Wang Jinglin*, Ren Haozhen*, Yuan Xianwen, Ma Hucheng, Shi Xiaolei#, Ding Yitao#
Abstract
Aims: Recently, the benefit of mesenchymal stem cells (MSCs) as a cell-based therapy for acute liver failure (ALF) has gained much attention, although the mechanism of action of MSCs in the treatment of ALF remains elusive. Pyroptosis is a novel form of programmed cell death with an intense inflammatory response. The aim of the present study was to explore the soluble cytokines secreted by MSCs and their therapeutic effects via inhibiting pyroptosis in ALF. Methods: MSCs obtained from C57BL/6 mice were isolated and cultured according to an established protocol. MSCs were transplanted into mice with D-galactosamine (Gal)-induced ALF. Liver function, survival rate, histology and inflammatory factors were determined. Exogenous recombinant rat interleukin (IL)- 10, ShIL-RNA and MCC950 (NLRP3 inhibitor) were administered to explore the therapeutic mechanism of MSCs. Statistical analysis was performed with SPSS version 19.0, and all data were analyzed by independent-samplest test. Results: Injection of IL- 10 or MSC transplantation ameliorated D-Gal-induced increase in alanine aminotransferase, aspartate aminotransferase, total bilirubin, NH3 and inflammatory cytokines. Blockage of IL- 10 confirmed the therapeutic significance of this cytokine. Conclusion: Pyroptosis was inhibited after IL- 10 infusion and inhibition of NLRP3 by MCC950 reversed liver dysfunction.
Keywords: Immunoregulation; Liver Disease; Pyroptosis; Stem Cell; Transplantation
Introduction
Acute liver failure (ALF) is characterized by abrupt onset of severe liver injury, with massive hepatocyte dysfunction, and the mortality rate is as high as 90%. Liver transplantation is the most effective treatment, but its widespread clinical application is limited by the shortage of donor liver,multiple post-operative complications and lifetime immunosuppressant treatment1. In recent years, the benefit of mesenchymal stem cells (MSCs) as a cell-based therapy for ALF has gained much attention 2, 3 .In addition to their differentiation ability, MSCs have the biological characteristics of low immunogenicity and immune regulation, and have shown therapeutic potential in the inflammatory diseases due to their modulation of both innate and adaptive immune systems 4.MSCs can produce a variety of cytokines such as transforming growth factor (TGF)-β,indoleamine 2,3-dioxygenase, inducible NO synthase (iNOS),prostaglandin E2 (PGE2),interleukin (IL)- 10 and TNF-α-stimulating gene (TSG) 6 5. These properties have made MSCs a potential therapeutic choice for immune and inflammatory disorders. However, the mechanism of action of MSCs in the treatment of ALF remains elusive. ALF destroys the balance of the liver immune microenvironmen 6. Pro-inflammatory cytokines are released, such as IL- 1β, IL-6, tumor necrosis factor (TNF)-α, chemokines, and leukotrienes. The uncontrolled immune response plays an essential role in the pathological process of ALF 7. How RNA virus infection to reduce the pro-inflammatory cytokines and dysfunctional hepatocytes is the key to curing ALF.
Pyroptosis was originally described as caspase- 1-dependent cell death. In contrast to apoptosis, pyroptosis has an inherently pro-inflammatory nature. The activation of caspase- 1 along with the inflammasome leads to the release of the pro-inflammatory cytokines (IL- 1β, IL- 18) and occurs before any morphologic expression of cell death 8. Pyroptosis has been shown to play an important role in the occurrence and development of infection, nervous system diseases and atherosclerosis. Pyroptosis is also involved in liver diseases. Hepatic endoplasmic reticulum stress can activate NLRP3 inflammasomes, leading to inflammation-mediated liver injury and hepatocyte pyroptosis 9. Inhibition of pyroptosis is a potential therapeutic approach for the aggressive inflammatory response of acute liver diseases. Considering the anti-inflammatory effects of MSCs, we infer that they are involved in the inhibition of hepatocyte pyroptosis.Male C57BL/6 mice aged 3-4weeks (weighting 15-20 g) were used as MSCs donors. Male C57BL/6 mice aged 6-7weeks (weighing 25-35 g) were used as MSCs recipients. All the mice were purchased from the laboratory Animal Center of the Affiliated Drum Tower Hospital of Nanjing University Medical School. This study was approved by the Institutional Animal Care and Use Committee of Nanjing University, China under the NIH Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize suffering. MSCs obtained from C57BL/6 mice were isolated and cultured according to an established protocol10. We used the Flow cytometry (BD FACSAria Ⅱ, USA) to identify the MSCs, the antibodies used were against mice antigens CD29, CD34, CD44, CD45, CD90 (BD Biosciences, CA, USA).Positive cells were counted and compared to the signal of corresponding immunoglobulin isotype.
Acute liver failure was induced by an intraperitoneal injection of 0.6g/kg D-galactosamine(D-Gal). 12 hour after ALF induction, the mice were randomly divided into six groups: (1) Control (saline); (2) PBS+D-Gal; (3) MSCs+ D-Gal; (4) MCC950(NLRP3 inhibitor) + D-Gal;This article is protected by copyright. All rights reserved. (5) ShIL- 10 MSCs+ D-Gal; (6) 10 μg/ kg recombinant mouse IL- 10 (R & D Systems, Minneapolis, MN, USA) + D-Gal 11. Either IL- 10, 1×106 MSCsin a volume of 0.5 ml PBS were transfused into the caudal vein over a period of 3 minutes. MCC950 were administrated intraperitoneally to mice 1h before the induction of liver failure. All groups of mice (n = 20 in each group) were sacrificed at 1, 2, 3, 4, 5, 6 or 7 d after administration of D-Gal (Supplementary Fig. 1).Primary hepatocytes were harvested by a two-step in situ collagenase perfusion technique with our modification12. The viability of the isolated hepatocytes determined by trypan blue exclusion was more than 95%. Nonparenchy- mal cells were verified by immunocytochemical analysis of albumin and cytokeratin 18.Hepatocytes (2 × 105) were co-cultured with MSCs (2×105) separated by a porous membrane in six-well culture plates for 24 h. 12. The total cell density remained constant at 1×106 cells per well. In these experiments, hepatocytes homoculture was performed as controls. The cytotoxicity of the D-Gal complexes against hepatocytes homoculture and co-culture medium was assessed using the CCK8 assay. This article is protected by copyright. All rights reserved. Shorthair(Sh) RNA-mediated RNA interference: We choose shRNA target sequence for IL10-RNAi-Forward: 5′-GATCCGCTTCCAAACTGGATATAA TTCAAGAGAT; IL10-RNAi-Reverse: 5′-AGCTTAAAAAAGCTTCCAAACTGGATATAATCTCTTGAAT’. (Ambion, Austin, TX).
The protein lysates extracted from the stable clone of IL- 10 knocked-down MSCs and scrambled shRNA-transfected MSCs were quantified by Western blot to confirm the effect of RNA interference. 20 mice in each group were used for the survival study. Mice that had lived or more than 7 days after transplantation were considered to be survivors.To evaluate the anti-inflammatory effect of MSCs, we measured the serum concentration of the following cytokines by ELISA (Abcam Ltd, Cambridge, UK) on day 3 after induction of ALF: IL-6, IL- 1α, IL- 1β, IL- 10, IL- 18, TNF-α. The plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) after treatment were measured with an automated biochemical analyzer. The levels of plasma inflammatory cytokines were detected using a commercially available ELISA kit according to the manufacturers’ instruments (ebioscience, USA).The liver sample was fixed in 4% paraformaldehyde for 24 h before processing. Fixed liver samples were dehydrated, paraffin-embedded and cut into small pieces (<3 mm thick, three from each liver). Sections were stained with hematoxylin and eosin for pathological assessment. All images presented in the results are representative of at least 3 images per liver. Images were analyzed and semi-quantified using Image Pro Plus software (Media Cybernetics, Bethesda, MD). We conducted the Western blot the procedure as described previously13, primary antibodys were rabbit anti-caspase- 1, IL- 1beta, IL- 18 polyclonal antibody (1:1000, Abcam Ltd, Cambridge, UK), GAPDH (Abcam Ltd, Cambridge, UK) expression was used as a loading control.All data arepresented as mean ± standard deviation. They were compared via ANOVA followed by a Student’st-test. In the mortality study, time-to-survival data were analyzed by the Kaplan-Meier method and compared via the log-rank test. Differences between values were considered significant at P < 0.05.
Results
Culture and identification of MSCs
MSCs from mouse bone marrow exhibited a spindle-shaped, a fibroblastic morphology (Fig. 1A). The immunophenotypical characterization by flow cytometry showed positive stromal marker expression (CD29, CD44 and CD90), but little or no hematopoietic marker expression (CD34 and CD45), which means a high purity after the third passage (Fig. 1B). MSCs were injected intravenously 12 hours after D-Gal injection to determine the therapeutic efficacy of MSCs in ALF.The engraftment MSCs retained in the liver was 0.5% approximately (Supplementary Fig. 2). In the D-GalN-treated group, 70% of mice did not survive for 3 days. By contrast, in the MSC-treated group, 60% of mice survived >7 days after ALF induction (Fig. 2A). As expected, in the MSC-treated group, there was a significant decrease in ALT, AST levels at each time point (P < 0.05), while the injection of saline showed no difference (Fig. 2B). All of them reached a peak on day 3. H&E staining was conducted to investigate the liver histology of mice with ALF after transplantation of MSCs. The ALF group showed extensive large nodules, severe infiltration of lymphocytes, hepatic cell necrosis and hemorrhaging involving entire lobules, and the hepatocytes had swollen cytoplasm. However, MSCs transplantation improved the histological damages (Fig. 2C). It seems that intravenous injection of MSCs could attenuate liver injury.Mesenchymal stem cells regulate inflammatory cytokines To investigate the change in inflammatory microenvironment, we detected the liver levels of inflammatory factors (interferon (IFN)-γ, IL-6, IL- 1α, IL- 1β, IL- 10, IL- 18, TNF-α) by ELISA on day 3 after injection of MSCs. Pro-inflammatory molecules such as IFN-γ, IL- 1β, and IL- 18 showed a significant reduction after administration of MSCs, while anti-inflammatory cytokine IL- 10 showed higher expression.These data indicate that MSCs play an anti-inflammatory role in secretion of anti-inflammatory cytokines (Fig. 2D).
Hepatocytes pyroptosis exacerbates ALF via the production of NLRP3-Caspase-1 inflammasomes IL- 1β and IL- 18 acted as pro-inflammatory factors and were markedly increased in acute liver injury. They have been reported as significant cytokines in the process of pyroptosis 8. We treated mice intraperitoneally with 50 mg/kg MCC950 or vehicle control (PBS) 1 h before intraperitoneal injection of D-Gal 14. Inhibition of NLRP3 also increased the survival rate significantly(Fig.3A). ALT, AST all decreased significantly and hepatic inflammation was resolved, in contrast to ALF mice (Fig.3B-C). MCC950 treatment also decreased the concentration of IL- 18 and IL- 1β in liver(Fig.3D). These data clearly demonstrate that MCC950 effectively and specifically blocks NLRP3 activation in vivo. When hepatocytes from a normal mouse were stimulated with D-Gal, NLRP3, caspase- 1 and IL- 1β processing was detected in the cell supernatants. However, when treated with 1μM MCC950 12h14 before D-Gal, NLRP3, IL- 1β and caspase- 1 processing was dose-dependently inhibited (Fig. 4A). CCK8 assay indicated that cell viability in the MCC950-treated group was increased significantly (Fig.4B). Thus, NLRP3-Caspase- 1 inflammasomes accelerate the hepatocytes death via pyroptosis. NLRP3–caspase- 1 inflammasome activation stimulated liver injury, thus, inhibition of hepatocyte pyroptosis may play a role in liver repair.NLRP3-Caspase- 1 inflammasomes were markedly reduced when hepatocytes were co-cultured with MSCs (Fig. 4C). These results suggested that MSCs can attenuate pyroptosis in D-Gal treated hepatocytes. To investigate pyroptosis in ALF and whether MSCs reverse injury through inhibition of pyroptosis, we examined the levels of NLRP3, IL- 1β, IL- 18 and caspase 1 in the liver on day 3. Consistently, D-Gal-treated group exhibited high levels, while MSCs-treated group showed a dramatic decreased (Fig. 4D).
It indicates that MSCs can reduce liver injury through inhibition of hepatocytespyroptosis.The data in our previous study on an ALF model 13 showed that IL- 10 released by MSCs has therapeutic potential in ALF. To block IL- 10 secretion from MSCs, we use ShIL- 10 RNA to infect MSCs (Fig. 5A). ALF mice were treated intravenously in four different groups to detect therapeutic efficacy of IL- 10 derived from MSCs: ALF, MSCs, ShIL- 10 MSCs and IL- 10 group. Three days later, ALT, AST showed no significant improvement after treatment with ShIL- 10 MSCs. However, administration of IL- 10 resulted in a significant decrease in ALT, AST levels compared with the MSC-treated group (Fig. 5B). H&E staining and survival rate showed similar results (Fig. 5C-D). This indicates that MSCs can release IL- 10, which plays an important role in reducing ALF. As we mentioned above, MSCs relieved liver injury through pyroptosis inhibition. Since IL- 10 is the most important anti-inflammatory cytokine released from MSCs, the efficacy of IL- 10 in hepatocyte pyroptosis needs to be determined. In vitro, D-Gal-treated hepatocytes medium within 0.02 μg/mLIL- 10 had little caspase- 1 activation and secreted low levels of NLRP3, IL- 1β and IL- 18 in contrast with D-Gal-treated hepatocytes medium (Fig. 5E). IL- 10 decreased the production of pro-inflammatory cytokines. In vivo, we treated four groups of ALF mice: PBS, MSCs, ShIL- 10 MSCs and IL- 10 group. The levels of Mass spectrometric immunoassay NLRP3, IL- 1β, IL- 18 and caspase- 1 activation in the ShIL- 10 group showed no significant change (P > 0.05) compared with the PBS group, whereas MSCs decreased the levels markedly (P < 0.05), and levels in the IL- 10 group were slightly higher than in the MSC group (Fig. 5F). Consistent with our expectations, mortality rate, liver function indexes (ALT, AST) and inflammation (H&E staining) increased again after administration of ShIL- 10 MSCs (Fig. 5B-D). These data demonstrated that MSCs inhibit hepatocytespyroptosisto mediate their therapeutic effect in ALF via releasing anti-inflammatory cytokine IL- 10.
Discussion
ALF is a critical disease with rapid dysfunction of hepatocytes accompanied by uncontrollable systemic inflammatory. After the establishment of the D-Gal-induced ALF model, 70% of mice could not be survive in 3 days. We found a sharp increase in ALT, AST.The expressions of pro-inflammatory cytokines (IFN-γ, IL-6, IL- 1β, IL- 18, TNF-α) were significantly enhanced while anti-inflammatory cytokines (IL- 1α and IL- 10) decreased dramatically. Our results suggest that ALF is a state of robust cellular immune response.Immune microenvironment changes are consistent with clinical symptoms, which suggests a role for the immune microenvironment in the process of ALF. MSCs have been studied for the therapeutic effect in ALF for several years. The mechanism remains controversial. It has been reported that stem cells facilitate liver repair by transdifferentiation into primary hepatocytes 15, 16 . However, this possibility has been challenged in other studies. Puppi etal. and Khan et al. had reported that only a small number of donor-derived cells were detected in vivo, which was not enough to replace the damaged hepatocytes 17. Recently, immunoregulation by cytokine secretion has been supported as a novel mechanism of MSC transplantation. These released factors play an important role in attenuating systemic inflammation, inhibiting apoptosis and repairing tissues18-20. In both our previous and current studies 13, we found that MSCs significantly improved the liver function (including ALT, AST) as well as the survival rate in D-Gal-induced ALF. We have also demonstrated that MSCs-derived IL- 10 was most distinct among the anti-inflammatory cytokines, suggesting a crucial role in the treatment of ALF with MSCs. Consistently, improvement of inflammatory environment was abolished after inhibition of IL- 10 by ShRNA. IL- 10 reduced the expression of pro-inflammatory cytokines and played an important role in reducing the inflammatory response. Therefore, MSC treatment could improve ALF by maintaining longer and higher IL- 10 expression than endogenous induction by D-Gal. Pyroptosis was originally described as caspase- 1-dependent cell death, it can occur in vitro21 and in vivo22.
Recently, the inherently pro-inflammatory nature of pyroptosis has been recognized. An inflammasome is a cytosolic, multiprotein platform that enables the activation of pro-inflammatory caspases, chiefly caspase- 1, through the induction of caspase- 1cleavage and release of IL- 1β, IL- 18 and other pro-inflammatory cytokines, which eventually produce inflammation23. Three types of inflammasomes are known. NLRC4 and NLRP1 contain CARD domains that directly interact with the Caspase- 1 CARD28. NLRP3 contains a Pyrin signaling domain which binds the Pyrin domain of the adaptor protein ASC. ASC recruits Caspase- 1 via CARD-CARD interactions29. AIM2 contains a HIN200 domain and a Pyrin domain that recruits ASC and activates Caspase- 130.Each of these inflammasomes can activate Caspase- 1 to trigger pyroptosis31. As expected, NLRP3 blockage markedly alleviates hepatic dysfunction in D-Gal-treated mice. To confirm the role of pyroptosis progress in acute hepatocytes damage, we cultured mouse hepatocytes stimulated with MCC950 and D-Gal. NLRP3 deficiency resulted in less death in response to D-Gal exposure. The levels of pro-inflammatory IL- 1β, IL- 18 and caspase- 1 decreased markedly. It seems that NLRP3 stimulates the occurrence of pyroptosis while its inhibition can ameliorate liver inflammation R 41400 and injury. ALF leads to a large number of antigens, a series of immune cell, inflammatory factors and chemokines are in high-stress state. Imbalance of the liver immune microenvironment can result in a lot of excessive activated immune cells, and form feedback immune damage to liver. Pyroptosis is often associated with a high inflammatory state in contrast to the silent apoptotic death, and frequently occurs upon infection24. NLRP3-Caspase- 1 inflammasome are a key inflammatory mediator driving the host response to infection25, injury26, and diseases21, 27.
In our work, MSCs repaired ALF through IL- 10 production,while inflammasomes aggravate liver injury. To determine whether MSCs alleviate pyroptosis to cure liver dysfunction through the same mechanism, we use ShIL- 10 RNA to inhibit MSCs secretion. We found that IL- 10 has the capacity to inhibit expression of NLRP3.IL- 10 negatively regulates the expression of NLRP3 inflammasome components and provides a link to degenerative liver function.The current study was preliminary and included in vivo and in vitro studies. In the near future, we will carryout further preclinical studies to validate the present findings. It is necessary to establish the whole network of IL- 10-induced anti-inflammatory factors, which may help to discover novel biomarkers for predicting the efficacy of MSCs in ALF. In conclusion, MSCs can release anti-inflammatory factors IL- 10, which can reduce the levels of NLRP3-caspase- 1 inflammasomes and play an important role in anti- pyroptosis in treatment of ALF.