Immunomodulatory components of Trichinella spiralis excretory-secretory products with lactose-binding specificity

Abstract

The immunomodulatory potential of Trichinella spiralis muscle larvae excretory-secretory products (ES L1) has been well documented in vitro on dendritic cells (DCs) and in animal models of autoimmune diseases. ES L1 products possess the potential to induce tolerogenic DCs and consequently trigger regulatory mechanisms that maintain immune homeostasis. The use of ES L1 as a potential treatment for various inflammatory disorders proved to be beneficial in animal models, although the precise immunomodulatory factors have not yet been identified. This study aimed at the isolation and characterization of ES L1 components that possess galectin family member properties. Galectin-1-like proteins (TsGal-1-like) were isolated from ES L1 based on the assumption of the existence of a lactose-specific carbohydrate-recognition domain and were recognized by anti-galectin-1 antibodies in Western blot. This TsGal-1-like isolate, similar to galectin-1, induced DCs with tolerogenic properties and hence, the capacity to polarize T cell response towards a regulatory type. This was reflected by a significantly increased percentage of CD4⁺CD25⁺Foxp3⁺ regulatory T cells and significantly increased expression of IL-10 and TGF-β within this cell population. Proteomic analysis of TsGal-1-like isolate by mass spectrometry identified nineteen proteins, seven with annotated function after blast analysis against a database for T. spiralis and the UniProt database. To our surprise, none of the identified proteins possesses homology with known galectin family members. Nevertheless, the isolated components of ES L1 possess certain galectin-1 properties, such as specific lactose binding and the potential to elicit a regulatory immune response, so it would be worth further investigating the structure of sugar binding within isolated proteins and its biological significance.

Keywords: Trichinella spiralis; galectin-1; immunomodulation; proteomics.

Table 1. Identification of proteins in ES L1 fraction by mass spectrometry

Figure 1. Anti-galectin-1 Ab-reactive proteins in T. spiralis muscle larvae. Immunohistochemical (a) and indirect immunofluorescence (IIF) staining (c, d) of the sections of isolated T. spiralis muscle larvae with anti-human galectin-1 Ab and (b, e) negative control, without anti-galectin-1 Ab. Arrows indicate binding of anti-galectin-1 Ab to subcuticular region - SC, stichocytes - ST, reproductive tracts - RT and gut - G

Figure 2. T. spiralis proteins recognized by anti-galectin-1 antibodies. Western blot of T. spiralis crude extract (a, line 1) and ES L1 products (a, line 2) with anti-human galectin-1 Ab. Mw markers are indicated by arrows. (b) SDS-PAGE of the components of ES L1 isolated on Lactose-Sepharose 4B column. st - Mw standards; line 1: ES L1 proteins; line 2: Lactose eluted proteins. Proteins were stained with silver staining kit. (c) Western blot of lactose eluted proteins with anti-human galectin-1 Ab-HRP (line 1) and anti-mouse galectin-1 Ab-HRP (line 2); st - Mw standards. (d) Binding of TsGal-1-like proteins to Matrigel. Serial dilutions of TsGal-1-like proteins in MePBS or PBS (0-20 μg/ml) were added to microtiter plate wells coated with Matrigel. Bound TsGal-1-like proteins were detected with anti-galectin-1 Ab, followed by anti-rabbit IgG-HRP.

Figure 3. Phenotype of DCs treated with TsGal-1-like proteins. Immature DCs were treated with T. spiralis ES L1 antigens (50 μg/ml), TsGal-1-like proteins (5 μg/ml) and LPS/IFNγ and the expression of maturation markers was analyzed by flow cytometry. The summarized results of HLA-DR, CD83, CD86, CD40, CCR7 and ILT3 expression by DCs are shown as mean ± SD from three different experiments. (Representative experiment for the expression of maturation markers is given in Supplementary Figure 1). *p < 0.05, **p < 0.01, ***p < 0.005 compared as indicated (one-way ANOVA with Tukey's posttest).

Figure 4. Cytokine expression of TsGal-1-like proteins-pulsed DCs. The summarized results of IL-12p40/p70, IL-10, and transforming growth factor (TGF)-β expression are presented as mean value of percentage (%) of the expression ± SD from three different experiments. *p < 0.05, **p < 0.01, ***p < 0.005 compared as indicated (one-way ANOVA with Tukey's posttest).

Figure 5. T helper polarization influenced by treated DCs. Treated DCs were thoroughly washed and then cocultured with magnetic-activated cell sorting-purified allogenic T cells (1 × 10⁵/well) for 6 days in 1:20 DC:T cell ratio. (a) Representative analysis of IL-4⁺, IL-10⁺ and IFN-γ⁺ cells within CD4⁺ T cells from one experiment are shown. (b) Presented are the summarized results shown as mean % ± SD of three experiments with different DCs donors. *p < 0.05, **p < 0.01, ***p < 0.001 compared to control, or as indicated by line (one-way ANOVA with Tukey's posttest).

Figure 6. Tolerogenic properties and functions of TsGal-1-like proteins-treated DCs. DCs treated with ES L1 antigens (50 μg/ml), TsGal-1-like proteins (5 μg/ml) and LPS/IFNγ were washed thoroughly and then cocultured with magnetic-activated cell sorting-purified allogenic T cells (Tly) (1 × 10⁵/well) for 3 days in 1:50 DC:T cell ratio and then re-stimulated with interleukin (IL)-2 (2 ng/ml) for another 3 days. (a) Representative analysis of CD25⁺Foxp3⁺ cells within CD4⁺ T cells and IL-10 and transforming growth factor (TGF)-β within CD4⁺CD25⁺ T cell population is shown, (b) the summarized results for the expression of CD4⁺CD25⁺FoxP3⁺ T regulatory cells and IL-10 and TGF-β within CD4⁺CD25⁺ T cells are shown as the mean % ± SD from three different experiments. *p < 0.05, **p < 0.01, ***p < 0.005 compared as indicated by line (one-way ANOVA with Tukey's posttest).