Immunmodulatory Mechanisms of Butyrate

The four-carbon fatty acid butyrate plays a central role in the homeostasis of the gastrointestinal tract. Apart from its well known function as an essential energy source for colonocytes and its potential cancer-suppressing capabilities, recent data convincingly demonstrate that butyrate has clear antiinflammatory effects in mucosal inflammation by modulating critical effector molecules of the immune system in vivo as well as affecting distinct immune cells in vitro. Investigating the detailed molecular and functional mechanisms of butyrate in various immunological models will help to understand the fundamental role of this molecule in basic physiology as well as in pathology. Furthermore, the detailed knowledge of the principles governing the actions of this SCFA will contribute to exploit new antiinflammatory drugs having more selectivity and fewer side effects than currently used immunotherapeutics. We, therefore, propose the following aims to be investigated in the course of this project: 1) To determine the impact of the bacterial product butyrate on all important stages of the dendritic cell life cycle, since these cells are responsible for the initiation of T cell-mediated immunity. The experiments in this section are designed to reveal whether butyrate interferes with the phenotypical and functional characteristics of immature as well as mature DC. Emphasis will be given on critical intracellular events of DC function, such as NF-kB activation, a recently reported target of butyrate in several cell types. 2) To characterize the functional capability of butyrate to alter the priming capacity of DC. We will determine cytokine production with regard to Th1 and Th2/3 cytokines in T cells that are exposed to butyrate-treated DC. The nature of the evolving T cell response will be studied in restimulation cultures using naive T cells. 3) To determine the action of butyrate on the production of COX-2 and PGE2 in both human monocytes and DC by the analysis of intracellular signaling and DNA-binding elements. 4) To establish the molecular basis of butyrate-based T-cell tolerance. It shall be elucidated whether the influence on transcriptional regulation by butyrate is responsible for downregulating T-cell responsiveness. 5) To define the role of butyrate and bacteria in human patients suffering from IBD. Since several reports point to a utilization defect of butyrate in mucosal inflammation, the interesting possibility exists that distinct components of the human immune system are resistant to the antiinflammatory effects of butyrate in IBD.

In the present project our knowledge about the immunomodulatory role of n-butyrate has been significantly extended by adding new insights into the effect of this short chain fatty acid on the functionality of a variety of cells relevant to the immune system like dendritic cells, monocytes, mast cells, T cells and endothelial cells. In parallel experiments inhibitors for selected signaling events were tested in order to compare the impact of such interference with the effects observed after butyrate treatment. It has been shown that dendritic cell differentiation as well as maturation was severely repressed by n-butyrate as reflected by impaired cytokine and surface marker expression characteristic for mature dendritic cells and reduced allostimulatory capacity. The effects were associated with reduced transactivation of a central transcription factor (NF-kB). Selectively interfering with this transcription factor resulted in similar alterations in the life cycle of dendritic cells. Here we were particularly successful also to demonstrate induction of allospecific hyporesponsiveness in primary T cells by pretreated dendritic cells. Interestingly, this effect was not impaired by the concomitant presence of calcineurin inhibitors or in patients receiving such immunosuppressive treatment. This might have important implications for future tolerance protocols, since they will have to be established under the cover of the presently administered immunosuppressive regimens. Investigation of the impact of interfering with the intracellular signaling molecule Janus kinase 3 revealed this molecule to be a very promising candidate for immunosuppression aimed at acting at different stages of immune cell activation but at the same time sparing other cells of the body because of its expression restricted to the immune cell compartment. Both T cell function and dendritic cells were profoundly repressed, which was in perfect agreement with promising in vivo experiments by others who showed remarkably improved of transplant survival rates. Finally, the most abundant urinary glycoprotein, uromodulin, has been tested for its impact on dendritic cell function and was shown to act as an endogenous activator of a pattern recognition molecule (TLR-4) known to activate the innate arm of immunity for priming specific immune responses. Therefore uromodulin, the function of which has been unknown for decades, appears to be an important candidate involved in the resolution of urinary tract infections.