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Title: The effect of human cytomegalovirus on neutrophil survival, autophagy, and extracellular traps
Author: Storisteanu, Daniel Matthew L.
ISNI:       0000 0004 7227 1045
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2018
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Neutrophils provide a rapid first response to invading pathogens and orchestrate the immune response. They are able to employ potent antipathogenic mechanisms such as phagocytosis, reactive oxygen species (ROS) generation, protease release from granules, and formation of neutrophil extracellular traps (NETs). Despite this, certain pathogens have evolved mechanisms to benefit from neutrophil effector functions. Human cytomegalovirus (HCMV) is a clinically important pathogen that infects the majority of the human population. Monocytes are considered the main vehicle of HCMV dissemination throughout the body, but little research has been done on its interaction with neutrophils. The virus encodes a range of immunomodulatory proteins including an IL-8 homologue that acts as a powerful neutrophil chemoattractant. Viral conservation of a protein that recruits neutrophils to the site of HCMV infection suggests that the interaction between neutrophils and HCMV provides an overall advantage to the virus, but little evidence exists so far to suggest this is the case. Here I report that human peripheral blood neutrophils exposed to a clinical strain of HCMV display a profound survival phenotype, as assessed by morphology, phosphatidylserine exposure, cell permeability, and caspase-3/7 activity. This occurs in the absence of viral gene production. Neutrophils also upregulated their release of inflammatory cytokines in response to HCMV, with higher concentrations of IL-6, IL-8, and MIP-1α detected in the secretomes of infected neutrophils. These secretomes induced monocyte chemotaxis and increased monocyte permissivity to HCMV infection, as well as augmented survival in healthy, uninfected neutrophils. These experiments were confirmed with clean HCMV after the discovery of contaminating Mycoplasma spp. in the viral inocula of the initial experiments. Mycoplasma-HCMV coinfection induced an autophagic phenotype in neutrophils, as assessed by Western blotting and qPCR of autophagy-related components. Inhibition of autophagy using 3-MA reversed a profound survival effect. The unintended inclusion of Mycoplasma spp. further led to the serendipitous discovery of yet another pathogenic ability to overcome neutrophil immune functions: contaminating Mycoplasma spp. as well as Mycoplasma pneumoniae profoundly degraded NETs. These extracellular chromatin structures were stimulated using PMA or pyocyanin, and their release was dependent on the generation of ROS: severely ROS-deficient murine bone marrow neutrophils were unable to generate NETs. However, small amounts of ROS were sufficient for NETs generation, as neutrophils from acute respiratory distress syndrome patients, including many that had attenuated ROS-responses, were still capable of NETs generation. The NETs-degradative properties of mycoplasma were confirmed by fluorescence confocal and scanning electron microscopy, as well as spectrophotometry and agarose gel electrophoresis. This study demonstrates that two pervasive pathogens, HCMV and M. pneumoniae, both frequently found in coinfections in clinical contexts, are able to overcome neutrophil antipathogenic mechanisms to potentially enhance pathogen dissemination. These data provide not only a novel example of manipulation of an anti-viral response in a cell not productively infected, but also a novel example of pathogenic NETs degradation. These findings may have implications on our understanding of mycoplasma and HCMV pathogenesis and provide new targets for the generation of therapies.
Supervisor: Chilvers, Edwin Sponsor: Gates Cambridge
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: cytomegalovirus ; neutrophil ; autophagy ; extracellular traps ; NETs ; apoptosis ; CMV ; HCMV ; Mycoplasma