Intestinal Epithelium-Derived Luminally Released Extracellular Vesicles in Sepsis Exhibit the Ability to Suppress TNF-a and IL-17A Expression in Mucosal Inflammation

Abstract

Sepsis is a systemic inflammatory disorder induced by a dysregulated immune response to infection resulting in dysfunction of multiple critical organs, including the intestines. Previous studies have reported contrasting results regarding the abilities of exosomes circulating in the blood of sepsis mice and patients to either promote or suppress inflammation. Little is known about how the gut epithelial cell-derived exosomes released in the intestinal luminal space during sepsis affect mucosal inflammation. To study this question, we isolated extracellular vesicles (EVs) from intestinal lavage of septic mice. The EVs expressed typical exosomal (CD63 and CD9) and epithelial (EpCAM) markers, which were further increased by sepsis. Moreover, septic-EV injection into inflamed gut induced a significant reduction in the messaging of pro-inflammatory cytokines TNF-a and IL-17A. MicroRNA (miRNA) profiling and reverse transcription and quantitative polymerase chain reaction (RT-qPCR) revealed a sepsis-induced exosomal increase in multiple miRNAs, which putatively target TNF-a and IL-17A. These results imply that intestinal epithelial cell (IEC)-derived luminal EVs carry miRNAs that mitigate pro-inflammatory responses. Taken together, our study proposes a novel mechanism by which IEC EVs released during sepsis transfer regulatory miRNAs to cells, possibly contributing to the amelioration of gut inflammation.

Keywords: IL-17A; TNF-a; extracellular vesicles; inflammation; intestinal epithelial cells; miRNAs; sepsis.

Figure 1

Characterization of EVs isolated from luminal washes in large intestines of control and septic mice. (A) Flow cytometry analysis of EVs isolated from luminal washes in large intestines of control and sepsis mice. EVs were isolated by differential UC and adsorbed on 4 μm poly-L-lysine microbeads overnight. Immobilized EVs (20 μg) were stained with indicated monoclonal antibodies (MAb) and subjected to flow cytometry to evaluate their expression. Representative histograms show changes in expression of indicated marker. Black line, isotype (C-EV); gray line, isotype (S-EV); blue line, MAb (C-EV); and red line, MAb (S-EV). (B) Representative NanoSight LM10 images showing sizes of EVs from control (top) and septic (down) mice. (C) Particle number of EVs as quantified by NTA. (D) Size distribution of gut- derived C-EVs (N = 15) and S-EVs (N = 16) mice groups as measured by dynamic light scattering (DLS) device. C-EV, control extracellular vesicle; and S-EV, sepsis extracellular vesicle.

Figure 2

RT-qPCR analysis for gene expression of TNF-α and IL-17A. Gut inflammation was induced in the mice by 2.5% DSS-containing drinking water for seven days. Direct injection of either vehicle or differential UC-isolated EVs into the lumen of the tied ileum was done on day 7 and the tissues were separated after 18 h for RNA extraction. Relative expression of genes to β-actin in ileal-loop tissues of mice was accessed. Lapa, laparotomy; PBS, phosphate-buffered saline (200 μL); C-EV, control EVs (50 μg/200 μL) injected; S-EV, sepsis EVs (50 μg/200 μL) injected into ileal space; and DSS, dextran sulfate sodium. Other abbreviations: TNF-α, tumor necrosis factor-α; IL-17A, interleukin-17A. N = 8–12. * p < 0.05.

Figure 3

miRNA profiling of gut luminal EVs. (A) Venn diagram of microRNA distribution in control and septic gut luminal EVs. (B) Pie chart of differential expression of gut luminal EV microRNAs based on fold-change (S-EV/C-EV) in miRNA RPKM values. (C) TNF-α- and IL-17A-targeting miRNAs among all the sepsis-upregulated miRNAs. The miRNAs upregulated during sepsis indicate those which showed more than a two-fold increase in RPKM values in S-EV compared with C-EV. All miRNAs were chosen based on a criteria of >1 RPKM value. The miRNAs detected only in S-EVs are in italic. S-EV, sepsis EVs; and C-EV, control EVs.

Figure 4

Sepsis EVs (S-EVs) enrich 12 chosen miRNAs expected to target TNF-α and IL-17A. (A) Among sepsis-augmented miRNAs in S-EVs, 12 miRNAs shown in Venn diagram were selected to further analyze their expressions. (B) RT-qPCR analysis for miRNA expressions in the EVs. These miRNAs were tested for their increased enrichment in S-EV. As a control, U6 was used to normalize miRNA levels. Total RNAs were isolated from EVs pooled from 5 mice in each group. At least four separate experiments were performed. Results are shown as the mean ± SEM. S-EV, sepsis EVs; and C-EV, control EVs. * p < 0.05; *** p < 0.001; **** p < 0.0001; and ns, not significant.

Figure 5

A proposed mechanism illustrating the dynamic role of septic IEC-derived luminal EVs in regulating gut inflammation. Sepsis induces gut-barrier dysfunction and epithelial injury. Sepsis-induced luminal release of EVs increases their epithelial trait. The luminal EVs possessing different miRNA expression levels relocate themselves to IECs in either a paracrine or autocrine fashion. Thus, the EVs secreted from IECs are thought to contribute to dampening pro-inflammatory responses in sepsis-damaged gut mucosa via the transfer of regulatory miRNAs.