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Autophagy inhibition via Becn1 downregulation improves the mesenchymal stem cells antifibrotic potential in experimental liver fibrosis

Abstract Liver fibrosis (LF) is the result of a vicious cycle between inflammation‐induced chronic hepatocyte injury and persistent activation of hepatic stellate cells (HSCs). Mesenchymal stem cell (MSC)‐based therapy may represent a potential remedy for treatment of LF. However, the fate of transplanted MSCs in LF remains largely unknown. In the present study, the fate and antifibrotic effect of MSCs were explored in a LF model induced by CCl4 in mouse. Additionally, MSCs were stimulated in vitro with LF‐associated factors, tumor necrosis factor‐α (TNF‐α), interferon‐γ (IFN‐γ), and transforming growth factor‐β1 (TGF‐β1), to mimic the LF microenvironment. We unveiled that MSCs exhibited autophagy in response to the LF microenvironment through Becn1 upregulation both in vivo and in vitro. However, autophagy suppression induced by Becn1 knockdown in MSCs resulted in enhanced antifibrotic effects on LF. The improved antifibrotic potential of MSCs may be attributable to their inhibitory effects on T lymphocyte infiltration, HSCs proliferation, as well as production of TNF‐α, IFN‐γ, and TGF‐β1, which may be partially mediated by elevated paracrine secretion of PTGS2/PGE2. Thus, autophagy manipulation in MSCs may be a novel strategy to promote their antifibrotic efficacy.


| INTRODUCTION
Liver fibrosis (LF), characterized by extracellular matrix (ECM) protein production and accumulation, is a vicious cycle between chronic inflammation-induced persistent parenchymal cell injury and activation of consequent wound-healing responses executed mainly by activated myofibroblasts, which define the LF microenvironment (Tsuchida & Friedman, 2017). Genetic techniques have provided strong evidence that primed hepatic stellate cells (HSCs) are the prominent cellular origin of matrix protein-secreting myofibroblasts (Lee, Wallace, & Friedman, 2015;Trautwein, Friedman, Schuppan, & Pinzani, 2015). Genetic lineage-tracing analysis performed by Kramann et al. (2015) strongly suggested that glioma-associated oncogene homolog 1 (Gli1) is an in vivo marker of myofibroblast progenitors; thus, HSCs can be traced in vivo using a Gli1 genetic lineage-tracing technique. In addition, both the occurrence and progression of LF are closely related to T-cell-mediated immune responses (Pesce et al., 2006;Wynn, 2004).
Multiple researches have consolidated the beneficial efficacy of mesenchymal stem cells (MSCs) on treatment of varied inflammatory disease (L. Bai et al., 2012;Rocheteau et al., 2015;Swart et al., 2015), and the therapeutic effects of MSCs are largely attributable to their profound immunoinhibitive capacity (Horton et al., 2013;Wang, Chen, Cao, & Shi, 2014). Nevertheless, the uncertainty of the transplanted MSCs poses critical challenge for obtaining predictable therapeutic effects in vivo. The in vivo inflammatory microenvironment is a pivotal necessity for MSCs to execute their immunomodulatory functions (Ren et al., 2008). The interactions between MSCs and inflammatory microenvironment regulate the immunomodulatory properties of MSCs (Y. Liu et al., 2011). However, few studies have evaluated the mechanisms through which the inflammatory microenvironment of LF controls the immunoregulatory capability of MSCs.
Autophagy is a highly conservative eukaryotic degradative process, which is a requisite for replying and adapting to environmental alterations (Cadwell, 2016). Autophagy-related genes (Atgs), including Atg7, Atg12, Atg5 as well as Becn1, regulate autophagosome maturation (Antonioli, Di Rienzo, Piacentini, & Fimia, 2017). Emerging evidence has confirmed a close link between autophagy and immunomodulation has been confirmed by emerging evidence (Shibutani, Saitoh, Nowag, Münz, & Yoshimori, 2015). Some studies unveiled that inflammatory microenvironment hindered the immunosuppressive properties of MSCs by priming autophagy (Dang et al., 2014). Therefore, we hypothesized that the LF microenvironment could also interact with the autophagic and immunoregulatory potential of MSCs and that modulation of autophagy may in turn influence the therapeutic effect of MSCs in LF.
In the current study, we assessed the autophagy activity in transplanted MSCs triggered by hepatic microenvironment in vivo.
We also examined the effect of stimulating MSCs by LF-related cytokines, including tumor necrosis factor (TNF)-α, interferon-γ (IFNγ), and transforming growth factor-β1 (TGF-β1) on autophagy-related gene expression. Furthermore, we evaluated the effect of autophagy inhibition on the antifibrotic activity of MSCs. The findings of this study are expected to provide novel insights into the relationship between autophagy and the immunomodulatory effects of MSCs, and indicate whether autophagy could be considered as a promising therapeutic target for MSCs for treating LF.
The cells were postfixed with 1% osmic acid for another 2 hr at 4°C, followed by dehydration using ethanol in graded concentrations.
Subsequently, the sections were incubated with uranylacetate and lead citrate (cat. no. 15326; Sigma), and images were acquired using an Olympus EM208S transmission electron microscope (Olympus, Tokyo, Japan).

| LF model induction and MSC transplantation
Male SPF C57BL/6 mice (2 months of age, average body weight: 25 g) were obtained from Chengdu Dossy Experimental Animals Co. Ltd.
(Chengdu, China). Gli1CreER t2 (cat. no. 007913) and Rosa26tdTomato (cat. no. 007909) mice were obtained from Jackson Laboratories (Bar Harbor, ME). For lineage-tracing studies, 6-7-week-old male mice underwent three intraperitoneal injections of tamoxifen (cat. no. 06734; Sigma) at a dose of 0.1 mg/kg body weight in corn oil/3% ethanol 10 days before LF induction. Detailed information on identification of Gli1 genetic lineage-tracing mice is given in the Supporting Information Materials. LF mice were induced by CCl 4 olive oil solution (CCl 4 :olive oil = 1:3) via intraperitoneal injection at a concentration of 4 ml/kg twice per week for 5 consecutive weeks (CCl 4 mice). Accordingly, the control group mice were then subjected to the same concentration of olive oil by intraperitoneal injection (oil mice).
MSCs of passage 5 (P5) infected with lentivirus-control shRNA were transplanted into oil mice and CCl 4 mice at a density of 1 × 10 6 cells in 100 μl normal saline per mouse via the tail vein (on Week 5, n = 8). Mice were euthanized at 0 and 72 hr after cell administration. In subsequent in vivo experiments, 32 mice were randomly designated into four groups with comparable mean body weights; the groups were as follows: oil + PBS, CCl 4 + PBS, CCl 4 + shNC, and CCl 4 + shBecn1 groups (n = 8 each). In the CCl 4 + PBS, CCl 4 + shNC, and CCl 4 + shBecn1 groups, CCl 4 -induced LF model mice were subjected to weekly administration of PBS, shNC-MSCs (1 × 10 7 /ml, P5), or shBecn1-MSCs (1 × 10 7 /ml, P5) from Weeks 3 to 5 via the tail vein. The oil + PBS group underwent PBS administration with the same volume and sequence as the treatment group after oil treatment. The mice were killed 3 days after cell transplantation. In some in vivo experiments, 32 mice were randomly allocated to four groups, that is, oil + shNC, oil + shBecn1, CCl 4 + shNC, and CCl 4 + shBecn1 groups (n = 8 each). shNC-MSCs or shBecn1-MSCs were administered to oil mice or CCl 4 mice (on Week 5) via tail vein. The mice were euthanized 3 days after cell transplantation for subsequent evaluation of the localization of exogenous MSCs in the liver.
All animal operations were conducted in line with the Chinese

| Western blot analysis
Total protein from 100 mg mouse liver tissues and MSCs subjected to the above-mentioned treatments was obtained using RIPA lysis buffer with the protein inhibitor phenylmethylsulfonyl fluoride (cat. no. ST506; Beyotime). The total protein was quantified using a BCA quantification kit (cat. no. P0010; Beyotime). Proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins in gel were shifted to PVDF membranes (cat. Millipore) overnight at 4°C. Membranes were then thoroughly rinsed by TBS-T and subjected to incubation of peroxidase-labeled secondary antibodies (cat. no. SA00001-1; Proteintech) for 1 hr at room temperature. The membranes were rinsed by TBS-T and then incubated with chemiluminescent HRP substrate (cat. no. WBKLSO100; Millipore). Proteins were detected with a chemiluminescence apparatus (Bio-Rad, Hercules, CA).

| Measurement of liver hydroxyproline content
Following the manufacturer's instructions, liver hydroxyproline levels were quantified by hydroxyproline detection kit (Nanjing JanCheng Biochemical Institute, Nanjing, China).

| Measurement of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels
Serum concentrations of AST and ALT were detected to estimate the degree of hepatic injury caused by CCl 4 administration. Mice were anesthetized as described above, and 0.5 ml blood was obtained from the eye sockets of mice, followed by 10 min of centrifugation at 3,000 rpm.
Based on the provided protocol, serum levels of AST and ALT were assessed by diagnostic quantification kits (cat. nos. C010-2 and C009-2; Nanjing JianCheng Bioengineering Institute, Nanjing, China).

| Coculture of MSCs and HSCs
MSCs and HSCs were cocultured in a Transwell coculture system (12-mm diameter; 0.4-mm pore size, cat. no. G3401; Corning, Corning, NY) with DMEM-low glucose containing 10% FBS. Briefly, HSCs at a density of 5 × 10 4 cells/well were cultivated in the bottom chambers of 24-well plates, and MSCs at a density of 2.5 × 10 3 cells/ well were seeded on the Transwell membrane inserts. Three groups were evaluated, that is, control, HSCs + shNC-MSC, and HSCs + shBecn1-MSC groups, with four wells per group. The HSCs were collected for analysis of proliferation and procollagen type I α1 secretion after coculturing for 24, 48, and 72 hr.

| Real-time PCR
no. 172-5271; Bio-Rad). GAPDH was utilized an internal control. The primer sequences are listed in Table 1.

| Statistical analysis
All data are expressed as means ± SEMs, and were statistically analyzed using GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA). Means of two groups were compared by Student's t tests. For multiple comparisons, statistical analysis was performed by analysis of variance. A p < .05 was considered statistically significant.

| Autophagy of MSCs occurred in a LF microenvironment
To determine whether autophagy of MSCs occurred during LF, EGFP-control lentivirus-transfected MSCs (shNC-MSCs) were transfused into oil mice or CCl 4 mice via the tail vein. Interestingly, the number of EGFP + MSCs and the extent of MAP1LC3 puncta in the Rv 5′-CCTCCCCGATCAGAGTGAA-3′ Abbreviations: Fw, forward primer; PCR, polymerase chain reaction; Rv, reverse primer.
3.2 | LF-related cytokines triggered autophagy in MSCs via upregulation of BECN1 To determine whether the autophagy activated by LF-related cytokines was triggered by upregulating BECN1, MSCs were stimulated with the three cytokines alone or in different combinations for 6 hr. All treatments significantly upregulated mRNA and protein expression of BECN1 (Figure 1f,g) downregulated p62 protein expression (Figure 1g).
To further explore the function that BECN1 played in autophagy, lentivirus-expressing shRNA specific to Becn1 (shBecn1-MSCs) was

| 2729
livers were significantly reduced after transplantation of shBecn1-MSCs (Figure 3c,d). These results unveiled that the promoted antifibrotic capability of shBecn1-MSCs in LF might be attributed to their inhibitory effects on HSCs proliferation.

| shBecn1-MSCs attenuated infiltration of CD4 + and CD8 + T lymphocytes in CCl 4 -induced fibrotic livers
The involvement of CD4 + and CD8 + T lymphocyte infiltration in LF has been confirmed (Pesce et al., 2006;Wynn, 2004). Therefore, The expression levels of PTGS2, p62, MAP1LC3-I, and MAP1LC3-II in MSCs were determined by western blot analysis analysis. shNC-MSCs were incubated in presence or absence of 3-MA, and shBecn1-MSCs were incubated in presence or absence of rapamycin, under combined stimulation with tumor necrosis factor-α (TNF-α, 20 ng/ml), interferon-γ (IFN-γ, 50 ng/ml), and transforming growth factor-β1 (TGF-β1, 10 ng/ml) for 12 hr. (c) shNC-MSCs and shBecn1-MSCs underwent single or combined treatment with TNF-α (20 ng/ml), IFNγ (50 ng/ml), or TGF-β1 (10 ng/ml) for 24 hr, and PGE 2 secretion in the supernatant was assayed using enzyme-linked immunosorbent assay (ELISA). The results are presented as means ± SEMs. (d) Mice were divided into the oil + PBS, CCl 4 + PBS, CCl 4 + shNC, and CCl 4 + shBecn1 groups and euthanized. Subsequently, serum levels of PGE 2 were quantified using ELISA, and (e) TNF-α, IFN-γ, and TGF-β1 messenger RNA expression in the mice liver were measured by quantitative polymerase chain reaction. (f) Serum concentrations of TNF-α, IFN-γ, and TGF-β1 were determined using ELISA. The findings are expressed as means ± SEMs. PGE 2 , prostaglandin E 2 ; PTGS2, prostaglandin-endoperoxide synthase 2. *p < .05, **p < .01, ***p < .001 Nikolic -Paterson, & Lan, 2016). TGF-β1 induces the production and inhibits the degradation of ECM proteins. Indeed, our results confirmed that TNF-α, IFN-γ, and TGF-β1 treatment induced marked increases in autophagosome formation, accompanied by significant increases in the MAP1LC3-II/MAP1LC3-I ratio. TNF-α or IFN-γ stimulated autophagy in MSCs at about 4 hr, while TGF-β1 induced increased autophagy in MSCs later than TNF-α and IFN-γ. This may be attributed to early inflammatory factors of liver damage such as TNF-α and IFN-γ, which activate immune cells and induce inflammation (Ge, Huang, & Yao, 2018). TGF-β1 is an important profibrotic factor and an immunomodulatory molecule (Fujio et al., 2016). Due to the different effects of these factors, there may be differences in the timing of induction of autophagy in MSCs. In vitro studies in MSCs further confirmed the crosstalk between autophagy and the LF microenvironment, consistent with our in vivo findings and with the previous findings demonstrating that TNF-α and IFN-γ could activate autophagy in various cell types (Yuan et al., 2018). Moreover, our results were further supported by a study confirming that TGF-β1 stimulates autophagy, suggesting that autophagy may participate in fibrosis via the TGF-β1 pathway (Ghavami et al., 2015). Taken together, these findings suggested that the microenvironment of LF, as defined by chronic inflammatory responses involving the proinflammatory factors TNF-α and IFN-γ and the subsequent repair process regulated by the profibrotic factor TGF-β1, triggered autophagy in MSCs both in vivo and in vitro.
We also found that the three LF-related cytokines upregulated both the mRNA and protein expression of BECN1, and downregulated p62 protein expression in MSCs. Consistent with this, Becn1 knockdown reduced the MAP1LC3-II/MAP1LC3-I ratio and increased P62 expression in MSCs. These findings suggested that the autophagy triggered by the three LF-related cytokines might be attributed to BECN1 upregulation in MSCs, which supported the past research demonstrating that IFN-γ and TNF-α trigger BECN1 expression to induce autophagy (Djavaheri-Mergny et al., 2006;Tu et al., 2011) and that autophagy is inhibited by reducing TGF-β1 secretion and TGF-β1/Smad signaling (T. Liu et al., 2018).
As an evolutionarily highly conserved cellular process, autophagy functions to maintain intracellular homeostasis (Cadwell, 2016).
Emerging evidence has shown close crosstalk between autophagy and immunomodulation (Clarke & Simon, 2018). Moreover, autophagy plays pivotal role in T-cell homeostasis and can modulate Th1/ Th2 T-cell responses (Deretic, 2012). In the present study, we revealed the promoted antifibrotic capability of shBecn1-MSCs in LF in contrast to that in shNC-MSCs. Importantly, proinflammatory cytokines are a prerequisite for MSCs to exert their immuneinhibitory functions (Ren et al., 2008 (Dang et al., 2014) found that inflammatory condition hampered the immunoinhibitive potential of MSCs by priming autophagy in a mouse model of experimental autoimmune encephalitis, supporting our findings. Similar results indicating protective effect of bone MSCs on n-hexane-induced neuropathy through autophagy inhibition were also found in another study (Hao et al., 2018). Collectively, our results demonstrate that counteracting LF microenvironment-induced autophagy via stable knockdown of Becn1 remarkably promotes the antifibrotic effects of MSCs.
Interestingly, a genetic lineage-tracing analysis performed by Kramann et al. (2015) strongly suggested that Gli1 is a specific biomarker for myofibroblast progenitors. Our results suggested that shBecn1-MSCs inhibited the proliferation of HSCs both in vivo and in vitro and decreased the secretion of procollagen I α1 in HSCs in an in vitro coculture system. These findings affirmed that the inhibitive function on HSCs proliferation might contribute to the augmented therapeutic consequence of shBecn1-MSCs in LF.
The onset and progression of LF are closely related to T-cellmediated immune responses (Pesce et al., 2006;Wynn, 2004), and the regulatory effect of MSCs on T lymphocyte proliferative capability confers them attractive candidates for antifibrotic treatment (Ren et al., 2008). Moreover, autophagy is imperative for both survival and immune function of CD4 + and CD8 + T lymphocytes (Jia et al., 2015). In this regard, the roles of autophagy played in the immunosuppressive properties of MSCs were exploited. Our results showed that transplanted MSCs were selectively recruited to the fibrotic portal area in the liver. Furthermore, shBecn1-MSCs obviously promoted the immunosuppressive capability of MSCs in CD4 + and CD8 + T lymphocyte proliferation. This finding suggested that the inhibitive effect of shBecn1-MSCs on infiltration of CD4 + and CD8 + T lymphocytes might partially account for the elevated antifibrotic effects on the fibrotic liver. Our study corroborated previous research by Ding et al. (2014), who showed that autophagy alleviates kidney fibrosis by regulating TGF-β1 expression. However, one study showed that MSCs exert immunosuppressive effects in CD4 + T lymphocytes via TGF-β1 secretion and activation of autophagy (Gao et al., 2016). The cause for this discrepancy may be associated with the molecular interplay between autophagy and immunity; indeed, autophagy functions in both activation and inactivation of immune signaling, thus balancing immune responses (Saitoh & Akira, 2010). Moreover, these discrepancies may be related to differences in the study designs, including tissue and species origins, cell isolation methods, T cell/MSC ratios, cell culture conditions, and time of data analysis.
The therapeutic effects of MSCs in inflammatory diseases is largely due to their unique capacity to secrete immunoregulatory factors (Ranganath, Levy, Inamdar, & Karp, 2012). Moreover, the colocalization of exogenous MSCs, T lymphocytes, and HSCs in the portal fibrotic area in CCl 4 -induced fibrotic livers indicated that the inhibition of exogenous shBecn1-MSCs on proliferative capacity of HSCs and T lymphocytes might affect their immunosuppressive capability via a paracrine pathway, but not via hepatocyte regeneration. PGE 2 is an inflammation-induced immunosuppressive agent secreted by MSCs, which mediates MSC-dependent inhibition on T lymphocytes (M. Bai et al., 2018). Notably, we found that knockdown of Becn1 suppressed inflammation-induced autophagy and generated more PTGS2/PGE 2 in MSCs, consistent with other studies. As outlined by Dang et al. (2014)  interactions with components of the immune system (Alfaifi et al., 2018). MSCs can secrete various immunoinhibitive cytokines, including indoleamine 2,3-dioxygenase, IL-6, nitric oxide, and IL-10 (Shi et al., 2012). Therefore, we believe that there are other molecules involved in the mechanisms through which shBecn1-MSCs modulate T cells and PGE 2 , and these factors should be investigated in the future. Blocked autophagy may cause cellular senescence (Ma et al., 2018) and increase the risk of tumor formation (Aita et al., 1999;Levy et al., 2014;Qu et al., 2003;Wang et al., 2019).
Autophagy levels are significantly reduced in aging bone marrow MSCs. In turn, elevated activity of autophagy can partially reverse this senescence and restore bone loss in aged mice. Under certain conditions, loss of the Becn1 gene in mice promotes tumorigenesis (Qu et al., 2003). In many human tumors, including breast, ovarian, and prostate cancers, the Becn1 gene is often partially deleted (Aita et al., 1999). These findings indicate that controlling the autophagic activity by genetic manipulation may affect the stem-like characteristics and differentiation functions of MSCs and increase the risk of tumor formation. Therefore, in future studies, it will be necessary to develop a safe and stable method for regulating autophagy.
F I G U R E 7 Schematic illustration of the proposed model depicting how autophagy suppression enhanced the antifibrotic effect of mesenchymal stem cells (MSCs) under the context of liver fibrosis (LF). LF involves chronic inflammation-induced parenchymal cell injury and activation of consequent repair processes executed by primed myofibroblasts progenitors. Based on our findings, the transplanted MSCs undergo autophagy in the LF microenvironment, and counteracting the microenvironment-induced autophagy by Becn1 knockdown promotes the antifibrotic effects of MSCs on CCl 4 -induced LF. The enhanced antifibrotic potential primed by autophagy inhibition in MSCs may be attributed to their dual inhibitory effects on CD4 + and CD8 + T lymphocyte infiltration and hepatic stellate cells (HSCs) and Gli1 + cell proliferation, which may be regulated by elevated paracrine secretion of PTGS2/PGE 2 . PGE 2 , prostaglandin E 2 ; PTGS2, prostaglandinendoperoxide synthase 2

| CONCLUSIONS
In summary, our results confirmed, for the first time, that the LF microenvironment impeded the antifibrotic effects of MSCs by inducing autophagy. However, counteracting microenvironmentinduced autophagy promoted the antifibrotic effect of MSCs in CCl 4 -induced LF. The boosted antifibrotic potential primed by autophagy inhibition in MSCs may be attributed to their suppressive effect on CD4 + and CD8 + lymphocytes infiltration and HSCs proliferation, which were regulated by elevated PTGS2/PGE 2 via a paracrine pathway. Our data support autophagy as a putative new therapeutic target for MSC-based remedy in LF and other inflammatory disorders (Figure 7).

ACKNOWLEDGMENTS
The authors