Exosome-based delivery of super-repressor IκBα relieves sepsis-associated organ damage and mortality

Abstract

As extracellular vesicles that play an active role in intercellular communication by transferring cellular materials to recipient cells, exosomes offer great potential as a natural therapeutic drug delivery vehicle. The inflammatory responses in various disease models can be attenuated through introduction of super-repressor IκB (srIκB), which is the dominant active form of IκBα and can inhibit translocation of nuclear factor κB into the nucleus. An optogenetically engineered exosome system (EXPLOR) that we previously developed was implemented for loading a large amount of srIκB into exosomes. We showed that intraperitoneal injection of purified srIκB-loaded exosomes (Exo-srIκBs) attenuates mortality and systemic inflammation in septic mouse models. In a biodistribution study, Exo-srIκBs were observed mainly in the neutrophils, and in monocytes to a lesser extent, in the spleens and livers of mice. Moreover, we found that Exo-srIκB alleviates inflammatory responses in monocytic THP-1 cells and human umbilical vein endothelial cells.

Fig. 1. Generation and characterization of engineered exosomes.

(A) Schematic of DNA constructs used for the production of Exo-srIκB (top). Schematic showing fusion proteins and their proposed activities (bottom). (B) Morphological characterization of Exo-Naïve and Exo-srIκB through transmission electron microscopy. (C) HEK293T cells that stably express mCherry or srIκB, and exosomes from these HEK293T cells, were lysed and immunoblotted against the indicated proteins. IB, immunoblot.

Fig. 2. The protective effects of Exo-srIκB in endotoxemia and CLP-induced sepsis.

(A) Survival curves of phosphate-buffered saline (PBS)–, Exo-Naïve–, and Exo-srIκB–treated septic mice. LPS C57BL/6 mice (n = 5 to 6 per group), LPS BALB/c mice (n = 10 per group), and CLP C57BL/6 mice (n = 14 to 15 per group). **P < 0.01 and *P < 0.05 compared with the PBS-treated sepsis group. (B) Levels of TNF-α, IL-6, IL-1β, and CCL4/macrophage inflammatory protein-1β in the plasma of exosome-treated mice were measured 24 hours after LPS injection or CLP. **P < 0.01 and *P < 0.05 compared with the PBS-treated sepsis group. †P < 0.05 compared with the Exo-Naïve–treated sepsis group. (C) Representative images of cortical tubular cells in kidney sections from sham, CLP with PBS, CLP with Exo-Naïve, and CLP with Exo-srIκB mice. Normal brush border (*) of proximal tubules or loss of brush border (○), chromatin condensation (white arrows), denuded basement membrane (white arrow heads), and vacuolization (yellow arrows). Scale bars, 100 μM. (D) Pathological kidney injury scores of representative kidney samples of each group. *P < 0.05 compared with the PBS-treated sepsis group. †P < 0.05 compared with the Exo-Naïve–treated sepsis group. GFP, green fluorescent protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Fig. 3. The biodistribution of exosomes in LPS-injected mice.

(A) Intravital imaging of mCLING-labeled exosome (red) uptake into neutrophils (LysM^gfp/+, green; Ly6G⁺, blue) in the liver of sham mice. (B) Representative time-lapse imaging of mCLING-labeled exosomes (red) inside the Ly6G⁺ neutrophil cells (green) of the liver in LPS-treated C57BL/6 mice. (C) Sequential images of flowing mCLING-labeled exosomes (red) inside the spleen of sham- and LPS-treated LysM^gfp/+ mice. Elapsed time is indicated. Magenta, autofluorescence. Scale bars, 50 μm.

Fig. 4. Inhibitory effect of Exo-srIκB on NF-κB signaling in vitro.

(A) HEK293–NF-κB–luciferase cells (2 × 10⁴ cells) were cultured either with Exo-Naïve or with Exo-srIκB (2 × 10⁵ particles). After 24 hours, the cells were treated with TNF-α (0.5 ng/ml) for an additional 18 hours. Luciferase activities were measured and normalized. (B) Exo-srIκB dose-dependently repressed NF-κB activation in NF-κB–luciferase cells. (C) THP-1 cells (5 × 10⁵ cells) were stimulated with LPS (1 μg/ml) and then treated with Exo-srIκBs (5 × 10⁶ particles). The supernatants were collected and assayed for the production of TNF-α and MCP-1. JSH-23 (50 μM) was used as the positive control. **P < 0.01. (D) Immunofluorescence of HUVECs incubated with mCLING-labeled Exo-srIκB. Representative images are shown. Nuclei were labeled with Hoechst. (E) HUVECs were stimulated with LPS (300 ng/ml) for 24 hours. Cells were harvested into a single-cell suspension and assessed through flow cytometry using specific phycoerythrin (PE)–conjugated antibodies against human ICAM-1. DMSO, dimethyl sulfoxide. *P < 0.05; **P < 0.01; ***P < 0.001.