Natural killer cell responses to influenza A virus in the human lung

Abstract

Influenza infection is a major contributor to global mortality and morbidity, with high potential for pandemic and vaccine evasion. Natural Killer (NK) cells are innate anti-viral effector cells but their functions in respiratory influenza infection are poorly understood. NK cells have been implicated in the control of influenza in mouse models but their regulation in the human respiratory environment is still an area of study. For instance, phenotypically and functionally unique resident NK cell populations have been described in the liver and uterus, but have not yet been explored in the human lung.

The aim of this thesis was to investigate the human lung NK cell phenotype and function during respiratory infection and disease, primarily focusing on influenza infection. To do this, NK cells were characterized from macroscopically normal human lung tissue by multi-parameter cytometry and NK cell function characterized in an ex vivo tissue explant model of influenza infection. NK cell function was further characterized by autologous co-culture of peripheral NK cells with influenzainfected monocyte-derived macrophages (MDMs) and primary bronchial epithelial cells (PBECS). Cytotoxic and cytokine responses of NK cells were measured by a combination of flow cytometry and enzyme-linked immunosorbent assay (ELISA). Mechanisms of NK cell activation to IAV-infected macrophages were further explored including activating and inhibitory contact with infected cells and cytokine release.

Both lung and peripheral NK cells were found to mediate strong anti-viral responses to influenza infection; with increased IFN-γ, Gzm-B and measures of degranulation. Furthermore, mechanistic studies demonstrated that physical contact between NK cells and influenza-infected MDMs was essential to NK cell activation, despite a strongly pro-inflammatory environment. However, NKp46 and NKG2D ligands were redundant in stimulating NK cell activation towards infected MDMs, demonstrating complicated and extensive regulation of NK cells activation by infected macrophages. The majority of human lung NK share a phenotype with circulating NK cells, however, this thesis identified novel lung resident NK cell populations. Lung resident (CD49a+) NK cells were found to respond more potently to influenza infection than non-resident (CD49a-) NK cells. This suggests that resident, trained NK cell populations are present in the human lung and may provide early and important control of viral infection. CD49a+ lung NK cells were also increased in donors with more severe COPD, indicating possible modulation of lung resident NK cells in chronic disease.

Overall this thesis presents evidence that NK cells play an important role during human influenza infection through both cytotoxicity and IFN-γ production in the human lung. This work has also highlighted the differences between lung resident and non-resident NK cells in terms of phenotype and function, which may have important implications for understanding mucosal immunity in the lung.

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Identifiers

ORCID for Karl Staples: orcid.org/0000-0003-3844-6457

ORCID for Salim Khakoo: orcid.org/0000-0002-4057-9091

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