Dargahi, Narges (2019) Immune modulation using probiotics and multiple sclerosis peptides. PhD thesis, Victoria University.

Abstract

Changes in physiological, immunological and gut microbiome can lead to a range of chronic conditions in humans. The ‘hygiene hypothesis’ identifies the increasing trend of immune-mediated disorders to possibly be a consequence of intestinal dysbiosis, that in turn results in a dysfunctional immune system leading to conditions such as, eczema, asthma, allergies and autoimmune diseases. Therefore, utilization of beneficial probiotic bacteria can increase their abundance within the gastrointestinal lumen, and subsequently modulate immune cells, such as, T helper (Th)-1, Th2, Th17, regulatory T (Treg) cells, B cells, macrophages, dendritic cells and monocytes. Modulation of immune cells are directly related to human health and pathogenesis of immune disorders. Chapter 1a describes the cross talk between probiotics and the gastrointestinal immune system, and their effects in relation to inflammatory bowel disease, multiple sclerosis (MS), allergies and atopic dermatitis. MS is one of the debilitating autoimmune disease of the central nervous system which has been increasing during the past decades. MS severely affects patients’ health, work and quality of life, and its treatment has changed over the last 20 years. In chapter 1b the immunopathology of MS and various available treatments have been investigated. As all the MS immunotherapeutic drugs target relapsing remitting MS (RRMS), in particular developing a treatment for progressive forms of MS is a medical challenge and medical specialists and clinicians are in constant battle with serious treatment challenges for MS. Although β- interferons 1a or 1b and glatiramer acetate are accounted as the most commonly used injectable disease modifying therapies in RRMS, however, one of the major challenge of these types of therapies has been the lack of devotion to treatment among MS patients, with approximately 50% of patients ceasing their therapy plan within the first year. This chapter revisits the basics of the immune-pathophysiology of MS to gain insights in the development of innovative improved drug treatments and presents current drug treatments and new and emerging immune modulating approaches for the immunotherapy of MS. This chapter provides groundwork for vaccine (or immune modulation) development research and the investigation of new potential vaccines (immune modulators) against MS which are used in chapters 5a and 5b. Backtracking probiotics beneficial effects to the host that occur through their contribution to the development and maintenance of a healthy immune system, tracked the steps to the use of some probiotics in the food industry as starter or secondary starter cultures to ferment dairy products. These probiotics include Streptococcus thermophilus (ST); in chapter 2, ST1275, ST285 and ST1342 bacteria were used to determine their modulatory effects on U937 human promonocytic cell line which exhibited differential cytokine induction, in particular, increased secretion of anti-inflammatory IL-4 and IL-10 cytokines were noted. ST also stimulated an increase in the production of CXCL8 and GM-CSF, as well as expression of cell surface markers, CD11c, CD86, C206, CD209, MHC-1. ST285 was determined the most potent probiotic, therefore was considered to investigate further in chapters 3, 4 and 6. The main objective of next study was to assess modulatory and anti-inflammatory properties of ST285 using human peripheral blood mononuclear cells (PBMC) from healthy donors. To fulfil this objective, modifications in the mRNA expression of genes related to innate and adaptive immunity were assessed and results showed strong immune modulatory effects of ST285 to human PBMC with an array of anti-inflammatory properties. ST285 reduced mRNA expression of IL-18, IFNγR1, CCR5, CXCL10, TLR-1, TLR-2, TLR-4, TLR-8, CD14, CD40, CD86, C3, GATA3, ITGAM, IRF7, NLP3, LYZ, TYK2 and IFNR1. ST285 upregulated IL- 1α, IL-1β, IL-6, IL-8, IL-10, IL-23, IFNγ, TNFα, CSF-2 to human PBMC; no changes to mRNA expression of IFNA1, IFNB1, IL-4, IL-5, IL-13, CCL2, CCL5, CCL8, CCR4, CCR8, CXCR3, TLR-3, TLR-5, TLR-6, TLR-9, CD4, CD80, FOXP3, STAT3, CD40LG, HLA-A, HLA-E and RORC were noted. These data demonstrated a predominant anti-inflammatory profile exhibited by ST285, hence, ST285 was validated for further investigation on human monocytes. Some of the beneficial effects attributed to probiotics may be through modulation of the immune system; the effect of ST285 to human monocytes was assessed and a range of immune modulating effects of ST285 by human monocytes was demonstrated. This included significant downregulation in the mRNA expression of IL-1R, IL-18, IFNγR1, IFNαR1, CCL2, CCR5, TLR-1, TLR-2, TLR-4, TLR-5, TLR-6, TLR-8, CD14, CD86, CD4, ITGAM, LYZ, TYK2, IFNR1, IRAK-1, NOD2, MYD88, ITGAM, SLC11A1, and significant upregulation in the mRNA expression of IL-1α, IL-1β, IL-2, IL-6, IL-8, IL-23, IFNγ, TNFα, and CSF-2. ST285 is used in the dairy industry, survives during cold storage, well tolerates upon ingestion, and their consumption may have beneficial effects with potential implications in inflammatory and autoimmune disorders, such as, multiple sclerosis. In order to determine a suitable autoimmune setting to investigate the effects of ST285 in an animal model, it was required to revisit MS treatments to determine the effects of recently developed agonist and antagonist MS vaccines. Encephalitogenic T cells are greatly implicated in the pathogenesis of MS, stimulation of these T calls is triggered by the formation of a tri- molecular complex among the human leukocyte antigen (HLA), an immunodominant myelin basic protein (MBP) epitope, and the T cell receptor (TCR). This next study (chapter 5) concentrated on the rational design and synthesis of non-peptide mimetic molecules, based on the immunodominant MBP83–96 epitope that is recognized by the TCR in complex with HLA, with a focused attention on the inhibition of the tri-molecular complex formation which can consequently lead to the inhibition of proliferation of activated T cells. In view of the interactions between the TCR and the HLA-MBP83–96 complex, a structure-based pharmacophore model was generated and the newly candidate molecules were obtained through the ZINC database, six molecules were synthesized and further evaluated in vitro as TCR antagonists. Analogues 15 and 16 were able to inhibit the stimulation of T cells by the immunodominant MBP83–99 peptide from immunized mice to some extent, and to a lesser degree by analogues 17 and 18 and then by analogue 19, presenting the lead compounds 15 and 16 may be used for immunotherapy against MS. In addition in chapter 5b, the immune modulatory effects of MBP83-99 peptide conjugated or not conjugated to carrier mannan, in either linear or cyclic forms were determined. It was shown that MBP83-99 modulated the immune responses in SJL/J immunized mice which resulted in cytokine secretion by immunized spleen cells which was protective in an experimental autoimmune encephalitis model and protection against axonal spinal damage. Molecular modelling was used to gain insights into the binding mode of the peptide to MHC class II. In the chapter 6, the effects of ST285 to agonist MBP83-99 peptide immunized mouse spleen cells was determined. Agonist peptide induced a Th1 profile, however in the presence of ST285 a significant increase in the expression of anti-inflammatory IL-4, IL-5, IL-10 cytokines,and decreased pro-inflammatory IL-1β and IFN-γ were noted. Regular consumption of probiotic bacteria such as ST285 in the form of capsules, fermented food or dairy products may therefore be beneficial in the management and treatment of autoimmune diseases such as multiple sclerosis. Consumption of probiotics contributes to a healthy microbiome of the GIT leading to many health benefits. They also contribute to the modulation of the immune system and are becoming popular for the treatment of a number of immune and inflammatory diseases.

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