Sepsis is one of the leading causes of death in the worldwide, particularly in the developing countries, and the costs to health care systems are huge (1). Sepsis is the most common cause of death in the intensive care unit, and sepsis-associated acute kidney injury (SA-AKI) is an independent risk factor for mortality. Its occurrence increases the complexity and costs of treatment (2). Currently, there are no effective methods to treat or prevent SA-AKI. Therefore, new ways to prevent SA-AKI are urgently needed.
The pathophysiology of sepsis includes interactions between the microbial pathogen and the host immune system, as well as inflammatory and coagulation responses (3). However, our understanding of the pathogenesis of SA-AKI is still limited. In the past few years, most related studies have focused on hemodynamic factors after bacterial invasion. Recently, new mediators, such as interleukin 17 (IL-17), have been discovered in sepsis. Interleukin 17 is mainly produced by T cells known as TH17, γδT cells (4). It has been demonstrated that IL-17 is involved in neutrophil recruitment via the production of several cytokines and CXC chemokines, such as CXCL1, CXCL5, and macrophage inflammatory protein 2 (CXCL2) (5–8). The receptor for IL-17 is expressed in various types of cells, such as endothelial cells, macrophages, and stromal cells. These cells produce diverse proinflammatory cytokines and chemokines in response to IL-17, which mediates inflammation and induces neutrophil recruitment to inflammatory sites (8). This suggests that IL-17 plays a uniquely powerful role in the inflammatory system. Thus, it is not surprising that the IL-17 family has been shown to be involved in various physiological and pathophysiological processes including sepsis (9, 10). The level of IL-17 gradually increases in serum during the inflammatory responses to sepsis, further suggesting that IL-17 could be an important pathogenic factor in SA-AKI. Inhibition of the action of IL-17 may therefore be a new direction for the treatment of SA-AKI. However, little is known about the proinflammatory effect of IL-17 in SA-AKI.
Mesenchymal stem cells (MSCs) have been shown to secretion of paracrine-acting angiogenic, trophic, anti-inflammatory, and immunomodulatory factors. Accordingly, MSC-based therapies are being evaluated for treatment of ischemic, inflammatory, and immunological disorders (11). Previous studies indicated that injection of MSCs both ameliorates renal injury and accelerates repair, such as ischemia-reperfusion (IR) or cisplatin-induced damage (12, 13). Several lines of evidence suggest that MSCs may also exhibit immunosuppressive or immunomodulatory properties, such as inhibition of αβT-cell and natural killer cell proliferation induced by mitogens or cognate peptides (14, 15). Mesenchymal stem cells also inhibit the differentiation and maturation of dendritic cells (16, 17). Moreover, MSCs are potent suppressors of γδT-cell proliferation, cytokine production, and cytolytic responses in vitro (18). Previous studies have shown that MSCs can ameliorate experimental sepsis and sepsis-induced acute lung injury (19, 20). Because acute lung injury, SA-AKI, and sepsis are all associated with an exaggerated inflammatory response, we hypothesized that MSCs would exert a beneficial effect in polymicrobial model of sepsis and SA-AKI. This study was designed to examine the beneficial effect of MSCs in SA-AKI mice. To investigate the mechanism by which MSCs exert their therapeutic effect in SA-AKI mice, we examined the inflammatory status of serum and kidney tissue, specifically the levels of the cytokine IL-17 and its related chemokines.
MATERIALS AND METHODS
Male C57Bl/6 mice weighing 25 to 30 g were housed in an animal facility with 12-h light-dark cycle at 22°C and free access to food and water. They were acclimated for 1 week before conducting experiments. All experimental procedures were approved by the Ethics Committee of The General Hospital of the People’s Liberation Army.
Culture of MSCs
C57Bl/6 mouse bone marrow–derived MSCs/red fluorescent protein (RFP) were purchased from Cyagen Biosciences (Sunnyvale, Calif). The culture process was initiated according to the manufacturer’s instructions. The sixth, eighth, and tenth passage MSCs were used for experiments.
Mouse model of cecal ligation and puncture
Cecal ligation and puncture (CLP)–induced sepsis was generated as described previously (21). Mice were anesthetized with 2% pentobarbital, and a 1- to 2-cm midline incision was made along the linea alba of the abdominal muscle to isolate and exteriorize the cecum. Seventy-five percent of the cecum was ligated with a 4-0 silk suture, and the cecum was punctured twice with a 21-gauge needle. A small amount (droplet) of feces was gently extruded from the penetration holes to ensure patency. The cecum was then returned to the peritoneal cavity, and the abdominal incision was closed with 4-0 silk sutures. After the operation, 1 mL prewarmed normal saline was administered into the peritoneal cavity. In the sham group, mice underwent the same procedure but were neither ligated nor punctured.
After 3 h of CLP, mice were injected 1 × 106 MSCs in 0.2 mL normal saline by tail vein (22). As a control, the CLP mice were administered only 0.2 mL normal saline. Mice were divided into three groups: sham, CLP, and CLP + MSCs.
Survival after surgery was assessed every 24 h for 7 days with n = 20 in each group.
Bacterial load determination
Blood was obtained from the vena cava at 24 h after CLP surgery (n = 6 in each group). Following 1:3 dilution in sterile phosphate-buffered saline, 100 µL samples were plated on 5% sheep blood agar plates (Remel, Lenexa, Kans). Plates were incubated for 24 h at 37°C under aerobic conditions; colony enumeration was then counted and multiplied by the dilution factor.
Assessment of kidney injury
Kidney injury was assessed by measuring the levels of serum creatinine and blood urea nitrogen and calculating the changes in levels. Blood samples were collected from the vena cava at the indicated time points (n = 10 in each group), and the serum was separated by centrifugation at 3,000 revolutions/min for 15 min at 4°C and then stored at −20°C until use.
Kidneys were harvested at 24 h after CLP. Specimens were fixed in 10% formaldehyde for at least 24 h. After dehydration in a series of ascending concentrations of ethanol, the samples were embedded in paraffin and then sectioned at 3-mm thickness for staining with periodic acid–Schiff. Stained sections were observed by light microscopy (Olympus IX71, Tokyo, Japan) at a magnification of 400× and were evaluated by three independent pathologists. Histological examinations for acute tubular necrosis were semiquantified using the following scoring system as described previously (22): 0 = none, 1 = less than 10%; 2 = 11% to 25%, 3 = 26% to 45%, 4 = 46% to 75%, and 5 = greater than 76%.
Enzyme-linked immunosorbent assay
Animals were anesthetized and killed at 24 h (n = 6 per group). Specific detection of IL-6, IL-17, tumor necrosis factor α (TNF-α), interferon γ (IFN-γ), and IL-10 was accomplished by sandwich enzyme-linked immunosorbent assay according to the manufacturer’s instructions (R&D Systems, Minneapolis, Minn).
Real-time polymerase chain reaction
Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, Calif) following the manufacturer’s instructions. The TaqMan Reverse Transcription Kit (Applied Biosystems, Foster City, Calif) and a Gene Amp polymerase chain reaction (PCR) System 9700 (Applied Biosystems) were used for generation of cDNA. Polymerase chain reaction was performed with the SYBR Green Mastermix and a 7500 Real-time PCR System (Applied Biosystems). Results were analyzed by the 2−ΔΔCT method with normalization to glyceraldehyde 3-phosphate dehydrogenase expression (n = 6 for each group). The primers used are listed in Table 1.
Immunofluorescence studies of kidney samples
For use in immunofluorescence studies, 4-µm-thick sections were cut and then incubated overnight with rat anti-Ly6G (1:100; Santa Cruz Biotechnology, Santa Cruz, Calif) antibody in a humidified chamber. After washing with phosphate-buffered saline, secondary antibodies conjugated with fluorescein isothiocyanate goat anti–rat immunoglobulin G (1:50; Jackson ImmunoResearch Laboratories; West Grove, Pa) were applied for 1 h at room temperature in a darkened humidified chamber. Nuclei were stained with DAPI (Zhongshan Goldenbridge Biotechnology, Beijing, China). Each tissue section was observed with light microscopy at a magnification of 400× (n = 6 for each group).
Two-photon fluorescence confocal imaging of MSCs after injection
We traced the distribution of MSCs after injection with a Leica two-photon fluorescence confocal imaging TCS SP5 system (Leica Microsystems, Mannheim, Germany). Mice were killed at 3, 6, 12, 18, and 24 h (n = 3 in each group at each time point; experiments repeated three times). Fresh, thick tissues of the right lung, right lower liver, spleen, and peritoneal cavity lymph node were sampled.
Data are expressed as mean ± SEM. Multiple comparisons of parametric data were performed using one-way analysis of variance followed by Student-Newman-Keuls post hoc tests. Expression levels of mRNA were calculated using the 2−ΔΔCT method. Survival of the two subgroups was estimated using a Kaplan-Meier analysis. Analyses were completed using SPSS for Windows (version 10.1; SPSS, Chicago, Ill). Statistical significance was defined as P < 0.05.
Effect of treatment with MSCs on the survival of CLP-induced septic mice
In the absence of antibiotic therapy, we attempted to investigate whether the mice injected with MSCs show any improvement in terms of survivability. The survival rate of the CLP group was only 30% at 7 days, whereas it increased by nearly 100% in the MSC treatment group (58% mice surviving) (Fig. 1). Treatment with MSCs significantly improved the survival rate compared with that in septic mice that received normal saline (P < 0.05).
Effect of treatment with MSCs on bacterial load of CLP-induced septic mice
To understand the mechanism of intravenous injection of MSCs on CLP-induced septic mice, we examined blood bacterial load in CLP mice at 24 h after CLP injury. Mice that received MSCs 3 h after CLP surgery exhibited significantly lower blood bacterial load compared with mice that received normal saline (P < 0.05; Fig. 2).
Effects of treatment with MSCs on SA-AKI
Sepsis-associated AKI was determined by measuring biochemical indicators in blood samples taken at 24 h. Levels of blood urea nitrogen and serum creatinine increased in mice with CLP-induced sepsis (269 ± 46 and 0.82 ± 0.16 mg/dL, respectively) compared with the sham group (25.2 ± 3.83 and 0.11 ± 0.01 mg/dL, respectively), indicating the development of AKI. Mesenchymal stem cell treatment dramatically reduced serum creatinine and blood urea nitrogen levels (P < 0.05; Fig. 3, A and B), compared with mice that received normal saline. Morphological changes in the CLP group were scored 24 h after MSC treatment by brush border loss, tubular degeneration, and vacuolization in the proximal tubules. Treatment with MSCs ameliorated the morphological damage and reduced the tubular injury score (P < 0.05; Fig. 3, C–F). Taken together, our data showed that CLP-induced polymicrobial sepsis resulted in AKI, and treatment with MSCs reduced the degree of AKI.
Effect of treatment with MSCs on the expression of cytokines in plasma
The CLP surgery led to significant increases in plasma levels of proinflammatory cytokines, including IL-6, IL-10, IL-17, IFN-γ, and TNF-α (Fig. 4), which are all indicators of the immune inflammatory status. Treatment with MSCs significantly decreased the level of IL-17, IL-6, IFN-γ, and TNF-α in plasma at 24 h (P < 0.05) compared with the CLP group. However, the level of IL-10 was significantly increased (P < 0.05) compared with that in the CLP group.
Effect of treatment with MSCs on renal mRNA expression of cytokines and chemokines
To investigate whether MSC treatment could directly affect local renal production of cytokines and chemokines, we examined the renal level of these cytokines using reverse transcriptase–PCR. Our results showed that the mRNA expression levels of IL-17, IL-6, IFN-γ, TNF-α, and IL-10 were significantly increased in both the CLP and MSC treatment groups at 24 h (Fig. 5). Compared with the untreated CLP group, treatment with MSCs decreased the expression levels of IL-17, IL-6, IFN-γ, and TNF-α (P < 0.05), but increased the expression level of IL-10 (P < 0.05; Fig. 5).
Previous studies showed that IL-17 in peritoneal fluid plays a critical role during severe polymicrobial sepsis. We speculated that IL-17 might play an important role in SA-AKI. We examined the mRNA expression levels in the kidney of the chemokines related to IL-17 function using reverse transcriptase–PCR. The levels of CXCL1, CXCL2, CXCL5, CCL2, and CCL3 were dramatically increased in the CLP group and were significantly decreased by treatment with MSCs (P < 0.05; Fig. 5).
Effect of treatment with MSCs on the infiltration of inflammatory cells
To test whether neutrophil infiltration plays a role in our model, we examined the degree of neutrophil infiltration in the kidney using immunofluorescence. In the sham group, there was no detectable Ly-6G–positive cell infiltration (Fig. 6A). In the CLP group, there were many Ly-6G–positive cells in the renal tissue (Fig. 6B). These cells were mainly detected in the renal cortex, including the glomerulus and tubulointerstitium. Compared with the CLP group, the MSC-treated group exhibited less neutrophil infiltration (Fig. 6, C and D).
Distribution of intravenously infused MSCs
The location of MSCs should aid in the elucidation of the molecular mechanism by which MSCs alleviate the damage that occurs during SA-AKI. Two-photon microscopy was used to evaluate the distribution of RFP-labeled MSCs after injection into the mice with CLP surgery. Images were acquired at 3, 6, 12, and 24 h after cell injection. Mesenchymal stem cells were mainly found in the spleen, lymph nodes, and lung after injection and persisted for at least 24 h with nearly no signal detected in the kidney throughout this period (Fig. 7). These results suggest that MSCs perform their protective function through paracrine secretions rather than cell-to-cell contacts.
The results from the present study show that bone marrow–derived MSCs moderate SA-AKI and reduce mortality in a clinically relevant model of polymicrobial sepsis induced by CLP. The improvement in SA-AKI was associated with an overall reduction in the inflammatory response, specifically a reduced production of IL-17.
Mesenchymal stem cells have been shown previously to preserve renal function after CLP in mice when administered up to 24 h before CLP (23). Those studies were extended here by determining the effects of MSCs when administered 3 h after the onset of CLP. Mesenchymal stem cells injected 3 h after CLP injury still have beneficial effects. Because treatments for septic patients usually begin after the onset of symptoms, Therefore, our results might be more relevant in guiding the clinical treatment of sepsis and SA-AKI.
In our study, we found that MSCs significantly reduced CLP-induced blood bacterial load, consistent with previous findings that MSCs have the ability to reduce bacterial burden in inflammation model. Mei et al. (24) showed that MSCs have the ability to reduce organ and peritoneal space bacterial burden in CLP model. Krasnodembskaya et al. (25) reported that MSCs as well as their conditioned medium possess direct antimicrobial activity in Escherichia coli pneumonia mice model, which is mediated in part by the secretion of LL-37. Although we did not examine the change of phagocytic capacity after MSC injection in CLP mice, we found reduced neutrophil infiltration in kidney and protective effect of SA-AKI after MSC treatment. Therefore, we speculated that MSCs might secrete antimicrobial peptides to reduce circulating bacterial loads in CLP mice model.
The pathogenesis of SA-AKI is not well understood. The inflammatory response inherent to sepsis has been examined as a direct mechanism of AKI. The kidney is particularly sensitive to mediator-induced damage. In this study, we demonstrated that not only the plasma levels of TNF-α, IL-6, IFN-γ, IL-17, and IL-10 significantly increased in parallel with the kidney injury, but also the mRNA levels of these cytokines in renal tissue increased at 24 h after CLP injury. These results show that the status of inflammation plays an important role in the pathogenesis of sepsis and SA-AKI. Interleukin 17 has been found to be elevated in a variety of inflammatory conditions. Our results show that the level of IL-17 in serum was significantly increased along with the CLP operation, especially at 24 h after CLP. The mRNA level of IL-17 in the kidney was also significantly increased at this time point. The infiltration of neutrophils in AKI has been demonstrated in previous studies (26, 27). Neutrophil infiltration was increased in the kidneys in our model. Levels of chemokines related to IL-17, such as CXCL1, CXCL2, CXCL5, CCL2, and CCL3, were also increased. Interleukin 17 acts on various cell types, including neutrophils, fibroblasts, epithelial cells, and endothelial cells. It also induces the expression of proinflammatory mediators such as IL-6, CXC chemokines, and matrix metalloproteinases (28, 29). Moreover, IL-17 has been reported to be a major orchestrator of sustained neutrophil mobilization and its associated activity through the induction of specific cytokines and colony-stimulating factors in resident lung cells (30). Collectively, this recruitment of neutrophils by IL-17 seems, in part, to be regulated by enhanced expression of chemokines (31). In our study, the levels of IL-17 and the recruitment of neutrophils in the kidney were all significantly increased. Therefore, IL-17 may be an important pathogenic factor in SA-AKI, and inhibiting IL-17 could be a new therapeutic target for the treatment of SA-AKI.
Interleukin 10 is a potent inhibitory cytokine that downregulates the monocyte inflammatory and procoagulant responses. Elevated levels of IL-10 have been detected in serum of patients with sepsis and after injection of lipopolysaccharide in mice. In our study, we also found increased levels of IL-10. It has been proposed that endogenously produced IL-10 serves a protective function in models of endotoxic shock. Thus, induction of IL-10 may be a protective measure.
Treatment with MSCs in an IR injury model resulted in significantly decreased expression levels of proinflammatory cytokines, such as IL-1β, TNF-α, and IFN-γ 24 h after IR injury and improved renal function (32, 33). Here we found that injection of MSCs 3 h after CLP led to decreased expression of TNF-α, IFN-γ, and IL-6, but increased IL-10 levels in both the plasma and renal tissues. Treatment with MSCs can reduce the serum and kidney tissue expression of IL-17 and IL-17–related chemokines. It can also reduce the infiltration of neutrophils in the CLP kidney. In this regard, our results are in line with previous reports that MSCs decrease proinflammatory cytokines and increase anti-inflammatory cytokines, which may explain their beneficial effects in SA-AKI. However, the detailed mechanisms of MSCs are unknown. Previous study showed that MSCs were able to suppress the proliferation and activation of CD4+ T cells and suppress CD4+ T cells differentiate into TH1 and TH17 cells. These suppressive effects were associated with the increase in CD4+CD25+Foxp3+ regulatory T cells and IL-10 secretion (34). Mesenchymal stem cells also could inhibit TH17 cell differentiation by IL-10 secretion (35). Duffy et al. (36) showed that MSC inhibition of TH17 cell differentiation is triggered by cell-cell contact. Jung et al. (37) showed that MSCs protected pancreatic function through reducing the infiltration of CD3+ T cells and upregulating Foxp3+ regulatory T cells into the injured pancreatic tissue in pancreatitis rat models. In our study, even though we did not examine the changes of immune cells after the MSC intervention, we found that MSCs could balance the status of inflammation and MSCs mainly homed to lymphoid organs. Therefore, we speculate that MSCs may directly interact with lymphocytes and regulate the secretion of IL-17 and IL-10. Our results may provide new insights into the endogenous immune responses after SA-AKI in mice treated with MSCs.
How MSCs exert their beneficial effects is presently poorly understood. Studies suggest that the distribution of MSCs might be essential for its beneficial effect. A previous study reports that MSCs can home to the damaged kidney to protect the kidney from further injury (33). However, recent studies found that intravenously injected MSCs mainly homed to the lungs and liver (22, 23). In this study, intravenously infused MSCs mainly accumulated in the lungs as “emboli” and in the spleen and lymph node as “debris.” The reason that the vast majority of intravenously injected MSCs accumulated in the lungs is unknown. We propose that the tissue-specific distribution may be due to the size of MSCs relative to pulmonary capillaries, which may prevent MSCs from passing through the pulmonary circulation. Mesenchymal stem cells accumulated in the spleen and lymph node as “debris” because MSCs were damaged by local macrophages. Although MSCs can protect against SA-AKI, no MSCs were observed in the injured kidney. In this study, we also demonstrated for the first time that intravenously injected MSCs accumulated in the peritoneal cavity lymph node. Our previous studies found that MSCs attenuated ischemic AKI by inducing regulatory T cells through splenocyte interactions (22). Therefore, we speculate that the protective effect of MSCs in SA-AKI occurs by paracrine secretions, rather than cell-to-cell contacts. This may also occur through interactions with lymphoid organs, including the spleen and peritoneal cavity lymph node, which modulate inflammatory or immune reactions.
There are some limitations in our study. Although we observed decreased levels of IL-17 and related cytokines and chemokines in renal tissues after treatment of CLP mice with MSCs, the mechanism through which MSCs regulate IL-17 remains unknown; MSCs did not home to kidney, so it is unclear how they could protect against SA-AKI. Our group has reported that MSCs might interact with splenocytes and protect against ischemic AKI, although the exact crosstalk between MSCs and splenocytes is unclear (22).
In summary, our study demonstrates that treatment with MSCs could protect against SA-AKI. Interleukin 17 may be involved in the pathogenesis of SA-AKI. Injection of MSCs can reduce the expression level of IL-17 in the kidneys, which could reduce the infiltration of inflammatory cells and maintain the balance between proinflammatory and anti-inflammatory status. Based on our work, MSCs may be a potentially effective treatment for SA-AKI.
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