Metabolic and immunologic control of intestinal cell function by mTOR

Abstract

The intestinal epithelium is one of the most quickly dividing tissues in our body, combining the absorptive advantages of a single layer with the protection of a constantly renewing barrier. It is continuously exposed to nutrients and commensal bacteria as well as microbial and host-derived metabolites, but also to hazards such as pathogenic bacteria and toxins. These environmental cues are sensed by the mucosa and a vast repertory of immune cells, especially macrophages. A disruption of intestinal homeostasis in terms of barrier interruption can lead to inflammatory bowel diseases and colorectal cancer, and macrophages have an important role in restoring epithelial function following injury. The mammalian/mechanistic target of rapamycin (mTOR) signalling pathway senses environmental cues and integrates metabolic responses. It has emerged as an important regulator of intestinal functions in homeostasis and disease. In this review, we are going to discuss intestinal mTOR signalling and metabolic regulation in different intestinal cell populations with a special focus on immune cells and their actions on intestinal function.

Introduction

The gastrointestinal (GI) tract is responsible for food uptake and digestion while withstanding mechanical abrasion, extreme pH variations and bacterial colonisation. It performs these tasks by a combination of the particular crypt–villus structure and its continuous epithelial regeneration (1). In fact, the intestinal epithelium is one of the most proliferative mammalian tissues, renewing approximately every five days (2,3). The duodenum has the longest villi, which subsequently decrease in length along the digestive tract until they are completely absent in the colon. The crypt is formed by an invagination of the intestinal wall and represents a highly protective environment for intestinal stem cells (ISCs). At its bottom, the ISCs give rise to progenitor cells (i.e. transit-amplifying cells) that migrate upwards while dividing and differentiating into functional cells. In that way, only post-mitotic cells are exposed for a short time to the hazards of the digestive tract (1,4).

Table 1 contains a summary of the different cell types and their respective functions found in the GI tract. The ISC niche provides factors (e.g. Wnt, Notch, Bmp) necessary for stem cell maintenance and differentiation (1); this niche consists of an epithelial component [Paneth cells (PCs)] in the small intestine (SI) (5) and deep crypt secretory cells (DCS cells) in the colon (6) as well as mesenchymal telocytes (7–13). Immune cells, such as macrophages, are also part of the niche and macrophage-depleted animals have a reduction in both PCs and stem cells (14). Macrophages have an important role in restoring epithelial function following injury (15–17).

Table 1. Cell types present in the GI tract and their functions.

Cell type	Function	Markers
Enterocytes	Absorptive lineage. Nutrient uptake.	Alkaline phosphatase
M-cells	Absorptive lineage. Overlie Peyer’s patches and sample the intestinal lumen to transport antigens to the lymphoid cells underneath.	Glycoprotein 2 (GP2)
Goblet cells	Secretory lineage. Mucus production, mucosal immune responses. Their number increases along the GI tract.	Mucins (Muc2)
Paneth cells (PCs) in small intestine (SI); deep crypt secretory cells (DCS cells) in colon	Secretory lineage. Maintain intestinal stem cell niche by nurturing and protecting ISCs. Release antimicrobial products as well as Wnt ligands, EGFs and Notch). In contrast to the SI, in colon no epithelial Wnt-producing cell has been identified although WntR are expressed on colonic stem cells. The colonic crypt seems to rely exclusively on non-epithelial sources for Wnt ligands.	Lysozyme (Lyz1), cKit, Reg4, Dll1, Dll4, Cd44, Cd24, etc.
Intestinal stem cells (ISCs)	Are located between PCs and DCS cells. The main markers are Lgr5, Ascl2 or Olfm4. As they start to differentiate, they express markers such as Bmi1, Hopx or mTert. The majority of papers found in the literature are based on studies of PCs in [Au: OK?] SI.	Lgr5, Ascl2, Olfm4, Cd44
Enteroendocrine cells	Secretory lineage. Release hormones.	Chromogranin A (ChgA)
Tuft cells	Secretory lineage. Defense against helminths.	double cortin- like kinase 1 (DCLK1)

EGFs, epidermal growth factors; WntR, Wnt receptor.

The GI tract is under repeated regenerative pressure during the lifespan of an organism because of the continuous exposure of the intestinal epithelium to pathogenic bacteria and food-derived or environmental toxins and, therefore, stem cell dysfunction can increase during aging and is particularly evident in the barrier epithelium, resulting in dysplasia, degenerative diseases and cancers (1,18,19). Epithelial injury leads to cellular damage with the formation of erosions and ulcers, which are the basis for the formation of inflammatory bowel diseases (IBDs), such as Crohn’s disease (CD) and ulcerative colitis (UC) (3). Interestingly, the malfunction of macrophages is also associated with IBD formation (20). IBDs are characterized by both acute and chronic inflammation of the intestine with multifactorial ethology (21) and individuals that suffer from these inflammation disorders have higher risks of developing colorectal cancer (CRC) (22–24).

Mammalian/mechanistic target of rapamycin (mTOR) is an evolutionarily conserved serine/threonine protein kinase that senses various intracellular and extracellular stimuli, integrating them to coordinate energy metabolism, proliferation, survival (25,26) and inflammation (27). mTOR signalling is linked to IBD pathogenesis (28–30), being involved for example in the regulation of immune cell differentiation (31), autophagy (32) and tissue recovery (33). By regulating the differentiation state of the intestinal epithelium, mTOR was identified as a potential driver of inflammation (34). In this review, we are going to discuss the role of mTOR signalling in the intestine in different cell populations at steady state and during disease. A particular focus will be given on intestinal cell proliferation and its link to mTOR activity (28,29,33,35–39). As mTOR is a well-known rheostat of cellular metabolism (25,27), we will also discuss some recent data about how metabolism and specific metabolites contribute to intestinal homeostasis (28,40–46).

mTOR signalling

mTOR exists in two complexes and regulates a broad set of basic cellular and metabolic processes. mTOR complex 1 (mTORC1) and mTORC2 have distinct functions regarding cell growth, proliferation, survival, energy homeostasis and inflammation (25,27,47–50). They are strongly implicated in pathological conditions, such as cancer (47,48,51–55), diabetes, obesity or neurodegenerative diseases (47,48,56). Interestingly, the repression of mTOR extends lifespan in different organisms, including mice and flies (57). Our review mainly focuses on mTORC1 signalling, a major sensor of the organismal nutritional state that promotes anabolic processes (biosynthesis of proteins, lipids and organelles) and limits catabolic processes (autophagy) (26) (Fig.1).

Fig. 1. The central role of mTORC1 in general and intestine-related metabolism.

Akt is one of the main upstream activators of mTORC1 and is a downstream effector of mTORC2. It is recruited after binding of growth factors or insulin to the cell surface via PI3K and then phosphorylates and inactivates TSC2 allowing Rheb to accumulate in the active (GTP-bound) state to trigger mTORC1 activation. This leads to phosphorylation of ribosomal S6K1 and 4E-BP1, mediators of protein translation and cell growth. In the case of nutrient deficiency, AMPK gets activated and inhibits mTORC1 directly or acting via Rheb. Therefore, autophagy is induced. For complete mTORC1 activation, amino acids are indispensable. They promote the translocation of mTORC1 to the lysosomal surface, where its activator Rheb resides. SAMTOR is a SAM sensor that links methionine and one-carbon metabolism to mTORC1 signalling. General effects of mTORC1 are listed in the upper box, whereas specific observations regarding the intestine are listed in the box below. For a detailed description, see the main text.

EECs, enteroendocrine cells; GCs, goblet cells; Mϕ, macrophages; PDK1, phosphoinositide-dependent kinase 1; PIP2, phosphatidylinositol (4,5)-bisphosphate; PIP3, phosphatidylinositol (3,4,5)-trisphosphate; PKC□, protein kinase C□; PTEN, phosphatase and tensin homolog; Raptor, regulatory associated protein of mTOR; SGK1, serum/ glucocorticoid-regulated kinase 1.

Furthermore, mTORC1 is known to be a major regulator of the cellular energy metabolism including glycolysis and oxidative phosphorylation (oxphos) (25). Protein synthesis is promoted by mTORC1-mediated phosphorylation of the eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) and the p70 ribosomal S6 kinase a (S6K1) (26,48) whereas lipid synthesis is increased through mTORC1-mediated regulation of sterol regulatory element binding protein1 (SREBP1) and peroxisome proliferator-activated receptor-γ (PPARγ) (26,48). mTOR is generally associated with an increase in mitochondrial metabolism and biogenesis (25–27,48). Tuberous sclerosis complex (TSC), consisting of a heterodimer (TSC1–TSC2), represents a negative regulator of mTORC1 and functions as a GTPase-activating protein (GAP) for the small Ras-related GTPase Rheb (Ras homolog enriched in brain) found on lysosomal surfaces. The active form of Rheb (GTP bound) directly stimulates mTORC1 activity, which then binds to the lysosome (26,48).

Growth factors stimulate mTORC1 through the activation of the canonical insulin and Ras signalling pathways. Phosphatidylinositol 3-kinase (PI3K) is activated and recruits AKT (also known as protein kinase B, PKB) at the plasma membrane, which causes TSC2 phosphorylation at multiple sites and therefore its inhibition. mTORC1 also senses the energy status of a cell through AMP-activated kinase (AMPK), a master sensor of the intracellular energy status. In response to energy depletion, AMPK is activated and phosphorylates TSC2 on Thr1227 and Ser1345 thereby reducing mTORC1 activity (25–27,48). An interesting point in mTORC1 activation, at least in vitro, is the absolute requirement for amino acids that interact with Rag proteins and are necessary for full mTORC1 activation (26).

mTOR signalling during homeostasis

Generally, the activity of mTORC1 and mTORC2 is low during homeostasis if compared with situations of acute damage or tumours (34,36,58–63). Studying mTOR signalling in the fly intestine and mouse tracheal epithelium shows that mTORC1 is transiently activated in stem cells upon regenerative stimuli. Stochastically, some cells fail to downregulate mTORC1 activity after regeneration and are driven to differentiation, resulting in the loss of stem cells (64). Repeated regenerative episodes result in the loss of tissue stem cells because of transient activation of the growth regulator mTORC1, and rapamycin is sufficient to prevent this loss (64). Mice with intestine-specific deletion of mTOR have reduced body weights compared with control animals. Their intestines are dilated and contain large amounts of liquid stools. In fact, organ-transplant patients on rapamycin treatment sometimes suffer from non-infectious episodes of diarrhoea, suggesting that mTOR plays an important role in maintenance of gut homeostasis (65).

Complete deletion of the mTORC1-activating component RagA (Ras-related GTP-binding protein A) in embryos or adult animals leads to rapid death through apoptosis and atrophy, among other factors, in the SI (38). Depleting RagA specifically in epithelial cells (ECs), on the other hand, causes no differences in body weight, villus length, crypt architecture or differentiation capacity, although apoptosis and chromosomal instability (CIN) are increased (28). This was confirmed by deleting mTORC1 specifically in ECs, or by a two-week treatment of wild-type mice with the pharmacological mTOR inhibitor ridaforolimus. Therefore, mTORC1 inactivation induces CIN and impairs intestinal crypt proliferation and regeneration (28).

Constitutive activation of mTORC1 in ECs (by deleting Tsc2) causes an increased proliferation as well as a faster migration of ECs towards the villi in the SI. Interestingly, the number of PCs is significantly reduced at the crypt base but increased in the villi of the SI. This suggests that mTORC1 and Tsc2 in ECs control the correct clustering and migration of PCs in the SI (29). On the other hand, by disrupting mTORC1 signalling in ECs, Sampson et al. showed that mTORC1 is not essential for crypt cell proliferation at homeostasis although it is important for the formation of proper intestinal epithelial structure by regulating the migration and functioning of the main cell populations (33).

Interestingly, some reports link mTORC1 inhibition to improved cell proliferation (39,66,67). In response to either rapamycin or calorie restriction, mTORC1 is reduced in PCs and, consequently, stem cells increase their proliferation. Therefore, mTOR inhibition in PCs can also improve intestinal regeneration in patients affected by intestinal atrophy (66). This was confirmed by Igarashi and Guarente, who describe mTORC1 to be reduced in PCs while being increased in ISCs after calorie restriction (67). PCs directly interact with stem cells to induce SIRT1 and enhance phosphorylation of mTORC1, thereby increasing protein synthesis and maintaining proliferation of ISCs (67).

Pentinmikko et al. found that intestines of old mice and humans have reduced regenerative capacities (39). Although old mice maintain an unchanged ratio of proliferating cells, they present fewer Lgr5⁺ ISCs but more PCs with an increased expression of the Wnt inhibitor Notum that was mTORC1-dependent. By treating old crypts with rapamycin, the regenerative function was restored and in vivo treatment with rapamycin resulted in a marked rejuvenation of intestinal regenerative capacity, attributable to effects on both PCs and ISCs. Targeting Notum, or reducing mTORC1 signalling in aged PCs, could therefore promote the regeneration of aged tissues (39).

Hence, these studies generally suggest that mTORC1 signalling is rather low during homeostasis in ECs, although long-term inhibition of mTORC1 will negatively affect EC function. During homeostasis, mTORC1 is mainly active in PCs and limits EC proliferation.

mTOR signalling during acute damage

Injury and excessive cell death accompanied by an increased proinflammatory milieu are the basis for the formation of IBDs, represented by CD and UC (3). They are characterized by epithelial damage and the submucosal accumulation of immune cells (68).

Activated mTOR and impaired autophagy play pivotal roles in intestinal inflammatory responses and oxidative stress injury (30). The majority of findings link mTORC1 inhibition to reduced EC proliferation during these inflammatory conditions suggesting that mTORC1 plays an important role in re-establishing homeostasis after injury (28,29,35,36). After stress-induced injury or irradiation, mTOR is essential for stem cell maintenance and proliferation, thereby promoting tissue recovery (33). Irradiated mice lacking RagA, mTOR or mTORC1 in ECs have significantly reduced regenerative capacity (28). Similarly, ablation of mTORC1 in ECs promotes DNA damage, chromosomal aberrations and apoptosis in a murine colitis model (28). The proliferative capacity of the crypt is impaired by an inability to activate the cyclin-dependent kinases CDK4/CKD6 (28).

As mentioned above, mTORC1 signalling is reduced in most ECs (with the exception of ISCs) by keeping mice on a calorie restriction diet (66,67). Interestingly, irradiated wild-type mice kept on protein-free chow have significantly reduced intestinal regeneration and mTORC1 signalling when compared with mice on normal chow (28). Thus, a protein-free diet may impair intestinal regenerative capacity by inhibiting mTORC1. Fittingly, if mTORC1 was overexpressed in ECs, faster colon regeneration was achieved after acute dextran sulfate sodium (DSS) treatment (28). These data underline mTORC1’s importance for intestinal tissue homeostasis and regeneration during acute damage. Inhibition of mTOR signalling via AZD8055 (30,69) or rapamycin (30,36,63) treatment either attenuates (30,69) or deteriorates (36,63) DSS-induced colitis showing both protecting and devastating effects.

Other studies indirectly concluded that mTOR is involved in controlling gut homeostasis and regenerative processes (63,70). By using mice devoid of Regnase-1 in ECs, an unknown role of this endoribonuclease in controlling mTORC1 was discovered. These mice are resistant to experimental colitis and maintain colon length, suffer from reduced weight loss and display fewer epithelial erosions and inflammatory infiltrations. In fact, regnase-1 controls colon epithelial regeneration by regulating mTORC1, purine metabolism and energy metabolism (63).

mTORC1 signalling and CRC

IBDs represent the main risk factors for CRC formation (22,23,71–73) whose incidence is closely linked to the western lifestyle of developed countries, where more than 2 million patients are diagnosed with CRC every year and more than 600,000 die from the disease (68). Despite a hereditary component, most cases of CRC are sporadic and develop slowly over several years (68). Mutations that provide niche independency while increasing proliferative fitness are suitable “first hits” (74). Pathways involved in this process included signal transducer and activator of transcription 3 (STAT3), nuclear factor _KB (NF-_KB) (75,76) and also mTOR (36,58–61,63).

Acting as a master switch of cellular catabolism, anabolism, proliferation, cell-cycle control, autophagy, angiogenesis and apoptosis, mTOR has been linked to oncogenic activities (77). Its activity gradually increases from steady state to mild and severe inflammation, being highest in cancer (36,58–63). mTORC1 signalling occurs as an early event in the process of tumorigenesis, thereby enhancing cancer cell survival, growth and proliferation (58–61). Rapamycin was initially developed as an immunosuppressive agent in transplant patients but has demonstrated anti-proliferative and anti-tumour effects in preclinical models and clinical malignancies (25,27,47–49,78). It activates autophagy via mTORC1 inhibition (79) but is not completely free of side effects, causing, among other symptoms, diarrhoea (65) or lung toxicity (80).

There are several reports showing an improved survival in cancer patients (77) and mice (62) treated with mTOR inhibitors. Nevertheless, various clinical trials on mTOR inhibition have shown modest success or even worsened the outcome (28,55,78,81). Therefore, a better understanding of mTOR’s roles in intestinal disorders, CRC and, especially, of its effects in different cell types is needed to develop optimal personalized therapeutic regimens.

The complexity of mTOR signalling is also underlined by the studies of Brandt et al., which describe both oncogenic as well as tumour-suppressive roles of mTORC1 in CRC, depending on the inflammation state (28). Induction of mTORC1 in C57/BL6 mice is essential for preventing colitis-induced CRC and mice with deleted epithelial mTORC1 have more polyps and aggressive tumours (correlating with increased DNA damage and CIN) whereas mice overexpressing mTORC1 have fewer tumours (28). Reducing inflammation diminishes crypt hyper-proliferation, restores intestinal homeostasis and prevents chronic inflammation and CRC in mice with inactivated mTORC1 (28). On the other hand, in the adenomatous polyposis coli (APC)^Min mouse model, deletion of epithelial mTORC1 protects the mice and lowers tumour burden. Here, mTORC1 inactivation has tumour-preventative activities and protects against Wnt/□-catenin activation-dependent CRC (28).

In summary, mTORC1 has both oncogenic and tumour-suppressive activities depending on the CRC tumour type, the inflammatory environment and association with canonical Wnt activation. The roles of mTOR in the GI tract are summarized in Table 2.

Table 2. Role of mTORC1 in different cell populations of the GI tract.

Genetic modela	Condition	Effects	Reference
Tsc2 deletion in ECs	Steady	↑mTORC1, ↑proliferation of ECs, ↑migration of ECs along crypt–villus axis ↑apoptosis of ECs	(29)
HEK293 and HEK293T cells	Glucose starvation / rapamycin	↑AMPK, ↓mTORC1 ↑autophagy (MEFs)	(79)
Wild-type	Calorie restriction / rapamycin	↓mTORC1 in PCs ↑ISC function	(66)
Wild-type	Calorie restriction	↓mTORC1 in PCs ↑ISC function	(67)
Wild-type (dogs and rats)	Rapamycin	↓regeneration of intestine, liver and kidney	(35)
Patients, rats, mTOR disruption in ECs of mice	Rapamycin, post-transplant	↓expression of NHE3 and Na+/H+ exchange activity ↑diarrhoea	(65)
Haplo-insufficiency of Rheb	Rapamycin, DSS- and TNBS- induced colitis	↓ mTORC1 in ECs ↓intestinal cell proliferation ↑ apoptosis ↑mortality in DSS- and TNBS-induced colitis ↓ IL-6-induced STAT3 activation	(36)
C57BL/6	Rapamycin, AOM-DSS colorectal cancer model	↓mTORC1 ↓STAT3 ↓ malignant transformation ↓expression of both pro- and anti-inflammatory cytokines	(58)
Raptor, Rictor or Raptor/Rictor deletion in ECs	Steady, post 10 Gy irradiation	↓mTORC1 and/or mTORC2 in ECs mTORC1 is necessary for ISC/progenitor maintenance and crypt regeneration post-injury, but is not of big importance at steady state	(33)
Inducible mTOR, RagA or MCRS1 deletion in ECs	Steady, post 14 Gy irradiation, DSS	↓mTOR or mTORC1 in ECs ↓ crypt cell proliferation (via CDK4/CDK6/rBP/e2F) ↑ apoptosis, ↑ CIN, ↑ barrier permeability	(28)
C57BL/6	Protein-free chow, then 14 Gy irradiation	↓mTORC1 ↓ intestinal regeneration	(28)
MCRS1 overexpression in ECs	DSS, AOM/DSS	↑ mTORC1 ↑ intestinal regeneration ↓ tumours	(28)
Inducible MCRS1 deletion in ECs	AOM/DSS	↓ mTORC1 in ECs ↑ tumour burden, ↑ IL-6, ↑ DNA damage/CIN	(28)
Deletion of both APC and MCRS1	AOM/DSS	↓ mTORC1, ↑ b-catenin accumulation. double mutants had increased survival and less tumours compared to APC mutants only	(28)
Double mutants for APC and Cdx2	Steady, mTOR activation, mTOR inhibition (LY294002)	↓ APC and CDX2 in distal colon ↑ CIN ↑ colonic polyps mTORC activation caused ↑ CIN and tumur initiation mTOR inhibition caused ↓ CIN	(59)
APC deletion	Everolimus	↓ mTOR ↓ mortality	(62)
C57BL/6	mTOR inhibition (AZD8055), DSS	↓ mTOR ↓ weight loss, ↓ colitis the anti-inflammatory effect is mediated via T helper cell polarization and proliferation (↓ INFγ, IL-17A, IL-1β, IL-6, TNFα and ↑ IL-10)	(69)
C57BL/6	mTOR inhibition (AZD8055) AOM/DSS	↓ mTORC2 in macrophages ↑ tumorigenesis	(55)
RagA deletion in embryos	Steady	↓ mTORC1 severe growth effects succumbed within ~10 days of embryonic development	(38)
RagA deletion in liver	Steady	↓ mTORC1 I most cells although some escaped cre deletion ↑ response to insulin	(38)
Inducible full-body deletion of RagA	Steady	↓ mTORC1 ↓ of 20% body weight 1 week after tamoxifen deletion of RagA in adult mice is lethal: 50% died 2–3 weeks after tamoxifen atrophy and apoptotic figures in SI ↑ of myeloid cells in spleen and bone marrow ↓ B lymphocytes	(38)
Full-body Raptor deletion	Steady	↓ mTORC1 atrophy and apoptotic figures in small intestine ↑ of myeloid cells ↓ B lymphocytes	(10)*
Gp130 knock-in	Steady	↑ GP130/STAT3 activation in response to IL-6/IL-11 Spontaneous gastric tumour development by 4 weeks of age	(61)
Patients	Gastric tumors	coactivation of mTORC1 and STAT3 congruent gene expression signatures between human intestinal-type gastric cancers and Gp130 knock-in mice (humans)	(61)
Gp130 knock-in	Everolimus	↓ mTORC1, no reduction in STAT3 phosphorylation ↓ cancer growth due to decreased tumour proliferation and vascularisation	(61)
C57BL/6	AOM-DSS everolimus	↓ tumour burden, ↑ tissue hypoxia ↓ STAT3 and mTORC1 activation no changes in □-catenin (Wnt)	(61)
Organoids (human and mouse)	Steady	age-induced reduction in the oranoid-forming capacity of colonic crypts ↑ organoid growth when co-cultured with young PCs	(39)
Lgr5–EGFP reporter mice, Tsc1 deletion in ECs	Steady, everolimus	↓ Lgr5hi ISCs in old mice and ↑ of PCs in old mice andxy3humans that contained ↑ Notum expression that was mTORC1 dependent	(39)
Gatm	DSS	↑ cell death and immune infiltrates, ↑colon shrinkage ↓ proliferation, ↓ mTOR, ↑AMPK	(70)
Regnase-1 deficiency in ECs	Steady, DSS	↑ mTORC1, ↑ purine metabolism, Intracellular: ↑ ATP, ↓ ADP, ↓ AMP ↑ proliferation, ↑ goblet cells ↓ weight loss, ↓ apoptosis Maintenance of intact barrier integrity	(63)
Regnase-1 deficiency in ECs	AOM/DSS	↑ mTORC1, ↑purine metabolism ↓ tumour burden	(63)

^aIf not stated differently, studies were performed in mice.

MCRS1, microspherule protein 1; MEFs, mouse embryonic fibroblasts; Raptor, regulatory associated protein of mTOR; TNBS, trinitrobenzenesulfonate.

mTORC1-related control of immune cells in the intestine

The GI tract represents the largest immune compartment of the human body (82) and innate immune cells such as dendritic cells (DCs) and macrophages are critical for maintaining the function of the intestine. DCs translocate from the lamina propria to mesenteric lymph nodes where they present antigens to naive T-cells thereby establishing the adaptive immune system (83). Intestinal macrophages (defined by CX₃CR1) establish the local homeostatic immune cell network and maintain the epithelial integrity by secreting factors important for stem cell renewal (e.g. prostaglandins, growth factors, Wnt ligands) (20). They furthermore contribute to the local clearance of bacteria and apoptotic cells and are even critically involved in epithelial wound healing (83).

Most intestinal macrophages are constantly replenished from circulating monocytes (84) and are characterized by an M2-like phenotype (CD206⁺) that secretes the cytokine IL-10 (83). Intestinal IL-10 is a pleiotropic anti-inflammatory cytokine and essential for the control of inflammation in the colon. One of its main functions is the maintenance of Foxp3 expression in T regulatory cells (T_regs), which become functionally defective in its absence (85). Already in 1993, Kühn et al. showed that IL-10 deficient mice develop spontaneous colitis (86). The connection of IL-10 and its receptor with colitis was confirmed in humans (87–90) and mice (87,91). Myeloid IL-10 production and mTOR signalling are closely linked (27) and a lack of mTORC1 signalling in DCs results in the suppression of IL-10 production, along with enhanced CD86 expression (92). These mice, in fact, are highly susceptible to DSS-induced colitis, underlining the importance of myeloid mTORC1 signalling in protecting from colitis (92).

In the case of injury, CD14^hi monocytes/macrophages are recruited and become activated (15,84) promoting epithelial regeneration (15) and releasing cytokines, thereby shaping T-cell responses (83). In the case of IBD, they support pathogenic T-cell function, amongst other effects, through IL-23 production (93–95). Consistent with this idea, macrophage-derived IL-23 supports effector T-cell differentiation during Helicobacter hepaticus-induced colitis (20,95,96), assisting the generation of highly pathogenic Th17 cells that co-express IFN-γ (96). Interestingly, mTORC1 inhibition by rapamycin or specific mTOR deletion in CX₃CR1⁺ macrophages inhibits intestinal expression of IL-23 and IL-22 and increases autophagic activity, thereby ameliorating intestinal fibrosis, a condition frequently seen in CD (97).

High doses of NaCl create a pro-inflammatory environment that enhances M1 polarisation of macrophages as well as Th17 frequencies in the lamina propria in DSS-treated mice (98). Importantly, the underlying mechanism involves an up-regulation of p38 in lamina propria mononuclear cells and the importance of macrophages in this process was shown by their depletion, which led to an alleviated DSS-induced colitis (98). Interestingly, in monocytes and macrophages, p38α signalling activates the mTOR pathway both in vitro and in vivo, being involved in increased IL-10 and decreased IL-12 levels (99).

In the APC^Min mice that represent a non-inflammatory based system of tumour induction, reduced IL-10 production (due to Tpl2 ablation) correlates with defects in mTOR activation and STAT3 phosphorylation in Toll-like receptor stimulated macrophages. This causes a defect in the generation and function of inducible T_regs with the consequence that these mice suffer increased intestinal inflammation and tumorigenesis (100). Furthermore, mTORC1 inhibition by rapamycin also reduces phospho-STAT3 signalling (36,58) and animals that harbour a STAT3 transcription factor deficiency in myeloid cells (intestinal macrophages and neutrophils) succumb to colitis (101). Experiments based on either genetic or pharmacological inhibition have shown that STAT3 phosphorylation and IL-10 induction by Toll-like receptor signals depend on mTOR activation (102). Deletion of STAT3 in myeloid cells causes a similar phenotype to the knockout of IL-10, underscoring the importance of STAT3 activation in myeloid cells for the induction of IL-10 (101).

We recently showed that mTORC2 signalling in macrophages is also important during colitis to prevent CRC. mTORC2 was active only in intestinal macrophages but not in ECs during DSS-induced colitis (55). This mTORC2 activity in macrophages was important to reduce inflammation and stimulate repair, as myeloid-specific deletion of rapamycin-insensitive companion of mTOR (Rictor), an essential component of mTORC2, worsened colitis and colitis-induced tumour formation by induction of the cytokine osteopontin. Moreover, treatment with the second-generation mTOR inhibitor AZD8055 reduced mTORC2 signalling in these macrophages and, as a consequence, the tumour burden in an azoxymethane (AOM)/DSS model was significantly increased (55).

Metabolic pathways in immune cells controlling intestinal function

The regulation of cellular metabolic processes in immune cells emerges as important factor to regulate inflammatory processes. Glycolysis is of high relevance for the inflammatory response of macrophages (103) and T-cells (104). Inhibiting glycolysis causes a decreased inflammatory response in macrophages (103) and selectively impairs Th17 proliferation and survival, causing a reduction in T cell-mediated inflammation in models of IBDs (104).

Ip et al. underlined this concept and the importance of macrophages by discovering another link between IL-10 production and metabolic regulation by the mTOR pathway (45). They found that IL-10 suppresses mTOR activity hours after lipopolysaccharide (LPS)-induced activation of macrophages by inducing the expression of the mTOR inhibitor DDIT4. This induces mitophagy to remove dysfunctional mitochondria that are generated in response to LPS-induced glycolysis and reactive oxygen species (ROS) production. Thereby, IL-10 maintains mitochondrial fitness and limits the need to switch to glycolysis. In the absence of IL-10 signalling, macrophages accumulate damaged mitochondria in a colitis mouse model and in IBD patients (45). Another demonstration of the importance of intestinal mTOR signalling was given recently by Waise et al. who showed that inhibition of intestinal mTOR is sufficient to lower glucose production and enhance glucose homeostasis, being a possible therapy for diabetes (56).

Commensal bacteria shape the immune system of the intestine, particularly at the colon level, by producing a variety of microbial metabolites such as short-chain fatty acids (SCFAs) including acetate, propionate and butyrate (105,106). Interestingly, reduced numbers of butyrate-producing bacteria are found in faecal samples from IBD or CRC patients (107). SCFAs are able to support the development of Th1 and Th17 effector cells as well as T_regs, depending on the cytokine milieu and immunological context. Because of their chemical properties, SCFAs are able to directly enter cells, although some cells also express surface receptors (such as GPR41 and GPR43) to increase their uptake (105).

In T-cells, SCFAs inhibit histone deacetylases (HDACs) to stimulate mTOR pathway activation that is required for their differentiation and cytokine expression (105). Nonetheless, these studies did not exclude whether this function on T-cells is mediated in an indirect manner through other cells, such as macrophages, DCs or ECs. In this regard, Schulthess et al. reported that macrophages are strongly influenced by SCFAs, especially by butyrate that promotes metabolic and transcriptional changes with enhancement of their bactericidal functions (106). Interestingly, the enhanced microbicidal function induced by butyrate is a consequence of increased glycolysis and suppressed mTOR activation (106).

Metabolic pathways in ECs

While the signalling pathways required for epithelial stem cell maintenance are well described (1), less is known about how metabolism contributes to epithelial homeostasis. There is a fine-tuned balance in responding to or tolerating commensal bacteria, and perturbations in this balance can result in IBDs (70,108). The crypt architecture protects the stem cell by creating a metabolic barrier formed by differentiated colonocytes that consume butyrate produced by commensal bacteria. ECs may use SCFAs including butyrate as nutrients to maintain intestinal homeostasis (109). However, if butyrate reaches the niche in the case of injury, it inhibits proliferation and delays wound repair (108).

The intestine is a highly plastic tissue with marked atrophy under fasting and rapid recovery after refeeding (40) and a connection between nutrient availability, inflammation and CRC does exist (28,68). Despite the fact that the intestine directly faces the nutritional environment, the critical dietary molecules that control ISCs are only beginning to be identified and it is still unknown to what extent diet quality and quantity modulate ISC behaviour. Calorie restriction generally has negative effects on mTORC1 activation (28,66,79), although this is not true for stem cells, as discussed above (66,67). ISCs appear to be isolated from the direct effects of calorie restriction and instead respond solely to the cyclic ADP ribose signal from PCs, which themselves are entrained by nutrients (66,67).

Creatine is a rapid source of energy that replenishes cytoplasmic ATP. Its levels are maintained by diet and endogenous synthesis from arginine and glycine, in which glycine amidinotransferase (GATM) catalyses a rate-limiting step (70). By using Gatm-deficient mice, Turer et al. showed that creatine maintains intestinal homeostasis and protects against colitis by rapid replenishment of cytoplasmatic ATP within colonic ECs, which restores barrier integrity. The loss of creatine in vivo leads to increased EC death and colitis, directly linking energy metabolism to intestinal homeostasis (70).

As one of the essential amino acids, methionine is a dietary requirement for most animals (41). Furthermore, it is essential to generate S-adenosylmethionine (SAM), the universal methyl donor required for all methyltransferases in our body. Therefore, methionine and SAM are key mediators in maintaining tissue homeostasis. Methionine depletion from the culture medium of mouse SI organoids decreases stem cell proliferation while promoting cell differentiation (41). In Drosophila, SAM is essential for protein synthesis in gut stem cells as it governs ISCs through methyltransferases involved in translation (40). SAM therefore represents a nutrient-sensing mechanism essential for dietary regulation of intestinal homeostasis. Loss of dietary methionine reduces SAM, thereby triggering cell-type-specific starvation responses in ISCs and in differentiated enterocytes (40).

Interestingly, a connection between mTORC1 and SAM was recently described (110). SAMTOR was identified to be an inhibitor of mTORC1 signalling by interacting with the Ragulator complex, necessary for mTOR activation. SAM disrupts this interaction by directly binding to SAMTOR. Methionine starvation, consequently, leads to SAM reduction and therefore SAMTOR activation and mTORC1 inhibition (110) (Fig. 1).

Apart from amino acids, changes in mitochondrial pyruvate carrier (MPC) and pyruvate also orchestrate proliferation and homeostasis of ISCs (42,43). During glycolysis, which takes place in the cytoplasm, glucose is converted into pyruvate that enters the mitochondria via the MPC to be oxidized by mitochondrial respiration. MPC expression is low in ISCs (permitting increased glycolysis) and increases during differentiation (permitting complete oxidation of pyruvate). MPC deletion in Lgr5⁺ cells expands the ISC compartment by increased proliferation. MPC therefore is necessary and sufficient to supress stem cell proliferation and plays a direct role in regulating ISC proliferation (42).

Deletion of MPC in differentiated ECs similarly resulted in an increase in ISC proliferation, as this represents a stress signal in ECs, which consequently induces ISC proliferation and EC regeneration (43). Lactate might be one signal that is released by ECs and used by ISCs as a nutrient to fuel mitochondrial respiration (43). Similar results were obtained with PCs and ISCs from the SI (44). ISCs have higher mitochondrial activity whereas PCs have high glycolysis. PCs support stem cell function by providing lactate to sustain the enhanced mitochondrial oxphos in the ISCs (44).

Conclusions

Because of its key roles in cell metabolism, mTORC1 is one of the most studied protein kinases. Its general effector functions and different activating or suppressive pathways are well established in vitro. The in vivo regulation and functions of mTORC1 are more complex, necessitating the elucidation of its role in the cellular context of the individual cell populations of the intestine. In the cases of IBDs and CRC, general mTOR inhibition can lead to opposing and unexpected results that are influenced by the spatiotemporal inflammatory milieu of the distinct cell types that may also change over time. Genetic models to interrupt or increase mTOR activation in different cell types have shed light on the individual roles of mTORC1 during homeostasis, IBD and CRC.

Although the intestine is the site of our body where nutrients are absorbed, only recently emerging evidence identifies how diverse metabolites and dietary molecules control intestinal homeostasis. Future studies and single-cell analysis will be able to illuminate the complex functions of our intestine during health and disease.

Abbreviations

AMPK

AMP-activated kinase

AOM

azoxymethane

APC

adenomatous polyposis coli

CAC

colitis-associated carcinoma

CD

Crohn’s disease

CIN

chromosomal instability

CRC

colorectal cancer

DCS cell

deep crypt secretory cell

DSS

dextran sulfate sodium

ECs

epithelial cells

EGF

epidermal growth factor

EEC

enteroendocrine cell

eIF4E

eukaryotic initiation factor 4E

4E-BP1

eukaryotic initiation factor 4E binding protein

GAP

GTPase-activating protein

GATM

glycine amidinotransferase

HDAC

histone deacetylase

IBD

inflammatory bowel disease

ISCs

intestinal stem cells

MCRS1

microspherule protein 1

Mϕ

macrophages

MPC

mitochondrial pyruvate carrier

mTORC1/2

mammalian/mechanistic target of rapamycin complex 1/2

Oxphos

oxidative phosphorylation

PCs

paneth cells

PKB

protein kinase B

PKCα

Protein kinase Cα

PPARγ

peroxisome proliferator-activated receptor-γ

Raptor

regulatory associated protein of mTOR

Rheb

Ras homolog enriched in brain

Rictor

rapamycin-insensitive companion of mTOR

S6K1

p70 ribosomal S6 kinase

SAM

S-adenosylmethionine

SCFA

short chain fatty acid

SGK1

Serum/ glucocorticoid-regulated kinase 1

SI

small intestine

SREBP1

sterol regulatory element binding protein1

T_regs

T regulatory cells

TSC

Tuberous sclerosis complex

UC

ulcerative colitis