Save your gut save your age: The role of the microbiome in stem cell ageing

Abstract The tremendous importance of microbiota in microbial homoeostasis, alterations in metabolism and both innate and adaptive immune systems has been well established. A growing body of evidence support that dysbiosis or compositional changes in gut microbiota is linked to the ageing of stem cells in terms of dysregulations of metabolism, aberrant activation of the immune system as well as promoting epigenetic instability of stem cell. In this concise review, we elucidate recent emerging topics on microbiotic alterations and underlying mechanisms in stem cell ageing.


| BACKG ROU N D
Ageing is an inevitable process in the majority of organisms. Ageing is associated with the occurrence of a spectrum of diseases including Alzheimer's disease, osteoporosis, type II diabetes and cancer. The problems of ageing and its related healthcare and economic issues result in a heavy burden all around the world. 1,2 Therefore, it is requisite to explore a novel strategy against ageing.
Stem cells are thought to demonstrate high potential in the field of anti-ageing. During ageing, the decline in the self-renewal and differentiation ability of stem cells and exhaustion of stem cells pool have been widely shown in various organs. Interference with ageing-driven dysregulation on self-renewal and differentiation ability of stem cells by remodulation of intrinsic and extrinsic factors should be a solution in the development of anti-ageing drugs.
Factors contributing to stem cell ageing are classified as systemic and micro-environment including hormones, systemic inflammation, host microbiome, local immune system and niche structure. Emerging studies suggest a close relationship between diversity, a constituent of host microbiota with the ageing process and ageing-related diseases. For instance, skeletal mass, bone formation and bone growth are progressively declined with age, while modulation of gut microbiota by administration of antibiotics treatments in conventional mice or administration of specific-gut microbiota in germ-free mice results in a decrease and an increase bone mass and bone growth respectively. The molecular events underlying are commensal bacteria secreted short-chain fatty acids to regulate the hormone for skeletal growth namely insulin-like growth factor-1. 3 Another study also mentioned that disruption of microbiota interferes our normal circadian clock, consequently provoking metabolic disorders accompanied by exaggerating the ageing. 4,5 In mammals, the change in diversity of gut microbiota has been linked to the different age groups. In general, elderly peoples result less Bacteroidetes in terms of quantity and quality and more Firmicutes compared with healthy middle-aged people In southwest China cohort. 6 In experimental models, host microbiota has associated with the lifespan of Drosophila. Studies demonstrated that modulation of gut microbiota in drosophila by antibiotic treatment or stool transplantation from old Drosophila to young Drosophila increases or decreases the lifespan of Dorosphila respectively. 7 However, how host microbiota affects the stem cell functions in term of ageing is still vague.
In this Review, we describe the contributions of host microbiota in stem cell ageing through modulation of metabolism, epigenetic changes as well as the inflammatory responses by the host immune system. We also introduce the possible microbiota-mediated signalling pathways in stem cell ageing.

| Host microbiome and metabolic changes in stem cell ageing
Ageing causes metabolic changes in stem cells. The metabolic changes in ageing stem cells contribute accumulation of mitochondrial damage accompanied with the imbalance between glycolysis and oxidative phosphorylation(OXPHOS) and accumulation of reactive oxygen species (ROS) resulting in depletion of stem cells pool. 8 Metabolic changes in stem cell niches are attributed to the microbiota and its derived metabolites. A recent report has linked microbiota and haematopoietic stem cells(HSCs) differentiation via alteration of metabolic stress. The composition of gut microbiota is reconstituted by a high-fat diet (HFD) in mice and alteration in gut microbiota leads to an increase the ratio of lymphoid cells to myeloid cells, indicating ageing haematopoiesis. 9,10 A similar phenomenon also exhibits in the intestinal stem cells. Acetobacter pomorum, a commensal bacterium residing in Drosophila, regulates host metabolic homoeostasis through insulin/insulin-like growth factor signalling, resulting in enrichment of basal intestinal stem cells numbers. 11 The possible mechanism for gut microbiota modulating host metabolism activity is gut microbial metabolites.
One of the gut microbial metabolites is short-chain fatty acid (SFCA) including acetate, butyrate and propionate. 12 Under normal homoeostasis, a handful amount of SCFA improves the lifespan of the host. For example, Beta-hydroxybutyrate (β-HB) improves the lifespan of C elegans by suppressing histone deacetylase (HDAC) activity and activation of skinhead-1(SKN-1)/NF-E2-related factor (Nrf) pathway, subsequently facilitating the TCA cycle metabolism and ultimately increasing Forkhead box protein (FOXO) activity for stem cell proliferation. 13 Nevertheless, under the conditions of "leaky" gut permeability caused by severe tissue damages and senescence, SFCA exerts their metabolic regulations on host stem cells through binding to G-protein coupling receptors, subsequently suppressing insulin signalling and causing malfunctions of mitochondrial electron transport chain activity accompanied with the imbalance of NAD+/ NADH ratio and dysregulation of NAD-dependent deacetylase sirtuin-1(SIRT1)/peroxisome proliferator activated receptor gamma coactivator 1 alpha(PGC1α) pathway. 14,15 As a result, more damaged mitochondria results along with an accumulation of ROS and imbalance between glycolysis and OXPHOS, eventually erroneous differentiation and proliferation of stem cells and in turn depletion of stem cell. 16 Evidence in support of this notion comes from old HSCs ex- transcriptional co-activator with a PDZ-binding domain (TAZ). 21 In agreement with HMEC, dysfunctions of autophagy in bone marrowderived mesenchymal stem cells result in accelerating ageing as exemplified by diminishing osteogenic differentiation and proliferation capacity, whereas autophagy activator rapamycin antagonizes the autophagy malfunction-provoked senescence. 22 Another type of microbial metabolites is endogenous ethanol.

Bacterially derived ethanol is produced by Proteobacteria including
Escherichia coli and other Enterobacteriaceae. 23 The elevated endogenous ethanol causes the inhibitory effects on proliferating stem cells and depletion of stem and progenitor cells in the human hippocampus. 24 Besides, more ethanol accumulates in the gut resulting in the increase of gut permeability by disruption of epithelial tight junctions especially zone occludins. 25 This allows the translocation of pathogenic bacteria and its endotoxins and ethanol exert more directly destructive effects on tissues. 25 As a consequence, depletion of stem cells pool occurs in order to compensate the damage tissues and accelerating ageing.
In Drosophila, the host microbiota also regulates the self-renewal

| Host microbiome and epigenetic alterations in stem cell ageing
A growing body of evidence showed that modulation of global DNA methylation has been linked to ageing in various stem cells such as HSCs and other tissues. 30,31 Besides, muscle and haematopoietic stem cells exhibit the enhancement of histone repressive marks, such as histone H3 lysine 9 trimethylation (H3K9me3) and histone H3 lysine 27 trimethylation (H3K27me3) during the ageing process. 32 Microbiota may exacerbate the ageing process by releasing more their metabolites butyrate in guts. Butyrate has known as an enhancer for H3K27me3 in vivo model. 33 The possible underlying mechanism for enhanced H3K27me3-modulated epigenetic modulation is increasing H3K27me3-producing enzymes polycomb repressive complexes 2, subsequently facilitating the loss in epigenetic marking as well as drift of H3K27me3, diminishing the genes in glycolysis along with a decline of energy and NADH to NAD + ratio, thereby the stem cells become more susceptible to ROS and propel itself from quiescent state into an active state for differentiation and proliferation. 34 Consistently, repressing the H327Kme3 in both C elegans and D melanogaster exhibited anti-ageing effects. 35,36 Apart from histone modulation in gene expressions, microbiota Moreover, another enhanced transcriptional factor IRFs has been reported in promoting senescence and impairing self-renewal and differentiation of stem cells and consequently shorting lifespan in vivo. 40 In an opposite way, depletion of microbiota reduces the relative methylation level of certain genes such as the toll-like receptor 4 gene, which is a gene associated with chronic inflammation in ageing. 41 Besides the responses of age-associated genes STAT and IRFs are diminished in antibiotic-treated mice. 42

| Host microbiome and immune system responses in stem cell ageing
In addition to modulating host metabolism and epigenetic alterations, host microbiome also affects local immune system during ageing. 49

| Host microbiome and ageing-associated developmental pathway
Host microbiome leads ageing-associated development on stem cells through several pathways including Wnt, Notch, transforming growth factor beta(TGFβ), mitogen-activated protein kinases (p38-MAPK) and JUN N-terminal kinase(JNK) signalling pathways. reporter activity by its HDAC inhibitor characteristic. 71 As shown in Table 1

| Notch and TGF-beta signalling pathways
The Notch signalling pathway is a key regulator to control intestinal epithelium homoeostasis by modulation of self-renewal rate of stem cell and differentiation rate of progenitor cells into various intestinal cell types such as paneth cells, goblet cells. 84 Ageing has been recog-

| p38-MAPK signalling pathway
Ageing associated activation of p38-MAPK and suppression of

| CON CLUS I ON S AND FUTURE PER S PEC TIVE S
The role of stem cells in ageing is repairing damaged tissues and regenerate specific tissues in order to maintain tissues homoeostasis.
During ageing, dysregulation of metabolism, alterations Reconstruction of gut microbiota and in turn restoring gut permeability is a crucial strategy in dealing with the stem cell-associated ageing process. To date, faecal microbiota transplantation brings our attention to the reconstruction of gut microbiota. There is evidence that faecal microbiota transplantation improves the growth performance by reducing intestinal permeability in piglets and maintaining intestinal morphology. 99 Since increasing intestinal permeability has been known in ageing-associated diseases, restoring intestinal permeability by faecal microbiota transplantation may be an effective and regenerative medicine strategy in stem cell development for aged. However, it remains to be investigated whether fecal microbiota transplantation from young donors to old receivers restores the self-renewal, differentiation and regenerative capacity of stem cells in order to ameliorate the health span. In conclusion, more investigations in the cross-talk between microbiota and intestinal stem cells are required to pave the way for the development of therapeutic drugs in prolonging lifespan and tackling ageing-associated diseases.

CO N FLI C T O F I NTE R E S T S
The authors declare that they have no competing interests.