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. 2009 Aug 3;206(8):1681-90.
doi: 10.1084/jem.20082462. Epub 2009 Jul 27.

The major component in schistosome eggs responsible for conditioning dendritic cells for Th2 polarization is a T2 ribonuclease (omega-1)

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The major component in schistosome eggs responsible for conditioning dendritic cells for Th2 polarization is a T2 ribonuclease (omega-1)

Svenja Steinfelder et al. J Exp Med. .

Abstract

Schistosoma mansoni eggs contain factors that trigger potent Th2 responses in vivo and condition mouse dendritic cells (DCs) to promote Th2 lymphocyte differentiation. Using an in vitro bystander polarization assay as the readout, we purified and identified the major Th2-inducing component from soluble egg extract (SEA) as the secreted T2 ribonuclease, omega-1. The Th2-promoting activity of omega-1 was found to be sensitive to ribonuclease inhibition and did not require MyD88/TRIF signaling in DCs. In common with unfractioned SEA, the purified native protein suppresses lipopolysaccharide-induced DC activation, but unlike SEA, it fails to trigger interleukin 4 production from basophils. Importantly, omega-1-exposed DCs displayed pronounced cytoskeletal changes and exhibited decreased antigen-dependent conjugate formation with CD4(+) T cells. Based on this evidence, we hypothesize that S. mansoni omega-1 acts by limiting the interaction of DCs with CD4(+) T lymphocytes, thereby lowering the strength of the activation signal delivered.

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Figures

Figure 1.
Figure 1.
S. mansoni egg culture supernatants promote bystander Th2 polarization. (A) Protein composition of SEA and egg supernatants evaluated by Coomassie blue–stained SDS-PAGE. (B and C) Naive DO.11.10 Tg CD4+ lymphocytes were cultured with DC and OVA peptide with or without 40 μg/ml SEA, 30 μg/ml egg supernatant, or control supernatants from mock cultures containing no eggs. After restimulation with PMA/ionomycin, cells were stained for CD4 and T1/ST2 (B) or CD4 and IL-4 plus IFN-γ (C), respectively. The FACS dot plots shown are gated on CD4+ T lymphocytes (percentages are shown). The experiment shown is representative of more than five performed. (D) The frequency of IL-4+ DO.11.10 T cells determined by intracellular cytokine staining (ICS) in response to gel filtration fractions of egg culture supernatants. (E) Coomassie blue–stained SDS-PAGE of fractions 22–24 from the column shown in D. Results are representative of two independent experiments performed with different egg culture supernatant preparations. (F) In situ ribonuclease activity of a 32-kD protein in both SEA and egg supernatants detected by zymogram gel.
Figure 2.
Figure 2.
Identification of omega-1 as the major component with Th2-inducing activity in SEA. (A) The elution profile of SEA from an SP-Sepharose column indicating the pools (pools A–I) tested for biological activity. The diagonal line indicates the gradient of NaCl. (B) Th2 response of DO.11.10 Tg cells induced by the different pools in A was measured by ICS and IL-4 secretion. The results from an assay performed at one fixed protein concentration (20 μg/ml) for each pool are shown, along with 40 μg/ml SEA as a positive control. Bars represent the percentage of IL-4+ CD4+ T lymphocytes, and the means ± SD of the IL-4 concentrations. The same pattern of activity was observed when three additional twofold serial dilutions were compared for each pool (not depicted). (C) The elution profile of pool I after separation on a Superdex 75 gel filtration column. (D and E) The indicated fractions were tested in Th2 polarization (D) and basophil (E) assays. Bars represent means ± SD for IL-4 measured by ELISA. (F) Coomassie blue–stained SDS-PAGE of SEA and fraction 21 from the column in C. (G) Western blot of SEA and fraction 21 developed with mAb specific for omega-1. (H and I) Flow cytometry of ICS performed on DO.11.10 Tg T cells stimulated with OVA/DCs in the presence of omega-1 or IL-4 preincubated with or without anti–omega-1 mAb (H) or in the presence of egg supernatants preincubated with control or anti–omega-1 mAb (I). The dot plots are gated on CD4+ T lymphocytes (percentages are shown). (J) Loss of Th2 activity in SEA depleted of omega-1 measured by ICS and IL-4 secretion in a DO.11.10 Tg T cell assay as described. The experiments shown in A–G and H–J are representative of two and three experiments, respectively.
Figure 3.
Figure 3.
Omega-1–pulsed DCs promote Th2-type responses in vivo. BALB/c mice were injected s.c. with BMDCs unstimulated or incubated overnight with 30 μg/ml SEA, 2 μg/ml omega-1, or irradiated T. gondii (RH strain) tachyzoites (1:1). 1.5 ×ばつ 106 popliteal LN cells/ml from individual animals (n = 3) were pooled by mixing equal numbers of cells from each animal within a group and stimulated with 0.2 μg/ml anti-CD3 mAb, 30 μg/ml SEA, 0.5 μg/ml omega-1, or 10 μg/ml of soluble tachyzoite antigen (STAg). Cytokines were measured by ELISA in 72-h supernatants. Data shown are means ± SD of ELISA values for each pool. ICS was performed after supernatant removal. When restimulated with SEA, the frequency of IL-4+ CD4+ T cells in pooled LN cultures from mice that received SEA- or omega-primed DCs was 14 and 8.8%, respectively, and when the same cultures were restimulated with omega-1 the frequencies were 7.2 and 7.9%. One representative experiment out of three performed is shown. When omega-1–pretreated B cells were injected instead of DCs, no immune response was detected (not depicted).
Figure 4.
Figure 4.
The Th2-polarizing function of both SEA and omega-1 is sensitive to ribonuclease inhibition. (A and B) Equal amounts of DEPC-treated or -untreated unfractionated SEA (40 μg/ml) or purified omega-1 (0.5 μg/ml) were added to the DC/DO.11.10 Tg CD4 T cell cultures, and IL-4 secretion was measured on day 3 by ELISA (A) and the frequency of IL-4+ cells was assayed by ICS on day 7 after restimulation with anti-CD3 mAb (B). Bars represent means ± SD for IL-4. The dot plots shown are gated on CD4+ T lymphocytes (percentages are shown). When DEPC-treated SEA or DEPC-treated omega-1 was added to the cultures in the presence of exogenous rIL-4, to evaluate possible nonspecific inhibitory effects of carryover DEPC, no decrease in the frequency of IL-4+ DO.11.10+ T cells was observed (not depicted). The data shown are representative of two experiments performed.
Figure 5.
Figure 5.
Omega-1 suppresses TLR ligand–mediated DC activation and induces cytoskeletal changes in DCs. (A and B) BMDCs were incubated in medium, 40 μg/ml SEA, or 1 μg/ml omega-1 with or without 40 ng/ml LPS. (A) Levels of p40 IL-12/23 and p70 IL-12 measured by ELISA. When tested in the presence of LPS, twofold dilutions of omega-1 (1–0.06 μg/ml) were assayed. Bars represent mean cytokine concentrations ± SD. *, P < 0.05; and NS refer to IL-12 secretion induced by LPS alone versus in the presence of SEA or omega-1. (B) Surface expression of CD40 and CD54 on DCs in the same cultures. Bars represent geometric mean fluorescences ± SD of two experiments. (C and D) BMDCs were cultured in medium, 40 ng/ml LPS, 40 μg/ml SEA, or 0.5 μg/ml omega-1 for 12 h. (C) The number of viable cells was determined in adherent versus nonadherent populations after culture on nontreated (left) or tissue culture–treated (right) plates. Bars indicate the frequency of cells in the two subpopulations for each culture condition. Results are from one representative experiment out of three performed. (D) CD11c+ BMDCs cultured on multichamber glass slides under the same conditions were stained with phalloidin (green) and DAPI (blue) and analyzed by confocal microscopy. (left) Images demonstrate the homogeneity of cell populations; (right) A zoom-in view, which is a projection of a z-stack. The photomicrographs shown are representative images from five fields examined in one experiment out of three performed. Bars, 5 μm. MFI, mean fluorescence intensity.
Figure 6.
Figure 6.
Omega-1–exposed DCs are less efficient in forming conjugates with CD4+ T cells. (A and B) BMDCs were cultured on nontreated plates with the same stimuli as in Fig. 5 C, pulsed with increasing amounts of OVA peptide, and incubated with OT-II Tg CD4+ T cells. The frequency of CD4+ T cell–forming conjugates with DC was measured by FACS. (A) A representative set of contour plots gated on CD4+ T lymphocytes (percentages are shown). (B) Quantification of T cells forming conjugates with DCs (means ± SD for duplicates) from one experiment (top). Fold increase in the frequency of OT-II Tg CD4+ T cells in conjugates with DCs was calculated as a function of peptide concentration for each type of DC based on the results from four independent experiments performed, of which two included omega-1 (bottom). (C and D) OT-II Tg T cells were cultured in the presence of DCs and increasing concentrations of OVA peptide for 36 h. Cultures with 1 μg/ml of peptide were tested with and without 0.5 μg/ml omega-1. (C) Viability of CD4+ T cells determined by PI staining of ungated populations (top). The contour plots gated on the CD4+ PI− population shows the percentage of cells that have completed the first cell cycle as determined by CFSE dilution (bottom). Cell size is indicated by forward scatter (FSC) on the y axis. (D) IL-2 in corresponding cultures measured by ELISA. Data shown are the mean concentrations ± SD. The data shown in C and D are representative of two experiments.

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