Pregnancy-specific glycoprotein 1 (PSG1) activates TGF-β and prevents dextran sodium sulfate (DSS)-induced colitis in mice

Abstract

Transforming growth factor-βs (TGF-βs) are secreted from cells as latent complexes and the activity of TGF-βs is controlled predominantly through activation of these complexes. Tolerance to the fetal allograft is essential for pregnancy success; TGF-β1 and TGF-β2 play important roles in regulating these processes. Pregnancy-specific β-glycoproteins (PSGs) are present in the maternal circulation at a high concentration throughout pregnancy and have been proposed to have anti-inflammatory functions. We found that recombinant and native PSG1 activate TGF-β1 and TGF-β2 in vitro. Consistent with these findings, administration of PSG1 protected mice from dextran sodium sulfate (DSS)-induced colitis, reduced the secretion of pro-inflammatory cytokines, and increased the number of T regulatory cells. The PSG1-mediated protection was greatly inhibited by the coadministration of neutralizing anti-TGF-β antibody. Our results indicate that proteins secreted by the placenta directly contribute to the generation of active TGF-β and identify PSG1 as one of the few known biological activators of TGF-β2.

Figure 1

LAP-β1 is present in recombinant and native PSG1-TGF-β complexes. (a) Different concentrations of recombinant PSG1 or control protein CEACAM9-Fc generated in HEK-293T and HeLa cells and PSG1 purified from pooled sera of pregnant women (nPSG1) were tested by ELISA for the presence of LAP-β1 as indicated in Methods. (b) HeLa-PSG1 and the parental HeLa cells transfected with empty plasmid were incubated with PE-labeled anti-human LAP-β1 mAb (full grey) or PE-labeled isotype control (empty white). (c) PSG1-Fc was generated in CHO-K1 (lane 1), HEK-293T (lane 2), HeLa (lane 3), Mouse embryonic fibroblast cells derived from TGF-β1 null-mice (lane 4). The supernatant of the transfected cells was purified on a protein A column. PSG1-His-FLAG was generated in CHO-K1 cells (lane 5) and was purified with a HisTrap column, followed by an anti-FLAG column. The control proteins Ceacam9-Fc (lane 6) and FLAG-Fc (lane 7) were purified from the supernatant of transfected CHO-K1 cells on a protein A column. After purification, proteins were concentrated, buffered exchange into PBS and run on SDS-PAGE. The GelCode Blue stained-gel shown here is a composite of lanes run in different gels. (d) PSG1 was purified from pooled serum of pregnant women by affinity chromatography with anti-PSG1 mAb #4. The eluted material was concentrated, buffered exchange into PBS run on SDS-PAGE and stained with GelCode Blue.

Figure 2

Recombinant and native PSG1 activate TGF-β secreted in latent form by TGF-β reporter cell lines. HEK-SMAD cells were treated with different concentrations of PSG1-Fc made in TGF-β1-null MEFs (a); native PSG1 (d) or mature TGF-β1 (g) in DMEM-0.1% ITS and light units were measured in the lysates after 16h. TMLECs were incubated with different concentrations of PSG1-Fc made in TGF-β1-null MEFs (b); native PSG1 (e) or mature TGF-β2 (h) in DMEM-0.1% ITS. Light units were measured in the cell lysates 16h post-treatment MFB-F11 cells were incubated with different concentrations of PSG1-Fc made in TGF-β1-null MEFs (c); native PSG1 (f) or mature TGF-β2 (i) in DMEM. The reporter (SEAP) was measured in the cell supernatant 24h post-treatment. The three reporter cell lines were also incubated with the control protein CEACAM9-Fc at the same concentration or with DMEM with 0.1% ITS (HEK-SMAD and TMLECs) or DMEM only (MFB-F11 cells). Results are expressed as Luciferase activity fold induction (FI) or SEAP activity fold induction (FI) after normalization with values obtained upon treatment of the cells with DMEM-0.1% ITS or DMEM for MFB-F11 cells. Treatments with PSG1 and CEACAM-Fc were performed in triplicate wells. Treatments with recombinant mature TGF-βs were performed in duplicate wells. All experiments were performed three independent times with similar results. In all figures, significant differences are noted as *p<0.05 as analysed by Student’s t –test.

Figure 3

PSG1-mediated activation of TGF-β is inhibited by the pan TGF-β-neutralizing 1D11 mAb and the TGF-β receptor I kinase inhibitor SB 431542. (a) TMLECs, (b) HEK-SMAD, and (c) MFB-F11 cells were treated with 7.5μg/ml recombinant PSG1 in the presence of 60μg/ml anti-TGF-β 1D11 Ab, isotype control Ab at the same concentration, 5 μM SB 431542 or DMSO. (d) HEK-SMAD were treated with 20 μg/ml native PSG1 (nPSG1) in the presence of 60 μg/ml anti-TGF-β 1D11 Ab, 5 μM SB431542 and the respective controls. The cells were also incubated with the control protein CEACAM9-Fc at 7.5 μg/ml (a – c ) and 20 μg/ml in (d). Results are expressed as Luciferase activity or SEAP activity after subtraction of the values obtained upon treatment of the cells with DMEM-0.1% ITS or DMEM for MFB-F11 cells. All treatments were performed in triplicate in three independent experiments. In all figures, significant differences are noted as *p<0.05 as analysed by Student’s t-test.

Figure 4

Recombinant and native PSG1 activate the small latent complex (SLC) of TGF-β1 in a cell-free system. PSG1-Fc generated in MEFs derived from TGF-β1-null mice (a); native PSG1 (b) or the control protein CEACAM9-Fc were incubated in triplicate at increasing concentrations with PBS or with SLC (0.5nM) for 1h at 37 °C, after which they were added to wells coated with TGF-β RII-Fc. The presence of active TGF-β1 was quantified after incubation with biotin-labelled anti-mature TGF-β1 Ab and streptavidin-HRP. (c) The amount of TGF-β1 already in the active form in the recombinant SLC and the concentration of active TGF-β1 that was obtained upon acid activation of the SLC were determined in parallel. All treatments were performed in triplicate in three independent experiments. In all figures, significant differences are noted as *p<0.05 as analysed by Student’s t-test.

Figure 5

PSG1 prevents DSS-induced colitis in mice. (a) Acute colitis was induced by administration of 2.5–3% DSS to the drinking water starting from day one to day 7 of the experiment. In addition mice were injected (i.p.) either with Flag-Fc (control protein, 30μg/day) or PSG1 (100μg/day) on day 0, 1, 3, 5 and 7 of the experimental day (ED) as described in Methods. (b) Weight loss (%) in control (Flag-Fc) and PSG1 treated mice (n=5/ group) was monitored every day. Colon length was recorded on ED 10. (c) Haematoxylin- and eosin-(H&E) stained colon sections on ED10 (original magnification ×50; scale bar= 500μm). Short arrows, epithelium (top arrow, right panel) and smooth muscle (bottom arrow, right panel); large arrows, cellular infiltrates (left panel).(d) IL-6, TNF-α, IFN-γ and IL-10 production by lymphocytes isolated from mesenteric lymph nodes (MLN) and stimulated with CD3ε and CD28 for 48h as described in Methods. (e) IL-6, TNF-α and IL-10 production by lymphocytes isolated from MLN and stimulated with IFN-γ and Pansorbin (SAC) for 48h as described in Methods. In Figures (d) and (e) cytokine concentrations were determined in culture supernatants by Cytometric Beads Array (CBA). (f) IFN-γ, TFN-α, IL-17, IL-10 and TGF-β1 expression in colon tissues on ED10 as analysed by qPCR. In all figures, significant differences are noted as †p<0.01 and ‡p<0.001 as analysed by one-way ANOVA followed by Bonferroni test. Data shown are mean values ± SD derived from five mice per group each analysed in duplicate.

Figure 6

Administration of anti-TGF-β abrogates the protective effect of PSG1 in DSS-induced colitis. (a) Experimental design. DSS-treated mice were injected (i.p.) either with Flag-Fc (control protein, 30μg/day) or PSG1 (100μg/day) on ED0, 1, 3, 5 and 7. In addition the mice received anti-TGF-β (1mg/day) or isotype control antibody on ED0 and ED3 as described in Methods. (b) Weight was monitored every day. Data are presented as weight loss (%). (c) Colon Length recorded on ED10. (d) H&E stained colon sections on ED10. (e) IL-6, TNF-α and IFN-γ and (f) IL-17 production by lymphocytes isolated from mesenteric lymph nodes (MLN) and stimulated with CD3ε and CD28 for 48h as described in Methods. (g) IL-6 and TNF-α production by lymphocytes isolated from MLN and stimulated with IFN-γ and Pansorbin (SAC) for 48h as described in Methods. In figures (d) and (e) cytokine concentrations were determined in culture supernatants by Cytometric Beads Array (CBA). In all panels, significant differences are noted as *p<0.05 and †p<0.01 as analysed by one-way ANOVA followed by Bonferroni test. Data shown are mean ± SD derived from five mice per group each analysed in duplicate.

Figure 7

Administration of PSG1 increases the frequency of CD4⁺FoxP3⁺LAP⁺ lymphocytes in the lamina propria. (a) Representative plots of FoxP3 and LAP stained CD4⁺ lymphocytes isolated from lamina propria (LP) were analysed by flow cytometry (FACS). (b) Percentages and absolute numbers of FoxP3⁺LAP⁺ LP lymphocytes isolated from Flag-Fc or PSG1 DSS-treated mice are depicted. (c) Percentages and absolute numbers of FoxP3⁺LAP⁺ LP lymphocytes isolated from Flag-Fc or PSG1 DSS-α-TGF-β treated mice are shown. All treatments were performed in triplicate in two independent experiments using 5 mice per group. In all figures, significant differences are noted as †p<0.01 and ‡p<0.001 as analysed by one-way ANOVA followed by Bonferroni test. Data shown are mean ± SD derived from five mice per group each analysed in duplicate.