Mechanisms and function of autophagy in intestinal disease

Autophagy and the control of inflammatory cytokines

In order to control homeostasis, cells that form the epithelial barrier, and immune cells in the intestine, must maintain host defenses against pathogenic microbes while maintaining tolerance to commensals. Numerous studies have highlighted the critical role of autophagy in limiting inflammatory cytokine responses and demonstrated how defects in autophagy lead to uncontrolled inflammation in the gut. Initial investigations focused on the role of core autophagy proteins such as ATG16L1 in the release of IL1B/IL-1β and IL18 after stimulation with bacterial products.⁸ This increased cytokine release has subsequently been demonstrated in both humans and mice harboring the ATG16L1^T300A susceptibility polymorphism, consistent with impaired selective autophagy in cells expressing this risk variant.^9–11 Increased CASP3 (caspase 3)-mediated cleavage of ATG16L1^T300A resulting in decreased protein levels and disruption of selective autophagy was found to be the mechanism underlying this risk polymorphism.^9,10 Furthermore, ATG16L1^T300A may act in a dominant-negative manner, suggesting a mechanism by which individuals harboring only one copy of this risk allele might exhibit increased susceptibility to CD.¹² Together, these data underscore a critical role in CD susceptibility for increased IL1B secretion after bacterial challenge resulting from impaired autophagy. Autophagy-mediated control of IL1B might also be a key factor underlying other genetically controlled diseases such as familial Mediterranean fever.¹³ Interestingly, the enhanced cytokine response associated with impaired ATG16L1 function is also beneficial in the context of extracellular enteric bacterial infection and colorectal cancer, suggesting potential explanations for why this polymorphism is highly prevalent in some populations.^14,15

Subsequent investigations have expanded our understanding of the role of autophagy in inflammation and also highlighted gaps in our understanding of the underlying processes. Autophagy is required for every cell type to function properly; however, impaired autophagy has different phenotypic effects in each cell type in the gut mucosa. Emerging data suggest that the cytokines affected by impaired autophagy and the associated phenotypic outputs are cell-type dependent, stimulation dependent, and autophagy-gene dependent highlighting the complexity of this incompletely understood mechanism. For example, acute amino acid starvation ameliorates intestinal inflammation in an autophagy-dependent manner.¹⁶ In this context, impaired autophagy enhances reactive oxygen species (ROS) production, inflammasome activation, Th17 responses, and IL1B production.¹⁶ Other CD-associated polymorphisms in distinct genetic loci also result in impaired autophagy and enhanced inflammatory response. For example, polymorphisms in MTMR3 (myotubularin related protein 3) lead to impaired autophagy, increased stimulation-dependent CASP1 activation, and increased IL1B release.¹⁷ Additionally, a CD-associated polymorphism within PTPN2 (protein tyrosine phosphatase, non-receptor type 2) alters autophagosome formation and bacterial clearance resulting in increased inflammatory cytokine secretion in intestinal cells.^18,19 A number of genes within IBD susceptibility loci were recently found to function in antibacterial autophagy and bacterial clearance, including the E3 ligase SMURF1 and the peroxisomal protein PEX13 (Fig. 1).^5,20,21 Finally, genetic polymorphisms in autophagy-related genes also influence the efficacy of anti-TNF therapies in CD.²²

Autophagy in intestinal epithelial cells

Epithelial cells in the intestine act as a first line of defense against bacterial invasion and represent an important control in limiting microbial interactions with the host. A specialized type of epithelial cell, Paneth cells in the small intestine, secrete antimicrobial peptides, including lysozyme, which function as antibiotics to restrict bacteria in close proximity.²³ Autophagy is critical in the development and function of Paneth cells. Recent studies have identified a key role for cellular metabolic changes and ROS production in Paneth cell maturation.²⁴ Importantly, genetic polymorphisms that disrupt pathways underlying Paneth cell function are thought to be critical in the development of CD.^9,10,25–27 It is likely that impaired autophagy could directly alter this pathway through ROS or changes in metabolic substrate availability, thus leading to Paneth cell dysfunction and disease. Paneth cells are particularly susceptible to disruptions in autophagy, with the most prominent effects centered on antimicrobial peptide-containing granule formation within these cells.^28,29 In infected mice. Paneth cells undergo autophagy-mediated secretion of lysozyme. Mice harboring the ATG16L1^T300A polymorphism exhibit aberrant packaging and secretion of lysozyme during infection resulting in impaired bacterial killing.³⁰ Mechanistically, sensing of commensal bacteria through NOD2 mediates LRRK2-dependent vesicular cargo sorting and underlies Paneth cell function in response to bacterial stimulation (Fig. 1).^31,32 Thus, Paneth cells are critical for host-microbe interactions in the intestine, and autophagy is a key pathway underlying proper Paneth cell function.

Goblet cells are another specialized epithelial cell type in the intestine that function in the innate defense against intestinal microbes. Goblet cells help form the mucous layer in the intestine by secreting mucins. Together these cells, in conjunction with the intestinal epithelial barrier, regulate the microecology of the gut. Autophagy controls the development and function of goblet cells,^33,34 and the ATG16L1^T300A polymorphism alters goblet cell morphology.⁹ Recently, a population of sentinel goblet cells has been identified in the intestine that senses pathogens in an endocytosis-mediated and ROS-dependent pathway resulting in the release of MUC2, a key intestinal mucin.³⁵ This event triggers MUC2 secretion from bystander cells and protects cells in the intestinal crypt from bacterial invasion.³⁵ These data highlight how genes within distinct CD-associated loci can functionally converge on the same pathway, and thus pinpoint critical cellular pathways underlying pathogenesis.

Autophagy and the control of intestinal microbes

Host-microbe symbiosis is critical in maintaining intestinal homeostasis. Dramatic shifts in the composition and diversity of the intestinal microbiota have been observed in CD patients and it is thought that some of these changes are mediated by host genetics and autophagy.^36,37 A number of genes in IBD susceptibility loci restrict intracellular bacteria, including ATG16L1, NOD2, IRGM, CALCOCO2/NDP52, and GPR65, suggesting that antibacterial autophagy is a major pathway underlying intestinal homeostasis in vivo.^5,31,38–41 These genes function in various steps of the antibacterial autophagy cascade, including bacterial sensing (NOD2), bacterial targeting (IRGM, CALCOCO2), autophagosome elongation (ATG16L1), and lysosomal function (GPR65). The interaction between NOD2 and ATG16L1 in antibacterial autophagy has been of particular interest and this interaction is thought to be important for bacterial clearance and increased cytokine release.^14,42 These findings highlight the ways in which autophagy controls the direct elimination of bacteria and the phenotypes associated with disruptions at multiple steps in this pathway (Fig. 1).

Alterations to autophagy can disrupt mucosal immunity in both cell-autonomous and cell non-autonomous manners. In myeloid cells, loss of ATG16L1 or the presence of the ATG16L1^T300A polymorphism alters the clearance of intracellular bacteria and antigen processing and presentation.^43,44 Proper presentation of components derived from commensal bacteria and impaired antigen presentation in the context of ATG16L1^T300A results in impaired induction of regulatory T cells, demonstrating cell nonautonomous effects of autophagy dysregulation in myeloid cells.⁴⁵ Furthermore, impaired autophagy in T cells has recently been shown to alter T cell metabolism with direct consequences on T cell activation.⁴⁶ In addition, autophagy plays a direct role in promoting the survival of regulatory T cells and in limiting proinflammatory TH2 cell expansion.⁴⁶ Recently, a role for TAX1BP1-driven autophagy in controlling cellular metabolism underlying T cell activation has been elucidated, further underscoring the critical relationship between immune activation, autophagy, and cellular metabolism.⁴⁷ Further research is required to determine how disruptions to autophagy alter cellular metabolism and immune outputs in other immune cell types.

Lysosomal dysfunction and pathogenesis

Lysosomes are dynamic organelles that control numerous cellular processes including the innate immune response, signaling, tissue repair, lipid homeostasis, iron homeostasis, and cellular energetics.⁴⁸ Contents of autophagosomes are delivered to lysosomes for degradation, and, thus, lysosomes are the fundamental endpoint of autophagy. Furthermore, lysosomes are critical cellular sites for MTOR (mechanistic target of rapamycin) complex 1 (MTORC1) kinase activation. Lysosomes are therefore key integration sites for the breakdown of nutrients and autophagy regulation.⁴⁹ Numerous genes control lysosomal positioning as well as precise lysosomal function, and these genes are critical for autophagic flux.⁵⁰ Additionally, the transcription factors TFEB and ZKSCAN3 simultaneously control autophagy and lysosomal function by regulating the expression of numerous autophagy and lysosomal genes.⁵¹ TFEB is an activator of autophagy, whereas ZKSCAN3 is an inhibitor. Activation of PRKC (protein kinase C) leads to activation of TFEB and concurrent inactivation of ZKSCN3.⁵¹ TFEB activation can also be triggered by release of lysosomal Ca²⁺ and activation of the protein phosphatase PPP3/calcineurin.⁵² Together these data highlight the tightly linked control of autophagy and lysosomal function, and suggest that impairment of either of these pathways could contribute to pathogenesis by similar mechanisms.

Lysosomal storage disorders (LSDs) are characterized by mutations in genes encoding lysosomal proteins, which result in accumulation of undigested macromolecules and progressive disease. Given that autophagy and lysosomes are inextricably linked, accumulating evidence suggests that LSDs can also be considered to be disorders of autophagy dysfunction. In support of this concept, LSDs preferentially affect cell types such as neurons that are particularly susceptible to disruptions to autophagy.^53,54 In people with Niemann-Pick disease type C1, the lipid signaling molecule sphingosine accumulates in lysosomes, which dampens sphingosine-mediated Ca²⁺ release and TFEB activation.⁵⁵ Additionally, a recent study demonstrated that NPC1 (NPC intracellular cholesterol transporter 1) protein and the solute carrier SLC38A9 coordinate cholesterol export from lysosomes and cholesterol sensing to modulate MTORC1 activation.⁴⁹ Loss of NPC1 disrupts this process and leads to sustained activation of MTORC1. Autophagy is also disrupted in Danon disease, a heritable lysosomal disease resulting from mutations in LAMP2.⁵⁶ Danon disease is one of the best-characterized autophagic vacuolar myopathies, a group of heritable myopathies characterized by the accumulation of autophagic vacuoles.⁵⁷ Taken together, these data highlight the ways in which autophagy and lysosomes are cross-regulated and how this regulation is impaired in disease.

Emerging data suggest that the role of lysosomal function in mucosal immunity and CD has not been fully appreciated. For instance, some patients with the LSDs Niemann-Pick disease type C1 and Hermansky-Pudlak syndrome develop a Crohn-like ileitis.⁵⁸ Mevalonate kinase deficiency (MKD) is a heritable, autoinflammatory disease that results in a reduction of prenylated proteins, and some MKD patients develop severe colitis.⁵⁹ Impaired autolysosomal turnover and increased IL1B secretion have been observed in the context of MKD mutations, suggesting these pathways are critical in disease.^60,61 This observation suggests that lysosomal function directly affects intestinal homeostasis, potentially through an autophagy-dependent mechanism. Furthermore, recent studies have identified a role for GPR65, an H⁺-sensing G protein-coupled receptor, in maintaining lysosomal pH and thus lysosomal function.⁵ Polymorphisms in GPR65 are associated with IBD, including 1 polymorphism, I231L, which results in a missense mutation. Loss of Gpr65 impairs autophagy-mediated intracellular bacterial clearance and disrupts mucosal immunity, suggesting that lysosomal function is critical for intestinal homeostasis.⁵ A study of patients with Niemann-Pick disease type C1 who developed IBD also identified defects in the ability to clear and respond to intracellular bacteria.⁵⁸ These studies underscore how the study of LSDs can shed light on the physiological role of autophagy disruption in intestinal homeostasis.

Given the critical role of autophagy in intestinal homeostasis and the impact of autophagy dysfunction in LSDs and inflammatory bowel diseases there is much therapeutic interest in finding molecules to activate selective autophagy. Such therapeutics could offer the critical balance of suppressing inflammation in immune cells such as macrophages and T cells while maintaining pathogen defense in critical epithelial cell types such as Paneth cells and goblet cells. Further understanding of the role of autophagy and autophagy-dependent pathways in these cells will likely identify appropriate therapeutic targets for this critical pathway.