Figure 1.
EmTIP: a T-cell immunomodulatory protein homologue from E. multilocularis.
(A) Alignment of EmTIP with human and mouse TIPs. Bioedit software, version 7.0.9, was used to align the amino acids sequences. Accession numbers for the sequences are as follows: EmTIP, HF912277; human TIP, Q8TB96; mouse TIP, Q99KW9. Identical residues are displayed in white on black background, biochemically similar residues in black on grey background. Gaps introduced to maximize the alignment are represented by dashes. Numbers at the end of each line correspond to the amino acid numbers in each respective sequence. EmTIP has 36% identity/48% similarity with Human TIP and 34% identity/42% similarity with Mouse TIP. The N-terminal signal sequences are shown in an open solid black box. Two FG-GAP repeats within EmTIP as predicted by SMART (http://smart.embl-heidelberg.de/) are shown within open solid red boxes. The C-terminal transmembrane domains are depicted in an open solid blue box. (B) Comparative structural architecture of E. multilocularis, human and mouse T-cell immunomodulatory proteins. Red boxes: signal sequences; Grey boxes: FG-GAP repeats; Blue boxes: Transmembrane domains. Sequence accession numbers are as described in (A), with structural architecture predicted with SMART (http://smart.embl-heidelberg.de/).
Figure 2.
Expression pattern of EmTIP and localization within E. multilocularis larvae.
(A) RT-PCR strategy for the amplification of the emtip transcript. Shown is an intron (red line)-exon (black boxes) arrangement of the EmTIP genomic locus. A 400 bp product specific for emtip was amplified using the primers FG-GAP_Dw and FG-GAP_Up spanning from exons 6–9. (B) E. multilocularis larvae (PC and MV) were separately used for qualitative assessment of emtip expression. 1 µl of cDNA of each larva was used as template for PCR with a high fidelity DNA polymerase (High-Fidelity DNA Polymerase, New England Biolabs). 2 µl of each PCR amplicon were resolved on a 1.5% agarose gel and stained with Ethidium bromide prior to visualization under a UV transilluminator. (C) Western blots of E. multilocularis larvae lysates probed with pre-immune rabbit serum (1) or anti-EmTIP immune-serum (2) followed by ECL detection and autoradiography. Whole larvae lysates (PC and MV) and E. multilocularis-infected jird liver (iL) were probed with pre-immune rabbit serum (1) or rabbit anti-EmTIP immune-serum followed by ECL detection and autoradiography. (D) Western blot of E/S products from E. multilocularis larvae after immunoprecipitation. Samples were probed with affinity-purified anti-EmTIP antibody followed by ECL detection and autoradiography. The positions of the molecular mass markers (in kilodaltons) are shown on the left. Arrow indicates the position of EmTIP around 58 kDa in primary cell E/S products. The blot is representative of two experiments with different biological isolates with similar results. (E) Staining with affinity-purified anti-EmTIP antibody followed by peroxydase-coupled anti-rabbit-IgG (Jackson ImmunoResearch) against haematoxilin counterstain of representative tissue sections from healthy jird liver (i), infected jird liver (ii), in vitro cultivated E. multilocularis primary cells (iii) and metacestode vesicles (iv). Note the EmTIP signal within the parasite intercellular spaces. Legend: a, E. multilocularis intra-cystic compartment with protoscoleces; b, germinal layer; c, laminated layer; d, periparasitic jird liver tissue; e, central cavities surrounded by aggregates of E. multilocularis primary cells as previously defined [36]; f, EmTIP signal; g, germinal layer; h, laminated layer. Scale bars: 40 µm.
Figure 3.
Anti-EmTIP antibody impairs E. multilocularis stem cell proliferation and metacestode vesicle formation in vitro.
(A) Dose-response curve of the antiproliferative effect of EmTIP inhibition using affinity-purified anti-EmTIP antibody on E.multilocularis primary cell cultures. E. multilocularis stem cell proliferation was assessed by measuring the level of BrdU incorporation (see material and methods for details of the BrdU cell proliferation assay) here expressed as proliferation index. (B) 60 µg/ml of anti-EmTIP antibody limits E. multilocularis stem cell proliferation whereas an equal amount of total rabbit IgG failed to do so. (C) Effect of anti-EmTIP antibody (60 µg/ml) on rat hepatoma cell line proliferation. (D) Effect of anti-EmTIP antibody vs. rabbit IgG on the de novo formation of E. multilocularis metacestode vesicles from primary cell cultures. (A, B, C) Bars represent mean ± SD for 4–6 replicates of two independent experiments. (D) Bars stand for mean levels. Data represent 4–8 biological primary cell isolates assayed individually. #, statistical trend p<0.1;*, statistical significance p<0.05; NS, not significant p>0.1.
Figure 4.
Elevated secretion of IFN-γ by CD4+ T-cells activated in the presence of EmTIP in vitro.
(A) Freshly isolated CD4+CD25− T-cells (2×105/ml) were stimulated with α-CD3 (0.1 µg/ml) and α-CD28 (5 µg/ml) in the presence of E/S products of E. multilocularis primary cells (PCE/S) or metacestode vesicles (MVE/S). Three days later, the T-cell supernatants were collected and probed for IFN-γ and IL-10 concentrations by Elisa. (B) Freshly isolated CD4+CD25− T-cells (2×105/ml) were stimulated with α-CD3 (0.1 µg/ml) and α-CD28 (5 µg/ml) in medium supplemented with normalized supernatants of Mock-, control- (pSecTag2), Emtip- (pSecTag2-Emtip) or Emtip_myc (pSecTag2-Emtip_myc)- transfected HEK-293T cells. Three days later, the T-cell supernatants were collected and probed for IFN-γ and IL-10 concentrations by Elisa. (A, B) Individual results are displayed. Bars represent the mean from 4–8 independent biological replicates assessed within 3–4 independent experiments. (C) Freshly isolated CD4+CD25− T-cells (2×105/ml) were stimulated with α-CD3 (0.1 µg/ml) and α-CD28 (5 µg/ml) in the presence of conditioned medium of primary cells (PCE/S) or metacestode vesicles (MVE/S) supplemented with total rabbit IgG or purified anti-EmTIP (anti-TIP) antibody at 30 µg/ml. Three days later, the T-cell supernatants were collected and probed for IFN-γ and IL-10 concentrations by Elisa. Individual results are displayed. Bars represent the mean from experiments with 4 biological replicates. (D) Freshly isolated CD4+CD25− T-cells (2×105/ml) were stimulated with α-CD3 (0.1 µg/ml) and α-CD28 (5 µg/ml) in medium supplemented with normalized supernatants of parasite larvae (PCE/S or MVE/S) or transfected HEK-293T cells (Mock, Control or rEmTIP). At day 3 of culture, 5 µg/ml Brefeldin A was added for an additional 6 hours. Then the cells were harvested and the intracellular IFN-γ levels were determined by flow cytometry. Numbers in upper gates indicate the percentage of IFN-γ+ CD4+ cells whereas the lower gate shows the percentage of IFN-γ+ CD4− cells. One representative experiment of 3 conducted with similar results is shown (Left) and data from all three experiments are summarized as bar graphs (Right). * (p<0.05), ** (p<0.01) (D) Bars represent mean ± standard deviations from three independent experiments with 3–7 biological replicates.