For body physiology to function properly, interplay betweenorgan systems is essential. Dysfunction in one organ or organ system can leadto abnormal bodily functions, and subsequent pathology. Abnormal functioning in an organ system can have a knock-oneffect in another organ system, and lead to further dysbiosis. Thebrain-gut-microbiota axis is one such example of systems in balance.
It is awell-established bidirectional communication network between the centralnervous system, the gastrointestinal tract, and the gut microbiota1.The gut microbiota is a collection of billions of micro-organisms that coloniseour gastrointestinal tract. While the relationship between the CNS andgastrointestinal tract is relatively well established in scientific literature,the role of the gut microbiota in regulating brain and gut behaviour is nowbecoming an area of interest. Recent research shows the number of gut microbiotamatch the number of cells in our body, if not slightly outnumber them2,and contain as much as 150 times as many genes as in our human genome2.It is logical, therefore, to assume it is a significant mediator on both thecentral nervous system, and the gastrointestinal tract Thereis a wealth of information describing many direct and indirect pathways throughwhich the gut microbiota influences both the central nervous system and thegastrointestinal tract.
Studies3 show that the gut microbiota isessential for normal gut motility, and barrier permeability. The immune system,through the release of pro-inflammatory cytokines, can alter the gutmicrobiota. The hypothalamic-pituitary-adrenal axis and cortisol secretion alsoinfluence the gut microbiota composition4. Cortisol can change gutpermeability, and alter the absorption of nutrients and short-chain fatty acids5.
The gut microbiota, through the anaerobic metabolism of dietary fibre, releaseshort chain fatty acids (butyrate, proprionate, and acetate), which are animportant energy source for humans6. Anothermechanism of action through which the gut microbiota influence the centralnervous system is through neurotransmitter modulation, particularly viaregulation of tryptophan metabolism.1.1 Tryptophan Tryptophanis an essential amino acid sourced in the diet. In the host, it exists in twocirculating forms; a free form and an albumin-bound form.
Tryptophan can bemetabolised down two pathways; about 10% of circulating tryptophan takes partin serotonin synthesis7, while most of it is metabolised tokynurenine. This kynurenine pathway of tryptophan metabolism is mediated by theenzymes tryptophan 2,3-diogygenase (TDO) and indoleamine 2,3-dioxygenase (IDO).These two enzymes make up the first and rate limiting step of kynureninesynthesis, and are stress and immune mediated, respectively.
Under pro-inflammatoryand stress conditions, IDO and TDO activity is upregulated by cytokines andcortisol. This increases the metabolism of tryptophan down the kynureninepathway. This increasein kynurenine synthesis has potentially negative down-stream effects in twoways; firstly, it decreases the amount of tryptophan available for serotoninsynthesis. Secondly, it increases kynurenine metabolites- quinolinic acid, whichhas neurotoxic effects at increased levels, and kynurenic acid, which, althoughit has neuroprotective effects against quinolinic acid toxicity8, can induce cognitive impairment whenabnormally elevated9. Researchhas been carried out previously to understand the microbial regulation oftryptophan. Studies have established that the gut microbiota is essential fornormal brain development and behaviour, and affects tryptophan synthesis. Forexample, Clarke et al10 have shown that male germ free mice haveincreased plasma tryptophan concentrations compared to their conventionallycolonised controls. It has also been shown in the same study that plasma kynurenine:tryptophan ratios (used as a marker for IDO and TDO activity) were decreased inboth male and female germ-free cohorts compared to conventionally colonisedcontrols.
Koreckaet al11 looked at the mechanisms behind the regulation of tryptophanavailability and metabolism. They measured the levels of expression of severalhepatic genes involved in tryptophan metabolism, and compared them to theexpression of the same genes in conventionally colonised mice. They foundreduced levels of IDO in the liversof germ-free mice as compared to conventionally colonised controls. They alsofound reduced levels of AhR and AhRR, two genes upon which thedownstream metabolites of kynurenine act.
However, they neglected to assess TDOactivity. TDO is the key hepatic enzyme involved in tryptophan metabolism, andmay be one of the mechanisms through which the gut microbiota influencetryptophan. 1.2 Butyrate Butyrateis a short chain fatty acid produced by anaerobic metabolism of dairy productsand fibre by the gut nicrobiota. It is partly absorbed in the intestine,travels via the enterohepatic circulation to the liver, and is processed here,where it may affect metabolic processes in hepatocytes, and subsequentlyregulate host physiology12. For example, butyrate stimulatesserotonin secretion from enterochromaffin cells in the gut, thus stimulatinggut motility13. Whilenot directly exerting an effect on the brain, butyrate influences CNSfunctioning indirectly, for example via the immune system or the enteric orvagal nervous system14. Butyratehas also been shown to exert significant effects on CNS functioning whenadministered at high levels, namely increasing neuronal plasticity andimproving long term memory function14.
Koreckaet al showed that butyrate could marginally induce the expression of AhR and AhRR in germ free mice11. Therefore, we decided to alsosupplement our mice with butyrate, to determine the influence it may have on thegenes selected for this study. We also supplemented conventionally colonisedmice with butyrate, to observe the influence it has on basal gene expressionlevels.