Ogbodo, Ekene N.
(2021)
Immunoregulatory properties of Helicobacter pylori derived molecules.
PhD thesis, University of Nottingham.
Abstract
Helicobacter pylori (H. pylori) is a micro-aerophilic, spiral-shaped Gram-negative bacterium which colonises the human stomach of approximately 50% of the population worldwide (1). The infection is asymptomatic in the vast majority of cases, however, about 10-15% result in peptic ulcer disease and 1-2% result in gastric adenocarcinoma. H. pylori prevalence in developed countries is decreasing as the incidence of asthma and allergy is increasing. H. pylori infections are usually established in early childhood when the immune system is developing and at a common age for asthma onset. The infection induces cellular immune responses of types known to inhibit those that drive allergy. Previously, we showed that H. pylori secreted component stimulated CD4+CD25hi regulatory T cells (Tregs) expressing the anti-inflammatory cytokine interleukin-10 (IL-10) more strongly than whole cell lysate. SDS-PAGE was used to resolve the H. pylori and a whole-cell lysate to determine which proteins from the supernatant were enriched and Mass spectrometry (MALDI-TOF) was used to identify the major proteins by peptide mass fingerprinting (PMF). From the result of the MALDI-TOF, vacuolating cytotoxin A (VacA) catalase (KatA), γ- Glutamyl transpeptidase (GGT) and Peptidyl prolyl cis-trans isomerases (PPT) were selected based on the few pieces of evidence of their immunoregulatory abilities, their solubility and potential to be cloned and purified in the past.
The main aim of this study is to investigate candidate H. pylori protein factors that are involved in the induction of the immunoregulatory response and how these effects could be utilised in the treatment of allergy and autoimmune response. Four H. pylori candidate proteins (VacA, KatA, GGT and PPT), previously selected on the basis of their immunoregulatory potential, were used in in vitro, in vivo or ex vivo investigations.
Three of these candidate proteins factors, KatA, GGT and PPT were first investigated in vivo. To do this the genes for each H. pylori candidate protein were cloned and expressed in ClearColi® BL21(DE3) E. coli to minimise effects from LPS. The proteins were purified and characterised. LPS content in the recombinant proteins was assayed, using an E-TOXATE assay, and shown to be <0.1 EU/ml. Jurkat T-cells, THP-1 monocytic cells and AGS epithelial were incubated for 1 hour with 10, 25 and 50 µg/ml of the recombinant proteins, prior to activation with PMA/Ionomycin, LPS or TNF-. After 24 hours of treatment IL-2 (Jurkat cells), IL-6 (THP-1) and IL-8 concentrations were quantified by ELISA. All three proteins induced a dose-dependent reduction in cytokine production, compared to controls treated only with PMA/Ionomycin, LPS or TNF-. KatA most strongly suppressed IL-2 secretion by Jurkat cells (79.5% reduction with 50 g/ml, p<0.05), whereas GGT was most effective in suppressing IL-6 from THP-1 cells (68.07% reduction with 50 µg/ml, p<0.05), there were no changes in the IL-8 production in AGS. There was no accompanying decrease in cell viability.
Likewise, VacA was investigated in vivo and ex vivo to compare the Treg response induced in vivo, by H. pylori mutants expressing different forms of VacA. Groups of 18 female C57BL/6 mice were infected orally with isogenic H. pylori SS1 mutants expressing the s1/i1 or s2/i2 form of VacA. A control group received plain Brucella broth as a placebo. Mice were killed at 3-, 6- and 9-weeks post-infection and their infection status confirmed. Spleen cells were isolated, stimulated with mitogens for 6 hours and stained with fluorochrome-conjugated antibodies. Treg populations were quantified by flow cytometry. Treg cells were purified and assayed for suppressive functional activity in vitro. Mitogen stimulation resulted in significantly increased frequencies of IL-10+ Tregs at all time-points and all groups (p<0.001). No statistically significant differences were found in frequencies of IL-10+ Tregs between the groups. There were also no differences in the functional suppressive activity of purified Tregs. Despite previously finding markedly increased Treg populations in peripheral blood from infected patients, we were unable to find increased Treg numbers in the spleen of infected mice. It is recommended that further investigation frequencies of Tregs in the gastric mucosa of the mice should be studied.
H. pylori infection exerts immunomodulation, through the Treg induction notwithstanding some of the protein factors such as GGT and KatA also have a direct immune regulatory effect on the immune and could be harnessed for the development of anti-inflammatory therapy.
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