Research ArticleIMMUNOLOGY

Exosomal delivery of NF-κB inhibitor delays LPS-induced preterm birth and modulates fetal immune cell profile in mouse models

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Science Advances  22 Jan 2021:
Vol. 7, no. 4, eabd3865
DOI: 10.1126/sciadv.abd3865
  • Fig. 1 Characterization of engineered exosomes to contain the NF-κB inhibitor SR using EXPLOR technology.

    (A) Morphological characterization of naïve and SR exosomes using TEM shows exosomes with classic cup-shaped morphology. (B) Representative graph of NTA demonstrating similar size distributions in diluted samples of naïve and SR exosomes to determine size and concentration of exosome preparations. (C) HEK293T cells stably express the SR IκB, while naïve cells do not. Exosomes isolated from SR-producing cells express SR, mCherry, green fluorescence protein (GFP), as well as exosome markers TSG101 and CD63. Exosomes were negative for GM130.

  • Fig. 2 SR exosomes delay LPS-induced preterm labor.

    (A) Experimental model for infection-induced preterm labor in CD-1 and C57BL/6J mouse models. (B) Survival curve comparing PBS (blue), LPS (red), LPS + naïve (gray), and LPS + SR (green) treatment groups. LPS and LPS + naïve injections caused PTB within 24 hours, while LPS + SR delayed PTB by an average of 24 hours, compared to controls. For all groups, n ≥ 4. (C) Maternal weight (left) and average pup weight (right) after LPS treatment were improved with SR treatment. For all groups, n ≥ 7. Data were analyzed using a one-way analysis of variance (ANOVA) with a Tukey’s post hoc test. (D) Images of pups from PBS, LPS, and LPS + SR at the time of collection on E16. (E to G) Fluorescence microscopy was used to colocalize mT expressing cells with the neutrophil marker Ly6G (green membrane fluorescence) in the fetal membranes of (E) PBS-injected, (F) LPS-injected, and (G) LPS + SR–injected mice. Scale bars, 10 μm. (H) Quantitation of colocalization for Ly6G+ neutrophils in the fetal membranes show significant increase of neutrophils in LPS-injected mice compared to PBS. Neutrophil infiltration was significantly reduced when treated with SR exosomes. For all groups, n ≥ 5. Data were analyzed using a one-way ANOVA with a Tukey’s post hoc test. Photo credit: Samantha Sheller-Miller, Talar Kechichian, and Phyllis Gamble, University of Texas Medical Branch at Galveston.

  • Fig. 3 Fetal macrophages are localized in maternal tissues.

    (A to C) Mɸ marker F4/80 (green) was colocalized with mT (red), indicating fetal Mɸ in the maternal cervix (A), uterus (B), and decidua (C). Arrows in three-dimensional (3D) model of colocalization indicate fetal macrophages (mT+ F4/80+).

  • Fig. 4 Flow cytometry analysis of fetal and maternal neutrophils and NK cells in fetal and maternal tissues.

    (A) Gating strategy used to identify maternal (mT) and fetal (mT+) neutrophils using Ly6G, NK cells using NK1.1, and DX5+ NK cells using NK1.1 and DX5. (B) Neutrophils in the placenta. mT/Ly6G+ cells in the placenta did not change, regardless of treatment. Total Ly6G+ and mT+/Ly6G+ specific neutrophils in the placenta were significantly increased in LPS-injected mice compared to PBS- and LPS + SR–injected mice. (C) Neutrophils in the fetal membranes. Total and mT+/Ly6G+ cells were significantly increased in LPS-injected mice compared to PBS- and LPS + SR–injected mice. mT/Ly6G+ cells did not change, regardless of treatment. (D) NK1.1+ and NK1.1+/DX5+ cells in the cervix. Total NK1.1+, mT+/NK1.1+, and total NK1.1+/DX5 cells increased in LPS compared to PBS- and LPS + SR–injected mice. mT+/NK1.1+/DX5+ cells decreased in LPS + SR– compared to LPS-injected mice. No changes were seen in mT cells, regardless of treatment. (E) NK1.1+ and NK1.1+/DX5+ cells in the placenta. Total NK1.1+, mT+/NK1.1+, total NK1.1+/DX5, and mT+/NK1.1+/DX5+ cells increased in LPS compared to PBS- and LPS + SR–injected mice. No changes were seen in mT cells, regardless of treatment. (F) NK1.1+ and NK1.1+/DX5+ cells in the fetal membranes. Total NK1.1+, mT+/NK1.1+, total NK1.1+/DX5, and mT+/NK1.1+/DX5+ cells increased in LPS compared to PBS- and LPS + SR–injected mice. No changes were seen in mT cells, regardless of treatment. Blue, PBS; red, LPS; green, LPS + SR. For all groups, n ≥ 4. Data are shown as means ± SEM. P values were calculated using a two-way ANOVA with a Tukey correction for multiple analyses. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

  • Fig. 5 SR reduces inflammation in maternal plasma.

    (A) Plasma IL-10 concentrations were significantly increased in all treatments compared to PBS mice. SR injection significantly increased plasma IL-10 concentrations compared to LPS alone. (B) Plasma IL-6 concentration was significantly decreased in PBS- and LPS + SR–injected compared to LPS-injected mice. (C) The plasma IL-8 concentration was significantly decreased in PBS compared to LPS-injected mice. (D and E) Plasma TNF-α and IL-1β concentrations, while not significant, were reduced in all treatments compared to LPS. Blue, PBS; red, LPS; gray, LPS + naïve; green, LPS + SR. For all groups, n ≥ 4. Data are shown as means ± SEM. P values were calculated using one-way ANOVA with a Tukey’s post hoc test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

  • Fig. 6 LPS-induced immune cell infiltration seen in PTB in a mouse model is predominantly a fetal response and is reduced at the feto-maternal interface with SR exosome treatment.

    LPS: Comparison between LPS and PBS. Cervix: LPS-injected mice had increased total NK1.1+ and DX5+ NK cells and increased fetal NK1.1+ cells. Uterus: LPS-injected mice had increased total and maternal M1 and M2 Mɸ. Decidua: LPS-injected mice had increased total neutrophils and decreased total M1 and M2 Mɸ, as well as decreased maternal M2 Mɸ. Placenta: LPS-injected mice had increased total neutrophils, NK cells, and DX5+ NK cells. In addition, fetal neutrophils, NK cells, and DX5+ NK cells were also increased in LPS-injected mice. Fetal membranes (FM): LPS-injected mice had increased total and fetal neutrophils, increased total and fetal NK cells, and increased total and fetal DX5+ NK cells. Arrows indicate increase or decrease in number of cells. *P = 0.07. Comparison between LPS and LPS + SR: Cervix: LPS + SR had decreased total and fetal NK cells and DX5+ NK cells. Uterus: LPS + SR had decreased total M1 and M2 Mɸ and decreased maternal M2 Mɸ. Decidua: No changes were seen in the decidua with SR treatment. Placenta: LPS + SR had decreased total DX5+ NK cells, as well as decreased fetal NK cells and DX5+ NK cells. Fetal membranes: LPS + SR–injected mice had decreased total and fetal neutrophils, decreased total and fetal NK cells, and decreased total and fetal DX5+ NK cells. Arrows indicate increase or decrease in number of cells. **P = 0.06.

  • Fig. 7 EXPLOR technology.

    (Top) Schematic of DNA constructs used to produce the SR IκB–loaded exosome. (Bottom) Schematic showing fusion proteins and their proposed activities of light-dependent protein-protein interactions to create SR-containing exosomes.

Supplementary Materials

  • Supplementary Materials

    Exosomal delivery of NF-κB inhibitor delays LPS-induced preterm birth and modulates fetal immune cell profile in mouse models

    Samantha Sheller-Miller, Enkhtuya Radnaa, Jae-Kwang Yoo, Eunsoo Kim, Kyungsun Choi, Youngeun Kim, Yu Na Kim, Lauren Richardson, Chulhee Choi, Ramkumar Menon

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