The gut microbiome describes a diverse range of bacteria, viruses, and other microbes that reside in the gut. It possesses many functions including digestion, vitamin production, and maintaining immunity [1]. The gut microbiome can generate short chain fatty acids, or SCFAs, that influence how the host’s immune cells behave. Some examples of SCFAs are acetate, propionate, and butyrate. They are synthesized in the large intestine from complex carbs, like dietary fiber, through the process of bacterial fermentation [2]. It has been shown that increasing dietary fiber in the diet of mice increases SCFA levels, as expected, and defends the mice from autoimmunity and allergies [3].
Because SCFAs are able to cross intestinal tissue to eventually affect mucosal immune responses, they have been discussed as a novel therapy for inflammatory immune responses associated with autoimmune diseases or as an anti-cancer therapy. As SCFAs can manipulate the function of various types of immune cells, it has been shown that a high dietary fiber diet supplemented with intestinal butyrate suppresses colorectal cancer [4]. It has also been shown that butyrate, a specific SCFA, can stimulate programmed cell death, or apoptosis, of cancer cells [5].
The main question that the authors of this paper are asking is how do SCFAs regulate immune responses in order to optimize therapeutic potential of SCFAs. More specifically, they are asking if they are capable of regulating CD8+ T lymphocytes (CTLs). A CTL is a type of white blood cell, a T cell, that kills infected or damaged cells via antigen-T cell receptor binding, seen below. An antigen is a toxin that causes an immune response. CTLs then release toxins, called cytotoxins, that cause apoptosis.
Source: http://what-when-how.com/acp-medicine/immunologic-tolerance-and-autoimmunity/
Typically, SCFAs serve two functions:
1. Interact with receptors on the T cell surface
When SCFAs bind to their receptors, various signaling pathways are stimulated. Some lead to the production of cytokines, like granzyme B and IFN-gamma.
2. Inhibit HDAC enzymes intracellularly
HDAC enzymes are histone-deacetylating meaning they remove a chemical group, called an acetyl, from proteins associated with DNA, allowing it to become more condensed. SCFAs are HDAC-inhibiting meaning they prevent the enzymes from removing the acetyl group and thus promote transcription of those regions of the DNA. Transcription means the genes in that region of the DNA are expressed.
Source: https://www.quora.com/Why-does-histone-acetylation-generally-increase-protein-transcription
The researchers then wanted to see if a specific cytotoxin, granzyme B, was influenced by SCFA treatment as it is a CTL-associated effector molecule and a commonly released cytokine [6]. They again exposed the CTL cells to acetate, propionate and butyrate for 3 days but this time measured production of granzyme B. They found that butyrate greatly increased the frequency of granzyme B producing TCLs.
Their next major finding was that SCFA-receptors GPR41 and GPR43 are not involved in butyrate’s regulation of CTLs. As described above, this is usually one of SCFAs’ two functions. The authors discovered this by testing CTLs that lacked the receptors to see if the cells increase cytotoxin production once exposed to butyrate. They saw no difference in the frequency of IFN-gamma cells in the population lacking the receptors compared to the cells with the receptors, meaning the receptors are not involved in the butyrate-dependent regulation. They also saw this lack of difference when they performed an equivalent experiment in mice.
The increase in cytotoxin expression is regulated by HDAC-inhibition by SCFAs rather than by their binding with their receptors. However, the increase in IFN-gamma expression is dependent upon concentration of butyrate. There is a threshold concentration that causes apoptosis. Low(er) concentrations of butyrate (0.25mM) are able to start the increase of IFN-gamma. Butyrate had an greater ability to influence gene expression compared to the other SCFAs tested.
They then looked at the at specifically where the acetylation occurred in CTLs. They looked at the acetylation of two genes, called Tbx21 and Ifn-gamma (known to be associated with CTLs) after butyrate exposure. They found that the butyrate caused more acetylation of two genes so they can be expressed. Results lead to the idea that the HDAC inhibitory function of butyrate may be involved in CTL-associated gene activation in CTLs.
A major component of cytotoxin expression in CTLs is epigenetics, or altering what genes are expressed without changing the actual genetic information. HDAC inhibition, an epigenetic mechanism, led to the increase in cytotoxin expression. Thus, enhancing CTL reactivity may be a be a way to improve T cell anti-tumor therapy. They are suggesting that the SCFA butyrate may offer an anti-cancer function by boosting CTL function. The authors saw that when butyrate was introduced, the expression of other key molecules involved with the combat of tumors increased. While low concentrations of butyrate is naturally occurring, the authors suggest that increasing this concentration may be useful as an anti-cancer therapy.
A future direction for this area of research may be more genetics-focused. While it is known that Tbx21 and Ifn-gamma encode cytokines, how do they function in the context of T cell therapy? Are there other important regulatory genes involved? Research could be focus on why the receptors are not involved in butyrate-mediated regulation of CTLs. As this receptor-SCLA interaction has been previously identified, why or how is it not involved in this process? Additionally, as the microbiome has direct effects in the gut-brain axis, it may be interesting to see how SCLA therapeutics effects the central nervous system.
[1] https://www.gutmicrobiotaforhealth.com/en/about-gut-microbiota-info/
[2] Koh, A., De Vadder, F., Kovatcheva-Datchary, P. & Backhed, F. From Dietary Fiber to Host Physiology: Short-Chain Fatty Acids as Key Bacterial Metabolites. Cell 165, 1332–1345, https://doi.org/10.1016/j.cell.2016.05.041 (2016).
[3] Trompette, A. et al. Gut microbiota metabolism of dietary fiber influences allergic airway disease and hematopoiesis. Nature medicine 20, 159–166, https://doi.org/10.1038/nm.3444 (2014);
[4] Singh, N. et al. Activation of Gpr109a, receptor for niacin and the commensal metabolite butyrate, suppresses colonic inflammation and carcinogenesis. Immunity 40, 128–139, https://doi.org/10.1016/j.immuni.2013.12.007 (2014).
[5] Domokos, M. et al. Butyrate-induced cell death and differentiation are associated with distinct patterns of ROS in HT29-derived human colon cancer cells. Digestive diseases and sciences 55, 920–930, https://doi.org/10.1007/s10620-009-0820-6 (2010).
[6] https://www.ncbi.nlm.nih.gov/gene/3002
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