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Title: Activation of mucosal Th-17 cells in human nasopharynx-associated lymphoid tissue by Staphylococcus aureus and S. pneumoniae
Author: Zaki, Lualuaa
ISNI:       0000 0004 9347 2444
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2019
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Bacterial colonisations are common and can lead to invasive diseases, including pneumonia, meningitis and bacteraemia. The common nasopharyngeal carriages include Staphylococcus aureus (S. aureus) and Streptococcus pneumoniae (S. pneumoniae). S. aureus colonises 20% of the human population persistently and infects around 60% of the population during their lifetime. S. pneumoniae can cause significant morbidity and mortality in humans especially in younger children and elderly. There is evidence to show an age-associated inverse correlation between these two bacterial carriages in the human nasopharynx. As S. pneumoniae carriage rate peaks around one to two years and then decrease, S. aureus carriage rate is low at younger children but increase with age to older children around 10-16 years old and adults. Both innate and adaptive immunity may mediate bacterial carriage. Innate lymphoid cells 3 (ILC3) were shown to be responsible for Nasopharyngeal maintaining tissue homeostasis and for providing early protection against bacterial colonisations, which may be particularly important in early life. With age, adaptive cellular immunity including Th17 cells becomes more mature and play a more important role in protection against pathogen colonisation. In this PhD project, Firstly, the carriage rates of both S. aureus and S. pneumoniae were analysed, using nasopharyngeal swab samples and the relationship between the bacterial carriage (e.g. S. aureus) and local Th17/IL-17A activation by S. aureus was examined in children and adults. Secondly, to determine if some staphylococcal antigens activate Th17 cells in NALT, the capacity of a number of S. aureus recombinant protein antigens to activate Th-17/IL-17A responses was investigated. Finally, the relationships between ILC3 and Th17 in NALT and bacterial carriage were examined. The bacterial carriage was examined by culturing nasopharyngeal swabs on blood agar plates. S. pneumoniae carriage rate was the highest in younger children aged around 2 years old and then gradually decreased to into adults. On the other hand, S. aureus carriage rate was lowest in young children and then increased in adolescents. The relationship between salivary IL-17A levels and the carriage rates revealed a significantly higher salivary IL-17A concentrations in S. aureus carriagepositive samples. This indicated that a local Th-17 response was activated in patients with S. aureus bacterial carriage. This hypothesis was supported by subsequent experiment using flow cytometry that showed stimulation of tonsillar cells (MNC) by S. aureus culture supernatant (CCS)elicited a marked increase in Th-17 frequency when compared to unstimulated controls. To examine the potential S. aureus recombinant proteins to activate a Th-17 immune response, flowcytometric analysis of CFSE labelled adenotonsillar MNC were stimulated with nine S. aureus recombinant proteins (IsdA, ClfB, SdrC, SdrD, CnaBE3, EsxAB, HlaH35L, Csa1A and FhuD2). Results suggested that HlaH35L, a non-toxic a-haemolysin derivative, was capable of stimulating CD4+ T cell proliferative response and activate Th-17 response in the tonsillar MNCs. The measurement of cytokines in stimulated MNC supernatant by both ELISA and cytometric beads array (CBA) indicated that HlaH35L elicited the production of Th-17 related cytokines IL-17A and IL-17F with IL-6. The ability of HlaH35L to stimulate CD4+ T cell proliferation and Th-17 response may indicate a vaccine potential of this antigen. The finding that a stronger Th-17 response after stimulation by the wild-type S. aureus CCS (containing Hla) than that by CCS derived from its isogenic Hla-/- mutant, indicating a Th17-activating property of Hla protein. Induction of Th-17 response from naïve T cells of tonsillar MNC was also studied using tonsillar MNC depleted of activated and memory T cells. Stimulation with S. aureus CCS induced Th-17 and IL-17A production from naïve T cells in tonsillar MNC. A subsequent study was carried out to examine the relationship between ILC3 and Th-17 cells and their effect on S. pneumoniae. Frequency of Th-17 cells in tonsillar tissues was shown to be significantly higher in adults than children, which may be related to the accumulation of memory Th-17 cells in NALT. The frequency of IL22+ ILC3 was compared in children and adults, results showed that the frequency of IL-22+ ILC3 in children was significantly higher than in adults. A significant increase in the number of NKp44+ ILC3 was shown to be associated with S. pneumoniae carriage. To determine whether the potent Th-17 response activated by S. aureus CCS could be inhibited, GSK805 (a ROR?t antagonist) was used to be co-cultured with MNC, followed by stimulation with S. aureus CCS. The results showed that GSK805 selectively reduced the Th-17 activation but not ILC3. This may have therapeutically implications in reduction of Th17-mediated inflammatory pathology. In summary, the activation of Th-17 response in human NALT (e.g. adenotonsillar tissues) may be common in humans during bacterial carriage in the nasopharynx. While the balance of the ILC3/Th-17 cells may contribute to the bacterial carriage status in the nasopharynx, and ILC3 may promote carriage and Th17 promote clearance of carriage. In addition, ILC3/Th-17 relationship development in different age groups may also impact the pattern of bacterial colonisation in the human nasopharynx. Non-toxic recombinant protein HlaH35L was shown to activate and promote Th-17 cells so maybe a vaccine candidate against bacterial carriage in the human nasopharynx. Understanding the development of natural immunity against S. aureus and to S. aureus carriage may provide important information on the development of protein-based vaccines against respiratory tract infections in humans.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral