The role of trained immunity against Streptococcus pneumoniae

Alem, Tamadhour (2026) The role of trained immunity against Streptococcus pneumoniae. PhD thesis, University of Glasgow.

Abstract

Background: Streptococcus pneumoniae is a gram-positive bacterium associated with multiple serious illnesses. According to the CDC antimicrobial resistance reports, pneumococcal bacteria are resistant to one or more antibiotics in more than 30% of cases. This high percentage necessitates the need for alternative therapeutic targets that stem from a better understanding of immune defences against the bacteria. The bacteria possess several virulence factors and can cause significant morbidity and mortality in humans. This thesis examines the innate immune responses activated in defence against Streptococcus pneumoniae. It investigates whether these responses involve the secretion of type I interferons and other pro-inflammatory cytokines, such as tumour necrosis factor alpha and interleukin-1 beta. Another aspect of this thesis is to investigate the phenomenon of trained immunity, in which innate immune cells produce a more robust response to a secondary infection by the same or a different pathogen. To do this, multiple cell models were used, including primary dendritic cells, macrophages, and cell lines. These cells were pre-treated with innate immune stimulants such as β-glucans, Zymosan derived from fungi, and LPS derived from E. coli, a gram-negative bacterium. Subsequently, the secretion of pro-inflammatory cytokines, TNF alpha and IL-1 beta, was measured in response to the pre-treatment of these cells and their infection with Streptococcus pneumoniae. This was done to explore whether pretreatment impacts cytokine release following infection in innate immune cells.
In other chapters, mRNA transcription of TNF alpha gene in response to pre-treatment with LPS and infection with Streptococcus pneumoniae was assessed. Also, the epigenetic changes underlying trained immunity were explored, specifically DNA methylation in response to LPS pre-treatment and infection with Streptococcus pneumoniae to compare the methylation percentages between different conditions. Finally, cellular proteins involved in toll-like receptor 4 (TLR4) signalling were examined using protein lysates, through gel electrophoresis and western blotting. These were done to determine which proteins are responsible for the pro-inflammatory effects of LPS pre-treatment and Streptococcus pneumoniae infection.
Main results: None of the cell models used in the study, including primary monocyte-derived dendritic cells, primary monocyte-derived macrophages, the BEAS-2B bronchial epithelial cell line, and U937 monocyte-derived macrophages, produced type 1 interferons following infection with Streptococcus pneumoniae. However, both primary cells and the U937 cell line produced pro-inflammatory cytokines, such as TNF alpha and IL-1 beta, in response to Streptococcus pneumoniae infection.
Innate immune stimulants, including beta-glucan, zymosan, and LPS, all appeared to stimulate the release of TNF alpha and IL-1 beta 24 hours after infection with Streptococcus pneumoniae. Additionally, LPS pretreatment of U937 cells and subsequent infection with Streptococcus pneumoniae appear to boost the transcription of TNF-alpha mRNA. The mRNA expression is at similar levels in cells that were pre-treated only. However, ELISA results indicate that the pretreated infected cells produced more TNF alpha compared to the pretreated only cells.
TNF alpha gene methylation following pre-treatment was studied. The first segment examined is in the promoter region. DNA methylation percentages revealed patchy results. However, it seems there’s a trend of increased methylation in LPS pre-treated and infected cells compared to pre-treated only cells, but this difference didn’t reach statistical significance. Increased methylation in the promoter region of a gene is associated with gene silencing. Similar results are seen in the second segment, which is in the gene’s first exon.
Lastly, cellular proteins involved in TLR4 signalling were examined. It appears that LPS pretreatment combined with infection boosts JNK phosphorylation.
Conclusion: Based on these results, it appears that Streptococcus pneumoniae infection doesn’t lead to IFN secretion. However, it boosts the secretion of the pro-inflammatory cytokines TNF alpha and IL-1 beta. Pretreatment of innate immune cells with innate immune stimulants prior to infection increases the secretion of TNF alpha and IL-1 beta 24 hours following infection. When combining the ELISA results with qPCR, it appears that both pre-treated infected cells and pre-treated-only cells undergo increased TNF alpha mRNA transcription; however, the infection seems to work post-transcriptionally to enhance mRNA translation through an unknown mechanism.
Regarding the TNF alpha methylation data, it appears that LPS pretreatment may cause increased methylation and gene silencing. However, when this result is added to previous findings, it could suggest that LPS pretreatment works through a different mechanism to induce TNF alpha gene transcription.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Supported in part by Imam Abdulrahman bin Faisal University.
• Subjects: Q Science > QR Microbiology > QR180 Immunology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Infection & Immunity
• Funder's Name: Imam Abdulrahman bin Faisal University
• Supervisor's Name: Evans, Professor Tom
• Date of Award: 2026
• Depositing User: Theses Team
• Unique ID: glathesis:2026-85947
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 22 May 2026 09:37
• Last Modified: 22 May 2026 09:38
• Thesis DOI: 10.5525/gla.thesis.85947
• URI: https://theses.gla.ac.uk/id/eprint/85947