ATG16L1 (Autophagy-Related 16 Like 1) was the first identified autophagy-related CD susceptibility gene. ATG16L1 functions as a scaffold protein that helps to assemble the autophagy machinery, and is required for the formation of autophagosomes ^[1][31]. The ATG16L1 T300A mutation is associated with an increased risk of developing CD, and several studies have suggested that these mutations may impair autophagy in intestinal epithelial cells and immune cells, contributing to the development of this disease ^[1][31]. The T300A variant in ATG16L1 results in aberrant Paneth cell architecture and disordered granularity ^[2][20]. Some studies have found that ATG16L1 deficiency leads to impaired autophagy-mediated clearance of intracellular bacteria in intestinal epithelial cells, resulting in increased bacterial load and the activation of inflammatory signaling pathways ^[1][3][31,38]. Further, ATG16L1 mutations reduce the expression of antimicrobial peptides in intestinal epithelial cells, impairing the ability of these cells to defend against pathogenic bacteria ^[1][31].

IRGM (Immunity-Related GTPase M) has been shown to interact with components of the autophagy machinery, such as ULK1 and Beclin 1, to promote the formation of autophagosomes in response to infection with intracellular pathogens ^[3][4][5][38,40,41]. IRGM-deficient mice have impaired clearance of intracellular bacteria in intestinal epithelial cells, leading to increased inflammation and tissue damage ^[6][42]. IRGM dampens inflammatory signaling via selective autophagy of the Nucleotide-binding domain, Leucine-rich Repeat-containing Family, and Pyrin Domain Containing 3 (NLRP3) inflammasome, thereby inhibiting its activation and downstream signaling ^[7][43]. In addition, IRGM1-deficient mice demonstrate Paneth cell abnormalities and elevated susceptibility to intestinal inflammation ^[8][44]. IRGM SNPs rs1000113, rs11747270, rs9637876, rs13361189 and rs180802994 are associated with CD ^[9][45]. Some studies have reported that IRGM SNPs may increase fecal lactoferrin (inflammatory biomarker) from the ileum and TNF in whole blood in humans with CD ^[4][40]. Further studies are warranted to explore more functional alterations due to IRGM SNPs in CD.

NOD2 (Nucleotide-Binding Oligomerization Domain-Containing Protein 2) encodes a protein involved in the regulation of the immune system and the maintenance of the intestinal barrier function ^[10][11][12][46,47,48]. NOD2 is an intracellular receptor that recognizes muramyl dipeptide motifs, a component of the bacterial cell wall, and upon activation elicits responses to eradicate invading pathogens including activation of NFκB inflammatory signaling. Recent studies have shown that NOD2 interacts with several proteins involved in autophagy, including ATG16L1 and IRGM, to recruit autophagy machinery to the cellular site of invading bacteria and incorporate bacteria into the forming autophagosome. This has been shown to be important in intestinal epithelial cells at the interface with the gut microbiome ^[3][13][38,49]. Genetic variation in NOD2 accounts for 20% of CD genetic risk, with three common variants, R702W, G908R, and L1007fs, particularly associated with ileal CD ^[14][50]. NOD2 mutations impair NFκB and autophagic responses to intracellular bacteria, leading to the accumulation of bacteria in intestinal epithelial cells ^[15][51]. NOD2 mutations have also been shown to reduce the expression of ATG16L1 in intestinal epithelial cells as an additional mechanism whereby autophagy-mediated clearance of intracellular bacteria is impaired ^[16][52]. Furthermore, NOD2 has been implicated in the Paneth cell-mediated responses against intestinal bacteria-induced inflammation ^[17][53].

LRRK2 (Leucine-Rich Repeat Kinase 2) plays a role in several cellular processes, including autophagy, the immune response, and the regulation of mitochondrial function ^[18][19][54,55]. LRRK2 has been demonstrated to regulate the formation of autophagosomes in intestinal epithelial cells and to maintain functional Paneth cells in CD ^[20][56]. LRRK2 plays a role in regulating the formation of autophagosomes by interacting with several proteins involved in this process. One such protein is Rab29, which is a small GTPase that regulates the recruitment of the autophagy-related protein ATG9 to the site of autophagosome formation ^[21][57]. In addition, LRRK2 has been shown to phosphorylate Rab29, which enhances its ability to recruit ATG9 to the site of autophagosome formation ^[21][57], and to phosphorylate ULK1, enhancing its activity and promoting the initiation of autophagy ^[22][58].

Mutations in the LRRK2 gene have been identified as a genetic risk factor for several diseases, including Parkinson’s disease and CD ^[18][23][54,59]. LRRK2 mutations impair the autophagic response to intracellular bacteria in intestinal epithelial cells, leading to an accumulation of bacteria and the activation of inflammatory signaling pathways ^[24][60]. In addition to its role in autophagy regulation, LRRK2 has also been shown to regulate the immune response and the expression of several toll-like receptor (TLR)-mediated inflammatory cytokines and chemokines ^[25][61]. LRRK2 mutation (M2397T polymorphism), associated with CD, alters CD14⁺ monocyte-derived type II interferon (IFN) response ^[26][62]. Moreover, different variants of LRRK2 are linked to CD and Parkinson’s disease. In CD, the exact mechanisms by which LRRK2 variants contribute to CD pathology are not yet fully understood. However, it is speculated that LRRK2 variants, such as LRRK2 G2019S, may impact immune responses, and intestinal barrier functions, or alter gut microbial composition ^[27][28][29][63,64,65]. In addition, a mutation in the LRRK2 N2081D allele results in elevated kinase activity while the LRRK2 R1398H variant increases its enzymatic activity altering LRRK2 functionality in CD and Parkinson’s disease ^[30][66]. In Parkinson’s disease, several pathogenic LRRK2 variants, such as G2019S and R1441C/G, have been linked to an increased risk of developing this disease ^[28][31][64,67]. These variants are thought to lead to increased LRRK2 kinase activity, which can contribute to neuronal dysfunction and degeneration. Dysregulated kinase activity may affect various cellular processes, including mitochondrial function, protein degradation pathways, and synaptic transmission, ultimately leading to the development of Parkinson’s disease symptoms ^[31][32][67,68].

ULK1 (Unc-51-Like Autophagy Activating Kinase 1) is a protein kinase that plays a critical role in the initiation of autophagy. ULK1 is part of a protein complex known as the ULK1 complex, which also includes ATG13, FIP200, and ATG101 ^[33][69]. This complex is activated in response to cellular stress, such as nutrient deprivation or the accumulation of damaged cellular components ^[33][69]. Once activated, the ULK1 complex phosphorylates several downstream targets, including the protein Beclin 1, which triggers the formation of autophagosomes ^[33][69]. In addition to its role in autophagy initiation, ULK1 also has several other cellular functions, including the regulation of cell growth and differentiation, metabolism, and the response to oxidative stress ^[34][35][36][70,71,72]. Dysregulation of ULK1 has been implicated in several diseases, including CD, cancer, neurodegenerative disorders, and metabolic disorders ^[37][73]. In regards to CD, studies have shown that ULK1 may play a role in regulating the differentiation and function of immune cells ^[38][74]. Dysregulation of ULK1 has also been linked to increased intestinal permeability, which can lead to the infiltration of bacteria and other harmful substances into the intestinal tissue, triggering an inflammatory response ^[39][75]. The SNP rs12303764 in ULK1 is associated with CD ^[38][74].

ATG4 (Autophagy-related protein 4) is a cysteine protease that plays a critical role in the regulation of autophagy via processing of LC3 ^[40][76]. Missense variants in ATG4C associated with CD are N75S, R80H, C367Y, K371R, R389X, and a frameshift mutation specifically enriched in Finland (1:62819215:C:CT) ^[41][9]. Four of these are truncating variants, suggesting loss-of-function variants in ATG4C increase the risk of CD. Several studies have suggested that dysregulation of ATG4-mediated processing of LC3 may contribute to the development and progression of CD. A study reported that mice lacking ATG4B, one of the isoforms of ATG4, had impaired autophagy and were more susceptible to the development of inflammation ^[42][77]. It has also been reported that ATG4B is downregulated in the intestinal mucosa of patients with CD ^[43][78], suggesting that dysregulation of ATG4-mediated autophagy may contribute to the development of this disease.

TCF4 (Transcription Factor 4) is a member of the basic helix--loop--helix (bHLH) family of transcription factors that play an important role in the regulation of gene expression and cell survival ^[44][45][79,80]. As a Wnt signaling pathway transcription factor, TCF4 plays an important role in the health of Paneth cells and production of antimicrobial peptides Defensin-5 and Defensin-6 in a Wnt-pathway-dependent manner ^[46][81]. TCF4 deficiency leads to impaired autophagy in intestinal epithelial cells, resulting in the accumulation of intracellular bacteria and the activation of inflammatory signaling pathways ^[47][82]. SNP rs3814570 in the TCF4 promoter region is associated with ileal CD, with the strongest association in patients with stricturing disease ^[48][83].

Xenophagy is a specialized form of autophagy that involves the degradation and elimination of invading microorganisms, such as bacteria, viruses, and parasites ^[49][50][98,99]. During xenophagy, the microorganisms are engulfed by an autophagosome and degraded in the autolysosome. Xenophagy is an important innate immune defense mechanism. It is activated by specific cellular receptors, such as TLRs, NOD-like receptors (NLRs), pyrin domain containing 3 (NLRP3), RIG-I-like (RLRs), and C-type receptors (CLRs), that recognize the presence of microbial components, such as bacterial cell wall components or viral nucleic acids ^[51][52][100,101]. TLRs are expressed on the surface of immune cells and basolaterally on intestinal epithelial cells and recognize specific pathogen-associated molecular patterns (PAMPs) on the surface of pathogens. TLR activation triggers downstream signaling cascades that can activate xenophagy and other immune responses ^[53][102]. NLRs, including NOD2, are cytosolic pattern recognition receptors that surveil the intracellular environment and activate inflammatory cascades and inflammasomes in response to infection ^[54][55][103,104]. NLRP3 recognizes various PAMPs and danger-associated molecular patterns (DAMPs), including bacterial cell wall components and damaged or dying host cells, respectively ^[56][105]. In addition to NLRP3 and TLRs, other receptors involved in xenophagy include RLRs, which recognize viral RNA, and the cGAS-STING pathway, which senses cytosolic DNA. These receptors activate signaling pathways that converge on the autophagy machinery to promote the clearance of invading pathogens and pathogenic DNA ^[57][106]. Dysregulation of xenophagy has been linked to various infectious diseases, including tuberculosis, Salmonella infection, and viral infections such as influenza and HIV ^[52][58][101,107].

Xenophagy has been shown to play a critical role in modulating the function of macrophages and DCs in several ways. Xenophagy regulates the production of proinflammatory cytokines, such as interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) in macrophages and DCs ^[59][60][108,109]. Xenophagy can regulate inflammasome activation and subsequent IL-1β production by controlling the degradation of bacterial or viral components and regulating the activation of signaling pathways downstream of cytokine receptors in response to bacterial infection ^[61][110]. Xenophagy can dampen the production of type I IFNs in response to viral infection by restricting the replication of viral pathogens in infected cells. Conversely, xenophagy can enhance the activation of interferon-stimulated genes (ISGs), including ISG15, UbE1L, UbcH8, and Herc5 ^[62][63][111,112], important for the innate immune response. Xenophagy can modulate cytokine receptor signaling by regulating the turnover of cytokine receptors. For example, xenophagy can mediate the degradation of the interleukin-1 receptor (IL-1R) and the type I IFN receptor (IFNAR), leading to the downregulation of IL-1β and type I IFN signaling, respectively ^[64][113]. Xenophagy also plays a critical role in modulating antigen presentation in macrophages and DCs ^[65][114]. Xenophagy can facilitate the processing of antigens from intracellular pathogens for presentation on MHC II molecules, leading to the activation of CD4+ T cells ^[64][113]. Xenophagy can also regulate the turnover of MHC II molecules, which can impact the efficiency of antigen presentation ^[66][115]. Through these mechanisms, xenophagy plays a critical role in modulating functional responses of macrophages and DCs, impacting the innate immune response to intracellular pathogens.

Multiple studies have suggested that defects in xenophagy may contribute to the development and progression of CD. Studies have shown that ATG16L1-, ATG4-, ULK1-deficient mice exhibit defective xenophagy and are more susceptible to bacterial infections in the gut ^[67][68][69][116,117,118]. Studies have also shown that IRGM expression is reduced in patients with CD ^[70][119], and that IRGM-deficient mice exhibit defective xenophagy and are more susceptible to bacterial infections in the gut ^[8][44]. In addition, mice lacking IRGM have been shown to exhibit defective Paneth cells accompanied by intestinal injury ^[71][120]. Loss of functional mutations in NOD2, associated with CD, result in decreased response to invading bacteria and abnormal immune responses driving chronic intestinal inflammation ^[54][55][103,104]. Mutations in LRRK2 have been associated with an increased risk of developing Parkinson’s disease, and some studies have suggested that LRRK2 may also play a role in regulating xenophagy in the gut ^[72][121].

ERphagy is a selective form of autophagy that involves the degradation of endoplasmic reticulum (ER) by autophagy. The ER is a complex network of interconnected tubules and flattened sacs that plays a critical role in protein synthesis, lipid metabolism, and calcium homeostasis. ERphagy is initiated by the recognition and isolation of a specific region of the ER by a protein called FAM134B, which acts as an ER-phagy receptor ^[73][122]. FAM134B binds LC3II and promotes the incorporation of the targeted ER region into an autophagosome that is forming.

ER stress occurs when there is an imbalance between protein folding demand and capacity in the ER ^[74][123]. ER stress can be induced by a variety of factors, including inflammation, oxidative stress, and genetic mutations. In CD, chronic inflammation in the intestinal mucosa can induce ER stress in various cells, including Paneth cells and goblet cells ^[75][124]. In CD, ER stress in Paneth cells has been shown to impair the secretion of antimicrobial peptides, leading to dysbiosis and increased susceptibility to bacterial infections ^[2][76][20,125]. Similarly, ER stress in goblet cells has been shown to impair mucus secretion and increase susceptibility to bacterial adhesion and invasion in CD ^[76][77][125,126]. The unfolded protein response (UPR) is a cellular stress response pathway that is activated in response to ER stress ^[78][127]. The UPR is initiated by three transmembrane proteins: PERK, ATF6, and IRE1. In CD, the UPR pathway is activated in Paneth cells and goblet cells, leading to increased expression of genes in humans (HSPA5 encoding BIP/HSP70, DERL1, EDEM, ATF6, XBP1, and IRE1) that are involved in protein folding, degradation, and secretion ^[75][124].

ERphagy plays an important role in dampening ER stress and maintaining ER homeostasis by removing damaged or excess ER. Dysfunction of ERphagy has been implicated in several diseases, including CD, neurodegenerative disorders, and cancer ^[79][80][128,129]. ERphagy is regulated by several key proteins, including X-box binding protein 1 (XBP1) and ATG16L1 ^[81][82][130,131]. XBP1 is a transcription factor that regulates the expression of genes involved in protein folding, secretion, ER stress response, and UPR ^[83][84][132,133]. Dysregulation of the UPR has been implicated in the pathogenesis of CD ^[85][134], and studies have suggested that XBP1 plays a role in the regulation of autophagy in response to ER stress ^[86][135]. Mice with deletion of Xbp1 in the intestinal epithelium (Xbp1^ΔIEC) developed ER stress, Paneth cell impairment, and mild-to-moderate spontaneous ileitis ^[87][18]. Loss of autophagy via crossing to mice with deletion of Atg7 in the intestinal epithelial cells (Atg7/Xbp1^ΔIEC), resulted in unresolved ER stress and a worsening of ileitis compared to Xbp1^ΔIEC mice ^[87][18]. Later studies demonstrated that prolonged activation of the UPR driving accumulation of IRE1α aggregates contributed to the development of transmural ileitis when not dampened by autophagy ^[88][136]. Studies have shown that XBP1 plays a role in the regulation of ERphagy by promoting the expression of FAM134B, the ERphagy receptor, and that ATG16L1 interacts with FAM134B and promotes ERphagy ^[81][89][130,137]. Similar to Paneth cells, impairment of ERphagy in mice by ATG16L1, IRGM1, or NOD2 deficiency results in goblet cell mucus secretion defects and compromised barrier function ^[8][77][90][44,126,138]. ER stress unresolved by ERphagy may be especially deleterious in intestinal secretory cells, such as Paneth cells or goblet cells, due to their high reliance on ER for their protein secretory functions.

Mitophagy is a specialized form of autophagy that involves the selective degradation and recycling of damaged or dysfunctional mitochondria, the energy-producing (ATP) organelles within cells. Damaged or dysfunctional mitochondria can produce harmful reactive oxygen species and contribute to cellular dysfunction and disease ^[91][139]. During mitophagy, mitochondria are targeted for degradation by specific receptors, such as Nix/Bnip3L or PINK1 recruitment of Parkin, and engulfed by the autophagosome. Dysregulation of mitophagy has been linked to various diseases, including neurodegenerative disorders, IBD, cancer, and metabolic disorders ^[92][93][94][140,141,142].

Similar to ER stress, studies investigating mitochondrial stress in the intestine have revealed the role of mitochondrial health as being crucial to Paneth cell health and functionality. A recent study induced intestinal epithelial cell-specific mitochondrial dysfunction via deletion of the mitochondrial chaperone protein Prohibitin 1 ^[95][19]. The mice developed spontaneous ileitis that was preceded by mitochondrial dysfunction in all epithelial cells, crypt cell death, and defects in Paneth cells. Importantly, IBD patients exhibit intestinal epithelial mitochondrial dysfunction and decreased expression of PHB1 ^[93][141]. Paneth cell abnormalities, including loss of antimicrobial peptide expression, and ileitis could be ameliorated in PHB1 deficient mice by the administration of a mitochondrial-targeted antioxidant, Mito-Tempo, suggesting that mitochondrial reactive oxygen species contributed to disease progression in this model of intestinal epithelial cell mitochondrial dysfunction ^[95][19]. A specific role of Paneth cell mitochondrial dysfunction in driving ileitis was demonstrated using mice with Paneth cell-specific deletion of PHB1 ^[95][19]. Further studies in these mice revealed a loss of Nix/Bnip3L-mediated mitophagy in the intestinal epithelium during PHB1 deficiency ^[96][143], suggesting mitochondrial dysfunction coupled with inefficient removal by mitophagy.

Collectively, these results identify Paneth cells as highly susceptible to mitochondrial dysfunction. Deficiency in Atg16L1, Atg7, Atg5, or Irgm1 in mice causes Paneth cell defects and CD autophagy-related susceptibility genes, or their disease-linked variants (ATG16L1 T300A, LRRK2, IRGM), are associated with Paneth cell abnormalities in the ileum ^[97][98][99][144,145,146]. Recent studies demonstrated mitochondrial impairment in abnormal CD Paneth cells that correlated with ATG16L1 T300A mutation ^[100][147]. Furthermore, LRRK2 serves as a significant risk factor for both Parkinson’s and Crohn’s disease, conditions associated with impairments in mitophagy and/or mitochondrial health ^[28][64]. Mitochondrial health may be especially important in Paneth cells due to their long-lived nature (30–60 days), compared to other intestinal epithelial cells ^[101][17], and their extensive ER for secretory functions coupled with the known communication between mitochondria and ER ^[102][148]. Future studies are needed to fully understand the complex interplay between mitochondria and ER in the intestine during health and disease and how it impacts specific epithelial and immune cell types.