Exosomes Derived from <i>Schistosoma japonicum</i> Cystatin-Treated Macrophages Attenuated CLP-Induced Sepsis in Mice

Huang, Feifei; Qian, Yayun; Li, Huihui; Chu, Liang; Wan, Chen; Shen, Qili; Li, Qianqian; Li, Xiuxiu; Wu, Xinyue; Zhan, Bin; Zhou, Rui; Yang, Xiaodi

doi:https://doi.org/10.1155/2024/8626082

Journal of Immunology Research

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Abstract Introduction Materials and Methods Results Discussion Conclusions Data Availability Disclosure Conflicts of Interest Authors’ Contributions Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2024 | Article ID 8626082 | https://doi.org/10.1155/2024/8626082

Exosomes Derived from Schistosoma japonicum Cystatin-Treated Macrophages Attenuated CLP-Induced Sepsis in Mice

Feifei Huang,^1,2,3Yayun Qian,^1,2Huihui Li,^2,4Liang Chu,^2,5Chen Wan,^2,4Qili Shen,^2,5Qianqian Li,²Xiuxiu Li,²Xinyue Wu,²Bin Zhan,⁶Rui Zhou,¹and Xiaodi Yang^2,4

Academic Editor: Payam Behzadi

Received04 Jan 2023

Revised13 Jul 2023

Accepted13 Nov 2023

Published23 Jan 2024

Abstract

Sepsis is a disease caused by multiple microbial infections resulting in multiple organ failure. Schistosoma japonicum secreted cystatin (Sj-Cys) is a strong immunomodulator that stimulates M2 macrophages and alleviates inflammatory damage caused by sepsis. To determine whether the therapeutic effect of Sj-Cys on sepsis can be conveyed by the exosomes released by Sj-Cys-stimulated macrophages, RAW264.7 macrophages were stimulated with rSj-Cys in vitro, the exosomes were obtained from the cell culture supernatant by ultracentrifugation. Sepsis was induced in BALB/c mice by cecal ligation and puncture (CLP). The septic mice were treated with exosomes derived from Sj-Cys-treated macrophages. The treatment effect of exosomes on sepsis was assessed by examining the survival rate of mice up to 72 hr and measuring serum levels of inflammatory cytokines, liver/kidney damage biomarkers, and observing pathological changes in tissue sections. The tissue levels of M1, M2 macrophage surface markers, and TRL2/MyD88 were measured to explore possible mechanisms. Results. Exosomes derived from Sj-Cys-treated macrophages exhibited significant therapeutic effect on CLP-induced sepsis in mice with prolonged survival rate and less damage of critical organs by downregulating the proinflammatory factors TNF-α and IL-6 and upregulating the anti-inflammatory factor TGF-β. The therapeutic effect of exosomes is associated with macrophage polarization from M1 to M2 in the infected tissues via downregulating TRL2/MyD88 inflammatory pathway. Conclusions. Exosomes derived from Sj-Cys-treated macrophages attenuated sepsis in mice through promoting macrophage polarization from M1 to M2 and reducing inflammatory responses, possibly via downregulating TLR2/MyD88 inflammatory signaling pathway. This offers new approaches for immunotherapy of sepsis.

1. Introduction

Sepsis is a systemic inflammatory syndrome attributed to the dysregulated host immune responses to bacterial infections and a life-threatening medical emergency [1]. The variability of sepsis between patients makes treatment challenging, despite the level of care and medical instruments being updated [2, 3] the mortality rate remains as high as 40% [4]. The evolution of sepsis is broadly categorized into two phases, systemic inflammatory response syndrome (SIRS) and compensatory anti-inflammatory response syndrome (CARS) [5]. SIRS is originated by various innate immune cells, which are activated by pathogen signaling when a pathogen invades, stimulating tumor necrosis factor-α (TNF-α) production, and speeding up the activation of other inflammatory cytokines and exacerbating tissue damage. This active phase of inflammatory factors is the main cause of organ damage in sepsis [6, 7]. Another aspect, CARS is a mechanism of systemic immune system by secreting anti-inflammatory factors such as IL-4, IL-10, and TGF-β in order to balance the inflammatory state and reduce inflammation [8]. Therefore, the timing and balancing of the SIRS and CARS have become crucial for the treatment of prognosis of sepsis.

Macrophages are innate immune cells that play a key role in initiating, amplifying, and clearing inflammation and are major target cells in the treatment of sepsis [9–11]. Macrophages exhibit different phenotypes and functions depending on the surrounding microenvironment [12]. Macrophages are distinguished into two types according to phenotype: the classical activation (M1) type inspired by type-1T helper cytokine (Th1), which secretes proinflammatory factors such as TNF-α and IL-6 and is engaged in the development of inflammation; and the alternative activation (M2) antigen induced by T helper type-2 (Th2) cytokine, which secretes transforming growth factor (TGF-β) and stimulates tissue repair [12]. All studies indicated that the proportion of M1 to M2 macrophages affects the development and consequence of inflammatory diseases.

Exosomes are nanoscale vesicles produced by various cells and play an important role in mediating intercellular signaling and communicating [13, 14]. Under the inflammatory response, the donor cells release active substances such as proteins, genes, and lipids into exosomes to the recipient cells resulting in the receptor phenotypic variation [15, 16]. In recent years, the function and possible application of exosomes in sepsis has attracted growing attention. It has been shown that exosomes are released from diverse effector cells and associated with various tissue or organ damage or repair in sepsis. For example, exosomes released by macrophages from TLR-9 knockout mouse ameliorated sepsis-induced mitochondrial oxidative stress and apoptosis in cardiomyocytes [17]. Adipose-derived stem cells released exosomes inhibited inflammation and oxidative stress in mice with lipopolysaccharide (LPS) induced acute kidney injury [18]. Mouse bone marrow mesenchymal stem cells released serum amyloid A1 (SAA1) protein in exosomes that reduced sepsis-induced lung injury in mice [19]. On the contrary, exosomal miR-155 stimulated macrophage proliferation and inflammation in lung tissue and mediated septic lung injury [20].

A large body of experimental evidence demonstrates that helminth infections modulate host immune responses by releasing a number of functional proteins to diminish immune attack on parasites as a survival strategy during infection, on the other hand, host may acquire the beneficial effect of the helminth-mediated immunomodulation to reduce inflammatory responses to some exogenous allergens or indigenous autoantigens [21, 22]; therefore, these helminth-derived immunomodulatory proteins have been experimentally tested as therapy agents for the treatment of allergic or inflammatory diseases [23]. In particular, cysteine protease inhibitors (cystatin) from various parasitic helminths are strong immunomodulatory proteins to reduce host inflammatory immune responses [24–26]. Schistosoma japonicum secreted cystatin (Sj-Cys) has been identified as a strong immunomodulatory protein [27]. It has been determined that proinflammatory factors released from LPS-stimulated macrophages can be inhibited by Sj-Cys. The recombinant protein of Sj-Cys (rSj-Cys) reduced the seriousness of collagen-induced arthritis [28] and cecal ligation and puncture (CLP)-induced bacterial sepsis in mice [29]. Further investigation revealed that the therapeutic effect of rSj-Cys on sepsis was taken placed through activating regulatory macrophages [29]. It is still unknown the therapeutic mechanism of rSj-Cys on sepsis. In this study, the exosomes extracted from Sj-Cys-treated macrophages (Sj-Cys-EXO) were used to treat CLP-induced bacterial sepsis in mice. We found that Sj-Cys-EXO conducted similar therapeutic effect on CLP-induced bacterial sepsis with reduced organ injury as our previous study with treatment of rSj-Cys in vivo in septic mice [29], demonstrating at the first time that Sj-Cys induced M2 polarization can be transferred to macrophages in septic tissue through exosomes to mitigate sepsis caused tissue or organ damage.

2. Materials and Methods

2.1. Expression of Recombinant Sj-Cys Protein (rSj-Cys)

The coding DNA for Sj-Cys (GenBank: FJ617450) was amplified from cDNA of S. japonicum adult worms and cloned into pPIC9k yeast expression vector, as described previously. The expression of rSj-Cys was induced with 0.5% methanol and the expressed rSj-Cys was purified using an immobilized metal affinity chromatography (IMAC) (Thermo, USA) [30]. The contaminated endotoxin in the purified rSj-Cys was removed using a Endotoxin Removal Kit (BioVision, USA) and confirmed by a LAL Chromogenic Endotoxin Quantitation Kit (Thermo Fisher Scientific, Waltham, USA). The concentration of rSj-Cys was determined with bicinchoninic acid (BCA) protein quantification kit (Biosharp, Hefei, China). Purity and molecular weight of rSj-Cys were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

2.2. Exosomes Isolation from Macrophages Treated with rSj-Cys

The RAW264.7 cells were cultivated in DMEM media supplemented with 10% fetal bovine serum (Sijiqing, Hangzhou, China) and 1x penicillin/streptomycin (Sigma–Aldrich, Steinheim, Germany). When the cell growth density reached about 80%, the culture media was changed to media with exosome-free serum (System Biosciences, UK). Then rSj-Cys was added into the culture up to 2 µg/ml and the culture was continued for additional 48 hr. The culture supernatant was harvested, the cells and debris were removed by centrifugation at 300×g; 2,000×g and 10,000×g, respectively and then filtrated through 0.22 μm filter. The filtered supernatant was centrifuged at 120,000×g for 90 min, the pellet was washed once with 1x phosphate-buffered saline, pH 7.4, and recovered by centrifugation at 120,000×g for another 90 min to obtain exosomes (EXO). The protein concentration of obtained EXO was quantified by BCA and the exosome-positive proteins (CD9, TSG101) in EXO were measured by Western blot with specific antibodies (Abcam, UK) [30].

Negative-staining transmission electron microscopy (TEM) was conducted to analyze the shape and size of exosomes. The exosomes were suspended in 2% glutaraldehyde, loaded on a copper grid and negatively stained with 1% uranyl acetate, then imaged with a HT-7700 TEM (Hitachi, Japan) at 100 kV [31]. The collected exosomes were also analyzed using a nanoflow cytometer (nFCM), as described in Tian et al.’s [32] study. Briefly, the concentration of each extracellular vesicle (EV) sample was determined by employing 100 nm orange FluoSpheres of known particle concentration to calibrate the sample flow rate. Two single-photon counting avalanche photodiodes were used for the simultaneous detection of the side scatter and orange fluorescence of individual EVs. The sample nFCM data were collected for 1 min at a sample pressure of 1.0 kPa. The particle concentrations and particle size distributions were calculated using NanoFCM software (Xiamen, China) [32].

2.3. Animals and Ethics

Male BALB/c mice at 6 weeks old and weight of 18–22 g were purchased from the Animal Centre of Henan Province. All animal experiments were conducted with protocol approved by Ethics Review Committee of Bengbu Medical College (approval number: LAEC-2022-413).

2.4. Murine Model of Sepsis

Sepsis model was established in mice through CLP that causes polymicrobial infection to mimic the pathophysiological characteristics of peritonitis and sepsis in patients in a clinical setting [33, 34]. As described previously, mice were kept fasted for 12 hr with access to water only. Under anesthesia with isoflurane inhalation, mice were stabilized and the abdominal cavity was open, the cecum was located and tied at the top 1.0 cm of the cecum. The cecum was punctured with an 18-gauge needle, a small amount of faeces was squeezed out. The cecum was placed back into the cavity and the abdomen was closed by suturing level by level. The control mice were subjected to incision without any manipulation of the cecum (sham surgery). The survival rate of the mice was observed for 72 hr.

2.5. Therapeutic Effect of Sj-Cys-EXO on Septic Mice

Total 75 BALB/c mice were randomly assigned to five groups with 15 each. Three groups of mice have performed CLP surgery, 30 min after surgery mice in each group were injected intraperitoneally with 10 µg of EXO derived from rSj-Cys-treated macrophages (CLP + Sj-Cys-EXO) or 10 µg EXO from nontreated macrophages (CLP + EXO) or PBS only (CLP + PBS) in the total volume of 100 μl, respectively. Another two groups performed sham surgery and treated with the same amount of Sj-Cys-EXO (Sham + Sj-Cys-EXO) or PBS only as control (Sham + PBS). Twelve hours after treatment, five mice in each group were sacrificed. Blood was drawn and sera were separated for testing the levels of cytokines and some biochemical makers for liver and kidney functions described below, liver, lung, and kidney were collected from each mouse for histopathological examination. The survival rate of the remaining 10 mice was monitored for 72 hr. The experiment was repeated for three times.

2.6. Serological Test

Sera from five mice euthanized 12-hr posttreatment were tested for the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), the liver function biomarkers, blood urea nitrogen (BUN) and creatinine (Cr) (kidney function markers), using an automatic chemistry analyzer (Beckman, USA). The proinflammatory IL-6, TNF-α, and anti-inflammatory TGF-β cytokine were detected in the sera by ELISA with specific antibodies (Dakewe, China). The standard range of IL-6 was 0–1,000 pg/ml with a serum dilution of 1 : 40, and the standard range of TGF-β was 0–500 pg/ml with a serum dilution of 1 : 100.

2.7. Histopathological Examination in Tissues of Liver, Lung, and Kidney

The livers, lungs, and kidneys were obtained from five mice euthanized 12-hr posttreatment and fixed with formalin. Tissue sections were cut and stained with hematoxylin and eosin (H&E) and examined for histopathological changes under the microscope (Olympus, Tokyo, Japan). Haemorrhage, hepatocyte necrosis, inflammatory cell infiltration, cytoplasmic vacuolation, and nuclear condensation were assessed in liver injury and pathological score was evaluated, as shown in Table 1 [35]. Alveolar wall thickening, vascular congestion, interstitial, and alveolar leukocyte infiltration were evaluated for lung injury, as shown in Table 2 [36]. Injured renal tubules and shrunk glomerulus were assessed in kidney injury, as shown in Table 3, based on the previous study [37].

2.8. Detection of Macrophage Phenotypic Markers

The levels of inducible nitric oxide synthase (iNOS), the macrophage M1 marker, and arginase-1 (Arg-1), M2 marker, were measured in the homogenate of liver, lung, and kidney tissues of treated mice using the corresponding ELISA kits (Elabscience, China).

Total RNA was extracted from liver, lung, and kidney tissues of each mouse using TRIzol-up (TransGen Biotech, China) and quantified by measuring A260/280. Total 2 µg of total RNA from each sample was used to reverse transcribed into cDNA using a reverse transcription kit (TransGen, China) on S1000 Thermal Cycler (Bio-Rad) in a total volume of 50 µl. The relative mRNA expression of macrophage polarization-related markers (iNOS and Arg-1) against housekeeper control GAPDH in these tissues was determined by a reverse transcription quantitative polymerase chain reaction (RT-qPCR) by using SYBR® Green I fluorescent dye to quantitatively detect amplified double-strand DNA in LightCycler® 96 Real-Time PCR System, Switzerland. The primers for each target gene used for the real-time PCR (Table 4) were designed and optimized to achieve similar efficiency for the targets and the internal control gene (GAPDH) [38]. Total 2 µl of total cDNA was used in the qPCR reaction for each sample, the reaction condition is 95°C for 5 s, 55°C for 15 s, and 72°C for 10 s with total 45 cycles. The final value was determined by the 2^−ΔΔCt formula.

2.9. Detection of TLR2 and MyD88 in the Liver, Kidney, and Lung Tissues of Mice by Western Blotting

The expression levels of TLR2 and MyD88 in the tissues of liver, kidney, and lung were determined by Western blot using specific antibodies. Briefly, tissues obtained from treated mice were lysed with lysis solution and the tissue lysate supernatant was collected by centrifugation. The protein concentration in the lysate supernatant was measured using the BCA method. The same amount of tissue lysate (20 µg) was separated on a 10% acrylamide gel and transferred onto polyvinylidene difluoride membranes. The membrane was then blocked with skimmed milk for 3 hr and incubated overnight at 4°C with rabbit anti-TLR2 antibody (1 : 2,000) (Abcam, USA), or rabbit anti-MyD88 antibody (1 : 1,000) (Cell Signaling Technology, USA), or rabbit anti-β-actin antibody (1 : 5,000) (Cell Signaling Technology, USA). The washed membrane was followed by reincubation with HRP-conjugated goat antirabbit IgG secondary antibody (Biosharp, Hefei, China) (1 : 7,000). The bands were visualized and density of the target bands was scanned by Image Lab (Bio-Rad). Results are expressed as a ratio of TLR2 and Myd88 to β-actin control.

2.10. Statistical Analysis

Statistical analysis was performed using GraphPad Prism 7.0 software (GraphPad Software, USA) for one-way or two-way analysis of variance for multiple group comparisons. All data are expressed as mean and standard deviation. Kaplan–Meier survival analysis was used to compare differences in survival between groups. was considered to be statistically significant.

3. Results

3.1. Expression, Purification, and Identification of rSj-Cys

The Sj-Cys was expressed successfully as a soluble recombinant protein. SDS-PAGE revealed that the purified rSj-Cys had a size of approximately 11 kDa as expected based on the sequence (Figure 1). The endotoxin level in the purified rSj-Cys protein was less than 0.06 EU/ml.

3.2. Successful Isolation of Exosomes from rSj-Cys Treated or Nontreated Macrophages

RAW264.7 macrophage cell line was incubated with 2 µg/ml rSj-Cys for 48 hr. The exosomes released by the rSj-Cys-treated or untreated macrophages were obtained by ultracentrifuging the culture supernatants. The obtained exosomes showed vesicles of 40–150 nm in diameter with a typical bilayer structure measured by TEM (Figure 2(a)). Flow cytometry analysis confirmed the size of 40–150 nm for those vesicles in the exosomes (Figure 2(b)). The exosome-specific markers, TSG101 and CD9, were detected in the obtained EXO via Western blotting using corresponding specific antibodies. There is no significant difference between rSj-Cys stimulated macrophage exosomes (Sj-Cys-EXO) or exosomes derived from untreated macrophages (EXO) in terms of morphology and intensity of TSG101 or CD9 (Figure 2(c)).

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3.3. Treatment with Sj-Cys-EXO Improved the Survival Rate of Mice with CLP-Induced Sepsis

To test the therapeutic effect of exosomes derived from rSj-Cys-treated macrophages on sepsis, septic mice were each injected intraperitoneally with 10 µg of Sj-Cys-EXO or the same amount of EXO from untreated macrophage. The survival rate of mice up to 72 hr in each group was observed. As seen in Figure 3, all mice receiving PBS in the CLP group (CLP + PBS) died within 48 hr. However, the 72 hr survival rate in CLP + Sj-Cys-EXO group increased to 30% that is significantly improved compared with CLP + PBS group (). The 72 hr survival rate for mice in the CLP + EXO group was only 10%, without statistic difference compared to control group of CLP + PBS. All mice in the sham-operated group with or without Sj-Cys-EXO treatment survived up to 72 hr.

3.4. Sj-Cys-EXO Improved Visceral Organs Function of Septic Mice

The sera collected from different groups of mice were analyzed using an automatic chemistry analyzer to measure the levels of ALT, AST (liver function), BUN, and Cr (kidney function). The findings showed that serological ALT, AST, BUN, and Cr levels were significantly elevated in the CLP group compared to the Sham group (Figure 4), indicating that sepsis caused severe damage to the liver and kidney. However, treatment with Sj-Cys-EXO in mice with sepsis (CLP + Sj-Cys-EXO) significantly reduced the levels of ALT, AST, BUN, and Cr in their sera compared to mice receiving PBS only. Mice treated with normal macrophages-derived EXO (CLP + EXO) also showed some extent of reduced levels of these liver or kidney injury markers, but the reduced level is not as significant as Sj-Cys-EXO. These results indicate that exosomes from macrophages, especially from Sj-Cys-treated macrophages, significantly improve the liver and kidney functions of septic mice. Sj-Cys-EXO has no effect on mice with sham surgery.

3.5. Sj-Cys-EXO Attenuates the Pathological Injury Caused by Sepsis

The liver, lung, and kidney tissue showed significant inflammation and damage in mice with CLP-induced sepsis without treatment (CLP + PBS), exhibiting inflammatory cell infiltration, edema, hemorrhage, tissue structure damage, and cell death. After being treated with exosomes from Sj-Cys-treated macrophages (CLP + Sj-Cys-EXO), the tissue damage in liver, lung, and kidney was significantly improved with less inflammation cell infiltration and edema and less hemorrhage and tissue structure distortion (Figure 5(a)). The organ tissue damage was also improved in septic mice treated with normal macrophage exosome (CLP + EXO); however, the improvement was not as significant as mice treated with Sj-Cys-EXO. The pathological injury scores in liver, lung, and kidney showed the similar level of improvement in septic mice treated with Sj-Cys-EXO or normal macrophage EXO, but with more significance with the former (Figure 5(b)). The above results showed that treatment with Sj-Cys-EXO greatly reduced the damage and pathology of important organs including liver, lung, and kidney in mice with CLP-induced sepsis.

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Figure 5

Treatment with Sj-Cys-EXO reduced liver, kidney, and lung tissue damage caused by CLP-induced sepsis. (a) The representative histopathology results of liver, kidney, and lung stained with H&E from mice 12 hr after CLP or sham surgery treated with Sj-Cys-EXO, EXO, or PBS. (b) The pathological score in liver, lung, and kidney from different groups of mice. The magnification ×200, scale bar = 100 µm. For (b), n = 5 per group. The results are presented as mean ± SD. , , .

3.6. Sj-Cys-EXO Regulated Macrophage Polarization in Liver, Kidney, and Lung Tissues of Septic Mice

To further investigate whether therapeutic effect of Sj-Cys-EXO on sepsis is taken placed through modulating macrophage polarization, the protein and mRNA expression levels of M1 macrophage marker iNOS and M2 macrophage marker Arg-1 were measured in liver, kidney, and lung tissues using ELISA and RT-qPCR, respectively. The results showed iNOS was significantly increased in those tissues of mice with CLP-induce sepsis in both protein and transcriptional levels compared to mice with sham surgery. However, the protein level of Arg-1 was significantly reduced, although the mRNA level was slightly increased. These results suggest that the serious polymicrobial infection significantly induces M1 macrophage polarization in infected organs. Treatment with Sj-Cys-EXO significantly reduced iNOS but increased Arg-1 in both protein and mRNA levels in liver, lung, and kidney of mice with CLP-induced sepsis (CLP + Sj-Cys-EXO), indicating that Sj-Cys-EXO induces the macrophage polarization from M1 to M2 in these organs (Figure 6). Treatment with normal macrophage-derived exosome (EXO) also promoted the M2 polarization in these organs but not so significant as Sj-Cys-EXO.

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3.7. Sj-Cys-Induced Macrophage-Derived Exosome Reduced Proinflammatory Cytokines and Induced TGF-β in Mice with CLP-Induced Sepsis

Inflammatory cytokines TNF-α, IL-6, and regulatory cytokine TGF-β have a major impact on the pathogenesis of sepsis. To measure whether exosomes from rSj-Cys-treated macrophages regulate cytokine production in mice with sepsis, the concentrations of inflammatory cytokins TNF-α and IL-6 and anti-inflammatory cytokine TGF-β were examined in sera by ELISA. The results demonstrated that both TNF-α and IL-6 levels were highly elevated in CLP-induced septic mice (CLP + PBS) relative to sham-operated mice. The regulatory cytokine TGF-β was also reduced in septic mice. However, septic mice treated with Sj-Cys-EXO had significantly lower levels of these proinflammatory cytokines compared to septic mice receiving PBS. These proinflammatory cytokines were also reduced in mice treated with EXO from normal macrophages; however, the decreased level was not as significant as mice treated with Sj-Cys-EXO (Figure 7(a)). In contrast, the serological level of TGF-β was significantly increased in septic mice treated with Sj-Cys-EXO (CLP + Sj-Cys-EXO) compared with mice received with PBS. Septic mice received with normal macrophage-derived EXO showed slightly increased TGF-β level (CLP + EXO) compared to nontreated CLP + PBS (Figure 7(b)). These outcomes indicate that exosomes from rSj-Cys-stimulated macrophages have ability to downregulate Th1 inflammatory response and upregulate regulatory response.

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3.8. Sj-Cys-EXO Inhibits the Expression of TLR2 and MyD88

To verify whether Sj-Cys-EXO regulates macrophage polarization in septic mice through TLR2/MyD88 signaling pathway, TLR2 and MyD88 expression level was measured in liver, lung, and kidney of septic mice. As shown in Figure 8, CLP-induced sepsis resulted in consistent activation of the TLR2/MyD88 signaling pathway in liver, lung, and kidney tissues. However, while the acute damage to these vital organs caused by sepsis was alleviated by the treatment with Sj-Cys-EXO, the TLR2 and MyD88 protein expression levels were also reduced in these tissues of treated mice. The reduced TLR2 and MyD88 level in EXO treated group was not as significant as those in Sj-Cys-EXO treated group (Figure 8).

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4. Discussion

Sepsis is a life-threatening disease induced by an overwhelming immune responses to bacterial infection, including overreactive cellular immune response and proinflammatory cytokine storm that cause injury and disfunctions in key organs including liver, lung, and kidney, the major cause of death in sepsis [39]. Therefore, there is an acute need to develop new approach targeting the inflammatory drivers of sepsis. Although the mechanism of overwhelming immune responses has not been fully understood, macrophages are major producers of inflammatory mediators in sepsis and other inflammatory diseases [40]. Our previous study has determined that Sj-Cys was a strong immunomodulator that reduces Th1 proinflammatory responses and mitigates the severity of sepsis-induced organ injury and damage mostly through polarizing M1 to M2 macrophages in a mouse model [29]. In this study, we used rSj-Cys to stimulate RAW264.7 macrophage cell line in vitro to obtain Sj-Cys stimulated macrophage-derived exosomes (Sj-Cys-EXO). These exosomes were used to intraperitoneally treat mice with CLP-induced sepsis to determine whether exosomes derived from Sj-Cys-treated macrophages can convey the therapeutic efficacy of rSj-Cys to mice with sepsis. Indeed, treating with Sj-Cys-EXO significantly decreased the severity of sepsis and enhanced the survival rate of septic mice up to 30% within 72 hr observation period compared to mice without treatment that all died within 48 hr after CLP surgery (Figure 3), which is similar to the results from the direct treatment with Sj-Cys recombinant protein itself in our previous study [29]. The increased survival rate seems to be Sj-Cys-EXO specific since exosomes derived from naïve RAW264.7 macrophage displaced less therapeutic effect in terms of survival rate. The improved survival rate was also reflected with reduced pathology damage of key organs such as liver, lung, and kidney, with less inflammatory cell infiltration, less congestion and edema, less dead cell, and structural disruption (Figure 5). The reduced organ injury caused by the sepsis was also confirmed by reduced levels of ALT and AST (biomarkers of liver cell damage) and reduced levels of BUN and Cr (biomarkers for kidney function damage) in sera (Figure 4). All results suggest that exosomes derived from rSj-Cys treated macrophage can transfer the anti-inflammatory and therapeutic effect of rSj-Cys to mice with sepsis. We also observe some therapeutic effects of exosomes derived from naïve macrophages on sepsis, possibly because the exosomes from normal macrophage cell line may contain some components directly from natural M2 cells that may partially reduce inflammation.

Further investigation in this study revealed that the immunological mechanism behind the reduced inflammation and tissue damage was related to the polarization of macrophage from M1 to M2 induced by the Sj-Cys-EXO, evidenced by the reduced expression levels (protein and mRNA) of macrophage M1-related markers iNOS and increased expression levels of M2 marker Arg-1 in liver, lung, and kidney tissues. The cytokine profile in sera was consistent with the M2 macrophage polarization in mice treated with Sj-Cys-EXO, exhibiting higher level of regulatory cytokine TGF-β and reduced levels of proinflammatory cytokines TNF-α and IL-6 (Figure 7). Therefore, the results clearly show that exosomes derived from Sj-Cys treated macrophages transfer the signal to macrophages in mice with CLP-induced sepsis to induce macrophage polarization from M1 to M2 resulting in the inhibition of proinflammatory reaction caused by the bacterial sepsis.

It has been identified that exosomes released into the extracellular environment by different types of immune cells play important roles in intercellular communication by transferring biological information between cells [41–43]. LPS-stimulated M1-type macrophage-secreted exosomes that promote the occurrence of inflammation and cause acute lung injury in mice [44]. However, M2-type macrophage-derived exosomes (M2EXO) played important roles in vascular tissue repair and skin wound healing possibly through reprograming M1-type macrophages to M2-type macrophages [45, 46]. Our findings are in agreement with above investigations and demonstrated that Sj-Cys-induced M2-type macrophage polarization that can transfer signals to the local macrophages by releasing exosomes to amplify the anti-inflammatory response and reduce immunopathology in septic mice.

In pathogenesis of sepsis, TLRs are critical in maintaining the delicate equilibrium between immune tolerance and activation [47, 48]. TLR2 is a lipoprotein-specific receptor for host defence against bacterial infection. During bacterial infection, TLR2 is activated that triggers downstream MyD88 signaling pathway and the activation of inflammatory cells. This activation results in the greater synthesis and release of cytokines causing SIRS or multiorgan dysfunction syndrome, the major complication and consequence of bacterial sepsis [49, 50]. There was evidence that Staphylococcus aureus released lipoprotein via EV to stimulate TLR2 on innate immune cells to expand inflammatory responses [51]. It was also found that macrophages infected with Mycobacterium could release exosomes to stimulate resting macrophages and activate the TLR2/TLR4/MyD88 signaling pathway, thereby promoting the expression of proinflammatory cytokines [52]. Exosomes were also involved in the transcellular transcytosis of peptidoglycan between intestinal epithelium cells to regulate intestinal immune response and homeostasis [53]. To investigate whether exosomes derived from rSj-Cys-treated macrophage alleviate sepsis in mice by inhibiting TLR2/MyD88-dependent signaling pathway, we measured the protein expression levels of TLR2 and MyD88 in liver, lung, and kidney tissues of septic mice after being treated with Sj-Cys-EXO. As expected, the TLR2 and MyD88 expressed were significantly elevated in mice with CLP-induced sepsis, indicating the TLR2/MyD88 signaling pathway is activated in septic mice. However, after being treated with Sj-Cys-EXO, the protein expression levels of TLR2 and MyD88 in these tissues were significantly reduced, suggesting Sj-Cys-EXO regulates macrophage polarization and reduces inflammatory immune responses by inhibiting TLR2/MyD88 signaling pathway.

The results identified in this study clearly demonstrate that the therapeutic effect of Sj-Cys on inflammatory sepsis can be conducted by the exosomes released by Sj-Cys-activated M2 macrophages. The specific components in the exosomes that conduct immunomodulatory efficacy are under investigation.

5. Conclusions

The findings of this study demonstrated that exosomes derived from Sj-Cys treated macrophage (Sj-Cys-EXO) attenuate the severity of CLP-induced sepsis in mice by promoting macrophages polarization from M1 to M2 and inhibiting TLR2/MyD88 signaling pathway.

Data Availability

All research data involved in this article were obtained from the article and the submitting authors.

Disclosure

A previous version of this manuscript was published as a preprint [54].

Conflicts of Interest

The authors declare that there are no conflicts of interest regarding the publication of this paper.

Authors’ Contributions

Xiaodi Yang, Rui Zhou, and Feifei Huang conceived and designed the study. Feifei Huang, Yayun Qian, Qianqian Li, Xiuxiu Li, Xinyue Wu, and Qili Shen performed the experiments. Huihui Li, Liang Chu, and Chen Wan analyzed the data. Feifei Huang and Yayun Qian wrote the manuscript. Bin Zhan and Xiaodi Yang critically revised the manuscript. Feifei Huang and Yayun Qian contributed equally to this work and shared first authorship.

Acknowledgments

This research was supported by the Natural Science Foundation of Anhui Province (no. 2208085MH238), Program of Natural Science Foundation of the Anhui Higher Education Institutions (no. KJ20210687), Key Research Platform Open Project of Bengbu Medical College (no. AHIAI2022K02), 512 Talents Development Program of Bengbu Medical College (no. by51201205), and Postgraduate Scientific Research Innovation Program of Bengbu Medical College (nos. Byycxz21092 and Byycxz22064).

References

M. Huang, S. Cai, and J. Su, “The pathogenesis of sepsis and potential therapeutic targets,” International Journal of Molecular Sciences, vol. 20, no. 21, 2019.
View at: Publisher Site | Google Scholar
M. Komorowski, “Clinical management of sepsis can be improved by artificial intelligence: yes,” Intensive Care Medicine, vol. 46, no. 2, pp. 375–377, 2020.
View at: Publisher Site | Google Scholar
P. Young, D. Mackle, R. Bellomo et al., “Conservative oxygen therapy for mechanically ventilated adults with sepsis: a post hoc analysis of data from the intensive care unit randomized trial comparing two approaches to oxygen therapy (ICU-ROX),” Intensive Care Medicine, vol. 46, pp. 17–26, 2020.
View at: Publisher Site | Google Scholar
M. D. Font, B. Thyagarajan, and A. K. Khanna, “Sepsis and septic shock—basics of diagnosis, pathophysiology and clinical decision making,” Medical Clinics of North America, vol. 104, no. 4, pp. 573–585, 2020.
View at: Publisher Site | Google Scholar
M. Singer, C. S. Deutschman, C. W. Seymour et al., “The third international consensus definitions for sepsis and septic shock (sepsis-3),” JAMA, vol. 315, no. 8, pp. 801–810, 2016.
View at: Publisher Site | Google Scholar
A. Copaescu, O. Smibert, A. Gibson, E. J. Phillips, and J. A. Trubiano, “The role of IL-6 and other mediators in the cytokine storm associated with SARS-CoV-2 infection,” Journal of Allergy and Clinical Immunology, vol. 146, no. 3, pp. 518–534.E1, 2020.
View at: Publisher Site | Google Scholar
Y. Ge, X. Xu, Q. Liang, Y. Xu, and M. Huang, “α-Mangostin suppresses NLRP3 inflammasome activation via promoting autophagy in LPS-stimulated murine macrophages and protects against CLP-induced sepsis in mice,” Inflammation Research, vol. 68, no. 6, pp. 471–479, 2019.
View at: Publisher Site | Google Scholar
S. Soldano, A. C. Trombetta, P. Contini et al., “Increase in circulating cells coexpressing M1 and M2 macrophage surface markers in patients with systemic sclerosis,” Annals of the Rheumatic Diseases, vol. 77, pp. 1842–1845, 2018.
View at: Publisher Site | Google Scholar
J. A. Ardura, G. Rackov, E. Izquierdo, V. Alonso, A. R. Gortazar, and M. M. Escribese, “Targeting macrophages: friends or foes in disease?” Frontiers in Pharmacology, vol. 10, Article ID 1255, 2019.
View at: Publisher Site | Google Scholar
E. Karakike and E. J. Giamarellos-Bourboulis, “Macrophage activation-like syndrome: a distinct entity leading to early death in sepsis,” Frontiers in Immunology, vol. 10, Article ID 55, 2019.
View at: Publisher Site | Google Scholar
V. Kumar, “Targeting macrophage immunometabolism: dawn in the darkness of sepsis,” International Immunopharmacology, vol. 58, pp. 173–185, 2018.
View at: Publisher Site | Google Scholar
M. Orecchioni, Y. Ghosheh, A. B. Pramod, and K. Ley, “Macrophage polarization: different gene signatures in M1(LPS+) vs. classically and M2(LPS–) vs. alternatively activated macrophages,” Frontiers in Immunology, vol. 10, Article ID 1084, 2019.
View at: Publisher Site | Google Scholar
A. S. Greenberg and A. R. Reeves, “The good and bad of adipose tissue macrophage exosomes in obesity,” Cell Metabolism, vol. 33, no. 4, pp. 700–702, 2021.
View at: Publisher Site | Google Scholar
A. Takahashi, R. Okada, K. Nagao et al., “Publisher correction: exosomes maintain cellular homeostasis by excreting harmful DNA from cells,” Nature Communications, vol. 9, Article ID 4109, 2018.
View at: Publisher Site | Google Scholar
S. Sahoo, M. Adamiak, P. Mathiyalagan, F. Kenneweg, S. Kafert-Kasting, and T. Thum, “Therapeutic and diagnostic translation of extracellular vesicles in cardiovascular diseases: roadmap to the clinic,” Circulation, vol. 143, no. 14, pp. 1426–1449, 2021.
View at: Publisher Site | Google Scholar
Y. Zhao, M.-F. Zhao, S. Jiang et al., “Liver governs adipose remodelling via extracellular vesicles in response to lipid overload,” Nature Communications, vol. 11, no. 1, Article ID 719, 2020.
View at: Publisher Site | Google Scholar
X. Li, J. Luo, Y. Li et al., “Macrophage-derived exosomes in TLR9^−/− mice ameliorate sepsis-induced mitochondrial oxidative stress and apoptosis in cardiomyocytes,” Oxidative Medicine and Cellular Longevity, vol. 2022, Article ID 5719974, 13 pages, 2022.
View at: Publisher Site | Google Scholar
W. Zhang, J. Zhang, and H. Huang, “Exosomes from adipose-derived stem cells inhibit inflammation and oxidative stress in LPS-acute kidney injury,” Experimental Cell Research, vol. 420, no. 1, Article ID 113332, 2022.
View at: Publisher Site | Google Scholar
Z. Lv, S. Duan, M. Zhou et al., “Mouse bone marrow mesenchymal stem cells inhibit sepsis-induced lung injury in mice via exosomal SAA1,” Molecular Pharmaceutics, vol. 19, no. 11, pp. 4254–4263, 2022.
View at: Publisher Site | Google Scholar
K. Jiang, J. Yang, S. Guo, G. Zhao, H. Wu, and G. Deng, “Peripheral circulating exosome-mediated delivery of miR-155 as a novel mechanism for acute lung inflammation,” Molecular Therapy, vol. 27, no. 10, pp. 1758–1771, 2019.
View at: Publisher Site | Google Scholar
C. Flohr, R. J. Quinnell, and J. Britton, “Do helminth parasites protect against atopy and allergic disease?” Clinical and Experimental Allergy, vol. 39, no. 1, pp. 20–32, 2009.
View at: Publisher Site | Google Scholar
R. M. Maizels, H. H. Smits, and H. J. McSorley, “Modulation of host immunity by helminths: the expanding repertoire of parasite effector molecules,” Immunity, vol. 49, no. 5, pp. 801–818, 2018.
View at: Publisher Site | Google Scholar
K. J. Erb, “Can helminths or helminth-derived products be used in humans to prevent or treat allergic diseases?” Trends in Immunology, vol. 30, no. 2, pp. 75–82, 2009.
View at: Publisher Site | Google Scholar
L. Chen, B. He, W. Hou, and L. He, “Cysteine protease inhibitor of Schistosoma japonicum—a parasite-derived negative immunoregulatory factor,” Parasitology Research, vol. 116, no. 3, pp. 901–908, 2017.
View at: Publisher Site | Google Scholar
Y. Chen, B. Wei, P. Xu et al., “Schistosoma japonicum cystatin suppresses osteoclastogenesis via manipulating the NF-κB signaling pathway,” Molecular Medicine Reports, vol. 23, no. 4, 2021.
View at: Publisher Site | Google Scholar
S. Hartmann, B. Kyewski, B. Sonnenburg, and R. Lucius, “A filarial cysteine protease inhibitor down-regulates T cell proliferation and enhances interleukin-10 production,” European Journal of Immunology, vol. 27, no. 9, pp. 2253–2260, 1997.
View at: Publisher Site | Google Scholar
B. He, G. Cai, Y. Ni, Y. Li, H. Zong, and L. He, “Characterization and expression of a novel cystatin gene from Schistosoma japonicum,” Molecular and Cellular Probes, vol. 25, no. 4, pp. 186–193, 2011.
View at: Publisher Site | Google Scholar
F. Liu, W. Cheng, F. Pappoe et al., “Schistosoma japonicum cystatin attenuates murine collagen-induced arthritis,” Parasitology Research, vol. 115, pp. 3795–3806, 2016.
View at: Publisher Site | Google Scholar
H. Xie, L. Wu, X. Chen et al., “Schistosoma japonicum cystatin alleviates sepsis through activating regulatory macrophages,” Frontiers in Cellular and Infection Microbiology, vol. 11, Article ID 617461, 2021.
View at: Publisher Site | Google Scholar
W. Zhang, X. Bai, B. Zhao et al., “Cell-free therapy based on adipose tissue stem cell-derived exosomes promotes wound healing via the PI3K/Akt signaling pathway,” Experimental Cell Research, vol. 370, no. 2, pp. 333–342, 2018.
View at: Publisher Site | Google Scholar
C. Lässer, M. Eldh, and J. Lötvall, “Isolation and characterization of RNA-containing exosomes,” Journal of Visualized Experiments, vol. 59, Article ID e3037, 2012.
View at: Publisher Site | Google Scholar
Y. Tian, M. Gong, Y. Hu et al., “Quality and efficiency assessment of six extracellular vesicle isolation methods by nano-flow cytometry,” Journal of Extracellular Vesicles, vol. 9, no. 1, Article ID 1697028, 2020.
View at: Publisher Site | Google Scholar
L. Dejager, I. Pinheiro, E. Dejonckheere, and C. Libert, “Cecal ligation and puncture: the gold standard model for polymicrobial sepsis?” Trends in Microbiology, vol. 19, no. 4, pp. 198–208, 2011.
View at: Publisher Site | Google Scholar
F. V. Sjaastad, I. J. Jensen, R. R. Berton, V. P. Badovinac, and T. S. Griffith, “Inducing experimental polymicrobial sepsis by cecal ligation and puncture,” Current Protocols in Immunology, vol. 131, Article ID e110, 2020.
View at: Publisher Site | Google Scholar
S. Ito, Y. Tanaka, R. Oshino, S. Okado, M. Hori, and K.-I. Isobe, “GADD34 suppresses lipopolysaccharide-induced sepsis and tissue injury through the regulation of macrophage activation,” Cell Death & Disease, vol. 7, Article ID e2219, 2016.
View at: Publisher Site | Google Scholar
L. Wang, Y. Cao, B. Gorshkov et al., “Ablation of endothelial Pfkfb3 protects mice from acute lung injury in LPS-induced endotoxemia,” Pharmacological Research, vol. 146, Article ID 104292, 2019.
View at: Publisher Site | Google Scholar
Y.-F. Li, B.-Y. Xu, R. An et al., “Protective effect of anisodamine in rats with glycerol-induced acute kidney injury,” BMC Nephrology, vol. 20, Article ID 223, 2019.
View at: Publisher Site | Google Scholar
K. J. Livak and T. D. Schmittgen, “Analysis of relative gene expression data using real-time quantitative PCR and the 2^−ΔΔC_T method,” Methods, vol. 25, no. 4, pp. 402–408, 2001.
View at: Publisher Site | Google Scholar
J.-M. Cavaillon, M. Singer, and T. Skirecki, “Sepsis therapies: learning from 30 years of failure of translational research to propose new leads,” EMBO Molecular Medicine, vol. 12, no. 4, Article ID e10128, 2020.
View at: Publisher Site | Google Scholar
P. Qiu, Y. Liu, and J. Zhang, “Review: the role and mechanisms of macrophage autophagy in sepsis,” Inflammation, vol. 42, pp. 6–19, 2019.
View at: Publisher Site | Google Scholar
D. M. Pegtel and S. J. Gould, “Exosomes,” Annual Review of Biochemistry, vol. 88, pp. 487–514, 2019.
View at: Publisher Site | Google Scholar
E. Willms, C. Cabañas, I. Mäger, M. J. A. Wood, and P. Vader, “Extracellular vesicle heterogeneity: subpopulations, isolation techniques, and diverse functions in cancer progression,” Frontiers in Immunology, vol. 9, Article ID 738, 2018.
View at: Publisher Site | Google Scholar
M. E. Marcus and J. N. Leonard, “FedExosomes: engineering therapeutic biological nanoparticles that truly deliver,” Pharmaceuticals, vol. 6, no. 5, pp. 659–680, 2013.
View at: Publisher Site | Google Scholar
T. Gong, X. Zhang, Z. Peng et al., “Macrophage-derived exosomal aminopeptidase N aggravates sepsis-induced acute lung injury by regulating necroptosis of lung epithelial cell,” Communications Biology, vol. 5, Article ID 543, 2022.
View at: Publisher Site | Google Scholar
H. Kim, S. Y. Wang, G. Kwak, Y. Yang, I. C. Kwon, and S. H. Kim, “Exosome-guided phenotypic switch of M1 to M2 macrophages for cutaneous wound healing,” Advanced Science, vol. 6, no. 20, Article ID 1900513, 2019.
View at: Publisher Site | Google Scholar
W. Yan, T. Li, T. Yin et al., “M2 macrophage-derived exosomes promote the c-KIT phenotype of vascular smooth muscle cells during vascular tissue repair after intravascular stent implantation,” Theranostics, vol. 10, no. 23, pp. 10712–10728, 2020.
View at: Publisher Site | Google Scholar
V. Kumar, “Toll-like receptors in sepsis-associated cytokine storm and their endogenous negative regulators as future immunomodulatory targets,” International Immunopharmacology, vol. 89, Part B, Article ID 107087, 2020.
View at: Publisher Site | Google Scholar
H. Yang, H. Li, W. Chen et al., “Therapeutic effect of Schistosoma japonicum cystatin on atherosclerotic renal damage,” Frontiers in Cell and Developmental Biology, vol. 9, Article ID 760980, 2021.
View at: Publisher Site | Google Scholar
A. Castoldi, T. T. Braga, M. Correa-Costa et al., “TLR2, TLR4 and the MYD88 signaling pathway are crucial for neutrophil migration in acute kidney injury induced by sepsis,” PLOS ONE, vol. 7, no. 5, Article ID e37584, 2012.
View at: Publisher Site | Google Scholar
K. A. Fitzgerald and J. C. Kagan, “Toll-like receptors and the control of immunity,” Cell, vol. 180, no. 6, pp. 1044–1066, 2020.
View at: Publisher Site | Google Scholar
K. Schlatterer, C. Beck, D. Hanzelmann et al., “The mechanism behind bacterial lipoprotein release: phenol-soluble modulins mediate toll-like receptor 2 activation via extracellular vesicle release from Staphylococcus aureus,” mBio, vol. 9, no. 6, 2018.
View at: Publisher Site | Google Scholar
S. Bhatnagar and J. S. Schorey, “Exosomes released from infected macrophages contain Mycobacterium avium glycopeptidolipids and are proinflammatory,” Journal of Biological Chemistry, vol. 282, no. 35, pp. 25779–25789, 2007.
View at: Publisher Site | Google Scholar
H.-F. Bu, X. Wang, Y. Tang, V. Koti, and X.-D. Tan, “Toll-like receptor 2-mediated peptidoglycan uptake by immature intestinal epithelial cells from apical side and exosome-associated transcellular transcytosis,” Journal of Cellular Physiology, vol. 222, no. 3, pp. 658–668, 2010.
View at: Publisher Site | Google Scholar
F. Huang, Y. Qian, H. Li et al., “Exosomes derived from Schistosoma japonicum cystatin treated macrophages attenuated CLP-induced sepsis in mice,” Research Square, 2022.
View at: Publisher Site | Google Scholar

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Copyright © 2024 Feifei Huang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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