HBV感染对肝脏自然杀伤细胞和固有淋巴样细胞22的影响

目的探讨HBV感染对肝脏自然杀伤细胞(NK细胞)和固有淋巴样细胞(ILC)22的影响,为阐明HBV感染诱导机体固有免疫应答的机制提供理论和实验依据。方法选取6~8周龄雄性BALB/c小鼠10只,随机分为实验组和对照组,每组各5只。实验组小鼠经尾静脉高压注射含10μg HBV全基因组质粒的生理盐水(体积相当于9%小鼠体质量);对照组小鼠仅注射生理盐水。4 d后处死小鼠,分离肝脏内淋巴细胞,应用流式细胞术检测肝脏内淋巴细胞中NK细胞亚群和ILC22细胞亚群的比例。组间比较采用t检验。结果高压尾静脉注射HBV全基因组质粒可诱导小鼠产生高水平的HBs Ag和HBe Ag,伴有血清ALT水平升高。实验组小鼠肝脏内NK细胞的比例较对照组小鼠显著升高[(25.90±4.92)%vs(12.98±2.13)%,t=3.811,P=0.003],但CD127+NK细胞和CD127-NK细胞亚群在NK细胞中所占比例在2组之间差异均无统计学意义(P值均0.05)。实验组小鼠肝脏内NKp46+ILC22细胞亚群比例较对照组明显升高[(36.05±6.85)%vs(10.22±3.54)%,t=7.372,P0.001],但NKp46-ILC22细胞亚群的比例在2组之间差异无统计学意义(P0.05)。结论 HBV感染可诱导肝脏内NK细胞和NKp46+ILC22水平升高,从而促进肝脏固有免疫应答。     

Differential expression of innate immune response genes in clinical phases of chronic hepatitis B infection

We investigated innate immune gene expression in clinical phases of chronic hepatitis B infection, including immune tolerant (IT), immune active (IA), inactive carrier (IC) and hepatitis B e antigen (HBeAg)‐negative phases, as well as healthy controls. Expression levels of interferon types I, II and III, their receptor subunits, IRFs, TLRs and other IFN‐induced genes in peripheral blood mononuclear cells were compared. Forty HBsAg‐positive treatment‐naïve subjects without co‐infection with HIV, HCV or HDV were enrolled. To complement the viral load, the expression levels of 37 innate immune genes were measured by qPCR. The highest response of the innate immune system was observed in the IT and HBeAg‐negative phases, and the IC phase had the lowest response; 31 of the 37 studied genes reached their maximum mRNA expression levels in the IT and HBeAg‐negative phases, and the minimum expression levels of 23 genes were found in the IC phase. The highest mRNA expression levels of IFNs, IFN receptor subunits, IRFs and TLRs genes in all clinical phases were IFN‐λ2 and 3, IFN‐γR2, IRF7 and TLR7, and the lowest levels of mRNA expression were observed for IFN‐α, IFN‐λR1, IRF8 and TLR2. We conclude that innate immune response genes are expressed differentially among chronic HBV phases, and this difference may help to develop new precise and noninvasive methods to determine the progression of disease in chronic HBV patients.     

Clinical immune characterization of hepatitis B virus infection and implications for immune intervention: Progress and challenges

The host immune response plays an important role in mediating hepatitis B virus (HBV) control and induction of liver damage, which determines the outcome of infection. However, interactions between HBV, the immune system, and the liver microenvironment, remain poorly understood. This review briefly outlines what we know about innate and adaptive immune responses to HBV, as well as the liver immunology in infected patients. It addresses how our knowledge of the anti-HBV immune response might aid the development of adoptive immune therapeutic strategies against HBV. This review also highlights the challenges we are facing in understanding the cellular and molecular mechanisms bywhich the innate, adaptive and liver immune responses exert a synergistic antiviral function and influence disease progression. It concludes by addressing future directions and unanswered questions regarding the use of clinical immunotherapy. We hope this review will help hepatologists and gastroenterologists to understand the anti-HBV immune response, as well as current challenges and potential immunotherapeutic strategies against this disease.     

HBV天然免疫的研究进展

HBV入侵人体时,天然免疫首先发挥重要的作用。成人感染HBV后,常常会被自发清除,然而在出生或者年幼时期感染HBV时,病情更容易慢性化并且迁延终生。在HBV感染初期,天然免疫应答对病毒复制的抑制发挥了重要作用,HBV感染的转归取决于宿主和病毒共同作用。简述了近年来有关HBV与宿主天然免疫中发挥重要作用的细胞类型、细胞因子、信号通路及其作用机制的研究进展,并指出了其在临床治疗方面的潜在价值。     

Critical coordination of innate immune defense against Toxoplasma gondii by dendritic cells responding via their Toll-like receptors

Toll-like receptors (TLRs) play an important role in host defense against a variety of microbial pathogens. We addressed the mechanism by which TLRs contribute to host defense against the lethal parasite Toxoplasma gondii by using mice with targeted inactivation of the TLR adaptor protein myeloid differentiation primary response gene 88 (MyD88) in different innate cell types. Lack of MyD88 in dendritic cells (DCs), but not in macrophages or neutrophils, resulted in high susceptibility to the T. gondii infection. In the mice deficient in MyD88 in DCs, the early IL-12 response by DCs was ablated, the IFN-γ response by natural killer cells was delayed, and the recruited inflammatory monocytes were incapable of killing the T. gondii parasites. The T-cell response, although attenuated in these mice, was sufficient to eradicate the parasite during the chronic stage, provided that defects in DC activation were compensated by IL-12 treatment early after infection. These results demonstrate a central role of DCs in orchestrating the innate immune response to an intracellular pathogen and establish that defects in pathogen recognition by DCs can predetermine sensitivity to infection.     

HBV介导的肝星状细胞和天然免疫细胞的相互作用

肝脏非实质细胞在HBV相关的肝脏疾病中发挥着至关重要的作用,而肝星状细胞(HSC)的激活以及与肝内细胞之间的相互作用是导致肝纤维化的主要原因。主要介绍了HBV诱导的肝脏炎症以及天然免疫细胞与HSC的相互作用,简述了HBV作用下单核/巨噬细胞和自然杀伤细胞(NK细胞)对HSC的激活和杀伤作用以及HSC的肝脏免疫调节作用。     

不同免疫状态慢性HBV感染患者的T淋巴细胞及NKG2D表达差异研究

目的探讨慢性HBV感染者在不同免疫状态下的T淋巴细胞及自然杀伤细胞G2D(NKG2D)表达差异。方法根据免疫状态的不同将80例慢性HBV感染患者分为免疫耐受组(28例)、免疫清除组(30例)、低(非)复制组(22例),另选取30名健康体检者作为对照组。对4组研究对象进行外周血T淋巴细胞亚群、NK细胞、NKG2D细胞频率及血清肿瘤坏死因子-α(TNF-α)、γ干扰素水平检测。结果4组研究对象的CD3^+T淋巴细胞百分比,差异均无统计学意义(P>0.05)。免疫清除组的CD4^+T淋巴细胞、CD4^+/CD8^+、NK细胞百分比分别为(30.14±5.36)%、(1.10±0.33)%、(9.67±4.31)%,均显著低于另外三组,CD8^+T淋巴细胞、NKG2D+/NK细胞百分比分别为(33.56±5.37)%、(12.96±3.42)%,均显著高于另外三组(P<0.05)。免疫耐受组和低(非)复制组的NKG2D^+/NK细胞百分比分别为(3.46±1.15)%和(3.55±1.07)%均显著低于对照组,免疫清除组的NKG2D+/NK细胞百分比显著高于对照组(P<0.05)。免疫清除组的TNF-α、γ干扰素水平分别为(69.62±16.46)ng/L和(69.85±12.30)ng/L,均显著高于免疫耐受组、低(非)复制组及对照组(P<0.05)。免疫耐受组、低(非)复制组及对照组的TNF-α、γ干扰素水平比较,差异均无统计学意义(P>0.05)。结论慢性HBV感染患者在不同免疫状态下,外周血淋巴细胞及NKG2D细胞表达有明显差异,临床可通过测定HBV感染者外周血T淋巴细胞、NKG2D细胞水平来评估患者的免疫状态,指导临床治疗。     

慢性乙型肝炎免疫致病机制和免疫治疗现状

核苷类似物和干扰素等抗病毒药物已被广泛应用于慢性乙型肝炎(CHB)的临床治疗,并取得了一定疗效.然而现有的抗病毒药物虽可显著抑制HBV复制,但却不能完全清除体内的共价闭合环状DNA(cccDNA),难以持久恢复患者的抗病毒免疫功能,导致HBsAg长期存在.因此,要实现HBsAg血清学转化,清除慢性HBV感染患者体内cccDNA,必须依靠患者特异性抗病毒免疫能力的恢复.     

NK cells in HIV‐1 infection: evidence for their role in the control of HIV‐1 infection

Increasing evidence supports the notion that the innate immune response, and in particular, natural killer cells play a central role in determining the quality of the host immune response to infection. In this review we highlight recent evidence that suggests that NK cells influence the clinical fate of HIV‐infected individuals.     

HBV感染者自然杀伤细胞活性分析

目的 探讨不同临床病期的慢性HBV感染者外周血单个核细胞(PBMC)中T淋巴细胞亚群及NK细胞活性变化.方法 收集慢性乙型肝炎、慢性重型乙型肝炎、乙型肝炎肝硬化、肝癌四组患者及健康人群的外周血,应用流式细胞术检测CD8+T细胞、CD4+T细胞及NK细胞的表达量和分泌细胞因子的能力,并检测NK细胞的杀伤活性.结果 各组患...     

Regulation of innate immunity against hepatitis C virus infection.

Chronic hepatitis C virus (HCV) infection is a global public health problem. HCV infection is treated with type I interferon (IFN), a natural product that is produced by cells during virus infection as a result of innate immune signaling events. The secreted IFN alert the surrounding cells to turn on an "antiviral state" that resists infection. In general, the role of innate immune response is to suppress viral replication and to induce cytokines and other factors that promote adaptive immunity and the resolution of infection. The mechanisms by which the innate immune response and IFN actions limit HCV infection are not well defined, but are likely to involve the function of specific IFN-stimulated genes. HCV also copesintensively with immune responses in order to establish persistent infection. Recent studies reveal that a other viruses use similar tactics to regulate the antiviral innate immune response. In the case of HCV, innate immune signaling is strictly controlled by the viral NS3/4A protease, resulting in the disruption of IFN production. Here, we summarize the current understanding of how HCV evades the innate immune system.     

NK细胞与丙型肝炎

自然杀伤细胞（NK细胞）无需抗原预先致敏,即可直接杀伤某些肿瘤细胞和病毒感染细胞,是人类抗感染免疫及抗肿瘤、清除异己细胞的第一道天然防线。HCV感染的患者NK细胞数量和功能降低可能是造成疾病慢性化的重要原因。本文就NK细胞与丙型肝炎的关系作一综述。     

p38 mitogen‐activated protein kinase impairment of innate immune cells is a characteristic feature of HBeAg‐negative chronic hepatitis B

The mitogen‐activated protein kinase p38 (MAPK) is implicated in the induction of immune responses by regulating the differentiation of T lymphocytes and production of cytokines. Our aim was to investigate p38MAPK phosphorylation in different stages of the natural history of hepatitis B virus (HBV) infection. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll‐Hypaque density‐based centrifugation from 10 patients with HBeAg‐negative chronic hepatitis B [HBeAg(?) CHB;HBV‐DNA>2000IU/mL], eight patients with HBeAg‐negative chronic HBV infection [HBeAg(?) CI;undetectable HBV‐DNA] and 8 healthy controls (HCs). p38MAPK phosphorylation was assessed by phospho‐specific flow cytometry in PBMCs and cell subsets (CD3+,CD3?,CD56+,CD56?) after stimulation with cytokines (IL‐12+IL‐2 and IL‐12+IL‐18) or nonspecific stimuli [arsenite, phorbol 12‐myristate 13‐acetate (PMA) and ionomycin] at 0,30,60,120 and 240 minutes using p38 phospho‐specific conjugated antibodies. ΙFN‐γ was determined by ELISA in PBMCs culture supernatants after stimulation with rhIL‐2, rhIL‐12 and rhIL‐18, with and without pre‐treatment with the p38 MAPK inhibitor, SB203580. HBeAg(?) CI patients showed the highest expression of phosphor‐p38 MAPK in total PBMCs and subpopulations compared to HBeAg(?) CHB and HCs. A striking impairment in p38 phosphorylation was noted in CD56+ cells and in especially in NK cells (CD3‐CD56+). SB203580‐induced inhibition of p38MAPK phosphorylation was associated with suppression of IFN‐γ production in all groups. The universal lack of p38 MAPK activation in CD56+ and in particular in NK cells from HBeAg(?) CHB patients during viremia suggests a potential cell‐dependent implication of this pathway in the natural history of HBV infection.     

乙型肝炎病毒感染时的骨髓造血干细胞

目的研究乙型肝炎患者骨髓造血干细胞中HBV的感染、复制及对造血干细胞增殖的影响。方法收集乙型肝炎患者9例,健康者7例骨髓液,磁珠分离仪分离纯化骨髓液CD34＋细胞,取部分干细胞进行免疫组化和原位杂交,以感染HBV的干细胞（2.2.15细胞）为阳性对照,并设健康人骨髓干细胞为阴性对照。余两组干细胞分别分为两组,一组在含有干细胞生长因子（SCF）、酪氨酸激酶受体家族Ⅲ的配体（FLT3）、促血小板生成素（TPO）、白介素-3（IL-3）和10%FBS的IMDM培养基中孵育,另一组在无细胞因子的同样培养基中孵育。第0、1、6、12d进行PCR病毒载量检测并做细胞计数。结果患者组骨髓干细胞经免疫组化染色后为变为棕黄色与阳性对照组一致,经原位杂交后细胞染色为蓝紫色与阳性对照组一致,而阴性对照组均未染色。对患者组骨髓干细胞加细胞因子和无细胞因子第1、6、12d细胞计数分别比较,均明显少于正常对照组（t=0.818,P〈0.05;t=3.599,P〈0.05;t=2.967,P〈0.05）。加细胞因子患者组第6、12d细胞内病毒载量明显高于不加细胞因子患者组（t=3.36,P〈0.05;t=5.71,P〈0.01）。结论 HBV可以感染骨髓造血干细胞并且可随干细胞的增殖不断复制。感染了HBV的干细胞增殖能力减弱。     

Innate antiviral immune responses to hepatitis B virus

Hepatitis B virus (HBV) is a major cause of acute and chronic hepatitis in humans. As HBV itself is currently viewed as a non-cytopathic virus, the liver pathology associated with hepatitis B is mainly thought to be due to immune responses directed against HBV antigens. The outcome of HBV infection is the result of complex interactions between replicating HBV and the immune system. While the role of the adaptive immune response in the resolution of HBV infection is well understood, the contribution of innate immune mechanisms remains to be clearly defined. The innate immune system represents the first line of defense against viral infection, but its role has been difficult to analyze in humans due to late diagnosis of HBV infection. In this review, we discuss recent advances in the field of innate immunity to HBV infection.     

急性乙型肝炎患者自然杀伤细胞的变化与疾病进展的关系

目的 探讨急性乙型肝炎(acute hepatitisB,AHB)患者外周血中自然杀伤细胞(natural killer cells,NK细胞)频率、功能变化及与疾病进展的关系.方法 入组15例AHB患者(AHB组)和14名健康者(健康对照组),采集外周血,用流式细胞仪分析NK细胞频率和活化指标,并与临床病毒学和生化指标进行相关性分析,同时观察NK细胞对K562细胞刺激的应答.结果 AHB组NK细胞频率低于健康对照组(P＜0.05).与健康对照组相比,AHB组NK细胞呈现高活化状态,且与转氨酶呈正相关.进一步研究发现,AHB组NK细胞分泌细胞因子IFNγ及脱颗粒活性(CD107a)的能力显著增强.结论 AHB组NK细胞高活化、分泌细胞因子能力和细胞毒能力显著增强,与临床指标有相关性,提示NK细胞在加速病毒清除的同时可引起肝损伤.     

慢性HBV感染的天然免疫与获得性免疫应答

HBV感染人体后,宿主的抗病毒免疫应答是决定感染结局的关键因素。目前普遍认为,在慢性HBV感染过程中宿主的天然免疫及获得性免疫功能受损,从而导致病毒持续无法清除。要实现对HBV感染持久免疫控制,需要更加全面地理解HBV感染慢性化引起的机体天然免疫和特异性免疫应答功能障碍的机制。总结了国内外研究团队在HBV感染慢性化免疫应答参与机制中取得的进展。     

免疫细胞在慢性乙型肝炎免疫耐受机制中的作用

机体对乙型肝炎病毒（HBV）的免疫耐受是HBV感染慢性化的主要机制。急性、自限性HBV感染过程中存在强烈的、多特异性的细胞免疫应答。而在慢性感染者这种反应明显减弱,存在免疫耐受。本文旨在阐述免疫细胞在HBV免疫耐受机制中可能发挥的作用。     

Cellular immune responses in hepatitis B virus e antigen negative chronic hepatitis B

The immunopathogenesis of hepatitis B e antigen (HBeAg) negative chronic hepatitis B (CHB) virus (HBV) infection has not been adequately investigated. We studied the cellular immune responses of peripheral lymphocytes using proliferating assays, intracellular cytokine staining (ICS) and ELISPOT interferon-gamma (IFN-gamma) assays after non-specific and specific stimulation with whole HBV proteins and synthetic peptides. Thirty patients with HBeAg negative CHB, eleven HBsAg inactive carriers, nine patients with acute hepatitis B and 22 healthy controls were included in the study. Patients with HBeAg negative CHB demonstrated an increased number of peripheral CD8+ T cells while their peripheral blood mononuclear cells showed increased proliferation after in vitro stimulation with overlapping hepatitis B core derived peptides and an envelope derived epitope (HBs 182-191 aa), similar to those observed in acute hepatitis B. Using ICS, we found an expanded population of IFN-gamma producing T lymphocytes, CD4+ and CD8+, after non-specific stimulation, in HBeAg negative CHB compared to all other groups. HBeAg negative CHB and acute hepatitis B patients had a similarly increased number of core specific T cells measured by the IFN-gamma assays. Inactive HBsAg carriers showed minimal proliferative responses overall while they exhibited an increased number of envelope specific effector T cells (measured by ICS). In conclusion, we showed that overall CD4+ T cell responses from patients with HBeAg negative CHB were comparable to those of acute hepatitis B, while inactive HBsAg carriers despite their limited proliferative capacity the effector activity of their peripheral T cells was maintained.