IL4I1 overexpression protects against nonalcoholic fatty liver disease in part by inhibiting the AKT/FOXO1 pathway-mediated Th17 cell differentiation

Abstract

Nonalcoholic fatty liver disease (NAFLD) has posed a huge threat to public health globally, but there are currently no approved drugs available. Growing evidence has proved the close association between increased Th17 cells and NAFLD progression. Interleukin-4 induced protein 1 (IL4I1), an amino acid oxidase secreted by immune cells, was reported to regulate the Th17 cells, but its exact role in NAFLD progression has not been fully explained yet. We found that IL4I1 was highly expressed in the liver of C57BL/6J mice with NAFLD induced by an 8-weeks western diet. To explore the IL4I1’s effect, mice were injected with AAV8 encoding IL4I1 1 week before western diet administration. The results showed that IL4I1 overexpression inhibited the NAFLD progression, demonstrated by relieved liver damage and lipid accumulation. The underlying mechanism in which IL4I1 acts on NAFLD might be attributed to the inactivated AKT/forkhead box protein O1 (FOXO1) signaling pathway–mediated decrease of Th17 cells in liver tissues. Subsequently, by culturing naive CD4⁺ T cells isolated from the spleen of mice in Th17 cell-polarizing conditions, we determined that IL4I1 overexpression inhibited Th17 cell differentiation by inactivating the AKT/FOXO1 pathway, whereas its knockdown exhibited opposite effects. Further, the AKT activator SC79 reversed the effect of IL4I1 overexpression on Th17 cell differentiation. Collectively, our study supports that compensatory upregulation of IL4I1 protects against liver damage and lipid accumulation in NAFLD progression, partially by inhibiting the activated AKT/FOXO1 signaling pathway-induced Th17 cell differentiation.

Nonalcoholic fatty liver disease (NAFLD) is mainly responsible for chronic liver disease and involves systematic metabolic disorders (1, 2). It covers liver abnormalities ranging from simple hepatic steatosis to nonalcoholic steatohepatitis (NASH) (3). Excessive fat accumulation in hepatocytes is the first pathological step and most prominent feature of NAFLD (4, 5). According to statistical data, the global prevalence of NAFLD is approximately 25% (6). Due to the worldwide obesity pandemic, the incidence of NAFLD presents a continuously increasing trend (7). However, there is currently no approved drug specifically for NAFLD treatment. It is urgent to identify the driving factors for the onset of NAFLD and provide a theoretical basis for effective NAFLD therapies.

Inflammation is a key biological process in the development of NAFLD. Excessive accumulation of free fatty acids in the liver tissues results in lipid toxicity to hepatocytes, manifested as an inflammatory response and subsequent liver damage. Growing evidence suggests that immunomodulatory Th17 cells play a crucial role in the inflammatory response under the pathological state of NAFLD. The hepatic steatosis microenvironment could produce specific inflammatory Th17 cell subsets in the liver, promoting tissue inflammation and accelerating the development of NAFLD (8). The liver inflammation during the development of NASH is related to the differentiation of T cells into Th17 cells (9). The increased number of Th17 cells and the expression of IL-17 were found in the liver tissues of patients and mice with NASH (10). Therefore, regulating Th17 cells might be a potential strategy for NAFLD relief.

Interleukin-4 induced protein 1 (IL4I1) is an amino acid oxidase secreted by immune cells, belonging to the L-amino acid oxidase family of flavin adenine dinucleotide binding enzymes (11). Santalasci et al. revealed that knockdown of IL4I1 significantly increased Th17 cell proliferation (12). Injection of recombinant IL4I could reduce the expression of IL-17 in the central nervous system and the percentage of Th17 cells in central nervous system and spleen, alleviating the severity of autoimmune encephalomyelitis in mice (13). Furthermore, by searching clinical datasets, we found the upregulated IL4I1 expression in NAFLD samples. However, the exact role of IL4I1 in NAFLD remains unknown.

Forkhead box protein O1 (FOXO1) is a transcription factor of the FOX protein family regulating lipid metabolism (14). Nevertheless, the function of FOXO1 was found in modulating lipid metabolism. FOXO1 overexpression increased the expression of adipose triglyceride lipase, promoting lipolytic activity and inhibiting triglyceride accumulation in the vitro NAFLD model (15). However, Kim et al. pointed out that FOXO1 activation was associated with lipid accumulation in the liver tissues of aged rats (16). Interestingly, it has been reported as a negative regulator of Th17 cell differentiation. In addition, activated AKT could lead to FOXO1 phosphorylation, thereby reducing FOXO1 activity. Sadik et al. suggested the role of IL4I1 in the aryl hydrogen receptor (AHR) activation. A published study confirmed the increased AKT phosphorylation level in lung fibroblasts of AHR knockout mice. Therefore, we speculate that IL4I1 can regulate Th17 cell differentiation through the AKT/FOXO1 signaling pathway.

The purpose of this study is to explore the IL4I1’s effects on NAFLD as well as the underlying mechanism. Here, we found that the compensatory upregulation of IL4I1 protected mice from NAFLD, partially by inhibiting the Th17 cell differentiation via the AKT/FOXO1 signaling pathway.

Results

The IL4I1 expression was upregulated in NAFLD samples

To elucidate the role of IL4I1 in the development of NAFLD, we first explore the expression profiles of IL4I1 under the pathological condition of NAFLD. Through searching Gene Expression Omnibus (GSE) datasets (GSE185051 and GSE135251), we found the increased IL4I1 expression in liver tissues of individuals with NAFLD (Fig. 1A). Then, the mouse model of NAFLD was established by the intake of a western diet (WD) for 8 weeks (Fig. 1B). The significantly elevated body weight, usually accompanied by NAFLD, was found in mice fed with a WD (Fig. 1C). As shown in Fig. 1D, the morphology of liver tissues collected from mice exhibited the obvious fatty liver characteristics, including dramatic liver enlargement and discoloration (Fig. 1D). Accordingly, mice with NAFLD had the higher liver weight and liver organ index (the liver/body weight ratio) compared with those controls (Fig. 1, E and F). Oil Red O staining was used to evaluate lipid droplet accumulation in tissues or cells. Representative images and quantitative analysis revealed that a WD induced lipid droplet accumulation in liver tissues of mice, as evidenced by the increased percentage of Oil Red O positive-staining area in the WD group (Fig. 1, G and H). In the present study, we supposed that IL4I1 might exert its effects on NAFLD by regulating Th17 cells, which are differentiated from CD4⁺ T cells. Therefore, we next localized the CD4 and IL4I1 expression in the liver of mice using double immunofluorescence staining. The results showed the enhanced percentage of CD4 and IL4I1 double-positive cells in liver tissues from NAFLD-like mice (Fig. 1, I and J). Together, these results indicated the successful establishment of the NAFLD model in mice and the upregulated IL4I1 expression in NAFLD samples.

Figure 1.

IL4I1 was highly expressed in mice with NAFLD.A, the expression value of IL4I1 in the liver of patients with NAFLD in Gene Expression Omnibus (GSE) datasets (in GSE185051, n = 4 for Con, n = 51 for NAFLD; in GSE135251, n = 10 for Con, n = 206 for NAFLD). B, the flow chart of the establishment of the NAFLD mouse model. C, the body weight of mice (n = 6). D, the morphology of liver tissues collected from mice. E, the liver weight of mice (n = 6). F, the liver organ index (liver/body weight ratio) of mice (n = 6). G, images of Oil Red O staining of liver tissues collected from mice. H, quantification of the percentage of Oil Red O-positive staining area (n = 6). I, images of double immunofluorescence staining of liver tissues collected from mice. J, quantification of the percentage of CD4 and IL4I1 double-positive cells (n = 6). Data were presented as mean ± SD and analyzed by Mann-Whitney test or unpaired t test. The black horizontal line in bar graphs represents the mean difference between the Con group and the NAFLD group or between the Con group and the WD group. IL4I1, interleukin-4 induced protein 1; NAFLD, nonalcoholic fatty liver disease; WD, western diet.

IL4I1 overexpression prevented against a WD-induced NAFLD in mice

Previous experimental data confirmed that IL4I1 might be associated with the decreased number of Th17 cells. Based on that, we speculated that the increased IL4I1 expression in subjects with NAFLD might be attributed to a compensatory mechanism. Next, to investigate whether IL4I1 would impact NAFLD phenotype, we overexpressed IL4I1 in liver tissues of mice by tail vein injection of AAV8 vector encoding IL4I1 (Fig. 2A) followed by the induction of NAFLD in mice. We found that IL4I1 overexpression reduced the body weight of WD-fed mice (Fig. 2B). It also improved the undesired morphology of liver tissues of mice with NAFLD (Fig. 2C). Similarly, the decreased liver weight and liver organ index were observed in NAFLD-like mice after IL4I1 overexpression (Fig. 2, D and E). Moreover, lipid metabolism disorder is one of the key pathological features of NAFLD (17). Serum levels of triglycerides and total cholesterol were raised by a WD, indicating the dysregulated lipid metabolism in mice with NAFLD. However, IL4I1 overexpression inhibited the increase of these two indicators (Fig. 2, F and G). Consistently, the increased levels of triglycerides and total cholesterol in the liver of WD-fed mice were reversed by IL4I1 overexpression (Fig. 2, H and I). Western blot assay verified the elevated IL4I1 protein expression in liver tissues of mice with a WD and IL4I1 overexpression compared to mice with only a WD, suggesting the close association between the highly expressed IL4I1 and the relief of NAFLD (Fig. 2J). These findings demonstrated that compensatory upregulation of IL4I1 alleviated the symptoms of NAFLD in mice.

Figure 2.

Overexpression of IL4I1 alleviated NAFLD in mice. A, the flow chart of IL4I1 overexpression in mice with NAFLD. B, the body weight of mice (n = 6). C, the morphology of liver tissues collected from mice. D, the liver weight of mice (n = 6). E, the liver organ index (liver/body weight ratio) of mice (n = 6). F, the triglyceride (TG) level in the serum of mice (n = 6). G, the total cholesterol (T-CHO) level in the serum of mice (n = 6). H, the TG level in the liver of mice (n = 6). I, the T-CHO level in the liver of mice (n = 6). J, the protein expression of IL4I1 in the liver tissues of mice. Data were presented as mean ± SD and analyzed by one-way ANOVA. The black horizontal line in bar graphs represents the mean difference between the Con group and the WD group, and the blue horizontal line in bar graphs represents the mean difference between the WD + AAV8^EV and WD + AAV8^IL4I1. IL4I1, interleukin-4 induced protein 1; NAFLD, nonalcoholic fatty liver disease; WD, western diet.

IL4I1 overexpression alleviated liver damages and lipid accumulation in NAFLD-like mice

NAFLD is characterized by a wide range of liver damages resulting from abnormal lipid deposition in the liver (18). Thus, we continued to check how IL4I1 would alter liver damage parameters. The alanine transaminase (ALT) and aspartate aminotransferase (AST) activities were measured to indicate whether liver damages have occurred. The results showed that a WD induced liver damages in mice, evidenced by the markedly increased ALT and AST activities, which were decreased by overexpression of IL4I1 (Fig. 3, A and B). H&E staining revealed the increased fat vacuoles in mice fed a WD compared with mice with a standard diet, while IL4I1 overexpression inhibited this alternation in the mouse liver (Fig. 3C). NAFLD activity score for images of H&E staining showed that WD-fed mice had a markedly higher score compared with those controls, but there was no significant difference between the WD + AAV8^EV group and the WD + AAV8^IL4I1 group. Of note, we observed a decrease in NAFLD activity score (the WD + AAV8^IL4I1 group versus the WD + AAV8^EV group = 4.445 versus 8.165). No statistical difference might be attributed to the small sample size used for scoring (Fig. 3F). After that, Sirius Red staining was performed to assess the degree of liver fibrosis. The representative images and quantitative analysis showed that the increased Sirius Red-positive staining area in the liver of NAFLD-like mice was reduced post IL4I1 overexpression, indicating that WD-induced liver fibrosis was reversed by IL4I1 overexpression (Fig. 3, D and G). Consistently, images of Oil Red O staining and the corresponding quantification further verified that IL4I1 overexpression declined the lipid accumulation in liver tissues of NAFLD-like mice, demonstrated by the significantly decreased Oil Red O-positive staining area in WD-fed mice with IL4I1 overexpression (Fig. 3, E and H). Moreover, IL4I1 is a phenylalanine oxidase catabolizing phenylalanine (19), but the phenylalanine metabolism is regulated by other enzymes such as phenylalanine hydroxylase and phenylalanine ammonia-lyase (20, 21). We next detected the phenylalanine level in the liver of mice. The results showed that WD resulted in the increased level of phenylalanine in liver tissues of mice with NAFLD, which was reversed by IL4I1 overexpression (Fig. 3I). Collectively, these data proved that IL4I1 overexpression exerted an inhibitory role in liver damages and lipid deposition caused by a WD.

Figure 3.

Overexpression of IL4I1 reduced liver damages and lipid metabolism disorder in mice with NAFLD. A, the serum ALT activity of mice (n = 6). B, the serum AST activity of mice (n = 6). C, images of H&E staining of liver tissues collected from mice. Black arrows indicated fat vacuoles. D, images of Sirius Red staining of liver tissues collected from mice. E, images of Oil Red O staining of liver tissues collected from mice. F, NAFLD activity score for images of H&E staining (n = 6). G, quantification of the percentage of Sirius Red O-positive staining area (n = 6). H, quantification of the percentage of Oil Red O-positive staining area (n = 6). I, the phenylalanine level in the liver of mice (n = 6). Data were presented as mean ± SD and analyzed by one-way ANOVA, Brown–Forsythe and Welch ANOVA tests, or Kruskal–Wallis test. The black horizontal line in bar graphs represents the mean difference between the Con group and the WD group, and the blue horizontal line in bar graphs represents the mean difference between the WD + AAV8^EV and WD + AAV8^IL4I1. ALT, alanine transaminase; AST, aspartate aminotransferase; IL4I1, interleukin-4 induced protein 1; NAFLD, nonalcoholic fatty liver disease; WD, western diet.

IL4I1 overexpression regulated Th17 cells in the liver of NAFLD-like mice by the AKT/FOXO1 pathway

We have verified the ameliorative effect of IL4I1 overexpression on NAFLD in the above section. To validate our hypothesis that IL4I1 would modulate the pathology of NAFLD by regulating Th17 cells, CD4⁺ T cells were enriched by the magnetic beads-based isolation from the liver tissues of mice and subjected to double immunofluorescence staining, flow cytometry analysis, and Western blot assays. As shown in Fig. 4A, abundant Th17 cells was found in the liver of NAFLD-like mice compared with the controls, demonstrated by the increased IL-17A-expressing cells. In contrast, IL4I1 overexpression produced the opposite effect on the enrichment profile of Th17 cells in the mouse liver (Fig. 4A). The IL-17A protein expression in CD4⁺ T cells isolated from mice with NAFLD was more than those derived from healthy controls, but the opposite trend was found in the IL4I1-overexpressing mice (Fig. 4B). Consistent to the results of immunofluorescence staining, flow cytometry analysis provided support for that IL4I1 overexpression reduced the number of Th17 cells (Fig. 4, C and D). AKT was evidenced to phosphorylate the negative regulator of Th17 cells FOXO1. We then sought to survey the underlying mechanism in which IL4I1 overexpression regulated Th17 cells by the AKT/FOXO1 pathway. Western blot assays showed that a WD induced the increase in the phosphorylation levels of AKT and FOXO1 was considerably reduced by IL4I1 overexpression (Fig. 4, E and F). Phosphorylated FOXO1 could lead to the nuclear export of FoxO1. The nuclear FOXO1 expression was increased in NAFLD-like mice receiving AAV-8 vector encoding IL4I1 (Fig. 4G). These results suggested that IL4I1 overexpression inhibited Th17 cells in the liver of NAFLD-like mice, partly by suppressing the AKT/FOXO1 signaling pathway.

Figure 4.

Overexpression of IL4I1 decreased the percentage of Th17 cells in liver tissues of mice with NAFLD.A, the protein expression of IL-17A in the liver tissues of mice. B, flow cytometry analysis for the Th17 cells (CD4⁺ IL-17A⁺) in the liver tissues of mice. C, the percentage of IL-17A⁺ cells in in CD4⁺ T cells isolated from the liver tissues of mice (n = 6). D, the protein expression of p-AKT and AKT in the liver tissues of mice. E, the protein expression of p-FOXO1 and FOXO1 in the liver tissues of mice. F, the nuclear protein expression of FOXO1 in the liver tissues of mice. Data were presented as mean ± SD and analyzed by one-way ANOVA. The black horizontal line in bar graphs represents the mean difference between the Con group and the WD group, and the blue horizontal line in bar graphs represents the mean difference between the WD + AAV8^EV and WD + AAV8^IL4I1. IL4I1, interleukin-4 induced protein 1; NAFLD, nonalcoholic fatty liver disease; WD, western diet; FOXO1, forkhead box protein O1.

The AKT activator eliminated the role of IL4I1 overexpression in Th17 cells in vitro

We then determined whether the IL4I1’s effect on Th17 cells could be confirmed in vitro. CD4⁺ T cells were purified from the spleen tissues, and the purity of enriched CD4⁺ T cells was more than 95% (Fig. 5A). Following that, CD4⁺ T cells (Th0 cells) were infected with lentiviral vector encoding IL4I1 and further allowed to polarize into Th17 under the specific culture conditions (Fig. 5B). The IL4I1 expression was found to increase in Th17 cells by comparison to Th0 cells (Fig. 5C). Expression of transcription factor RORγt is a characteristic of Th17 cells (22). Western blot assay showed that Th17 cells had a higher RORγt expression compared to Th0 cells, while IL4I1 overexpression inhibited its expression in Th17 cells (Fig. 5D). Flow cytometry analysis attested the inhibitory effect of IL4I1 overexpression on Th17 differentiation (Fig. 5, E and F). Next, to explore the role of IL4I1 knockdown in Th17 differentiation, CD4⁺ T cells were purified from the spleen tissues (purity > 95%), infected with lentiviral vector carrying shRNA targeting IL4I1, and stimulated under the Th17 polarization conditions (Fig. 5, G and H). Inconsistent with the overexpression experiments, IL4I1 knockdown reduced the IL4I1 expression but elevated the RORγt expression in Th17 cells (Fig. 5, I and J). Moreover, IL4I1 knockdown promoted Th17 differentiation, as evidenced by the higher proportion Th17 cells following LV^shIL4I1 infection (Fig. 5, K and L).

Figure 5.

Overexpression of IL4I1 inhibited Th17 differentiation in vitro. A, flow cytometry analysis for the purity of CD4⁺ T cells isolated from the spleen tissues of mice. B, the flow chart of polarizing CD4⁺ T cells infected with Lv^EV or Lv^IL4I1 to Th17 cells. C, the protein expression of IL4I1 in Th17 cells with IL4I1 overexpression. D, the protein expression of RORγt in Th17 cells with IL4I1 overexpression. E, flow cytometry analysis for the Th17 cells (CD4⁺ IL-17A⁺) differentiated from CD4⁺ T cells. F, the percentage of IL-17A⁺ cells in CD4⁺ T cells (n = 3). G, flow cytometry analysis for the purity of CD4⁺ T cells isolated from the spleen tissues of mice. H, the flow chart of polarizing CD4⁺ T cells infected with Lv^shNC or Lv^shIL4I1 to Th17 cells. I, the protein expression of IL4I1 in Th17 cells with IL4I1 knockdown. J, the protein expression of RORγt in Th17 cells with IL4I1 knockdown. K, flow cytometry analysis for the Th17 cells (CD4⁺ IL-17A⁺) differentiated from CD4⁺ T cells. L, the percentage of IL-17A⁺ cells in CD4⁺ T cells (n = 3). Data were presented as mean ± SD and analyzed by one-way ANOVA. The black horizontal line in bar graphs represents the mean difference between the Th0 group and the Th17 group, and the blue horizontal line in bar graphs represents the mean difference between the Th17+Lv^EV and Th17+Lv^IL4I1. Lv^EV, empty lentiviral vector; Lv^IL4I1, lentiviral vector expressing IL4I1; Lv^shNC, lentiviral vector carrying nontargeting shRNA; Lv^shIL4I1, lentiviral vector carrying shRNA targeting IL4I1; IL4I1, interleukin-4 induced protein 1.

By performing the Western blot assay, we found the increased phosphorylated AKT expression and the decreased nuclear FOXO1 expression in Th17 cells compared with Th0 cells. IL4I1-overexpressing Th17 cells had the contrary expression profiles of phosphorylated AKT and nuclear FOXO1 in Th17 cells (Fig. 6, A and B). However, IL4I1 knockdown activated AKT and reduced nuclear FOXO1 expression, resulting in the opposite trends in Th17 cells (Fig. 6, C and D). Afterward, an AKT activator SC79 was added during the process of Th17 polarization, which was used for the rescue experiment. We observed that SC79 treatment eliminated the inhibitory effect of IL4I1 overexpression on the phosphorylated AKT and nuclear FOXO1 expression in Th17 cells (Fig. 6, E and F). In addition, the AKT activation increased percentage of IL-17A⁺ cells of IL4I1-overexpressing CD4⁺ T cells under the polarizing conditions (Fig. 6, G and H). In conclusion, these results indicated that IL4I1 overexpression inhibited Th17 cell differentiation in part by inactivating the AKT/FOXO1 pathway.

Figure 6.

Overexpression of IL4I1 regulated Th17 differentiation by the AKT/FOXO1 pathway.A, the protein expression of p-AKT and AKT in Th17 cells with IL4I overexpression. B, the protein expression of FOXO1 in Th17 cells with IL4I overexpression. C, the protein expression of p-AKT and AKT in Th17 cells with IL4I knockdown. D, the protein expression of FOXO1 in Th17 cells with IL4I knockdown. E, the protein expression of p-AKT and AKT in Th17 cells after SC79 treatment. F, the protein expression of FOXO1 in Th17 cells after SC79 treatment. G, flow cytometry analysis for the Th17 cells (CD4⁺ IL-17A⁺) differentiated from CD4⁺ T cells after SC79 treatment. H, the percentage of IL-17A⁺ cells in CD4⁺ T cells after SC79 treatment (n = 3). Data were presented as mean ± SD and analyzed by one-way ANOVA. The black horizontal line in bar graphs represents the mean difference between the Th0 group and the Th17 group, and the blue horizontal line in bar graphs represents the mean difference between the Th17+Lv^EV and Th17+Lv^IL4I1. FOXO1, forkhead box protein O1; IL4I1, interleukin-4 induced protein 1; Lv^EV, empty lentiviral vector; Lv^IL4I1, lentiviral vector expressing IL4I1; Lv^shNC, lentiviral vector carrying nontargeting shRNA; Lv^shIL4I1, lentiviral vector carrying shRNA targeting IL4I1.

Discussion

Fatty liver disease is the most common cause of chronic liver disease, consisting of alcoholic fatty liver disease (ALD) and NAFLD (23, 24). ALD is caused by the abnormal metabolism in the liver after alcohol intake, while NAFLD is related to systematically metabolic disorders (25, 26). The overall prevalence of ALD and NAFLD is 6% and 25% (27), respectively. Considering the wider impact of NAFLD, we paid our attention on NAFLD in the present study. The mouse model of NAFLD was then constructed by a WD, a major risk factors for NAFLD (28). In the present study, only male mice were included in animal experiments. Men had a higher overall prevalence of NAFLD than women (29). Moreover, male mice fed with a WD showed more severe NAFLD symptoms than those female mice (30). More importantly, this study aimed to explore the potential therapeutic target for NAFLD and the underlying mechanism. Therefore, all animal study were performed in male mice. We first found that the highly expressed IL4I1 in the pathological state of NAFLD was a compensatory mechanism. Accordingly, IL4I1 overexpression inhibited Th17 cell differentiation by inactivating of the AKT/FOXO1 signaling pathway, thereby ameliorating a WD-induced NAFLD.

Most preliminary studies concerning IL4I1 have focused on inflammatory diseases or cancer. IL4I1 was responsible for the inhibitory effect of muscle stem cells on the infiltration of neutrophils into damaged lungs (31). On the contrary, in the tumor progression, IL4I1 was implicated in immune escape of cancer cells (32). The opposite effects of IL4I1 on cancers compared to other inflammatory diseases might be caused by the unique tumor microenvironment. NAFLD is also an inflammatory liver disease. Similar to these studies of inflammatory diseases, we also confirmed that IL4I1 suppressed the NAFLD by regulating Th17 cell differentiation. Of note, a previous study reported that IL4I1 knockdown could alleviate high glucose-induced inflammation and lipotoxicity in HepG2 cells by inhibiting the activation of AHR. However, AHR exerts the completely opposite effects on NAFLD progression. Wada et al. pointed out that liver-specific AHR knockout mice exhibited more severe liver steatosis, inflammation, and damage following a high-fat diet compared to those controls (33). In contrast, an AHR agonist was found to alleviate fatty liver in high fat–induced obese mice. A prospective research suggested that IL4I1 was capable of activating AHR (34). Furthermore, Ahr deficiency contributed to the increased Th17 cells in the intestine (35). Activation of AHR contributed to the IL-17-induced inflammation in HeLa cells (36). Based on this, we supposed that the high expression of IL4I1 in NAFLD samples might be a compensatory upregulation. Consistent with our conjecture, overexpression of IL4I1 had a protective effect on a WD-induced NAFLD.

IL4I1 is an enzyme that catalyzes phenylalanine. Our results showed that mice with NAFLD had a higher phenylalanine in the liver, which was reversed by IL4I1 overexpression. Since the level of phenylalanine does not only depend on IL4I1, we supposed that the phenylalanine metabolism is regulated by other enzymes under the NAFLD pathology. A previous study has pointed out that patients with NAFLD had a higher phenylalanine level in serum samples (37). Phenylalanine administration induced hepatic lipid deposition and inhibited mitophagy in the liver, promoting liver steatosis (38). The results of our investigation are consistent with these previous studies. Moreover, we determined that IL4I1 overexpression reduced the Th17 proportion in the liver tissues of WD-fed mice and found that the proportion of Th17 cells was relatively low in all experimental groups. Gomes et al. reported that mice on a high-fat diet had 3.09% Th17 cells in their livers, whereas mice on a normal diet had only 1.80% (39). Additionally, Herck et al. found that mice fed a high-fat, high-fructose diet exhibited approximately 5% Th17 cells in their livers, compared to around 1% in those on a control diet (40). Our results are similar to these previously published studies. Further, we isolated splenic CD4⁺ T cells to investigate the effects of IL4I1 on Th17 differentiation in vitro, rather than using hepatic CD4⁺ T cells. The spleen is the largest secondary lymphoid organ and contains a high abundance of T cells (41), which makes it an ideal source for in vitro studies. Moreover, isolating CD4⁺ T cells from the liver is a more complicated process than isolating them from the spleen. In a previous high-quality literature, in vitro studies have also been conducted on CD4⁺ T cells isolated from the spleen to explore Th17 cell differentiation under the pathological state of NAFLD (8). More importantly, the present study mainly focuses on exploring the effects of IL4I1 on the differentiation of Th17 cells. According to the above reasons, splenic CD4⁺ T cells were isolated and used for in vitro differentiation.

Numerous literature has shown the activation of AKT in the pathological state of NAFLD, although the activation mechanisms of AKT are different. AKT is an upstream regulator of FOXO1 and can phosphorylate FOXO1 in three conserved sites (Thr 24, Ser 256, and Ser 319), thereby inhibiting the activity of FOXO1 (42, 43). After being phosphorylated by AKT, FOXO1 binds to the 14-3-3 protein and exports from the nucleus to the cytoplasm (44). FOXO1 is capable of regulating the cell development and function of Th17 cells (45). Nuclear FOXO1 could inhibit Th17 cell differentiation by downregulating the RORγt activity (46). In the present study, we also confirmed the activated AKT, increased phosphorylated FOXO1, the nuclear translocation of FOXO1, and the increased Th17 cells in the liver of NAFLD-like mice. Following the in vitro rescue experiment, the AKT activator SC79 was added in the process of Th17 polarization, we observed SC79 eliminated the Th17 cell differentiation-suppressing effect of IL4I1 overexpression and determined that IL4I1 inhibited Th17 differentiation in part by promoting the inactivation of the AKT/FOXO1 signaling pathway. In addition, a prior study implied that the elevated AKT phosphorylation level in lung fibroblasts of AHR knockout mice (47), which once again prompted us that AHR might play an important role in the regulation of Th17 cell differentiation by IL4I1 overexpression.

To sum up, our findings showed that the IL4I1 expression was upregulated in the pathological condition of NAFLD, and IL4I1 overexpression alleviated NAFLD progression by inhibiting Th17 differentiation via the AKT/FOXO1 signaling pathway. This study first ascertained the upregulated IL4I1 expression is a protective and compensatory response to alleviate NAFLD. However, some limitations still existed in the current study and urgently need to be solved in the further research plan. Only male mice were used for animal study in the present study, and including female mice in further research would ensure the effects of IL4I1 on potential sex differences in NAFLD. Besides, AHR was associated with NAFLD progression and might be seriously involved in AKT inactivation and subsequent Th17 differentiation. However, we did not explore whether AHR would be activated by IL4I1 overexpression and how it would affect the AKT/FOXO1 signaling pathway. Furthermore, our research only demonstrated that the AKT/FOXO1 signaling pathway is one of the possible mechanisms by which IL4I1 protects NAFLD. Whether IL4I1 would affect Th17 differentiation in the pathology of NAFLD by regulating other signaling pathways is worth in-depth exploration. The more detailed mechanism in which IL4I1 modulated NAFLD progression need to be explored in depth in the future plan.

Experimental procedures

Animal study

Male C57BL/6J mice (8-week-old) were used for the construction of the in vivo NAFLD model. All mice were kept under a controlled environment (12 h light/12 h dark cycle, 22 ± 1 °C temperature, and 45∼55% humidity) and allowed to eat a standard diet and drink ad libitum. After 1 week of acclimatization, mice were randomly divided into two groups, including the control group and the WD group. Following that, the diet of mice in the WD group was replaced with a WD for 8 weeks, while mice in the control group remained on the standard diet for 8 weeks. After fasting for 12 h, all mice were euthanized followed by the collection of serum and liver tissue samples.

Another batch of male C57BL/6J mice (8-week-old) was used to explore the role of IL4I1 in NAFLD. After 1 week of acclimatization, mice were randomly divided into four groups, including the control group, the WD group, the WD + AAV8^EV group, and the WD + AAV8^IL4I1 group. Then, the empty AAV8 vector and the AAV8 encoding IL4I1 were given to mice in the WD + AAV8^EV group and the WD + AAV8^IL4I1 group via the tail vein injection (3 × 10¹¹ vg per mouse), respectively. After another week, the controls were still served a standard diet for 8 weeks, while mice in the other three groups were fed with a WD for 8 weeks. Following fasting for 12 h, mice were then euthanized for the collection of serum and liver tissues.

A total of 36 mice were used for animal study. Animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals.

Western blot

The whole protein was extracted from liver tissues or cells using RIPA lysis buffer (Cat#PR20001, Proteintech) containing common protease inhibitor cocktail (Cat#PR20032, Proteintech) and phosphatase inhibitor cocktail (Cat#PR20015, Proteintech). However, nucleoprotein was obtained using a nucleoprotein extraction kit (Cat#PK10014, Proteintech). The concentrations of extracted proteins were determined by the BCA method. Boiled protein samples were separated by SDS-PAGE and then transferred onto PVDF membranes (Cat#LC2005, Thermo Fisher Scientific). Next, the membranes were blocked with the blocking solution (5% skimmed milk powder dissolved by the TBST buffer) (Cat#PR20011, Proteintech), followed by the incubation of primary (4 °C, overnight) and secondary antibodies (37 °C, 40 min). Protein bands were visualized using an Ultrasensitive Enhanced Chemiluminescence Detection Kit (Cat#PK10003, Proteintech). The primary antibodies for Western blot are as follows: IL4I1 antibody (1:500, Cat#bs-6841R, Bioss), IL-17A antibody (1:1000, Cat#bs-1183R, Bioss), p-AKT^Thr308 antibody (1:500, Cat#341790, ZEN-BIOSCIENCE), AKT antibody (1:1000, Cat#342529, ZEN-BIOSCIENCE), p-FOXO1^Ser256 antibody (1:1000, Cat#310198, ZEN-BIOSCIENCE), FOXO1 antibody (1:1000, Cat#380978, ZEN-BIOSCIENCE), RORγt antibody (1:500, Cat#bs-23109R, Bioss). Their corresponding secondary antibody was Goat-anti-rabbit IgG-HRP (1:10,000, Cat#SA00001–2, Proteintech). β-actin antibody (1:20,000, Cat#66009-1-Ig, Proteintech) was used as the internal reference of total protein, and Goat-anti-mouse IgG-HRP (1:10,000, Cat#SA00001–1, Proteintech) was its secondary antibody. LaminB1 antibody (1:500, Cat#R24825, ZEN-BIOSCIENCE) was used as the internal reference of nuclear protein, and Goat-anti-rabbit IgG-HRP (1:10,000, Cat#SA00001–2, Proteintech) was its secondary antibody. The original images of western blot assays are provided in Supporting information.

Oil red O staining

Fixed liver tissues were placed in 20% and 30% sucrose solutions for dehydration. Completely dehydrated tissues were treated with an optimal cutting temperature compound embedding agent and sectioned into 10-μm slices. After being washed with the distilled water for 2 min, sections were successively immersed into the 60% isopropyl alcohol (Cat#80109218, Sinopharm Chemical Reagent Co., Ltd) for 2 min and Oil Red O stock solution (Cat#O0625, Sigma-Aldrich) for 5 min. Thereafter, sections were differentiated with the 60% isopropyl alcohol and stained with hematoxylin (Cat#H8070, Solarbio) for 1 min. Glycerin–gelatin mounting medium was used to mount sections, which were imaged under a microscope (OLYMPUS).

Double immunofluorescence staining

Fixed tissues were flushed with the running water, dehydrated in gradient ethanol solution, and cleared with xylene. Then tissues were subjected to paraffin embedding and section preparation of 5-μm slices. For immunofluorescence staining, sections were deparaffinized with xylene and rehydrated in ethanol solution of decreasing concentrations. After antigen retrieval and blocking, sections were incubated with the primary (4 °C, overnight) and secondary antibodies (room temperature, 90 min). Nuclear staining was achieved by DAPI (Cat#D106471-5 mg, Aladdin). Finally, sections were mounted with anti-fluorescence quencher (Cat# S2100, Solarbio) and observed under a microscope (BX53, OLYMPUS). The antibodies for double immunofluorescence staining are as follows: IL4I1 antibody (1:200, Cat#bs-6841R, Bioss) and its secondary antibody Goat-anti-rabbit IgG-FITC (1:200, Cat#ab6717, Abcam), IL17A antibody (1:200, Cat#bs-1183R, Bioss) and its secondary antibody Goat-anti-rabbit IgG-FITC (1:200, Cat#ab6717, Abcam), CD4 antibody (1:100, Cat#sc-52385) and its secondary antibody Goat-anti-mouse IgG-Cy3 (1:200, Cat#ab97035, Abcam).

Sirius Red staining

Dehydrated tissues were cleared in xylene solution (Cat#1330–20–7, Aladdin) for 30 min followed by paraffin-embedding and tissue slicing (5 μm). Following deparaffinization and rehydration, sections were stained with Sirius Red (Cat#G1472, Solarbio) dissolved in saturated aqueous solution of picric acid for 5 min. An increasing gradient of ethanol and xylene were used to treat sections. After being mounted with neutral gum, sections were observed under a microscope (OLYMPUS).

Measurement of liver damages

The activities of ALT and AST were detected by the commercially available kits (Cat#C009 for ALT, Cat#C010 for AST, Nanjing Jiancheng Bioengineering Institute). The experimental protocols were conducted in according to the manufacturer’s instructions.

Detection of phenylalanine level

The phenylalanine level in the liver tissues of mice was determined using a commercially available kit (Cat#E-BC-K846-M, Elabscience) following the manufacturer-provided procedures.

Assessment of lipid metabolism

The levels of triglyceride and total cholesterol in the serum and liver tissues of mice were measured using the commercial kits (Cat#A110 for triglyceride, Cat#A111 for total cholesterol, Nanjing Jiancheng Bioengineering Institute). The detailed procedures complied with the protocols provided by the manufacturer.

Hepatic immune cell isolation

Isolation of hepatic immune cells was carried out as the previously described (48). Briefly, liver tissues were minced and digested with 1 mg/ml collagenase type VIII at 37 °C for 20 min and filtered. After being washed with phosphate buffered saline solution, single cell suspension were mixed with the 37.5% Percoll, which was centrifuged at 350g for 30 min. The cell pellet was collected and then washed and treated with red blood cell lysis buffer. Hepatic immune cells were isolated and collected for further use.

CD4⁺ T cell sorting

Hepatic immune cells (1 × 10⁷) were resuspended in 40 μl buffer solution and incubated with the 10 μl biotin-antibody cocktail at 2∼8 °C for 5 min. Subsequently, 30 μl buffer solution and 20 μl Anti-Biotin MicroBeads (CAT#130–104–454, Miltenyi Biotec) was added to incubate cells at 2∼8 °C for 10 min. Cell suspension was then loaded onto the LS column, which was placed in the field of a magnetic MACS Separator and rinsed with 3 μl buffer solution. The effluent passing through the LS column was collected, and it contained the unlabeled cells, namely CD4⁺ T cells.

Flow cytometry analysis

The enriched CD4⁺ T cells were treated with 10 μg/ml Brefeldin A (Cat#B802056, Macklin Inc.), 50 ng/ml phorbol 12-myristate 13-acetate (Cat#P849986, Macklin Inc.), and 1 μg/ml ionomycin (Cat#I838446, Macklin Inc.). After 4 h of culture, cells were collected, mixed with 100 μl flow cytometry buffer, and incubated with FITC-conjugated anti-CD4 antibody (Cat#100405, BioLegend) at 4 °C in dark for 30 min. Cells were washed and treated by the fixative solution at room temperature for 20 min. Centrifugation was performed to discard the supernatant, and cells were collected and mixed with penetration enhancer. After being centrifuged, the cell pellet was mixed with flow cytometry buffer and incubated with APC-conjugated anti-IL-17A antibody (Cat#506915, BioLegend) at 4 °C in dark for 30 min. Flow cytometry buffer-washed cells were subjected to flow cytometry analysis.

Splenic CD4⁺ T cell isolation, cell transfection, differentiation, and treatment

Spleen tissues were picked out from mice under the sterile condition and cut into small pieces, which were minced and filtered to prepare cell suspension. Subsequently, immune cell isolation and CD4⁺ T cell sorting were conducted as the previously mentioned (49). Isolated CD4⁺ T cells were cultured in the RPMI-1640 medium (Cat#31800, Solarbio) supplemented with 15% FBS (Cat#11011–8611, Tianhang). The purity of enriched CD4⁺ T cells generally more than 95%. CD4⁺ T cells were cultured in the culture medium containing the empty lentiviral vector (Lv^EV), the lentiviral vector expressing IL4I1 (Lv^IL4I1), the lentiviral vector carrying nontargeting shRNA (Lv^shNC), or the lentiviral vector carrying shRNA targeting IL4I1 (Lv^shIL4I1) in a 37 °C incubator with 5% CO₂.

After 24 h of infection, CD4⁺ T cells were incubated with plate-bound anti-CD3 (5 μg/ml, Cat#BE0002, BioXcell) and anti-CD28 (2 μg/ml, Cat#BE0328, BioXcell) containing the Th17 polarization cytokines (2 ng/ml TGF-β3, Cat#RP00645, ABclonal; 30 ng/ml IL-6, Cat#Z03189, Genscript; 10 ng/ml IL-1β, Cat#RP01340, ABclonal; 20 ng/ml IL-23, Cat#RP02928LQ, ABclonal; 10 μg/ml anti-IFN-γ, Cat#BE0055, BioXcell; and 10 μg/ml anti-IL-4, Cat#BE0045, BioXcell) for 5 days (49). During the last 4 h of incubation, 10 μg/ml Brefeldin A, 50 ng/ml phorbol 12-myristate 13-acetate, and 1 μg/ml ionomycin were added to stimulate cells. Subsequently, cells were collected after centrifugation, fixed by fixative solution, and treated using the penetration enhancer. After being incubated with APC-conjugated anti-IL-17A antibody at 4 °C in dark for 30 min, cells were washed and analyzed by flow cytometry.

To explore whether AKT activation would involve the IL4I1’s effects, an AKT activator SC79 (0.5 μg/ml, Cat#S80614, Yuanye) was added during the last 24 h of Th17 polarization.

Statistical analysis

All data were analyzed by GraphPad Prism 9 and presented as mean ± SD. Mann–Whitney test and unpaired t test were used to compare differences between two groups. Ordinary one-way ANOVA and Brown-Forsythe and Welch ANOVA tests were used to compare differences among groups. The p values less than 0.05 was considered statistically significant.

Data availability

Data available on reasonable request from the corresponding author.

Supporting information

This article contains supporting information.

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article.

Reviewed by members of the JBC Editorial Board. Edited by Qi-Qun Tang