Cell-based therapies have emerged as potential alternatives to liver transplantation for the treatment of ALF due to their feasibility and low-invasive nature.10 Among the cells, human hepatocytes were isolated from the donor organ livers that are either unused surplus or rejected for human transplantation. Although the isolation techniques have been optimized to improve the viability and metabolic function, the cell yield after thawing still remained low, and the detrimental effect of the freezing process on metabolic function and cell attachment property is still inevitable.(29) Recently, the novel therapeutic strategies have been developed to promote the function of transplanted hepatocytes, including the co-transplantation strategies.(30) Previous studies have demonstrated that co-transplantation of hepatocytes and MSCs exerted a significant therapeutic effect on ALF animal models, in which the co-transplanted MSCs exerted differentiation abilities towards hepatocytes in PLGA (poly-lactic-co-glycolic acid) scaffolds to support hepatocellular metabolism and stabilization in ALF. However, there seem on the effect of MSCs on ameliorating inflammatory response during ALF is rarely investegated.9 Furthermore, paracrine factors secreted by MSCs were documented to be responsible for preventing the deleterious cytokine/chemokine/receptor responses in ALF.(21, 31) Of note, HNF4α is a key regulator of morphological and functional differentiation of hepatocytes.(32) Therefore, in the current study, HNF4α was overexpressed in UMSCs to investigate the protective effects of UMSCs in ALF mice. The results showed that HNF4α-UMSCs promoted the synthesis and secretion of human primary hepatocytes and enhanced the viability and function of hepatocytes in ALF(Fig.1D-1E). Furthermore, to prevent possible immune reaction induced by transplanted cells, the microcapsules were used to construct an immuno-isolation membrane to eliminate the entry of immunocytes into the host immune system, tolerant from immune recognition and immune attack. Microcapsules have a porous structure that allows oxygen and other nutrients to nourish the encapsulated cells. Moreover, they also provide a diffusive control of surrounding vasculature or tissue by the paracrine factors secreted by the encapsulated cells.17 A previous study showed that MSCs in the microcapsules with porcine hepatocytes enhanced the viability and function of porcine hepatocytes.(33) Therefore, we used APA to microcapsule HNF4α-UMSCs with human primary hepatocytes and transplanted these microcapsules into ALF mice to observe their effects on ALF in the present study (Fig.1C and Fig.2A). The results showed that transplantation of HNF4α-UMSC-HEP has significantly attenuated liver injury, resulting in improved survival rate of LPS/D-gal-induced ALF mice (Fig.2B-2E). Moreover, hepatocytes co-transplanted with HNF4α-UMSCs in APA microcapsule can compensate the function of injured liver, preserving as substitute cellular source of large scale of quantities for liver function.
ALF is characterized by hepatic encephalopathy, coagulopathy, and progressive multiorgan failure.(34) Acute inflammation along with dysregulation of immune cells contributes to the pathogenesis of ALF.(35) For better simulation of the acute inflammatory condition, LPS combined with D-gal was often used to induce liver failure model in mice.(36) LPS is known to drive the process of inflammation by activating Toll-like receptors (TLRs) on macrophages and following a promotion on the secretion of inflammatory cytokines and infiltration of inflammatory cells.(37) D-gal triggers oxidative stress in hepatocytes, causing lipid peroxidation.(38) Of note, MSCs have been reported to suppress oxidative stress and neutrophil inflammatory response in rats with hepatic ischemia-reperfusion injury(8) and ALF.(39) In this study, HNF4α-UMSC-HEP significantly alleviated neutrophil and macrophage infiltration in the liver (Fig.3A-3D), and the inflammatory cytokine secretions in the serum as well as mRNA levels of inflammatory cytokines in the liver (Fig.3E-3F).
In addition, polarization of macrophages was also critically involved in the inflammatory responses of ALF.(35, 40) Macrophages are highly plastic, and has the ability to switch between two functional phenotypes, pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages, in response to microenvironment changes.(41) The activated M1 macrophages exert phagocytic activity and act as initiators of inflammation, accompanied by the release of pro-inflammatory cytokines, including IL-1β, iNOS, and TNF-α. In contrast, M2 macrophages upregulate the expression of anti-inflammatory factors and other factors, such as IL-10, TGF-β, and other molecules, that are involved in cell proliferation, wound healing, tissue remodeling, and immnuomodulation,(42, 43) and these in turn contribute to the resolution of M1 macrophage-mediated inflammatory response. Therefore, a phenotypic switch from M1 to M2 has been suggested as a novel therapeutic approach to reduce inflammation in spleen, adipose tissue and liver.(44-46) We herein found the dramatically increased portion of M1 macrophages in the livers of LPS/D-gal-induced ALF mice. Moreover, the number of M1 macrophages was reduced and the number of M2 macrophages was increased in the livers of ALF mice in HNF4α-UMSC-HEP group (Fig.4A-4D). The CM of HNF4α-UMSC-HEP decreased the LPS-induced M1 polarization and enhanced M2 polarization of macrophages in Raw264.7cells in vitro (Fig.4E-4F). Taken together, HNF4α-UMSC-HEP switched the macrophage functional phenotype from pro-inflammatory M1 to anti-inflammatory M2 to alleviate ALF.
Mounting evidence showed that MSCs secretions exerted a beneficial effect by reducing the inflammatory response, promoting the survival and proliferation of injured cells, and ameliorating liver injury, by the mediation of paracrine factors including IL-10, TIMP-1, and MCP-1.(47-49) In the present study, microcapsules were used as carriers to encapsulate UMSCs and hepatocytes to persistently release the secretory factors contributing to the repair of impaired liver in ALF. Firstly, the transplanted microcapsules of HNF4α-UMSC-HEP significantly reduced the inflammatory response, repaired the liver injury and improved the survival rate of mice with ALF (Fig.2 and Fig.3). Secondly, HNF4α-UMSC-HEP decreased the LPS/D-gal-induced M1 polarization and enhanced M2 polarization of macrophages both in vitro and in vivo (Fig.4). Thirdly, the CM of HNF4α-UMSC-HEP promoted cell viability and reduced liver injury induced by D-gal (Fig.5A and 5B). To further determine the secretory proteins of HNF4α-UMSCs, a protein chip assay was performed. The results showed that 38 proteins were significantly upregulated in HNF4α-UMSCs when compared with those of UMSCs. Analysis of GO categories revealed that the proteins in the CM of HNF4α-UMSCs are more closely associated with some pathways that regulate fundamental cellular processes, such as proliferation, differentiation, motility, stress response, apoptosis, and survival (Fig.5D).(50) Of note, heat map analysis showed that the levels of growth factors (FGF-9, HGF, HB-EGF, PDGF and TGF-β) and anti-inflammatory cytokines (IL-1ra, IL-10, IL-11 and IL-13) were considerably higher in the CM of HNF4α-UMSCs compared to those in UMSCs (Fig.5C). Thus, the proteins including FGF9, HB-EGF, HGF, PDGF and TGF-β were selected to perform antibody neutralization experiments to determine their protective effects on injured hepatocytes. The results showed that only HB-EGF has significantly relieved hepatocyte injury, and no significant effects were observed in other groups (Fig.5E). Furthermore, previous studies have shown that HB-EGF could protect intestine tissues from inflammatory damage by promoting the M2 polarization of macrophages in necrotizing enterocolitis injury.27 In addition, HB-EGF combined with HGF obviously inhibited BDL-induced cholestatic liver injury by exerting acute cytoprotective effects and enhancing the anticholestatic effects and liver regeneration during the chronic phase.(51) Interestingly, in the present study, we found that HB-EGF neutralization antibody in mice has reversed the protection effects against ALF (Fig.6A-6D) and the switch of polarization of macrophages from M1 to M2 that was exerted by HNF4α-UMSC-HEP on ALF mice (Fig.6E-6F). These data suggested that HNF4α-UMSC-HEP played the therapeutic effects mainly mediated by HB-EGF. More importantly, HNF4α binds to the HB-EGF promoter and directly upregulates the expression of HB-EGF (Fig.6G-6I). Together, these results indicated that HNF4α-UMSC-HEP ameliorated the ALF mice, mainly mediated by activating the expression of HB-EGF by HNF4α.
In conclusion, the present study revealed that the overexpression of HNF4α in UMSCs has effectively increased the synthesis and secretion of human primary hepatocytes. Co-encapsulation of HNF4α-UMSCs and hepatocytes attenuated LPS/D-gal-induced liver injury and improved the survival rate of ALF mice. The molecular mechanism includes the reduction in the inflammatory response and promotion of macrophages polarization switch from M1 to M2, and the paracrine, in which HNF4α-UMSC-HEP promoted survival, proliferation, and metabolism of hepatocytes. In addition, HB-EGF upregulated by HNF4α plays a key role in macrophages polarization and a direct protection from the cytoprotective effects. These findings provide novel insights into the approach of cell-based therapy for ALF(Fig.7).