Exosome-Derived From Sepsis Patients' Blood Promoted Pyroptosis of Cardiomyocytes by Regulating miR-885-5p/HMBOX1

Background Septic myocardial depression has been associated with increased morbidity and mortality. miR-885-5p has been shown to regulate cell growth, senescence, and/or apoptosis. Published studies demonstrated that Homeobox-containing protein 1 (HMBOX1) inhibits inflammatory response, regulates cell autophagy, and apoptosis. However, the role of miR-885-5p/HMBOX1 in sepsis and septic myocardial depression and the underlying mechanism is not fully understood. Materials and Methods Exosomes (exos) derived from sepsis patients (sepsis-exos) were isolated using ultracentrifugation. Rats were subjected to cecal ligation and puncture surgery and treated with sepsis-exos. HMBOX1 was knocked down or overexpressed in AC16 cells using lentiviral plasmids carrying short interfering RNAs targeting human HMBOX1 or carrying HMBOX1 cDNA. Cell pyroptosis was measured by flow cytometry. The secretion of IL-1β and IL-18 was examined by ELISA kits. Quantitative polymerase chain reaction (PCR) or western blot was used for gene expression. Results Sepsis-exos increased the level of miR-885-5p, decreased HMBOX1, elevated IL-1β and IL-18, and promoted pyroptosis in AC16 cells. Septic rats treated with sepsis-exos increased the serum inflammatory cytokines is associated with increased pyroptosis-related proteins of hearts. MiR-885-5p bound to the three prime untranslated regions of HMBOX1 to negatively regulate its expression. Overexpressing HMBOX1 reversed miR-885-5p-induced elevation of inflammatory cytokines and upregulation of NLRP3, caspase-1, and GSDMD-N in AC16 cells. The mechanistic study indicated that the effect of HMBOX1 was NF-κB dependent. Conclusion Sepsis-exos promoted the pyroptosis of AC16 cells through miR-885-5p via HMBOX1. The results show the significance of the miR-885-5p/HMBOX1 axis in myocardial cell pyroptosis and provide new directions for the treatment of septic myocardial depression.


INTRODUCTION
Sepsis is a serious inflammatory disease caused by the systemic immune response to infections (1). About 15% of sepsis patients undergo septic shock, leading to 10% admissions to intensive care units and a 50% death rate (2). It has been estimated that 15% of sepsis-related mortality is secondary to myocardial depression (3). The best treatment for myocardial dysfunction is sepsis management. Although myocardial depression in sepsis has been the focus of many investigations, its etiology and molecular mechanism remain unclear.
Exosomes (exos) play an important role in intercellular communication (4). Exos can deliver various bioactive substances to target cells to regulate different biological processes (5). Wang et al. indicated that miR-223 in exos derived from mesenchymal stem cells inhibited macrophage inflammation in sepsis (6). Upregulated levels of serum exosomal miR-885-5p were found in hepatitis C virus-infected blood donors and preeclampsia women (7,8). Besides, miR-885-5p was reported to be a tumor-suppressive factor in inhibiting cell cycle progression and cell survival (9). Therefore, further clarifying the function of serum exosomal miRNAs especially miR-885-5p in patients with sepsis will help in the understanding of the pathogenesis of sepsis affecting myocardial function.
Cardiomyocytes involve in the contractile function of the heart (10). Sepsis is characterized by overwhelming inflammation followed by immunosuppression (11). Pyroptosis, first reported in 1992, is a form of necrotic and inflammatory programmed cell death mediated by masterminds and inflammatory caspases (12). Pyroptosis could be activated via the canonical caspase-1 inflammasome pathway (13). The activated caspase-1 not only elevates inflammatory cytokines but also cleaves gasdermin D (GSDMD) to form membrane pores, leading to pyroptosis (14,15). Numerous studies have proved the correlation between inflammation and pyroptosis. For example, Zeng et al. have shown that pyroptosis tightly controls the release of inflammatory cytokines, and inflammation (16). It has also been demonstrated that inflammation and subsequent myocardial dysfunction, were dramatically increased in caspase-1 −/− mice (17). Therefore, the pyroptosis of myocardial cells may be one of the reasons for myocardial dysfunction caused by sepsis. It has been reported that homeobox-containing protein 1 (HMBOX1) is a key immunosuppressive factor that protects hepatocytes from inflammatory responses by inhibiting the infiltration and activation of macrophages (18). In addition, HMBOX1 is also involved in regulating autophagy and apoptosis of vascular endothelial cells (19). Importantly, data collected from the Targetscan database (http://www.targetscan.org/) indicated that HMBOX1 is a potential target for miR-885-5p. However, the detailed relationship between HMBOX1 and miR-885-5p is still unclear in cardiomyocytes.
Although many studies have been carried out, the function of miR-885-5p and HMBOX1 in cardiomyocytes and the role of sepsis in affecting myocardial function are still not fully understood. Therefore, in this study, using AC16 cells, we elaborated the roles of miR-885-5p/HMBOX1 in cardiomyocyte pyroptosis.

Blood Samples
Blood samples of sepsis patients and corresponding healthy individuals (n = 15) were recruited from the Zhongshan Hospital, Fudan University from June 2017 to October 2019, including 22 males and 8 females, age range 37-67 years old, mean age 52.1 ± 6.0 years old). Septic shock was defined as persisting hypotension requiring vasopressors to maintain mean arterial pressure (MAP) ≥ 65 mmHg and a serum lactate level > 2 mmol/L (18 mg/dl) despite adequate volume resuscitation (20). We excluded patients under 18 years old; those with pregnancy, severe anemia, active bleeding, platelet disorders, or chemotherapy; or those using full-dose heparin or any other medications that interfere with platelet function. The enrolled patients had 30 ml blood samples collected from a central venous catheter. Healthy volunteers provided blood samples that served as controls. All the patients were given informed consent. The study has the approval from the ethics committee of Zhongshan Hospital (B2021-390R), Fudan University.

Cell Culture and Transfection
Human myocardial cells (AC16) were from ATCC. Cells were cultured using DMEM with 10% FBS under a 5% CO 2 atmosphere at 37 • C. Oligonucleotides including miR-885-5p mimic and the miR-885-5p inhibitor anti-miR-885-5p were used (Thermo Scientific, Lafayette, CO, USA) for overexpression or inhibition of miR-885-5p, respectively. For cell transfection assays, the synthetic oligonucleotides were transfected into cells using a Lipofectamine RNAiMAX Kit (Invitrogen, Carlsbad, CA, USA) at about 50% confluence. The media were changed 24 h post transfection and the indicated cells were used for further investigations. In some experiments, the Nuclear factor kappa B (NF-κB) inhibitor PDTC (10 µM; S1809, Beyotime Biotechnology, China) was dissolved in DMSO (Sigma-Aldrich, St. Louis, USA) and used to treat cells.

Exosome Collection
Exosomes from sepsis patients (sepsis-exos) or controls (controlexos) were collected as described previously (21). In brief, blood serum was centrifuged at 2,000 g for 20 min to separate cellular debris, and the supernatant was further centrifuged twice at 1,000 g for 30 min twice using a 100-kDa MWCO hollow fiber membrane (Millipore, Bedford, MA, USA). Then, the supernatant was diluted, underlaid on 30% sucrose/D 2 O cushion (density 1.210 g/cm 3 ), and ultracentrifugated at 100,000 g for 1.5 h at 4 • C. Exosome-enriched fractions were centrifuged thrice at 1,000 g for 30 min, and filtered. In some experiments, AC16 cells were seeded (50,000 cells/well) and provided with PKH67-labeled exos for 4 h. Cells were observed using a fluorescence microscope.

Transmission Electron Microscopic (TEM) Analysis
The exosome pellets were suspended in PBS and then fixed in paraformaldehyde (4%) and glutaraldehyde (4%) in PBS (0.1 M, pH 7.4) at 4 • C for 5 min. After adding a drop of the exosomal sample, the carbon-coated copper grid was immersed in a phosphotungstic acid solution (2%, pH 7.0) for 30 s. The morphology and size of exos were observed using TEM analysis (JEM-1200EX; Jeol Ltd., Akishima, Japan).

Cecal Ligation and Puncture (CLP) Surgery Model
Cecal ligation and puncture-induced sepsis model was established as previously described. In brief, rats (200-250 g) were anesthetized with 1% pentobarbital, and a 2-3 cm incision was made to expose the caecum, which was punctured once with an 18-gauge needle. A small amount of feces was extruded from the hole to ensure patency. The abdominal incision was closed by applying sample running sutures. Rats underwent the same procedure except CLP was used as the sham group. All the rats were kept in warm water immediately after surgery. In some experiments, sepsis-exos (100 µg/kg) were i.v. injected into rats after surgery. Serum and heart tissues were harvested for study 72 h after surgery.

Quantitative PCR (QPCR)
Total RNA was extracted from AC6 cells using TRIzol R reagent (Thermo Fisher Scientific, Bremen, Germany). Total RNA (0.5 µg) in 10 µl volume was reverse transcribed into cDNA using the RevertAid TM First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Bremen, Germany) according to the manufacturer's protocol. RNAs were extracted, reverse transcribed into cDNA for qPCR with the following conditions: 95 • C, 10 min followed by 40 × (95 • C, 15 s; 60 • C, 45 s).

Enzyme-Linked Immunosorbent Assay (ELISA)
The levels of IL-1β and IL-18 in supernatants of AC16 cell and rat serum were assessed with ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

Luciferase Reporter Assay
The wild type (WT) human HMBOX1 3 ′ UTR sequence was amplified via PCR and cloned into the Dual-Luciferase Reporter Assay System (psiCHECK-2 vector; Promega, Madison, WI, USA). To construct the mutant (mut) plasmid, the complementary sequences for miR-885-5p in the 3 ′ UTR of HMBOX1 were mutated. AC16 cells were co-transfected with HMBOX1-WT or HMBOX1-mut and miR-885-5p mimic or miR-control. At 36 h after transfection, luciferase activities were detected using the dual-luciferase reporter assay system.

Data Analysis
Data (mean ± SD) were analyzed by Prism 7.0 (GraphPad Software, San Diego, CA, USA). For comparative analyses of normally distributed variables, Student's t-tests and ANOVA were used. The nonparametric Mann-Whitney test was used when the distribution was not normal. P < 0.05 was defined as statistically significant.

Sepsis-Exos Promoted the Pyroptosis of Human Myocardial Cells
To study the effect of spesis-exos on pyroptosis, sepsis-exos, and control-exos were successfully isolated. The exos morphology was observed using TEM (Supplementary Figure 1A) and confirmed by exosomal markers CD9, CD63, and CD81 (Supplementary Figure 1B). Results also suggested that exos could be uptaken by AC16 cells (Supplementary Figure 1C). Sepsis-exos dramatically increased pyroptosis of AC16 cells compared with control-exos ( Figure 1A). Sepsis-exos also enhanced the secretion of IL-1β and IL-18 ( Figure 1B) and decreased HMBOX1 expression in mRNA ( Figure 1C) and protein levels ( Figure 1D). The data suggest that sepsisexo treatment increased the pyroptosis, elevated inflammatory cytokines, but inhibited HMBOX1 in human myocardial cells.

Sepsis-Exos Promote the Severity of Sepsis in Rats
To further elucidate the impact of sepsis-exos in vivo, septic rats underwent CLP surgery were treated with sepsis-exos. Sham rats served as control. Treatment of sepsis-exos significantly increased the serum levels of IL-1β and IL-18 72 h after surgery ( Figure 3A). Moreover, levels of HMBOX1 in heart tissues were dramatically decreased, while expression of pyroptosisrelated proteins NLRP3, caspase-1, and GSDMD-N were all upregulated after treatment of sepsis-exos ( Figure 3B). These  data demonstrated that sepsis-exos increased the inflammatory cytokines is associated with increased pyroptosis-related proteins of hearts in septic rats.

Nuclear Factor Kappa B Inhibitor PDTC Rescued the Function of HMBOX1 in AC16 Cells
To understand the role of NF-κB in pyroptosis, NF-κB inhibitor PDTC was introduced in this study. Silencing of HMBOX1 caused significant increase of pyroptosis in AC16 cells, which was blocked by the administration of NF-κB inhibitor PDTC ( Figure 7A). Silencing of HMBOX1 also resulted in the elevated secretion of IL-1β and IL-18, which was diminished by PDTC  Figure 7B). Administration of NF-κB inhibitor PDTC also blocked HMBOX1 silencing-caused downregulation of HMBOX1 and NF-κB (cytoplasm), and upregulation of NF-κB (nuclear), NLRP3, caspase-1, and GSDMD-N in AC16 cells (Figures 7C,D). These findings demonstrated that the administration of NF-κB inhibitor PDTC rescued the effect of HMBOX1 silencing on AC16 cells.
MicroRNAs  (23). HMBOX1 is characterized by containing homeobox domain (24). Studies revealed that HMBOX1 promotes the proliferation of gastric cancer cells (25). Loss of HMBOX1 has been demonstrated to promote LPS-induced apoptosis of vascular endothelial cells (26). Our data indicated that miR-885-5p negatively regulated HMBOX1. Overexpressing HMBOX1 reversed miR-885-5p-induced elevation of inflammatory factors, and upregulation of nuclear NF-κB, NLRP3, caspase-1, and GSDMD-N, and promotion of AC16 pyroptosis. The results increase our knowledge of miR-885-5p/HMBOX1 in the injury of cardiomyocytes and broaden our understanding of septic myocardial depression.
Pyroptosis involves different diseases. For example, dysregulation of pyroptosis led to tissues damage (27). Pyroptosis has also been related to type II diabetic mellitus through activated caspase-1 and elevated IL-1β/IL-18 (28). In gastric cancer cells, increased GSDMD inhibits tumor development through cell cycle arrest (29). An in vivo study by Lei et al. demonstrated that pyroptosis contributes to cardiomyocyte injury in myocardial infarction (30). We showed that sepsis-exo or miR-885-5p mimic not only elevated IL-1β and IL-18, but also increased NLRP3, caspase-1, and GSDMD-N, leading to promotion of pyroptosis. These results demonstrated the significance of pyroptosis in regulating miR-885-5p/HMBOX1-mediated cardiomyocyte injury. We also further showed that miR-885-5p/HMBOX1mediated cardiomyocyte injury is NF-κB-dependent. It has been confirmed that activation of the NF-κB-dependent pathway induces NLRP3-mediated pyroptosis (31). In this study, our results indicated that overexpression of HMBOX1 inhibited the translocation of NF-κB from the cytoplasm to nuclear in sepsisexo-treated AC16 cells. Importantly, the NF-κB inhibitor PDTC rescued the function of HMBOX1 in siHMBOX1 transfecting cells. Hence, these findings demonstrated that HMBOX1 is involved in the network of NF-κB-dependent pathways and suppressed the translocation of NF-κB from the cytoplasm to nuclear. Moreover, these findings indicate the functional role of miR-885-5p/HMBOX1 in cardiomyocyte injury, and therefore, improve our knowledge of septic myocardial depression. Although future studies based on animals or clinical specimens can provide more relevant and pervasive data, we show a novel mechanism underlying septic myocardial depression. However, although our present data showed that exsomal miR-885-5p plays a critical role in human myocardial cell pyroptosis, we still cannot exclude that miR-885-5p might work in cell apoptosis. The cellular and molecular mechanisms of exsomal miR-885-5p in other forms of cell death need further investigation.
In short, this study revealed a new function of miR-885-5p/HMBOX1 signaling, showing that sepsis-exos-induced cardiomyocytes damage via miR-885-5p and HMBOX1 (Figure 8). These findings identified an important role of the miR-885-5p/HMBOX1 axis which is relevant to cardiomyocytes pyroptosis, and might contribute to septic myocardial depression treatment.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Ethics Committee of Zhongshan Hospital, Fudan University. The patients/participants provided their written informed consent to participate in this study.