The Association between Expression of NK Cells and Prognosis of Patients with HBV Acute-on-Chronic Liver Failure in Advanced Phase

Acute-on-chronic liver failure (Acute-on-chronic liver failure, ACLF) is acute liver function decompensation on the basis of chronic liver disease. The progression of ACLF develops from advanced phase, plateau phase to remission phase. The pathophysiological basis of ACLF in different phases is various. In advanced phase, immune imbalance and systemic inflammatory reaction plays key roles. In this study, we try to assess the association between expression of NK cells and its receptors and prognosis of patients with ACLF in advanced phase. A total of 35 inpatients with HBV acute-on-chronic liver failure in advanced phase were recruited. They were divided into case group (n = 18) and control group (n = 17) according to whether the patients was dead in the 12 weeks. PBMC were detected for the frequency and expression of NK cell receptors by flow cytometric analysis. Our results demonstrated that patients who died had lower expression of NK cells and inhibitory receptor KIR3DL1, higher levels of FASL. During 12-week follow-up in those case alive, we found that NK cells increased, while expression of FASL decreased. High short-term mortality of ALCF was associated with NK cell, especially related to KIR3DL1 and FASL (PNK = 0.036, PKIR3DL1 = 0.0265, PFasL = 0.0008).

short-term mortality and causes a series of symptoms including jaundice, coagulopathy, ascites, hemorrhage, cholestasis, hepatic encephalopathy, hepatorenal syndrome [1].In China, the most common cause for liver failure is hepatitis B virus infection.The progression of ACLF can be divided into several stages: advanced phase, plateau phase and remission phase.In different phase, patients undergo various pathophysiology [2].In advanced phase, immune damage and hypoxic-ischemic damage may be dominant [3].
Currently, no criteria are available for the precise definition of different phases.According to whether their total bilirubin within 7 days of average daily rise is ≥17.1 umol/L or not, we differentiated patients with ACLF into advanced phase and not-advanced phase.
NK cells are important immune cells which are closely related to antitumor, antiviral and immune regulation in vivo [4] [5].In the early stage of acute HBV infection, the cytotoxicity of NK cells increases, IL-2 levels rise, both play the clearance of the virus.In chronic hepatitis B, cirrhosis, hepatocellular carcinoma patients, the number and activity of NK cells were suppressed [6].The persistence of HBV may be associated with viral suppression of NK cell as the representative of the innate immune system.
In this study, we try to assess the association between expression of NK cells and its receptors and prognosis of patients with ACLF in advanced phase.Dynamic observation of immunological characterization of natural killer cell was tested in those case alive.1).

Flow Cytometric Analysis
Peripheral blood mononuclear cells (PBMC) were isolated from fresh hepari- FITC-conjugated anti-Perforin and PE-conjugated anti-FASL were obtained from eBioscience (San Diego, CA).Stained cells were analyzed by the BD FACSCalibur (BD Bioscience).The lymphocyte gate was established using forward and side scatter parameters.The positive gate was set using isotype controls (all from BD Bioscience).The percentages of NK cells expressing activating receptors, inhibitory receptors, FASL, Perforin, and Granzyme B were determined individually according to instructions, analyzed using FlowJo software.

Statistical Analysis
All data were analyzed using Windows software SPSS version 20.0.Data of clinical and biochemical features were expressed as frequency and mean ± standard deviation, and compared using the chi-square and t tests.The frequency was compared using Chi-square, and the quantitative data was compared using t tests.Spearman's correlation was used between variables.One-way ANOVA analysis was used between quantitative data.P-values <0.05 were considered statistically significant.

Lower Levels of NK Cells and Inhibitory Receptor KIR3DL1 in Case Group
The comparison of NK cells showed lower levels in case group compared to control group (Z = 26, P = 0.036).There were no markedly differences between case group and control group, after comparison of activating NK receptors NKG2D, NKp30, NKp46 (P > 0.05).But the percentage of inhibitory NK receptor KIR3DL1 in case group was lower than in control group (Z = 17, P = 0.0265).

Higher Levels of FASL in Case Group
The expression of FASL in case group was much higher than that in control group (Z = 0.00, P = 0.0008).Significant deviations were not observed in Perforin and Granzyme B (P > 0.05; Figure 2).

Increased NK Cells Expression during 12-Week Follow-up in Control Group
Dynamic observation of immunological characterization of NK cells in case

Discussion
The progression of ACLF can be divided into several stages: advanced phase, plateau phase, and remission phase.The pathophysiological basis of ACLF in different stages is various.Immune imbalance and systemic inflammatory reaction plays a key role in the development of liver failure.Immune damage may be dominant in early advanced phase, then hypoxic-ischemic damage and endotoxin begin to take effect [8].During the progress of ACLF, immunosuppression played a major role in plateau phase.When it comes to remission phase, immune reconstruction and rebalance begin to work [9].According to whether their total bilirubin within 7 days of average daily rise is ≥17.1 μmol/L, we differentiated patients with ACLF into advanced phase and investigate their relationship between immunological situation and prognosis.
Some previous studies had documented NK cells activated is related with HBV-ACLF [10] [11] [12].Liver injury induced by HBV mainly depends on the host immunological response to the virus.Immune injury caused by HBV is mainly in cellular immunity, including dendritic cells, NK cells and CTL cells [13] [14].Some studies had found that activity of NK cells in patients with ACLF was significantly increased, the progression of disease was associated with the increase of NKp30 receptor and NKp46, and liver injury mediated by NK cells through FAS/FASL and NKG2D/NKG2D ligand pathway [15]

Conclusions
In summary, results from this study demonstrated that prognosis of patients with ACLF in advanced group had higher expression of NK associated immunological situation, including more NK cells gathered in liver, lower expression of inhibitory receptor KIR3DL1 and higher expression of FASL.If patients stayed alive during progress of ACLF, their immunological imbalance tended to recover and reconstruct.We think NK cells play a major role during ACLF process, and its function is mainly passed by FAS/FASL pathway which causes hepatocytes apoptosis and necrosis.

Figure 1 .
Figure 1.Lower levels of NK cells and inhibitory receptor KIR3DL1 in case group.1) *: The percentage of NK cells in case group patients was lower than that in Control patients (Z = 26, P = 0.036); 2) *: The percentage of inhibitory NK receptor KIR3DL1 in case group was lower than in control group (Z = 17, P = 0.0265).There were no significantly differences in activating NK receptors NKG2D, NKp30, NKp46, inhibitory NK receptors NKG2A, KIR2DL1, KIR2DL3, P > 0.05.

Figure 2 .
Figure 2. Higher levels of FASL in case group.*: The expression of FASL in case group was much higher than that in control group (Z = 22.5, P = 0.0008).Such difference was not observed in Perforin and Granzyme B (P > 0.05).

Figure 3 .
Figure 3. Increased NK cells expression during 12-week follow-up in control group.*: The percentage of NK cells gradually increased during the follow-up, had statistical difference in baseline and 12-week (Z = −2.06,P = 0.039).The inhibitory receptor KIR3DL1 gained fluctuation during follow-up, but had no statistical difference in each time point (P > 0.05).The expression of FASL decreased during 12 weeks observation, though all-time point had no significant differences (P > 0.05).

Table 1 .
Comparison of clinical, biochemical and virologic characteristics of patients in the case group and control group.
needed.So, further research requires more investigation and continuous cooperation between clinicians, researchers, and patients.
progression of ACLF, immunological changes in plateau phase and immunological intervention during advanced phase.A deeper level of immune investigation W. Z. Weng et al.DOI: 10.4236/aid.2017.74012124 Advances in Infectious Diseases is also