Sepsis remains one of the leading causes of mortality in the world (1–3). Vigorous immune responses in this circumstance are beneficial to eradicate invading pathogens; however, the hyperactive and imbalanced cytokine network may lead to tissue damage, multiple organ dysfunction, and even death. In recent decades, many biomodulators have been developed to suppress inflammation; however, none have been demonstrated to be beneficial for patients with sepsis (4). Therefore, it is important to develop innovative and efficacious strategies to bring the immune responses back into balance to ultimately improve the outcomes of septic patients.
Mesenchymal stem cells (MSCs) are considered a promising platform for cell-based therapy. Given their immunomodulatory properties, MSCs are being investigated to prevent and treat clinical diseases associated with aberrant immune responses (5–9). With plasticity in immunomodulatory function, MSCs orchestrate pathogen clearance during sepsis through promotion of immune cell survival and function followed by attenuating systemic inflammation to preserve host integrity and augment tissue repair (7–10). In vivo benefits of MSC administration in the context of sepsis have been demonstrated (11–15); however, the mechanism of MSC-mediated regulation during sepsis has yet to be elucidated.
In pathogen-associated molecular patterns, the activation of Toll-like receptors (TLRs) in the early phase of infection has been reported to be essential for immediate host responses to microbial invasion and also linked to innate and adaptive immunity (16–18). To date, 11 members of the TLR family have been identified in mammals (19). TLR4 has been reported to have specificity for bacterial lipopolysaccharides and related compounds present on the outside of gram-negative bacteria. Sensing the presence of infection by TLR4 has been reported to lead to production of inflammatory cytokines through two different signaling cascades: myeloid differentiation factor 88 (MyD88)-dependent and MyD88-independent pathways (19,20). The MyD88-dependent pathway involves the early phase of nuclear factor-κB (NFκB) activation, and the MyD88-independent pathway activates interferon regulatory factor 3 (IRF3) and involves the late phase of NFκB activation.
The aim of this study was to examine the immunomodulatory effect of MSCs on mice with sepsis. Despite the important role of TLR4 signaling in sepsis, no studies have reported the effects of MSCs on this signaling pathway in this circumstance. Therefore, we investigated the mechanism of MSC-mediated immunomodulation by determining the time-series expression of TLR4 signaling in the liver and circulating cytokines in mice after cecal ligation and puncture (CLP)-induced sepsis.
MATERIALS AND METHODS
Isolation and identification of UCMSCs
This study was approved by the Institutional Review Board of Chung Shan Medical University Hospital (CS 14103), and written informed consent was obtained from the donor. The umbilical cord-derived MSCs (UCMSCs) were collected, isolated, and identified as in our previous reports (21–24), and the procedure was described in the supplement (Supplemental Digital Content 1, https://links.lww.com/SHK/A355).
CLP model of sepsis in mice
The experimental protocol was approved by the Institutional Animal Care and Use Committee of the Chung Shan Medical University Experimental Animal Center (IACUC Approval No: 1598). Six-week-old male C57BL/6 mice were provided by the National Science Council. A well-established CLP model of polymicrobial sepsis, which closely resembles the process of septic peritonitis in humans (25), was used in this study. The detailed procedure of CLP is described in the supplement (Supplemental Digital Content 1, https://links.lww.com/SHK/A355).
To evaluate the effects of UCMSC administration after CLP-induced sepsis, mice in the UCMSC group received intraperitoneal injections of one million UCMSCs in 0.5 mL sterile phosphate-buffered saline (PBS; Gibco, Gaithersburg, MD) immediately after CLP. In vitro cultured UCMSCs of passage 5 were used. Mice in the sham control group and PBS group received sterile PBS in a volume of 0.5 mL with no cells at the same time point.
Survival after CLP or sham operation was assessed every 6 h for 4 days. For antimicrobial therapy of polymicrobial bacterial infections, subcutaneous injections of imipenem (0.5 mg/day) were given immediately after surgery and then repeated every 24 h for 4 days. All mice were sacrificed at the end of the fourth day.
Assessment of TLR4 activation in the liver
In response to infections, TLR4-mediated reactions are an important part of the innate immune defense. After activation, TLR4 signals through two signaling pathways, MyD88-NFκB and Toll receptor-associated molecule (TRAM)-IRF3. To evaluate the role of TLR4 signaling in sepsis, the mice were euthanized at 0, 1, 2, 3 and 6 h after surgery depending on the experiment. The time-series expressions of MyD88, NFκB, TRAM, and IRF3 mRNA and protein levels in the liver tissues were measured. The detailed procedure is described in the supplement (Supplemental Digital Content 1, https://links.lww.com/SHK/A355).
Determination of serum cytokine levels
To determine the levels of circulating cytokines, serum was separated by centrifugation at 10,000 g for 10 min at 4°C, aliquoted, and stored at −80°C until assayed. The concentrations of monocyte chemotactic protein (MCP)-1, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin (IL)-6, and IL-10 were measured by cytometric bead array immunoassay (BD CBA Mouse Soluble Protein Flex Set System; BD Biosciences, San Jose, CA), according to the manufacturer's instructions. Data were analyzed using flow cytometry (FACSCanto; BD Biosciences, San Jose, CA) with FCAP Array software.
Data analysis was performed using SPSS 16.0 for Windows. Survival was analyzed with Kaplan–Meier survival curves, and the log-rank test was used to compare the survival curves of the PBS and UCMSC groups. For continuous variables, intergroup differences were tested by one-way ANOVA, and the Games–Howell test for post hoc analysis. A value of P <0.05 was considered to be statistically significant.
In vitro, UCMSCs adhered to culture plates and showed a spindle-shaped fibroblastic morphology (Fig. 1A). They expressed CD29, CD44, CD73, CD90, CD105, HLA-A, HLA-B, and HLA-C, and were negative for CD34, CD45, CD14, and HLA-DR. Under induction conditions, the UCMSCs were capable of achieving osteogenic and adipogenic differentiation (Fig. 1, B and C). These findings were in accordance with the International Society for Cellular Therapy criteria (26).
The effects of UCMSC administration on survival in the mice after CLP-induced sepsis with and without antimicrobial therapy were then investigated (Fig. 2). In the context of antibiotic co-administration, UCMSC administration significantly improved survival of the mice after CLP compared with those receiving PBS only (P = 0.032; Fig. 2A). In the absence of appropriate antimicrobial therapy, the beneficial effects on survival from UCMSC administration disappeared (P = 0.374; Fig. 2B).
Assessment for TLR 4 activation and histopathologic changes in the liver
TLR4-mediated reactions are important for innate immune defense, and TLR4 activation in response to infections involves the MyD88-NFκB and TRAM-IRF3 signaling pathways. To evaluate the time course of TLR4 activation and the effects of UCMSC administration during sepsis, the expression levels of MyD88, NFκB, TRAM, and IRF3 in the liver were measured at 0, 1, 2, 3, and 6 h after CLP or sham operation. Using Q-PCR, the expression level of MyD88 mRNA was significantly increased at 6 h in the mice after CLP or sham operation (Fig. 3A), implicating that the activation of the MyD88-NFκB pathway started between 3 and 6 h after surgery. At 6 h after surgery, the expression level of MyD88 mRNA was significantly elevated in the mice of the PBS group compared with the sham-operated mice (P = 0.010). In contrast, the administration of UCMSCs after CLP decreased the expression of MyD88 mRNA compared with the mice receiving PBS only (P = 0.041). Consistent with these results, the down-stream activated transcription factor, NFκB, had an increased ratio of phosphorylation as measured by ELISA 6 h after surgery in the mice in the PBS group (Fig. 3B). Furthermore, the expression of MyD88 protein in the liver was evaluated by Western blotting (Fig. 4) and immunohistochemical staining (Fig. 5). Consistently, the expression of MyD88 protein at 6 h after CLP was significantly decreased in the mice receiving UCMSCs compared with those receiving PBS only. These results suggested that UCMSCs may exert their immunomodulatory influence on septic mice via the MyD88-NFκB pathway.
The activation of TRAM-IRF3, the other signaling pathway after TLR4 activation in response to an infection, was also assessed by Q-PCR, Western blotting, and immunohistochemical analysis (Figs. 3C, D, 4, and 5). By 6 h after CLP, there was no significant difference in the expression levels of TRAM or IRF3 between the mice receiving UCMSCs and those receiving PBS only. We speculated that UCMSC-mediated immunomodulation during sepsis may not be via the TRAM-IRF3 pathway in this animal model.
Hepatic injury after CLP-induced sepsis was also evaluated histopathologically at each time point. There were no obvious histopathologic findings of hepatic injury by 6 h after surgery in any of the mice (Fig. 5), suggesting that the activation of the MyD88-NFκB pathway in the liver tissues and the immunomodulatory effects from UCMSCs occurred prior to hepatic injury.
Time-series changes of serum cytokine profiles
To assess the time-series changes in circulating inflammation-associated cytokines in the mice after CLP-induced sepsis and the influence of UCMSC administration, serum concentrations of MCP-1, TNF-α, IFN-γ, IL-6, and IL-10 were measured at 0, 1, 2, 3 and 6 h after CLP or sham operation (Fig. 6). Among these cytokines, elevation of serum TNF-α levels occurred earlier in response to CLP-induced sepsis. Compared with the sham-operated mice, serum TNF-α levels were significantly elevated in the mice 3 h after CLP, whether or not they received UCMSCs. Of note, the serum TNF-α levels continued to increase in the mice of the PBS group, whereas the TNF-α levels were not higher 6 h after CLP in the mice that received UCMSCs. There are two possible explanations for these findings. First, UCMSCs could rescue septic mice from the exacerbated inflammatory status, and second, UCMSCs began to exert an immunomodulatory effect between 3 and 6 h after injection. Consistent with these hypotheses, serum levels of MCP-1, IFN-γ, and IL-6 at 6 h after CLP were significantly lower in the mice receiving UCMSCs than in those receiving PBS only. In addition, a significant increase in serum levels of IL-10, an anti-inflammatory cytokine, was noted in the mice of the UCMSC group at the same time point.
As TLR4 is the first-line effector molecule in innate immunity to infections, we investigated the effects of UCMSC administration in mice with sepsis by studying the time-series expression of TLR4 signaling after CLP. We found increased expression levels of MyD88 mRNA and protein in the liver at 6 h but not before 3 h after CLP operation, suggesting that activation of the MyD88-NFκB pathway started between 3 and 6 h after CLP-induced sepsis. Of interest, MyD88 expression was lower at 6 h after CLP in the mice receiving UCMSCs compared with those receiving PBS only, indicating the immunomodulatory effect of UCMSCs during sepsis. Accordingly, UCMSCs also began to exert an effect via this pathway between 3 and 6 h after administration. Consistently, the proportion of phosphorylated NFκB, which is a down-stream activated transcription factor, was significantly decreased in the mice receiving UCMSCs compared with those receiving PBS only after CLP. This study is the first to show in vivo evidence for the association of the MyD88-NFκB pathway and MSC-mediated immunomodulation during sepsis. This study is also the first to show the time course of TLR4 activation during sepsis.
Activation of TLR4 signaling in response to infections leads to synthesis and secretion of a variety of inflammatory cytokines through two different pathways (19,20). Signaling through MyD88 results in activation of the transcription factor NFκB and the production of inflammatory cytokines such as IL-6, TNF-α, and IFN-γ. IFN-γ is also called type II interferon. On the other hand, TLR4 signaling through TRAM-IRF leads to the production of type I interferons. A multiorgan NFκB-dependent response was demonstrated to be readily activated upon challenge of lipopolysaccharides (27). Several studies found that deficient NFκB activation resulted in immunosuppression which triggers and maintains inflammation (28,29), suggesting that inhibition of massive NFκB activation could reduce inflammatory responses. In the present study, lower serum levels of TNF-α, MCP-1, IFN-γ, and IL-6 were noted in the mice receiving UCMSCs compared with those receiving PBS only at 6 h after CLP. These findings indicate that the administration of UCMSCs into the septic mice brought the hyperinflammatory immune responses back into balance and the MyD88-NFκB pathway played an important role in response to the immunomodulatory signals from the UCMSCs.
It is interesting to observe that administering UCMSCs into the septic mice had a positive impact on survival with the co-administration of antibiotics; however, there was no benefit on survival from UCMSC administration when no appropriate antibiotics were used. Indeed, supportive care with antimicrobial agents to control infections is the foundation of management in patients with sepsis (30,31). However, pathophysiological processes of a vast array of pro-inflammatory and counterinflammatory cytokines occur during sepsis, and the disordered and chaotic relationship of these cytokines is harmful (4). As not unilaterally immunosuppressive, the immunomodulatory activity of MSCs can be modified with active crosstalk from their microenvironment (10,32). MSCs have been demonstrated to augment antimicrobial responses, reduce microbial burden, abridge pro-inflammatory and damage responses, ameliorate host tissue injury, and prolong host survival in the context of sepsis (33). Our results suggested that UCMSC-associated immunomodulation could provide a microenvironment with relatively steady concentrations of inflammation-associated cytokines for bacterial clearance, and also attenuates collateral self-tissue damage from hyperinflammation during sepsis.
The impact of MSCs of various origins in septic animals has been studied, and MSCs have been found to decrease in vivo infectious burden and prolong host survival regardless of whether they were derived from allogeneic/xenogeneic or bone marrow/non-bone-marrow (11–15). Bone marrow is considered to be the traditional source of MSCs; however, an important issue in cellular therapy is to find alternative cell sources with better availability. Umbilical cords are rich in MSCs which can be easily collected and cultured (34). Compared with bone marrow MSCs that require an invasive and painful procedure to harvest, obtaining UCMSCs is safe for both mother and baby. In our previous study, UCMSCs were found to have a greater proliferative potential and faster growth rate in vitro(21), indicating the advantages of rapid expansion and consequent downstream application. In addition, we also demonstrated a higher immunomodulation potential from UCMSCs (21,23). In humans, we found that UCMSCs can promote hematopoietic engraftment after hematopoietic stem cell transplantation and treat refractory graft-versus-host disease effectively and safely (22,23,35,36). Therefore, the umbilical cord could represent a feasible source of MSCs for clinical application.
In conclusion, this study provides the first in vivo evidence for the association of the MyD88-NFκB signaling pathway and MSC-mediated immunomodulation during sepsis. UCMSCs started to exert an immunomodulatory effect between 3 and 6 h after administration, and the MyD88-NFκB pathway played an important role in response to immunomodulatory signals from the UCMSCs. UCMSCs were beneficial in decreasing mortality associated with CLP-induced sepsis, and this was probably because UCMSCs could bring the hyperinflammatory immune responses back into balance in the septic mice. Owing to the effectiveness in the CLP model and successful application in humans, the use of UCMSCs may open up new perspectives for cell-based therapy of sepsis.
This study was supported by Chung Shan Medical University Hospital (CSH-2015-C-021), China Medical University Hospital (DMR-104-030), the Research Laboratory of Pediatrics, Children's Hospital, China Medical University, and the Taiwan Ministry of Health and Welfare Clinical Trial and Research Center of Excellence (MOHW104-TDU-B-212-113002).
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