Guts for Self-Eating: Role of Autophagy in Gastrointestinal Health and Disease

Abstract

Autophagy, a highly conserved cellular degradation pathway promotes cell survival via lysosomal degradation of aberrant cellular proteins and recycling of the nutrients. A variety of human diseases are now linked to defective autophagy and there is ever-growing interest in the application of autophagy in healthy living as well as disease prevention and therapies. Autophagy plays very important and complex functions in the gastrointestinal tract which are an intense focus of the current research efforts. Autophagy maintains cellular homeostasis mainly through proteostasis, lipid regulation, mitigation of damaged mitochondria, removal of intracellular infectious agents and foreign material, and reduction in reactive oxygen species and inflammasome. Recent studies show that although autophagy is mostly beneficial, it can induce damaging effects depending upon the cellular contexts such as homeostatic or inflammatory conditions. We summarize that this double-edge effect of autophagy is associated with cell-specific and cell-autonomous functions of autophagy, noncanonical autophagic effects, and autophagy-independent functions of autophagy-related proteins. We review opposing effects of autophagy pathway and its differential cellular functions specifically relevant to gastrointestinal homeostasis. We highlight the impacts of autophagy-related genetic defects in inflammatory bowel disease and the evolving role of autophagy in gastrointestinal and liver diseases including fibrosis and neoplastic processes. We also provide a contemporary perspective on the clinical studies related to autophagy and highlight the context-specific outcomes of autophagy and their relevance. The growing knowledge of the diverse autophagy regulatory mechanisms will provide further insights into how this life-friendly, self-cleansing cellular process can be harnessed for therapeutic advantages in gastrointestinal and liver diseases.

Autophagy: Pathway and Cellular Functions

Autophagy (auto: oneself, phagy: to eat, latin) came into the limelight when Yoshinori Ohsumi was awarded the 2016 Nobel Prize in Physiology or Medicine for his discoveries of mechanisms for autophagy. Autophagy refers to the mechanisms that help the host cells to eliminate misfolded, aggregated proteins and organelles via lysosomal degradation.¹ In a well-coordinated, multistep process of macroautophagy (commonly referred as autophagy), more than 15 autophagy-related genes (ATGs)/proteins take part in initiation (formation of the phagophore mediated by 2 key protein kinase complexes, ULK1/2-ATG13-RB1CC1/FIP200 complex, and the BECN1-PIK3C3/Vps34-PIK3R4/Vps15-ATG14 complex), expansion of the phagophore (involving ubiquitin-like proteins ATG12 and GABARAP/light chain 3 [LC3]), autophagosome formation (closure of phagophore which requires lipidation of cytosolic ATG8 or LC3-I to LC3-II), and the fusion of autophagosome with the lysosome to form an autolysosome where the cargo proteins are selectively degraded (Figure 1).² Besides the core ATG proteins, additional proteins required for this degradative pathway include cytoskeletal motor proteins, tethering factors, soluble N-ethylmaleimide–sensitive factor attachment protein receptor proteins, and membrane adapter proteins.³

Figure 1.

The canonical autophagy pathway, which is largely regulated by mTOR and AMPK, is initiated by assembly of ULK1 complex and recruitment of PI3KC3 complex to form phagophore. Recruitment of ATG12–ATG5–ATG16L1 complex helps phagophore expansion and promote LC3 lipidation on the phagophore membrane. During LC3 lipidation, LC3/Atg8 is cleaved by ATG4 protease to generate cytosolic LC3-I which is conjugated to phosphatidylethanolamine in a ubiquitin-like reaction to form LC3-II, a process that requires Atg7 and Atg3 (E1 and E2-like enzymes, respectively). The lipidated form of LC3, known as LC3-II, is attached to the autophagosome membrane. With the help of endosomal sorting complexes required for transport machinery and N-ethylmaleimide–sensitive factor attachment protein receptor complexes (not shown) the autophagosomes are fused with lysosomes to form an autolysosome where the cargo proteins are degraded.

The fundamental cellular function of autophagy is 2-fold. Autophagy maintains cellular homeostasis by degradation of toxic misfolded and aggregated proteins and provides nutrients to cells, particularly under stress. Based on the cargo, the autophagy functions in numerous cell homeostatic processes including proteostasis and removal of misfolded proteins, damaged mitochondria (mitophagy), lipids (lipophagy), intracellular infectious agents, and foreign material (xenophagy). Though, by and large, autophagy is a degradative pathway, under certain conditions autophagy can deliver the cargo to the plasma membrane and extracellular environment. A classic example of this secretory autophagy is the extracellular release of proinflammatory cytokine interleukin (IL)-1β which is delivered to extracellular space via incorporation into the autophagic membrane.^4,5 Moreover, autophagy-related proteins may have autophagy independent functions such as ATG5-mediated regulation of cytokines during intestinal inflammation⁶ or ATG6-mediated steady state regulation of intestinal tight junction (TJ) barrier.⁷

Autophagy as a Lifestyle

Autophagy is the major catabolic mechanism used by eukaryotic cells to maintain nutrient homeostasis and adaptation to stress, and also complementarily referred to as ‘recycling’ or ‘self-cleansing’ process. These days, autophagy is a common word in our health-conscious society. Search for “autophagy” on commercial shopping platforms yields hundreds of hits that include books, diet supplements, cooking recipes and more. Currently, potential autophagy modulation is achieved through means such as various modes of fasting, calorie restriction, and physical activity.

In terms of fasting, intermittent fasting is a common practice and has different short- and long-term diet programs with regular cycles between the time of eating and fasting.8, 9, 10 In alternate-day fasting protocol, fasting is done every other day with unrestricted feeding on feeding days. In time-restricted programs, food consumption, with or without calorie restriction, is limited to a certain window of few hours a day. Calorie-restricted programs consist of providing up to 25%–30% of total energy demands for a period ranging from a day to several weeks.¹¹ Due to the experimental evidence of calorie restriction having beneficial impact on metabolic health, inflammation, and age-related diseases, it is being explored in clinical studies as a treatment for inflammatory diseases.¹² Intermittent fasting has also been shown to be beneficial for metabolic dysfunction–associated steatotic liver disease (MASLD), especially when periods of fasting exceed 12–18 hours.¹³

Autophagy has also recently emerged as an important process involved in the adaptive responses to exercise.¹⁴ The outcomes of exercise in terms of autophagy include both increased autophagy flux as well as transcriptional activation of autophagy genes by various transcriptional factors to increase the autophagic capacity in human skeletal muscles.^15,16 Furthermore, autophagy seems to mediate many of the positive effects of exercise such as increase in muscle mass, better glucose regulation, and reduction in reactive oxygen species (ROS).¹⁴ However, the exact mechanism behind induction of autophagy may depend on factors such as intensity/duration of exercise, feeding, and timing of biological sampling,^14,17,18 which may add variability to the autophagic responses observed after exercise. The proteostasis (regulation of protein through the biogenesis, folding, trafficking, and degradation) function of autophagy is thought to mediate the clearance of damaged proteins and organelles due to heat, altered pH, and mechanical stress, and maintain cellular function in myocytes.^17,19,20 Though precise autophagic response to exercise in different cell types in intestine has not been documented in humans, both calorie restriction and exercise are known to be capable of instigating a global autophagic response.^21,22 Also, at least in part, exercise and calorie restriction initiate autophagy through the common cellular pathways, such as energy sensing AMP-activated protein kinase (AMPK) and histone modifying Sirtuin 1, resulting in inhibition of mammalian target of rapamycin (mTOR).^12,19 Moreover, in experimental models, intestinal autophagy is required for the beneficial effect of dietary restriction on health span, longevity, and stem cell regeneration.²³ Overall, intestinal autophagy appears to be an important component of the global impact of autophagy on the host health and disease. At the same time, considering the constant exposure of the intestinal lining to microbes, luminal antigens, and nutrients, the autophagic responses might be different in the intestine compared to other parenchymal organs such as liver and muscular organs such heart and skeletal muscles.

Autophagy in Health and Disease

A variety of human diseases are now linked to defective autophagy pathway or mutations in ATGs that regulate various signaling pathways.²⁴ An example is Crohn’s disease (CD) where several ATG mutations can dysregulate major cellular defenses such as xenophagy, inflammasome, and antimicrobial secretions. On the other hand, selective autophagy deficiencies due to mutations in autophagy adapter proteins or master kinases is linked to amyotrophic lateral sclerosis.^25,26 While genetic mutations disrupting autophagolysosomal fusion are associated with congenital neurodevelopmental problems,²⁷ mutations disabling lysosomal functions are linked to Alzheimer’s disease²⁸ and Parkinson’s disease.²⁹ The defects in autophagolysosomal degradation or loss of degradative capacity often lead to toxic accumulation of cargo such as α-synuclein³⁰ or amyloid precursor protein.³¹ Recent evidence suggests autophagy is as a primary mechanism of lipid metabolism in mammalian hepatocytes, making it an important therapeutic target in MASLD.³²

Autophagy has a broad influence on cellular senescence. Under homeostatic conditions, autophagy plays antisenescence role by mitigating dysfunction of mitochondria, ROS, and endoplasmic reticulum (ER) stress, and maintaining lysosomal integrity. However, in certain conditions such as advanced senescence, autophagy may function as prosenescence mechanisms by supporting cell survival. In the later case, autophagy supports cell survival by degrading antisenescence factors and supporting synthesis of senescence-associated factors.³³ At organismal level, autophagy has been consistently identified as a declining pathway during aging in several organisms while upregulation of autophagy by genetic or pharmacological means promotes longevity in animals. Rapamycin treatment or overexpression of autophagy genes has been shown to have geroprotective effects in Drosophila and mice.^34,35 In particular, intestinal autophagy has been important in promoting longevity and health span and maintaining intestinal barrier in model organisms.^36,37

Clinical Studies on Autophagy

In line with the relevance of autophagy for human health, several clinical studies have been initiated to investigate the effect of autophagy inducing lifestyles or autophagy inducing pharmacologic agents on human health. More than 100 of these clinical trials have been investigating the effect of autophagy modulation on diverse conditions such as diabetes, aging, autoimmune disorders, infections, and cancers (ClinicalTrials.gov). Autophagy modulation along with primary therapeutic agents has also been investigated in several clinical conditions, including MASLD, and cancers. The most relevant questions being investigated in these clinical trials are when (timeline) is autophagy induced, how to detect it (peripheral sampling or native target tissue), and if it has clinical effect (impact on target clinical parameters) after an intervention. Various forms of restrictive diets including nutrient-dense fasting mimicking diet (FMD) has been shown to induce autophagy and promote multisystem regeneration and reduce inflammatory bowel disease (IBD)–associated inflammation.^38,39 In a randomized controlled trial, 4 weeks of strict alternate day fasting improved markers of general health in middle-aged humans (including cardiovascular markers, fat mass, fat-to-lean ratio, and levels of proaging amino-acid methionine (NCT02673515).⁴⁰ In other clinical trials (NCT02158897 and NCT04150159), periodic plant-based FMD showed beneficial effects including reduced insulin resistance and lower hepatic fat, along with a decrease of 2.5 years in median predicted biological age.^41,42 The effect of FMD on autophagy in humans (NCT06115551) as well as the status of autophagy in pediatric CD (NCT05842564) is currently awaited. Similarly, the effect of Dapagliflozin, a selective sodium–glucose cotransporter 2 inhibitor and potentially autophagy inducer is being investigated in mild-to-moderate ulcerative colitis (NCT05986136).

Role of Autophagy in Gastrointestinal Tract

Our improved understanding of the remarkable ability of autophagy to regulate intestinal epithelial architecture and metabolism,^43,44 functions of specific intestinal epithelial subsets,45, 46, 47 and inflammatory pathways and gut microbiota⁴⁸ is recent and evolving. Autophagy promotes several intestinal homeostatic processes including Paneth cell function,⁴⁹ mucin production,⁴⁵ stem cell regeneration,⁴³ antibacterial defense,⁵⁰ and paracellular barrier.51, 52, 53

The interest in the autophagic functions in intestine was mostly fueled by the findings of the genome-wide association studies that identified mutations in ATG genes as risk factors for CD.^54,55 The most commonly involved ATG variant in CD is ATG16L1 ^T300A and the risk allele (G allele) is significantly associated with both adult-onset and pediatric-onset CD, as well as perianal CD.⁵⁶ Via its interaction with multiple autophagy-related proteins, ATG16 plays an important role in phagophore formation and its maturation into autophagosome, which subsequently fuse with the lysosome. The ATG16L1 ^T300A variant expression has been associated with decreased antimicrobial peptide production, impaired intracellular bacterial clearance, defects in antigen presentation and increased proinflammatory cytokine production.^46,49,57 The T300A variation makes ATG16L1 more susceptible to cleavage by caspase 3, resulting in a reduction in the autophagic flux.⁵⁸ Similarly, mutations in other ATGs, which are known to increase the risk for IBD, affect cellular defenses and the intestinal barrier (listed in the Table).

Table.

Autophagy Genes Involved in IBD Risk

Gene	Mechanism and function
ATG16L1	T300A polymorphism promotes caspase 3-mediated cleavage.58 ATG16L1 plays an important role in Paneth cell lysozyme secretion,59 bacterial clearance,46 homeostasis of ER stress,60 and cytokine and T cell responses.46,48
IRGM	Regulates autophagy organization for antibacterial defense.61
LRRK2	Variant allele in CD (and Parkinson’s disease) increases LRRK activity and inhibits autophagy.62
GPR65	Variant of proton-sensing G protein–coupled receptor affects lysosomal pH and decreases intracellular bacterial clearance.63
MTMR3	Mutation in this PI3P phosphatase decreases autophagy and increases proinflammatory response in macrophages.64
PTPN2	Single-nucleotide polymorphism in PTPN2 gene that encodes protein tyrosine phosphatase nonreceptor type 2 impairs autophagosome formation, diminishes bacterial clearance, and increases apoptosis.65PTPN2 also regulates epithelial-macrophage crosstalk to promote intestinal barrier.66
CALCOCO2	Mutation disrupts function of this autophagy adapter and enhances inflammatory gene response.67
SMURF1	This ubiquitin ligase plays a role in selective autophagy of Mycobacterium tuberculosis and antituberculous host defense.68
XBP1	XBP1 is involved in activation of autophagy genes and hypomorphic variant is a genetic risk for IBD.69

LRRK2, leucine-rich repeat kinase 2; PTPN2, protein tyrosine phosphatase nonreceptor type 2; XBP1, X-box binding protein 1.

Autophagy and Cellular Pathogenic Mechanisms

Common presence of aberrant structure and function of Paneth cell in mice mutant for different ATGs,⁷⁰ indicates importance of an intact autophagy pathway in cellular homeostasis. Diverse developmental, infectious, metabolic, and pathophysiologic processes lead to accumulation of misfolded proteins and induce ER stress which is followed by the unfolded protein response (UPR) aimed at restoration of cellular homeostasis. However, persistent unmitigated ER stress overwhelms UPR and disrupts intestinal cellular homeostasis. Mutations in the genes involved in the UPR as well as autophagy are known to be strongly associated with the risk for IBD development,⁶⁹ and elevated ER stress has been detected in intestinal epithelial cells in IBD^71,72 as well as several IBD models,^69,71, 72, 73, 74 indicating a pathologic role of ER stress in human intestinal diseases. Autophagy is thought to be an alternate way of degradation of misfolded proteins.⁷⁵ Also, a major UPR pathway, inositol-requiring enzyme 1 upregulates several cell survival autophagy genes via activation of X-box binding protein 1.⁷⁶ Thus, deficiency of autophagy exaggerates ER stress.^47,69 In another example, nuclear factor erythroid 2-related factor 2 (Nrf2) which is a basic-leucine-zipper transcriptional factor and a principal antioxidant mechanism, controls the expression of antioxidant cytoprotective genes during cellular stress.⁷⁷ Normally, Nrf2 is ubiquitinated or kept in the cytoplasm by cysteine rich Keap1, an E3 ubiquitin ligase adapter. In canonical pathway, oxidative stress oxidizes cysteine residues in Keap1, thereby interrupting the Nrf2 ubiquitination and activating Nrf2. The exciting discoveries, a decade ago, connected autophagy to redox. In these noncanonical pathways, the autophagy substrate p62 activates Nrf2 via competitive binding to Keap1^78,79 or degradation of Keap1,⁸⁰ indicating an important role of autophagy in alleviating oxidative stress, particularly since Nrf2 levels are reduced in IBD.⁸¹

Autophagy and Gut Permeability

Increased gut permeability due to the loss of intestinal interepithelial TJ barrier is a key feature of IBD. In a recent large prospective study, increased intestinal permeability, as early as 3 years before the clinical diagnosis, was found to be associated with the later development of CD.⁸² Moreover, persistent increase in intestinal permeability predicts poor clinical outcome, while normalization of intestinal permeability correlates with long-term clinical remission.⁸³ The increased intestinal permeability in IBD is partly attributed to an increase in the levels of channel-forming TJ protein claudin-2 as well as a decrease in the levels of barrier-forming occludin.^84,85 We have shown that autophagy profoundly enhances intestinal TJ barrier function via its effect on intracellular trafficking of TJ proteins. Autophagy promotes endocytosis and lysosomal degradation of pore-forming claudin-2 and ATG7 dependent LC3 lipidation is required for internalization of claudin-2 into autophagosomes.^51,86 On the other hand, autophagy enhances barrier-forming occludin protein levels by inhibiting its constitutive degradation and protects against inflammation-induced TJ barrier loss.^7,52 Furthermore, acute deletion of Atg7 in mice increased colonic claudin-2 levels and decreased occludin levels and promotes the susceptibility to experimental colitis. This effect of TJ barrier enhancement is also evident upon activation of autophagy pathway in human colonic explants.^51,52 Numerous studies show that a variety of compounds that have beneficial effects against TJ barrier disruption and intestinal inflammation also induce autophagy. The specific role of autophagy in modulating the TJ barrier and inflammation in the disease ameliorating effects of these compounds needs further investigations.

Role of Autophagy in Gut Immunity

Autophagy plays essential cell-specific functions in distinct gut immune cells to promote gut mucosal homeostasis. In dendritic cells and macrophages, autophagy processes antigens for major histocompatibility complex I and II presentation, maintains major histocompatibility complex II expression, and regulates cytokine secretion and inflammasome. Many of these functions are carried out via autophagy-mediated intracellular trafficking of immune molecules and degradation of damaged mitochondria, thus limiting ROS production and direct degradation of inflammasome.⁸⁷ In the gut, autophagy (via xenophagy) restricts systemic dissemination of enteric pathogens such as Salmonella,⁵⁰ Shigella,⁸⁸ and Listeria.⁸⁹ It must be noted though that several pathogens subvert autophagic machinery and use it for their survival and replication.⁹⁰

There is significant evidence that autophagy helps survival and proliferation of distinct T cell population in intestinal mucosa.⁸⁷ In the absence of autophagy, Treg cell population is decreased while Th2 population is significantly increased along with aberrant immune responses in intestinal mucosa. However, disease-associated Th1 and Th17 population is decreased in mice with Atg16l1 deficiency.⁹¹ Autophagy may also contribute to the regulation of signaling cascades downstream of the T cell receptor in activated T cells, particularly via regulation of NFκB pathway.^87,92 The specific functions of autophagy in other unique leukocyte cells such as intraepithelial lymphocytes are not clear while autophagy is known to support maturation of innate lymphoid cells.⁹³ Analogous to the requirement of autophagy in highly secretory Paneth cells and goblet cells, autophagy is needed for long survival of plasma cells.⁹⁴ Thus, autophagy plays a nuanced function in gut immunity.

Autophagy and Intestinal Fibrosis

Autophagy also impacts intestinal fibrosis, which is an undesirable outcome in IBD. By and large, most previous studies show an antifibrotic role of autophagy,95, 96, 97, 98 although autophagy has also been shown to promote intestinal fibrosis.⁹⁹ This is analogous to the unclear role of autophagy in fibrosis in other organs. Though autophagy inhibits fibrosis via collagen degradation in primary fibroblasts,⁹⁵ inhibition of IL-23/IL-22-mediated intestinal fibrosis,⁹⁶ Rho-associated protein kinase inhibition,⁹⁷ and prevention of epithelial-to-mesenchymal transition,⁹⁸ it has also been shown to activate myofibroblasts.⁹⁹ It is possible that the adverse cellular microenvironment during inflammation decimate autophagy and it’s antifibrotic role as inhibition of NACHT, LRR and PYD domains-containing protein 3 in intestinal fibroblasts from IBD patients promotes autophagy while reducing proliferation and increasing collagen degradation.¹⁰⁰ Thus, the role of autophagy in fibrosis may be context, model, and stage dependent.

Effect of Autophagy on Gut Microbiota

The autophagic process in the host intestinal cells also regulates luminal microbiota. Experimental studies show that lack of autophagy in intestinal epithelial cells increases bacterial burden, reduces microbiota diversity, and alters the balance of gut microbiota from anti-inflammatory to proinflammatory bacteria.^101,102 The CD ATG16L1^T300A mutant expression in mice leads to promotion of Th1 and Th17 differentiation and also steady state and disease associated increase in Bacteroidetes species.⁴⁸ In humans, autophagy-related IBD variants are associated with reduction in butyrate-producing species,¹⁰³ and increase in IBD-promoting Ig-A coated bacteria¹⁰⁴ and pathosymbiont groups such as Enterobacteriaceae, Bacteroidaceae, and Fusobacteriaceae.¹⁰⁵ On the other hand, the gut microbiota and bacterial metabolites are able to influence intestinal autophagy, mostly via affecting the energy metabolism of intestinal epithelial cells.^106,107 Though the mechanisms are not completely understood, it is certain that autophagy in host intestinal cells regulate gut microbiota while defective autophagy is associated with establishment of pathobionts,¹⁰⁸ and that the complex interplay between gut autophagy, microbiota, and inflammation have implications for several systemic diseases besides IBD.

Autophagy in Neoplastic Process

Considering the facts that autophagy promotes cell homeostasis and ameliorate intestinal inflammation, it is thought that autophagy may reduce colitis-associated colon cancer (CAC) initiation. The experimental evidences for specific mechanisms through which autophagy can inhibit CAC development include suppression of ROS-induced chromosomal instability,¹⁰⁹ inflammasome,¹¹⁰ oxidative stress,¹¹¹ colonization of CAC-promoting bacteria,¹¹² etc. However, just like any other biological process, autophagy has double edge. Thus, being a cell survival mechanism, autophagy has undesirable outcomes once the tumor transformation has taken place. By providing nutrition and immune escape for the cancer cells, autophagy may promote tumor development in advanced stages.¹¹³ Thus, during early hepatocarcinogenesis, autophagy inhibits early-stage tumor formation¹¹⁴ while it supports cell proliferation during advanced stages of cancer.¹¹⁵ This protumorigenic and antitumorigenic role of autophagy may also be associated with other concomitantly active cellular mechanisms such as p53.¹¹⁶ Autophagy also forms the basis of embryonic development pause-like stage or diapause that tumor cells acquire during the chemoresistance.¹¹⁷ However, autophagy has more to offer as an anticancer tool in the form of autophagy-dependent cell death and autophagy-mediated cell death.¹¹⁸ During autophagy-dependent cell death, more than single ATG proteins promote cell death by mechanisms such as excessive ER phagy, mitophagy, or autosis in the presence of active autophagic flux.^118,119 In autophagy-mediated cell death, autophagy proteins interact with cell death molecules to trigger various types of cell death such as apoptosis, ferroptosis, and necrosis in specific cellular context of availability of adenosine triphosphate or depletion of iron and lipids.¹¹⁸ Thus, though current clinical applications in cancers are focused on inhibition of autophagy to decimate cell survival, there is also scope to use autophagy to induce cancer cell death.

Role of Autophagy in Chronic Liver Diseases (CLDs) and Fibrosis

Autophagy has several beneficial effects in chronic liver diseases (CLDs) such as viral hepatitis, alcohol-related liver disease, MASLD, and metabolic dysfunction–associated steatohepatitis (MASH). Autophagy promotes insulin sensitivity,¹²⁰ degrades intracellular lipids in the hepatocytes (lipophagy),¹²¹ and prevents cell injuries caused by oxidative stress and inflammatory cytokines.^122,123 The hepatocyte injury during CLD is followed by progressive replacement of liver parenchyma by fibrotic tissue with excessive deposition of collagen-rich extracellular matrix. Numerous studies unequivocally demonstrate that compounds that induce autophagy in the liver have an antifibrotic role through various mechanisms such as inhibition of AKT/mTOR,¹²⁴ modulation of Sirtuin 1/transforming growth factor-β/Smad3 pathway,¹²⁵ and accumulation of senescence-inducing transcription factor, GATA6.¹²⁶ Other autophagy-mediated antifibrotic mechanisms include apoptosis in hepatic stellate cells (HSCs),^127,128 as well as reduction in the accumulation and release of exosome-bound profibrotic mediators.^129,130 In fact, aging or obesity, which inhibit autophagy, are known to predispose to CLD.¹³¹ Also, the activity of autophagy-inducer AMPK are low and autophagy-repressor mTOR is high is MASH and our previous work shows that moderate exercise in patients with biopsy-proven MASH reduces phospho-ribosomal protein S6 levels, implicating autophagy in the beneficial effect of exercise in MASH.¹³²

Persistent liver injury activates the quiescent lipid-storing HSCs which transdifferentiate into proliferative fibrogenic myofibroblasts and become the primary source of liver fibrosis.¹³³ Despite significant research, the role of autophagy in HSCs remains ambiguous. Several studies depict autophagy as a profibrogenic process due to its association with HSC activation. The main mechanism underlying autophagy-mediated HSC activation and fibrosis is autophagy-mediated lipid droplet (LD) depletion which has been shown to concur with HSC activation.¹³³ It is postulated that LD loss supports the fibrogenic phenotype of HSC by supplying extra energy for extracellular matrix production and deposition.¹³³ However, the promotion of fibrogenic responses by autophagy in HSCs, as shown by many prior studies does not fit well with the fact that antifibrotic agents such as statin and metformin are known autophagy inducers. Moreover, interventions such as weight loss, calorie restriction, extended fasting, and physical activity that are recommended to patients with metabolic syndromes like MASLD and MASH promote autophagy.^131,134, 135, 136 Overall, the role of autophagy in liver homeostasis and disease appears to be model, experimental agent, cell type, and context-specific, and the dichotomous role of autophagy in hepatocytes (cytoprotective, antifibrogenic) and HSCs (profibrogenic) needs further investigation. Also, results of a clinical trial studying whether autophagy pathway–related genetic polymorphisms in ATG16l1 affect MASLD (NCT01988441) will shed light on the role of autophagy in liver diseases.

Context Specific Outcomes of Autophagy

Outcomes of autophagy depend on the contexts including but not limited to the cell type, cargo targeted by autophagy, extent of autophagy induction, stage of disease, etc. Besides the differential outcomes of autophagy in hepatocytes vs stellate cells as discussed above, cell-specific effect of autophagy is exemplified by the autophagic immune responses. Autophagy has been shown to promote Treg and decimate Th2 responses. The basis of this differential regulation of T cell repertoire is thought to be the metabolic differences. Treg and memory CD8⁺ cells being dependent on fatty acid metabolism suffer glycolytic shift in the absence of autophagy while Th2 cells, which can sustain prolonged glycolysis, resist deficiency of autophagy.^91,137 We have previously demonstrated the substrate specific outcome of autophagy induction in the model Madin-Darby Canine Kidney (MDCK) epithelia where autophagy induction reduced the paracellular permeability and increased the transepithelial resistance in claudin-2 high MDCK II cells, and this effect of autophagy was not evident in claudin-2 deficient MDCK I cells.⁸⁶ Autophagy can be cytodestructive when sustained AMPK-mediated activation of extracellular signal-regulated kinase mitogen-activated protein kinase causes disassembly of mTORC2 complex.¹³⁸ Similarly, autophagy is rapidly and excessively upregulated in necrotizing enterocolitis mice model and erythropoietin, a breast milk component downregulated autophagy via the Akt/mTOR signaling pathway to preserve the intestinal barrier function and reduce the incidence of necrotizing enterocolitis.¹³⁹ Furthermore, AMPK mediated cytokine-induced TJ barrier modulation independent of intracellular energy levels, indicating that autophagy regulators may have autophagy-independent role in various cellular processes.¹⁴⁰ A recent study showed that CYP1B1 is upregulated during HSC activation and causes metabolic depletion of trehalose in HSCs. In this context, trehalose, which is a known inducer of autophagy in several other cell types including hepatocytes, inhibits autophagy in HSCs.¹⁴¹ Thus, autophagy modulators may have cell-specific effects.

The role of autophagy in the host defense responses during an infectious process can be very complicated. For instance, H. pylori infection induces autophagy as a host defense mechanism and the outcomes of the interaction of autophagy and H. pylori pathogenic mechanisms are defined by the various virulence factors of H. pylori and the magnitude of apoptosis, ER stress, and lysosomal damage induced by H. pylori.¹⁴² Also, several microRNAs regulate the H. pylori invasion and survival in the gastric mucosa by either promoting or decimating expression of various ATGs.¹⁴² The outcome of autophagic modulation can also be defined by other concomitantly active cellular mechanisms. In a humanized mouse model of activated oncogenic allele of Kras-driven pancreatic ductal adenocarcinoma, autophagy has protumorigenic and antitumorigenic roles in the presence and absence of tumor suppressor p53, respectively. Thus, autophagy inhibitor hydroxychloroquine, which is currently being targeted in several clinical trials, promotes pancreatic ductal adenocarcinoma in mice containing oncogenic Kras but lacking p53.¹¹⁶ Also, the interaction between an environment and autophagy genes can result into cellular defects, for instance Atg16l1 T300A together with smoking leads to Paneth cell defects,¹⁴³ and such interaction can also affect Paneth cell function in a noncell-autonomous way via proinflammatory hyperactivity of LRRK2 kinase in macrophages.¹⁴⁴

Conclusion

Autophagy has been extensively studied in the biomedical field due to its importance in homeostasis as well as several human diseases. In spite of the use of autophagy-inducing agents or tools in several human health areas such as life style modifications (calorie restriction, fasting, exercise), neurodegenerative diseases (clearance of toxic protein aggregates), geroprotection (eg, luteolin supplement, NCT04489017), cancers (chemosensitization) etc., the application of autophagy in intestinal and hepatic inflammation is unclear. There are several aspects of autophagy that need to be clarified before autophagy can be fully employed in clinics. Though largely beneficial for the host, autophagy may be detrimental due to its prosurvival effects in cancer progression,¹⁴⁵ drug resistance in cancer,¹⁴⁶ or autophagic cell death caused by excessive autophagic activation.¹⁴⁷ Cell autonomous effects of autophagy is another reason for confusing outcomes at the tissue, organs, and host level, indicating need for the clear resolution of cell-specific and cell-independent functions of autophagy.

It is also recognized that the detection of autophagy can be a ‘moving target’ due to cell specificity and the dynamic nature of the autophagic process, as well as due to the new knowledge and techniques being constantly developed.¹⁴⁸ There is also lack of reliable, noninvasive autophagy biomarkers which can be used to assess the effectiveness of autophagy-based interventions. The current measurement of autophagy in terms of turnover of autophagy proteins, degradation of autophagy substrates, and detection of autophagic structures requires invasive tissue sampling or genetic modulation of autophagy in the animal models. Few innovative studies have used human blood samples to demonstrate autophagy flux,^149,150 but these methods need ex-vivo test sample incubations.

Autophagy being an integral part of cellular homeostasis, it is essential to know the impact of any particular IBD therapeutic tool on intestinal autophagy. The major IBD therapeutic agents such as corticosteroids, amino salicylates, thiopurines, immunomodulators, and biologics are known to be autophagy modulator.¹⁵¹ Previous studies show that functional autophagy induces regulatory macrophages during anti-tumor necrosis factor therapy and thus may be required for optimal therapeutic outcomes.¹⁵² Similarly, as cytoplasmic STAT3 can inactivate global transcription machinery which drives autophagy genes under stress,¹⁵³ autophagy may play a role in determining the outcomes of the new class of JAK-STAT inhibitors IBD therapy. Moreover, ability of rapamycin analogs in successful treatment of refractory CD^154,155 but failure in maintaining CD remission in a clinical study,¹⁵⁶ indicates necessity for context-based, personalized, and more nuanced examination of autophagy modulators in IBD therapeutics. Overall, the modulation of the autophagy pathway may have context dependent outcomes and not uniform responses across the various disease conditions. Thus, future studies should consider personalized, cell type–specific, physiological vs pathologic, tissue- vs organ-level outcomes in exploring the immense potential autophagy has in transforming the therapeutic efforts against gastrointestinal and liver diseases (Figure 2) as well as many more autoimmune and systemic diseases.

Figure 2.

Autophagy plays several beneficial roles in maintaining homeostasis, as indicated. The beneficial effects of autophagy are boosted by exercise, calorie restriction, and rapamycin and its analogs and statins. In certain disease states, autophagy induction may have undesirable outcomes mainly due to its prosurvival functions in a cell and tissue-specific contexts.