycin-treated mice were significantly higher, compared with those of the PBS-treated mice (Fig. 3B). The hepatic protein expression of FoxP3 was then examined using western blot analysis. Consistent with the results of the flow cytometric analysis, the expression of FoxP3 in the liver from the rapamycin-treated mice was markedly enhanced, compared with that from the PBS-treated mice (Fig. 3C). The relative protein levels are shown in Fig. 3D.


Rapamycin ameliorates CCl4-induced liver fibrosis in mice through reciprocal regulation of the Th17/Treg cell balance.

Gu L, Deng WS, Sun XF, Zhou H, Xu Q - Mol Med Rep (2016)

Rapamycin treatment upregulates the suppressive efficacy of Tregs in mice with liver fibrosis. (A) Cells were isolated from the spleen and liver tissues of mice in each group and subjected to intracellular FoxP3 staining. Treg cell frequency was analyzed using flow cytometry and (B) quantified. Data represent one experiment of three with similar results and values are expressed as the mean ± standard error of the mean (n=5). *P<0.01, vs. PBS-treated group. (C) Protein levels of FoxP3 in the liver were determined using western blot analysis and (D) expressed as relative change, compared with the control animals. Data are presented as the mean ± standard error of the mean (n=6). *P<0.01, vs. PBS-treated group. (E) CD4+CD25+ T cells were purified from the spleen tissues of the rapamycin- or PBS-treated mice. The titrated CD4+CD25+ T cells were co-cultured with 2×105 CD4+CD25- T cells, as responder cells, which were obtained from PBS-treated mice in the presence of anti-CD3/CD28 antibody. The co-cultured cells were maintained for 72 h, and 1 µCi [3H]-thymidine was added to the culture 18 h prior to harvesting. The ratios of CD4+CD25− T cells: CD4+CD25+ T cells are shown. Data are presented as the mean ± standard error of the mean (n=3 in each group). *P<0.05, vs. PBS-treated group. Tregs, regulatory T cells; CCl4, carbon tetrachloride; PBS, phosphate-buffered saline; FoxP3, forkhead/winged helix transcription factor P3.
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f3-mmr-14-02-1153: Rapamycin treatment upregulates the suppressive efficacy of Tregs in mice with liver fibrosis. (A) Cells were isolated from the spleen and liver tissues of mice in each group and subjected to intracellular FoxP3 staining. Treg cell frequency was analyzed using flow cytometry and (B) quantified. Data represent one experiment of three with similar results and values are expressed as the mean ± standard error of the mean (n=5). *P<0.01, vs. PBS-treated group. (C) Protein levels of FoxP3 in the liver were determined using western blot analysis and (D) expressed as relative change, compared with the control animals. Data are presented as the mean ± standard error of the mean (n=6). *P<0.01, vs. PBS-treated group. (E) CD4+CD25+ T cells were purified from the spleen tissues of the rapamycin- or PBS-treated mice. The titrated CD4+CD25+ T cells were co-cultured with 2×105 CD4+CD25- T cells, as responder cells, which were obtained from PBS-treated mice in the presence of anti-CD3/CD28 antibody. The co-cultured cells were maintained for 72 h, and 1 µCi [3H]-thymidine was added to the culture 18 h prior to harvesting. The ratios of CD4+CD25− T cells: CD4+CD25+ T cells are shown. Data are presented as the mean ± standard error of the mean (n=3 in each group). *P<0.05, vs. PBS-treated group. Tregs, regulatory T cells; CCl4, carbon tetrachloride; PBS, phosphate-buffered saline; FoxP3, forkhead/winged helix transcription factor P3.
Mentions: The present study subsequently investigated whether the effect of rapamycin was associated with modulation of Treg cell function in CCl4-induced hepatic fibrosis. The percentages of Tregs in the spleen and liver were analyzed using flow cytometry (Fig. 3A). The frequencies of Tregs in the spleen and liver from the rapamycin-treated mice were significantly higher, compared with those of the PBS-treated mice (Fig. 3B). The hepatic protein expression of FoxP3 was then examined using western blot analysis. Consistent with the results of the flow cytometric analysis, the expression of FoxP3 in the liver from the rapamycin-treated mice was markedly enhanced, compared with that from the PBS-treated mice (Fig. 3C). The relative protein levels are shown in Fig. 3D.

Bottom Line: It was found that rapamycin treatment led to a significant reduction in the number of Th17 cells and in the expression levels of ROR‑γt in the liver tissues.Consequently, rapamycin treatment prevented the development of CCl4-induced hepatic fibrosis, which was shown by its histological appearances.These results suggested that the immunosuppressive effect of rapamycin on liver fibrosis was associated with the suppression of hepatic fibrogenesis and regulation of the Th17/Treg cell balance.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastrointestinal Surgery, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200127, P.R. China.

ABSTRACT
Previous investigations have suggested that the activation of Th17 cells and/or deficiency of regulatory T cells (Tregs) are involved in the pathogenesis of liver fibrosis. The aim of the present study was to investigate the effect of rapamycin on immune responses in a carbon tetrachloride (CCl4)-induced murine liver fibrosis model. Liver fibrosis was induced by intraperitoneal administration with CCl4. Following injection of CCl4, the mice were treated intraperitoneally with rapamycin (1.25 mg/kg/day) for 8 weeks. Hematoxylin and eosin staining and Masson's trichrome staining were used for histological examination. The protein levels of forkhead/winged helix transcription factor P3, retinoic-acid-related orphan receptor (ROR)‑γt in liver tissue were determined by western blotting, the frequency of Th17 and Treg cells in the liver was evaluated by flow cytometry, and a suppression assay was measured by incorporating [3H]‑thymidine. In addition, to explore the effect of Tregs expanded with rapamycin on hepatic stellate cells (HSC), HSCs were co‑cultured with Tregs from rapamycin or phosphate‑buffered saline‑treated mice. It was found that rapamycin treatment led to a significant reduction in the number of Th17 cells and in the expression levels of ROR‑γt in the liver tissues. Simultaneously, the results of the present study showed a significant increase in the frequency of Tregs and a marked enhancement in the expression of forkhead/winged helix transcription factor P3 in the rapamycin‑treated mice. Furthermore, the Tregs in rapamycin‑treated mice had significantly higher suppressive effects, compared with the cells from mice treated with phospphate‑buffered saline. Consequently, rapamycin treatment prevented the development of CCl4-induced hepatic fibrosis, which was shown by its histological appearances. These results suggested that the immunosuppressive effect of rapamycin on liver fibrosis was associated with the suppression of hepatic fibrogenesis and regulation of the Th17/Treg cell balance.

No MeSH data available.


Related in: MedlinePlus