Fig 1.
Dose- and time-dependent effect of IL-1β treatment on HBMEC barrier function using TEER.
Cell index was normalised at the time point immediately prior to the addition of varying concentrations of IL-1β and medium (black line) was set as baseline. Changes in baseline normalised cell index are plotted at the respective time points (mean ± SD of 4 independent experiments).
Fig 2.
Morphology of THP-1 and IT4var14 IE after co-culture for 24 hrs.
Images from a representative co-culture experiment in the absence (left, A-E) or presence of xanthine oxidase (right, F-J). A and F: THP-1 cells only; B and G: THP-1 + uRBC; C, D, E, H, I and J: THP-1 + IE. THP-1 cells were activated upon 24 hour co-culture with IE with highly vacuolised cytoplasm, enlarged euchromatic nucleus and nucleoli (C, D, H and I). Phagocytosis of IE occurred (arrows in C, D, H and I) and in co-cultures with xanthine oxidase, parasites did not progress beyond the trophozoite stage and appeared pyknotic (compare J with E). The scale bar is 10 μm.
Fig 3.
HBMEC barrier function is not altered by THP-1/IE co-culture supernatants.
(A) Schematic of the co-culture and TEER procedure. (B) Representative TEER trace of HBMEC with the different culture conditions. Cell index was normalised at the time point immediately prior to the addition of the (co-)culture medium (black arrow) with media mix (MM, bright green) set as baseline (baseline normalised cell index, BNCI). (C) ΔBNCI of HBMEC for all the IT4var14/THP-1 (co-)culture conditions in the presence (red dots) or absence (grey squares) of xanthine oxidase (XO) after 24 hours. (D) Same as C with ΔBCNI at 48 hours. Mean ± SEM of 6 independent experiments, no significant difference was detected between the conditions as calculated by two-way ANOVA.
Fig 4.
IL-1β levels in xanthine oxidase treated THP-1/IE co-cultures.
IL-1β levels were measured in 24 hour (co-)culture medium in the presence of xanthine oxidase with LPS + nigericin treatment as control. Mean ± SEM from 11 (8 for uninfected RBC only condition) independent experiments is plotted, of which 9 have also been used in TEER assays. Statistical significance is not shown, as most of the IL-1β concentrations are lower than the sensitivity of the ELISA (3.6 pg/ml).
Fig 5.
Uric acid detection in THP-1/IE co-culture medium.
Uric acid levels were measured in supernatants from 24 hour (co-)cultures in the presence or absence of xanthine oxidase (XO). Mean ± SEM from 9 independent experiments is plotted; statistical significance between the +XO conditions was calculated by two-way ANOVA with ** p < 0.01, *** p < 0.001 and **** p < 0.0001.