Metabolic crosstalk between skeletal muscle cells and liver through IRF4-FSTL1 in nonalcoholic steatohepatitis

Inter-organ crosstalk has gained increasing attention in recent times; however, the underlying mechanisms remain unclear. In this study, we elucidate an endocrine pathway that is regulated by skeletal muscle interferon regulatory factor (IRF) 4, which manipulates liver pathology. Skeletal muscle specific IRF4 knockout (F4MKO) mice exhibited ameliorated hepatic steatosis, inflammation, and fibrosis, without changes in body weight, when put on a nonalcoholic steatohepatitis (NASH) diet. Proteomics analysis results suggested that follistatin-like protein 1 (FSTL1) may constitute a link between muscles and the liver. Dual luciferase assays showed that IRF4 can transcriptionally regulate FSTL1. Further, inducing FSTL1 expression in the muscles of F4MKO mice is sufficient to restore liver pathology. In addition, co-culture experiments confirmed that FSTL1 plays a distinct role in various liver cell types via different receptors. Finally, we observed that the serum FSTL1 level is positively correlated with NASH progression in humans. These data indicate a signaling pathway involving IRF4-FSTL1-DIP2A/CD14, that links skeletal muscle cells to the liver in the pathogenesis of NASH.

found that skeletal muscle-specific IRF4 knockout (F4MKO) mice fed a chow diet exhibited normal body weight and insulin sensitivity, but increased exercise capacity 19 .However, when put on a high-fat diet (HFD), F4MKO mice appeared to be protected from diet-induced obesity, and showed improved glucose tolerance and increased insulin sensitivity 20 .Further investigations are needed to determine whether the ablation of IRF4 in skeletal muscle would affect liver metabolism, given that insulin resistance in skeletal muscles has been linked to increased hepatic de novo lipogenesis and hepatic steatosis in the elderly 21 .
Follistatin-like protein 1 (FSTL1), a secretory glycoprotein, is a potential target for steatosis-associated liver fibrosis 22 .Macrophage FSTL1 has been shown to promote liver fibrosis by inducing M1 polarization and inflammation 23 .The overexpression of Fstl1 in the liver could promote the expression of proinflammatory cytokines TNF-α, IL-1β, and IL-6 24 .Silencing Fstl1 using shRNA can reduce Col1a1 mRNA expression and macrophage accumulation in carbon tetrachloride injury-mediated liver fibrosis in mice 25 .Meanwhile, FSTL1 has been identified as a muscle-derived secretory myokine 7,26 , and skeletal muscle is a major source of circulating FSTL1 27 .Dynamic resistance exercise was found to increase the expression of skeletal muscle-derived FSTL1, which can supplement the insufficiency of cardiac FSTL1 expression, induce cardiac angiogenesis, and promote cardiac rehabilitation in myocardial infarction rats 26 .Although FSTL1 has various functions, it remains unclear which receptors are required for FSTL1-mediated inflammatory responses, lipid metabolism, and fibrosis in the liver.
Inter-organ crosstalk has gained increasing attention recently.In this study, we proposed a signaling pathway that potentially regulates the communication between skeletal muscle and the liver, especially in NASH models.First, we found that IRF4 expression was significantly upregulated in the skeletal muscle of NASH mice.Second, F4MKO mice showed ameliorated hepatic pathology when administered a NASH diet.Third, proteomics analysis and dual luciferase assays results indicated that FSTL1 acts as a myokine that mediates this crosstalk.Moreover, findings from cell co-culture experiments suggested that DIP2A and CD14 could be receptors of FSTL1 in the liver to mediate its function.Lastly, the serum FSTL1 level was increased in patients with NASH compared with that in healthy individuals.Taken together, these results revealed a signaling pathway from skeletal muscles to the liver via the IRF4-FSTL1-DIP2A/CD14 pathway in the pathogenesis of NASH.

Ablation of IRF4 expression in skeletal muscles ameliorated NASH
To confirm whether IRF4 expression was altered in the skeletal muscles of the NASH model, we measured the mRNA and protein expression levels in skeletal muscle and liver tissues from high-glucose and high-fat-diet-fed NASH mouse models.IRF4 protein and mRNA expression was increased in the gastrocnemius (GAS) (Fig. 1a, b), without significant changes in the liver of NASH mice (Supplementary Fig. 1a, b).As exercise can attenuate the NASH phenotype (Supplementary Fig. 1c-e), IRF4 expression was found to be decreased in GAS in NASH mice subjected to exercise (Fig. 1c, d).These results indicated that skeletal muscle IRF4 expression was associated with the hepatic pathology of NASH.
To investigate whether the ablation of IRF4 in skeletal muscles ameliorated NASH progression, we generated skeletal muscle-specific IRF4 knockout (F4MKO) mice, as previously described 19 .Male mice were then fed a Western diet and high-fructose diet for 24 weeks to induce NASH, as reported before 28 .No differences in body weight and liver weight were observed between F4MKO and Flox mice (Supplementary Fig. 1f-h) fed the NASH diet.Liver tissue morphology and H&E staining results showed a mild difference in hepatic steatosis (Fig. 1e, f, Supplementary Fig. 1i).Moreover, unlike HFD-fed mice 20 , the F4MKO mice showed no changes in fat and muscle depots compared with Flox mice (Supplementary Fig. 1j, k).Similarly, no changes were observed in oxygen consumption, carbon dioxide production, respiratory exchange ratio (RER), and energy expenditure (EE) (Supplementary Fig. 1l-o).GAS fiber type-related genes and H&E staining also did not reveal any changes in the muscle fiber type or muscle cross-sectional area (Supplementary Fig. 1p, q).This was further illustrated by the NASH/NAFLD activity score (NAS score) in the liver of Flox and F4MKO mice (Fig. 1f).In addition, we observed that F4MKO mice showed lower serum ALT and AST levels (Fig. 1g, h), which were indicators of liver damage.Collectively, these data suggested that deletion of IRF4 in skeletal muscles could improve NASH progression.The amelioration of hepatic steatosis and liver damage was also observed in female mice (Supplementary Fig. 1r-t).
F4MKO mice showed a reduction in lipid accumulation, lower inflammation levels, and decreased fibrosis when fed the NASH diet To further investigate the potential hepatic pathologies, we assessed lipid metabolism, inflammation, macrophage infiltration, and fibrosis in F4MKO and control mice.As anticipated, the levels of triglycerides and cholesterol were lower in the liver of F4MKO mice (Fig. 2a, Supplementary Fig. 2a, b), whereas the level of plasma triglycerides and cholesterol did not change (Supplementary Fig. 2c, d).Concomitant with this, lipogenic genes (Srebp1c, Scd1, Nr1h3, Fas) were downregulated in the liver of F4MKO mice compared with that in control mice, whereas fatty acid oxidation genes (Pparα, Cpt1a) were upregulated (Fig. 2b).To address macrophage infiltration in the liver, we measured F4/80 + Kupffer cells/macrophages in the liver.F4/80 + staining showed that there were fewer F4/80 + -positive cells in F4MKO mice than in control mice (Fig. 2c).In addition, the M1 macrophage maker gene Nos2 was decreased and M2 marker genes Clec10a was upregulated.In line with this, the inflammatory genes Il-6, Il-1β, and Tnf were downregulated and anti-inflammatory gene Il-10 was upregulated in samples obtained from F4MKO mice (Fig. 2d).Furthermore, liver fibrosis was suppressed in the samples of F4MKO mice assessed by Sirius Red staining, along with the downregulation of key genes of fibrosis (Acat2, Col1a1, Col1a2, Timp2, and Tgfβ) (Fig. 2e, f).
Next, we assessed whether skeletal muscle tissues from F4MKO mice could directly regulate liver function by using a co-culture system (Supplementary Fig. 2e).We first generated cells to mimic NASH in vivo by treating hepatocytes and macrophages with palmitic acid (PA), and treating HSC-T6 cells with TGFβ (Supplementary Fig. 2f-i).When primary hepatocytes were co-cultured with GAS from F4MKO or control mice, lipid droplet accumulation was found to decrease (Fig. 2g), and the cellular TG level also decreased significantly in the F4MKO group relative to that in the control group (Fig. 2h).This phenomenon was also observed in HepG2 and AML12 cells (Supplementary Fig. 2j-m).In addition, the expression of inflammation-related genes in macrophages and fibrosis in HSC-T6 cells decreased when the cells were co-cultured with GAS from F4MKO mice (Fig. 2i, j).These data indicated that IRF4-specific knockout in skeletal muscles led to a reduction in lipid accumulation, inflammation, and fibrosis in liver tissues and in cells in vitro.
The expression of FSTL1, a myokine, was decreased in F4MKO mice and transcriptionally regulated by IRF4 The co-culture system indicated that skeletal muscles can directly communicate with liver tissues.We performed serum proteomics using samples from F4MKO and control mice and identified 376 differentially expressed proteins corresponding to a fold change >1.5 or fold change <0.67.This included 194 upregulated proteins and 182 downregulated proteins (Supplementary Fig. 3a).We then compared genes encoding these proteins with those differently expressed genes identified in our previous F4MKO muscle RNA-seq data 20 , and identified 15 overlapping downregulated and three overlapping upregulated genes (Supplementary Fig. 3b).Next, we overlapped these 18 proteins with myokines obtained from public literature 29 , identifying 12 downregulated and one upregulated myokine (Supplementary Fig. 3c, d).To further identify changes in these 13 myokines secreted by skeletal muscles, we examined these genes expression in the GAS of F4MKO NASH mouse models.We just found that Decorin, Fibronectin1, Fstl1, and Mmp2 were significantly downregulated in the GAS of F4MKO mice (Supplementary Fig. 3e).Fstl1, Mmp2, and Decorin expression was reported to be associated with NASH 23,30,31 .However, Mmp2 and Decorin exhibit a protective role on NASH, downregulation is likely to worsen NASH pathology 30,31 .Fstl1 was reported to promote fibrosis in the liver and Fibronectin 1 was expression important for the extracellular matrix 23,32 .We thus choose Fstl1 as a candidate that linked the muscle and liver tissues in our models.Meanwhile, we found that the protein expression of FSTL1 did not change in the GAS F4MKO mice fed a normal chow diet, whereas it decreased significantly in the GAS, soleus, and quadriceps of F4MKO mice under NASH model conditions (Fig. 3a, Supplementary Fig. 4a-c); the same was observed with plasma FSTL1 (Fig. 3c).Consistent with this, FSTL1 expression was higher in the GAS of NASH mice but lower in the GAS of NASH mice subjected to exercise (Fig. 3b).
We then assessed whether Fstl1 expression was transcriptionally regulated by IRF4.We constructed luciferase reporter genes containing different segments of the FSTL1 promoter and co-transfected them into 293T cells with pCDH-GFP or pCDH-IRF4.IRF4 increased the activity of the FSTL1 promoter in the −1520 to +76 region, whereas IRF4 had no effect on the activity of the promoter in the −1122 to +76 region, indicating that the element responsible for the action of IRF4 on FSTL1 promoter was positioned between bases −1520 and −1122 (Fig. 3d).Next, we analyzed the FSTL1 promoter between −1520 and −1122 using an online tool for predicting the interaction between promoters and transcription factors (http://jaspar.genereg.net).We subsequently identified three potential binding sites of IRF4 (ISRE) within this region of the FSTL1 promoter.We then constructed three mutant reporter genes with a disrupted ISRE motif, and re-conducted the dualluciferase reporter assay.IRF4 exerted no effect on the FSTL1 promoter with the three different mutations (Fig. 3e).The data suggested that IRF4 manipulates FSTL1 expression by enhancing the activity of the FSTL1 promoter.Furthermore, the −1520 to −1122 region is responsible for this effect.Chip-qPCR results further confirmed that IRF4 was bound to the −1520 to 1122 region of FSTL1 promoter (Fig. 3f).

Skeletal muscle-derived FSTL1 was responsible for IRF4mediated NASH progression
To verify whether FSTL1 mediates IRF4's effects in liver cells in vivo, we induced the overexpression of FSTL1 directly in the GAS of mice using adenovirus-associated virus (AAV) delivery.Flox and F4MKO mice were first modeled with NASH.After 24-week NASH modeling, AAV-FSTL1, tagged with flag, was injected into the GAS.After 4 weeks of the AAV injection, the mRNA expression of FSTL1 in GAS was increased significantly in the AAV-FSTL1-treated group (Supplementary Fig. 4d), and the FSTL1 protein levels in GAS and serum also increased significantly (Fig. 4a, b).The circulating FSTL1 level also increased significantly (Fig. 4c).We observed that the FSTL1 level in the liver increased after AAV injection (Supplementary Fig. 4e), but the FSTL1 mRNA expression in the liver remained unchanged (Supplementary Fig. 4f), indicating that FSTL1 was secreted from the GAS and moved to the liver.There were no differences in the body, GAS, and liver weight   among Flox, F4MKO, and F4MKO + FSTL1 mice (Supplementary Fig. 3g-k) fed the NASH diet.Liver tissue H&E staining showed that the degree of steatosis and inflammation in F4MKO mice injected with AAV-FSTL1 were similar to that observed in Flox mice (Fig. 4d), which was further illustrated by the NAS scores of the liver tissue of Flox, F4MKO, and F4MKO + FSTL1 mice (Fig. 4e).In addition, the F4MKO + FSTL1 mice exhibited higher serum ALT and AST levels (Fig. 4f, g).These data suggested that skeletal muscle-derived FSTL1 contributed to the amelioration of hepatic pathology in F4MKO mice.Furthermore, we induced FSTL1 knockdown in both the liver and GAS using AAV.FSTL1 knockdown in the GAS (GFSTL1-KD) did not result in any significant improvement in circulating triglyceride level, hepatic lipid accumulation, or hepatic damage (Supplementary Fig. 5a,  c-e).However, we observed a mild reduction in liver fibrosis in GFSTL1-KD mice assessed by Sirius Red staining (Supplementary Fig. 5b).In addition, the expression of key genes associated with fibrosis (Acat2 and Col1a1) was also found to be lowered (Supplementary Fig. 5f).However, we did not observe any significant effects on the NASH phenotype (Supplementary Fig. 5g-k) upon FSTL1 knockdown in the liver.These results provided further confirmation that FSTL1 derived from skeletal muscle plays a dominant role in the NASH pathology phenotype.

FSTL1 overexpression of counteracted the effects on the liver induced upon the ablation of skeletal muscle IRF4
To test whether FSTL1 mediates the effect of disrupted IRF4 expression in skeletal muscles on NASH progression, we performed the transcriptome analysis of liver tissues in Flox, F4MKO, and F4MKO + FSTL1 mice.KEGG pathway analysis revealed that the non-alcoholic fatty liver disease (NAFLD) pathway was repressed in F4MKO mice compared with that in control mice.Upon injection with AAV-FSTL1, the NAFLD pathway was activated relative to that in AAV-GFP-injected F4MKO mice (Fig. 5a, b).Next, we verified that skeletal muscle-derived FSTL1-mediated NASH progression with regards to hepatic lipid metabolism, fibrosis, and inflammation in vivo when specific IRF4 deletion was induced in skeletal muscles.First, we found that triglyceride content was significantly lowered in the liver of F4MKO mice but increased in the liver of AAV-FSTL1-injected F4MKO mice (Fig. 5c, Supplementary Fig. 6a).Consistent with this, key genes associated with lipogenesis were downregulated in F4MKO mice, but their expression  was restored in F4MKO + FSTL1 mice (Fig. 5d).Second, we checked the degree of inflammation in the liver.F4/80 staining showed that there were less kupffer cells in F4MKO than in control mice, whereas the number of cells was greater in F4MKO mice injected with AAV-FSTL1 (Fig. 5e).Moreover, the expression of inflammatory genes, such as Il-6, Il-1β, Tnf, and Il-10 was reversed in F4MKO mice injected with AAV-FSTL1 (Fig. 5f).Third, Sirius Red staining showed that the degree of fibrosis in the liver was similar to that in control and F4MKO + FSTL1 groups but decreased in the F4MKO group (Fig. 5g).QPCR analysis revealed the same changes in the expression patterns of fibrosisrelated genes (Fig. 5h).
In addition, recombinant protein-FSTL1 (rFSTL1) increased the TG concentration in primary hepatocytes and HepG2 cells treated with PA, but not the basal levels (Supplementary Fig. 6b, c).Since FSTL1 was downregulated in F4MKO mice, we added rFSTL1 into the co-culture system.In the primary hepatocyte steatosis cell model, rFSTL1 restored the cellular TG levels and lipid accumulation in cells cocultured with GAS from F4MKO mice (Fig. 5i).rFSTL1 exerted the same effect in an HepG2 steatosis cell model (Supplementary Fig. 6d).Meanwhile, we tested the effects of rFSTL1 in macrophage cells (Supplementary Fig. 6e-h).rFSTL1 counteracted inflammatory gene expression in macrophages co-cultured with GAS from F4MKO mice (Fig. 5j).Consistent with this, rFSTL1 reversed liver fibrosis gene expression in HSC-T6 cells co-cultured with GAS from F4MKO mice (Fig. 5k, Supplementary Fig. 6i-m).In conclusion, FSTL1 overexpression could counteract the effects of skeletal muscle IRF4 ablation in steatosis, fibrosis, and inflammation in NASH mice.

Skeletal muscle IRF4 regulated liver metabolism via FSTL1-DIP2A/CD14
To screen the receptors of FSTL1 that is/are responsible for the liver phenotypes in F4MKO mice, we measured the expression of their transcripts.Twelve receptors were reported to be related to FSTL1 [33][34][35][36] .
According to sc-RNA-Seq data from human and mouse livers, these 12 receptors were expressed in different patterns in different cell types (Supplementary Fig. 7a-d) [37][38][39][40] .We then measured the expression of these receptors in our models.The expression of seven receptors was altered in F4MKO mice; these were including Cd14, Dip2a, Eng, Bmpr2, Tgfbr1, Tgfbr2, and Bmpr1b (Supplementary Fig. 7e).Given that the Bmpr1b transcript was not detected in mouse NASH liver sc-RNA-Seq (Supplementary Fig. 7d), we designed the shRNA of the remaining six receptors to further clarify how FSTL1 regulates liver metabolism.We used mouse AML12 hepatocytes, hepatic stellate HSC-T6 cell, and a macrophage cell line, which were infected with an shRNA lentivirus to knockdown the receptors of FSTL1 (Supplementary Figs.8a, 9a, 10a).Surprisingly, rFSTL1 increased lipid accumulation in AML12 cells, but the effects were only blocked by shDip2a and shCd14 and not by other receptors (Supplementary Fig. 8b-g).The blockade of Dip2a and Cd14 in AML12 cells did not result in any changes in the expression of the other receptors (Supplementary Fig. 8h, i).Again, we found that inflammatory gene expression was increased in macrophage cells treated with rFSTL1, whereas the expression of these genes normalized when the cells were infected with shCd14 (Supplementary Fig. 9b-g).
Similarly, when Cd14 knockdown in macrophage cells did not cause changes in other receptors (Supplementary Fig. 9h).Combining these findings with the sc-RNA-Seq data, we selected four to six receptors (Eng, Bmpr2, Tgfbr2, and Dip2a) that showed a change in expression patterns in NASH HSCs.The fibrosis-related genes were upregulated in rFSTL1 supplement cells but downregulated only in shDip2a infected cells (Supplementary Fig. 10b-e).However, the expression of other receptors did not change in HSC-T6 cells infected with shDip2a (Supplementary Fig. 10f).
To identify the receptor that contributes to the effects of the skeletal muscle IRF4-FSTL1 axis on the liver, co-culture experiments were carried out.FSTL1 could not restore cellular lipid accumulation and lipid metabolism gene expression in Dip2a or Cd14 knockdown AML12 steatosis cells co-cultured with GAS from F4MKO mice (Fig. 6a-h).Moreover, following Cd14 knockdown in macrophages and Dip2a knockdown in HSC-T6, rFSTL1 was unable to reverse inflammation-related gene expression in macrophages and fibrosisrelated gene expression in HSC-T6 cells co-cultured with GAS from F4MKO mice (Fig. 6i-r).Collectively, these data suggest that skeletal muscle IRF4 regulates lipid metabolism, fibrosis, and inflammation via FSTL1-DIP2A/CD14.To further validate these results in vivo, we used AAV delivery to directly block both Dip2a and Cd14 expression in the liver inducing the overexpression of FSTL1 in the GAS.FSTL1 could not rescue hepatic damage, lipid accumulation, and fibrosis when both Dip2a and Cd14 were knocked down in the liver (Fig. 6s-w).In summary, these data revealed that skeletal muscle IRF4 regulates NASH progression through the FSTL1-DIP2A/CD14 pathway.

The serum FSTL1 level was associated with hepatic steatosis and fibrosis in humans
To determine whether the serum FSTL1 level associated with liver pathology in humans, we analyzed specimens from 43 healthy participants, 43 patients with steatosis, 41 patients with early NASH and 53 with advanced NASH.The demographic characteristics are presented in Fig. 7a.Although the FSTL1 level did not vary by age, BMI, plasma TG level, free fatty acid (FFA) levels, or cholesterol levels, we observed a significant positive correlation between FSTL1 expression and liver pathologies (steatosis, ballooning, fibrosis, and NAS) (Fig. 7b-d).Notably, the plasma FSTL1 level was also correlated with glucose levels or HbA1c (Fig. 7e-g).Nonetheless, the Fstl1 mRNA level in liver biopsies from patients with NASH was significantly lower than that in controls (Fig. 7h, i).This, suggested that the increased plasma FSTL1 level in patients with NASH might be owing to FSTL1 secretion from other tissues, such as skeletal muscles, as shown in the present study.Consistent with the level of FSTL1 in the liver, the expression of its receptors was decreased in the liver from patients with NASH in our study as well as in other studies 41 (Fig. 7h, i, Supplementary Fig. 11).Our data thus strongly suggested a model in which skeletal muscle IRF4 transcriptionally regulates FSTL1, and the latter is secreted in the liver to mediate liver metabolism through DIP2A/CD14, eventually affecting the pathology of NASH (Fig. 7j).

Discussion
The association between the pathogenesis and natural course of NASH and skeletal muscle dysfunction has been recognized widely in recent times.Here, we showed that skeletal muscle plays a role in the progression of NASH via the IRF4-FSTL1-DIP2A/CD14 axis (Fig. 7j).
Fig. 5 | Overexpression of FSTL1 counteracts the liver effects induced by ablation of skeletal muscle IRF4.a, b Gene set enrichment analysis of Flox, F4MKO, and F4MKO + FSTL1 liver from RNA-seq data.c Oil red O staining of liver in Flox+GFP, F4MKO + GFP, and F4MKO + FSTL1 mice (n = 3 biologically independent animals.Scale bars, 50 μm).d Relative mRNA expression of lipid metabolismrelated genes in NASH liver of Flox+GFP, F4MKO + GFP, and F4MKO + FSTL1 mice (Flox+GFP, n = 6 biologically independent animals; F4MKO + GFP, n = 5 biologically independent animals; F4MKO + FSTL1, n = 6 biologically independent animals).e F4/80 staining in NASH liver of Flox+GFP, F4MKO + GFP, and F4MKO + FSTL1 mice (n = 3 biologically independent animals.Scale bars, 50 μm).f Relative mRNA expression of inflammatory genes in NASH liver of Flox+GFP, F4MKO + GFP, and F4MKO + FSTL1 mice (n = 6 biologically independent animals).g Sirius red staining of NASH liver in Flox+GFP, F4MKO + GFP, and F4MKO + FSTL1 mice (n = 3 biologically independent animals.Scale bars, 50 μm).h Relative mRNA expression of fibrosis genes in NASH liver of Flox+GFP, F4MKO + GFP, and F4MKO + FSTL1 mice (n = 6 biologically independent animals).i Cellular TG level of primary hepatocytes co-cultured with indicated gastrocnemius (n = 3 biologically independent cell experiments).Primary hepatocytes cells treated with 25 mM glucose and 200 μM PA were co-cultured with gastrocnemius from Flox or F4MKO mice for 48 h.j Relative mRNA expression of inflammatory genes in macrophages co-cultured with gastrocnemius (Flox+GFP, n = 6 biologically independent cell experiments; F4MKO + GFP, n = 5 biologically independent cell experiments; F4MKO + FSTL1, n = 4 biologically independent cell experiments).Macrophages were treated with 200 μM PA for 24 h, then macrophages were co-cultured with GAS from Flox and F4MKO mice for 48 h and treated with or without 100 ng/ml rFSTL1.k Relative mRNA expression of fibrosis genes in HSC-T6 cells co-cultured with GAS (n = 4 biologically independent cell experiments).HSC-T6 cells were treated with 10 ng/ ml rTFGβ for 24 h, then HSC-T6 cells were co-cultured with GAS from Flox and F4MKO mice for 48 h and treated with or without 100 ng/ml rFSTL1.All results were shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, compared with the Flox+GFP group; # p < 0.05, ## p < 0.01, ### p < 0.001, compared with the F4MKO + GFP group, a two-way ANOVA followed by Bonferroni post-tests was used for statistical analysis.Source data are provided as a Source data file.Surprisingly, IRF4 expression was higher in skeletal muscles, but not in the liver tissues, in NASH models.IRF4-specific deletion in skeletal muscle alleviated steatosis, inflammation, and fibrosis in the liver.These effects were mediated by a myokine named FSTL1, which exerts its function through DIP2A and CD14 in the liver.Importantly, serum FSTL1 expression is positively correlated with NASH progression in humans.Skeletal muscles are a major reservoir for secreted FSTL1, as addressed in other studies 27 .Our findings suggest a novel pathway that plays a role in the communication between skeletal muscles and the liver, which are potential drug targets in metabolic diseases.
Of note, IRF4 was shown to play a key role in the development and function of immune cells 14,42 .We have described that IRF4 expression in fat induces catabolic effects (increased lipolysis, anti-lipogenesis, and increased M2 macrophage abundance) 15,43 , and the overexpression of IRF4 in skeletal muscle favors diet induced-obesity 20 .Recently, we addressed that F4MKO mice are obesity-resistant and exhibit enhanced energy expenditure, insulin sensitivity, and exercise capacity 19,20 .Weight loss can help alleviate lipid-induced hepatocellular injury by mobilizing fat from the liver.However, limited information is available about such phenomena in the absence of weight loss, especially with respect to alterations in hepatic fat metabolism.Here, F4MKO mice fed the NASH diet showed an improvement in hepatic injury without body weight changes.Upon further investigation, IRF4 was found to transcriptionally regulate FSTL1 expression in skeletal muscle, the latter being secreted in the liver to exert its function on liver cells.Skeletal muscles act as endocrine organs, secreting various myokines that affect other metabolic tissues, including the liver 7 .The insufficient or excessive secretion of myokines may regulate steatosis, inflammation, or fibrosis in the liver.FSTL1 is a myokine that mediates the function of IRF4s function in the liver.The serum FSTL1 level is positively correlated with NASH in humans.rFSTL1 treatment increases lipid accumulation in hepatocytes, enhances fibrosis in HSCs, and augments inflammatory gene expression in macrophages.However, FSTL1 expression is reported to be induced by dynamic resistance exercise and promotes cardiac rehabilitation in MI rats 26 .The controversial function of FSTL1 may be attributed to the post-translational modification of FSTL1.For example, in the sequence of mouse Fstl1, three potential sites are present for N-glycosylation and two potential sites are present for O-glycosylation.Glycosylation at these sites leads to cell-type specificity 44 .The activation of various signaling pathways through different receptors is likely responsible for that: the protective role of FSTL1 in the heart is mediated via DIP2A in MI rats 26 .In this study, not only DIP2A but also CD14 was found to responsible for the role of FSTL1 in the liver.However, the fact that additional endogenous or exogenous factors are involved in the regulation process cannot be ignored.
In addition, the circulating levels of FSTL1 were found to be elevated in patients with various diseases, such as cardiovascular diseases 35,45,46 , and liver disease 22,41 .In line with previous findings, our findings showed that FSTL1 concentrations were increased in patients with NASH.However, the mRNA levels of Fstl1 in the liver of patients with NASH were found to be lowered in our study, which was consistent with previous findings from transcriptome sequencing experiments 41 .Collectively, our findings, combined with previous results, suggest that muscle-derived FSTL1 is involved in the regulation of NASH development.This hypothesis is further supported by the following lines of evidence from in vitro and in vivo experiments.First, FSTL1 knockdown in the liver did not affect NASH progression.
Second, FSTL1 knockdown in muscle partially alleviated NASH phenotypes, indicating that muscle-derived FSTL1 plays an essential role in the development of NASH.Third, findings from in vitro studies showed that recombinant FSTL1 protein could rescue the steatosis cell phenotype.Finally, in our model, replenishing FSTL1 in muscle effectively restored the NASH phenotypes induced by the reduction of FSTL1 expression resulting from IRF4 knockout in muscle.Thus, musclederived FSTL1 plays a major role in the regulation of NASH.In conclusion, our findings demonstrate that IRF4 expressed in skeletal muscle plays a critical role in the pathogenesis of NASH through the FSTL1-DIP2A/CD14 pathway.Targeting this signaling pathway could serve as an attractive strategy for NASH management.
However, there are several limitations of this study.First, our study used high cholesterol content (1.25%) in the NASH diet, whether other NASH diets can obtain the same conclusion is worthy of further investigation.Second, we knockdown FSTL1 only in GAS but no other skeletal muscles, which may be not enough to mimic the effect of Mly1cre/FSTL1.Third, we investigated the effect of FSTL1 on aggravated NASH induced by IRF4 muscle knockout mice, but the precise molecular mechanism of how FSTL1-DIP2A/CD14 pathway regulates liver metabolism need to be investigated in the future.

Animals
Mice were maintained under a 12-h light/12-h dark cycle at constant temperature (23 °C) and humidity (50-60%) with free access to food and water.The muscle-specific IRF4 knockout mice were generated as previous reported, detailed protocols and information regarding the establishment have been described previously 19 .All animals received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals.The animal experiments abide by the ARRIVE guidelines.All animal studies were approved by the Institutional Animal Care and Use Committee of Shanghai University of Sport (102772022DW019).

Animal experiment design
C57BL/6 mice were obtained from Gempharmatech Co., Ltd (Nanjing, China).The mice were allowed to acclimate to the living environment for 7 days prior to the start of all experiments.
Animal experiment design 2#.To investigate whether ablation of IRF4 in skeletal muscle would ameliorate NASH progression, we generated skeletal muscle-specific IRF4 knockout (F4MKO) mice.The male and female Flox and F4MKO mice were established NASH model for   24-week.After the NASH modeling period, the liver tissues were collected for the examination of diet-induced NASH in Flox and F4MKO mice.
Animal experiment design 3#.To specifically overexpressed FSTL1 in GAS in vivo experiments, the adeno-associated virus serotype 8 (AAV8)-muscle creatine kinase (MCK) encoding full-length FSTL1 sequences (AAV-FSTL1) delivery system was established.After 24-weeks NASH modeling, AAV-MCK promoter-FSTL1 tagged with Flag, was injected to gastrocnemius.After 4-weeks AAV injection, the liver tissue samples were collected from mice to detect corresponding histological, biochemical and molecular biological analysis.
Animal experiment design 4#.To construct a special knockdown of FSTL1 in vivo, AAV packaged a short hairpin RNA target FSTL1 was generated with a standard molecular procedure.And injected into male NASH mice via tail vein or directly into the gastrocnemius to achieve FSTL1 knockdown in liver or gastrocnemius, respectively.After 4-weeks AAV injection, the blood and liver tissues were collected to assess the effect of FSTL1 knockdown in the liver and muscle on the progression of NASH.
Animal experiment design 5#.To further validate that skeletal muscle IRF4 regulates lipid metabolism, fibrosis, and inflammation via FSTL1-DIP2A/CD14 in vivo, we constructed a special knockdown of Dip2a and Cd14 in vivo as described in model 4#.And the AAV of Dip2a and Cd14 were injected into male F4MKO mice via tail vein, based on overexpressed of FSTL1 in the GAS.After recovery for 4 weeks, the blood and liver tissues were subjected to histological analysis and biochemical analysis.

Human subjects
All subjects were from Zhongshan Hospital, Fudan University.A liver biopsy was performed according to the EASL-EASD-EASO clinical practice guidelines to evaluate the severity of liver histology 51 .NAFLD was histologically diagnosed by the presence of ≧5% hepatic steatosis; NAFL was histologically diagnosed by the presence of steatosis without ballooning or lobular inflammation; NASH was histologically diagnosed by the joint presence of steatosis, ballooning, and lobular inflammation 51 .The NAFLD activity score (NAS) and the steatosis, activity, and fibrosis score (SAF) were used to quantify the severity of liver steatosis, ballooning, inflammation, and fibrosis based on liver histology 52,53 .NASH with fibrosis score <2 was defined as early NASH (eNASH) and NASH with fibrosis score ≧2 was defined as fibrotic NASH (fNASH) 51 .The competing etiologies (chronic viral hepatitis, hypothyroidism, excessive alcohol consumption, drugs leading to steatosis) of steatosis were ruled out based on laboratory examination and liver histology.Subjects with (1) age <17 years old; (2) type 1 diabetes mellitus (T1DM), gestational diabetes, and other specific types of diabetes; (3) acute complications of diabetes; (4) severe renal disease or abnormal renal function (Cr≧115 µmol/L); or (5) history of malignant tumor, severe mental illness, or parenteral nutrition were excluded.
Finally, a total of 180 adult subjects (43 control, 43 NAFL, 41 eNASH, and 53 fNASH subjects) were included in this study.All subjects underwent routine physical measurements, blood biochemical tests, and medical history records as previously described 54 .All protocols were performed in accordance with the Declaration of Helsinki of 1975 and approved by the ethics committee of the Zhongshan Hospital, Fudan University, and each subject provided written informed consent.

Isolation of primary hepatocytes
Primary hepatocytes were isolated by the collagenase perfusion method as described previously 55 .The isolated hepatocytes were enriched by Percoll gradient centrifugation.Then cultured in William's E Medium containing 10% FBS and antibiotics on collagen-coated plates.After overnight incubation (16 h), the culture medium was refreshed.

Fig. 1 |
Fig. 1 | Ablation of IRF4 in skeletal muscle attenuates NASH.a, b Western blot (n = 3) and QPCR (CON, n = 5 biologically independent animals; NASH, n = 6 biologically independent animals) analysis of the expression of IRF4 in skeletal muscle of control (CON) and NASH mice.c, d Western blot (n = 3) and QPCR (NASH, n = 6 biologically independent animals; NASH+Exe, n = 5 biologically independent animals) analysis of the expression of IRF4 in skeletal muscle of NASH and NASH+ Exercise (NASH+Exe) mice.e H&E staining of liver in Flox and F4MKO mice on NASH diet (Flox, n = 8 biologically independent animals; MKO, n = 7 biologically independent animals.Scale bars, 100 μm).f Quantification of steatosis, lobular

Fig. 7 |
Fig. 7 | Serum FSTL1 level is associated with NASH progression in humans.a Clinical characteristics of subjects.b Associations between serum FSTL1 and serum lipid profile.c Associations between serum FSTL1 and liver histology score.d FSTL1 relative level in human serum (NC, n = 44 biologically independent samples; NAFL, n = 44 biologically independent samples; eNASH, n = 42 biologically independent samples; fNASH, n = 53 biologically independent samples).e-g Associations between serum FSTL1 and FPG, 2HPG and HbA1c.h Heatmap of Fstl1 and its receptors from human liver biopsy RNAseq.i Relative mRNA