Bin Gulam Ali, Shamsul

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The helminth parasite Fasciola hepatica (liver fluke) causes fasciolosis in mammals. It causes significant economic loss to the agricultural industry, primarily affecting cattle and sheep. It is also a zoonotic disease affecting humans worldwide. Streptococcus pneumoniae, a commensal bacterium of humans, is asymptomatically carried in the nasopharynx of healthy individuals. However, if the bacterium escapes from its natural niche to other anatomical loci, it can cause pneumonia, middle ear infection or a range of disseminated infections collectively termed invasive pneumococcal disease. The pneumococcus is a global killer, especially in children under 5 years old and in the elderly. F. hepatica is known to release excretory/secretory (FhES) and tegumental coat (FhTEG) molecules that drive Th2 and regulatory responses with concomitant suppression of protective Th1/Th17 immune responses. These are associated with the switching of macrophage (MΦ) phenotypes from classically activated (M1/CAMΦ) to alternatively activated (M2/AAMΦ). Helminth-bacterial co-infections have been reported to result in harmful consequences, due to the effects of the helminth on host immune responses. Previous studies have demonstrated that hosts, co-infected with liver fluke and bacterial respiratory pathogens, such as Bordetella pertussis and Mycobacterium bovis, fail to clear these bacterial infections, which require Th1 or Th17 immune responses. Recent findings in animal models have shown that chronic helminth infections may have implications for vaccine efficacy against Streptococcus pneumoniae. The aim of this project was to investigate the impact of F. hepatica on pneumococcal infection utilising FhES antigens to emulate F. hepatica–S. pneumoniae co‐infection. Chapter 3 describes the modulation of the immortalised murine macrophage cell line, J774.2, in response to F. hepatica antigens and different bacterial stimulants (Mycobacterium bovis sonicate extract (MbSE) and different S. pneumoniae D39 preparations). Macrophage activity was assessed by measuring nitric oxide (NO) (CAMΦ marker) and arginase (AAMΦ marker) levels in cell culture supernatants and cell lysates. Significant down-modulation of NO was observed in both FhES+ and FhTEG+MbSE co-exposed cultures compared to MbSE alone, suggesting the J774.2 phenotype was modulated towards AAMΦ. Co-exposure to FhES+MbSE augmented arginase production further supporting the evidence of AAMΦ polarisation caused by F. hepatica. Neither F. hepatica antigen alone affected arginase production in J774.2 MΦs. Whilst live Ply-deficient mutant D39Δply and wild type D39 failed to induce NO production, culture supernatant derived from D39 (D39SN) alone induced significant levels of NO. Significantly up-modulated NO production was detected in FhES+D39SN co-exposed cultures suggesting CAMΦ activation. Data presented in Chapter 4 demonstrates the capacity of FhES to modulate the immune response to live D39 using ex vivo murine BMDMΦs. Co-exposed outbred CD1 BMDMΦs had a mixed CAMΦ and AAMΦ phenotype. BMDMΦs derived from BALB/c mice, co-exposed to FhES and D39 infection, showed evidence of CAMΦ activation, supported by up-modulation of pro-inflammatory cytokines and chemokines. In contrast, C57BL/6 BMDMΦs showed evidence of AAMΦ activation during co-exposure. In older BALB/c mice there was evidence of declining CAMΦ function in D39 infection. In Chapter 5, the effect of FhES on S. pneumoniae nasopharyngeal carriage was investigated using an in vivo CD1 mouse model. Counts of viable bacteria in the nasopharynx and lungs were determined, cytokines were quantified and immune cell populations were analysed. Exposure to FhES antigens promoted a favourable environment for nasopharyngeal colonisation with D39 in the co-exposed animals. Lower levels of TGF-β1 production were observed in D39 alone infected mice compared to FhES treated and co-exposed mice. The involvement of AAMΦ in the co-exposed mice was supported by the recruitment of mannose receptor (MR) expressing populations of MΦ and monocytes. The nasopharynx was the focal point of immune responses to pneumococcal carriage compared to the lungs, indicated by the apparent influx of innate and adaptive immune cells. Chapter 6 describes gene expression in ex vivo BALB/c BMDMΦ in response to F. hepatica-pneumococcus co-exposure. The combination of both fluke and D39 stimulants up-regulated Th1 associated genes. The pathogen response gene IL-1β induce CAMΦ activation likely resulted from NLRP3 inflammasome activation, possibly mediated by Ply released by D39 and cathepsins in FhES. Toll-like receptor gene TLR2 but not TLR4 and TLR7 genes were activated in the co-exposed BALB/c BMDMΦs. Down-regulation of CCL6, CXCL14 and TGF-β1 in co-exposed cultures could alter the immune response in the real co-infection scenario in human hosts. Overall, this study provides a valuable insight into the potential of F. hepatica-pneumococcus interaction to subvert immune system responses. The results presented here demonstrated the capacity of F. hepatica ES antigens to modulate immune responses to pneumococcal infection, although the differences observed in different models systems suggests host factors including age and genetics play a role in determining how this modulation manifests. The findings suggest that an active F. hepatica infection in the liver may impact on pneumococcal survival and dissemination in the human host, a topic to be comprehensively addressed in future studies.

Item Type: Thesis (PhD)
Divisions: Faculty of Health and Life Sciences
Faculty of Health and Life Sciences > Institute of Infection, Veterinary and Ecological Sciences
Depositing User: Symplectic Admin
Date Deposited: 03 Sep 2021 15:46
Last Modified: 18 Jan 2023 22:49
DOI: 10.17638/03121654