Extracellular vesicle-encapsulated IL-10 as novel nanotherapeutics against ischemic AKI

Abstract

Recently, extracellular vesicles (EVs) have been attracting strong research interest for use as natural drug delivery systems. We report an approach to manufacturing interleukin-10 (IL-10)-loaded EVs (IL-10⁺ EVs) by engineering macrophages for treating ischemic acute kidney injury (AKI). Delivery of IL-10 via EVs enhanced not only the stability of IL-10, but also its targeting to the kidney due to the adhesive components on the EV surface. Treatment with IL-10⁺ EVs significantly ameliorated renal tubular injury and inflammation caused by ischemia/reperfusion injury, and potently prevented the transition to chronic kidney disease. Mechanistically, IL-10⁺ EVs targeted tubular epithelial cells, and suppressed mammalian target of rapamycin signaling, thereby promoting mitophagy to maintain mitochondrial fitness. Moreover, IL-10⁺ EVs efficiently drove M2 macrophage polarization by targeting macrophages in the tubulointerstitium. Our study demonstrates that EVs can serve as a promising delivery platform to manipulate IL-10 for the effective treatment of ischemic AKI.

Fig. 1. Preparation and characterization of the IL-10⁺ EVs.

(A) RAW cells were transfected with RFP-tagged IL-10 and then were stimulated with dexamethasone. The intracellular distribution of IL-10 was examined by immunostaining against RFP. The right box is a heatmap visualization of the boxed region. Scale bar, 10 μm. (B) Immunostaining of IL-10 (green) and the EV marker CD63 (red) in engineered RAW cells. The yellow box is a higher magnification of the boxed region in the merged image. Scale bar, 10 μm. (C) Western blotting analysis of EV-associated (Alix, CD63, and CD81) and macrophage-associated markers (CD68 and CD206) in IL-10⁺ EVs. (D) Size distribution and representative TEM images of IL-10⁺ EVs. (E) A heatmap showing the protein levels (normalized to array reference) of each cytokine obtained from antibody array analysis. M0 EVs, EVs from untreated RAW cells. G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN-γ, interferon-γ; M-CSF, macrophage colony-stimulating factor; TNF-α, tumor necrosis factor–α. (F) ELISA analysis of IL-10 in EVs, and Western blotting analysis of IL-10 and IL-10 receptor in IL-10⁺ EVs (n = 3 or 6). (G) IL-10 mRNA in IL-10⁺ EVs was measured by real-time quantitative PCR (n = 3). (H and I) Comparison of the stability of IL-10⁺ EVs and free IL-10 under different conditions, including put in 37° or − 80°C for a week, suspended in pH 5.5 solution for 12 hours. Then, IL-10 concentration was detected by ELISA analysis at different time points (n = 3). Data are presented as means ± SD. **P < 0.01 and ***P < 0.001, two-tailed t test (F, G, and I) and one-way analysis of variance (ANOVA) (H). BLC, B lymphocyte chemoattractant; IP-10, interferon gamma induced protein 10; I-TAC, interferon-inducible T-cell alpha chemoattractant; KC, C-X-C motif chemokine 1; JE, monocyte chemoattractant protein-1; MCP-5, monocyte chemoattractant protein-5; MIG, monokine induced by interferon-gamma; MIP-1a, macrophage inflammatory protein 1α; RANTES, regulated on activation, T-cell expressed, and secreted; SDF-1, stromal cell-derived factor 1; TARC, thymus and activation regulated chemokine; TIMP-1, tissue inhibitor of metalloproteinases 1; TREM-1, triggering receptor expressed on monocytes 1.

Fig. 2. Kidney targeting of IL-10⁺ EVs.

(A) LC-MS/MS analysis of the protein composition of IL-10⁺ EVs, with the parental RAW cells as the control. Integrin expression identified in IL-10⁺ EVs and parental cells was shown. (B) Western blotting analysis of ITGα₄, ITGα₅, ITGα_L, ITGα_M, ITGβ₁, and ITGβ₂ on IL-10⁺ EVs. (C to E) For in vivo distribution, mice were injected intravenously with DID-labeled IL-10⁺ EVs (n = 3). (C) Imaging of fluorescence intensity of indicated organs at 6, 12, 24, 48, and 96 hours after injection (35 min ischemic time). (D) Imaging of fluorescence intensity at 12 hours in IRI kidneys of 20-, 28-, and 35-min ischemic time. (E) Representative confocal images showing the accumulation of DID-labeled IL-10⁺ EVs in tubules including proximal tubule [aquaporin 1 (AQP1)] and distal tubule [NaCl cotransporter (NCC)], endothelia cells (CD31), and macrophages (CD68) in frozen kidney sections. DT, distal tubule; PT, proximal tubule. Scale bars, 10 μm. (F and G) Cellular uptake of PKH67-labeled IL-10⁺ EVs in H/R-induced TECs (n = 3). (F) Representative confocal images and quantification of PKH67-labeled IL-10⁺ EVs in each cell after 12 hours of incubation. Immunostaining of TNF was used to indicate the cellular inflammation. Scale bar, 10 μm. (G) Flow cytometry analysis of the CD54⁺PKH67⁺ cells. PE, phycoerythrin. Data are presented as means ± SD. #P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t test (C, E, and F) and one-way ANOVA (D and G).

Fig. 3. IL-10⁺ EVs protect against renal I/R injury in mice.

(A) Schematic diagram of the experimental design. Briefly, mice were concurrently treated with IL-10⁺ EVs (100 and 200 μg) or vehicle every 24 hours after renal I/R injury and were euthanized at 3 days after disease induction. (B) Effects of IL-10⁺ EVs on serum creatinine (n = 10). (C) The quantification of tubular injury based on H&E staining (n = 10). (D) Representative images of H&E staining of renal cortex and medulla. Scale bars, 100 μm. (E and G) Representative images of TUNEL staining and quantification of the apoptotic cells (n = 6). Scale bars, 50 μm. HFP, High power field; (F and H) Representative confocal images and quantification of KIM-1⁺ tubules (n = 6). Scale bars, 20 μm. (I) Western blotting analysis of caspase-3 in kidney tissues (n = 3). HPF, high power field. (J) real-time quantitative PCR analysis of inflammatory cytokine mRNA levels in kidney tissues (n = 6). Data are presented as means ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 versus IRI group, #P < 0.05, ##P < 0.01, and ###P < 0.001, one-way ANOVA.

Fig. 4. IL-10⁺ EVs inhibit mTOR signaling and induce mitophagy to maintain mitochondrial fitness.

(A) Western blotting analysis of mTOR signaling and LC3 in kidney tissues (n = 3). (B) Representative TEM images of autophagic events in renal tubules. The number of autophagosomes and autolysosomes in each cell were quantified (n = 3). Red arrowheads, autophagy; green arrowheads, mitophagy. Scale bars, 2 μm. (C to E) Assessment of the effects of IL-10⁺ EVs on mitochondrial function in cultured TECs (n = 3). (C) Real-time changes in the OCR of TECs were detected by Seahorse assay. OCR, oxygen consumption rate; BR, basal respiration; MRC, maximal respiratory capacity; N.S., not significant. (D) Flow cytometry analysis of the mitochondrial ROS production in TECs labeled with MitoSOX. (E) Flow cytometry analysis of the mitochondrial membrane potential in TECs labeled with JC-1. (F) Representative TEM images of mitochondria in renal tubules. Scale bars, 1 μm. (G) Immunohistochemical analysis of cytochrome c oxidase subunit I (MT-CO1) expression in kidney sections. Scale bars, 100 μm. (H) Mitochondrial respiratory chain complex I enzymatic activity (n = 6). Data are presented as means ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 versus IRI group or HR group, one-way ANOVA.

Fig. 5. IL-10⁺ EVs induce a shift in renal macrophages.

(A and B) BMDMs were stimulated with LPS (100 ng/ml), IL-4 (20 ng/ml) + IL-13 (20 ng/ml), or different doses of IL-10⁺ EVs (5, 15, and 30 μg) for 48 hours, respectively (n = 3). (A) Expression analysis of CD86 and CD206 by flow cytometry. (B) Dose-dependent effects of IL-10⁺ EVs on CD206 expression. (C to E) Exploration of the phenotype changes of renal macrophages between IRI group and IL-10⁺ EV group. (C) Flow cytometry analysis of F4/80⁺CD86⁺ or F4/80⁺CD206⁺ macrophages in the kidneys (n = 3). (D) Representative confocal images of CD68⁺CD86⁺ or CD68⁺CD206⁺ macrophages in kidney sections (n = 5). Scale bars, 10 μm. The shift of macrophages was indicated by CD206⁺/CD86⁺. (E) real-time quantitative PCR measuring M1-like (TNF and iNOS) and M2-like (CD206 and Arg-1) markers in macrophages isolated from the kidneys (n = 3). Data are presented as means ± SD. *P < 0.05 and ***P < 0.001 versus IRI group, one-way ANOVA (B), two-tailed t test (C to E).

Fig. 6. IL-10⁺ EVs attenuate AKI-CKD transition.

A 35-min unilateral IRI was adopted, and IL-10⁺ EVs (200 μg) were administered intravenously after reperfusion and continued every 24 hours, three times in total. Mice were euthanized at 4 weeks after disease induction. (A) Representative images of PAS staining (n = 6). The bottom was a higher magnification of the boxed region. Scale bars, 500 μm (top) and 200 μm (bottom). (B) Representative images of Masson trichrome staining on sagittal sections (n = 6). The bottom was a higher magnification of the boxed region. Scale bars, 1 mm (top) and 200 μm (bottom). (C) Western blotting analysis of collagen I (Col. I) and α–smooth muscle actin (α-SMA) in kidney tissues (n = 4). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (D) Representative confocal images of CD68⁺CD86⁺ or CD68⁺CD206⁺ macrophages in kidney sections (n = 6). Scale bars, 10 μm. (E) Schematic illustration of the preparation of IL-10⁺ EVs for the treatment of ischemic AKI. Data are presented as means ± SD. ***P < 0.001 versus IRI group, one-way ANOVA (A to C) and two-tailed t test (D).