Exosomal miR-216a-5p targeting TLR-4 alleviates liver ischemia-reperfusion injury by regulating M2 macrophage polarization and neutrophil inflammation

Abstract

Macrophages and neutrophil play a key role in the initiation and recovery of liver ischemia reperfusion injury (IRI) through transitions in the phenotype and induces inflammation, However, the mechanisms governing these damages have yet to be fully elucidated. Exosomes have emerged as an important mediator of cellular crosstalk in various physiological and pathological processes. This study explored the role of exosomal miRNA in macrophage polarization and liver IRI. Through high-throughput sequencing of small RNAs in exosomes, we identified the negative regulator miR-216a-5p in liver IRI. Mechanistically, miR-216a-5p skewed M2 macrophage polarization and inhibited neutrophil infiltration by targeting TLR4. In conclusion, we demonstrated that exosome-derived miR-216a-5p favors an anti-inflammatory environment by promoting the M2 polarization of macrophages and inhibiting the neutrophil inflammatory response by targeting the TLR4/NF-κB and PI3K/AKT signaling pathways, revealing the endogenous protective mechanism in liver IRI.

Introduction

During liver transplantation or hepatic resection, the liver inevitably suffers from ischemia reperfusion injury (IRI), which is a major risk factor for organ dysfunction and can result in acute and chronic rejection after transplantation, especially when using grafts from donors after circulatory death [1]. Alleviating liver IRI not only improves clinical outcomes but also expands the donor organ pool by using marginal liver grafts [2]. Despite its clinical importance, however, the mechanisms underlying liver IRI and effective prevention and treatment strategies remain unclear.

Liver IRI is a biphasic response that includes an acute phase of injury caused by oxidative stress from Kupffer cells and a later phase caused by an intense inflammatory response resulting from inflammatory cell infiltration and subsequent neutrophil-dependent liver parenchymal injury [3].

Kupffer cells, the major population of resident tissue macrophages in the body, play an important part in the innate immune response [4]. The activation of Kupffer cells and infiltration of bone marrow-derived macrophages are considered key events in the initiation and propagation of liver IRI, which has two modes: classical activation (M1) and alternative activation (M2) [5]. M1 macrophages, marked by TNFα, IL6, and iNOS, promote inflammation and tissue injury, whereas M2 macrophages, marked by CD206, IL10, and Arg1, resolve inflammation and promote tissue regeneration [4].

In hepatic IRI, multiple signaling mechanisms play key roles in the polarization of macrophages [6]. Our previous study found that the hepatocyte-derived exosomal miR-122-5p was able to induce Kupffer cell M1 polarization via PPARδ down-regulation and NF-κB pathway activation to mediate liver injury [7]. Macrophages switching phenotypes from M1 to M2 reflects the functional change from tissue injury to eventual repair/recovery [8]. However, the mechanisms governing this switch have yet to be fully elucidated.

During IRI, the liver undergoes an intense inflammatory process in which neutrophils play an important role, characterized by the initial activation of neutrophils by chemokines and their recruitment to the liver [9,10]. Neutrophils then induce the activation of resident hepatocytes, leukocytes, and Kupffer cells through the release of cytokines and chemokines and the production of danger signals [11,12]. Inhibition of neutrophil infiltration then protects hepatocytes from damage after IR.

Exosomes are tiny vesicles, 30–150 nm in diameter, secreted by cells [13]. Recent studies have shown that exosomes play an important role in mediating communication between cells, especially in liver diseases, for example, hepatocellular carcinoma cell-derived exosome miR-934 induces macrophage M2 polarization and promoted liver metastasis [14]. Lipotoxic hepatocyte-derived exosomal MicroRNA 192-5p activates macrophages associated inflammatory response in nonalcoholic fatty liver disease [15]. However, the role of exosomes in hepatic ischemia-reperfusion injury has not been clearly reported.

The present study therefore screened for the anti-inflammatory exosomal miR-216a-5p, and evaluated whether exosomal miR-216a-5p skewed M2 macrophage polarization and the neutrophil inflammatory response by targeting TLR4, which might suggest a promising strategy for protecting against IRI.

Methods and materials

Mouse and patient samples

Male C57Bl/6 mice (6–8 weeks old) were purchased from Shanghai Slac Laboratory Animal Co., Ltd, and housed under specific-pathogen-free conditions. The model of partial liver ischemia reperfusion injury was established as described previously [16]. The animals were randomly divided into four groups (n = 6 per group; total: 24 mice): Sham group (Sham), miR-216a-5p agomir group (agomir), miR-216a-5p antagomir group (antagomir), and miR-216a-5p negative control group (NC). miR-216a-5p agomir (20 nmol) or antagomir (200 nmol) was administrated by intravenous injection 1 h before the induction of liver IRI. The mice were sacrificed 24 h after reperfusion, and serum and liver tissues were collected for subsequent analyses. I confirm within the manuscript that we have followed ARRIVE guideline.

Human serum samples were obtained from five patients (Table 1) undergoing liver transplantation within the 6 h prior to transplantation and at 24h postoperatively with their written informed consent, these human serum samples were used to detect the level of exosome derived miR-216a-5p in Figure 1K.

Table 1.

The basic information of patients.

Number	Dignosis	Operation time (min)	MELD score	Blood loss (ml)	Bilirubin (μmol/L)	AST (u/l)	ALT (u/l)
Patient1	Cirrhosis	440	11	200	12.3	25	22
Patient2	Cirrhosis	505	16	500	51.6	55	52
Patient3	Cirrhosis	465	10	600	13.2	33	35
Patient4	Polycystic liver	510	14	500	56.1	14	18
Patient5	HCC	635	13	500	45.2	94	158

Figure 1.

miRNA Expression profile of serum-derived exosomes from mice with liver IRI. (A–B) The representative HE image of mice liver tissue in sham, IRI6h and IRI24h groups, demonstrating liver injury in the sham group and the ischaemia-reperfusion 6h and 24h groups. (C) Representative transmission electron microscopy photographs. (D) Analyzing the exosome particle size by a nanoparticle tracking analysis (NTA). (E) Exosomal protein markers CD9 and CD63 detected by Western blotting. (F) Volcano plot and (G) heat map showing the differentially expressed miRNAs in exosomes. (H) Functional enrichment of miR-216a-5p downstream target genes. (I) Levels of ALT in serum of mice in sham, IRI6h and IRI24h groups. Expression of miR-216a-5p in serum exosomes of mice suffering from (J) mice liver IRI model and (K) liver transplant patients by quantitative PCR.

Ethical approval statement

This study has obtained relevant ethical approval. For the part involving human subjects, the study has been approved by the Medical Ethics Committee of Liaocheng People’s Hospital (Approval number: 2018012), and written informed consent was obtained for each participant according toscientific ethical institutional guidelines. Participant’s information is be presented in Table 1.

For the part involving animal experiments, this study has obtained approval from the Animal Experiment Ethics Committee of the Experimental Animal Center of Taizhou Enze Medical Center (Approval number: tzy-2022016). Throughout the animal experimentation process, we will strictly adhere to animal welfare and ethical regulations, minimizing the pain and discomfort of the animals to the greatest extent possible, and ensuring the reliability and scientific validity of the experimental results.

Exosome extraction and identification

Serum samples collected from liver transplant patients and mice were used to purify exosomes thought ultracentrifugation [17]. Serum was centrifuged at 2,000 and 13,500 g for 20 min to eliminate cell debris and large vesicles. Pellets were then washed once with phosphate-buffered saline (PBS) and purified by centrifugation at 200,000 g for 2 h. Exosome samples were diluted 5-fold with PBS, stained with 2% phosphotungstic acid at room temperature, and examined by transmission electron microscopy (Hitachi, HT7700, Japan). The size and concentration of exosomes were measured by a nanoparticle tracking analysis (NTA) at 80 kV using a ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany).

Cell isolation, extraction, and culture

Bone marrow macrophages (BMMs) isolated from femurs and tibias of C57BL/6 mice (8–9 weeks old) were cultured for 6 days in a medium containing 10% FBS, penicillin (100 U/ml), streptomycin (100 μg/ml), and 10 ng/mL mouse M-CSF (Peprotech-In, Suzhou, China). All cells were cultured in a humidified incubator at 37 °C with 5% CO₂, and the medium was changed every 2 days. The human promyelocytic leukemia cell line HL-60 was cultured in RPMI medium (Invitrogen, Carlsbad, CA, USA) containing 10% FBS, 2 mM L-glutamine, and 1% penicillin and streptomycin. Dimethyl sulfoxide at 12.7 µL/ml/million cells was added for 5 days to induce HL-60 cells to differentiate into neutrophil like cells (dHL-60) [18].

Western blotting

Pretreated liver tissue and cells were added to ice-cold RIPA lysis buffer (Beyotime, Shanghai, China) with 1 mM PMSF. Protein concentrations were then determined with a BCA assay kit. Approximately 20 μg of protein samples was loaded onto 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to PVDF membranes (Millipore, Massachusetts, USA). After blocking with 5% skimmed milk for 2 h, the membranes were incubated at 4 °C with INOS (1:1000; Abcam, Waltham, USA), GAPDH (1:5000; Bioss, Suzhou, China), CD206 (1:1000; Proteintech, Wuhan, China), p-Akt (1:1000; Proteintech), p-PI3K (1:1000; Servicebio, Wuhan, China), P-p65 (1:1000; Cell Signaling, Massachusetts, USA), and TLR-4 (1:1000; Proteintech), followed by 3 washes in tris buffered saline with tween20 (TBST) and incubation with the corresponding secondary antibodies for 2 h at 4 °C. Specific proteins were visualized by applying the ECL kit (a Chemiluminescent Substrate for improving bright signals;Beyotime, Shanghai, China) to specific protein bands.

Polymerase chain reaction (PCR)

Total RNA was extracted from cells or liver tissue using the RNAiso plus reagent (Takara, Kyoto, Japan). miRNA was extracted from serum and exosomes using the miRNeasy Mini Kit (TIANGEN, Beijing, China). cDNA was then synthesized using the PrimeScript RT kit. Quantitative real-time PCR was performed using a 7300plus (Applied Biosystems, California, USA) real-time PCR instrument. GAPDH (mRNA) and miRNA external reference (TIANGEN) (miRNA in serum and exosomes), U6 (miRNA in tissues) were used to normalize the expression of target genes calculated relative to normal controls. All primer sequences are listed in Supplementary Table S1.

Sequencing and enrichment analyses

Serum exosomes were isolated from sham control (n = 3) and IRI (n = 3) mice, and total RNA was extracted using the miRNeasy Micro Kit (217084; Qiagen, North Rhine-Westphalia, Germany). The miRNA sequencing analysis was performed on an Illumina HiSeq 2500 system (Ribo Bio, Guangzhou, China). Differentially expressed miRNAs between the two groups were identified by the fold change and P-value. Hierarchies were clustered to show samples with distinguishable miRNA expression profiles between samples. Metascape was used to identify the functional and interaction networks of the miR-216a-5p target genes (Table S2).

Immunofluorescence staining of liver tissue sections

According to a previous published method [19], Firstly, the samples were fixed in ice acetone precooled at 4 C for 10 min, and washed with PBS 3 times, and then permeated with 0.3% TX-100 for 20 min and washed with PBS for 3 times. The samples were blocked by 3% BSA for 1 h, and the slides were incubated overnight at 4 °C with primary antibodies (F4/80, LY6G, MPO; Abcam). The next day, the nuclei were stained with DAPI after incubation with the corresponding secondary antibodies conjugated with Cy3 or FITC (Servicebio, Wuhan, China). They were then observed with a confocal laser microscope.

Histology

Liver tissue was fixed in 4% paraformaldehyde and then embedded in paraffin wax, and paraffin sections were soaked in 100% xylene and a mixture of xylene and alcohol (1:1) for 15 min and dewaxed. After soaking in 100, 95, and 75% alcohol solutions for 1 min, the sections were washed with 0.1 saline. Hematoxylin/eosin (HE) staining was used to assess pathological liver damage according to Suzuki’s criteria [20].

Flow cytometry

Macrophage polarization was detected by flow cytometry. Macrophages were centrifuged and resuspended cells in cold PBS and then incubated with antibodies against anti-CD86-PE (#105106, BioLegend, California, USA), anti-CD206-APC(#141706, BioLegend, California, USA)and anti-F4/80-PerCP (#123116, BioLegend, California, USA) for fluorescent staining for 30 min at 4 °C. A FACS analysis was performed after washing, and data were obtained using a BD Accuri^™ C6 flow cytometer (BD Biosciences, San Jose, CA, USA).

The dual luciferase assay

The potential binding sites for miR-216a-5p and TLR-4 were predicted by TargetScan. To verify the direct binding of miR-216a-5p to TLR-4, miR-216a-5p and wild-type TLR-4 3′UTR or mutant TLR-4 3′UTR were reconstructed with pmirGLO plasmid in 293 T cells, using Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc. Massachusetts, USA) according to the manufacturer’s protocol. After 48 h, the luciferase activity was measured using a dual luciferase reporter assay system (Promega, Wisconsin, USA) according to the manufacturer’s protocol.

Statistical analyses

Group sizes are indicated in the figure legends. Data are presented as the mean ± standard deviation unless otherwise noted. Statistical analyses were performed by either an analysis of variance (ANOVA) or t-test. A difference of p < 0.05 was considered statistically significant.

Results

miRNA expression profiles of serum-derived exosomes from mice with liver IRI

Representative HE staining images of liver tissue sham, liver ischemia and reperfusion at 6h and 24h (Figure 1A). We could observe that after the liver suffers from ischemia and perfusion, different degrees of damage to the liver tissue occur (Figure 1B/I). To explore the miRNA expression profiles in serum-derived exosomes after reperfusion, we constructed a mice model of liver IRI and isolated serum exosomes, which were then analyzed by transmission electron microscopy, NTA and Western blotting. Transmission electron microscopy displayed a typical size and round cup-shaped morphology (Figure 1C). Furthermore, the mean particle size of exosomes was about 100 nm as determined by an NTA (Figure 1D) and exosome-specific markers CD9 and CD63 were easily detected by Western blotting (Figure 1E). To identify the miRNAs involved in liver IRI, we performed RNA-seq of exosomes and identified the miRNAs with the most significant abundance difference (>2-fold, p < 0.05) illustrated in a volcano map (Figure 1F); the top miRNAs are shown in the heat map in (Figure 1G). miRNAs regulate biological functions primarily through interference with downstream target genes. The target genes of the top 10 miRNAs were thus predicted by TargetScan. GO analyses was then performed, showing that the downstream genes of miR-216a-5p were primarily enriched in the inflammation relevant pathways that exacerbated liver IRI (Figure 1H). Based on this target regulation by miRNA, we hypothesized that miR-216a-5p might play a protective role during liver IRI and may be a potential target for alleviating liver IRI. Therefore, we selected it for further study.

To confirm the expression of miR-216a-5p in serum-derived exosomes, we performed qRT-PCR analysis with serum samples from mice and human. The results showed a change in miR-216a-5p level after reperfusion at different time points of perfusion (Figure 1J-K).

Role of miR-216a-5p in liver IRI

To investigate the functional role of miR-216a-5p in liver IRI, miR-216a-5p agomir were transfected before liver IRI. Tissue damage was then evaluated according to Suzuki’s criteria by HE staining of liver tissue sections (Figure 2A/B) and damage (Figure 2I). The results showed that miR-216a-5p agomir significantly improved liver injury. To determine the cell type responsible for the miR-216a-5p-mediated hepatoprotection, miR-216a-5p tracing was performed, resulting in the detection of internalization by Kupffer cells and neutrophils (Figure 2C/E), and PCR results demonstrated that miR-216a-5p agomir promoted the expression level of miR-216a-5p in liver (Figure 2G).

Figure 2.

miR-216a-5p Alleviated liver IRI by promoting kupffer cell polarization to the M2 type and inhibiting the neutrophil inflammatory response. (A-B) HE staining (A) and suzuki’s criteria (B) for mice liver sections after reperfusion. Immunofluorescence demonstrates macrophage (C) and neutrophil (E) uptake of miR-216a-5p (agomir); immunofluorescence staining of F4/80 together with CD206 in the miR-216a-5p agomir and NC groups (D) and immunofluorescence staining of LY6G together with MPO (F); expression levels of miR-216a-5p in mouse liver tissues after miR-216a-5p (agomir) intervention (G) and quantification (H) of macrophages and neutrophils after reperfusion in miR-216a-5p agomir and miR-216a-5p NC groups; the ALT level in serum of sham and miR-216a-5p (agomir) intervention groups (I); the mRNA levels of M1 (iNOS, TNF-α, IL-6) and M2 (Arg1, CD206) (J) and ELISA and Western blotting results of iNOS, CD206, and IL-6 (K-M) after injection with miR-216a-5p (agomir) and miRNA (NC).

As the largest immune cell population in the liver, Kupffer cells and infiltrating macrophages play an important role in the initiation of IRI in the liver. Macrophages can polarize to different phenotypes and perform different functions, we therefore explored whether or not miR-216a-5p (agomir) could polarize macrophages from the M1 to the M2 phenotype. We analyzed the characteristic polarization of macrophages by immunofluorescence staining with F4/80 and M2-associated CD206. We observed a significant decrease in the number of M2 macrophages after reperfusion compared to the Sham group. Interestingly, miR-216a-5p (agomir) increased the number of CD206-positive macrophages compared to the NC group (Figure 2D/H).

As a major player among circulating leukocytes, neutrophils are recruited to the site of injury, leading to sterile inflammation and amplifying hepatocellular injury. Consistent with previous studies [21], the number of LY6G + MPO+ neutrophils was significantly increased after reperfusion, and miR-216a-5p (agomir) decreased the number of LY6G + MPO-positive neutrophils compared to the NC group (Figure 2F/H). In addition, decreased levels of pro-inflammatory factors (iNOS, TNF-α, IL-6) and increasing anti-inflammatory factors (Arg1, CD206) were detected compared to the NC group in mRNA (Figure 2J), which was consistent with these inflammation factors in protiens level on Western blotting and ELISA results (Figure 2K-M).

These results indicate that miR-216a-5p alleviated liver IRI by inducing the polarization of macrophages to the M2 phenotype and inhibiting the neutrophil inflammation response.

Effects of blocking miR-216a-5p

Contrary to the above results, when mice were transfected with antagomir to block miR-216a-5p before suffering IRI, there was a significant increase in liver injury in mice, as demonstrated by HE staining (Figure 3A), Suzuki’s criteria (Figure 3B), and liver inflammation and damage (Figure 3I-M). miR-216a-5p(antagomir) was intaked by macrophages and neutrophils (Figure 3C/E) which demonstrated that miR-216a-5p antagomir suppressed the level of miR-216a-5p in liver (Figure 3G) and decreased the number of CD206-positive macrophages compared to the NC group (Figure 3D/H). Similarly, the number of LY6G⁺MPO⁺ neutrophils was markedly increased after being transfected with miR-216a-5p antagomir before liver IRI reperfusion (Figure 3F/H). These results suggest that miR-216a-5p was associated with a reduced neutrophil infiltration and inflammatory response. This may be another important mechanism involved in the protective effect of miR-216a-5p during liver IRI.

Figure 3.

Blocking miR-216a-5p inhibited kupffer cell polarization toward the M2 type and promoted the neutrophil inflammatory response. HE staining and suzuki’s criteria for the mouse liver miR-216a-5p antagomir and NC groups (A–B). Immunofluorescence demonstrates macrophage (C) and neutrophil (E) uptake of miR-216a-5p(agomir), Immunofluorescence staining and quantification of macrophages (D/H) in liver tissue after reperfusion in the miR-216a-5p antagomir and NC groups. Immunofluorescence staining of LY6G together with MPO and quantification (F/H) of neutrophils after reperfusion in the miR-216a-5p antagomir and NC groups; expression levels of miR-216a-5p in mouse liver tissues after miR-216a-5p (antagomir) injection (G); the ALT level in serum of sham and miR-216a-5p (antagomir) intervention groups (I); the mRNA levels of iNOS, TNF-α, IL-6, Arg1, and CD206 (J) and the protein (K–M) levels of iNOS and CD206 in mouse liver in the miR-216a-5p antagomir and NC groups.

Effects of miR-216a-5p on BMMs and dHL-60 inflammation

To further verify whether or not miR-216a-5p mediated the polarization of macrophages to the M2 type and regulated the neutrophil inflammatory response, we co-cultured miR-216a-5p (agomir)+LPS with BMMs and dHL-60. The level of miR-216a-5p was significantly elevated in BMMs and dHL-60 (Figure 4A). miR-216a-5p (agomir) treatment significantly attenuated the LPS-induced inflammatory response in BMMs and dHL-60 compared to the control group. Inflammatory markers (iNOS, TNF-α, IL-6) were downregulated, and M2 (Arg1, CD206) markers were increased at the protein levels (Figure 4B–E) and mRNA (Figure 4F–G). In addition, flow cytometric detection found that miR-216a-5p-agomir promoted the polarization of BMMs toward M2 and alleviated the polarization of BMMs cells toward M1 induced by LPS (Figure 4H). In addition, after we blocked the levels of miR-216a-5p in BMMs and dHL-60 with antagomir (Figure 4I), we found that pro-inflammatory factors (iNOS, TNF-α, IL-6, IL-1) were elevated, the expression of M2 markers (Arg1, CD206) was inhibited at the protein levels (Figure 4J–M) and mRNA (Figure 4N–O). These results suggest that miR-216a-5p promoted macrophage polarization to the M2 type and alleviated the neutrophil inflammatory response.

Figure 4.

miR-216a-5p Induced macrophages towards M2 polarization and inhibited the neutrophil inflammatory response in vitro. (A) Levels of miR-216a-5p in BMMs and dHL-60 after co-culture with miR-216a-5p agomir; protein levels of iNOS, CD206, and IL-6 detected by Western blotting (B–D) and ELISA (E) in BMMs and dHL-60 after co-culture with miR-216a-5p agomir. M1 markers (iNOS, TNF-α, IL-6) and M2 markers (Arg1, CD206) detected by quantitative PCR in BMMs (F) and dHL-60 (G) after co-culture with miR-216a-5p agomir. (H) Flow cytometry to detect macrophage polarization. (I) qPCR detected the expression level of miR-216a-5p, after transfection of miR-216a-5p (antagomir) in BMMs and dHL-60. iNOS and CD206 levels according to Western blotting (J-L) and ELISA (M) after treatment with miR-216a-5p antagomir and in the NC group. (N–O) mRNA levels of M1 markers (iNOS, TNF-α, IL-6) and M2 markers (Arg1, CD206) after treatment with miR-216a-5p antagomir and in the NC group.

Effects of miR-216a-5p on M2 macrophage polarization and the neutrophil inflammatory response through targeting TLR4

Next, we further investigated the mechanisms underlying the miR-216a-5p-regulated inflammatory response. TLR-4 was predicted to be a downstream target gene of miR-216a-5p from the TargetScan database (Figure 5A). To test whether or not miR-216a-5p could directly bind to TLR-4 and regulate its expression, we performed a dual luciferase gene reporter assay and showed that the activity of the luciferase reporter was significantly reduced by the overexpression of miR-216a-5p, whereas the activity of the 3’UTR-mutant luciferase reporter of TLR-4 was not affected (Figure 5B). This demonstrated that TLR-4 was a direct target gene of miR-216a-5p. The mRNA expression of TLR4 were simultaneously inhibited in BMMs and dHL-60 after the overexpression of miR-216a-5p (Figure 5C). Conversely, knockdown of miR-216a-5p in BMMs and dHL-60 by antagomir resulted in increased TLR-4 levels compared to the control at the mRNA level (Figure 5D). In addition, we also constructed siRNA against TLR-4 and transfected siTLR-4 into BMMs and HL-60 cells, and we could find that the mRNA and protein levels of TLR-4 were significantly inhibited (Figure 5E–G). As shown in (Figure 5H–I), knockdown of TLR4 suppressed the release of anti-inflammatory cytokines (INOS, IL-6, TNF-α and IL-1) and promoted the levels of CD206 and Arg-1. Subsequently, we found that the detrimental effects of antagomir were counteracted by knockdown of TLR4 in BMMs and dHL-60. TLR-4 knockdown reversed the pro-inflammatory effect of antagomir knockdown of miR-216a-5p on BMMs and dHL-60 (Figure 5J–O). These findings are consistent with our hypothesis and confirmed that exosomal miR-216a-5p regulates M2 macrophage polarization and the neutrophil inflammatory response by targeting TLR4.

Figure 5.

miR-216a-5p Exerted a biological function by targeting TLR-4. (A) Binding sites for TLR-4 and miR-216a-5p predicted by TargetScan. (B) Validation of direct binding of TLR-4 and miR-216a-5p in a dual luciferase assay. (C–D) Expression of TLR-4 in BMMs and dHL-60 after co-culture with miR-216a-5p agomir and antagomir. Effect of siTLR-4 on knockdown of TLR-4 protein (E-F) and mRNA (G) levels in macrophages and HL-60 cells. RNA levels of IL-6, INOS, TNF-α, IL-1, CD206, and Arg-1 after knocking down expression levels of TLR-4 in macrophages (H) and HL-60 cells (I). Rescue experiments for miR-216a-5p inhibition were conducted by downregulating TLR4 in BMMs (J–L) and dHL-60 according to Western blotting (M–O).

Effects of miR-216a-5p on M2 macrophage polarization and the neutrophil inflammatory response through the TLR4/NF-κB and PI3K/AKT signaling pathways

TLR-4 positively regulates the NF-κB signaling pathway [22–24], which is a critical transcription factor regulating the M1 polarization of macrophage and inducing the neutrophil inflammatory response. Furthermore, previous studies have shown that there is a mutual regulation of TLR4/NF-κB and PI3K/AKT signaling pathways in several kinds of IRI [25–27].

We therefore explored whether or not miR-216a-5p exerted regulatory effect on the crosstalk between the NF-κB and PI3K/AKT signaling pathways by targeting TLR-4 in macrophages and neutrophils when liver suffer from IRI. Western blotting revealed that the levels of TLR4 and p-P65 were significantly downregulated, while those of p-AKT was markedly upregulated following agomir treatment in BMMs (Figure 6A and B) and dHL-60 (Figure 6C and D). However, the effects of the administration of antagomir in BMMs (Figure 6E and F) and dHL-60 (Figure 6G and H) on the expression of TLR4, p-P65 and p-AKT were the opposite. This demonstrates that miR-216a-5p affects macrophage polarization and the inflammatory response through the regulation of the NF-κB and PI3K/AKT signaling pathways.

Figure 6.

miR-216a-5p Regulated BMMs toward M2 polarization and inhibited the dHL-60 inflammation response through TLR4/NF-κB and PI3K/AKT signaling pathways. Representative images and quantification of Western blotting findings for TLR4 and the downstream NF-κB and PI3K/AKT signaling pathways in BMMs (A-B) and dHL-60 (C–D) on administration of LPS, between miRNA (NC) and miRNA (agomir). Representative images and quantification of Western blotting findings for TLR4 and the downstream NF-κB and PI3K/AKT signaling pathways in BMMs (E–F) and dHL-60 (G–H) on administration of LPS, between miRNA (NC), and miRNA (antagomir).

Discussion

Hepatic IRI is a complex, multifactorial process involving multiple cells, such as liver sinusoidal endothelial cells, hepatocytes, Kupffer cells, neutrophils, and a complicated intracellular signaling network [1]. This process includes damage-associated molecular patterns (DAMPs) activating signals through Toll-like receptors (TLRs) and subsequently recruiting and activating innate immune cells [2]. It has been well recognized that Kupffer cells and infiltrating macrophages are key contributors to the initial inflammation and later reparative process following liver IRI [28]. In our previous study, we found that exosomes after reperfusion function as DAMPs, exacerbating liver injury by inducing Kupffer cell polarization toward the M1 type. We also identified miR-122-5p as a key molecule mediating the pro-inflammatory effect of exosomes [7].

Early M2 polarization in KCs/macrophages is essential for inflammation resolution and hepatic repair [29]. However, the mechanisms and molecular cues regulating the phenotypic transitions of macrophages remain poorly defined. If Kupffer cells trigger IRI in the liver, neutrophils represent the main actor in causing injury [2]. Accumulated neutrophils release oxidants and proteases, directly damaging hepatocytes and endothelial cells.

Upon inflammatory stimulation, the organism initiates a stress response to help prevent uncontrolled inflammation. Several anti-inflammatory mediators have been shown to be expressed after IR and to play key roles in the resolution of the injury response [30]. In our study, we presented the first evidence that significantly increased exosomal miR-216a-5p levels after liver IR act as a potent anti-inflammatory mediator to skew M2 macrophage polarization and suppressed the neutrophil inflammatory response, negatively affecting the function of multiple pro-inflammatory factors.

Emerging data have highlighted the importance of exosomes as mediators of cellular crosstalk in various physiological and pathological processes by shuttling their bioactive cargo [31]. Almost all types of cells are exosome-releasing and/or exosome-targeting cells, including macrophages, hepatocytes and other cells [32].

The quantity and composition of exosomes released by donor cells are often altered in pathophysiological states. In our previous, we found that the level of exosomes increased by about 5-fold compared to control mice as early as 6 h after reperfusion. Exosome adoptive transfer causes liver injury and interstitial inflammation, suggesting a pathological role for exosome in hepatic IRI [7]. However, exosomes can positively or negatively exert modulatory effects, depending on their origin and cargo. Indeed, emerging evidence has suggested the therapeutic effects of MSC-derived exosomes [33–35]. Furthermore, through high-throughput sequencing of small RNAs in exosomes, we screened for the increased expression of miR-216a-5p, in previous study miR-216a-5p alleviated H₂O₂/LPS induced human bronchial epithelial cell injury during asthma[36,37], and exosomal miR-216a-5p released from hypoxia-prime ASCs prevented intestinal mucosal epithelium from inflammatory factor-induced injury in colitis[38], consistented with gene enrichment analysis performed in current study which revealed the involvement of the target gene of TLR4 in the inflammatory pathways, implying the hepatoprotective effect of miR-216a-5p. By blocking miR-216a-5p, antagomir worsened liver IRI and inflammation, and this effect was reversed by agomir. Taken together, these data reveal an important role of miR-216a-5p in preventing uncontrolled inflammation.

To further investigate the mechanism by which miR-216a-5p attenuates hepatic liver injury and inflammation, Cy5-labeled miR-216a-5p was shown to be taken up by Kupffer cells and neutrophils, triggering M2 polarization, inhibiting the neutrophil inflammatory response and decreasing the inflammatory response. Similar results were also confirmed in the cell model. In agreement with previous reports [26,39], we also verified the target gene of TLR4 by a luciferase report analysis.

It has been proven that the TLR4/NF-κB signaling pathway can elicit M1 activation [40]. TLRs, especially TLR4, are a key sentinel system responsive to M1 activation and the neutrophil inflammatory response, triggering cascades of signals to activate the inflammatory response through crosstalk between NF-κB and PI3K/AKT with downstream genes encoding inflammatory cytokines [40]. Inhibiting TLR4/NF-κB and activating the PI3K/AKT signaling pathway have been shown to shift the polarization of macrophages to the M2 phenotype from the M1 phenotype and inhabit the neutrophil inflammatory response [41].

In microglia, knockdown of TLR4 reversed the detrimental effects of the inhibition of miR-216a-5p. In contrast, the overexpression of TLR4 abolished the beneficial effects of miR-216a-5p by shifting microglial polarization. Accordingly, we also found that miR-216a-5p agomir treatment resulted in a significantly inhabited TLR4 expression and PI3K/AKT, while activated NF-κB pathway, thus inducing M2 macrophage polarization.

In septic mice, overexpression of miR-let-7b significantly downregulated the neutrophil TLR4 expression at the post-transcriptional level, targeting the NF-kB pathway, which in turn inhibited the decrease in levels of pro-inflammatory factors (IL-6, IL-8, TNF-α) and suppressed neutrophil recruitment, thereby attenuating liver and lung injury in CLP-induced septic mice [42]. In our current study, miR-216a-5p significantly inhibited neutrophil infiltration, reflected by LY6G + MPO immunofluorescence staining, which suggests another mechanism is involved in hepatoprotection. However, in in vitro experiments, the overexpression of miR-216a-5p in dHL-60 cells alleviated the LPS-induced inflammatory response of dHL-60, as evidenced by the decrease in pro-inflammatory factors, such as IL-6, IL-1, TNF-α, and iNOS.

Therefore, it is interesting to hypothesize that miR-216a-5p might attenuate liver inflammation injury by inducing macrophages towards M2 polarization and inhabiting the neutrophil inflammatory response through the TLR4-NF-κB and PI3K/AKT pathways. Further investigations are warranted to explore the origins of the increased circulating levels of exosomal miR-216a-5p.

In conclusion, we demonstrated that exosome-derived miR-216a-5p favors an anti-inflammatory environment by promoting the M2 polarization of Kupffer cells and the neutrophil inflammatory response targeting the TLR-4, NF-κB, and PI3K/AKT signaling pathways, thus clarifying the endogenous protective mechanism involved in liver IRI. Given these findings, miR-216a-5p is expected to be a new molecular target for mitigating liver IRI.

Funding Statement

This work was supported by Medical Science and Technology Project of Zhejiang Province (2021PY083& 2024KY1788), Major Research Program of Taizhou Enze Medical Center Grant (19EZZDA2), Program of Taizhou Science and Technology Grant (23ywa33), Open Project Program of Key Laboratory of Minimally Invasive Techniques and Rapid Rehabilitation of Digestive System Tumor of Zhejiang Province (21SZDSYS01).

Authors contributions

L L, Y-X H, Q W, X-H Z, Y-L Q and S-W L articipated in the design of the study. Y-X H, L L and Y-J Z performed the statistical analysis. L L, Y-X H, Q W drafted the manuscript. All authors read and approved the final manuscript.

Disclosure statement

The all authors have no conflict of interest to declare.

Informed consent statement

All study participants, or their legal guardians, provided informed written consent before enrollment.

Data availability statement

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.