Fig. 7: HM EVs regulate cell death in THP-1 monocytes.

Cells were treated with 40 µg/mL term or preterm human milk EVs for 6 h, followed by inflammatory activation with 1 ng/mL LPS for two hours, and 10 µg/mL nigericin for 1 h. Cell death was measured in THP-1 cells treated with (a) term or preterm HM EVs, or media only control; (b) term or preterm HM EVs with LPS and nigericin, or LPS and nigericin alone. Protection against nigericin and LPS induced pyroptotic cell death was measured by flow cytometry of annexin V and propidium iodide signal. n = 10–16, four replicate experiments. Tolerogenic response was present in THP-1 cells treated with either term (c) or preterm (d) HM EVs. Tolerogenic response was proposed for biological replicates that increased cell death in control samples but protected against pyroptotic cell death following inflammatory activation, indicated in brackets. e HM EV miRs predicted targets TLR4, MYD88, and NEK7 could result in downregulation of programmed cell death via upstream inhibition of inflammasome pathway. f Term, but not preterm HM EVs, reduced apoptosis. THP-1 monocytes were treated with 40 µg/mL term or preterm human milk EVs for 6 h, followed by etoposide (30 µM) for 4 h, with or without an emricasan (EM, 10 µM) pre-treatment for 30 min, or media only control. Geometric mean of fluorescence intensity (FITC) was plotted, n = 3. **p < 0.01, *p < 0.04, ns=non-significant, one-way ANOVA with multiple comparisons.