Recombinant PGRN promotes the induction of inducible regulatory T cells in vitro.

<p>Naïve CD4+GFP- cells in lymphocytes of spleen from Foxp3-GFP reporter mice were purified by using Mitenyi reagents and a MACS apparatus. The purity of cells was evaluated by FACS method. CD4+GFP- cells conversion assay was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112110#s2" target="_blank">Methods</a>, and GFP expression in TGF-β-unstimulated CD4+GFP- cells was taken as a control. After 3-4 days, cells were washed and GFP expression was analyzed by FACS. Data represent three independent experiments are shown. (A) No TGF-β and no PGRN. (B) No TGF-β plus 200 ng/ml of PGRN. (C) No TGF-β plus 1 µg/ml of PGRN. (D) 0.1 ng/ml of TGF-β and no PGRN. (E) 0.1 ng/ml of TGF-βplus 200 ng/ml of PGRN. (F) 0.1 ng/ml of TGF-β plus 1 µg/ml of PGRN. Data represent as a means ±SE of a representative experiment. *<i>p</i><0.05; **<i>p</i><0.01.</p

Wnt signaling components expression in wild type and PGRN-deficient CD4+CD25+ T cells measured by real-time PCR.

<p>All values are shown as a relative ratio to GAPDH measured by 2-ΔΔct method. Data represent as a means ±SE of a representative experiment. *<i>p</i><0.05; **<i>p</i><0.01.</p

PGRN deficiency does not alter the generation of CD4+CD25+Foxp3+ T cells in vivo.

<p>Flow cytometric evaluation of CD4+CD25+Foxp3+ T cells in one-, three-, and six-week-old wild type (WT) and PGRN-deficient mice. (A) The percentage of CD4+ and CD8+ T cells in thymus from one-week-old C57BL/6 mice and PGRN-deficient mice. (B) The percentage of CD4+CD25+Foxp3+ cells in thymus from one-week-old C57BL/6 mice and PGRN-deficient mice. (C) The proportion of CD4+CD25+Foxp3+ cells in spleen from three-week-old C57BL/6 mice and PGRN-deficient mice. (D) CD4+CD25+Foxp3+ cells in lymph nodes from three-week-old C57BL/6 mice and PGRN-deficient mice. (E) The proportion of CD4+CD25+Foxp3+ cells in spleen from six-week-old C57BL/6 mice and PGRN-deficient mice. (F) CD4+CD25+Foxp3+ cells in lymph nodes from six-week-old C57BL/6 mice and PGRN-deficient mice. All data was representative of three mice per group and indicated as mean ± SEM.</p

PGRN deficiency does not affect the conversion of naïve CD4+CD25- T cells into iTreg mediated by TGF-β.

<p>Naïve CD4+ CD25- T cells isolated from both wildtype (WT) and PGRN-deficient mice were stimulated with plate-bound CD3 Ab and solute CD28 Ab and cultured the cells for 3-4 days in the presence or absence of TGF-β, and the expression of GFP was measured by FACS. (A) The GFP levels in CD4+ T cells in the absence of TGF-β. (B) The GFP levels in CD4+ T cells in the presence of 1 ng/ml TGF-β. (C) The GFP levels in CD4+ T cells in the presence of 10 ng/ml TGF-β. All data were repeated three times with similar results.</p

In vivo proliferation of CD4+CD25+ T cells from spleen and lymph nodes analyzed by BrdU corporation.

<p>Wild type and PGRN-deficient mice were injected intraperitoneally with BrdU labeling reagents, and the degrees of BrdU incorporation by CD4+CD25+ T cells from lymphocytes of spleen and lymph nodes were determined by FACS. (A) The BrdU incorporation of wild type CD4+CD25+ T cells from splenocytes. (B) The BrdU incorporation of PGRN-deficient CD4+CD25+ T cells from splenocytes. (C) The percentage of wild type CD4+CD25+ T cells from lymphocytes of lymph nodes that incorporated BrdU. (D) The percentage of PGRN-deficient CD4+CD25+ T cells from lymphocytes of lymph nodes that incorporated BrdU. All data was representative of three mice per group and indicated as mean ±SEM.</p

PGRN treatment does not change the proportions of CD4+CD25+Foxp3+ cells in normal conditions.

<p>One week-old Foxp3-RFP reporter mice were divided into two groups, three mice per group. PGRN group mice were treated with 100 µg PGRN every two days for 1 week, and PBS group mice were injected with the same volume of PBS as a control. The lymphocytes of spleen, peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN), and Peyer's patches (PP) were isolated and analyzed by FACS. All data are representative of three independent experiments.</p

Fewer CD4+CD25+Foxp3+ Treg cells seen in PGRN-deficient CIA model.

<p>Wild type (n = 6) and PGRN-deficient mice (n = 6) were intradermally immunized with 100 µl of chicken type II collagen emulsified with an equal volume of complete Freund's adjuvant (CFA). 21 days post immunization, draining lymph nodes were extracted and CD4+CD25+Foxp3+ T cells were analyzed by FACS. Data represent as a means ±SE of a representative experiment. **<i>p</i><0.01.</p

PGRN-deficient CD4+CD25+ Treg decreased the suppressive capacity to Teff proliferation.

<p>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. *<i>p</i><0.05; **<i>p</i><0.01.</p

Additional file 2: Figure S2. of Progranulin derivative Atsttrin protects against early osteoarthritis in mouse and rat models

showing quantification of OARSI score based on Safranin O staining for PBS, PGRN, or Atsttrin-treated ACLT mice (DOCX 56 kb

Additional file 1: Figure S1. of Progranulin derivative Atsttrin protects against early osteoarthritis in mouse and rat models

showing expression of PGRN in cartilage at different ages, assayed by immunohistochemistr