Yoshioka Teppei, Tatsumi Tomohide, Miyagi Takuya, Mukai Kaori, Nishio Kumiko, Nishio Akira, Yokoyama Yoshinobu, Suda Takahiro, Kegasawa Tadashi, Shigekawa Minoru, Hikita Hayato, Sakamori Ryotaro, Takehara Tetsuo
Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
PLoS One. 2017 Mar 22;12(3):e0174103. doi: 10.1371/journal.pone.0174103. eCollection 2017.
Natural Killer (NK) cells are involved in the control of viral infection. However, the role of NK cells in chronic hepatitis B (CHB) remains unclear. This study investigated the frequencies and roles of NK cells in CHB, with a focus on activating receptor NKp46 and inhibitory receptor NKG2A.
PATIENTS/METHOD: Peripheral blood lymphocytes were obtained from 71 CHB patients and 37 healthy subjects (HS). The expressions of NKp46 and NKG2A were analyzed using flow cytometry. The role of NKp46-ligand was assessed using an in vitro co-culture system. Cytotoxicity and IFN-γ production in NK cells were evaluated using RT-PCR and flow cytometry.
CHB patients were classified into treatment-naïve patients with low HBV DNA titer (CHB-L; n = 28), high HBV DNA titer (CHB-H; n = 24) by the cut-off level of serum HBV DNA 4 log copies/ml, and patients receiving nucleos(t)ide analogue (CHB-NA; n = 19). The expressions of NKp46 and NKG2A were higher in CHB-H than in HS/CHB-L/CHB-NA. HepG2.2.15 had higher NKp46-ligand expression than HepG2. When NK cells from HS were co-cultured with HepG2.2.15, inhibition of the NKp46 and NKp46-ligand interaction by anti-NKp46 antibody significantly reduced cytolysis of HepG2.2.15 and IFN-γ production. However, those reductions were not observed in co-culture with HepG2. Additionally, NK cells that highly expressed NKp46 also highly expressed NKG2A (NKp46highNKG2Ahigh subset). The frequencies of NKp46highNKG2Ahigh subset in CHB-H were higher than those in HS/CHB-L/CHB-NA. Among treatment-naïve CHB patients, the frequencies of NKp46highNKG2Ahigh subset were positively correlated with serum ALT (P<0.01, r = 0.45) and HBV DNA (P<0.01, r = 0.59) levels. The expressions of Fas-L, STAT1, TRAIL and CD107a were higher and IFN-γ expression was lower in the NKp46highNKG2Ahigh subset than in the other subsets.
The NKp46 and NKp46-ligand interaction contributes to NK cell activation. A novel NK cell subset, the NKp46highNKG2Ahigh subset, may be associated with liver injury and HBV replication.
自然杀伤（NK）细胞参与病毒感染的控制。然而，NK细胞在慢性乙型肝炎（CHB）中的作用仍不清楚。本研究调查了CHB中NK细胞的频率和作用，重点关注激活受体NKp46和抑制性受体NKG2A。
患者/方法：从71例CHB患者和37名健康受试者（HS）中获取外周血淋巴细胞。使用流式细胞术分析NKp46和NKG2A的表达。使用体外共培养系统评估NKp46配体的作用。使用RT-PCR和流式细胞术评估NK细胞中的细胞毒性和IFN-γ产生。
根据血清HBV DNA 4 log拷贝/ml的临界水平，CHB患者分为未经治疗的低HBV DNA滴度患者（CHB-L；n = 28）、高HBV DNA滴度患者（CHB-H；n = 24）以及接受核苷（酸）类似物治疗的患者（CHB-NA；n = 19）。CHB-H中NKp46和NKG2A的表达高于HS/CHB-L/CHB-NA。HepG2.2.15的NKp46配体表达高于HepG2。当HS的NK细胞与HepG2.2.15共培养时，抗NKp46抗体对NKp46和NKp46配体相互作用的抑制显著降低了HepG2.2.15的细胞溶解和IFN-γ产生。然而，与HepG2共培养时未观察到这些降低。此外，高表达NKp46的NK细胞也高表达NKG2A（NKp46高NKG2A高亚群）。CHB-H中NKp46高NKG2A高亚群的频率高于HS/CHB-L/CHB-NA。在未经治疗的CHB患者中，NKp46高NKG2A高亚群的频率与血清ALT（P<0.01，r = 0.45）和HBV DNA（P<0.01，r = 0.59）水平呈正相关。NKp46高NKG2A高亚群中Fas-L、STAT1、TRAIL和CD107a的表达高于其他亚群，而IFN-γ表达低于其他亚群。
NKp46与NKp46配体的相互作用有助于NK细胞激活。一种新的NK细胞亚群，即NKp46高NKG2A高亚群，可能与肝损伤和HBV复制有关。
