Acute-on-chronic liver failure (ACLF) is defined as an acute deterioration in liver function on the basis of pre-existing chronic liver disease with high 28-d mortality. The pathogenic characters of ACLF are related to cytokine storm that induces systematic inflammatory response and subsequent immune paresis [4, 24]. Currently, liver transplantation remains the definitive treatment with a good outcome [2]. However, Rapid disease progression and limited donor supply is driving scientists to explore novel treatment strategies [9], including bone marrow–derived mesenchymal stem cells (MSCs) transplantation, which have achieved partial success in ACLF clinical treatment [25].
MSCs are non-hematopoietic progenitor cells with potential to differentiate into multiple lineages of the mesenchyme. MSCs show distinguishing features on immune-privileged, immunomodulatory and tissue regeneration capacities, which highlighted them into one of the most promising therapies for ACLF [13, 15, 18, 21]. Our recent clinical trials showed that infusion of allogeneic bone marrow-derived MSCs was safe and convenient for patients with HBV-related ACLF, and significantly increased the 24-week survival rate via an improvement of liver function and decreasing the incidence of severe infections [16]. But the underlying molecular and cellular machinery is unclear. Limited by availability of human liver tissue and suitable animal model, the regulation of MSCs on liver immune cells is rarely reported, although MSCs have showed to mainly migrate and reside in liver [26]. Therefore, we tried to probe the crosstalk between MSCs and liver immune cells in ACLF settings.
Our research revealed MSCs could increase the frequency of peripheral blood NK cells of patients with HBV associated ACLF, and the liver function improved synchronously, the same results were shown in mouse models. In ACLF mice, we found MSCs improved liver DX5− NKP46 cells proliferation, GM-CSF secreting, and these two cells showed stronger cell-cell adhesion.
NK cells are the main subpopulation of leukocytes in the liver, and their physiological function are still largely unknown. Conventional NK cells from peripheral blood, spleen or lymph nodes are recognized as a subset of innate cytotoxic large granular lymphocyte, which recognize and eliminate stressed tumor or virus-infected cells [27]. Recent progresses suggest that liver NK cells are heterogeneous, consists of circulating and local resident populations which have different frequency, phenotype, development pathway, gene and protein expression profile and functions characters [28]. Liver resident NK cells preferentially performed immunological regulation, while circulating NK cells execute effector functions [19, 29]. Previous reports confirmed cross-talk between human MSCs and NK cells in vitro [20, 21, 30]. These works guided us to assess the effect of MSCs on NK cells under ACLF context.
Limited by practical infeasibility of liver samples from ACLF patients, we only managed to look for surrogated peripheral blood NK cells changed by MSC infusion into HBV-ACLF patients. Consistent with previous reports, frequency of peripheral blood NK cells from ACLF patients was significantly decreased as compared with that of healthy volunteers [22]. This is mostly due to re-distribution of systemic leukocytes under several and acute inflammation in liver. After MSCs transfusion, the decrease of blood NK frequency was reversed, which nearly reached to healthy volunteers’ level in week8. This peripheral NK frequency change was earlier than inflammation remission, such as INR decreased significantly in week 12. This phenomenon suggested that NK change maybe a cause of inflammation remission, instead of a consequence, if there is some direct relationship between them. Furthermore, patients with less peripheral NK% had early and worse clinical outcome than those with higher NK%, which also echoed our assumption.
To get some clue of the relationship between blood NK frequency change and inflammation emission, typical NK phenotypic markers were assessed. KIR3DL1 frequency on blood NK cells was increased instantly by MSCs transplantation, NKG2A frequency showed slight upregulation in late stage, and all others (KIR2DL1/2/3, NKG2D, FasL, Perforin, Granzyme B) kept intact. To our knowledge, this unique profile of NK receptors change hasn’t been reported before, which implied that MSCs had unique impact on NK cells from ACLF patients.
To understand the cellular and molecular machinery of MSCs on liver NK cells in ACLF setting, we elaborately established chronic liver damage mouse model by intraperitoneal injection of low-dose CCL4 for eight weeks to induce cirrhosis. The ACLF occurs in the setting of a progressive chronic systemic inflammation, mostly liver cirrhosis [6]. To initiate an acute burst of liver damage and systemic inflammation, a sub-lethal dose of CCL4 was injected. Our data showed that the mouse death rate reached 70% within 6 hours after challenge, which is comparable to the high mortality of ACLF patients. In line with this, the liver histological structure was deformed and serum aminotransferase level was strikingly elevated, which strongly implied that CCL4 induced liver damage was responsible for the mouse death. Furthermore, more caspase 3 was cleaved after challenge indicated apoptosis involved in CCL4 mediated liver damage, which excluded the complete hepatic toxicity induced by CCL4 in this model. Significant lymphocytes infiltration in the liver after CCL4 challenge indicated the exacerbated systemic inflammatory responses, which matched the paramount pathogenic characters of human ACLF. According to widely-acknowledged criteria for ACLF mouse model [31, 32], our optimized experimental conditions produced adequate ACLF murine model.
Owing to tissue scarceness of primary MSCs, it is necessary to proliferate and expand them through cell culture. Their fate and function in culture is influenced by various external factors, including the specific cell source, donor age, plating density, passage number, plastic surface quality and external cytokines [33]. After long-term comparison and optimization, the MSCs under our culture conditions showed typical phenotype and functional recovery of ACLF mice. In line with the decreased death rate, autogenic MSCs alleviated liver damage and promoted hepatic regeneration in ACLF mice.
As expected, the expanded MSCs significantly increased survival rate of ACLF mice, which was consistent with other reports [12, 15, 34, 35] and our clinical trials [16]. The mechanism may attribute to MSCs’ capacity of immune regulation and regeneration [14]. Although it was found that MSCs could promote cell proliferation in liver, it is hard to tell these were hepatocytes or liver immune cells. However, there was significant inflammatory remission and less cell death after MSCs transplantation, which emphasized their role in immune regulation, and echoed our observation in HBV-ACLF patients. Hence, the NK cells were specifically investigated w/o MSCs in ACLF settings.
Similar to HBV-ACLF patients, MSCs infusion upregulated frequency of circulating peripheral blood NK cells in ACLF mice, which also showed in liver instead of spleen. Both liver and peripheral blood contain higher frequency of NK cells, but spleen keep intact after MSCs transfusion. It is more likely that there were more NK proliferation and development, instead of immune cells redistribution only after liver inflammatory remission.
More and more evidence proved that liver NK cells could be stratified into conventional circulating NK cells (NKP46+DX5+) and local resident liver NK cells (NKP46+DX5−). DX5 (or CD49b) has been recently characterized by 150 KD integrin, which non-covalently associates with CD29 (β1 integrin) to form the CD49b/CD29 complex regulating NK or T cells. A lot of proof showed that these two subpopulations differ in their development stage, phenotypic markers, transcription factor profile and functional characters [28]. Reports showed liver NKP46+DX5− cells involved in the pathogenesis of different liver disease [36–38], and they proliferated under specific circumstance [39].Strikingly, frequency of NKP46+DX5− cells upregulated by MSCs infusion along with higher NKG2A expression. Except for possible liver NKP46+DX5+ cells re-joined blood circulation after liver inflammatory remission, it was likely that new NK produced considering the change of NKG2A frequency. In vitro co-culture of purified NK and MSCs varified this assumption, the MSCs promoted proliferation of NKP46+DX5− liver cells, instead of NKP46+DX5+ liver cells or blood NK cells. Furthermore, more GM-CSF was produced from NKP46+DX5− liver cells by MSCs.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) was originally classified as a hematopoietic growth factor [40]. Recent rat model showed GM-CSF involved in biliary wound healing and hepatocellular regeneration [40, 41]. GM-CSF has also been used in the treatment of sepsis-associated immunosuppression, enhancement of lymphoid and myeloid subpopulations [42]. Considering the similar immunological characters between sepsis and ACLF, it is reasonable to propose that GM-CSF restored immune competency and/or enhance liver regeneration in ACLF patients.
To further investigate the potential machinery of MSCs’ preference on DX5− instead of DX5+ cells, purified NK cells were co-cultured with MSCs in vitro. The liver DX5− NK cells showed higher cell-cell conjugation potency with MSCs than DX5+ NK cells, which may be associated with significant proliferation of liver NK cells induced by MSCs. Previous reports showed NK cells killed MSCs in vitro, we didn’t get this [43].The reason may be due to the different experimental model and conditions.