key: cord-0955734-d6eoax7n
authors: Xiang, Ming; Liu, Tingting; Tian, Cheng; Ma, Kun; Gou, Jing; Huang, Rongrong; Li, Senlin; Li, Qing; Xu, Chuanrui; Li, Lei; Lee, Chih-Hao; Zhang, Yonghui
title: For the Topic – The New Therapeutic Approaches, Biomarkers and Molecular Mechanisms of Organ Fibrosis and COVID19 Rehabilitation Kinsenoside attenuates liver fibro-inflammation by suppressing dendritic cells via the PI3K-AKT-FoxO1 pathway
date: 2022-01-21
journal: Pharmacol Res
DOI: 10.1016/j.phrs.2022.106092
sha: 1db084c7c2d47e852b9832c136440d89a2fe1be2
doc_id: 955734
cord_uid: d6eoax7n

Kinsenoside (KD) exhibits anti-inflammation and immunosuppressive effects. Dendritic cells (DCs) are critical regulators of the pathologic inflammatory milieu in liver fibrosis (LF). Herein, we explored whether and how KD repressed development of LF via DC regulation and verified the pathway involved in the process. Given our analysis, both KD and adoptive transfer of KD-conditioned DC conspicuously reduced hepatic histopathological damage, proinflammatory cytokines release and extracellular matrix deposition in CCl(4)-induced LF mice. Of note, KD restrained the LF-driven rise in CD86, MHC-II, and CCR7 levels and, simultaneously, upregulated PD-L1 expression on DCs specifically, which blocked CD8(+)T cell activation. Additionally, KD reduced DC glycolysis, maintained DCs immature, accompanied by IL-12 decrease in DCs. Inhibiting DC function by KD disturbed the communication of DCs and HSCs with the expression or secretion of α-SMA and Col-I declined in the liver. Mechanistically, KD suppressed the phosphorylation of PI3K-AKT driven by LF or PI3K agonist, followed by enhanced nuclear transport of FoxO1 and upregulated interaction of FoxO1 with the PD-L1 promoter in DCs. PI3K inhibitor or si-IL-12 in DC could relieve LF, HSC activation and diminish the effect of KD. In conclusion, KD suppressed DC maturation with promoted PD-L1 expression via PI3K-AKT-FoxO1 and decreased IL-12 secretion, which blocked activation of CD8(+)T cells and HSCs, thereby alleviating liver injury and fibro-inflammation in LF.

Liver fibrosis (LF) 1 is a chronic condition with multifactorial pathology, and it poses a significant risk of progression into hepatocellular carcinoma [1] . In LF, hepatic immunity is altered and liver inflammation occurs, ultimately resulting in hepatic stellate cell (HSC) 2 activation, extracellular matrix (ECM) deposition and 1 LF: liver fibrosis 2 HSC: hepatic stellate cell J o u r n a l P r e -p r o o f 4 fibro-inflammation [2] . The animal treated with carbon tetrachloride (CCl 4 ) is the classic LF model. In the early stage of CCl 4 -induced LF, free radical compound (CCl 3 and Cl) in liver damages hepatocytes [3] . Then, injured hepatocytes disrupt immune homeostasis, and stimulate aberrant immune cell activity [4] . After being activated by damaged hepatocytes, innate immune cells, especially dendritic cells (DCs), recruit and activate CD8 + T effector cell [5] . CD8 + T cells in turn aggravate hepatocyte injury by releasing cytotoxic cytokines [6] . The secondary attack of immune cells results in the aggravation of hepatocyte injury and fibro-inflammation [7] . In addition, injured hepatocytes and activated immune cells transformed quiescent HSCs, the most important cell involved in LF, to myofibroblasts (MFB) via cell cross-talk [8] .

DCs are critical for the regulation of liver immunity, and irregular DC activity can induce the activation of T cells and HSCs to contribute to the pathological inflammation-rich environment and fibrogenesis LF [9] . The immaturation and immunosuppression of DCs could be mediated by programmed cell death ligand 1 (PD-L1), the ligand of programmed cell death 1 (PD-1) [10] . Immunosuppressed DCs produced fewer pro-inflammatory cytokines, such as IL-12. The IL-12 heterodimer containing IL-12 p40 and IL-12 p35 was reported to be correlated with LF and HSC activation [11] . Therefore, inducing DC immunosuppression may be an attractive approach to experimental therapeutics in fibro-inflammatory liver disease. Recently, DC function was reported to be associated with the phosphatidylinositol 3 3 KD: Kinsenoside J o u r n a l P r e -p r o o f 5 kinase/protein kinase B (PI3K-AKT) axis, which was implicated in reprogramming immune cell functions, controlling cellular responses (survival, proliferation, and metabolism), and regulating LF [12, 13] . The PI3K-AKT axis was activated in DCs stimulated by lipopolysaccharide (LPS), and LY294002 (PI3K inhibitor) blocked the activation of DCs [14] . One mechanism of PI3K-AKT-mediated regulation of cellular function involves the phosphorylated inactivation of the forkhead box protein O1 (FoxO1) [15] . Being a transcription factor, FoxO1 is capable of binding to gene promoters in the nucleus and regulating expression of target genes. However, the AKT-mediated phosphorylation of FoxO1 causes FoxO1 exit from the nucleus, along with the suppression of FoxO1 activity [16] . Multiple studies have highlighted FoxO1 involvement in LF progression, along with the maturity and function of DCs [17, 18] .

Anoectochilus roxburghii (A. roxburghii), widespread in tropical regions, is a traditional herb native to China [19] . Kinsenoside (KD 3 ,

roxburghii (Fig. S1A) . Emerging evidences revealed a wide array of pharmacological benefits of KD, including anti-hyperglycemia, anti-hyperlipidemia, anti-osteoarthritis, renoprotection and immunosuppression [20] [21] [22] [23] . Besides, KD ameliorated oxidative damage against retinal pigment epithelium and subsequent angiogenesis via ERK/p38/NF-κB pathway [24] , and protected advanced glycation end products (AGEs)-induced endothelial dysfunction through AGEs-RAGE-NF-κB pathway [25] .

A prior study demonstrated that KD exerted a hepato-protective effect on J o u r n a l P r e -p r o o f 6 CCl 4 -induced LF mice and in patients with liver disease, but the mechanism is currently unknown [26] .

Given these evidences, KD is possible to ease LF via enhancing DC immunosuppression. To further explore this, we detected the role of KD in LF therapy and mechanism in vivo, in vitro and applying cell adoptive transfer model. The results

suggested that KD exhibited fibro-inflammation relieve and LF prevention via inhibiting DC maturation and function by promoting FoxO1 binding to PD-L1

promoter via PI3K-AKT-FoxO1 axis. 

A. roxburghii was extracted by immersion in water four times at 80 ℃ until the crude of ethanol reached 95%. The supernatant was obtained and subjected to a silica gel chromatography column elution with chloroform-ethanol (4:1). The eluents with KD were combined under the direction of thin layer chromatography to produce a crude KD sample, which was further washed with ethanol to obtain pure KD. The purity of KD (>98%) was analyzed using a high-pressured liquid chromatography with an evaporative light scattering detector (Fig. S1B) , and identification was done with nuclear magnetic resonance (NMR), based on data from a prior report [27] .

As reported previously, 6-week-old C57BL/6J mice received intraperitoneal injection of 10% CCl 4 (dosage: 2 mL/kg) two times a week for 8 weeks to induce LF [28] 

DCs and T lymphocytes were prepared as described before [29] . .5-10 μg/mL). All cells were maintained at 37 ℃ in an incubator with 5% CO 2 . The cells were treated with agonists to simulate inflammation or injury microenvironment reflecting the in vivo conditions.

BMDCs were exposed to LPS (10 μg/ml, 24 h) to harvest the LPS-treated mature 

To characterize the KD-mediated regulation of JS-1 cross-priming response to DCs were assessed via quantitative real-time-PCR (qPCR), western blot, and immunofluorescence (IF).

AML-12 (mouse hepatocyte) was treated with 400 mmol/L alcohol for 24 h or 20 mmol/L CCl 4 for 48 h to produce the hepatocyte injury model [33, 34] . Cell apoptosis was examined with a FITC-Annexin V Apoptosis Detection Kit (BD Biosciences, Franklin, NJ, USA), as per operational guidelines. Upon 24 h or 48 h treatment, the cells were exposed to EDTA-free trypsin and re-suspended in 1×binding buffer, before exposure to FITC-Annexin V and PI at room temperature (RT) for 15 min.

Finally, the apoptotic rate was assessed via FCM. 11 

Liver sections underwent staining with hematoxylin and eosin (H&E) and sirius red for histopathological examination. Immunohistochemical staining was carried out 

Immune 

FoxO1 and negative siRNAs were acquired from the Viewsolid Biotech (Beijing, China). The following list belongs to the target sequences of these siRNAs: The siFoxO1-mus-1362-transfected cells exhibited optimal interference efficiencies (Data not shown). We, therefore, selected siFoxO1-mus-1362 for use in our experiment, and its interference effect is presented in Fig. S1D . 

ChIP assays were done with Simple ChIP Plus Enzymatic Chromatin IP Kit 

The cells were incubated in a 24-well Agilent Seahorse XF Cell Culture Microplate (Agilent Technologies, Inc., Santa Clara, CA, USA), at 10 4 cells per well. Next, the plate was placed in a 37 ℃ incubator to allow for cell adherence. Subsequently, the culture medium was removed and specific medium from the kit was introduced to the cells, which were then incubated without CO 2 . Detecting drugs were introduced to the cells, according to kit instructions. For oxygen consumption, the drugs used were oligomycin, carbonyl cyanide-4-trifluoromethoxyphenylhydrazone (FCCP), rotenone, and antimycin A. In addition, for the glycolysis stress test, we used glucose, oligomycin, and 2-Deoxy-D-glucose (2-DG).

BMDCs extracted from control, LF, and LF mice exposed to KD (30 mg/kg) were cultured as mentioned before. Spleen CD8 + T cells from normal mice and DCs subjected to different treatments were co-cultured at ratios of 10:2 and 10:1 for 48 h [36] . CD8 + T cell proliferation was examined with cell counting kit-8 (CCK8) assays at 450 nm using a multi-well plate reader.

Protein extracts were separated on 10% SDS-polyacrylamide gels, followed by 

Total RNA was isolated from liver tissues, DCs, and JS-1 with TRIzol, as per kit directions, and used as templates to form cDNA using the transcription kit (Promega, Madison, WI, USA). Subsequently, the cDNA was amplified via incubation at 95 ℃ Table S1 . 

In CCl 4 -induced LF mice, oral administration of 30, 20, or 10 mg/kg KD markedly diminished the extent of LF with substantial lymphocyte infiltration and collagen deposition (Fig. 1A ). In line with the macroscopic changes, the serum ALT and AST levels, along with the liver index conspicuously decreased with KD treatment (Fig.   1B) . Furthermore, KD lowered the production of pro-inflammatory cytokines like IL-2, IFN-γ, TNF-α, and IL-12, and raised the secretion of anti-inflammatory cytokines IL-10. KD also reduced NO release, thus, lowering inflammatory responses ( Fig. 1C and D) . Interestingly, the effects of KD were comparable to or better than those of silymarin, which was used in clinical settings against LF. In addition, KD had no effect on the serum ALT, AST levels or on the fibro-inflammatory histopathological damage and fibrosis agents in healthy mice ( Fig. S2A and B) . In additional, KD had no adverse effect on heart, lung, spleen, and kidney in healthy mice (Fig. S2C) , and did not alter the survival rate of normal hepatocyte AML-12 (Fig. S2D) . These data suggested that KD strongly suppressed fibro-inflammation in LF, without causing any serious adverse reactions.

Numerous immune cells regulate the development of hepatic fibro-inflammation [37] .

We quantitatively determined the proportions of various hepatic immune cells, involved in the CCl 4 -driven LF. After CCl 4 administration, DC maturity and CD8 + T cell differentiation were greatly enhanced, and M2-type macrophage polarization reduced slightly in the liver. KD reversed the CCl 4 -mediated regulation of DCs, CD8 + T cells, and M2-type macrophages, and had little effect on other subtypes of immune cells in the liver (Fig. S3 ).

Of note, in vitro, KD also strongly retarded BMDC maturation and function ( Fig. S4A-C) . However, KD repolarized M1 macrophages to the M2 phenotype J o u r n a l P r e -p r o o f 18 marginally (Fig. S4D) . And there was no obvious regulation of KD to magnetic bead sorting spleen CD8 + T cell proliferation (Fig. S4E) . The different effect of KD on CD8 + T cells in vivo and in vitro suggested that KD effect to CD8 + T cells might be indirect.

Given the vital role of hepatocyte injury and HSC activation in LF and to identify potential target cells of KD, the effect of KD on JS-1 (mouse HSCs) and

AML-12 (mouse hepatocytes) were further examined in vitro. Only high dose of KD lowered the expression and secretion of Col-I, TIMP-1, and TIMP-2, but did not alter the levels of α-SMA, as well as the apoptotic status of JS-1, compared to TGF-β 1 group (Fig. 2) . Compared to its predominant effect in vivo, KD exhibited a poor effect on HSC in vitro. Additionally, CCl 4 or alcohol induced apoptosis and ALT and AST release from AML-12, but KD did not relieve the injury of AML-12 directly (Fig. S5) . The apparent modulation loop involving interactions of DCs and CD8 + T cells might be important to maintain immune homeostasis during progression of LF.

In vivo, we observed that KD reduced the levels of MHC-II and co-stimulator CD86 on CD11c + BMDCs (Fig. 3A) . It inhibited CCR7, but substantially elevated levels of the negative regulatory molecule PD-L1 on CD11c + BMDCs ( Fig. 3B and C) . The level of indoleamine-2,3-dioxygenase (IDO), a tolerant DC-secreted anti-inflammatory agent, was also elevated upon KD exposure (Fig. 3C) . Moreover, J o u r n a l P r e -p r o o f 19 KD downregulated levels of IL-12, an essential DC-secreted inflammatory agent ( Fig. 3C and D) . In addition, using MLR, we revealed that KD significantly downregulated the DC-mediated induction of CD8 + T lymphocyte proliferation ex vivo (Fig. 3E ).

Following maturity, DC metabolism shift toward glycolysis [38] . We analyzed DC glucose metabolism after KD treatment. The results demonstrated that KD suppressed glucose utilization, Glut1 expression, glucose uptake, and glycolysis-related genes expressions in BMDCs. Additionally, oxygen consumption and ATP generation were weakened in BMDCs after KD treatment (Fig. S6 ). Since the maturation of DCs mainly depends on increased ATP flux via the glycolytic pathway [38] , it seems reasonable to consider that KD exerts its immunosuppressive effects in DCs at least in part by suppressing glycolysis in DCs. Immunogenic DCs serve a critical role in antigen presentation during early immune response. In all, these data suggest that the KD-mediated immunosuppressive and anti-inflammatory properties may largely ascribe to the suppression of DC maturation and function in LF.

To determine the immunomodulatory effect of KD-conditioned DCs on experimental LF and CD8 + T cells, LF mice were injected with KD (5 μg/mL)-treated DCs via tail vein. To estimate the distribution of injected DCs, DiR-labeled DCs were J o u r n a l P r e -p r o o f 20 administrated to mice in the same way, and high Dir fluorescence was found in the liver of mice (Fig. S7) . The results demonstrated that adoptive transfer DCs was abundant in the liver and adoptive transfer of KD-conditioned DCs to LF mice diminished the severity of fibro-inflammation in the liver. The expression of α-SMA, a HSC activation-related gene, in the liver was also been suppressed by KD-conditioned DCs ( Fig. 4A and B) . Moreover, we also observed alterations in the gene expression: α-SMA, TGF-β 1 , Col-I, and TIMP-1/-2 levels were reduced, whereas MMP-13 levels were elevated (Fig. 4C) . Additionally, in mice treated with KD-conditioned DCs, pro-inflammatory cytokines were diminished and anti-inflammatory cytokines were elevated in both serum and liver ( Fig. 4D and E) .

Hepatic CD8 + T cell levels were low in the LF mice with KD-conditioned DCs versus LF models (Fig. 4F) . Collectively, these results indicate that KD-conditioned DCs disrupts HSC and CD8 + T cell responses, and that DCs are indispensable to the action of KD on LF.

In our previous study, the PI3K-AKT axis was found to contribute to KD effect in autoimmune hepatitis [39] . Here, we revealed that KD strongly suppressed phosphorylation of PI3K (Tyr458) and AKT (Ser473), without affecting their mRNA and total protein expressions in BMDCs isolated from LF mice ( Fig. 5A and B) . Meanwhile, we pre-treated LPS-induced DCs with LY294002 (PI3K inhibitor), which retarded maturity and function of DCs. Adding KD did not enhance the effect of LY294002, which suggested that the main signal target of KD might have been blocked. Lastly, down-regulating FoxO1 using siFoxO1 also hindered the roles of KD in DCs (Fig. S8D-F) . We also found that 740 Y-P activated the phosphorylation of PI3K (Tyr458), AKT (Ser473), and FoxO1 (Ser256), while, KD treatment caused less phosphorylation of these molecules, and increased FoxO1 presence in the nucleus of DCs ( Fig. S9A and B) . Interestingly, blockade of PI3K-AKT axis using LY294002 diminished the effect of KD on PI3K-AKT-FoxO1 axis ( Fig. S9C and D) , and PD-L1 expression on DCs (Fig. S8E and F) .

To elucidate the mechanism whereby FoxO1 mediated the KD-induced immune tolerance of DCs, we further examined the direct interaction of FoxO1 and the PD-L1 promoter, using the ChIP assay. Based on our data, KD significantly increased FoxO1 binding to the PD-L1 promoter in LPS-activated DCs (Fig. 5F ). 

Inhibiting the activation and ECM secretion of HSCs is the center for treating LF [40] . In addition, HSCs are capable of interacting with immune cells [41] . And it is considered that the activated immune cells-secreted IL-6/IL-12 cytokine family may be crucial for the activation of HSCs [42] . 6 ). These data suggested that KD inhibited IL-12 generation in DCs, which impeded the interaction of JS-1 with DCs, thus reducing JS-1 activation.

Our data also revealed that both KD and LY294002 inhibited the expression and secretion of IL-12 in BMDCs, and the KD-mediated effect on IL-12 secretion was J o u r n a l P r e -p r o o f 23 abolished upon pretreatment with PI3K inhibitor (Fig. 3C and D, S4B and C, and S8F). Additionally, 740 Y-P raised IL-12 production in DCs, whereas KD reversed it (Fig. S8C) . Furthermore, FoxO1 deficiency invalidated KD-mediated regulation of IL-12 generation by BMDCs (Fig. S8F) . These results elucidated that the decreased expression and release of IL-12 in DCs by KD might rely on PI3K-AKT-FoxO1 axis.

Herein, we demonstrated a KD-mediated suppressive effect on fibro-inflammation and DC function in CCl 4 -induced LF. KD impeded the maturation of DCs and caused immunosuppression by promoting the binding of FoxO1 to the PD-L1

promoter via the PI3K-AKT pathway. Subsequently, the immunosuppressive DCs suppressed the CD8 + T cell differentiation by inducing PD-L1 and down-regulated HSC activity via secreting fewer IL-12 (Fig. 7) .

The liver gathers a large population of DCs which enter the liver as immature cells via the portal vein and continue to mature with the progression of LF [43] .

DCs and kupffer cells, which produces persistent inflammation and fibrosis [44] .

Hepatic inflammation during LF is regulated by immune cells, especially DCs. In patients with advanced LF, liver-infiltrating CD8 + T cells are abundant, and are likely abnormally induced by activated DCs [45] . DCs-regulated liver immunity also plays J o u r n a l P r e -p r o o f 24 a central role in the progress of LF. Abnormal DCs accelerate the development of fibrosis [46] . Inflammatory DCs, expressing CX3C chemokine receptor1 (CX3CR1), play a major role in inflammation in mice with liver injury and are associated with elevated TNF-α levels [46] . Results reported here indicated that KD had an indirect regulation on normal liver cells (AML-12). However, KD-exposed DCs exhibited immature phenotypes, decreased chemokine receptor CCR7 densities, increased PD-L1 levels, lowered secretion of pro-inflammatory cytokines, and suppression of T cell activation, especially CD8 + T cells, thus alleviating liver injury and fibro-inflammation. Furthermore, adoptive transferring KD-conditioned DCs ameliorated inflammation and LF, which suggests that DCs are the main target cells mediating the inhibited effect of KD on fibro-inflammation in murine LF.

HSCs are the most important cell type involved in LF [47] . In normal liver, HSCs exist in a quiescent non-proliferative state [48] . During acute and chronic liver injury, HSCs activate and transdifferentiate to MFB [49] , which are the main source of collagens and other ECM proteins [50] . The activation of HSCs could be stimulated by inflammatory cytokines, such as IL-1 and IL-6 [51] . Previous study reported IL-6/IL-12 cytokine family was able to transform quiescent HSCs into MFB via inducing STAT1 and STAT3 phosphorylation [42] . IL-12 is involved in the interaction between DC and HSC potentially. DC-mediated liver inflammation is predominantly reliant on IL-12, which contributes to the progression of LF [52] . Reports showed that The targeted effect of KD on fibrotic DCs might rely on PI3K-AKT axis in DCs.

The PI3K-AKT axis regulates a myriad of cellular responses like cell survival, proliferation, metabolism, and so on [12, 13] . Mice deficient in the PI3K display severely impaired DC migration in response to the chemokines. In lymphocytes, PI3K are important in regulating basal lymphocyte motility, and PI3K expression is required for B cell morphology [54] . The PI3K-AKT axis participates in the reprogramming of the activities of numerous immune cells, especially DCs and macrophages [12] . PI3K-AKT axis was reported to be able to regulate the progression of acute lung injury via altering the maturation and function of DCs [14] .

Additionally, the pathway is crucial for the glycolysis in DCs [55] . DC stimulation via toll-like receptor (TLR) agonists results in the acceleration of glycolysis. These alterations are produced by pathways that involves AKT, and are crucial for DC activation [56, 57] . Additionally, targeted inhibition of AKT strongly suppresses the However, the molecular targets of PI3K-AKT in the regulation of DCs remain elusive. PI3K-AKT axis influences intracellular events via multiple pathways, for example, PI3K-AKT axis regulates cell survival and proliferation by activating metabolic genes sterol-regulatory element-binding proteins (SREBPs) and AS160 [59, 60] . Notably, FoxO1 is also a significant modulator of cell survival, metabolism and function [17] . AKT phosphorylates FoxO1 and causes FoxO1 to exit from the nucleus.

This, in turn, regulates the transcription of targeted genes [61] . In addition, FoxO1

facilitates cell cycle arrest partly via the production of cell cycle inhibitory agents like p27Kip1 and p21, and suppression of cyclin D1 levels [62] . 

KD treatment improved the liver inflammatory microenvironment in LF and reprogrammed intracellular glycolysis to decrease DC migration and maturation.

Mechanistically, KD diminished the activity of the PI3K-AKT axis and increased 

The authors declare no competing financial interests.

Animal care and experimental procedures were carried out in accordance with the guidelines of the Tongji Medical College, Huazhong University of Science and Technology Institutional Animal Care and Use Committee (IACUC Number: 2505).

All data generated or analyzed during this study are included in this published article. Immunofluorescence of FoxO1 (green) in DCs. BMDCs isolated from control mice or LF mice treated with or without KD (30 mg/kg). (F) ChIP assay exploring the physical binding between FoxO1 and PD-L1 promoter in DCs. IgG was used as the matched control. LPS-stimulated DCs were either exposed to KD (5 μg/mL) or not.

Data expressed as mean ± SD (n=6). * P<0.05, ** P<0.01 relative to controls; # P<0.05, ## P<0.01 relative to CCl 4 or LPS groups. n.s., not significant. Data expressed as mean ± SD (n=6). ** P<0.01 relative to normal DCs group; ## P<0.01 relative to LF DCs group. n.s., not significant. 

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as assessed by the mRNA levels of FoxO1 via qPCR (left), and protein levels of FoxO1 via western blot (right). (E) The interference efficiency of si-IL-12, as assessed by the mRNA levels of IL-12 p40 via qPCR (left), and the levels of total IL-12 in DC suspension via ELISA (right)

Supplementary Figure 2. KD exerted nontoxic nature for healthy mice and normal hepatocytes and showed better effect on LF than silymarin. (A) Representative images of H&E, sirius red, and α-SMA immunohistochmical antibody stained liver sections. Original magnification, 200×; scale bar, 100 μm. (B) Levels of ALT and AST in serum. KD group mice received KD at a dose of 30 mg

Positive mice received Silymarin (50mg/kg) after CCl 4 treatment. (C) Representative images of H&E stained heart, lung, spleen and kidney sections. Original magnification, 200×; scale bar, 100 μm. (D) Survival rate of AML-12. Data expressed as mean ± SD (n=6)

05, ## P<0.01 relative to CCl 4 group. n.s., not significant

Supplementary Figure 3. The KD-mediated regulation of hepatic immune cells in LF mice. (A) Representative FCM images (left) and percentage (right) of CD86 and MHC-II molecules on the surface of DCs. MHC-II and CD86 were assessed on

The proportion of CD4 + T, CD8 + T cells, NK cells (CD3 -NK1.1 + ), and NKT cells (CD3 + NK1.1 + ). CD4 and CD8 levels were examined in CD3 + T cells. (C) The percentage and polarization of macrophages. (D) The proportion of MDSCs

Data expressed as mean ± SD (n=6). * P<0.01, ** P<0.01 relative to controls

# P<0.05, ## P<0.01 relative to CCl

The KD-mediated regulation of immune cells in vitro. (A) The proportion of MHC-II, CD86, and CCR7, as well as mean fluorescence intensity of PD-L1 on CD11c + DCs, as detected by FCM. (B) The transcript levels of IDO, IL-12 p40, and PD-L1 in DCs. (C) The secretion of total IL-12 in DC supernatant

D) The proportions of M1 and M2 macrophage, as evidenced by FCM (up), and the mRNA levels of cytokines in macrophages, as assessed by qPCR (down). (E) CD8 + T cell proliferation, as assessed by MTT. Data expressed as mean ± SD (n=6)

# P<0.05, ## P<0.01 relative to LPS

Apoptotic rate of AML-12 induced by CCl 4 . (B) Survival rate of AML-12 induced by CCl 4 . (C) ALT and AST level in AML-12 supernatant. (D) Apoptotic rate of AML-12 induced by alcohol. Data expressed as mean ± SD (n=6)

BMDCs isolated from normal mice were LPS-stimulated, with or without KD. (C) Glycolysis-related genes were analyzed using qPCR. (D) Glucose uptake. DCs extracted from LF mice that either received or did not receive KD. Data expressed as mean ± SD (n=6)

05, ## P<0.01 relative to LPS or CCl 4 groups

Supplementary Figure 7. The biodistribution of DiR-labeled DC in heart, lung, liver, spleen and kidney at different days after adoptive transfer (n=6)

KD suppressed DC maturation and activity via the PI3K-AKT-FoxO1 axis. (A) The percentage of MHC-II and CD86 on CD11c + DCs. (B) The percentage of CCR7 and the mean fluorescence intensity of PD-L1 on

Transcript levels of IDO, IL-12 p40, and PD-L1, and the secretion of total IL-12. DCs isolated from C57BL/6J mice were incubated with 740 Y-P or

D) The percentage of MHC-II and CD86 on CD11c + DCs. (E) The percentage of CCR7 and the mean fluorescence intensity of PD-L1 on CD11c + DCs

Transcript levels of IDO, IL-12 p40, and PD-L1, and the secretion of total IL-12

DCs isolated from C57BL/6J mice were incubated with LY294002, LY294002+KD, siFoxO1, or siFoxO1+KD, in the presence of LPS. Data expressed as mean ± SD

05, ** P<0.01 relative to controls

05, ## P<0.01 relative to LPS group. n.s., not significant

A) Effects of KD and 740 Y-P on the phosphorylated and total levels of PI3K, AKT, and FoxO1. (B) Effects of KD and 740 Y-P on the nuclear (left) and cytoplasmic (right) levels of FoxO1 protein. DCs isolated from C57BL/6J mice were incubated with 740 Y-P or 740 Y-P+KD. (C) Effect of KD and 1. KD alleviated fibro-inflammation in LF

KD enhanced FoxO1 binding to PD-L1 promoter in DC, inhibiting CD8 + T cell responses

KD decreased IL-12 secretion in DC to remit inflammation and block HSC

We gratefully acknowledge Prof. Robert L. Rodgers, Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, for providing constructive discussions and language editing.