Fig. 5: Q586B2 is released by parasites, exerts both pro- and anti-inflammatory activity on myeloid cells (BMDMs) in vitro and at the site of inoculation myeloid cells are the most prominent IL-10 producing cells mediating the attenuated first-peak parasitemia observed in Tb927.6.4140-KO parasite-infected mice.
a Schematic of the Nb-based sandwich ELISA whereby Nb39 was used as capturing Nb, biotinylated Nb39 was used as detecting Nbs and streptavidin-HRP was used to develop the ELISA. The illustration in (a) was created using Biorender.com by Maida Živalj. b Detection of native Q586B2 via a Nb-based sandwich ELISA in secretome of WT (90:13 strain) and Tb927.6.4140-KO parasites cultured in HMI-9 for 48 h in vitro. c Detection of native Q586B2 via a Nb-based sandwich ELISA in serum from naïve and T. b. brucei-infected C57BL/6 mice collected at day 6 p.i. For b and c, results are representative for 2 independent experiments (n = 4) and presented as mean ± SEM. Unpaired t-test (Two-tailed) was performed using Welch’s correction. d Production of TNF, IL-6, MIF, and IL-10 by BMDMs generated from C57BL/6 mice (2 × 105 cells/well) following 48 h incubation with a ½ serial dilution of LPS-free Q586B2 (red bars) starting from 0.25 µg/ml or buffer alone (gray bars). Negative controls: cells incubated with buffer alone or without any protein. Data are shown as means ± SEM and representative of 2 independent experiments (n = 3). A Multiple paired Student’s t-tests was used for comparison between experimental groups, with ns indicating not significant for p ≥ 0.05 and p < 0.05. e To assess which cells express IL-10 in vivo, IL-10-GFP (VeRT-X) mice were injected intraperitoneally with PBS (control) or 5 µg LPS-free Q586B2 and 18 h later sacrificed. Subsequently, the cellular source of IL-10 was analyzed via flow cytometry using the gating strategy described in Fig. S5. The expression of IL-10 was presented as delta median fluorescence intensity (delta MFI), by subtracting the MFI of PBS-injected mice from Q586B2-injected mice, for each of the different immune cells that were identified. Results are representative of 2 independent experiments (n = 3) and presented as mean ± SEM. ND Not detected. f Parasitemia of LysMCre × IL-10fl/fl C57BL/6 mice infected with Wild-type (WT: T. b. brucei parasite 90:13 pleiomorphic strain, black circles) or Tb927.6.4140-deficient (red circles) parasites. Results are representative of 2 independent experiments (n = 5) and presented as mean ± SEM. NS not significant, ND Not detectable. Parasitemia was analyzed using a Mann–Whitney U-test (only significance p < 0.05 is shown). Source data are provided as a Source Data file.
