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Progranulin facilitates conversion and function of regulatory T cells under inflammatory conditions.

Wei F, Zhang Y, Zhao W, Yu X, Liu CJ - PLoS ONE (2014)

Bottom Line: PGRN was required for the immunosuppressive function of Tregs, since PGRN-deficient Tregs have a significant decreased ability to suppress the proliferation of effector T cells (Teff).In addition, PGRN deficiency caused a marked reduction in Tregs number in the course of inflammatory arthritis, although no significant difference was observed in the numbers of Tregs between wild type and PGRN deficient mice during development.Furthermore, PGRN deficiency led to significant upregulation of the Wnt receptor gene Fzd2.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, New York University Medical Center, New York, New York, United States of America; Institute of Pathogenic Biology, Shandong University School of Medicine, Jinan, China.

ABSTRACT
The progranulin (PGRN) is known to protect regulatory T cells (Tregs) from a negative regulation by TNF-α, and its levels are elevated in various kinds of autoimmune diseases. Whether PGRN directly regulates the conversion of CD4+CD25-T cells into Foxp3-expressing regulatory T cells (iTreg), and whether PGRN affects the immunosuppressive function of Tregs, however, remain unknown. In this study we provide evidences demonstrating that PGRN is able to stimulate the conversion of CD4+CD25-T cells into iTreg in a dose-dependent manner in vitro. In addition, PGRN showed synergistic effects with TGF-β1 on the induction of iTreg. PGRN was required for the immunosuppressive function of Tregs, since PGRN-deficient Tregs have a significant decreased ability to suppress the proliferation of effector T cells (Teff). In addition, PGRN deficiency caused a marked reduction in Tregs number in the course of inflammatory arthritis, although no significant difference was observed in the numbers of Tregs between wild type and PGRN deficient mice during development. Furthermore, PGRN deficiency led to significant upregulation of the Wnt receptor gene Fzd2. Collectively, this study reveals that PGRN directly regulates the numbers and function of Tregs under inflammatory conditions, and provides new insight into the immune regulatory mechanism of PGRN in the pathogenesis of inflammatory and immune-related diseases.

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PGRN-deficient CD4+CD25+ Treg decreased the suppressive capacity to Teff proliferation.Freshly isolated, CFSE-labeled CD4+CD25- T cells from Thy1.1 mice were used as Teff and co-cultured with Tregs at ratios of 1∶2, 1∶1, 2∶1, 4∶1 and 8∶1. CFSE-based Teff proliferation suppression assay in vitro in which 5×105 CFSE-labeled Teff cells were stimulated with CD3 antibody in the presence of mitomycin-treated APC cells and different ratios of FACS sorted wild type or PGRN-deficient Tregs. The CFSE proliferation was evaluated by FACS. All data are representative of three separate experiments. (A) Negative control. (B) Positive control. (C) The CFSE dilution when Teff co-cultured with Tregs at ratios of 1∶2. (D) The CFSE dilution when Teff co-cultured with Tregs at ratios of 1∶1. (E) The CFSE dilution when Teff co-cultured with Tregs at ratios of 2∶1. (F) The CFSE dilution when Teff co-cultured with Tregs at ratios of 4∶1. (G) The CFSE dilution when Teff co-cultured with Tregs at ratios of 8∶1. Data represent as a means ±SE of a representative experiment. *p<0.05; **p<0.01.
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pone-0112110-g005: PGRN-deficient CD4+CD25+ Treg decreased the suppressive capacity to Teff proliferation.Freshly isolated, CFSE-labeled CD4+CD25- T cells from Thy1.1 mice were used as Teff and co-cultured with Tregs at ratios of 1∶2, 1∶1, 2∶1, 4∶1 and 8∶1. CFSE-based Teff proliferation suppression assay in vitro in which 5×105 CFSE-labeled Teff cells were stimulated with CD3 antibody in the presence of mitomycin-treated APC cells and different ratios of FACS sorted wild type or PGRN-deficient Tregs. The CFSE proliferation was evaluated by FACS. All data are representative of three separate experiments. (A) Negative control. (B) Positive control. (C) The CFSE dilution when Teff co-cultured with Tregs at ratios of 1∶2. (D) The CFSE dilution when Teff co-cultured with Tregs at ratios of 1∶1. (E) The CFSE dilution when Teff co-cultured with Tregs at ratios of 2∶1. (F) The CFSE dilution when Teff co-cultured with Tregs at ratios of 4∶1. (G) The CFSE dilution when Teff co-cultured with Tregs at ratios of 8∶1. Data represent as a means ±SE of a representative experiment. *p<0.05; **p<0.01.

Mentions: The proportion of normal CD4+CD25+ Tregs constitutes 5–10% of peripheral CD4+ T cells in mice and 1–2% in humans, and can potently suppress the proliferation of active CD4+CD25- and CD8+ T cells [36], [37]. To evaluate the role of PGRN signaling in regulation of CD4+CD25+ Tregs function, we performed in vitro CFSE-based proliferation suppression assay. 5×105 CFSE-labeling Teff cells were stimulated for 72 hours with CD3 antibody (5 µg/ml) in the presence of 1×105 APC cells and varying ratios of FACS purified WT or PGRN-deficient CD4+CD25+ Tregs, and CFSE dilution was evaluated by FACS. The CFSE proliferation in negative control and positive control group was 1.93±0.1% and 94.2±3.2%, respectively (Fig. 5A and B). Our results demonstrate that wild type and PGRN-deficient CD4+CD25+ Tregs significantly suppress the CFSE proliferation when Teff co-cultured with Tregs at rations of 1∶2, 1∶1, 2∶1 and 4∶1, compared with positive control group (p<0.05) (Fig. 5B–F). PGRN-deficient Tregs significantly decreased suppressive capacity when Teff co-cultured with Tregs at ratios of 1∶2 (66.3±3.2% CFSE dilution in KO versus 52.5±3.0% CFSE dilution in WT, p<0.01, Fig. 5C) and 1∶1 (79.6±3.78% CFSE dilution in KO versus 65.4±3.6% CFSE dilution in WT, p<0.01, Fig. 5D), compared with wild type Tregs. Suppressor cells were added at suppressor and responder cells rations of 2∶1, PGRN-deficient Tregs showed a slightly lower suppressive capacity than wild type Tregs (80.8±2.18% CFSE dilution in KO versus 75.5±1.5% CFSE dilution in WT, p<0.05, Fig. 5E). However, no significant difference were found between wild type and PGRN-deficient Tregs at ratios of 4∶1 (86.3±1.4% CFSE proliferation in KO versus 83.8±1.7% CFSE proliferation in WT, p>0.05, Fig. 5F) and 8∶1 (89.3±0.1% CFSE dilution in KO versus 87.6±2.0% CFSE dilution in WT, p>0.05, Fig. 5G). Collectively, PGRN is required for the immunosuppressive function of Tregs in vitro.


Progranulin facilitates conversion and function of regulatory T cells under inflammatory conditions.

Wei F, Zhang Y, Zhao W, Yu X, Liu CJ - PLoS ONE (2014)

PGRN-deficient CD4+CD25+ Treg decreased the suppressive capacity to Teff proliferation.Freshly isolated, CFSE-labeled CD4+CD25- T cells from Thy1.1 mice were used as Teff and co-cultured with Tregs at ratios of 1∶2, 1∶1, 2∶1, 4∶1 and 8∶1. CFSE-based Teff proliferation suppression assay in vitro in which 5×105 CFSE-labeled Teff cells were stimulated with CD3 antibody in the presence of mitomycin-treated APC cells and different ratios of FACS sorted wild type or PGRN-deficient Tregs. The CFSE proliferation was evaluated by FACS. All data are representative of three separate experiments. (A) Negative control. (B) Positive control. (C) The CFSE dilution when Teff co-cultured with Tregs at ratios of 1∶2. (D) The CFSE dilution when Teff co-cultured with Tregs at ratios of 1∶1. (E) The CFSE dilution when Teff co-cultured with Tregs at ratios of 2∶1. (F) The CFSE dilution when Teff co-cultured with Tregs at ratios of 4∶1. (G) The CFSE dilution when Teff co-cultured with Tregs at ratios of 8∶1. Data represent as a means ±SE of a representative experiment. *p<0.05; **p<0.01.
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Related In: Results  -  Collection

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pone-0112110-g005: PGRN-deficient CD4+CD25+ Treg decreased the suppressive capacity to Teff proliferation.Freshly isolated, CFSE-labeled CD4+CD25- T cells from Thy1.1 mice were used as Teff and co-cultured with Tregs at ratios of 1∶2, 1∶1, 2∶1, 4∶1 and 8∶1. CFSE-based Teff proliferation suppression assay in vitro in which 5×105 CFSE-labeled Teff cells were stimulated with CD3 antibody in the presence of mitomycin-treated APC cells and different ratios of FACS sorted wild type or PGRN-deficient Tregs. The CFSE proliferation was evaluated by FACS. All data are representative of three separate experiments. (A) Negative control. (B) Positive control. (C) The CFSE dilution when Teff co-cultured with Tregs at ratios of 1∶2. (D) The CFSE dilution when Teff co-cultured with Tregs at ratios of 1∶1. (E) The CFSE dilution when Teff co-cultured with Tregs at ratios of 2∶1. (F) The CFSE dilution when Teff co-cultured with Tregs at ratios of 4∶1. (G) The CFSE dilution when Teff co-cultured with Tregs at ratios of 8∶1. Data represent as a means ±SE of a representative experiment. *p<0.05; **p<0.01.
Mentions: The proportion of normal CD4+CD25+ Tregs constitutes 5–10% of peripheral CD4+ T cells in mice and 1–2% in humans, and can potently suppress the proliferation of active CD4+CD25- and CD8+ T cells [36], [37]. To evaluate the role of PGRN signaling in regulation of CD4+CD25+ Tregs function, we performed in vitro CFSE-based proliferation suppression assay. 5×105 CFSE-labeling Teff cells were stimulated for 72 hours with CD3 antibody (5 µg/ml) in the presence of 1×105 APC cells and varying ratios of FACS purified WT or PGRN-deficient CD4+CD25+ Tregs, and CFSE dilution was evaluated by FACS. The CFSE proliferation in negative control and positive control group was 1.93±0.1% and 94.2±3.2%, respectively (Fig. 5A and B). Our results demonstrate that wild type and PGRN-deficient CD4+CD25+ Tregs significantly suppress the CFSE proliferation when Teff co-cultured with Tregs at rations of 1∶2, 1∶1, 2∶1 and 4∶1, compared with positive control group (p<0.05) (Fig. 5B–F). PGRN-deficient Tregs significantly decreased suppressive capacity when Teff co-cultured with Tregs at ratios of 1∶2 (66.3±3.2% CFSE dilution in KO versus 52.5±3.0% CFSE dilution in WT, p<0.01, Fig. 5C) and 1∶1 (79.6±3.78% CFSE dilution in KO versus 65.4±3.6% CFSE dilution in WT, p<0.01, Fig. 5D), compared with wild type Tregs. Suppressor cells were added at suppressor and responder cells rations of 2∶1, PGRN-deficient Tregs showed a slightly lower suppressive capacity than wild type Tregs (80.8±2.18% CFSE dilution in KO versus 75.5±1.5% CFSE dilution in WT, p<0.05, Fig. 5E). However, no significant difference were found between wild type and PGRN-deficient Tregs at ratios of 4∶1 (86.3±1.4% CFSE proliferation in KO versus 83.8±1.7% CFSE proliferation in WT, p>0.05, Fig. 5F) and 8∶1 (89.3±0.1% CFSE dilution in KO versus 87.6±2.0% CFSE dilution in WT, p>0.05, Fig. 5G). Collectively, PGRN is required for the immunosuppressive function of Tregs in vitro.

Bottom Line: PGRN was required for the immunosuppressive function of Tregs, since PGRN-deficient Tregs have a significant decreased ability to suppress the proliferation of effector T cells (Teff).In addition, PGRN deficiency caused a marked reduction in Tregs number in the course of inflammatory arthritis, although no significant difference was observed in the numbers of Tregs between wild type and PGRN deficient mice during development.Furthermore, PGRN deficiency led to significant upregulation of the Wnt receptor gene Fzd2.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, New York University Medical Center, New York, New York, United States of America; Institute of Pathogenic Biology, Shandong University School of Medicine, Jinan, China.

ABSTRACT
The progranulin (PGRN) is known to protect regulatory T cells (Tregs) from a negative regulation by TNF-α, and its levels are elevated in various kinds of autoimmune diseases. Whether PGRN directly regulates the conversion of CD4+CD25-T cells into Foxp3-expressing regulatory T cells (iTreg), and whether PGRN affects the immunosuppressive function of Tregs, however, remain unknown. In this study we provide evidences demonstrating that PGRN is able to stimulate the conversion of CD4+CD25-T cells into iTreg in a dose-dependent manner in vitro. In addition, PGRN showed synergistic effects with TGF-β1 on the induction of iTreg. PGRN was required for the immunosuppressive function of Tregs, since PGRN-deficient Tregs have a significant decreased ability to suppress the proliferation of effector T cells (Teff). In addition, PGRN deficiency caused a marked reduction in Tregs number in the course of inflammatory arthritis, although no significant difference was observed in the numbers of Tregs between wild type and PGRN deficient mice during development. Furthermore, PGRN deficiency led to significant upregulation of the Wnt receptor gene Fzd2. Collectively, this study reveals that PGRN directly regulates the numbers and function of Tregs under inflammatory conditions, and provides new insight into the immune regulatory mechanism of PGRN in the pathogenesis of inflammatory and immune-related diseases.

Show MeSH
Related in: MedlinePlus