Fc Gamma RIIb Expression Levels in Patients With Chronic Hepatitis B Virus Infection

Background: Fc gamma receptor IIb (FcγRIIb), is an important inhibitory receptor which plays a vital role in regulating various immunoresponse processes and pathogenesis of many infectious disease. The purpose of our research is to show the FcγRIIb expression in serum and liver biopsy specimen of hepatitis B virus (HBV) suffers and explore its association with chronic HBV infection. Methods: ELISA assay was adopted to identify the serum FcγRIIb levels in 119 HBV suffers and 24 healthy controls. The immunohistochemical method was then employed to identify FcγRIIb expression in the biopsy specimens of patients with chronic hepatitis B (CHB). The integrated optical density (IOD) value was measured to represent FcγRIIb expression levels. Results: The serum FcγRIIb levels were decreased in CHB patients compared to controls (P < 0.001). FcγRIIb levels in CHB patient group were remarkably lower than in HBV carrier group (P< 0.001). In addition, the serum FcγRIIb levels were negatively associated with AST and ALT (r=-0.3936(cid:0)P=0.0063; r=-0.3459(cid:0)P =0.0097 respectively). The IOD values of FcγRIIb expression in moderate CHB and severity CHB group were signicantly lower (P=0.006, P<0.001, respectively). The FcγRIIb level tended to be lower with the pathological changes of hepatitis. Further, the correlation analysis revealed FcγRIIb had negative correlations with AST and ALT (r=-0.688(cid:0)P=0.0016; r=-0.686(cid:0)P=0.0017 respectively) but positively associated with platelet counts (r=0.6464(cid:0)P=0.0038). Conclusions: FcγRIIb levels are signicantly related to chronic HBV infection and progression of CHB. The change of FcγRIIb may affect the progression of liver inammation and brosis in CHB patients


Background
Chronic hepatitis B virus (HBV) infection has always been a prime public health challenge across the world. According to the WHO global hepatitis report, the quantity number of HBV cases in 2015 was 257 million, and the infection rate was 3.5% [1].Chronic HBV infection is a primary cause of liver cirrhosis and hepatocellular carcinoma (HCC). Moreover, it accounts for 786,000 HBV associated mortality annually, making it the tenth major cause of mortality death across the globe [2]. HBV can stimulate both innate and adaptive immunoresponse. The suppression of adaptive immunity is known as a crucial factor in maintaining HBV persistence infection through inhibition the killing effect of CD8 + T lymphatic cell [3,4]. Innate immune response has been con rmed involved in viral clearance, and interferon, a key effector of innate immunity, have long been used to treat HBV infections [5].
FcγRIIb is the sole inhibitory FcγR, which can negatively modulate downstream signaling pathway [6]. It has three common isoforms, b1, b2 and b3, and b3 is the only soluble isoform that lacks of transmembrane domain and rst cytoplasmic domain [7,8]. FcγRIIb is expressed on the surface of nearly all leukocytes as well as B cell and involved in regulating these cell mediated immunity [9,10]. Recent study shows that FcγRIIb also can be expressed in memory CD8 + T cells and regulate their activation and survival [11]. Another prominent feature of FcγRIIb is that it can be expressed on non-hematopoietic cells, such as airway smooth muscle cells [12] or liver sinusoidal endothelial cells(LESCs) [13]. FcγRIIb expressed in LESCs is responsible for removal of small immunocomplex in hepatic sinusoids [14], which is essential to sustain liver immunity homeostasis. Ishikawa et al conducted a research on nonalcoholic fatty liver disease biopsy samples that progression of liver in ammation and brosis was associated with the declined expression of FcγRIIb in LSECs, and may in uence its scavenger functions [15]. A study focused on HCC discovered that a decreased expression of FcγRIIb was more likely to have a higher cancer grade [16]. Moreover, brinogen like protein 2 (FGL2) is a member of the brinogen superfamily and secretes by the regulatory T (Treg) cells [17]. It has been reported capable of binding to FcγRIIb and exerting an immunosuppressive effect. [18].
FcγRIIb is a regulating molecule involved in various processes of both the innate and adaptive immune, such as antigen presentation, FcγR-mediated cellular activation, and apoptosis. It also regulates various signaling pathways such as TLR [19]and MAPK [20]. FcγRIIb is an important checkpoint of humoral tolerance in immune system of human. The hypofunction of FcγRIIb may result in activation of immunoresponse signaling and virus clearance [17]. Liver injury occurrence in HBV infection is strongly dependent on the host immune responses to the virus and is often accompanied by transaminases elevation. Thus FcγRIIb might be pivotal for immune reactions of the interaction between HBV and the host. Moreover, as a ligand of FcγRIIb, FGL2 participates in the immune response of persistent virus infections via regulating the FcγRIIb immune inhibitory pathway. Expression of FGL2 was associated with HBV infection and related to the clinical result of HBV associated liver diseases [21]. This study measured FcγRIIb expression levels in the HBV sufferers and analyzed the correlation with clinical parameters in attempt to elucidate the role of FcγRIIb in the immunoresponse process of chronic HBV infection.

Patients of serum samples
The serum samples come from 119 chronic HBV sufferers who have not taken any antiviral drugs, and 24 healthy individuals who attended physical health examination in the First Hospital of Jilin University between June 2019 and August 2020. According to the Guidelines for the Prevention and Treatment of Chronic Hepatitis B (2019 Edition) issued by the Chinese Medical Association, the patients were divided into HBV carriers and CHB patients. Patients who had other liver diseases, such as alcoholic hepatitis, fatty liver disease, acute or chronic infectious diseases, or autoimmune diseases were excluded. The basic and clinical data of patients and healthy controls were represented in Table 1.

ELISA experiment
The ELISA experiments (soluble FcγRIIb: Jingmei, Jiangsu, China) were conducted according to manufacturers' protocols. Brie y, incubating the serum samples in 96-well plates coated with primary antibodies. After reaction, washing the plate and adding horseradish peroxidase labeled secondary antibodies into each well of the plate. And after reaction, washing again. Detecting protein mass by secondary antibody speci c development reagents. Optical density of each well was identi ed via microplate reading device (Multiskan Sky, Thermo Fisher, USA).

Immunohistochemistry and morphometry
Immunohistochemical staining of FcγRIIb was performed on liver tissue specimens. The para n sections were de-waxed to water. Undergoing antigen unmasking with 10 mM citric buffering solution (PH6.0) for ten minutes at 95˚C. Adding 3% hydrogen peroxide into each section to block the activity of endogenous peroxidase. After washing with PBS, incubating the sections with a primary rabbit anti-human FcγRIIb antiserum (Abcam, USA). After washing with PBS, then incubating the sections with enzymes second anti-rabbit antibody. Detecting with diaminobenzidine and then counterstaining with hematoxylin. The integrated optical density (IOD) values of the sample sections in every group were determined via Image-Pro Plus 6.0 software after tissue images program posterior to the collection of the sample images with a light microscopical device (× 40). Selecting 5 elds of vision stochastically to identify the positive IOD values, and then calculating the average IOD values, of which were deemed as the relative expression of FcγRIIb.
The results of HBV-DNA were converted to log 10 IU/ml.

Statistics
All statistic assays were performed with GraphPad Prism 8. The experimental data are expressed as means ± standard deviation (SD). Comparisons of FcγRIIb expression levels were performed by the Kruskal-Wallis nonparametric test. Comparisons of IOD value of FcγRIIb expression were performed by one-way ANOVA test. Correlations were calculated using Spearman correlation analysis. A two-tailed pvalue < 0.05 was of statistical signi cance.

Result
Serum expression levels of FcγRIIb in chronic HBV sufferers Participate characteristics Table 1 summarized the information of participates enrolled in this study including age, gender and clinical parameters. In total, most of participates were men. No differences existed between two groups regarding the age. In HBV carrier group, there were more patients with positive hepatitis B e antigen (HBeAg). In CHB group, more patients were HBeAg negative while anti-HBeAg were positive.

FcγRIIb expression level in serum
The values of serum FcγRIIb were 201.9 ± 15.19ng/ml in healthy control group, 183.9 ± 33.47 ng/ml in HBV carrier group and 141.0 ± 37.14 ng/ml in CHB group. Compared with healthy control group, there was no signi cant difference of serum FcγRIIb level in HBV carrier group (P = 0.123), while FcγRIIb level in CHB patients was signi cantly lower (P < 0.001) ( Figure. 1A). The serum FcγRIIb level in CHB patient group were signi cantly lower than in HBV carrier group (P < 0.001). Among all the HBV sufferers, the serum FcγRIIb level of positive HBeAg sufferers was statistically signi cantly lower than that of negative HBeAg sufferers (P = 0.007) (Fig. 1B).

Correlations with biochemical parameters
The correlations of FcγRIIb expression levels with sera biochemistry in CHB patients are shown in Fig. 2.  Table 2 summarized the information of 18 participates enrolled in the study including age, gender and clinical parameters. With the biopsy proven pathologically inspection, ve patients were con rmed as severity CHB, four patients were con rmed as mediate CHB and ve patients as mild CHB. Four patients with normal liver tissue were control group.

FcγRIIb expression level in liver tissue
Typical optical microscope images of patients in different group are presnented in Fig. 3. The FcγRIIb signals were discovered merely in LSECs [13]. In the study group, the IOD values of FcγRIIb were 23696.08 ± 3847.33 in control group, 21392.93 ± 7536.20 in mild CHB group, 10287.25 ± 2878.82 in mediate CHB group and 5214.78 ± 1071.27 in severity CHB group. Compared with control group, no signi cant difference FcγRIIb level existed in mild CHB group, while FcγRIIb level in moderate CHB and severity CHB group were signi cantly lower (P = 0.006, P < 0.001, respectively). The FcγRIIb expression level in severity CHB and moderate CHB group were also lower than in mild CHB group (P < 0.001, P = 0.018, respectively). In other word, the FcγRIIb level tended to be lower with the pathological changes of hepatitis. (Fig. 4) Correlation with biochemical parameters and pathological stage. The correlations of FcγRIIb expression level of liver biopsy specimen with biochemical parameters and pathological stage are shown in Fig. 5. A correlation analysis showed that FcγRIIb had a negative relationship with AST (r=-0.688,P = 0.0016) and ALT (r=-0.686 P = 0.0017), and a positive relationship with platelets (r = 0.6464 P = 0.0038). The regressive lines of sera ALP, γ-GTP and HBV-DNA presented negative slopes, whereas no signi cant association existed. The regressive line presented positive slopes for ALB, though there was no signi cant correlation. The comparison of the pathological stage revealed that FcγRIIb had a signi cantly negative correlation with in ammation grade (r=-0.913 P < 0.001)and brosis state(r=-0.875 P < 0.001). It could be inferred that the decline of FcγRIIb levels in the development of chronic hepatitis is related to liver in ammation and brosis getting worse (Table 3).

Discussion
FcγRIIb is an inhibitory receptor, works as a regulation molecule of the immunosystem, and is vital for the progression of various autoimmune diseases and infectious diseases. In our previous study, we performed qRT-PCR assay to identify FcγRIIb mRNA expression in HBV sufferers [22]. The study showed that FcγRIIb expression was higher in the HBV carrier group in contrast to the CHB group. And we speculate that during the immune tolerance stage of HBV infection FcγRIIb expression played the role of immunosuppression. Thus declining in FcγRIIb expression in patients with CHB leads to a decreasing of the inhibition effect immune response and results in host immune activation. In this study, we discovered FcγRIIb levels of serum samples were signi cantly decreased compared to HBV carriers and healthy controls. It further suggested FcγRIIb could be a potential target of the immune response during chronic HBV infection.
During the early stage of virus infection, the innate immunity suppresses replicative activities and transmission, whereas the adaptive immunity mainly affects the virus removal during the late of infection period [23].HBV can be recognized by related receptors and trigger antiviral innate immunity. Then the envelope antigen and nucleocapsid antigen of the virus can stimulate the adaptive immune response, with the help of CD4 + T cell, antibody can be produced and combined with HBV to form an immune complex that can be phagocytized by monocyte and macrophages. FcγRIIb can regulate expression and function of immune cells in innate immunity, such as maturation and antigen presentation ability of dendritic cells and the phagocytic ability of monocytes and macrophages [6].A study has shown HBsAgspeci c B cells in CHB patients exhibited a rised expression of suppressive receptors like PD-1 and FcγRIIb to suppress B cell activation and anti-viral immunity [24]. The clearance of persistent HBV infection relies on the cytotoxic immune response of HBV-speci city CD8 + T cell. CD8 + T cells can induce cytotoxic reaction by recognizing antigen presented by APC and MHC-antigen, which can promote apoptosis of HBV infected hepatocytes [25]. However, the persistent HBV infection may result from HBV-speci c CD8 + T cells function impairment, in other words, the majority of HBV-speci city CD8 + T cells are able to activated but poorly proliferate and functionally exhausted [26,27]. The mechanism of this functional decline is very complex, such as the expression of suppressive receptors and relevant ligands on hepatocytes increased, like PD-1 and PD-L1 [28,29]. FcγRIIb can help dendrite cells acquire antigen from immune complexes and present antigen to Ag-speci city CD4 + and CD8 + T cell to induce robust immune reaction [24]. The vaccine chimegen HBV is a chimera immune therapy protein for treating chronic hepatitis B,and it is targeted dendritic cell. It could bind to immature dendrite cells and internalize FcγRII and mannose receptor (CD206) by endocytosis, which could lead to elevated MHC I and MHC II surface expression, induce T cell proliferation and restore HBV speci c T cell function [30]. Treg cells are speci c subgroup of CD4 + T cells which are crucial for constructing and sustaining immunotolerance. Tregs from CHB patients can suppress HBV speci city T cell immune response and result in a chronic, persistent HBV infection [31].Moreover, FcγRIIb can bind to FGL2 working as an effector molecule of Treg cells and has immunoregulatory activity [32]. Hoang and his colleagues discovered that FGL2 levels were diseases remarkably increased in patients with cirrhosis and HCC in contrast to control group [21]. Therefore, FcγRIIb is vital for immune responses and is involved in the pathogenesistic mechanism of virus infections, especially during HBV infection.
The levels of AST and ALT could denote the severe degree of liver damage during HBV infection, and the elevated liver enzyme can be viewed as a biomarker of immunity stimulation. In the early clinical studies, researchers found that patients with elevated ALT had a higher rate of HBV clearance than patients with normal ALT values. And the result highlights necessity of the activation of host immunosystem against virus antigens [33].In our study FcγRIIb levels in CHB patient group were signi cantly lower than in HBV carrier group and had a negative relationship with ALT and AST. Immune tolerance occurs frequently during chronic HBV infection. Therefore, FcγRIIb may be a checkpoint to break the immune tolerance state, restore effector T cells, clear infected hepatocytes, and eliminate circulating immune antigen by humoral immunity.
It has been known that FcγRIIb is expressed on LSEC and that the liver is the major site of small immune complex including blood bore SIC clearance [13,14]. FcγRIIb is a kind of scavenger receptor and the distributional feature of FcγRIIb has been presented in intrahepatic lobules of healthy livers [34]. Some researches revealed the association between FcγRIIb expressing change in LESCs and liver diseases. Ishikawa and his colleagues conducted a study on nonalcoholic liver disease biopsy samples to evaluate the FcγRIIb expressing on LSECs. The result showed an inverse proportion relationship between FcγRIIb expressing and brosis stages, and there are the highest expressing levels at the incipient stage of brosis and lowest at the third stage of brosis [15].In patients with HCC, Geraud et al discovered a decreased expression of FcγRIIb was more likely to have a higher cancer grade. And FcγRIIb vanished in 63% in the adjacent tissue specimens of a microarray of HCC tissue [16]. Our study showed that in contrast to the controls, the expressing of FcγRIIb in moderate CHB and severe CHB group decreased signi cantly. It also declined with the development of in ammation and brosis in CHB patients. Correlation analysis showed FcγRIIb expression levels on LSEs had a negative relationship with ALT and AST, which is consistent with serum results. Moreover, there was a positive relationship between FcγRIIb levels and platelet count. Platelets are remarkably related to liver in ammation and brosis. Gomez et al [35] discovered platelet count of patients with NASH decreased along with the development of liver brosis. And they reported platelet count could work as a new indirection marker of end-stage liver diseases and portal hypertension evaluation. Our study also showed a negative relationship between in ammation grade and brosis stage and FcγRIIb expressing levels via regression assay. In other words, patients with CHB that had a declined FcγRIIb expressing levels on LESCs may also have a lower platelet count, a higher in ammatory degree and a higher brosis phase. FcγRIIb may be related to the enhancement of liver in ammation and brosis in CHB patients. However, more researches are needed to evaluate the importance and to unveil the function of FcγRIIb in chronic HBV infection.

Conclusion
In summary, our research is supportive to the conclusion that