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. 2023 Jan 25;26(2):106042. doi: 10.1016/j.isci.2023.106042

Figure 1.

Figure 1

Internalization of Mab by macrophages requires tetraspanin CD81

(A and B) Uptake of Mab is reduced in THP-1 macrophages (A) and primary human macrophages (B) pre-treated with neutralizing antibodies raised against CD43, MR, CR3 or CD81. Macrophages were lysed with 1% Triton X-100 and then successive dilutions of the different suspensions were prepared and plated on LB agar before CFU counting. Data are mean values ±SD for six (A) or four (B) independent experiments, each time in triplicate (A) or quadruplicate (B), n = 18 (A) or 16 (B). One-tailed Tukey’s test: ns, non-significant > 0.05, ∗∗∗∗p < 0.0001.

(C) Blocking CD43 or MR or CR3 or CD81 receptors with neutralizing antibodies significantly reduces the percentage of Mab-infected THP-1 macrophages. Quantification of the percentage of host cells containing bacilli was performed using an epifluorescence microscope. Images were acquired focusing on the combined signals (CD43 in green and Mab in red) and scoring for the presence or absence of bacilli in macrophages using ImageJ. Data are mean values ±SD for four independent experiments (n = 80 fields). One-tailed Tukey’s test: ns, non-significant > 0.05, ∗∗∗p < 0.001.

(D) Percentage of Mab-infected THP-1 macrophage categories after pre-treatment with neutralizing antibodies raised against CD151 or CD81. The images illustrate a macrophage < 5 bacilli, 5 to 10 bacilli or > 10 bacilli (upper right). The nuclei were stained with DAPI (blue) and the surface of macrophages was stained with anti-CD43 antibodies (green). 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.

(E) Five immunofluorescent fields were taken at 3 h post-infection using a confocal microscope (40× magnification), showing the macrophages infected with the Mab (in red) following pre-treatment with indicated antibodies. The nuclei are in blue and the CD43 protein associated with the plasma membrane of macrophages is in green. White arrows indicate Mab inside the cells.