香港大学：流感病毒直接感染人类NK细胞， 在NK细胞复制，并诱导NK细胞凋亡

Influenza Virus Directly Infects Human Natural Killer Cells and Induces Cell Apoptosis

　



流感是一种急性呼吸道病毒性疾病，在感染的最初几天就会传播。规避宿主固有免疫防御系统，包括自然杀伤细胞，对于病毒作为人类和其他动物的病原体的成功发挥着重要作用。


NK细胞在感染细胞的微环境中遇到流感病毒，在流感病毒感染过程中对宿主先天免疫有重要作用。


因此，研究流感病毒对NK细胞的直接影响十分重要。


在这项研究中，我们首次证明流感病毒直接感染人的NK细胞，并在NK细胞中复制增殖。病毒进入NK细胞是通过网格蛋白和小细胞内吞作用介导的，而不是通过大胞饮作用，并且依赖于细胞表面的唾液酸。


此外，流感病毒感染导致NK细胞显著凋亡。


我们的研究结果表明，流感病毒可以直接靶向杀灭NK细胞，这是流感病毒规避NK细胞固有免疫防御的一种潜在的新策略，这种防御可能会促进病毒的传播，也可能有助于病毒的发病机制。

Influenza Virus Directly Infects Human Natural Killer Cells and Induces Cell Apoptosis

  1. 1Department of Pediatrics & Adolescent Medicine, Li Ka Shing Faculty of Medicine, the University of Hong Kong, Hong Kong SAR, People's Republic of China
  2. 2Department of Microbiology, Li Ka Shing Faculty of Medicine, the University of Hong Kong, Hong Kong SAR, People's Republic of China
  3. 3HKU-Pasteur Research Centre, the University of Hong Kong, Hong Kong SAR, People's Republic of China

ABSTRACT 

Influenza is an acute respiratory viral disease that is transmitted in the first few days of infection. Evasion of host innate immune defenses, including natural killer (NK) cells, is important for the virus's success as a pathogen of humans and other animals.

NK cells encounter influenza viruses within the microenvironment of infected cells and are important for host innate immunity during influenza virus infection.

It is therefore important to investigate the direct effects of influenza virus on NK cells.

In this study, we demonstrated for the first time that influenza virus directly infects and replicates in primary human NK cells. Viral entry into NK cells was mediated by both clathrin- and caveolin-dependent endocytosis rather than through macropinocytosis and was dependent on the sialic acids on cell surfaces.

In addition, influenza virus infection induced a marked apoptosis of NK cells.

Our findings suggest that influenza virus can directly target and kill NK cells, a potential novel strategy of influenza virus to evade the NK cell innate immune defense that is likely to facilitate viral transmission and may also contribute to virus pathogenesis.

 

Figure 1: Strategies of viruses to evade NK cell-mediated immune surveillance. A) Upon recognition of virus-infected cells by activating NK cell receptors, NK cells secrete cytokines and kill infected cells to control viral infection. However, viruses have evolved strategies to evade NK cell immune surveillance, such as listed in B-E. B) Viruses encode their own MHC class I homolog to inhibit NK cell function through binding to NK cell inhibitory receptors. C) Viruses encode anti-inflammatory cytokine IL-10 mimicry to suppress NK cell function. D) Viruses encode soluble ligands binding to NK cell activating receptor NKG2D to block NK cell recognition of infected cells. E) Viruses directly infect NK cells resulting impaired NK cell function and NK cell apoptosis.

　

Influenza is an acute respiratory virus infection that continues to pose endemic, zoonotic, and pandemic threats to human health, with significant morbidity and mortality (17). At the early phase of viral infection, innate immunity plays important roles in host defense by limiting viral replication and helping to initiate an adaptive immune response. Natural killer (NK) cells are key effector cells in innate immunity and play a critical role in the first line of host defense against acute viral infections by directly destroying infected cells without the need for prior antigen stimulation (7, 20). As influenza illness and virus transmission usually occur in the first few days of infection, the virus has to devise strategies to evade host innate immune responses, including NK cell immunity (15, 21).

 

NK cells can recognize and kill influenza virus-infected cells (2, 10, 23); to counteract this killing, however, influenza virus has developed an escape strategy that inhibits NK cell cytotoxicity by increasing the binding of two inhibitory receptors to the infected cells after infection (1). The individuals with complete NK cell deficiency developed life-threatening varicella zoster virus and cytomegalovirus infection, but no severe influenza virus infection occurred (30, 40). Indeed, the interaction between human NK cells and influenza virus remains poorly understood. After influenza virus infection, respiratory epithelial cells release inflammatory chemokines that recruit NK cells to the site of infection (12). As a lytic virus, numerous influenza virus particles are released from the infected epithelia and macrophages (5, 9, 33). In the infected microenvironment, NK cells undoubtedly encounter these infective virus particles. It is therefore important to investigate the direct interaction of NK cells with influenza virus. Patients with severe influenza virus infection were shown to have diminished NK cells in peripheral blood and an almost complete absence of pulmonary NK cells, together with marked apoptosis (13, 42). During influenza virus infection in mice, a transient increase of NK cytotoxicity is followed by a marked decrease in NK cell activity, with a virus dose-dependent effect (8, 28). These data suggest that influenza virus may directly target NK cells as part of its immunoevasion strategies. However, no reports of the direct effects of influenza virus on human NK cells have so far been available.

 

In this study, we demonstrated that influenza virus infects and replicates in primary human NK cells. Viral infection was dependent on sialic acids on the cells. The entry was mediated by both clathrin- and caveolin-dependent endocytosis rather than macropinocytosis. Influenza virus infection induced a marked apoptosis of NK cells, which contributed to reduced NK cell cytotoxicity. This, to the best of our knowledge, is the first paper to demonstrate that influenza virus can directly infect NK cells and induce cell apoptosis. These findings suggest that influenza virus may have developed a novel strategy to evade NK cell innate immune defenses, which is likely to facilitate viral transmission and may also contribute to virus pathogenesis.

　

Influenza virus infection is dependent on the sialic acids on NK cells.The sialic acid on target cells is now commonly recognized as the receptor for influenza virus infection. Upon binding to sialic acids on the cell surface, the virus is internalized by receptor-mediated endocytosis. We next determined the role of sialic acid in the influenza virus infection of NK cells by treating cells with serial concentrations of sialidase prior to viral infection, aiming to cleave the sialic acids on NK cells. Six hours after infection, intracellular viral antigen was detected by flow cytometry. As shown in Fig. 3A, the blockade of influenza virus infection by sialidase in NK cells was dose dependent. The frequency of virally infected NK cells decreased by around half at the concentration of 100 mU of sialidase/ml, and almost total blockade of the infection was found when 500 mU of sialidase/ml was applied. Cell viability was not affected by the sialidase treatment, as evidenced by no changes in annexin V binding and PI uptake of the treated NK cells (Fig. 3B). In addition, the treated cells also showed major histocompatibility complex (MHC) class I staining similar to that of mock-treated cells (Fig. 3C). These data suggested that influenza virus infection of NK cells is mediated by sialic acids. In addition, double staining against viral protein and anti-EEA1 was performed at 1 h after influenza virus infection. Confocal microscopy analysis showed that these two proteins colocalized (Fig. 3D), indicating that influenza virus entered NK cells via endocytosis.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3128146/