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Correspondence Address correspondence to: Philippe Gual, PhD, Inserm UMR1065/C3M, Bâtiment Universitaire ARCHIMED, Team "Chronic liver diseases associated with obesity and alcohol", 151 route Saint Antoine de Ginestière, BP 2 3194, 06204 Nice, France. fax: +33 4 89 06 42 60.
Spleen tyrosine kinase (SYK) signaling pathway regulates critical processes in innate immunity, but its role in parenchymal cells remains elusive in chronic liver diseases. We investigate the relative contribution of SYK and its substrate c-Abl Src homology 3 domain-binding protein-2 (3BP2) in both myeloid cells and hepatocytes in the onset of metabolic steatohepatitis.
Hepatic SYK-3BP2 pathway was evaluated in mouse models of metabolic-associated fatty liver diseases (MAFLD) and in obese patients with biopsy-proven MAFLD (n = 33). Its role in liver complications was evaluated in Sh3bp2 KO and myeloid-specific Syk KO mice challenged with methionine and choline deficient diet and in homozygous Sh3bp2KI/KI mice with and without SYK expression in myeloid cells.
Here we report that hepatic expression of 3BP2 and SYK correlated with metabolic steatohepatitis severity in mice. 3BP2 deficiency and SYK deletion in myeloid cells mediated the same protective effects on liver inflammation, injury, and fibrosis priming upon diet-induced steatohepatitis. In primary hepatocytes, the targeting of 3BP2 or SYK strongly decreased the lipopolysaccharide-mediated inflammatory mediator expression and 3BP2-regulated SYK expression. In homozygous Sh3bp2KI/KI mice, the chronic inflammation mediated by the proteasome-resistant 3BP2 mutant promoted severe hepatitis and liver fibrosis with augmented liver SYK expression. In these mice, the deletion of SYK in myeloid cells was sufficient to prevent these liver lesions. The hepatic expression of SYK is also up-regulated with metabolic steatohepatitis and correlates with liver macrophages in biopsy-proven MAFLD patients.
Collectively, these data suggest an important role for the SYK-3BP2 pathway in the pathogenesis of chronic liver inflammatory diseases and highlight its targeting in hepatocytes and myeloid cells as a potential strategy to treat metabolic steatohepatitis.
This work reports that SYK-3BP2 pathway activity enhances the LPS-TLR4 responses in both hepatocytes and resident liver macrophages. This initiates the onset of local inflammation and contributes to liver accumulation of leukocytes. Targeting of 3BP2 or SYK prevents metabolic steatohepatitis features.
and still presents an unmet medical need with no approved pharmacologic therapies. MAFLD is a broad term that encompasses the full spectrum of fatty liver diseases from simple hepatic steatosis to metabolic steatohepatitis, fibrosis/cirrhosis, and hepatocellular cancer. Obesity and metabolic syndrome are also associated with the development of MAFLD. Similarly, type 2 diabetes mellitus, insulin resistance, hypertension, and weight gain are risk factors for MAFLD progression.
In addition to genetic and environmental factors, the interaction of gut and adipose tissue with the liver enhances liver metabolic disorders (steatosis and insulin resistance), chronic inflammation, and injury-mediated fibrosis.
In MAFLD liver, a large number of innate immune cells are involved in the onset of the chronic inflammation, including resident and recruited macrophages and neutrophils but also parenchymal hepatocytes.
Stressed hepatocytes, such as fatty and sub-lethal hepatocytes, take on an immune function by sensing excessive metabolite levels and pathogen- and damage-associated molecular patterns (PAMPs and DAMPs, respectively), which in turn elicit inflammatory events associated with metabolic dysfunction.
Liver-resident macrophages (Kupffer cells) can initiate inflammation and help to recruit blood-derived monocytes. The polarization of both resident and recruited macrophages into pro-inflammatory phenotypes subsequently promotes MAFLD progression. The gut-derived endotoxins, lipids, and molecules associated with hepatocellular damage and death (DAMPs, extracellular vesicles, apoptotic bodies, etc) are the main factors contributing to macrophage and hepatic stellate cell (HSC) activation and the regulation of chronic hepatitis and fibrogenesis. To study pathways that are conserved in both hepatocytes and macrophages and involved in the steatosis-steatohepatitis transition, we focused on the role of the spleen tyrosine kinase (SYK) pathway in the regulation of inflammatory mediators.
SYK is a cytoplasmic non-receptor tyrosine kinase, and SYK-dependent signaling is known to regulate critical processes in innate and adaptive immunity downstream immunoreceptors (including B-cell receptors, T-cell receptors, and Fc receptors), C-type lectins receptors, integrins, and toll-like receptors (TLRs).
SYK inhibitors blocked HSC activation in vitro and in animal models of hepatic fibrosis, including toxin challenge (carbon tetrachloride, thioacetamide [TAA], N-nitrosodiethylamine)- and bile duct ligation-induced liver fibrosis models.
The regulation of macrophage activity by SYK in the context of liver injury and exacerbated by DAMPs could provide an attractive mechanism in chronic liver diseases. This exacerbated activation of macrophages by TLR ligands (DAMPs, PAMPs, and saturated lipids) was found to be a central pathogenic mechanism for chronic inflammation and MAFLD. In addition, TREM1, a member of the triggering receptors expressed on myeloid cells (TREM) family that mediates the inflammatory response through TLR4, DNAX-activation protein of 12 kDa (DAP12), and SYK, has been implicated in liver diseases.
In line with the important role of the SYK pathway in inflammatory diseases, gain-of-function mutations in SYK, leading to increased phosphorylation and hyperactivated downstream signaling, were recently associated with immune dysfunction and systemic inflammation.
was necessary for TLR-mediated activation of macrophages in response to PAMPs and DAMPs. Indeed, 3BP2-deficient macrophages exhibited dramatically reduced inflammatory responses to microbial challenge and reduced phagocytosis.
ameliorated the chronic inflammation driven by the mutation of 3BP2 in mice.
Although SYK and its signaling partner 3BP2 are expressed in liver tissue, many details are still elusive, such as their relative local expression in response to liver status and their relative pathogenic roles in hepatocytes and macrophages throughout the development of liver steatosis and progression to steatohepatitis. We thus investigated these open questions in different mouse models of MAFLD, human MAFLD samples, and transgenic animals deficient in 3BP2 or SYK signaling or with sustained activation of 3BP2 signaling (cherubic 3BP2 mutants).
Hepatic Expression of 3BP2 and SYK Correlated With Liver Complications in Mouse Models of MAFLD
We first evaluated the hepatic expression levels of Sh3bp2, Syk, and macrophage marker Emr1 (F4/80) in a dietary mouse model of obesity and hepatic steatosis. After 33 weeks of high-fat diet (HFD), the wild-type C57BL/6 (Wt) mice developed obesity, hepatic injury as evaluated by alanine aminotransferase (ALT) activity, and severe hepatic steatosis (Figure 1A–C). The hepatic expression levels of Syk and Emr1 were increased upon 33 weeks of HFD (Figure 1D) and hepatic expression of Syk correlated with hepatic steatosis, ALT activity, and hepatic expression of Emr1 (Figure 1E). The hepatic expression levels of Sh3bp2 tended to increase with liver complications (Figure 1D). To subsequently investigate the behavior of the 3BP2 and SYK in response to the progression and the severity of the MAFLD, we monitored hepatic expression in Wt mice fed a diet deficient in methionine and choline (MCDD) for 2 and 7 weeks. The duration of the MCDD challenge correlates with the severity of the steatohepatitis associated with the aggravation of liver injury and fibrogenesis. At 2 weeks, mice developed moderate hepatic steatosis with mild hepatic inflammation (as evaluated by the inflammatory foci number) and liver injury (Figure 2A–C). The long-lasting MCDD challenge (7 weeks) aggravated the liver complications with severe steatosis, inflammation, and liver injury (Figure 2A–C). Hepatic Sh3bp2 and Syk expression levels were progressively increased with the MAFLD severity (Figure 2D). Hepatic Sh3bp2 and Syk also strongly correlated with hepatic steatosis, inflammatory foci number, liver injury, and hepatic expression of Emr1 (Figure 2E). Upon 7 weeks of MCDD challenge, the increased Syk expression was due to the up-regulation of both isoforms of Syk (Syk-b (S) and Syk (L)) (the latter being the main isoform expressed in liver) (Figure 2F). The hepatic expression levels of SYK were also increased upon MCDD at the protein level (Figure 2G). We then evaluated which cell types in the liver were responsible for 3BP2 and SYK expression. Sh3bp2 and Syk expression at the mRNA level was detected in hepatocytes and non-parenchymal fraction from non-pathogenic liver in lean mice (Figure 2H). Sh3bp2 was expressed at higher levels than Syk in hepatocytes. In addition, Sh3bp2 and Syk were highly expressed in non-parenchymal cells compared with Syk expression in hepatocytes with higher expression of Syk than Sh3bp2. Therefore, these results indicate that 3BP2 and SYK could impact the functions of both hepatocytes and non-parenchymal cells. In addition, the up-regulation of 3BP2 and SYK with MAFLD severity suggests a potential role in the steatosis-steatohepatitis transition.
3BP2 Deficiency Partially Prevented Liver Injury, Hepatic Steatosis, and Steatohepatitis Induced by MCDD Challenge
The role of 3BP2 in liver injury and steatohepatitis induced by MCDD was then investigated using mice deficient for 3BP2. The MCDD challenge was associated with elevated hepatic Sh3bp2 expression in Wt mice (Figure 3A). The Sh3bp2-/- mice displayed less liver damage as shown by the lower ALT activity levels (Figure 3B). Furthermore, histologic analyses demonstrated less hepatic steatosis and inflammatory foci in the livers of MCDD-fed Sh3bp2-/- mice compared with livers of MCDD-fed Wt mice (Figure 3C and D). The decreased inflammatory foci number was associated with partial prevention of the recruitment of inflammatory macrophages (F4/80+ CD11bhigh cells) and neutrophils (Ly6G+ Ly6C- cells) into the liver as evaluated by flow cytometric analysis (Figure 3E and F). This was associated with less hepatic expression of Emr1 (encoding for F4/80) and the adhesion molecule Sele (encoding for E-SELECTIN) involved in neutrophil recruitment into the liver
(Figure 3G). In addition, MCDD-fed Sh3bp2-/- mice displayed less hepatic inflammation as evaluated by expression of inflammatory markers including Cd44, Tnf, Ccl2, and Syk compared with MCDD-fed Wt mice (Figure 3G). This effect of the hepatic inflammation was also confirmed by the decreased hepatic expression of CD44 and SYK at the protein level (Figure 3H) and decreased serum level of CCL2 (Figure 3I). In addition, the hepatic expression of Trem1, an important amplifier of TLR4 pathway via SYK activity,
decreased in MCDD-fed Sh3bp2-/- mice compared with MCDD-fed Wt mice (Figure 3G). In line with this, the expression of Dap12, the transmembrane adaptor required for TREM1 function, also decreased (Figure 3G). Because hepatic injury and inflammation are key actors in the fibrogenic process, the 3BP2 deficiency also prevented the priming of hepatic fibrosis as evaluated by the expression of Tgfb1, Timp1, and Col1a1 (Figure 3G). The protective effect of 3BP2 deficiency on liver complications was also associated with decreased expression of oxidative stress markers Cyba (encoding for P22PHOX) and heme oxygenase-1 (HMOX1) and increased Ppar-alpha, the master regulator of beta-oxidation (Figure 3G). Altogether, we show that by regulating hepatic steatosis, inflammation, and liver injury, 3BP2 plays an important role in the onset of MAFLD.
The SYK/3BP2 Axis Significantly Contributed to the Up-Regulation of Inflammatory Mediators in Lipopolysaccharide-Stimulated Hepatocytes
Because hepatocytes are key actors in the early onset of local inflammation, we investigated the role of 3BP2 and SYK in the expression of inflammatory mediators in hepatocytes. Indeed, hepatocytes sense pathogen products such as lipopolysaccharide (LPS) and contribute to the up-regulation of liver cytokines and chemokines leading to the recruitment of inflammatory cells into the liver.
In the steady state, the silencing of Sh3bp2 strongly decreased the expression of Syk in primary mouse hepatocytes. On the other hand, the down-regulation of Syk did not impact Sh3bp2 expression (Figure 4A). In response to LPS, the expression of Sh3bp2 and Syk was up-regulated (LPS vs PBS: Sh3bp2 = 1.62 ± 0.07, P = .0001; Syk = 1.54 ± 0.13, P = .0074, n = 4), and the silencing of Sh3bp2 prevented LPS-stimulated Syk expression (data not shown). In addition, the down-regulation of Sh3bp2 or Syk strongly decreased the up-regulation of inflammatory mediators including Tnf, Ccl2, Il1b, and Il6 in response to LPS (Figure 4B). In line with this, SYK inhibition by SYK inhibitor R406 (as evaluated by the phosphorylation levels of SYK and ERK) (Figure 4C) strongly prevented the up-regulation of these inflammatory mediators in response to LPS (Figure 4E). Interestingly, R406 treatment also reduced the expression of Sh3bp2 and Syk in the steady state (Figure 4D). These data indicate that 3BP2 regulates SYK expression in challenged hepatocytes and that the SYK/3BP2 axis contributes to the expression of inflammatory mediators in response to PAMPs (LPS).
The Knockdown of SYK in Myeloid Cells Partially Prevented Liver Injury and Inflammation Induced by MCDD Challenge
We investigated the contribution of SYK to the development of the liver complications mediated by MCDD feeding. Because SYK deficiency leads to perinatal lethality in mice
and because SYK was preferentially expressed in liver non-parenchymal cells (Figure 2H), we focused on the role of SYK in myeloid cells. The Sykflox/floxLysMcre/cre mice (mSykKO mice) displayed a significant decrease in SYK expression in hepatic inflammatory monocytes (Ly6G- Ly6Chigh cells) and neutrophils (Ly6G+ Ly6C- cells) without disruption of the SYK expression in lymphocytes as evaluated by flow cytometric analysis (Figure 5A). After 4 weeks of MCDD feeding, the mSykKO mice displayed lower hepatic expression of Syk than MCDD-fed Wt mice (Figures 5B and H at the gene and protein levels, respectively) without impacting the sh3bp2 expression level (Figure 5G). In addition, the MCDD-fed mSykKO mice exhibited less liver damage, as shown by the lower ALT levels (Figure 5C). Furthermore, histologic analyses showed less accumulation of inflammatory foci in the livers of MCDD-fed mSykKO mice compared with livers of MCDD-fed Wt mice without changing in hepatic steatosis (Figure 5D and E). The down-regulation of SYK in myeloid cells strongly prevented the recruitment of infiltrating neutrophils (Ly6G+ Ly6C- cells) and macrophages (F4/80+ CD11bhigh cells) into the liver (Figure 5F). Moreover, these were associated with reduced hepatic expression of inflammatory markers such as Cd44 (Figure 5G and H at the gene and protein levels, respectively) and Sele (E-Selectin) (Figure 5G). In line with the potential role of SYK in the regulation of macrophage activation/polarization,
the hepatic expression of M1 markers Itgax (CD11C) and Tnf decreased, whereas the expression of the M2 marker Clec10a (CD301) did not change in MCDD-fed mSykKO mice compared with MCDD-fed Wt mice (Figure 5G). As reported for 3BP2 deficiency (Figure 3G), the knockdown of SYK in myeloid cells strongly decreased the hepatic expression of Trem1 and Dap12, the priming of the fibrosis (Tgfb1 and Timp1), and oxidative stress (Cyba and Hmox1) (Figure 5G). This effect of the inflammation was also confirmed by the decreased serum level of CCL2 (Figure 5I). These results indicate that the conditional ablation of SYK in myeloid cells is sufficient to reduce liver inflammation, injury, and, in turn, the priming of fibrosis in MCDD mice.
SYK Deletion in Myeloid Cells Prevented Severe Hepatitis and Fibrosis in Homozygous Sh3bp2KI/KI Mice
To further demonstrate the key role of the SYK/3BP2 pathway in the development of chronic liver diseases, we revisited this pathway in the livers of cherubic mice. Cherubism is an autoinflammatory autosomal dominant bone dysplasia associated with point-mutations of the Sh3bp2 gene leading to the accumulation of proteasome-resistant mutant 3BP2. The accumulation of 3BP2 mutant proteins exacerbated activities of intracellular signaling components including SYK in myeloid cells.
We evaluated the liver status in homozygous cherubic animals (Sh3bp2 G418R knock-in mice [Sh3bp2KI/KI mice], equivalent to the G420R mutation in cherubism patients), which developed systemic inflammation without regression of the lesions with age.
The 14-week-old Sh3bp2KI/KI mice already displayed cherubic manifestation (kyphosis) and facial inflammation (closed eyes) (Figure 6A and B) in addition to elevated liver weight relative to body weight (Figure 6C) and aspartate aminotransferase (AST) to ALT ratio (Figure 6D). We also found that the Sh3bp2KI/KI mice developed severe hepatitis, as evaluated by the accumulation of large inflammatory foci in the livers without hepatic steatosis (Figure 6E and F). The SYK deletion in myeloid cells in cherubic mice (mSykKOSh3bp2KI/KI mice) prevented all these clinical and biological abnormalities at the early and late age (mean age, 16- versus 43-week-old mice) (Figure 6A–D, data not shown). Interestingly, the SYK deletion in myeloid cells in Sh3bp2KI/KI mice decreased the hepatic expression of SYK at the mRNA and protein levels (Figure 6G and H). The SYK deletion in myeloid cells in Sh3bp2KI/KI mice also prevented hepatic inflammatory foci accumulation even in old mice (Figure 6E and F). This was also associated with lower hepatic expression of macrophage marker Emr1 and inflammatory markers including Cd44, Tnf, Ccl2,Trem1, and Dap12 (Figure 6G). This effect of inflammation was also confirmed by the decreased hepatic expression of CD44 at the protein level (Figure 6H) and decreased serum level of CCL2 (Figure 6I). In addition to the prevention of liver inflammation, SYK deficiency in myeloid cells was also associated with the prevention of hepatic fibrosis as evaluated by collagen staining (picrosirius red stain, Figure 6E) and lower gene expression of fibrosis markers like Tgfb1, Timp1, and Col1a1 (Figure 6G). Finally, the hepatic expression of Sh3bp2 and Syk correlated with liver inflammation and fibrosis (Figure 6G). Altogether, we reported that the sustained activation of 3BP2-SYK pathway mediated by 3BP2 G418R mutant is thus associated with the development of significant fibrotic-hepatitis.
Hepatic Expression of SYK Increased With Metabolic Steatohepatitis in Obese Patients
We examined the relationship between hepatic 3BP2 and SYK expression with MAFLD in human liver biopsies from morbidly obese patients who had undergone bariatric surgery. Patients were classified into 3 groups: without MAFLD, with hepatic steatosis, and with metabolic steatohepatitis and severe steatosis (assessed on the basis of histopathological features; Table 1). Liver mRNA levels of SH3BP2 did not change with obesity and MAFLD (data not shown). By contrast, liver mRNA levels of SYK were strongly up-regulated with metabolic steatohepatitis (Figure 7A). This expression correlated with nonalcoholic fatty liver disease activity score, ALT activity, and hepatic expression of macrophage marker CD68 (Figure 7B). Consistent with our animal studies, the hepatic expression of SYK could be a local marker of hepatic inflammation in MAFLD patients.
NOTE. Without MAFLD: patients with normal liver histology; Steatosis: patients with steatosis; NASH: patients with severe steatosis and nonalcoholic steatohepatitis. Data are expressed as mean ± SD and compared using the non-parametric Mann-Whitney test.
BMI, body mass index; HOMA-IR, homeostatic model assessment for insulin resistance.
Here we report that hepatic SYK and 3BP2 contribute to the onset of hepatic inflammation by acting on both parenchymal and myeloid cells. In concert with liver macrophages, the SYK/3BP2 axis in hepatocytes could initiate the local inflammation and contribute to the significant accumulation of bone marrow–derived macrophages and neutrophils in the liver, a hallmark of metabolic steatohepatitis and severe hepatitis. Indeed, SYK/3BP2 regulates the LPS-TLR4 pathway in both hepatocytes and macrophages. The genetic deletion of 3BP2 or SYK in a context of metabolic steatohepatitis or deletion of SYK in myeloid cells in a context of a pro-inflammatory 3BP2 mutation significantly reduced liver inflammation, injury, and fibrosis (priming and/or collagen deposition). We also report that hepatic SYK is a marker of liver inflammation and correlates with hepatic macrophage recruitment in mouse and human metabolic steatohepatitis. We also provide evidence that an important mechanism by which 3BP2 participates to metabolic steatohepatitis is the regulation of the expression of its signaling partner SYK, leading to exacerbated SYK-mediated inflammatory pathway in liver. Together, these data provide novel evidence that targeting the SYK/3BP2 axis is a potential therapeutic approach in chronic liver diseases.
We first show that the SYK/3BP2 pathway in liver parenchymal cells plays an important role in the expression of inflammatory mediators in response to LPS. Remarkably, 3BP2 targeting lowers SYK expression and decreases the expression of tumor necrosis factor (TNF) α, CCL2, interleukin 1β, and interleukin 6 in response to LPS (Figure 4). Although SYK inhibition slightly decreases the expression of 3BP2, the targeting of SYK (silencing or inhibition) also significantly prevents the up-regulation of these inflammatory mediators upon LPS stimulation (Figure 4). In addition to its detoxifying and metabolic roles, hepatocytes also sense pathogens including bacteria products (for example, LPS) via their membrane and cytoplasmic pattern recognition receptors.
Importantly, it has been reported that deficiency or down-regulation of TLR4 in hepatocytes resolved the hepatic inflammation mediated by HFD and associated with decreased insulin resistance, oxidative stress, and hepatic steatosis.
These studies support the notion that TLR4 signaling in liver parenchymal cells plays a pivotal role during the early stage of MAFLD by initiating local inflammation. Our results emphasize the importance of the hepatocyte SYK/3BP2 signaling pathway to control the LPS/TLR4-mediated responses.
Among the inflammatory mediators, hepatocytes are a source of the chemokine CCL2, whose expression is modulated by the SYK/3BP2 pathway (Figure 4). The CCL2/CCR2 pair is a key player in the recruitment of inflammatory monocytes into the injured liver and drives hepatic fibrosis.
To illustrate this, it has been reported that pharmacologic inhibition of CCL2 or CCR2 in murine models of MAFLD and chronic liver injury (chronic CCl4 treatment) reduced monocyte recruitment into the liver and ameliorated liver inflammation, injury, and fibrosis.
In addition to therapeutic chemokine targeting, we show here that global deficiency of 3BP2 and SYK deletion in myeloid cells both prevent the recruitment of macrophages and neutrophils into the liver associated with reduced hepatic expression of CCL2 but also CD44 and E-selectin (Figures 3 and 5). CD44 and E-selectin (CD62E) are also important drivers of leukocyte recruitment into the inflamed liver,
From our experimental data, we also found that CD44, which interacts with extracellular matrix components including E-selectin, regulates the recruitment of macrophages and neutrophils into the liver. CD44 also regulates the macrophage activation mediated by TLR4 ligands including DAMPs, LPS, and saturated fatty acids in a context of steatohepatitis.
This pro-inflammatory polarization of resident and recruited liver myeloid cells can also be dependent on the SYK/3BP2 pathway. Indeed, we have recently reported that 3BP2 is necessary for LPS-induced activation of signaling pathways in macrophages.
Interestingly, we here report that the prevention of liver inflammation and injury mediated by the targeting of 3BP2 or SYK strongly decrease TREM1/DAP12 expression. Because of the central place of the SYK pathway in both TLR4 and TREM-1 signaling,
this suggests the establishment of an inflammatory loop in which SYK and its partner 3BP2 exacerbate liver inflammation.
The chronicity of liver inflammation is also an important driver of the progression of liver complications such as liver injury and fibrosis. From our different mouse models, the down-regulation of 3BP2 or SYK in myeloid cells reduces both liver injury and fibrosis (fibrosis priming and/or collagen accumulation) along with significant decrease in hepatic inflammation (Figures 3, 5, and 6). In line with these results, SYK deletion in the myeloid compartment confers protection against fibrogenic progression upon TAA-mediated liver injury.
Interestingly, 3BP2 and SYK mutant mice display a reduction in liver expression of TNF-α and markers of oxidative stress (P22phox and Ho-1), coupled with reduced liver injury (Figures 3 and 5). In addition to promoting liver inflammation and associated injury, the SYK pathway can also directly regulate the activity of the HSC. Indeed, it has been shown that SYK inhibitors prevented HSC activation in vitro and in animal models of hepatic fibrosis.
However, the protective effects of SYK targeting seem to be more related to an effect on myeloid cells than on HSC because its deficiency in HSC (LratCreSykflox/flox) did not mitigate the severity of fibrosis mediated by TAA treatment.
Interestingly, we also show that SYK deletion in myeloid cells prevents hepatic inflammation in cherubic mice expressing the 3BP2 G418R mutant (Sh3bp2 G418R knock-in mice, equivalent to the G420R mutation in cherubism patients). Indeed, SYK deletion in these mice mediates a drastic reduction in inflammatory foci number, macrophage recruitment, and hepatic expression of TNF-α, CCL2, and CD44. These protective mechanisms were also associated with restoration of hepatic 3BP2 and SYK expression to normal in Sh3bp2 G418R knock-in mice (Figure 6). Interestingly, both the protective effects of SYK deletion in the accumulation of inflammatory foci and the up-regulation of hepatic TNF-α expression have been recently reported in another mouse model of cherubic mice (Sh3bp2 P416R knock-in mice).
We further show in our Sh3bp2 G418R knock-in mice that the selective knockdown of SYK in myeloid cells is also sufficient to prevent the development of liver fibrosis with the quasi-absence of collagen deposition and changes to TGFβ, TIMP1, and COL1α1 expression (Figure 6). Because the SYK inhibitor treatment in cherubic mice reduces systemic and hepatic inflammation,
the inhibition of SYK activity could also ameliorate liver fibrosis in mouse cherubism.
We also report that the 3BP2 deficiency reduces liver steatosis upon MCDD challenge (Figure 3). Although additional studies to better understand the role of 3BP2 in hepatocyte metabolism and steatosis development are needed, the 3BP2 deficiency is associated with the prevention of decreased peroxisome proliferator activated receptor alpha (PPARα) upon MCDD challenge (Figure 3G). It is well-established that PPARα plays a key role in the regulation of hepatocyte metabolism including fatty acid uptake, beta-oxidation, and triglyceride turnover.
Mice with genetic deletion of PPARα fed MCDD developed more severe steatosis and necroinflammation compared with Wt mice. To the contrary, treatment with PPARα agonist Wy-14643 can prevent and reverse MCDD-induced hepatic steatosis, inflammation, and liver injury in a PPARα-dependent manner, likely via increased fatty acid oxidation.
Interestingly, the treatment of primary hepatocytes with the SYK inhibitor R406 also enhances PPARα and PGC1α expression (Ppara: ×1.8 ± 0.2, P = .029; Pgc1a: ×4.6 ± 1.0, P = .013, n = 4). Altogether, this suggests that SYK and its signaling partner 3BP2 could also be important regulators of hepatocyte metabolism and liver steatosis development.
Finally, we find that the hepatic expression of SYK is up-regulated with metabolic steatohepatitis and correlates with the presence of liver macrophage in obese patients (Figure 7). This is not dependent on hepatic fibrosis because our patients with liver biopsy-proven steatosis or metabolic steatohepatitis have only minimal scarring (F1: portal fibrosis without septa). Interestingly, different groups recently reported an association between the hepatic levels of SYK and liver fibrosis/cirrhosis in patients with hepatitis B virus infection, hepatitis C virus infection, alcohol abuse, cholestasis, and metabolic steatohepatitis.
Altogether, these results could indicate that liver SYK expression is a relevant new biomarker for metabolic steatohepatitis and its progression to cirrhosis.
In summary, the harmful functions of the SYK/3BP2 signaling pathway in chronic liver diseases occur at different stages of the disease (from steatosis to fibrosis) and in different liver cells (hepatocytes, macrophages, HSC, etc). In the early stages by enhancing LPS-TLR4 responses in hepatocytes and resident macrophages, the SYK/3BP2 pathway could initiate the onset of local inflammation and the up-regulation of factors involved in the recruitment of monocytes and neutrophils into the liver. The increase in pro-inflammatory polarization of recruited macrophages by SYK and 3BP2 could fuel a feed-forward mechanism whereby the amplified local inflammation subsequently contributes to liver injury and fibrosis. Inhibition of the SYK/3BP2 pathway in both hepatocytes and liver non-parenchymal cells (macrophages and HSCs) could represent a new therapeutic strategy to dampen inflammation, cell death, and disease progression in chronic liver diseases.
Materials and Methods
Animals and Study Design
Mice had a C57BL/6 background. Sh3bp2-/- (Sh3bp2KO) mice have previously been described.
were generated by the Institut Clinique de la Souris (Illkirch, France, http://www-mci.u-strasbg.fr) by knocking-in a GGA-to-CGA mutation in the Sh3bp2 exon 9, resulting in a Gly418-to-Arg substitution in 3BP2 protein, as previously described.
LysMcre/cre (B6.129P2-Lyz2tm1(cre)lfo/J) mice were provided by the Jackson Laboratory (Bar Harbor, ME). The Syk conditional knockout mouse (Syk cKO) model was generated by the Institut Clinique de la Souris (internal reference IR00002283/K504) by floxing exon 2 (containing the ATG starting site) and exon 3 of the Syk locus. Myeloid Syk conditional mutant mice (mSykKO) were obtained by crossing the Syk cKO strain with the LysMcre/cre transgenic mice
Seven-week-old Wt mice (Janvier Labs, Le Genest-Saint-Isle, France) were acclimated to our animal facilities under a 12-hour/12-hour light/dark cycle at a temperature of 21°C ± 2°C and were fed ad libitum either a HFD (45% fat D12451; Research Diet, New Brunswick, NJ) or control diet (Ctrl D) for 33 weeks.
Averaging 19-week-old male Wt, Sh3bp2KOand SykFlox/FloxLysMcre/cre(mSykKO) C57BL/6 male mice were acclimated to our animal facilities under 12-hour/12-hour light/dark cycle at a temperature of 21°C ± 2°C and were fed ad libitum either MCDD or control diet (Ctrl D) for 2, 4, and 7 weeks as indicated (diets from SSNIFF (MCDD # E15653-94; control diet # E15654-04).
Characterization of Sh3bp2KI/KI mice
Wt (mean age, 29 weeks, n = 6, 16% of male), Sh3bp2KI/KI (mean age, 14 weeks, n = 6, 33% of male), young mSykKOSh3bp2KI/KI (mean age, 16 weeks, n = 15, 58% of male), and old mSykKOSh3bp2KI/KI (mean age, 43 weeks, n = 6, 50% of male) were analyzed. At the end of the corresponding challenge, blood was collected, and mice were immediately killed, after which the liver was removed. One part of the liver was immediately frozen in liquid nitrogen and stored at –80°C until analysis. The second part was fixed in buffered formalin, paraffin-embedded, sectioned, and stained with hematoxylin-eosin and picrosirius red as indicated. The remaining part was used for a flow cytometry analysis. The guidelines of laboratory animal care were followed, and the local ethical committee approved the animal experiments (Comité Institutionel d'Ethique Pour l’Animal de Laboratoire, national agreement no. 28) (NCE/2013-108) (mouse models of MAFLD: APAFIS#5100-2015121110477413 v6) (Sh3bp2 project: APAFIS#6999-2016110416248139 v3) (SYK project: APAFIS#16775-2018090516097783 v2) (Authorization of the C3M animal facility: B06-088-20).
Morbidly obese patients (Nice) (gene expression: n = 33) were recruited through the Department of Digestive Surgery and Liver Transplantation (Archet 2, University Hospital, Nice, France) where they underwent bariatric surgery for their morbid obesity. Bariatric surgery was indicated for these patients in accordance with French guidelines. Exclusion criteria were presence of hepatitis B or hepatitis C infection, excessive alcohol consumption (>20 g/d), or another cause of chronic liver disease as previously described.
The characteristics of the study groups are described in Table 1. Before surgery, fasting blood samples were obtained and used to measure ALT and AST, glucose, insulin, and hemoglobin A1c. Insulin resistance was calculated using the homeostatic model assessment index.
Three histopathological features were semiquantitatively evaluated: grade of steatosis (0, <5%; 1, 5%–30%; 2, >30%–60%; 3, >60%), lobular inflammation (0, no inflammatory foci; 1, <2 inflammatory foci per ×200 field; 2, 2–4 inflammatory foci per ×200 field; 3, >4 inflammatory foci per ×200 field), and hepatocellular ballooning (0, none; 1, few balloon cells; 2, many cells/prominent ballooning). Nonalcoholic fatty liver disease activity score, which is the unweighted sum of scores of steatosis, lobular inflammation, and hepatocellular ballooning, was used to grade activity. Presence of metabolic steatohepatitis was defined as nonalcoholic fatty liver disease activity score 5.
Liver tissue was obtained from 5 lean subjects (5 women; age, 44 ± 9 years; body mass index, 21 ± 1.9 kg/m2) undergoing partial hepatectomy for benign tumors (neighbor tissues from 4 adenoma and 1 focal nodular hyperplasia; CHU Henri Mondor, Department of Pathology, AP-HP - Université Paris Est Créteil, Créteil, France). Three subjects underwent left lobectomy or bisegmentectomy without ischemic preconditioning, and 2 patients underwent right hepatectomy with potential ischemic preconditioning (no data). Liver samples did not display any hepatic steatosis, inflammation, or fibrosis. All subjects gave their informed written consent to participate in this study in accordance with French legislation regarding Ethics and Human Research (Huriet-Serusclat law). The Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale de Nice approved the study (07/04:2003, N° 03.017).
Serum Transaminases Assay and CCL2 Levels
Serum transaminases activity (AST/ALT) was determined using an in vitro test with pyridoxal phosphate activation on Roche/Hitachi cobas c systems (ASTPM, ALTPM; cobas, Meylan, France). Roche/Hitachi cobas c systems automatically calculate the concentration of each sample. Serum levels of CCL2 were evaluated with enzyme-linked immunosorbent assay (#DY479; Bio-Techne, Minneapolis, MN), as described in the manufacturer’s instructions (dilution to the fifth).
Flow Cytometric Analysis
A fraction of the mouse liver was crushed on a 100 μm cell strainer and washed with RPMI 1640 medium. The cellular suspension was centrifuged at 630g for 5 minutes at 4°C. The cell pellet was resuspended and centrifuged on a density cushion of Percoll (40% and 70%) at 780g for 20 minutes at room temperature. The non-parenchymal cell fraction between 40% and 70 % Percoll was collected, centrifuged at 630g for 5 minutes at 4°C, and resuspended in phosphate-buffered saline containing 3% fetal bovine serum and 5 mmol/L EDTA. Cell suspensions were incubated with purified anti-FcgRII/III monoclonal antibody blocking antibody before being stained at 4°C for 10 minutes to block Fc receptors. Cells were then stained with fluorochrome-coupled antibodies for 30 minutes at 4°C against CD45 (clone 30F11), F4/80 (clone BM8), CD11b (clone M1/70), Ly6G (clone 1A8), or Ly6C (clone AL21). All antibodies were purchased from BD Biosciences (Franklin Lakes, NJ), eBioscience (San Diego, CA), and Bio Rad ABD Serotec (Oxford, United Kingdom). For intracellular staining, cells were fixed and permeabilized using the BD Cytofix/Cytoperm (Ref 554714) according to the manufacturer’s instructions. Samples were first stained for cell surface markers as described above and then for intracellular markers using anti-SYK antibodies (clone D3Z1E XP, PE conjugate; Cell Signaling Technology, Danvers, MA). Samples were acquired using a fluorescence activated cell sorting (FACS) Canto II flow cytometer, and the data were analyzed using the FACS Diva software version 8.0.1 (BD Biosciences) or FlowJo V10 software (Treestar, Ashland, OR).
Hepatic Parenchymal and Non-parenchymal Cell Isolation
Mouse hepatocytes were isolated with a two-step collagenase procedure. Briefly, mouse livers were perfused with HEPES buffer containing 8 g/L NaCl, 33 mg/L Na2HPO4, 200 mg/L KCl, and 2.38 g/L HEPES, pH 7.5, supplemented with 0.5 mmol/L EGTA for 3 minutes at 3 mL/min, then with HEPES buffer for 3 minutes at 3 mL/min, and finally with HEPES buffer supplemented with 1.5 g/L CaCl2 and 0.026% collagenase type IV (Sigma-Aldrich; C5138, Saint-Quentin-Fallavier, France) for 7 minutes at 3 mL/min. Livers were then carefully removed and minced in Williams’ E medium (Thermo Fisher Scientific Inc, Waltham, MA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific Inc), 100 units/mL penicillin, 100 mg/mL streptomycin, 2 mmol/L L-glutamine, and 0.02 UI/mL insulin (Umulin; Lilly France, Neuilly-sur-Seine, France). The cell suspension was then filtered (100 μm), and hepatocytes were pelleted after 2 centrifugations at 50g for 5 minutes. The supernatants were collected at each step, which contained the non-parenchymal cell fraction. Pelleted hepatocytes were resuspended in 39% Percoll solution, centrifuged at 50g for 5 minutes at room temperature, and collected at the bottom of the tube. Viability was evaluated by trypan blue exclusion (Sigma-Aldrich).
Primary Hepatocyte Treatments
Down-regulation of 3BP2 and SYK was achieved using Lipofectamine RNAiMAX technologies (MSS216132, Mouse Sh3bp2; HSS238073, mouse Syk; Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. After 48-hour transfection, cells were incubated with Williams’ E medium supplemented with 0.5% bovine serum albumin for 1 hour and then treated with LPS (50 μg/mL) for 6 hours. For the R406 inhibitor, cells were incubated with (1) Williams’ E medium supplemented with 0.5% bovine serum albumin overnight and then successively treated with R406 (10 μmol/L) for 2 hours and then LPS (50 μg/mL) for 5 min as indicated (Western blot analysis) and (2) Williams’ E medium supplemented with 0.5% bovine serum albumin without or with R406 (10 μmol/L) for 1 hour and then treated with LPS (50 μg/mL) for 6 hours (gene expression analysis).
Total liver RNA was extracted using the RNeasy Mini Kit (74104; Qiagen, Hilden, Germany) and treated with Turbo DNA-free DNase (AM 1907, Thermo Fisher Scientific Inc) following the manufacturer’s protocol. The quantity and quality of the RNA samples were determined using the Agilent 2100 Bioanalyzer with RNA 6000 Nano Kit (5067-1511; Agilent Technologies, Santa Clara, CA). Total RNA (1 μg) was reverse transcribed with the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific Inc). Real-time quantitative polymerase chain reaction (PCR) was performed in duplicate for each sample using the StepOne Plus Real-Time PCR System (Thermo Fisher Scientific Inc) as previously described.
The proteins were probed with Anti-SYK (#2712; Cell Signaling Technology), Anti-CD44 (AF6127; Bio-Techne), Anti-phospho SYK (Tyr525/526) (#2710; Cell Signaling Technology), Anti-p44/42 MAPK (#4695; Cell Signaling Technology), Anti-phospho-p44/42 MAPK (Thr202/Tyr204) (#9101; Cell Signaling Technology), and anti-HSP60 (K-19) (sc-1722; Santa Cruz Biotechnology, Dallas, TX) antibodies at 1 μg/mL (Cell Signaling Technology) or at 1/1000 (Santa Cruz Biotechnology) as indicated.
Statistical significance between 2 human or mouse study groups was determined using the non-parametric Mann-Whitney test. Data from cell lines were analyzed using the Student t test. Spearman’s correlation test was used for the correlative analysis. Results were considered significant for P < .05.
The authors thank the INSERM U1065 animal facility staff and Dr V. Corcelle, the INSERM U1065 flow cytometry platform, Dr Jeanne Tran-Van-Nhieu (HU Henri Mondor, Department of Pathology, AP-HP - Université Paris Est Créteil, Créteil, France) for providing the liver tissues from lean subjects, and Professor Robert Rottapel (Princess Margaret Cancer Center, University of Toronto, Toronto, Canada) for providing the Sh3bp2 KO mice.
Conflicts of interest The authors disclose no conflicts.
Funding Supported by grants from INSERM (France), charities (Association Française pour l’Etude du Foie (AFEF) to PG, Société Francophone du Diabète (SFD) to PG, SFD/Roche Pharma to PG, and SFD and LifeScan to BBM. This work was also funded by the French Government (National Research Agency, ANR): #ANR-18-CE14-0019-01, #ANR-18-CE14-0019-02, #ANR-18-CE14-0022-01, #ANR-19-CE14-0044-01, and through the "Investments for the Future" LABEX SIGNALIFE (#ANR-11-LABX-0028-01) and the UCAJEDI Investments in the Future project (#ANR-15-IDEX-01). EV was supported by a PhD grant from the Fondation pour la Recherche Médicale and LL and MB by the ANR (#ANR-18-CE14-0019-01 and #ANR-18-CE14-0019-02, respectively). MB is currently supported by a doctoral fellowship from French Ministry of Research.