Mechanism and action site of tolerance induction by the short-chain fatty acid n-Butyrate

Several investigators have recently demonstrated effects of short-chain fatty acids on immune cells and established a new and unique function for this naturally occurring substance. We have proven the efficacy of n-butyrate in establishing tolerance against allogeneic antigens showing that addition of this compound to a human mixed lymphocyte culture induced specific downregulation of T-cell proliferation against priming alloantigens leaving the response against unrelated third-party antigens or polyclonal activators intact. Induction of hyporesponsiveness was preventable if Cyclosporin A was present during the priming period suggesting that specific downregulation of T- cell alloreactivity by n-butyrate depends on a calcium-dependent T-cell receptor-mediated signal. Pretreatment of APCs by n-butyrate led to a differential regulation of critical costimulatory receptors, induction of apoptosis and resulted in a dose- and time-dependent downregulation of their capability to stimulate T-cell responses. We now want to further elucidate the mechanisms underlying the observed antigenspecific downregulation by n- butyrate. We plan to investigate which type of cell affected by this compound is responsible for the induction of hyporesponsiveness and will focus our interest especially on activated human T-cells subjected to the influence of n-butyrate by characterizing in detail their phenotype, intracellular signalling pathways and pattern of cytokine production; antibody-based T-cell stimulation (OKT-3) and mixed lymphocyte cultures MLCs) will be used to define the mechanisms of the observed alloantigen-specific hyporesponsiveness (deletion, anergy, suppression, immune deviation). Furthermore it is our purpose to characterize the precise site of action of this compound using various molecular probes each known to share particular effects of the pleiotropic acting short-chain fatty acids. The proposed experiments should provide deeper insight into the requirements, mechanisms and consequences of antigen-specific nonresponsiveness by a substance, the importance of which is underlined by its physiological occurrence in the gastrointestinal tract, where an involvement in immune regulation might be envisaged as well. Finally a more detailed understanding of the mechanisms leading to tolerance phenomena in vitro could form the basis to establish new strategies for the induction of clinical tolerance.