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. 2012 Sep 24;209(10):1753-67, S1.
doi: 10.1084/jem.20111381. Epub 2012 Sep 10.

Schistosome-derived omega-1 Drives Th2 Polarization by Suppressing Protein Synthesis Following Internalization by the Mannose Receptor

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Free PMC article

Schistosome-derived omega-1 Drives Th2 Polarization by Suppressing Protein Synthesis Following Internalization by the Mannose Receptor

Bart Everts et al. J Exp Med. .
Free PMC article

Abstract

Omega-1, a glycosylated T2 ribonuclease (RNase) secreted by Schistosoma mansoni eggs and abundantly present in soluble egg antigen, has recently been shown to condition dendritic cells (DCs) to prime Th2 responses. However, the molecular mechanisms underlying this effect remain unknown. We show in this study by site-directed mutagenesis of omega-1 that both the glycosylation and the RNase activity are essential to condition DCs for Th2 polarization. Mechanistically, we demonstrate that omega-1 is bound and internalized via its glycans by the mannose receptor (MR) and subsequently impairs protein synthesis by degrading both ribosomal and messenger RNA. These experiments reveal an unrecognized pathway involving MR and interference with protein synthesis that conditions DCs for Th2 priming.

Figures

Figure 1.
Figure 1.
Generation and evaluation of glycosylation and RNase mutants of recombinant omega-1. (A) The amino acid sequence of omega-1 (NCBI Protein database accession no. ABB73003.1) is shown in which the mutation sites are depicted. The two conserved amino acid sequence (CAS) domains essential for catalytic activity are marked in gray, and the two N-linked glycosylation sites are boxed. (B) RNA from PBMCs was incubated for 1 h with the different omega-1 variants (500 and 100ng/ml) and analyzed on a 2% agarose gel for breakdown. RNase A was used as a positive control. One of two experiments is shown. (C) The omega-1 mutants were run under nonreducing conditions by SDS-PAGE and silver stained. A Western blot by staining with a specific anti–omega-1 monoclonal antibody was in line with the absence of glycosylation only on the omega-1 glycosylation mutant. (D) MALDI-TOF mass spectrum of glycopeptides from a tryptic digest of recombinant WT omega-1, covering the glycosylation site N176. Recombinant omega-1 was subjected to SDS-PAGE under reducing conditions and stained with Colloidal blue. Stained bands were excised, subjected to reduction and alkylation, and digested with trypsin. The MALDI-TOF-MS spectrum derived from the upper band in the SDS-PAGE pattern is depicted. Signals ([M+H+]) are labeled with monoisotopic masses. Composition of the glycan moieties are given in terms of hexose (H), N-acetylhexosamine (N), and fucose (F). Differences in fucose content are indicated by double-headed arrows. Signals that cannot be assigned to glycopeptides are marked with asterisks.
Figure 2.
Figure 2.
The glycosylation and RNase activity of omega-1 are essential for conditioning human DCs to prime Th2 responses. (A) Human moDCs were pulsed for 48 h with increasing concentrations of the mutant variants of recombinant omega-1 (ω-1) in combination with 100 ng/ml LPS as a maturation factor, and surface expression of CD86 was determined by FACS analysis. The expression levels, based on geometric mean fluorescence, are shown relative to the DCs stimulated with LPS alone, which is set to 1. Data are based on two independent experiments and shown as mean ± SD. (B) Conditioned DCs were co-cultured for 24 h with a CD40-L–expressing cell line to mimic the interaction with T cells. IL-12p70 cytokine expression levels are shown relative to the DCs stimulated with LPS alone, which is set to 1. Data are representative of triplicate wells from one of two independent experiments and shown as mean ± SD. (C) DCs conditioned as described in A were cultured with allogeneic naive CD4+ T cells for 12 d in the presence of staphylococcal enterotoxin B and IL-2. Intracellular cytokine production was assayed by FACS 6 h after the stimulation of primed T cells with PMA and ionomycin. Based on intracellular cytokine staining, the ratio of T cells single-positive for either IL-4 or IFN-γ was calculated relative to the control condition. Data are based on two independent experiments and shown as mean ± SD. (D) An example of T cell polarization assay as described in C induced by the different recombinant omega-1 variants. The frequencies of each population are indicated as percentages in the plot. One representative result from five independent experiments is shown. (E) T cell polarization assay as described in C but in the absence of LPS. Data are representative of three independent experiments. Bars represent mean ± SD. * and #, P < 0.05, for significant differences compared with control conditions (*) or between test conditions (#) based on paired analysis (two-sided paired Student’s t test). H58F, RNase mutant; N71/176Q, glycosylation mutant.
Figure 3.
Figure 3.
Glycosylation and RNase activity are essential for omega-1 to prime Th2 responses in vivo. 4get/KN2 IL-4 dual reporter mice were injected s.c. with 20 µg SEA or 3 µg WT or mutant recombinant omega-1 into the footpad. After 7 d, the frequency of GFP+ and huCD2+ within the CD4+CD44high effector T cell population was determined by flow cytometry in the draining popliteal LNs. (A–C) Depicted are concatenated FACS plots (A), frequencies of huCD2+ within the CD4+CD44high population (B), and total huCD2+ T cell numbers in draining LNs (C) of combined data of four mice per group. (A) The frequencies of each population are indicated as percentages in the plots. One of three independent experiments is shown. Bars represent mean ± SD. * and #, P < 0.05; ** and ##, P < 0.01; ***, P < 0.001 for values significantly different from the PBS control (*) or between test conditions (#) based on two-sided t test. H58F, RNase mutant; N71/176Q, glycosylation mutant.
Figure 4.
Figure 4.
MR mediates recognition and internalization of omega-1 by human DCs. (A) Human moDCs were incubated for 1 h with PF-647–labeled recombinant WT omega-1, the glycosylation mutant, or the RNase mutant and analyzed for uptake of antigens by FACS analysis. One representative experiment with duplicate samples out of two experiments is shown. Bars represent mean ± SD. (B) Human moDCs were incubated for 1 h with PF-647–labeled omega-1 and, where indicated, either preincubated (pre) with EGTA to prevent omega-1 binding to CLRs a priori or treated afterward with EGTA (post) to remove any CLR-bound omega-1 from the cell surface. One of two independent experiments is shown, and data represent mean ± SD of duplicates. (C and D) A binding/internalization assay of natural (C) and recombinant omega-1 (D) by immature moDCs was performed analogous to B after preincubation with the indicated reagents. Binding and internalization are shown relative to control pretreatment. (C) Data are based on five experiments and are shown as mean ± SD. (D) One of two independent experiments is shown, and data represent mean ± SD of duplicates. (E and F) 3T3 cell line–expressing MR (E) and K-SIGN–expressing DC-SIGN (F) or parental control cell lines (3T3 and K-562) were incubated with PF-647–labeled omega-1 and SEA in the presence or absence of EGTA to determine specificity. One representative experiment based on duplicate samples out of two is shown. Bars represent mean ± SD. * and #, P < 0.05; ** and ##, P < 0.01; ***, P < 0.001 for significant differences compared with control conditions (*) or between test conditions (#) based on two-sided Student’s t test. H58F, RNase mutant; MFI, mean fluorescence intensity; N71/176Q, glycosylation mutant.
Figure 5.
Figure 5.
Omega-1 suppresses protein synthesis through breakdown of rRNA and mRNA. (A) After human moDCs had been pulsed for 40 h with 125, 250, and 500 ng/ml omega-1 (ω-1) in combination with 100 ng/ml LPS, the cells were co-cultured for 24 h with the J558 cell line, expressing CD40-L, to mimic the interaction with T cells. Bars represent mean ± SD of triplicate wells of one of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 for values significantly different from the LPS control. (B) After16-h incubation of human DCs with a concentration range of indicated reagents in the presence of 100 ng/ml LPS, protein synthesis was assessed after a 2-h pulse with radioactively labeled methionine. Ricin, as potent inhibitor of protein synthesis, was taken along as positive control (Montanaro et al., 1973). One of two experiments is shown. Data points represent mean ± SD of duplicates. (C) As described in B, protein synthesis by human DCs was followed over time after exposure to 500 ng/ml omega-1 either in the presence or absence of 100 ng/ml LPS. Data are shown relative to unstimulated or LPS-stimulated controls as depicted by the dotted line. Data are representative of two independent experiments and are depicted as mean ± SD. (D) Protein synthesis by human moDCs after exposure to increasing concentrations of the recombinant omega-1 variants was assessed as described in B. Data are shown relative to LPS-stimulated DCs. Data are representative of two independent experiments and are depicted as mean ± SD. (E) DCs were stimulated with FITC-labeled recombinant omega-1 for 1 h, and uptake was visualized by confocal laser-scanning microscopy. Nuclei were stained with Hoechst. One of three experiments is shown. See Video 1 for z-stacked images. (F) Cytoplasmic fractions of human DCs stimulated for 3 h with omega-1 were run under nonreducing conditions by SDS-PAGE and analyzed by Western blot for the presence of omega-1 or silver stained to control for input. DCs incubated at 4°C were taken along as controls as these cells have surface-bound but not internalized omega-1. One of two experiments is shown. (G) Human DCs were stimulated with 1 µg/ml FITC-labeled omega-1 and after 2 h fixed and stained for rRNA. Subcellular localization of omega-1 was determined by confocal microscopy. One representative cell from three independent experiments is shown. Arrowheads depict areas where rRNA and omega-1 colocalize (yellow). Bars, 10 µm. (H) After rabbit reticulocyte lysate containing functional ribosomes was incubated for 1 h with 1 and 5 µg/ml omega-1, 1 and 5 µg/ml IPSE as negative control, or 25 µg/ml ES (schistosome egg excretory/secretory products), containing omega-1, isolated rRNA was analyzed for breakdown on a 2% agarose gel. The RNase α-sarcin was taken along as positive control as it should give a single rRNA cleavage product when incubated with functional ribosomes (arrowhead; Kao et al., 2001). One of three independent experiments is shown. (I) rRNA was isolated from 24-h omega-1–stimulated human DCs and was visualized by running a laboratory-on-a-chip picogel. One of three experiments is shown. (J and K) rRNA or mRNA expression of the indicated genes in DCs was assessed by real-time quantitative PCR at different time points after stimulation with 500 ng/ml and 2 µg/ml omega-1 in the presence or absence of 100 ng/ml LPS. Data are shown relative to unstimulated or LPS-stimulated controls, which were set to 1. RNA expression was normalized based on a genomic real-time quantitative PCR for ccr5. Data represent the mean of three independent experiments. H58F, RNase mutant; N71/176Q, glycosylation mutant.
Figure 6.
Figure 6.
MR mediates omega-1–induced DC modulation and Th2 polarization in vitro. After 1-h preincubation with blocking antibodies against MR, DC-SIGN, or an isotype control (20 µg/ml), human moDCs were pulsed for 16 (A) or 48 h (B–D) with 500 ng/ml natural omega-1 (ω-1) in combination with 100 ng/ml LPS. (A) Protein synthesis was assessed as described in Fig. 5 B. One representative experiment based on duplicate samples out of three experiments is shown. Data are shown as mean ± SD. (B) The expression levels of CD86 on human DCs assessed by FACS are based on geometric mean fluorescence, relative to the DCs stimulated with LPS alone, which is set to 1 (dashed line). Data are based on three independent experiments and shown as mean ± SD. (C) Conditioned human DCs were co-cultured for 24 h with a CD40-L–expressing cell line to mimic the interaction with T cells. IL-12p70 cytokine expression levels are shown relative to the DCs stimulated with LPS alone, which is set to 1 (dashed line). Data are based on three independent experiments and shown as mean ± SD. (D) Conditioned human moDCs were cultured with allogeneic naive CD4+ T cells for 12 d in the presence of staphylococcal enterotoxin B and IL-2, and T cell polarization was analyzed as described in Fig. 1. FACS plots of one representative experiment out of six are shown. Bar graphs are based on six independent experiments and represent mean ± SD. (E) Murine splenic WT or MR−/− DCs from a C57BL/6 background were co-cultured with naive BALB/c CD4+ T cells in the presence 2 µg/ml omega-1. After an expansion with rIL-2 at day 3, T cells were restimulated on day 6 with PMA and ionomycin and analyzed for intracellular cytokines. One representative experiment out of three is shown. * and #, P < 0.05; **, P < 0.01; *** and ###, P < 0.001 for significant differences with the LPS control (*) or between test conditions (#) based on paired analysis (two-sided paired Student’s t test).
Figure 7.
Figure 7.
MR is essential for omega-1–driven Th2 polarization in vivo. MR−/− and WT Bl/6 mice were injected s.c. with omega-1 (ω-1; 2 µg in 30 µl PBS) or PBS into the footpad. (A) After 7 d, the cells from the draining LNs were restimulated in vitro for 4 d with PBS, 2 µg/ml omega-1, or 10 µg/ml PHA, as polyclonal stimulus, after which cytokine production was determined by ELISA. (B) Intracellular cytokine production of the CD3+/CD4+ T cells from these LNs was assayed by FACS after an additional 6-h restimulation with PMA and ionomycin. FACS plots show concatenated data from four mice. The bar graphs represent the percentage of T cells single-positive for either IL-4 or IFN-γ. One of two independent experiments is shown. Data are means ± SEM of four mice per group based on pooled triplicate wells for each mouse. *, P < 0.05; **, P < 0.01 for significant differences based on paired analysis (two-sided paired Student’s t test).

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