The involvement of the spleen during chronic phase of Schistosoma mansoni infection in galectin-3-/- mice

Schistosoma mansoni synthesizes glycoconjugates which interact with galectin-3, eliciting an intense humoral immune response. Moreover, it was demonstrated that galectin-3 regulates B cell differentiation into plasma cells. Splenomegaly is a hallmark event characterized by polyclonal B cell activation and enhancement of antibody production. Here, we investigated whether galectin-3 interferes with spleen organization and B cell compartment during chronic schistosomiasis, using wild type (WT) and galectin-3-/- mice. In chronically-infected galectin-3-/- mice the histological architecture of the spleen, including white and red pulps, was disturbed with heterogeneous lymphoid follicles, an increased number of plasma cells (CD19-B220-/lowCD138+) and a reduced number of macrophages (CD19-B220-Mac-1+CD138-) and B lymphocytes (CD19+B220+/highCD138-), compared with the WT infected mice. In the absence of galectin-3 there was an increase of annexin-V+PI- cells and a major presence of apoptotic cells in spleen compared with WT infected mice. In spleen of WT infected mice galectin-3 was largely expressed in lymphoid follicles and extrafollicular sites. Thus, we propose that galectin-3 plays a role in splenic architecture, controlling distinct events such as apoptosis, macrophage activity, B cell differentiation and plasmacytogenesis in the course of S. mansoni infectio

Histological analysis of lymphoid follicles of MLNs of infected mice.

<p>(A) In wild type (WT) mice, section of lymphoid follicles showed scarce apoptotic bodies (arrow). In infected gal-3−/− mice (B), there was high number of cellular debris dispersed throughout the follicles (arrows). Immunofluorescence to MOMA-2+ macrophages. (C) Immunoreactivity for MOMA-2 Alexa 488 (green cells) in MLNs of WT and (D) in galectin-3−/− mice. (E) Detailed MOMA-2+ cell clusters in WT mice and (F) rare MOMA-2+ cells in the absence of galectin-3. The nuclei were stained with DAPI. Data are representative of three independent experiments, each carried out in three mice with chronic infection.</p

Absolute number of the cell subsets in the mesenteric lymph nodes of WT and galectin-3−/− mice infected with <i>S.mansoni.</i>

<p>Data are reported as means ± SEM, They are representative of three independent experiments, Statistical analysis: Tukey's multiple comparison test (*, <i>P</i><0.05).</p

Cell cycle analysis and apoptosis index in MLNs of WT and galectin-3^−/− infected mice.

<p>Histograms represent the stages of cell cycle in WT (A) and Gal-3^−/− mice (B) infected with <i>S.mansoni</i>. In both graphs, the phases were described as bellow: M1 - sub G1/G0; M2 – G1/G0; M3 – S phase; M4 – G2/M and M5 – hyperploid cells. (C–D) Quantification of Annexin-V⁺/Propidium iodide (PI) ⁻ apoptotic cells (gated in R2 region) and Annexin-V⁺/PI⁺ dead cells (gated in R3 region), in WT (C) and Gal-3^−/− mice (D). (E) Quantification of apoptotic cells induced by high temperature. Solid bars represent WT mice and open bars indicate Gal-3^−/− mice. Data are reported as means ± SEM and are representative of three independent experiments. Statistical analysis: Tukey's multiple comparison test (*, <i>P</i><0.05). A–B, original magnification, 400x.</p

Phenotypic analysis of B lymphocytes in the MLNs.

<p>B220+ CD19+ cells were selected and quantified in uninfected wild typeWT and galectin-3−/− mice (A and B, respectively), and in chronically-infected wild typeWT and galectin-3−/− mice (C and D, respectively). (D) The arrow pointed to distinct B220low subpopulation found in the absence of galectin-3. (E) Histograms reflect the surface expression of CD138, a plasma cell marker. Full histogram: WT mice. Empty histogram: galectin-3−/− mice. (F) Absolute number of plasma cells in MLNs of infected WT (solid bars) and infected galectin-3−/− mice (open bars). Data are reported as means + SEM and are representative of three independent experiments, each carried out in five mice with chronic infection. Statistical analysis: Tukey's multiple comparison test (*, P<0.05).</p

Relative number of cells during cell cycle events in MLNs of mice chronically-infected with <i>Schistosoma mansoni.</i>

<p>Data are reported as means ± SEM, They are representative of three independent experiments, Statistical analysis: Tukey's multiple comparison test (*, <i>P</i><0.05).</p

Immunohistochemistry to localize B lymphocyte and plasma cell niches in MLNs.

<p>(A) Immunoreactivity for B cells using anti-B220 antibody preferentially within of lymphoid follicles (LF) in chronically-infected WT wild type mice. (B) In galectin-3−/− mice, B220+ cells were randomly dispersed by the parenchyma forming numerous lymphoid follicles. In infected WT mice, CD138+ plasma cells and Blimp-1+ antibody-secreting cells were found in cellular cords in extrafollicular regions (C and E, respectively). In infected galectin-3−/− mice, CD138+ and Blimp-1+ plasma cells were randomly scattered throughout the parenchyma (D and F, respectively). A–F: Original magnification, 200x. Boxed images: original magnification, 400x. Data are reported as means + SEM and are representative of three independent experiments.</p