Exosome‐mediated pyroptosis of miR‐93‐TXNIP‐NLRP3 leads to functional difference between M1 and M2 macrophages in sepsis‐induced acute kidney injury

Abstract Sepsis is a systemic inflammatory response syndrome caused by infection, resulting in organ dysfunction. Sepsis‐induced acute kidney injury (AKI) is one of the most common potential complications. Increasing reports have shown that M1 and M2 macrophages both take part in the progress of AKI by influencing the level of inflammatory factors and the cell death, including pyroptosis. However, whether M1 and M2 macrophages regulate AKI by secreting exosome remains unknown. In the present study, we isolated the exosomes from M1 and M2 macrophages and used Western blot and enzyme‐linked immunosorbent assay (ELISA) to investigate the effect of M1 and M2 exosomes on cell pyroptosis. miRNA sequencing was used to identify the different miRNA in M1 and M2 exosomes. Luciferase reporter assay was used to verify the target gene of miRNA. We confirmed that exosomes excreted by macrophages regulated cell pyroptosis in vitro by using Western blot and ELISA. miRNA sequencing revealed the differentially expressed level of miRNAs in M1 and M2 exosomes, among which miR‐93‐5p was involved in the regulation of pyroptosis. By using bioinformatics predictions and luciferase reporter assay, we found that thioredoxin–interacting protein (TXNIP) was a direct target of miR‐93‐5p. Further in vitro and in vivo experiments indicated that exosomal miR‐93‐5p regulated the TXNIP directly to influence the pyroptosis in renal epithelial cells, which explained the functional difference between different phenotypes of macrophages. This study might provide new targets for the treatment of sepsis‐induced AKI.


| INTRODUC TI ON
Sepsis is a life-threatening organ dysfunction caused by the host's dysfunctional response to infection, 1 leading to multiple organ dysfunction syndrome including acute kidney injury (AKI). 2 AKI is defined as a group of clinical syndromes that refers to a sudden (1-7 days) and sustained (>24 hours) sudden decline in renal function. 3 It has been alleged that 50% of AKI cases are triggered by severe sepsis, resulting in a 50%-70% mortality in AKI. 4,5 The mechanism of sepsis-associated AKI includes haemodynamic hypothesis and inflammatory factor hypothesis, but these hypotheses cannot explain all AKI included in the process of sepsis. 4 The exact mechanism remains to be further verified.
AKI is characterized by damage or death of tubular epithelial cells, in which pyroptosis has been reported to play a role in the progression of AKI. 6 Pyroptosis is a newly discovered mode of programmed cell death in inflammatory cells that mediated by the activation of various caspases, mainly caspase-1, and the formation of inflammatory bodies. 7 A variety of Gasdermin family members shear and multimerize during the process of pyroptosis, leading to cell perforation, which in turn causes cell death. 7 Compared with apoptosis, pyroptosis occurs more rapidly and is accompanied by the release of a large number of pro-inflammatory factors. 8 Studies have discovered pyroptosis in glomerular epithelial cells and proximal tubule cells and declared that pyroptosis may be involved in the pathophysiological process of tubular epithelial cells injury in sepsis-associated AKI. [9][10][11] Macrophages (MФ) is one of the most important leucocyte species involved in AKI. 12 Monocyte-macrophages have been found to mediate the acute phase within the first 24 hours of AKI, promoting inflammatory cell infiltration. 13 Actually, at different stages of injury, macrophages differentiate into different phenotypes and play different roles. 12 In the early stages of AKI, the kidney tissue exhibits a 'sterile inflammation response'. At this stage, the phenotype of macrophage is mainly M1, which releases the pro-inflammatory mediator and causes damage to the proximal tubule of the outer layer of the renal medulla. As the disease progresses, macrophages gradually gather and engulf apoptotic cells or their released factors.
Meanwhile, the phenotype transforms from pro-inflammatory M1 macrophages to anti-inflammatory M2 macrophages. 14,15 Therefore, we speculate that the M1 macrophages may be the 'culprit' that promotes the cell death of renal epithelial cells and eventually causes tubular necrosis, while M2 macrophages can alleviate tubular necrosis.
Exosomes are phospholipid bilayer vesicles derived from the endosome pathway, ranging from tens to hundreds of nanometres in diameter. 16 They contain abundant protein and genetic information substances that mediate the exchange of substances between cells. 17,18 It was found that microRNAs (miRNAs) transmitted by exosomes can regulate as signal molecules. For example, monocytesderived exosomes transported miR-150 to endothelial cells to promote migration of endothelial cells or angiogenesis. 19 Studies also show that macrophage-derived exosomal miRNAs play important roles in kidney diseases, including AKI. 20,21 This project intends to explore whether M1 and M2 macrophages transmit miRNA through exosomes to influence the pyroptosis in renal epithelial cells. The specific molecular mechanisms involved in the functional difference between M1 and M2 macrophages in AKI were elucidated.

| Cell culture
Primary peritoneal macrophages were harvested from the peritoneal exudates of 6-to 8-week-old BALB/c female mice following the established protocol. 22 Briefly, 72 hours after injecting 2 mL of 3% proteose peptone per mouse into the peritoneal cavity, mice were killed by rapid cervical dislocation. Then, peritoneal fluid was withdrawn slowly. After centrifugation, cells were cultured at 37°C in 5% CO 2 in Dulbecco's modified Eagle medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% foetal bovine serum (FBS, Gibco), 100 U/mL penicillin, 100 µg/mL streptomycin and 20% L929-conditioned medium, with the cell concentration adjusted to 2-3 × 10 6 cells/mL. After 1-2 hours, non-adherent cells were removed by washing with PBS, and adherent cells left were uncommitted macrophages (MФ). TCMK-1 cells, a mouse kidney epithelial cell line (CCL-139), were purchased from the American Type Culture Collection (ATCC) and incubated in complete DMEM, at 37°C in 5% CO 2 . To mimic the inflammatory state, TCMK-1 cells were stimulated with 100 ng/mL of lipopolysaccharide (LPS, Sigma-Aldrich, Saint Louis, MO, USA) for 24 hours.

| Layered co-culture
The transwell co-culture model was established based on the modification of a previously published method. 23 In the present model, TCMK-1 cells and different phenotypes of macrophages were co-cultured on the bottom and top surface of transwell microporous membrane, respectively. Briefly, a transwell insert with microporous membrane was gently wrapped around the edge using the sterilized parafilm (Parafilm M®, Bemis, USA) to build a parafilm fence. TCMK-1 cells were then plated on bottom side of transwell insert membrane and were incubated for over 6 hours in medium at 37°C in 5% CO 2 to attach to the microporous membrane firmly. Next, the transwell insert was turned over and put back to allow TCMK-1-plated side to face down. Polarized macrophages were seeded onto the top side of membrane. Finally, the resultant transwell plate was incubated at 37°C in 5% CO 2 in ECM.
Thus, TCMK-1 cells formed the first layer on bottom side of the membrane, while macrophages formed the second layer on top side. The layers were isolated by the membrane, but the medium could diffuse freely across the membrane.

| Cell viability assay
Standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were conducted to detect the cell viability. At 24 hours after co-cultured, TCMK-1 cells were seeded into 96-well plates. MTT solution was added into the medium to treat cells for 4 hours. Then, the medium was removed softly, and 150 μL dimethylsulphoxide (DMSO, Sigma-Aldrich) was injected to dissolve the formazan. The absorbance was measured at 570 nm ± 10 using a plate-reader (Bio-Rad Laboratories). The cell viability index is expressed as relative value of control group.

| Enzyme-linked immunosorbent assay
The concentrations of TNFα, IL-12, IL-10, IL-18 and IL-1β in medium and serum were determined using commercial ELISA kits (R&D Systems) following the manufacturer's instructions.

| Isolation, characterization and analysis of exosomes
The treatment conditions for M1 and M2 macrophages polarization have been described previously. Subsequently, we collected exosomes from the supernatants of M1 and M2 cell cultures, respectively, without additional stimulation. Exosomes were isolated using ExoQuick (System Biosciences, Palo Alto, CA, USA) according to the manufacturer's instructions. Briefly, media were collected and centrifuged at 2000 g for 10 minutes at 4℃. To thoroughly remove cellular debris, media were centrifuged again at 10 000g for 30 minutes. Reagents were then added and the mixture was vortexed and put overnight at 4℃. After centrifuged at 1500 g for 30 minutes at 4℃, the pellet containing exosomes was resuspended in PBS or ultrapure water. The isolated exosomes were verified by TEM, particle analyser and specific protein markers. Exosomes were fixed in 2.5% buffered glutaraldehyde overnight at room temperature. It was subsequently stained by 1% osmium tetroxide for 2 hours. Then, exosomes were treated by gradient ethanol dehydration, embedded in resin and examined with a TEM. The exosome particle size and concentration were measured by nanoparticle tracking analysis (NTA) with ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany).

| miRNA sequencing
The differentially expressed miRNA level in the exosomes was screened by BGI Genomics (Shenzhen, China), as previously described. 24 Briefly, total RNA was isolated to prepare the libraries. After the single-strand DNA circle (ssDNA circle) was made to construct the final miRNA library, the DNA nanoballs (DNBs) were loaded into the patterned nanoarrays and single-end read of 50 bp were read through on the BGISEQ-500 platform. To obtain miRNA profiles that differently distributed in M1 and M2 macrophages, we set the fold change threshold of M2 group at |log2 (Fold Change)| >1 as compared to M1 group for further analysis. The differentially expressed miRNAs were determined using ANOVA.

| RNA extraction and qPCR
Total RNAs were extracted by using TRIzol reagent (Thermo Fisher Scientific) and were quantified using the NanoDrop ND-1000 (Thermo Fisher Scientific).
According to the results of high-throughput sequencing, specific complementary DNAs (cDNAs) were synthesized from 10 ng of RNA eluate using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Then, the quantitative real-time PCR (qRT-PCR) was performed by amplifying cDNA using TaqMan MicroRNA Assay Mix (Thermo Fisher Scientific) to confirm the expression level of the screened five microRNAs. Primers for qRT-PCR used are shown as following: Gapdh F: 5′-ATCAAGAAGGTGGTGAAGCGGAA-3′ R: 5′-TGGAAGAGTGGGAGTTGCTGTTGA-3′

| Luciferase reporter assay
Cells were co-transfected with plasmids containing 3′-UTR of wild or mutant fragments and miR-93 mimics or inhibitor using Lipofectamine 3000 (Invitrogen, Foster City, CA, USA). At 48 hours after co-transfection, firefly and renilla luciferase activities in TCMK-1 cells were detected consecutively using dual-luciferase reporter assay system (Promega, MA, USA). The firefly luciferase activity was normalized by renilla luciferase activity. Each assay was repeated in three independent experiments.

| Animals
A total of 60 female 8-to 12-week-old BALB/c mice (weighing 18-22 g) were purchased for this study. All mice were reared in plastic cages and given free access to food and water under standard conditionstemperature 25 ± 2°C, humidity 55 ± 5% and 12-hours light/dark cycle. The mice were randomly divided into six groups: a control group (abdominal injection of physiological saline), a 293T-exosomes group, a M1-exosomes group, a M1-miR-93-OE-exosomes group, a M2-exosomes group and a M2-miR-93-KD-exosomes group. To establish a sepsis-induced model, caecal ligation and puncture (CLP) was performed as described previously. 25 In brief, mice were anaes-

| Histological studies
The kidney tissue sections collected from mice were immediately fixed in 10% neutral-buffered formalin and were paraffin-embedded to generate tissue blocks. The tissue blocks were cut at 4 µm thickness. The sections were routinely dewaxed with xylene, washed with ethanol, and performed with haematoxylin and eosin staining (H&E) and periodic acid-Schiff (PAS) staining as previously described. 26 The pathomorphological changes of the renal tissues from each group were then observed under a microscope.

| Western blotting
After harvested and centrifuged, cells were lysed in loading buffer, which was prepared as previously described. 27 All samples includ-

| Data analysis
Statistically significant differences were assessed using the Student's t test or one-way analysis of variance tests in this study. The results were expressed as the mean ± SEM. The criterion for statistical significance was set at P <0.05 or P <0.01.

| M1 and M2 macrophages showed opposite impact on the LPS-induced pyroptosis of TCMK-1 cells in vitro
It is well known that macrophages have a strong plasticity in vivo and in vitro. During the process of AKI, macrophages polarized into different phenotypes in response to the microenvironment and exert diverse effects. 11,12 In the early stages of AKI, the phenotype of macrophage is mainly M1, which releases the pro-inflammatory mediator and causes damage to the proximal tubule of the outer layer of the renal medulla. Given the opposite effect of M2 and M1 macrophages, we hypothesized that M2 macrophage may be the key point to prevent the renal damage of AKI.
We then investigated the effect of M1 and M2 macrophages on the LPS-induced proptosis of TCMK-1 cells, promoting us to find out the mechanism of the difference effect. We first sorted the macrophages ( Figure 1A) and polarization into M1 and M2 macrophages ( Figure 1B). M1 macrophage produced more proinflammatory factor TNFα and IL-12 than M2 macrophage, and M2 macrophage released more IL-10 than M1 macrophage ( Figure 1D).
We then investigate the effect of M1 and M2 macrophage on LPS-induced TCMK-1 injury. M1 and M2 macrophages were, respectively, co-cultured with TCMK-1 cells. It was obvious that the TCMK-1 cells co-cultured with M1 macrophages showed evident swelling with characteristic large bubbles, indicating the marked cell damage ( Figure 1E). MTT assay showed that LPS-induced significant decrease of cell viability, which was aggregated by coculture with M1 macrophage; co-culture with M2 macrophage inhibited LPS-induced cell injury ( Figure 1F).  Figure 1H).
Meanwhile, it could be easily seen that the cell viability, the concentrations of IL-18 and IL-1beta and the pyroptosis-related proteins' level in TCMK-1 cells co-cultured with M2 macrophages showed the opposite trend compared with those co-cultured with M1 macrophages ( Figure 1G,H). Hence, we concluded that M1 macrophages could aggravate the level of pyroptosis of TCMK-1 cells, while M2 macrophages alleviated TCMK-1 cells on the contrary.

| M1-and M2-derived exosomes showed the opposite impact on the pyroptosis in TCMK-1 cells
Exosomes have been found to be the carriers of substances secreted by macrophages to participate in the cellular bioregulation. 28

| miR-93 exhibited the greatest different expression in macrophage-derived exosomes, contributing to the inhibition of pyroptosis in TCMK-1 cells
Macrophage exosomal miRNAs could play their roles through mediating the biological information among cells. 29 Here, we examined the differences of miRNAs expression between M1-and M2-derived exosomes ( Figure 3A; Table S1). We chose five most differentially expressed miRNAs and verified their expressions in M1 and M2 exosomes by using qPCR. As shown in Figure 3B Figure 3G).

| miR-93-5p inhibited pyroptosis pathway in tubular epithelial cells by directly targeting TXNIP
We then predicted the downstream target gene by using TargetScan, miRDB, mirDIP, miRTarbase and miRPathDB ( Figure 4A). The intersection showed 68 potential target gene, among which we found TXNIP, an activator of NLRP3 pathway.
TXNIP is an endogenous inhibitor of the thioredoxin antioxidant and is essential for nod-like receptor protein 3 (NLRP3) inflammasome activation, which could activate cysteine aspartate protease 1 to regulate the maturation and secretion of IL-1β and IL-18 and induce pyroptosis. 30 The conserved binding sites between miR-93-5p and TXNIP are shown in Figure 4B
Scheme figure in Figure 5A shows the process of the animal experiment. We constructed the AKI model in mice, and exosomes from M2 or M2 knocked down of miR-93-5p were administrated to mice, respectively. We found that M2-derived exosomes sig-  Figure 5H).
Hence, we drew a conclusion that the differences between the function of M1 and M2 macrophages in AKI were caused by miR-93/TXNIP axis mediated by exosomes ( Figure 6).

| D ISCUSS I ON
Previous studies have pointed out that macrophages play im- macrophages on AKI remains unclear. In the present study, we identified that exosomal miR-93-5p was responsible for the different effect of M1 and M2 macrophages, which regulated the cell pyroptosis in sepsis-induced AKI.
Pyroptosis is a necrotic-type cell death that was thought to occur exclusively in macrophages, 33 but recent reports find comparable features in T lymphocytes, neurons and tubular epithelial cells. [34][35][36] Emerging evidence points out that pyroptosis is involved in the mechanisms for AKI. 37,38 The inhibition of pyroptosis reduced the inflammatory changes in renal ischaemia/reperfusion injury and decreased the creatinine levels and ameliorated renal dysfunction. 37 In TXNIP was reported to interact with accumulated reactive oxygen species and stimulate inflammation and cell apoptosis. 31,43,44 Increasing studies have pointed out that the combination of TXNIP and inflammasome plays a causative role in ischaemia/reperfusion, 45,46 which is a critical risk factor for AKI. The knockdown of TXNIP could significantly inhibit the activation of NLRP3 inflammasome in I/R injured HK-2 cells as characterized by decreased IL-1β and IL-18 levels, which represent the level of pyroptosis. 43 TXNIP is suggested as an important future target to develop newer therapeutics for its ability to reduce AKI sensitivity of kidney tissues. 45 In the present study, we identified the potential targets of miR-93-5p and found that TXNIP was a candidate target of miR-93-5p. Following luciferase reporter assay and western blot further verified that miR-93-5p negatively regulated the TXNIP expression through binding to its 3′UTR. Overexpression of TXNIP significantly abolished the effect of miR-93-5p on restraining the cell pyroptosis. Our findings indicated that exosomal miR-93 excreted from macrophages exerted its regulatory role on TXNIP in TCMK-1 cells. To confirm that our hypothesis also makes sense in vivo, we generated the mouse model of sepsis-induced AKI by CLP. The results proved that exosomal miR-93-5p inhibited the pyroptosis during AKI progression through regulating TXNIP.
In summary, the present study demonstrated that different phenotypes of macrophages affected the pyroptosis of sepsis-induced AKI by regulating miR-93/TXNIP signalling via exosomes delivery.
Therefore, exosomal miR-93/TXNIP signalling plays a crucial role in the progress of sepsis-induced AKI, which provides potential targets for the treatment of AKI.