Investigation on the utilization of beneficial microbes in modulating the gut microbiota and metabolic profiles in ageing

Ageing is characterized by an inevitable, time-dependent decline in physiological functions and adaptive capacity. Specifically, altered gastrointestinal (GI) physiology can affect the nutrient absorption as well as intestinal microbiota. The intestinal microbiota is now considered as a key player in human health and well-being. Apart from host genetics, diet, antibiotic use, sex hormones, and circadian rhythm, age appears to be a vital factor in altering the GI microbial composition. Moreover, the distribution of the microbiota differs according to the location in the GI tract. Thus, imbalances in microbiota or dysbiosis during ageing may not be limited to fecal microbiota and extend to the other parts of the GI tract, especially, the cecum and colon. The age-related imbalances in microbiota may lead to changes in metabolic phenotype and may influence the development of the mucosal immune system. Given the potential for microbiota to change with age and its influence on host metabolism and immune system, modulation of microbial composition offers an opportunity to promote digestive health. Probiotics are natural, safe and beneficial modulators of the gut microbial composition and metabolic phenotype. Dietary supplementation of probiotics in all age groups has yielded promising outcomes. However, to be classified as a probiotic, a dietary compound must survive GI transit, adhere to the intestinal epithelium and demonstrate immune-modulatory effects. Therefore, the overall aim of the study was to understand microbial changes in ageing mice to provide dietary mechanisms utilizing probiotics to promote gut health. However, to confer any health benefits, probiotics need to survive through the hostile environment of the digestion process in sufficient numbers (at least 10\\(^7\\) colony forming units (CFU)), tolerating acids, bile, and pancreatic digestive enzymes, and finally, adhere to the intestinal epithelium in the colon. The objectives of this study were (i) to evaluate the in in vitro efficacy of different probiotics strains, (ii) to examine whether the role of Lactobacillus acidophilus DDS-1 can modulate the host metabolic phenotype under the condition of age-affected gut microbial shifts in young and ageing C57BL/6 mice, (iii) to investigate the effect of L. acidophilus DDS-1 supplementation on cecal and mucosal-associated microbiota, short-chain fatty acids (SCFA) and immunological profiles in young and ageing C57BL/6J mice. This thesis consists of seven chapters. Chapter 1 is an overall introduction to the rationale of the research, hypothesis, and aims of the research project. Chapter 2 is a review and critique of the literature and summarises the role of microbiota on the human lifecycle. Chapter 3 is a critical and systematic review of potential therapeutic interventions such as antibiotics, probiotics and fecal microbiota transplantation for dysbiosis in the ageing population. Chapter 4 provides information on in vitro probiotic efficacy parameters including digestion simulation, adhesion to colonic cells and immune-modulation property, which enabled to select the right probiotic strain, L. acidophilus DDS-1. Chapter 5 and chapter 6 details research experiments and data generated that characterize clinical parameters, histopathology, immunological profiling, integrated metabolomics and microbiota analysis with and without DDS-1 supplementation in young and ageing C57BL/6 mice. Chapter 5 present the effect of L. acidophilus DDS-1 on fecal microbiota and its associated metabolic phenotype. Chapter 6 describes the research experiments and data generated that detail the effect of L. acidophilus DDS-1 on cecal and colonic-mucosa associated microbiota, SCFA production, immune response in young and ageing C57BL/6 mice. The last Chapter 7 is a comprehensive summary of all the results obtained from the research objectives and includes potential directions for future work arising from this research project. Probiotic strains utilized in in vitro study include L. acidophilus DDS-1, Bifidobacterium animalis ssp. lactis UABla-12, L. plantarum UALp-05 and Streptococcus thermophilus UASt-09. Screening assays such as in in vitro digestion simulation, adhesion, cell viability, and cytokine release were used to evaluate the probiotic potential. All strains showed good resistance in the digestion simulation process, especially DDS-1 and UALp-05, which survived up to a range of 10\\(^7\\) to 10\\(^8\\) CFU/mL from an initial concentration of 10\\(^9\\) CFU/mL. Two human colonic mucus-secreting cells, HT-29 and LS174T, were used to assess the adhesion capacity, cytotoxicity/viability, and cytokine quantification. All strains exhibited remarkable adhesion capacity. No significant cellular cytotoxicity or loss in cell viability was observed. DDS-1 and UALp-05 significantly upregulated anti-inflammatory IL-10 and downregulated pro-inflammatory TNF-˜í¬± cytokine production. All the strains were able to downregulate IL-8 cytokine levels. Taken together, DDS-1 demonstrated superior survival rates, excellent adhesion capacity and strong immunomodulatory effect under different experimental conditions. The in vivo study was carried out to investigate whether four weeks of DDS-1 supplementation can modulate the host metabolic phenotype under the condition of age-affected gut microbial shifts in young and ageing C57BL/6J mice. Collected fecal samples were analyzed using 16S rRNA gene sequencing for identifying gut microbiota and untargeted gas chromatography-mass spectrometry metabolomics analysis. Gut microbial shifts were observed in the control groups (young and ageing) leading to an alteration in metabolism. Principal coordinate analysis of microbiota indicated distinct separation in both the DDS-1-treated groups. L. acidophilus DDS-1 increased the relative abundances of beneficial bacteria, such as Akkermansia muciniphila and Lactobacillus spp. and reduced the relative levels of opportunistic bacteria such as Proteobacteria spp. Metabolic pathway analysis (MetPA) identified ten key pathways involving amino acid metabolism, protein synthesis and metabolism, carbohydrate metabolism and butanoate metabolism. The key metabolic pathways identified by MetPA play a vital role in probiotic-induced gut microbiota-associated metabolic changes. To the best of our knowledge, this is the first detailed study that demonstrates an integrated pattern recognition approach to understand the changes in the ageing gut by exploring the metabolic network with and without probiotic supplementation. Furthermore, we observed microbial shifts in the cecum and colonic mucosal samples in the control groups (young and ageing), with higher inter-individual variation in the mucosal-associated microbiota, leading to an alteration in SCFA levels and immune responses. DDS-1 treatment increased the abundances of beneficial bacteria such as Akkermansia spp. and Lactobacillus spp. more effectively in cecal samples than in mucosal samples. DDS-1 also enhanced the levels of butyrate while downregulating the production of inflammatory cytokines (IL-6, IL-1˜í‚â§, IL-1˜í¬±, MCP-1, MIP-1˜í¬±, MIP-1˜í‚â§, IL-12, and IFN-˜í‚â•) in serum and colonic explants. To sum up, I have generated new data and knowledge on the role of microbiota in health and disease in the ageing murine model. Our results demonstrated a major shift in microbial composition from young to ageing (which may lead to dysbiosis), observed not only in fecal microbiota but also in the cecum and colonic-mucosa-associated microbiota. Our findings suggest that modulation of microbiota by L. acidophilus DDS-1 results in improvements in immunological and metabolic profiles in ageing mice. Our investigations highlight the crucial role of beneficial microbes/probiotics in determining and modulating the metabolic profile in the healthy-ageing gut. Similar approaches could be used to identify and target specific health-promoting metabolic pathways in young and ageing populations. This study highlights the importance of evaluating the efficacy of probiotics in in vitro as well as in clinically relevant experimental models before progressing to human clinical trials. Finally, L. acidophilus DDS-1 showed promising probiotic efficacy in in vitro as well as in in vivo and could be a right candidate for clinical studies in ageing individuals.