Recombinant GST-CD81-LEL or CD81-LEL derived peptides reduce Mab internalization by macrophages
(A) Uptake of Mab is reduced in THP-1 macrophages pre-treated with recombinant GST-CD81-LEL at different concentrations (2, 8 and 16 μg/mL). Macrophages were lysed with 1% Triton X-100 and serial dilutions were plated before CFU counting. Data are mean values ±SD for four independent experiments (each time in triplicate) (n = 12). One-tailed Tukey’s test: ns, non-significant > 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
(B) Pre-incubation of Mab with different concentrations of recombinant GST-CD81-LEL (2, 8 and 16 μg/mL) significantly reduces the bacterial uptake and percentage of infected macrophages. Quantification of the percentage of cells containing bacilli was performed as in Figure 1C. Data are mean values ±SD for six independent experiments (n = 120 fields). One-tailed Tukey’s test: ns, non-significant > 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.
(C) Percentage of macrophage categories after pre-treatment with recombinant GST-CD81-LEL at 16 μg/mL. The number of bacilli/macrophage drops when bacilli were pre-incubated with CD81-LEL. Values are means ± SD for three independent experiments performed each time in triplicate (n = 900 infected macrophages). One-tailed non-paired t test: ns, non-significant > 0.05, ∗∗∗p < 0.001.
(D) Two immunofluorescent fields taken at 3 h post-infection using a confocal microscope (40× magnification), showing the macrophages infected with Mab (in red) after pre-treatment with GST-CD81-LEL. The nuclei are shown in blue, the CD43 protein associated with the plasma membrane of macrophages is in green. White arrows indicate Mab inside the macrophage.
(E) Uptake of Mab is reduced in macrophages when bacilli were pre-treated with CD81-LEL-derived peptides (810 and 910) either in a linear form (reduced SH) or in a cyclic form (oxidized SS) at 50 or 100 μM. A scrambled (SCR) peptide in its reduced and oxidized form is used as negative control. To determine the number of internalized bacilli, cells were lysed with 1% Triton X-100 and lysates were plated on LB agar before CFU counting. Data are mean values ±SD for four independent experiments performed each time in triplicate (n = 12). One-tailed Tukey’s test: ns, non-significant > 0.05, ∗p < 0.1, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
(F) Four immunofluorescent fields taken at 3 h post-infection using a confocal microscope (40× magnification), showing the macrophages infected with Mab (in red) after pre-treatment with CD81-LEL-derived or SCR peptides. The nuclei are shown in blue, the CD43 protein associated with the plasma membrane of macrophages is in green. White arrows indicate Mab inside the cells.