Exosomes from adipose-derived mesenchymal stem cells alleviate sepsis-induced lung injury in mice by inhibiting the secretion of IL-27 in macrophages

Abstract

Acute lung injury (ALI) represents a frequent sepsis-induced inflammatory disorder. Mesenchymal stromal cells (MSCs) elicit anti-inflammatory effects in sepsis. This study investigated the mechanism of exosomes from adipose-derived MSCs (ADMSCs) in sepsis-induced ALI. The IL-27r^-/- (WSX-1 knockout) or wild-type mouse model of sepsis was established by cecal ligation and puncture (CLP). The model mice and lipopolysaccharide (LPS)-induced macrophages were treated with ADMSC-exosomes. The content of Dil-labeled exosomes in pulmonary macrophages, macrophages CD68⁺ F4/80⁺ in whole lung tissues, and IL-27 content in macrophages were detected. The mRNA expression and protein level of IL27 subunits P28 and EBI3 in lung tissue and the levels of IL-6, TNF-α, and IL-1β were measured. The pulmonary edema, tissue injury, and pulmonary vascular leakage were measured. In vitro, macrophages internalized ADMSC-exosomes, and ADMSC-exosomes inhibited IL-27 secretion in LPS-induced macrophages. In vivo, IL-27 knockout attenuated CLP-induced ALI. ADMSC-exosomes suppressed macrophage aggregation in lung tissues and inhibited IL-27 secretion. ADMSC-exosomes decreased the contents of IL-6, TNF-α, and IL-1β, reduced pulmonary edema and pulmonary vascular leakage, and improved the survival rate of mice. Injection of recombinant IL-27 reversed the protective effect of ADMSC-exosomes on sepsis mice. Collectively, ADMSC-exosomes inhibited IL-27 secretion in macrophages and alleviated sepsis-induced ALI in mice.

Fig. 1. IL-27 was highly expressed in mice with CLP-induced acute lung injury.

A The level of serum IL-27 of mice in the sham group and CLP group was detected using ELISA kit. B The mRNA expressions of EBI3 and P28 in mouse lung tissue were detected using RT-qPCR. C The protein levels of EBI3 and P28 in mouse lung tissue were detected using Western blot. D The mRNA expressions of IL-6, TNF-α, and IL-1β in mouse lung tissue were detected using RT-qPCR. E The protein contents of IL-6, TNF-α and IL-1β in lung tissues of mice in each group were detected using ELISA. F The lung injury was detected using HE staining, scale bar = 50 μm. G The wet/dry ratio was used to calculate the formation of pulmonary edema, N = 8. H: 24 h after CLP, Evans blue dye was injected to measure pulmonary vascular leakage, N = 8. Measurement data are depicted as mean ± SD. The t test was used for the comparisons between two groups. One-way ANOVA was employed for the comparisons among multiple groups, followed by Tukey’s multiple comparisons test. *p < 0.05 vs. Sham group; #p < 0.05 vs. CLP group.

Fig. 2. Isolation and identification of ADMSCs and ADMSC-exosomes.

A The morphological characteristics of primary ADMSCs were observed under the inverted microscope. B Adipogenic (left), osteogenic (middle), and chondrogenic (right) differentiation experiments of ADMSCs. C The surface markers of ADMSCs were detected using flow cytometry. D The diameter of ADMSC-exosomes was observed under the TEM. E The diameter distribution and concentration of exosomes were detected using NTA. F The exosome marker proteins (Alix, CD9, and TSG101) and endoplasmic reticulum protein (GRP94) were detected using Western blot. The cell experiment was repeated three times independently.

Fig. 3. ADMSC-exosomes inhibited the LPS-mediated release of IL-27 in macrophages.

A F4/80⁺ macrophages in lung tissue were detected using immunofluorescence (green staining) (nuclei were labeled by DAPI and presented blue staining). B The level of IL-27 in the supernatant of BMDMs treated with different concentrations of LPS for 12 h or LPS (100 ng/mL) for different times was detected using ELISA. C PKH67 (green)-labeled ADMSC-exosomes were co-cultured with BMDMs for 24 h, and then the fluorescence intensity of PKH67 in BMDMs was observed (DAPI-labeled nuclei were blue). D The time process of IL-27 release from BMDMs was detected using ELISA after the incubation of LPS (100 ng/mL) and ADMSC-exosomes (10 μg/μL). E The dose response of ADMSC-exosomes at different concentrations to IL-27 released by LPS-activated BMDMs within 12 h was detected using ELISA; Control indicated that the BMDMs were at rest. F BMDMs after LPS incubation were detected, with the addition of ADMSC-exosomes for 12 h or not. G The red staining of IL-27 in BMDMs was detected after 12 h-incubation, and Isotype represented the negative control. The cell experiment was repeated 3 times independently. Measurement data are depicted as mean ± SD. One-way ANOVA was employed for the comparisons among multiple groups, followed by Tukey’s multiple comparisons test. *p < 0.05.

Fig. 4. ADMSC-exosomes reduced the number of pulmonary macrophages and the release of IL-27 in sepsis mice.

A ADMSC-exosomes were isolated, purified and labeled with Dil dye; flow chart of injection in CLP sepsis mice. B The co-localization of exosomes (Dil pre-labeled red) and macrophages (F4/80, green) in lung tissue was detected using immunofluorescence staining, scale bar = 25 μm. C The number of macrophages CD68⁺ F4/80 ⁺ in single-cell suspension of whole lung tissue was detected using flow cytometry. D The content of IL-27 in serum of CLP mice treated with ADMSC-exosomes was detected using ELISA. E The mRNA expressions of P28 and EB13 in lung tissue of CLP mice treated with ADMSC-exosomes were detected using RT-qPCR. F The protein levels of P28 and EB13 in lung tissue of CLP mice treated with ADMSC-exosomes were detected using Western blot. G The content of IL-27 in macrophages after the treatment of ADMSC-exosomes or not detected using immunofluorescence. Blue fluorescence indicated DAPI, green fluorescence indicated F4/80, and red fluorescence indicated IL-27. N = 8. Measurement data are depicted as mean ± SD. One-way ANOVA was employed for the comparisons among multiple groups, followed by Tukey’s multiple comparisons test. *p < 0.05 vs. Sham group; #p < 0.05 vs. CLP group.

Fig. 5. ADMSC-exosomes alleviated sepsis-induced lung injury by mediating the secretion of IL-17.

A The survival rate of mice within 7 days (sham group: N = 10; other groups: N = 15). B The mRNA expressions of IL-6, TNF-α, and IL-1β in mouse lung tissue were detected using RT-qPCR 24 h after CLP induction, N = 8. C The protein contents of IL-6, TNF-α and IL-1β in mouse lung tissues were detected using ELISA. D The wet/dry ratio was used to calculate the formation of pulmonary edema, N = 8. E 24 h after CLP, Evans blue dye was injected to measure pulmonary vascular leakage, N = 8. F The morphological changes of lung were measured (scale bar = 25 μm) and the tissue injury was quantitatively analyzed (N = 8) using HE staining. Measurement data are depicted as mean ± SD. One-way ANOVA was employed for the comparisons among multiple groups, followed by Tukey’s multiple comparisons test. *p < 0.05 vs. CLP + PBS group; #p < 0.05 vs. CLP + Exo group. Kaplan-Meier method was used to calculate the survival rate. Log-rank test was used for univariate analysis.

Fig. 6. Mechanism diagram.

ADMSC-exosomes inhibited IL-27 secretion in macrophages, thereby delaying sepsis-induced lung injury in mice.