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. 2020 May 29;11:1034. doi: 10.3389/fimmu.2020.01034

Figure 2.

Figure 2

In vitro conditioning with plasma from long living individuals leads to polarization of LLIs and controls macrophages toward M2 phenotypes. Macrophages were generated from peripheral blood monocytes of controls (35–75 years, N = 10) and of long living individuals (range 95–99, N = 10) upon 7 days in vitro culture with 20% autologous plasma. (A) Panel shows FACS histogram profiles of CD206, CD163, and CD80 protein levels at the cell surface of recovered MPL-macrophages (viable gated CD68+ cells) from a representative control volunteer (upper plots, 67 years-old male) vs. a representative LLI (lower plots, 98 years-old male). Cell staining was gated using isotype control antibodies (gray histograms). (B) Bars graph in panel report the mean fluorescence intensity (MFI) values ± SD of CD163 and CD206 M2 marker on viable CD68+ gated cells from controls (N = 10) and LLIs (N = 10). Pairwise comparisons statistically significant are indicated (ANOVA; *P < 0.05 and **P < 0.01). (C) IL-12p70 and IL-10 secretion by control and LLIs macrophages after 1 μg/ml LPS stimulation for 24 h. Cell culture supernatants were collected and cytokines secretion was determined using bead-based multiplex ELISA. Results were expressed as the mean ± SD of all sample determinations conducted in duplicate. All pairwise comparisons are statistically significant (ANOVA; *P < 0.05, **P < 0.01). (D–F) Peripheral blood monocytes of control volunteers (35–75 years, N = 10) were 7 days-exposed to plasma from LLIs (N = 10) and autologous or allogenic control plasma as comparison, in the presence or absence of BPIFB4 (1:100) blocking antibodies for the last 72 h of culture. After 7 days in vitro culture, cytofluorimetric analysis of recovered MPL-macrophages was conducted. (D) FACS histogram profiles for CD163 M2 markers in MPL-macrophages of both control plasma- and 3 representative LLIs-treated cells are shown. (E) Representative flow cytometry CD206 vs. CD163 density plots for each experimental condition is presented. (F) Bars graph report the percentage ± SD of CD206+CD163+ of gated MPL-macrophages from ten independent experiments using different donors (ANOVA; **P < 0.01, ***P < 0.001).