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Intraamniotic infection and inflammation are associated with spontaneous preterm birth (PTB) and preterm premature rupture of the membranes (pPROM). In this study, we tested engineered extracellular vesicles, or exosomes, cargoing an inhibitor to pro-inflammatory transcription factor (NF-kB), called super repressor (SR) IkB, to prolong gestation in an infection (LPS) induced PTB mouse model.
HEK293T (human embryonic kidney cell) derived exosomes were engineered to contain SR using a protein loading via optically reversible protein-protein interaction (EXPLORs) method (Yim, et al 2016). In this method, SR is actively incorporated into exosomes during biogenesis. These exosomes were isolated, quantified and used for our studies. Intraperitoneal (IP) injection of either LPS (100 mg) or PBS were performed in CD-1 mice on gestational day 15 followed by injection of PBS, SR exosomes (5x1010) or naïve exosomes (exosomes derived from HEK293T cells under normal culture conditions, 5x1010) every 2 hours for a total of 4 injections. Treatment groups (Group 1-LPS+PBS; Group 2-LPS+SR; Group 3-LPS+naïve, and Group 4-PBS) were monitored for preterm birth. Upon delivery of at least one pup in Group 1, mice were euthanized, and maternal plasma, uterus and cervix were collected for cytokine analysis using Luminex (IL-1β, IL-8 and IL-10) and Western blot for NF-kB activation via RelA phosphorylation (P-NF-kB) respectively. Survival graphs were created in GraphPad and one-way ANOVA was performed to determine statistical significance (P <0.05).
Animals injected with PBS delivered at the expected gestational age (19.5 days). LPS and LPS+naïve induced PTB within 10 hours; however, injection of SR exosomes prolonged delivery by an average of 21 hours in this model (Fig 1). Consistently lower levels of pro-inflammatory cytokines, IL-1β and IL-8, were seen in maternal plasma of LPS+SR compared to LPS mice (Figure 2A), while anti-inflammatory cytokine, IL-10, levels were significantly increased in LPS+SR mice compared to LPS (P=0.01) and PBS controls (P<0.0001) (Figure 2A). In the cervix and uterus, P-NF-kB expression was significantly decreased in LPS+SR compared to LPS (P=0.005, P=0.03) (Figure 2B).
Exosomes can be engineered to carry pharmaceutical agents that can dampen the infection-induced inflammation associated PTB and pPROM.