Figure 10

Soluble native Entamoeba histolytica (Eh) Gal-lectin is immunostimulatory but does not activate the inflammasome. (a, b) PMA-differentiated THP-1 macrophages or lipopolysaccharide (LPS)-primed bone marrow-derived macrophages (BDMs) were exposed to live Eh or soluble Gal-lectin (1 μg ml⁻¹) for 30 min. (d, e) Increasing concentrations of soluble Gal-lectin were added to PMA-differentiated THP-1 macrophages and stimulated with 1.5 × 10³ live Eh for 30 min. Secretion of processed caspase-1 (a, c, d) and interleukin (IL)-1β (c) was detected in cell supernatants (SN) by immunoblot and IL-1β secretion was measured by enzyme-linked immunosorbent assay (ELISA; b, e). (f) Tumor necrosis factor α (TNF-α) release by PMA-differentiated THP-1 macrophages exposed to the indicated number of live ameba or soluble Gal-lectin for 3 h measured by ELISA. Specificity for the Gal-lectin was shown by pre-treating the Gal-lectin with adherence inhibitory mAb 1G7 or immunoglobulin G (IgG) control (1:100 dilution) for 10 min at 4 °C before incubation with macrophages. (g) Immunoblot showing the expression of pro-IL-1β, NLRP3, and pro-caspase-1 in the lysates of BDM macrophages primed with LPS (1 μg ml⁻¹) or Gal-lectin (1 μg ml⁻¹) for 4 h. (h) BDM were primed with Gal-lectin (1 μg ml⁻¹) for 4 h and nigericin (10 μM) was added in the final 30 min. Active caspase-1 and IL-1β were detected in cell SNs by immunoblot. **P<0.01, ***P<0.005 vs. negative control. n=4. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; LYS, lysates.

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