Immunomodulatory effects exerted by Lactobacillus salivarius strains on innate immune cells

Despite increased application of commensal bacteria for attempting to improve the symptoms of a variety of inflammatory conditions, including inflammatory bowel diseases, diarrhoea and irritable bowel syndrome, therapeutic approaches that involve live bacteria are hampered by a limited understanding of bacterium-host interactions. Lactobacilli are natural inhabitants of the mammalian gastrointestinal tract and many lactobacilli are regarded as probiotics meaning that they exert a beneficial influence on the health status of their consumers. Modulation of immune responses is a plausible mechanism underlying these beneficial effects. The aim of this thesis was to investigate the effect of 33 Lactobacillus salivarius strains on the production of inflammatory cytokines from a variety of human and mouse immune cells. Induction of immune responses in vitro was shown to be bacterial- and mouse strain-dependent, cell type-dependent, blood donor-dependent and bacterial cell number-dependent. Collectively, these data suggest the importance of a case-by-case selection of candidate strains for their potential therapeutic application. Toll-like receptors (TLRs) recognize microbe-associated molecular patterns (MAMPs) and play a critical role in shaping microbial-specific innate and adaptive immune responses. Following ligand engagement, TLRs trigger a complex network of signalling that culminate in the production of inflammatory mediators. The investigation of the molecular mechanisms underlying the Lb. salivarius-host interaction resulted in the identification of a novel role for TLR2 in negatively regulating TLR4 signalling originated from subcellular compartments within macrophages. Notably, sustained activation of JAK/STAT cascade and M1-signature genes in TLR2-/- macrophages was ablated by selective TLR4 and JAK inhibitors and by absence of TLR4 in TLR2/4-/- cells. In addition, other negative regulators of TLR signalling triggered by Lb. salivarius strains were found to be the adapter molecules TIRAP and TRIF. Understanding negative regulation of TLR signalling may pave the way for the development of novel therapeutics to limit inflammation in multiple diseases.

Stress-induced visceral pain in rodents: neurochemical, hormonal, immune and epigenetic mechanisms

Visceral pain is a debilitating disorder which affects up to 25% of the population at any one time. It is a global term used to describe pain originating from the internal organs, which is distinct from somatic pain. Currently the treatment strategies are unsatisfactory, with development of novel therapeutics hindered by a lack of detailed knowledge of the underlying mechanisms. The work presented in this thesis aimed to redress this issue and look in more detail at the molecular mechanisms of visceral pain in preclinical models. Stress has long been implicated in the pathophysiology of visceral pain in both preclinical and clinical studies. Here a mouse model of early-life stress-induced visceral hypersensitivity was validated. Moreover, mouse strain differences were also apparent in visceral sensitivity suggesting a possible genetic component to the underlying pathophysiology. Furthermore, gender and sex hormones were also implicated in stress sensitivity and visceral pain. Using the rat model of maternal separation, some of the epigenetic mechanisms underpinning visceral hypersensitivity, specifically the contribution of histone acetylation were unravelled. Glutamate has been well established in somatic pain processing, however, its contribution to visceral pain has not been extensively characterised. It was found that glutamate uptake is impaired in viscerally hypersensitive animals, an effect which could be reversed by treatment with riluzole, a glutamate uptake activator. Moreover, negative modulation of the metabotropic glutamate (mGlu) receptor 7 was sufficient to reverse visceral hypersensitivity in a stress sensitive rat strain, the Wistar Kyoto rat. Furthermore, toll-like receptor 4 (TLR4) was implicated in chronic stress-induced visceral hypersensitivity. Taken together, these findings have furthered our knowledge of the pathophysiology of visceral pain. In addition, we have identified glutamate transporters, mGlu7 receptor, histone acetylation and TLR4 as novel targets, amenable to pharmacological manipulation for the specific treatment of visceral pain.

Lost in translation? The potential psychobiotic Lactobacillus rhamnosus (JB-1) fails to modulate stress or cognitive performance in healthy male subjects

Background: Preclinical studies have identified certain probiotics as psychobiotics a live microorganisms with a potential mental health benefit. Lactobacillus rhamnosus (JB-1) has been shown to reduce stress-related behaviour, corticosterone release and alter central expression of GABA receptors in an anxious mouse strain. However, it is unclear if this single putative psychobiotic strain has psychotropic activity in humans. Consequently, we aimed to examine if these promising preclinical findings could be translated to healthy human volunteers. Objectives: To determine the impact of L. rhamnosus on stress-related behaviours, physiology, inflammatory response, cognitive performance and brain activity patterns in healthy male participants. An 8 week, randomized, placebo-controlled, cross-over design was employed. Twenty-nine healthy male volunteers participated. Participants completed self-report stress measures, cognitive assessments and resting electroencephalography (EEG). Plasma IL10, IL1β, IL6, IL8 and TNFα levels and whole blood Toll-like 4 (TLR-4) agonist-induced cytokine release were determined by multiplex ELISA. Salivary cortisol was determined by ELISA and subjective stress measures were assessed before, during and after a socially evaluated cold pressor test (SECPT). Results: There was no overall effect of probiotic treatment on measures of mood, anxiety, stress or sleep quality and no significant effect of probiotic over placebo on subjective stress measures, or the HPA response to the SECPT. Visuospatial memory performance, attention switching, rapid visual information processing, emotion recognition and associated EEG measures did not show improvement over placebo. No significant anti-inflammatory effects were seen as assessed by basal and stimulated cytokine levels. Conclusions: L. rhamnosus was not superior to placebo in modifying stress-related measures, HPA response, inflammation or cognitive performance in healthy male participants. These findings highlight the challenges associated with moving promising preclinical studies, conducted in an anxious mouse strain, to healthy human participants. Future interventional studies investigating the effect of this psychobiotic in populations with stress-related disorders are required.