Fc gamma RIIb expression levels in human liver sinusoidal endothelial cells during progression of non-alcoholic fatty liver disease

Liver sinusoidal endothelial cells (LSECs) play a pivotal role in hepatic function and homeostasis. LSEC dysfunction has been recognized to be closely involved in various liver diseases, including non-alcoholic steatohepatitis (NASH), but not much is known about the fate of the scavenger receptors in LSECs during NASH. Fc gamma receptor IIb (FcγRIIb), known as a scavenger receptor, contributes to receptor-mediated endocytosis and immune complexes clearance. In this study, to elucidate the fate of FcγRIIb in the progression of non-alcoholic fatty liver disease (NAFLD), we examined FcγRIIb levels in NAFLD biopsy specimens by immunohistochemistry, and investigated their correlation with the exacerbation of biological indexes and clinicopathological scores of NASH. The FcγRIIb expression levels indicated significant negative correlations with serum levels of blood lipids (triglyceride, total cholesterol, high-density lipoprotein-cholesterol), type 4 collagen and hyaluronic acid, which are involved in hepatic lipid metabolism disorder, fibrosis, and inflammation, respectively. However, there was no significant difference of FcγRIIb expression levels among the pathological grades of NAFLD. During NAFLD progression, inflammation and fibrosis may influence the expression of FcγRIIb and their scavenger functions to maintain hepatic homeostasis.


Introduction
Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome and is categorized into non-alcoholic fatty liver (NAFL), with simple fatty liver, and non-alcoholic steatohepatitis (NASH), with inflammation and fibrosis. NAFL is a benign disease, but NASH can lead to severe chronic hepatic diseases, such as advanced fibrosis [1], cirrhosis [2], and hepatocellular carcinoma (HCC) [3,4]. In 1998, a two-hit model [5] was proposed as a PLOS ONE | https://doi.org/10.1371/journal.pone.0211543 January 29, 2019 1 / 13 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 clearance of small immune complexes in hepatic sinusoids [27].Moreover, the expression of FcγRIIb in endothelial cells has been suggested to be associated with hepatic and cardiovascular diseases [28,29]. As a ligand for FcγRIIb, fibrinogen-like protein 2 (FGL2), which is known to have immunomodulatory function, has been reported to have an affinity for FcγRIIb [30]. Interestingly, the plasma level of FGL2 is higher in patients with NAFLD [31] and liver cirrhosis [32]. These scavenger receptors on LSECs share the clearance task, and their downregulation may lead to increased blood concentrations of their specific ligands: hyaluronan, oxLDL, type IV collagen (IVCol), IgG and perhaps FGL2. However, not much is known about the fate of the endocytic receptors during human NAFLD. In the present study, we aimed to elucidate the correlations of FcγRIIb expression levels in LSECs of NAFL and NASH patients with clinical biochemical and pathological phenotypes. Furthermore, we examined another scavenger receptor, stabilin-2 expression, in LSECs of NAFLD patients.

Patients
We analyzed 26 patients with NAFLD who had undergone liver biopsy for definitive diagnosis at Jikei University Hospital between 2011 and 2015 and were histologically diagnosed with NAFL or NASH. Fasting blood samples were collected from patients before liver biopsy. Patients who had been clinically diagnosed with other liver diseases or had consumed more than 20 g of alcohol per day were excluded from this study. Two patients with hepatitis B surface antigen (HBsAg) positive were included. This study was carried out with the approval of the Ethics Committee of the Jikei University School of Medicine, and the Ethics Committee of Ochanomizu University. Informed consent was obtained from all patients.

Routine histological and biochemical analysis
For routine histology, liver biopsy samples were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) and embedded in paraffin blocks. Sections, 3 μm thick, were stained with hematoxylin-eosin (H-E) and Masson's trichrome for routine examination. The pathological investigation was performed by a board-certified pathologist of the Japanese Society of Pathology, and the histological grade of steatosis, inflammation, ballooning, fibrosis stage and NAFLD activity score (NAS) were determined using NASH Clinical Research Network criteria proposed by Kleiner et al [33].

Immunohistochemistry and morphometry
Immunohistochemical staining of FcγRIIb was performed on liver biopsy samples. The paraffin sections were deparaffinized and treated with 10 mM citric buffer (pH6.0) for 10 min at 95˚C for antigen unmasking. Endogenous peroxidase activity was exhausted by 3% hydrogen peroxide. The sections were then pre-incubated with 5% normal goat serum-1%BSA in PBS, and incubated with a primary rabbit anti-human FcγRIIb antiserum (Peptide Institute, Osaka, JAPAN) or a rabbit anti-human stabilin 2 antiserum (abcam). Secondary antiserum from Histo Fine simple stain (MAX-PO Multi, Nichirei, Tokyo, JAPAN) was used, and detected with diaminobenzidine (DAB) and then counterstained with hematoxylin. The FcγRIIb expression level was morphometrically quantified on microphotographs taken with a 20 x objective lens on a BZ-X710 microscope and Hybrid Cell Count software (KEYENCE, Tokyo, JAPAN) as follows. For all specimens, as much fields as possible of the hepatic parenchymal area was photographed (12-35 fields per patient). The procedures to extract the whole tissue area and FcγRIIb positive area were determined, respectively, as shown in S1 Fig. Then the areas were automatically measured under the same conditions. To avoid observer-related bias, morphological observation and evaluation were blindly performed, i.e. the observer was unaware of the diagnosis and pathological scores.

Statistical analyses
Statistical analyses were performed using SPSS Statistics 23 software (IBM, NY, USA). Comparisons of FcγRIIb expression levels with sex, diagnosis, and clinicopathological scores were performed by the Mann-Whitney or the Kruskal-Wallis nonparametric test. A P-value <0.05 was considered to be significant. Investigation of associations between FcγRIIb expression and body mass index (BMI) or serum biochemical values were performed by the Pearson correlation coefficient test. R-values(±) >0.7, >0.4, >0.3 were considered to have a strong, medium, and small correlation, respectively. Table 1 summarized the age, sex, BMI, and pathological characteristics of the 26 patients analyzed in this study. The number of patients was 13 for both males and females, and the mean ages for males and females were 48.5 and 56.6 years, respectively. With the biopsy-proven pathological examination, two patients were diagnosed as NAFL and 24 patients as NASH. In Table 1, the two patients diagnosed with NAFL are listed from the top, followed by the 24 diagnosed as NASH, in ascending order of NAS and then ascending ALT value. This suggests that the serum ALT level is not a sufficient indicator of NASH progression, though it is used as one of the screening criterions for NAFLD.

FcγRIIb distribution
Representative light microscopic images of patients who were diagnosed as NASH (a: NAS, 2; b: NAS, 8) are shown in Fig 1. As it is well known, the FcγRIIb signals were found only in LSECs [25]. In a case with low NAS (= 2), FcγRIIb signals were strong and surrounded the entire circumference of the sinusoids (Fig 1A), whereas in a case with the highest NAS (= 8) among the cases we analyzed, FcγRIIb signals were very weak and were only irregularly expressed in LSECs ( Fig 1B).

FcγRIIb expression levels
For morphometric analysis, the FcγRIIb expression level was calculated as the ratio of the positive area to whole tissue area, using the morphometric procedure described in the materials and methods section (S1 Table). We also analyzed the FcγRIIb expression level as the intensity of positive signals (S1 Table), and this showed a similar tendency to the measurement results stated above. Hereinafter, the ratio of the FcγRIIb positive area is denoted as the FcγRIIb expression level in this paper.

Correlation with biological indexes and serum biochemical markers
The correlations of FcγRIIb expression levels with BMI, Plt, and serum biochemical markers are shown in Fig 2. The Pearson correlation test indicated that there was no correlation between FcγRIIb levels and BMI. The regression lines of serum AST, ALT, ALP, and γ-GTP showed negative slopes, but there was no significant correlation. The regression lines showed positive slopes for TB, Alb, and Plt, which decreased with liver disease deterioration, though there was no significant correlation. On the other hand, in the factors related to lipid dynamics, it was shown that the FcγRIIb levels had a small negative correlation (R = -0.357, -0.300) with TG and TC, and a medium negative correlation (R = -0.409) with HDL-C. With LDL-C, there was no significant correlation, but the regression line also showed a negative slope, suggesting that it is similar to other lipid-related factors. In FBS and HbA1c, there was no correlation between FcγRIIb levels, meaning there was no correlation of FcγRIIb levels with shortand medium-term blood glucose levels. Both IVCol and hyaluronan, which increase with progression of hepatic fibrosis, showed medium negative correlation (R = -0.490, -0.525).

Comparisons of diagnosis, sex, and pathological scores
The comparisons of diagnosis, sex, and pathological values are shown in Fig 3. FcγRIIb levels were significantly higher in females compared with in males (Mann-Whitney test, P = 0.009), whereas there was no significant difference in FcγRIIb expression levels of the liver biopsy specimen among the pathological grades of NAFLD. The mean FcγRIIb levels tended to be higher with the progression of steatosis, but no significant difference was observed. FcγRIIb levels became lower with the progression of inflammation and ballooning. Especially, it was significantly lower in patients with ballooning stage 3 as compared with those in stage 2 (Kruskal-Wallis test, P = 0.022). Interestingly, in the comparison between the fibrosis stages, which are closely related to the exacerbation of NASH, FcγRIIb levels tended to be higher at the early stage of fibrosis and became slightly lower at the 1B, 2 and 3 fibrosis stages. This is consistent with the findings that there were medium negative correlations between FcγRIIb levels and IVCol or hyaluronan. It is also suggested that the decline of FcγRIIb levels in the progression of NAFLD correlates with the exacerbation of fibrosis.

Stabilin-2 distribution
High amounts of HDL-C, IVCol, and hyaluronan in peripheral blood coincided with low expression of FcγRIIb. Interestingly, some of these molecules are ligands for LSEC receptors other than FcγRIIb. Then, we examined the distribution of stabilin-2, a hyaluronan receptor in liver from patients with NASH. Under the microscopic observation, stabilin-2 positive LSECs tended to be more frequent in patients with low level of blood hyaluronan (S2 Fig), but nonspecific signals were detected in some biopsy specimen. Thus, we were unable to elucidate the expression levels of stabilin-2 during NASH.

Discussion
In this study, we determined the expression levels of FcγRIIb, which is a scavenger receptor on the LSEC, in human NAFL and NASH biopsy specimens, and elucidated their correlations with individual biological indexes and pathological grades. The FcγRIIb expression levels indicated significant negative correlations with serum TG, TC, HDL-C, VICol, and hyaluronan concentrations, respectively. Capillarization of LSECs is one of the basic pathological features of hepatic cirrhosis and chronic hepatitis [34]. LSECs of cirrhosis patients produce IVCol, fibronectin, and laminin, lose their characteristic fenestrations, and the expression levels of LSEC receptors LYVE-1, FcγRIIb and stabilin are decreased [9]. The microenvironment caused by the transformed LSECs is involved in the onset and progression of hepatic fibrosis, and instructively dictates hepatic regeneration and healing [35,36]. In a rodent NASH model, capillarization arises at an early stage of NASH and exacerbates with NASH progression [11].
In human LSECs, the distribution pattern of FcγRIIb has recently been shown in intrahepatic lobules of normal livers [37]. In relation to hepatic diseases, the expression levels of LSEC markers, including FcγRIIb, of HCC patients were sequentially lost during tumor progression [22], and it disappeared in 63% of cases in the peritumoral tissue samples of a tissue microarray of HCC [7].
Interestingly, several of the serum components that showed negative correlations with the expression of FcγRIIb are ligands for LSEC receptors: hyaluronan, which is the first molecule shown to be endocytosed via stabilin in LSECs, HDL-C, which binds to a receptor in LSEC, and IVCol, which probably binds to the Mannose receptor in LSEC [17,38]. These serum components are well known to increase with the progression of NASH. The increase of individual serum components indicates the progression of hepatic injury in the NASH patients and probably reflects the functional status of scavenger receptors expressed in LSECs. Our results indicate that the decrease of FcγRIIb expression may also be closely related to its scavenger function, involving several LSEC receptors.
Hyaluronic acid synthesized by mesenchymal cells is generally eliminated by LSECs. Serum hyaluronic acid contents increase in alcoholic liver disease, chronic hepatitis C, and NAFLD [39][40][41]. Recently, it was demonstrated that the index of hyaluronic acid in patients can accurately predict the survival rate of liver diseases of varying severity [20].Here, we attempted to elucidate the expression of stabilin-2 proteins, one of the hyaluronan receptors, in liver biopsy tissue of NASH patients, but we were unable to elucidate its relationship with NASH progression. However, there are several reports indicating the relationship between stalibin-2 and FcγRIIb expression in HCC. Geraud and colleagues examined the expression of FcγRIIb and stabilin-2 by using a tissue microarray of HCC patients and revealed the loss of both receptor proteins in the majority of tumorous tissues [22]. In the peri-tumorous liver tissues, loss of stabilin-2 expression was significantly less likely to occur (38%) than loss of FcγRIIb expression (63%), and the loss of stabilin-2 in peri-tumorous tissues was associated with survival. They described that loss of stabilin-2 may enhance survival by preventing endothelial-tumor cell adhesive interactions and microvascular invasion. Schledzewski and colleagues revealed that serum hyaluronic acid levels were elevated in stabilin-1 and stabilin-2 double KO mice, but the liver showed only mild perisinusoidal fibrosis without dysfunction [42]. They also indicated that the expression levels of FcγRIIb and LYVE-1 in double KO mice were not different from WT mice. From these previous observations, the attenuation pattern of individual LSEC receptors appears to vary depending on the type of liver disease and the degree of progression. The attenuation of FcγRIIb in LSECs seems to be involved in inflammation and fibrosis in NASH patients, at the least. Platelets are also closely involved in inflammation and fibrosis, and the platelet count of NASH patients decreases with progressive fibrosis [43]. Platelets mediate binding of endothelial cells to T cells during hepatic injury [44], and promote various processes such as inflammation, fibrosis and liver repair by release of biological substances [45]. In this study, although there was no correlation between platelet count and FcγRIIb expression levels, the regression line indicates a positive slope. In other words, patients with low FcγRIIb expression levels exhibit a tendency to have a low platelet count (Fig 2), a high inflammation grade, or a high fibrosis stage (Fig 3). FcγRIIb expression levels may be related to the enhancement of inflammation and fibrosis in NASH patients.
The phenotypic changes of LSECs associated with various liver diseases have been clarified in detail. In the LSEC studies, contradictory results may be shown depending on species differences, phenotypic markers used for identification, and culture conditions [46]. Here, we showed that FcγRIIb expression may coincide with scavenger functions of several LSEC ligands related to inflammation, hepatic fibrosis, and hepatic lipid metabolism. These findings are useful to clarify details of transformation and dysfunction in LSECs during NAFLD. Further investigations are required to assess the significance and to address the role of scavenger receptors, including FcγRIIb, in LSECs during NASH progression.

Conclusions
In summary, the present study contributes to support the notion that the changes in LSEC phenotype are one of crucial events in human NASH progression. Our results suggest that progression of inflammation, hepatic fibrosis, and hepatic lipid metabolism disorder during NASH may influence the expression and scavenger functions of FcγRIIb in LSECs. Immunohistochmical signals of stabilin-2 in LSECs are detected (arrowheads). Unfortunately, nonspecific signals are also detected in b. Nuclei were counterstained with hematoxylin (blue). Scale bars represent 100 μm. (TIF) S1 Table. FcgR2b expression level. For the 26 patients, the two patients diagnosed with NAFL are listed from the top, followed by the 24 diagnosed as NASH, listed in ascending order of NAS and then ascending ALT value. The order of patients in this table is the same as Table 1. A comparison with the Fibrosis score and FcgR2b expression level is stated in Discussion. � FcgR2b positive area / whole tissue area. �� Intensity of FcgR2b positive area / whole tissue area. (XLSX)