MyD88 in Hepatic Stellate Cells Promotes the Development of Alcoholic Fatty Liver via the AKT Pathway

doi:10.21203/rs.3.rs-1030228/v1

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MyD88 in Hepatic Stellate Cells Promotes the Development of Alcoholic Fatty Liver via the AKT Pathway

https://doi.org/10.21203/rs.3.rs-1030228/v1

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Myeloid differentiation primary response gene 88 (MyD88), an adaptor protein in the TLRs signaling pathway, is expressed in various liver cells including hepatocytes, Kupffer cells and hepatic stellate cells (HSCs). However, the specific role of MyD88 in HSCs in alcoholic fatty liver (AFL) has not been well investigated. Here, to study the role of MyD88 in HSCs in the development of AFL and its related molecular mechanism, chronic-binge ethanol mice models were established in mice with specific MyD88 knockout in quiescent (MyD88^GFAP−KO) and activated HSCs (MyD88^SMA−KO), respectively. The results showed that the expression of MyD88 in HSCs was significantly increased in ethanol-induced liver tissues of wild-type mice. MyD88 deficiency in quiescent HSCs inhibited inflammation and lipogenesis, but in activated HSCs it only inhibited inflammation under the ethanol feeding condition. Further mechanism studies found that MyD88 promoted the osteopontin (OPN) secretion of HSCs, which further activated the AKT signaling pathway of hepatocytes and upregulated lipogenic gene expression to promote fat accumulation. In addition, OPN also promotes inflammation by activating p-STAT1. Thus, MyD88 may represent a potential candidate target for the prevention and targeted therapy in AFL, and MyD88 inhibitor can be also applied in inhibiting adipogenesis.

Molecular Biology

Internal Medicine

Molecular Genetics

Drug Discovery, Design, & Development

MyD88

hepatic stellate cells

alcoholic fatty liver

Osteopontin

The expression of MyD88 in HSCs was significantly increased in ethanol-induced liver tissues of wild-type mice.
MyD88 deficiency in quiescent HSCs inhibited inflammation and lipogenesis under the ethanol feeding condition.
MyD88 deficiency in activated HSCs only inhibited inflammation under the ethanol feeding condition.
MyD88 promoted the OPN secretion of HSCs, which further activated the AKT signaling pathway of hepatocytes and upregulated lipogenic gene expression to promote fat accumulation.
OPN also promotes inflammation by activating p-STAT1.

Excessive alcohol consumption is a leading cause of alcoholic liver disease (ALD), which is characterized by a spectrum of liver pathology ranging from fatty liver, alcoholic hepatitis, fibrosis, cirrhosis and hepatocellular carcinoma (HCC)[1, 2]. Alcoholic fatty liver (AFL) is the initial stage of the disease, and increasing evidences suggest that it has potentially pathologic condition[3, 4]. To date, several molecular mechanisms regulating hepatic lipid metabolism have been elucidated[5, 6]. Ethanol metabolism–associated oxidative stress and chemokines have been reported to play critical roles in the development of AFL[7, 8]. Although potential therapeutic methods for AFL have been developed for decades, effective therapies for AFL are still in great needs.

AFL is a kind of disorder in lipid metabolism, which is mainly manifested by hepatic steatosis. Steatosis is defined as intrahepatic fat of at least 5% of liver weight, which is characterized by the increase of triglyceride and cholesterol esters and is the first manifestation of chronic ethanol-induced fatty liver[9]. Enhanced lipid synthesis results from a higher expression of lipogenic enzymes and cytokines, such as fatty acid synthase (FAS), acyl CoA carboxylase (ACC), ATP citrate lyase (ACL) and malic enzyme (ME), which are regulated by sterol regulatory element binding protein-1c (SREBP-1c)[10, 11]. Hepatic ethanol oxidation triggers the translocation of SREBP-1c. SREBP-1c is matured by proteolysis and becomes a SREBP protein fragment with transcriptional activity, which enters the nucleus and enhances lipogenic gene expression. Regulation of lipid metabolism can be used as a target for prevention or treatment of fatty liver related steatosis.

Hepatic stellate cells (HSCs) are a kind of non-parenchymal cells in the liver. In a healthy liver, HSCs are in a quiescent state and contain a large number of lipid droplets storing vitamin A[12]. When the liver is damaged, HSCs are activated into myofibroblasts, express α-smooth actin(α-SMA), and secrete a large number of extracellular matrix proteins[13]. HSCs play a role in AFL, hepatitis, liver fibrosis, liver cirrhosis and liver cancer. Activated HSCs can promote liver fibrosis and regeneration of liver epithelial cells. In addition, activated HSCs release a variety of growth factors and promote tumor growth[14]. Activated HSCs can also recruit macrophages and promote the secretion of TNF-α, which binds to the corresponding receptors on hepatocytes to induce the expression of fat related genes. It has also been reported that alcohol stimulates HSCs to produce more 2-arachidonoylglycerol (2-AG), which can increase the expression of fat related genes in hepatocytes through paracrine pathway and promote the occurrence of AFL[15]. At present, the mechanism of HSCs regulating lipogenesis is not very clear.

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) in the form of dimers and further activate innate immune system[16, 17]. TLRs are expressed in many different cell types of liver with different expression patterns and play important roles in chronic liver diseases through the interaction between different cell types. Myeloid differentiation primary response gene 88 (MyD88) is a key adaptor molecule for the most TLRs (except TLR3) and IL-1 receptor families and is mainly responsible for transmitting signals to the downstream, activating NF-κB and MAPK family members and leading to the production of pro-inflammatory or anti-inflammatory cytokines and type I interferon[18]. MyD88 plays significant roles in inflammation related diseases. In addition, it is also involved in regulating the metabolism of energy, glucose and lipids in a variety of cells[19]. The physiological functions of MyD88 varied in different tissues and cell types.

Neuronal MyD88 dependent signal was a key regulator of diet induced insulin resistance in vivo. It has been reported that MyD88 specific deletion in the central nervous system could prevent peripheral glucose metabolism impairment induced by HFD, palmitate and blunted insulin resistance[20]. However, systemic knockout of MyD88 exacerbate the impaired glucose tolerance induced by high-fat diet[21] and increase the risk of diabetes in mice[22]. Hepatocyte specific deletion of MyD88 contributes to glucose intolerance, inflammation and liver insulin resistance independently of body weight and adiposity, which has been proved in the album-MyD88 KO mice with high-fat diet model and are partly due to differences in gene expression and transcription factor activity[23]. Furthermore, MyD88 in endothelial cells and myeloid cells also induces inflammation and insulin resistance in high-fat diet[24]. Nagy LE et al. found that deficiency of MyD88 in myeloid cells inhibited the occurrence of steatosis and inflammation in myeloid-specific MyD88-deficient (MyD88^LysM−KO ) mice with Lieber-DeCarli ethanol containing diet model[25]. MyD88 in macrophages(Kupffer cells)promotes ethanol-induced liver injury and steatosis in the high-fat diet model[26]. However, the role of MyD88 in HSCs in the development of AFL and its mechanism remains unclear.

For this purpose, mice with genetic deletion of MyD88 in quiescent HSCs (MyD88^GFAP−KO) and activated HSCs (MyD88^SMA−KO) matched with littermate controls were used in a chronic-binge ethanol model for studying the HSC-specific role of TLR signaling and its related molecular mechanism in ALD. We found that MyD88 was mainly expressed in HSCs and alcohol-induced liver injury and inflammation in mice. MyD88 in quiescent HSCs also promotes hepatic fat accumulation. Further mechanistic studies found that MyD88 in HSCs promotes liver fat accumulation mainly by secreting OPN, activating the AKT pathway and upregulating lipogenic gene expression. Taken together, these results indicated that MyD88 may represent a potential candidate for the prevention and treatment of AFL.

2.1 Mice

MyD88^flox/flox (MyD88^fl/fl), GFAP-Cre[27] and SMA-Cre mice[27-30] on a C57BL/6 background have been previously described. Mice with a conditional knockout of MyD88 in GFAP-expressing quiescent HSCs (MyD88^GFAP−KO) were generated by crossing MyD88^fl/fl and GFAP-Cre mice. And mice with a conditional knockout of MyD88 in SMA-expressing activated HSCs (MyD88^SMA−KO) were generated by crossing MyD88^fl/fl and SMA-Cre mice. Eight-week-old male mice were used. All mice were bred in specific pathogen-free and humidity- and temperature-controlled microisolator cages with a 12-hour light/dark cycle at the Institute of Biophysics Chinese Academy of Sciences. All animal studies were performed after being approved by the Institutional Laboratory Animal Care and Use Committee of the Institute of Biophysics, Chinese Academy of Sciences.

2.2 Chronic-binge ethanol mice models

Chronic-binge ethanol mice models was established as described previously[31]. Ten week old male C57BL/6 mice, MyD88^GFAP−KOand MyD88^SMA−KO mice were fed with liquid control diet (TROPHIC, Nantong, China) for 2 days to adapt liquid diet at 4pm every afternoon and then were divided into two groups randomly and five mice in each group: ethanol group was fed with liquid Lieber-DeCarli diet (TROPHID, Nantong, China) containing 1% ethanol for 2 days, increasing to 2% ethanol for 2 days, and 4% ethanol for 7 days and finally maintained at 5% ethanol for 10 days at 4pm every afternoon, followed by a single dose gavage of 31.5% ethanol (5 g/kg body weight) on the last day. Control mice were fed with pair-fed diet and finally given a single dose gavage of isocaloric dextrin maltose. Mice were always given a gavage in the morning and euthanized 9 h after receiving a gavage. For the ethanol feeding period, food intake of the ethanol-fed mice was recorded, and the weight-matched control feeding mice were pair-fed with the same volume of the control diet, so that ethanol- and pair-fed mice consumed equal amounts of diet and ingested the same energy. Besides the body weight of mice was recorded every day.

2.3 Cell culture

The LX-2 and HepG2 cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). These cells were cultured in DMEM (BI, Israel) supplemented with 10% fetal bovine serum (FBS, BI) and 1% penicillin/streptomycin at 37°C with 5% CO₂. In the experimental groups, LX-2 cells were exposed to ST2825 (10 µM) (MedChemExpress, USA) for 2 h, and then were challenged with TLR1/2 agonist (Pam3CSK4, 100 ng/mL), TLR4 agonist (LPS, 1 mg/mL) and palmitic acid (400 µM) (Sigma, USA) respectively for 24 h. In the control groups, LX-2 cells were directly challenged with TLR1/2 agonist (Pam3CSK4, 100 ng/mL), TLR4 agonist (LPS, 1 mg/mL) and palmitic acid (400 µM) (Sigma, USA) respectively for 24 h. To induce excessive lipid accumulation, HepG2 cells were cultured in DMEM medium with 20 µM AKT inhibitor (CSNpharm, USA) for 24h, then 400 µM palmitic acid and 200 ng/mL recombinant protein OPN (Cloud-Clone Corp, China) for 24h[32].

2.4 Isolation of HSCs

Isolation of primary hepatic stellate cells was performed as previously described[30]. 10-week-old mice were used to isolate primary hepatic stellate cells. Briefly, primary HSCs were isolated from livers by in situ perfusion and density gradient centrifugation. The mice were anesthetized with 3% ethyl carbamate and treated with phosphate belanced solution and sterile collagenase for liver perfusion. And the liver was removed, cut it into small pieces, and was placed in the digestive juice at 37°C for 30 minutes. Filtered cells were centrifuged at 50×g for 2 min to remove hepatocytes. The remaining non-parenchymal cell (NPC) fraction was collected, washed, and isolated by a 10% and 40% Optiprep (Axis-Shield, Scotland) gradient system, respectively[33]. The purity of the HSC fraction was estimated based on the autofluorescence signal. Cell viability was examined by Trypan blue exclusion. Both the cell purity and viability were greater than 90%.

2.5 Biochemical assays of blood and liver tissues

Blood samples of mice were collected by the eyeball and centrifuged at 3000⋅g for 10 minutes to obtain serum. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), cholesterol (Cho) and triglyceride (TG) levels were detected by Pony Testing International Group (Beijing, China). Liver tissues were homogenized in cold phosphate-buffered saline (PBS) and centrifuged at 2400 g for 10 minutes to obtain supernatant, and TG and Cho levels in supernatant were measured by Pony Testing International Group (Beijing, China).

2.6 Histology and immunostaining

Paraffin or cryostat sections of liver tissue were prepared as described previously[34]. The sliced liver paraffin sections were stained with hematoxylin and eosin (H&E) (Zhongshanjinqiao, Beijing, China) and Sirius Red, respectively. To detect hepatic fat accumulation, cryostat liver sections were stained with Oil Red O. For immunofluorescence detection, the cryostat sections were respectively incubated with anti-F4/80, anti-Gr-1, anti-CD11b (1:200; BD Pharmingen, San Diego, CA), anti-MyD88 and anti-GFAP antibodies (1:200; Abcam, Cambridge, UK), and followed by incubation with Alexa Fluor 488- or 594-conjugated secondary antibodies (1:500; Invitrogen, Carlsbad, CA, USA). Eventually, cell nuclei were stained with DAPI. Results were evaluated under the microscope (DP71, OLYMPUS) of both bright-field and fluorescence microscopy.

2.7 Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was isolated from frozen liver tissues and cells respectively by TRIzol reagent (Invitrogen, USA), and then up to 0.5µg of RNA was reverse transcribed by Prime Script RT Master Mix Kit (MedChemExpress, Princeton, NJ, USA). cDNA was duplicated by SYBR Premix ExTaqTM Kit (MedChemExpress, Princeton, NJ, USA). The primer sequences are listed in Supplementary Table 1. Data were analyzed using the 2^−ΔΔCt method and normalized to GAPDH expression[35].

2.8 Western blot analysis

Western blot was performed as previously described[36]. Tissue and cell extracts were analyzed with the following primary antibodies: anti-MyD88 (Abcam, Cambridge, UK), anti-AKT, anti-p-AKT, anti-STAT1 and anti-p-STAT1 antibodies (Cell Signaling Technology, USA). HRP-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were used as secondary antibodies. Blots were scanned using a Clinx Science Instrument. All specific bands were quantified with the ImageJ automated Digitizing System.

2.9 Quantitative and statistical analysis

All the data were expressed as means±SEM and analyzed using GraphPad Prism software. Differences between the two groups were compared using two-tailed unpaired Student’s t-test analysis. One-way ANOVA tests with a Bonferroni correction were used for multiple comparisons. For all tests, p < 0.05 was considered statistically significant. p < 0.01 was considered extremely significant.

3.1 Expression of MyD88 in HSCs is upregulated in the liver of AFL mice

To explore the relationship between the expression of MyD88 in mouse liver and the development of AFL, C57BL/6 mice were administered ethanol to generate a chronic-binge ethanol mice models, which is widely used for the pathogenetic study of AFL. Liver tissues were harvested after AFL was established (Fig. 1a). As shown in Fig. 1b, c, the protein expression level of MyD88 was significantly upregulated in the livers of ethanol-fed mice compared with control mice detected by Western blot. MyD88⁺ cells in the liver tissues were further detected by immunofluorescence staining. Consistently, ethanol feeding led to a significant increase of the number of MyD88⁺ cells. Double immunofluorescence staining revealed that most of HSCs (GFAP⁺ cells) of AFL mice expressed MyD88 (Fig. 1d). The results clearly demonstrated that MyD88 expression in HSCs of WT mice was upregulated under the ethanol feeding condition.

3.2 MyD88 deficiency in HSCs attenuates ethanol-induced liver injury

To investigate the contribution of MyD88 in HSCs in liver injury, MyD88 ^GFAP−KO and MyD88 ^SMA−KO mice were generated by Cre loxp system, and control littermates were used in a chronic-binge ethanol model (Supplementary Fig. 1). There is no significant difference of the body weight between MyD88 ^GFAP−KO and MyD88^fl/fl mice, MyD88 ^SMA−KO and MyD88^fl/fl mice under the ethanol feeding condition (Fig. 2a, b). To characterize the pathology associated with ethanol feeding in MyD88^GFAP−KO, MyD88^SMA−‍KO and MyD88^fl/fl mice, the liver tissues from each group were collected for further evaluation. The liver/body weights of the MyD88^GFAP−KO mice were significantly increased compared with those of MyD88^fl/fl mice (Fig. 2c). However, there was no significant differences of the liver/body weights between MyD88^SMA−KO mice and MyD88^fl/fl mice (Fig. 2d). We further examined the serum ALT and AST levels in MyD88^fl/fl, MyD88^GFAP−KO and MyD88^SMA−KO mice under the ethanol feeding condition. The results showed that the ALT level was significantly decreased in MyD88^GFAP−‍KO mice compared with that in MyD88^fl/fl mice (Fig. 2e, f) and the AST level was significantly decreased in MyD88^SMA−‍KO mice compared with that in control mice (Fig. 2g, h). Further H&E staining showed that both MyD88^GFAP−KO and MyD88^SMA−‍KO mice had reduced inflammatory cell infiltration, hepatocyte cell death and steatosis compared with MyD88^fl/fl mice (Fig. 2i, j). These results indicated that MyD88 knockout in HSCs significantly reduced liver injury.

3.3 MyD88 deficiency in HSCs attenuates ethanol-induced liver inflammation

To investigate the effect of MyD88 in HSCs on inflammation, the infiltration of immune cells was detected by immunofluorescence staining. The results showed that the infiltration of F4/80⁺ macrophages, Gr1⁺ neutrophil and CD11b⁺ monocyte was significantly reduced in MyD88^GFAP−KO mice and MyD88^SMA−‍KO compared with MyD88^fl/fl mice under the ethanol feeding condition (Fig. 3a-d). We further examined the gene expression of a series of pro-inflammatory cytokines in MyD88^fl/fl, MyD88^GFAP−KO and MyD88^SMA−‍KO mice under the ethanol feeding condition. As shown in Fig. 3e, f, mRNA levels of the proinflammatory cytokines interleukin-6 (IL-6), IL-1β, tumor necrosis factor α (TNF-‍α) and monocyte chemoattractant protein-1 (MCP-1) were significantly down-regulated in livers of MyD88^GFAP−KO and MyD88^SMA−‍KO mice compared with that of MyD88^fl/fl mice after ethanol feeding. In summary, MyD88 knockout in HSCs can significantly attenuates hepatic inflammation.

3.4 MyD88 deficiency in HSCs attenuates fat accumulation

AFL is mainly caused by continuous alcohol intake. The fat in the liver becomes small lipid droplets in the early stage of steatosis, and subsequently expands into large lipid droplets. Fat accumulation in the liver is often accompanied by elevated Cho and TG. The levels of Cho and TG in mouse serum and liver tissue were further detected by biochemical indicators.

The results showed that except serum TG, the levels of serum Cho,hepatic Cho and hepatic TG were significantly decreased in the MyD88^GFAP−KO mice compared with those in MyD88^fl/fl mice (Fig. 4a, b). However, there was no significantly difference in serum and hepatic TG and Cho between MyD88^SMA−KO and MyD88^fl/fl mice (Fig. 4c, d).

In order to further verify the production of TG and other neutral fats in the liver tissues, the liver tissues of MyD88^GFAP−KO, MyD88^SMA−KO and MyD88^fl/fl mice were stained with Oil Red O. As shown in Fig. 4e, the accumulation of lipid droplet in the liver tissues of MyD88^GFAP−‍KO mice was significantly reduced compared with MyD88^fl/fl mice. The accumulation of lipids occurred in hepatocytes. Consistently, the mRNA levels of lipogenesis genes such as SREBP1, SCD1, ACC1 and FANS were significantly downregulated in the liver tissues of MyD88^GFAP−KO mice compared with MyD88^fl/fl mice (Fig. 4f). However, there was no significant differences in hepatic fat accumulation and related lipogenesis genes between MyD88^SMA−KO and control mice (Fig. 4g, h). In addition, results from Sirius Red staining demonstrated that no obvious fibrosis occurred, and MyD88 deficiency in quiescent HSCs has no obvious effects on liver fibrosis in this model (Supplementary Fig. 2). These results indicated that MyD88 deficiency in HSCs significantly inhibited hepatic fat accumulation.

3.5 MyD88 deficiency in quiescent HSCs inhibits the expression of OPN

To study how the MyD88 in quiescent HSCs affected the ethanol-induced inflammatory and adipogenesis in liver cells, the GEO database (GSE151158) was used to analyze the gene expression levels in the livers of 21 healthy people and 40 fatty liver patients and found that the expression level of OPN gene in the livers of fatty liver patients was significantly increased (Fig. 5a). Related evidence also showed that OPN could promote adipogenesis[37]. Then the expression of OPN in the livers of MyD88^GFAP−KO and MyD88^fl/fl mice was detected by RT-qPCR. As shown in Fig. 5b, the expression level of OPN in the liver of MyD88^GFAP−KO mice was significantly decreased compared with MyD88^fl/fl mice, and there is no obvious change in MyD88^GFAP−KO mice (Fig. 5c). Immunohistochemical staining further confirmed that the OPN protein expression in the liver of MyD88^GFAP−KO mice was also significantly reduced (Fig. 5d).

To explore whether MyD88 deficiency in quiescent HSCs would affect the secretion of OPN, the HSCs line LX-2 was treated with MyD88 inhibitor ST2825 for 2h and then with TLR1/2 and TLR4 agonists for 24h. The results showed that the agonist treatment promoted the expression of OPN in LX-2 cells, and the OPN expression was significantly reduced after adding the MyD88 inhibitor ST2825, and the TLR4 agonist had the most significant effects (Fig. 5e). The above results showed that the TLR4 signaling pathway played a key role in regulating the expression of OPN. After activation of the TLR4 signaling pathway by palmitic acid, the expression of MyD88 and OPN genes were detected by RT-qPCR. The result indicated that ST2825 significantly inhibited the expression of MyD88 (Fig. 5f), and palmitic acid significantly promoted the expression of OPN, while ST2825 inhibitor significantly reduced the OPN expression (Fig. 5g). The results showed that the expression of OPN in HSCs was regulated by MyD88.

To further study the effect of OPN secreted by HSCs on the adipogenesis of HepG2 cells, HepG2 cells were treated with palmitic acid to induce fatty liver-like cells, and the expression of hepatic lipogenesis genes in HepG2 cells was detected by RT-qPCR. The mRNA levels of SREBP1, SCD1, ACC1 and FANS in HepG2 cells were significantly up-regulated after palmitic acid treatment (Fig. 5h). Subsequently, the recombinant protein OPN was added exogenously to explore its effect on the adipogenesis of HepG2 cells. RT-qPCR results showed that OPN significantly promoted the expression of adipogenesis-related genes SCD1、FANS、SREBP and ACC1 in HepG2 cells (Fig. 5h). The results indicated that OPN secreted by HSCs significantly promoted adipogenesis in HepG2 cells.

3.6 MyD88 promoted liver fat accumulation and inflammation via the AKT pathway and STAT1 pathway respectively

To elucidate the underlying mechanism responsible for ethanol-induced hepatic lipid accumulation, we tested the signaling pathways in which MyD88 may be involved during liver fat accumulation and inflammation. The results showed that there were no significant differences in the total protein level of AKT in the liver tissues between MyD88^GFAP−KO mice and MyD88^fl/fl mice (Fig. 6a), but the p-AKT expression was significantly increased in the liver of MyD88^GFAP−KO mice compared with MyD88^fl/fl mice (Fig. 6a, b). Meanwhile, it was found that there were no significant differences in the total protein level of STAT1 in the liver of MyD88^GFAP−KO mice and MyD88^fl/fl mice, but the phosphorylation level of STAT1 in MyD88^GFAP−KO mice was significantly lower than that of MyD88^fl/fl mice (Fig. 6a, b). The AKT and STAT1 signaling pathway was further analyzed in the human hepatoma cell line HepG2. HepG2 cells were pretreated with palmitic acid for 24 hours, and then OPN was added for 24h. The related protein expressions of AKT and STAT1 signaling pathway were detected by Western blot. The results showed that Akt phosphorylation level was significantly decreased and STAT1 phosphorylation level was significantly increased in OPN treatment group compared with the control group (Fig. 6c, d). To further explored whether OPN affected fat accumulation by regulating the phosphorylation of AKT, we added the AKT inhibitor MK-2206 to HepG2 cells to detect the expression of genes related to fat production. RT-qPCR results showed that the expression levels of adipogenesis-related genes SCD1、FANS、SREBP1 and ACC1 were significantly increased after AKT inhibitor treatment (Fig. 6e). The above results suggested that MyD88 enhanced liver fat accumulation via activating AKT pathway and promoted inflammation through STAT1 pathway.

MyD88 is a key player in ALD. However, due to its wide expression, the cell-type specific contribution of MyD88-mediated signaling to ALD is still unclear. In this study, the specific role of MyD88 in HSCs in AFL has been investigated. We found that deficiency of MyD88 in quiescent HSCs significantly reduced liver injury, inflammation and steatosis, while MyD88 deficiency in activated HSCs (myofibroblasts) could only inhibit the progression of liver injury and inflammation, and had no obvious effects on steatosis. Furthermore, mechanistic studies showed MyD88 regulated OPN secretion of HSCs to active AKT signaling pathways of hepatocytes and upregulate lipogenic gene expression to promote fatty accumulation (Fig. 7). Thus, MyD88 may represent a potential candidate target for the prevention and targeted therapy in AFL.

More evidences showed that TLRs mediated innate immune response was related to liver injury, inflammation, liver fibrosis and liver fat metabolism, which may be attributed to the expression of TLRs in a variety of liver cells, including liver parenchymal cells, Kupffer cells, sinusoidal endothelial cells, HSCs, bile duct epithelial cells and liver dendritic cells[38-40]. TLRs other than TLR3 can bind to corresponding ligands and transmit signals through adapter molecule MyD88, and further activate NF-κB and JNK signal pathway[18]. In this study, we also confirmed the relevance between TLR and liver lipogenesis. We found that MyD88 deficiency in HSCs attenuated ethanol-induced liver inflammation and fat accumulation, and MyD88/TLR4 signaling pathway played a key role in regulating the expression of OPN and is related to the formation of AFL. Nagy LE et al. reported that specific MyD88 deletion in myeloid cells reduced ethanol-induced increases of plasma ALT and hepatic steatosis. Ethanol-induced inflammation and the expression of pro-inflammatory mediators were also attenuated in the livers of MyD88^LysM−KO mice[25]. However, Cani PD et al. found that MyD88 deletion in hepatocyte induced profound changes in glucose and lipid metabolism, which specifically showed that liver glycogen and the phosphorylation of insulin-induced AKT decreased, liver lipid and the inflammation of visceral adipose tissue increased[41]. These studies indicate that MyD88 in hepatocytes, myeloid cells, and HSCs may have different effects and mechanism on hepatic steatosis and inflammation.

OPN is a highly phosphorylated glycoprotein and expressed in various cells and widely exists in extracellular matrix, which has important functions on cardiovascular disease, cancer, diabetes, cell viability and wound healing[42], and is also related to the immunity regulation, metabolism and inflammation process[41]. In present study, we found that OPN derived from HSCs acted on hepatocyte, promoted lipogenesis and inflammatory response by inhibiting the phosphorylation of AKT and increasing the phosphorylation of STAT1 respectively. In the meantime, the relevance between OPN and hepatic lipogenesis was also verified in clinical cases. In this study, the gene expression levels of 21 healthy people and 40 patients with fatty liver were analyzed by GEO database (gse151158). It was found that the expression level of OPN was significantly increased in the liver of patients with fatty liver. Consisted with our results, Smid V. et al. reported that OPN was able to promote lipogenesis and could be used as a potential marker of fatty liver[43]. Haber PS et al. also confirmed that OPN is overexpressed in the liver of patients with AFL, especially when HSCs are activated, the expression level of OPN will increase significantly[44]. In addition, MORALES-IBANEZ et al. found that OPN deficiency inhibited liver damage by constructing OPN-knockout mice and chronic-binge ethanol model[41]. Gómez-Ambrosi J et al. found that OPN deficiency prevented hepatic steatosis via reduction in the expression of molecules involved in the onset of fat accumulation such as Pparg, Srebf1, Fasn, Mogat1, Dgat2 and Cidec in OPN-knockout mice with by a high-fat diet (HFD)[45]. Furthermore, related evidence also reported that OPN participated in the AKT and STAT1 signaling pathways and played a role in steatosis and inflammation. However, GE et al. confirmed that OPN combined with lipopolysaccharide (LPS) reduced the activation of macrophages and the expression of TNF-α, and reduced liver damage and steatosis[46]. The mechanism of OPN on liver injury and steatosis still needs to be further explored. AFL has become a severe issue that threatened human health all over the world[47]. At present, abstinence, nutritional support and glucocorticoid therapy are still the main methods for the treatment of AFL which have respective limitations, effective targeted therapy are in needs. It is essential to deeply understand the mechanism of AFL and provide theoretical and experimental basis for the treatment of AFL[48]. In recent years, it had been found that both innate and adaptive immunity were involved in the development of AFL. TLRs, as a type of pattern recognition receptors, were expressed in various cells in the liver and played a key role in the activation of the immune system[49]. As an important adaptor protein in the TLRs signaling pathway, MyD88 not only regulated the immune response, but also affected the metabolic process. Various cell types are involved in the occurrence of AFL, such as hepatocytes, immune cells and HSCs. Among them, Kupffer cells was the most widely studied, which caused inflammation, liver damage and fatty liver by secreting a large number of inflammatory cytokines such as TNF-α, IL-1 and IL-17[50]. In addition, it had also been found that HSCs not only played a role in the development of liver fibrosis by secreting extracellular matrix, but also promoted lipogenesis by secreting factors such as endocannabinoids. Depending on these results, we further explored the role of MyD88 in HSCs in the development of AFL and its mechanism.

In conclusion, our results demonstrated a mechanism for the pathogenesis of AFL in which MyD88 promoted lipid accumulation and inflammation via the AKT and STAT1 pathway. The present study showed that MyD88 deficiency in quiescent HSCs significantly inhibited ethanol-induced hepatic lipid accumulation, liver injury and inflammation by inhibiting the secretion of OPN. MyD88 could be a target for AFL therapy. Further studies should evaluate if OPN protein treatment can inhibit the progression of AFL.

Funding: This work was supported by the National Natural Science Foundation of China (81972689 and 81772497).

Institutional Review Board Statement: All animal studies were performed after being approved by the Institutional Laboratory Animal Care and Use Committee of the Institute of Biophysics, Chinese Academy of Sciences.

Author Contributions:

Jinhua Zhang, Lingling Hou conceived and designed the study; Yukun Li carried out the experiments; Yukun Li and Lingling Hou wrote the manuscript; Yukun Li and Lingling Hou and Miaomiao Wei drafted the manuscript; Yukun Li, Miaomiao Wei, Qi Yuan, Yu Liu, Tian Tian, Lingling Hou and Jinhua Zhang read and approved the final manuscript. All authors have read and agreed to the published version of the manuscript.

Informed Consent Statement: Not applicable.

Data Availability Statement: All relevant data are presented in this paper.

Declaration of Interests: The authors declare no conflict of interest.

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Supplementalmaterial.docx
Supplementary Materials: Figure S1: Generation of mice with genetic deletion of MyD88 in HSCs. Figure S2: The effect of knocking out MyD88 in quiescent HSCs on hepatic fibrosis. Table S1: Primer sequences for RT-PCR.

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Editorial decision: Major Revisions Needed
09 Dec, 2021
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Reviewers invited by journal
02 Nov, 2021
Editor assigned by journal
29 Oct, 2021
First submitted to journal
28 Oct, 2021

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MyD88 in Hepatic Stellate Cells Promotes the Development of Alcoholic Fatty Liver via the AKT Pathway

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Abstract

Figures

Key Message

1. Introduction

2. Materials And Methods

2.1 Mice

2.2 Chronic-binge ethanol mice models

2.3 Cell culture

2.4 Isolation of HSCs

2.5 Biochemical assays of blood and liver tissues

2.6 Histology and immunostaining

2.7 Quantitative real-time polymerase chain reaction (qRT-PCR)

2.8 Western blot analysis

2.9 Quantitative and statistical analysis

3. Results

3.1 Expression of MyD88 in HSCs is upregulated in the liver of AFL mice

3.2 MyD88 deficiency in HSCs attenuates ethanol-induced liver injury

3.3 MyD88 deficiency in HSCs attenuates ethanol-induced liver inflammation

3.4 MyD88 deficiency in HSCs attenuates fat accumulation

3.5 MyD88 deficiency in quiescent HSCs inhibits the expression of OPN

3.6 MyD88 promoted liver fat accumulation and inflammation via the AKT pathway and STAT1 pathway respectively

4. Discussion

Declarations

References

Supplementary Files

Status:

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