1 Introduction

Crohn's disease, a chronic idiopathic inflammatory bowel disorder, is named after the American gastroenterologist Burrill B. Crohn. In 1932, he published a seminal paper outlining Crohn's disease symptoms for the first time: fever, diarrhea, and emaciation (Crohn et al. 1984). Crohn's disease can occur anywhere in the gastrointestinal tract, with recurrent transmural intestinal inflammation produced by immune responses, most commonly involving the terminal ileum in adults but the entire intestine in children (Alula and Theiss 2023). Although the etiology of Crohn's disease has not been defined, multiple studies have revealed that Crohn's disease may be linked to genetic susceptibility, environmental factors, immune system dysfunction, or a combination of these factors (Ruthruff 2007).

Autophagy is a vital cellular process that entails the recycling and degradation of superfluous or defective cellular components such as organelles and proteins (Mizushima and Komatsu 2011). The initiation of autophagy involves the formation of the phagophore, where the edges of isolation membranes elongate and encompass cytoplasmic cargos. Following the encapsulation of cytoplasmic cargos within the double-membrane structure, autophagosomes undergo maturation, resulting in the formation of a fully developed autophagosome structure. Subsequently, these autophagosomes fuse with late endosomes and lysosomes, initiating the degradation process of the enclosed cargos (Glick et al. 2010). Autophagy dysregulation has been linked to a variety of diseases, including cancer, infections, neurodegenerative disorders, and autoimmune diseases (Klionsky et al. 2021).

Single nucleotide polymorphisms (SNPs) in autophagy-associated genes have been identified as a susceptibility factor for Crohn's disease, providing the first indication that autophagy contributes to the genesis of the disease. This information was obtained by genome-wide association studies (GWAS) (Verstockt et al. 2018). One of the initial pieces of evidence came from the autophagy-related 16 like 1 (ATG16L1) study by Hampe et al., who discovered a statistically significant interaction for Crohn's disease risk between rs2241880, a coding SNP (T300A), and established nucleotide-binding oligomerization domain 2 (NOD2) susceptibility variants (P = 0.039) (Hampe et al. 2007). NOD2 was the first gene implicated in Crohn's disease susceptibility, and mutations in the NOD2 gene may lead to aberrant activation of the immune system, which can increase the likelihood of develo** Crohn's disease (Hugot et al. 2001; Ogura et al. 2001). As research progresses, more and more autophagy-related Crohn's disease susceptibility genes have been identified, such as IRGM, ULK1, LRRK2, TLR4, etc. (Lapaquette et al. 2012a). In addition, the therapeutic effects of some conventional drugs used to treat Crohn's disease may be mediated in part by modulation of the autophagy pathway, further confirming the link between autophagy and Crohn's disease (Hooper et al. 2017). Furthermore, several autophagy modulators are proposed to possess potential therapeutic effects on Crohn's disease (Zhang and Liu 2019).

Thoroughly analyzing the impact of autophagy-related gene mutations on Crohn's disease will be beneficial in deepening our understanding of the intricate interactions between autophagy and this disease. Subsequently, we discussed in detail the effect of autophagy-related gene mutations in Crohn's disease, elucidating that autophagy modulation may be a novel approach to treating Crohn's disease. In this review, we also emphasize the autophagy modulation involved in conventional small-molecule drugs for treating Crohn's disease and introduce autophagy modulators with potential therapeutic effects on Crohn's disease, which will help us to develop more drugs to treat Crohn's disease.

2 Overview of Crohn's disease

Crohn's disease is commonly recognized as an autoimmune disease marked by chronic inflammation in any area of the gastrointestinal tract (Beutler 2001). The disease's progression is devastating, and its prevalence is expanding globally (Roda et al. 2020). Crohn's disease’s clinical manifestation varies depending on the location of the disease, the intensity of inflammation, and the disease's behavior (Danese et al. 2015). Pain in the lower right abdomen, persistent diarrhea, and weight loss are common symptoms of the disease (Alula and Theiss 2023). Patients may also feel loss of appetite and malaise (Torres et al. 2017). For patients with colon involvement, rectal bleeding or bloody diarrhea may be the main manifestation. Furthermore, approximately one-third of individuals develop perianal disease (Eglinton et al. 2012). Lower health-related quality of life ratings are frequently associated with pain and fecal incontinence in those with perianal Crohn's disease (Parian et al. 2023). Extraintestinal manifestations, which can impact many body systems such as the eyes, skin, muscles, bones, mouth, and hepatobiliary system, are also reported in nearly half of Crohn's disease patients (Vavricka et al. 2011).

Although the exact etiology of Crohn's disease is unknown, it is believed that a combination of genetic susceptibility, environmental factors, and gut microbiota results in impaired epithelial barrier function and an abnormal immune response (Roda et al. 2020). GWAS has identified hundreds of loci associated with genetic susceptibility of Crohn's disease, including genes involved in innate immunity and bacterial sensing, such as ATG16L1, NOD2, IRGM, LRRK2, STAT3, IL23R, HLA, JAK2, and others (Sazonovs et al. 2022). Among them, ATG16L1, NOD2, IRGM, and LRRK2 are autophagy-associated genes, which highlights the key role of autophagy in genetic susceptibility of Crohn's disease (Alula and Theiss 2023). Environmental variables influence the onset and progression of Crohn's disease in genetically susceptible hosts. Smoking, one of the most studied environmental variables, doubles vulnerability to Crohn's disease (Mahid et al. 2006). The risk of Crohn's disease is further increased by early exposure to antibiotics (Ungaro et al. 2014). Statins (Ungaro et al. 2016) have been linked to a lower risk, while oral contraceptives (Cornish et al. 2008), aspirin (Ananthakrishnan et al. 2012), and nonsteroidal anti-inflammatory medicines are other medications that may raise risk. Micronutrients (zinc and iron) and vitamin D have also been linked to a higher risk of Crohn's disease (Ananthakrishnan 2015). Notably, individuals diagnosed with Crohn's disease frequently manifest a dysbiosis of the gut microbiota, and in particular, the adherent-invasive E coli (AIEC) pathotype is closely related to the pathogenesis of Crohn's disease (Palmela et al. 2018). This correlation underscores the significance of dysbiosis in the gut microbiota in the context of Crohn's disease development. Autophagy plays a role in modulating cellular responses to gut microbiota, and the anomalous proliferation of gut microbiota has the potential to initiate an immune response, thereby inducing inflammation and lesions. Paneth cells located at the base of the small intestinal crypts of Lieberkühn can clear invading pathogens and maintain a diverse gut microbiota by secreting secretory granules containing antimicrobial peptides (AMPs) and other peptides (Noah et al. 2011). Autophagy dysfunction of Paneth cell in Crohn's disease patients may lead to alterations in the composition of the gut microbiota, specifically manifested by the colonization of AIEC or Salmonella typhimurium on intestinal epithelial cells (IEC) (Garrett et al. 2010). Simultaneously, maintaining normal autophagic function is crucial for the production of pro-inflammatory cytokines in immune cells to sustain intestinal immune homeostasis. However, Crohn's disease patients with mutations in autophagy-related genes are unable to achieve this balance (Nguyen et al. 2013). These findings highlight that abnormal autophagy may be intertwined with multiple factors such as gut microbiota dysbiosis, immune system disorders, intestinal inflammation, and genetic mutations, which together contribute to the development of Crohn's disease. Because of the complexity of the underlying cause, Crohn's disease cannot currently be cured, but the patient's symptoms can be managed with the use of drugs, nutritional therapy, and surgical procedures. The most commonly used medications in pharmacotherapy are divided into four categories: corticosteroids (Dignass et al. 2010), immunomodulators (McDonald et al. 2014; Chande et al. 2015), 5-aminosalicylate (5-ASA) medications (Magro et al. 2020), and monoclonal antibody drugs (Chenna et al. 2023). Children with Crohn's disease can improve their symptoms through nutritional therapy because malnutrition and emaciation are often associated with this disease (Sasson et al. 2021). Surgery is indicated in a variety of scenarios, including structuring Crohn's disease with obstructive symptoms, fistulizing or perianal Crohn's disease with infectious complications or concerns linked to the draining of the fistula, and failure of medications (Feuerstein and Cheifetz 2017). All these medical interventions have their setbacks, even though they can all somewhat regulate Crohn's disease symptoms. For instance, immunomodulators can raise the risk of malignant tumors (Bourrier et al. 2016), corticosteroids can have adverse effects like diabetes, osteoporosis, high blood pressure, and infections (Uskudar Cansu et al. 2018), and the safety of upcoming monoclonal antibody drugs is unknown (Torres et al. 2017). Therefore, there is an imperative need to find innovative therapies to address the challenges of Crohn's disease. Our focus has recently centered on potential targets to modulate autophagy in treating Crohn's disease. MicroRNAs, particularly MicroRNA-106B and MicroRNA-143, have emerged as key players influencing intestinal autophagy and inflammatory responses by targeting susceptibility genes like ATG16L1 and ATG2B, potentially compromising autophagy-mediated bacterial clearance (Lu et al. 2014; Lin et al. 2018). Moreover, the 5-hydroxytryptamine (5-HT) receptor has emerged as a novel therapeutic target for Crohn's disease, as heightened intestinal 5-HT levels were found to inhibit autophagy and increase susceptibility to colitis (Haq et al. 2021).The discovery of these targets suggests that it is feasible to focus our research on the regulation of autophagy in Crohn's disease and reinforce our confidence in investigating the relationship between autophagy and Crohn's disease (Fig. 1).

Fig. 1
figure 1

Overview of Crohn's disease. The etiology of Crohn's disease is related to genetic susceptibility, environmental factors, and gut microbiota. Genetic susceptibility is associated with autophagy-related genes, including ATG16L1, NOD2, IRGM, etc. Symptoms of Crohn's disease is determined by the site of onset, the intensity of inflammation, and the behavior of the disease, and can be classified into major symptoms and extraintestinal manifestations. Owing to the complexity of the underlying etiology, Crohn's disease is currently incurable, but it is possible to manage the patient's symptoms with medications and surgical procedures. The most used drugs in pharmacologic therapy fall into four categories: corticosteroids, immunomodulators, 5-ASA medications, and monoclonal antibody drugs

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3 Regulation of autophagy

Autophagy is a multifaceted process that is separated into five stages: initiation, nucleation, extension, fusion, and degradation (Dikic and Elazar 2018). The activation of the Unc-51-like kinase 1 (ULK1) complex (consisting of ULK1, RB1-inducible coiled-coil protein 1 (FIP200), autophagy-related protein 13 (ATG13), and ATG101) is essential for autophagy initiation (Zachari and Ganley 2017). AMP-activated protein kinase (AMPK) directly activates ULK1 and induces autophagy under glucose starvation conditions by phosphorylating Ser317 and Ser777. In contrast, a rise in mTOR activity under nutritional conditions inhibits ULK1 activation because it leads to phosphorylation at Ser757 of ULK1, interrupting the connection between ULK1 and AMPK (Kim et al. 2011). To initiate phagophore nucleation, the ULK1 complex activates the class III PI3K (PI3KC3) complex I, which is made up of Beclin 1, ATG14, vacuolar protein sorting 34 (VPS34), activating molecule in Beclin 1-regulated autophagy protein 1 (AMBRA1), and general vesicular transport factor (p115) (Noda 2017; Zhou et al. 2017). When PI3KC complex I is activated, VPS34 generates phosphatidylinositol 3-phosphate (PI3P), facilitating the recruitment of autophagy-related PI3P-binding proteins such as WD-repeat protein that interact with PtdIns (WIPI) protein and Double FYVE-containing protein 1 (DFCP1) (Lamb et al. 2013). ATG9 vesicles were also recruited at this time to extend the phagophore (Shima et al. 2019). Furthermore, the extension of the phagophore membrane engages two ubiquitin-like systems, namely, the microtubule-associated protein light chain 3 (LC3) complex and the ATG12-ATG5-ATG16L1 complex (Itakura and Mizushima 2010). LC3-II plays a dual role in both elongating the phagophore membrane and sealing the phagophore (Jiang et al. 2021). Autophagosomes are double-layered vesicles that are produced when the phagophore membrane is sealed and eventually fuses with lysosomes. The autophagic cargo is digested by acidic hydrolases in the lysosome, and recycled nutrients are released back into the cytoplasm and utilized by the cell. Although autophagy was initially thought to be a non-selective process occurring under starvation conditions, it is now recognized that autophagy also plays a role in the targeted and selected removal of specific substrates, so-called selective autophagy, which includes processes such as mitophagy, xenophagy and aggrephagy. Selective autophagy encompasses distinct stages, involving the identification of a degradation signal, cargo recognition facilitated by selective autophagy receptors, ubiquitination, and the recruitment of autophagosomal machinery (Mancias and Kimmelman 2016).

Autophagy is a self-digestive process in which the cell clears unnecessary cellular components, such as damaged organelles or proteins, as their accumulation can be toxic to the entire system. Additionally, autophagy serves as a defense mechanism against invading pathogens, constituting an integral part of both innate and adaptive immunity (Azzman 2019). Its activation primarily occurs through sensors of innate immunity, namely pattern recognition receptor (PRR) signaling. The fundamental challenge faced by intestinal immunity is maintaining a delicate balance between tolerance and responsiveness to microbes. Research on polymorphisms in autophagy-related genes (ATG16L1, NOD2, IRGM) suggests that compromised sensing and handling of intracellular bacteria by innate immunity contribute to various inflammatory, immune, and metabolic disorders. Currently, autophagy is widely recognized as a key regulatory mechanism with the capacity to integrate multiple aspects of Crohn's disease pathogenesis (Muzes et al. 2013).

4 Autophagy-related genes are associated with Crohn's disease susceptibility

A substantial body of evidence implies that more than 30 different genomic loci are linked to genetic susceptibility to Crohn's disease, including epidemiologic data based on family study concordance data, linkage analysis, and GWAS (Van Limbergen et al. 2009). The genes encoded by these loci are involved in many autophagy-related genes, such as ATG16L1, IRGM, LRRK2, etc. (Table 1). Therefore, elucidating the function that autophagy plays in Crohn's disease could consolidate our understanding of Crohn's disease and offer promising avenues for future research.

Table 1 Autophagy-related genes involved in susceptibility to Crohn's disease
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4.1 ATG16L1

ATG16L1 forms an autophagy-important complex with ATG5 and ATG12 to localize the autophagosomes within the cell and to drive their elongation process (Kuma et al. 2002; Rioux et al. 2007; Fujita et al. 2008). ATG16L1 has a SNP that causes threonine to be replaced by alanine (T300A), which has been defined as a risk allele for Crohn's disease (Hampe et al. 2007; Rioux et al. 2007). The Crohn's disease-related ATG16L1 mutation T300A disrupts intestinal immune homeostasis by diminishing AMPs production in Paneth cells and over-secretion of the pattern recognition receptors (PRRs)-mediated pro-inflammatory cytokines by dendritic cells (DCs) and macrophages (Okai et al. 2022).

Schwalbe and Paneth provided the initial description of Paneth cells as columnar epithelial cells with noticeable eosinophilic granules in the late 1800s (Clevers and Bevins 2013). Paneth cells, found within the small intestinal crypts of Lieberkühn, are intricately specialized secretory epithelial cells. The dense granules made by Paneth cells are rich in AMPs and immunomodulatory proteins, which control the composition of the intestinal flora (Lueschow and McElroy 2020). AMPs induce bacterial cytoplasm to flow out of the cell by piercing the bacterial membrane and producing holes (Ulm et al. 2012; Hemshekhar et al. 2016; Ho et al. 2017). Cadwell et al. used lysozyme staining examinations to examine the effects of the ATG16L1 risk allele on Paneth cells. They found that the granule exocytosis pathway exhibited significant abnormalities in ATG16L1-deficient Paneth cells (Cadwell et al. 2008). Consequently, Paneth cell biology and distinct regulatory characteristics are modulated by ATG16L1, which in turn affects the intestinal epithelium of Crohn's disease patients.

Additionally, the progression of Crohn's disease is significantly influenced by pro-inflammatory cytokines (Strober and Fuss 2011). ATG16L1 is involved in PRRs-mediated signaling pathways in addition to autophagy (Fig. 2). PRRs are classified into four categories: Toll-like receptors (TLRs), C-type lectin receptors (CLRs), NOD-like receptors (NLRs), and RIG-I-like receptors (RLRs) (Takeuchi and Akira 2010). After recognition of gut microbiota by macrophages and DCs through PRRs, ATG16L1 negatively regulates their pro-inflammatory cytokine responses (Okai et al. 2022). ATG16L1-deficient macrophages, when stimulated with the TLR4 ligand lipopolysaccharide (LPS), release elevated levels of inflammatory cytokines IL-18 and IL-1β. The mechanism involves ATG16L1 deficiency inducing Toll/IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF)-dependent caspase activation in LPS-stimulated macrophages (Saitoh et al. 2008). Caspase-1 subsequently cleaves IL-18 and IL-1β precursors, amplifying their production (He et al. 2016). A follow-up study revealed that macrophages isolated from patients carrying the Crohn's disease-associated ATG16L1 mutant T300A produced more IFN-β when stimulated with the ligand poly (I: C) for TLR3 and the ligand LPS for TLR4. Simultaneous activation of TLR3 and TLR4 leads to accumulation of TRIF, which subsequently leads to sustained kinase phosphorylation of tank-binding kinase 1 (TBK1) and transient phosphorylation of the TRIF-dependent transcription factor interferon regulatory factor 3 (IRF3), resulting in increased IFN-β production (Samie et al. 2018). It was also found that ATG16L1 negatively regulates the pro-inflammatory cytokine response mediated by receptor-interacting serine-threonine kinase (RICK), which is a subsequent signaling molecule for TLR2 and NOD2. TLR2 and RICK engage in a physical interaction in the presence of Pam3CSK4 (PAM), and ATG16L1 and RICK form a complex upon activation of NOD2 by muramyl dipeptide (MDP). MDP is considered to be the smallest immunogenic component of bacterial cell wall peptidoglycan (PGN) (Stafford et al. 2022). It has been demonstrated that, in response to PAM stimulation, cells transfected with ATG16L1 siRNA generated higher levels of nuclear factor-κB (NF-κB)-dependent cytokines, IL-6, and IL-12p40, than cells transfected with control siRNA (Honjo et al. 2021). Furthermore, MDP activation of NOD2 negatively controls TLR2-mediated pro-inflammatory factor responses in DCs (Watanabe et al. 2008). These results suggest that ATG16L1 plays an important role in the control of intestinal inflammation.

Fig. 2
figure 2

ATG16L1 dysfunction and increased risk of Crohn's inflammation. At homeostasis in vivo (left panel), the dense granules produced by Paneth cells are enriched with AMP and immunomodulatory proteins that control the composition of the intestinal flora. Moreover, after sensing the gut microbiota via PRRs, ATG16L1 negatively regulates TRIF- and RICK-mediated pro-inflammatory cytokine responses to maintain gut homeostasis. In the ATG16L1 dysfunctional state (right panel), AMP production by Paneth cells is reduced. This leads to increased PRRs stimulation of macrophages and DCs, which are not well regulated by ATG16L1, resulting in intestinal dysbiosis

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4.2 NOD2

Since the discovery of NOD2 variation in Crohn's disease in 2001, it has been recognized that NOD2 is the most important gene in the pathogenesis of Crohn's disease. The critical role of NOD2 in Crohn's disease has been confirmed by GWAS, next-generation sequencing, and functional analyses (Ashton et al. 2023). NOD2 is composed of two caspase recruitment domains (CARD), a nucleotide-binding domain (NBD), and a leucine-rich repeat (LRR) region that extends from the carboxyl to the amino terminus (Hugot et al. 2001). R702W (rs2066845), G908R (rs2066845), and L1007fs (rs2066847) were discovered to be three SNPs that are specifically linked to Crohn's disease and are located in or near the LRR region of NOD2 (Mirkov et al. 2017). NOD2 mutations associated with Crohn's disease interfere with several aspects of immune homeostasis, such as inhibition of autophagy and reduced MDP sensing in macrophages and DCs, as well as a reduction of AMPs secreted by Paneth cells, leading to abnormalities in barrier function and bacterial clearance (Fig. 3).

Fig. 3
figure 3

Regulation of the immune system and autophagic processes by NOD2. The activation of NOD2 promotes the sensing of MDP and autophagy in macrophages and DCs, mediating bacterial clearance, reducing inflammation, maintaining immune balance, and consequently preserving intestinal barrier function

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Activation of the NOD2 protein results in the formation of active oligomers that recruit adaptor proteins and generate downstream signaling cascades (Bonen et al. 2003). The LRRs region of NOD2 directly recognizes MDP (Grimes et al. 2012). MDP can enter cells via intracellular bacterial phagocytosis, solute channels, and endosomes. The NOD2 monomeric receptor responds to MDP by changing its conformation, which is controlled by ATP, and then oligomerizing through contacts with other exposed NBD domains. The downstream aptamer RICK is subsequently recruited and bound via the CARD-CARD interaction (Hasegawa et al. 2008; Fridh and Rittinger 2012). Then, X-linked IAP (XIAP), cellular inhibitors of apoptosis (cIAPs), and tumor necrosis factor receptor-associated factor 6 (TRAF6) are recruited to RICK and attached to it (Abbott et al. 2007; Bertrand et al. 2009; Krieg et al. 2009). The NF-κB essential modulator (NEMO) is ubiquitinated once the linear ubiquitin chain assembly complex (LUBAC) is recruited by the ubiquitin ligase XIAP (Damgaard et al. 2012). The subsequent phase involves recruitment and activation of the IκB kinase (IKK) complex, triggering phosphorylation of IκB (inhibitor protein of NF-κB). The expression of pro-inflammatory and AMP genes is then activated when NF-κB translocates to the nucleus (Balasubramanian and Gao 2017). In addition, transforming-growth-factorβ-activated kinase-1 (TAK1) is also recruited by polyubiquitinated RICK (Hasegawa et al. 2008). TAK1 functions by assembling with TAK1-binding proteins (TAB1, TAB2, and TAB3) (Xu and Lei 2020). TAK1 then activates mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK), p38α MAPK, and c-Jun N-terminal kinase (JNK). These kinases phosphorylate activator protein 1 (AP-1) transcription factors upon their translocation into the nucleus, which comprises ATF, c-fos, c-Jun, and JDP family members. The production of pro-inflammatory cytokines and AMPs is subsequently mediated by these transcription factors binding to TPA DNA-response elements (TRE) (Windheim et al. 2007; Mukherjee et al. 2019). NOD2 activation primes NLRP3 inflammasome activity by promoting NF-kB pro-inflammatory transcription, triggering downstream IL18 and IL1β production (Ashton et al. 2023). Furthermore, it has been shown that in NOD2-deficient mice, MDP stimulation leads to increased NF-κB activation (Maeda et al. 2005).

Table 2 Potential autophagy inducers for the treatment of Crohn's disease
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Table 3 Modulation of autophagy by current small-molecule drug for the treatment of Crohn's disease
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Studies have revealed that NOD2 triggers autophagy activation in DCs, promoting major histocompatibility complex (MHC) class II antigen-specific CD4 + T cell responses and bacterial processing. Furthermore, it was discovered that the DCs derived from Crohn's disease patients who expressed NOD2 or ATG16L1 risk alleles associated with the disease were impaired in antigen presentation, bacterial transport, and autophagy induction (Cooney et al. 2010). In addition, another study showed that NOD2 can attract the autophagy protein ATG16L1 to the plasma membrane at the bacterial entry through a process that is not dependent on the transcription factor NF-κB or the adapter RICK. In pure cells with NOD2 shifter mutations associated with Crohn's disease, mutant NOD2 fails to recruit ATG16L1 to the plasma membrane, and autophagosome encapsulation of invading bacteria is impaired (Travassos et al. 2010). Moreover, it has been found that ATG16L1 and NOD2 both support the autophagy-dependent antimicrobial pathway, which is modified by Crohn's disease-associated mutations in a way that is specific to certain cell types (Homer et al. 2010). These findings demonstrate the connection between autophagy and two Crohn's disease-related susceptibility genes and support the involvement of NOD2 in impaired autophagic responses. Autophagy impairments can lead to an ineffective clearance of microbial infections, in turn exacerbates the inflammatory response, which may complicate the onset and progression of Crohn's disease and increase the severity of the disease.

4.3 IRGM

IRGM is a member of the immune-related GTPases (IRG) gene family and is located on chromosome 5q33.1 (Taylor 2007). IRGM can directly or indirectly regulate core autophagy (Fig. 4). The regulation of core autophagy by IRGM is multifaceted. IRGM controls core autophagy and exerts its anti-microbial activities through five discrete but convergent mechanisms. IRGM stimulates the phosphorylation of crucial autophagy regulators, assembling them into autophagy initiation complexes. It maintains AMPK in a phosphorylated state at Thr172 to promote its activation. Consistently, IRGM increases total activated ULK1 phosphorylated by AMPK at Ser317 and Ser555, as well as active Beclin 1 phosphorylated at Ser93 and Ser96 by AMPK and Ser15 by ULK1. Additionally, IRGM has the capacity to form complexes with ATG16L1 and NOD2, thereby enhancing the binding between ATG16L1 and NOD2 (Chauhan et al. 2015). NOD2 acts as a PRR that transduces microbial recognition signals to the IRGM, situating the IRGM in contact with various innate immune sensory inputs. Besides its role in autophagy initiation, IRGM can bind to LC3 and transport the SNAREcomponent STX17 to autophagosomes for lysosomal fusion (Kumar et al. 2018). IRGM has also been discovered to stimulate nuclear ectopic translocation of Transcription Factor EB (TFEB) via interacting with calcineurin, hence counteracting the action of mTOR as a negative regulator of TFEB (Kumar et al. 2020). TFEB is a protein that binds to the lysosomal periphery and is phosphorylated by mTOR. Under varying stress situations, it is maintained in the cytoplasm but can translocate to the nucleus and promote the expression of the lysosomal system (Medina et al. 2015). Once IRGM directed autophagy is set in motion, it clears microbes or their products, thus acting not only to contain infection but perhaps more importantly to prevent excessive inflammation (Chauhan et al. 2016).

Fig. 4
figure 4

Regulation of core autophagy by IRGM. IRGM controls core autophagy and exerts its anti-microbial activities through five discrete but convergent mechanisms. The regulation of the core autophagic process by IRGM can be categorized into five aspects. (1) IRGM stimulates the phosphorylation of key autophagy regulators. (2) IRGM promotes the co-assembly of ULK1 and Beclin 1. (3) IRGM promotes the interaction between NOD2 and ATG16L1. (4) IRGM binds to LC3 and transports STX17 to autophagosomes for lysosomal fusion. (5) IRGM induces TFEB nuclear expansion by interacting with calcineurin

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Two IRGM polymorphisms, including a 'silent' SNP C313T (rs10065172) inside the coding region and a 20-kb deletion upstream of the IRGM gene, are strongly associated with Crohn's disease risk (Parkes et al. 2007; McCarroll et al. 2008; Prescott et al. 2010). Polymorphisms in the IRGM gene that are associated with Crohn's disease risk could alter expression levels and disrupt cellular functions crucial for initiating and sustaining autophagy against resilient intracellular bacteria. A study by Prescott et al. found that lower levels of IRGM expression were detected in untransformed lymphocytes from Crohn's disease patients (Prescott et al. 2010). Subsequently, heightened expression of the microRNA family miR-196 was detected in inflamed intestinal epithelial cells of individuals with Crohn's disease. This increased expression resulted in the reduction of protective IRGM variants, leaving the disease-associated variants unaffected. Consequently, the diminished levels of IRGM expression resulted in compromised autophagy and amplified intracellular bacterial replication (Brest et al. 2011). Studies have further demonstrated that induced knockdown of IRGM using siRNA in human cells leads to dysfunctional autophagy mechanisms, which in turn facilitate bacterial persistence and intensify pro-inflammatory responses (Lapaquette et al. 2010; Lapaquette et al. 2012b).

4.4 LRRK2

Functional genetic variants in Leucine-rich repeat kinase 2 (LRRK2) are the largest known genetic contributor to Parkinson's disease, and these variants also increase the risk of Crohn's disease (Ridler 2018). A SNP (rs3761863) in LRRK2 has been found to be significantly associated with Crohn's disease susceptibility (Franke et al. 2010; Liu et al. 2011). In a noncanonical way that depends on Beclin 1 activation but is independent of mTOR and ULK1, chemical suppression of LRRK2 kinase activity promotes macroautophagy (Manzoni et al. 2016) (Fig. 5A). Apart from its involvement in autophagy, LRRK2 possesses the ability to influence innate immune responses and inflammatory pathways. LRRK2 is observed to be highly expressed in innate immune cells (Ahmadi Rastegar and Dzamko 2020). It can act as a novel positive regulator of RICK, TAK1, and TRAF6, activating downstream NF-κB and MAPK pathways through the NOD2-RICK pathway to promote the induction of inflammatory cytokines (Yan and Liu 2017; Takagawa et al. 2018). Another study showed that LRRK2 inhibits the activity of the transcription factor nuclear factor of activated T cells (NFAT), which activates the expression of inflammatory genes during transcription (Liu et al. 2011) (Fig. 5B). Furthermore, Paneth cell deficiencies are common in Crohn's disease patients, and LRRK2 is essential for maintaining Paneth cell function (Liu et al. 2017).

Fig. 5
figure 5

Other autophagy-related genes associated with Crohn's disease susceptibility. A Modulation of autophagy pathway. B Modulation of inflammatory pathway

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4.5 ULK1

ULK1 is a serine/threonine protein kinase in mammals that functions by modulating the phosphorylation status of other autophagy-related proteins during the initiation process of autophagy (Ganley et al. 2009) (Fig. 5A). ULK1 phosphorylates ATG16L1 at Ser278, thereby facilitating autophagy in wild-type ATG16L1. This mechanism, on the other hand, increases ATG16L1 (T300A) degradation thus inhibiting autophagy (Alsaadi et al. 2019). Two SNPs in ULK1, rs12303764 and rs3923716 were significantly associated with Crohn's disease susceptibility (Henckaerts et al. 2011; Randhawa et al. 2017). Polymorphisms in ULK1 result in defective macrophage-mediated AIEC clearance in Crohn's disease patients (Buisson et al. 2019).

4.6 ATG4

ATG4 is the only protease among dozens of ATG proteins, and since both the processing of LC3 by ATG4 and the deglutition reaction are important for the formation of autophagosomes, inhibition of ATG4 impedes autophagy at the stage of autophagosome formation (Fernandez and Lopez-Otin 2015) (Fig. 5A). It has been shown that ATG4B regulates intestinal homeostasis and protects mice from experimental colitis (Cabrera et al. 2013). Granulomas are a typical microscopic appearance in Crohn's disease and are linked to the autophagy gene variations ATG4A (rs5973822) and ATG4D (rs10439163; rs2304165; rs7248026) (Brinar et al. 2012).

4.7 MTMR3

Myotubularin-related protein 3 (MTMR3) is a member of the myotubularin family and possesses an active phosphatase structural domain that specifically dephosphorylates PI3P (Vergne and Deretic 2010) (Fig. 5A). Local PI3P levels at autophagosome formation sites determine the initiation of autophagy and the size of autophagosome membrane structures. While overexpressing wild-type MTMR3 led to noticeably smaller nascent autophagosomes and decreased autophagic activity, MTMR3 knockdown boosted autophagosome formation (Taguchi-Atarashi et al. 2010). An SNP (rs713875) in MTMR3 was found to be significantly associated with Crohn's disease susceptibility by GWAS (Franke et al. 2010). One important study showed that the rs713875 Crohn's disease risk polymorphism enhanced MTMR3 expression, which in turn increased NOD2-induced caspase-1 activation, NF-κB signaling, and cytokine secretion, and lowered PI3P thus inhibiting autophagy (Lahiri et al. 2015) (Fig. 5B).

4.8 PTPN2

Protein tyrosine phosphatase non-receptor 2 (PTPN2), also known as T cell protein tyrosine phosphatase (TCPTP), is a member of the PTP family of signaling proteins (Song et al. 2022). Tumor necrosis factor (TNF) and IFN-γ have been demonstrated to activate the Epidermal growth factor receptor (EGFR)-mTOR pathway, resulting in autophagosome formation and increased intracellular PTPN2 protein level. By suppressing EGFR activity and possibly indirectly suppressing mTOR activity, PTPN2 promotes autophagy (Fig. 5A). The presence of Crohn's disease-associated ATG16L1 (T300A) inhibits TNF and IFN-γ-induced PTPN2 protein increase. It has also been discovered that the Crohn's disease-associated PTPN2 variation (rs2542151) increases mTOR activity, which impairs autophagy (Scharl et al. 2012b). Another study showed that another SNP in PTPN2 (rs1893217) also influences autophagosome formation and induces NOD2-dependent MAPK phosphorylation (Scharl et al. 2012a) (Fig. 5B).

Overall, autophagy-related genes implicated in Crohn's disease have a significant role in the pathogenesis of the disease. Polymorphisms and mutations in these genes increase the genetic susceptibility to Crohn's disease, and further research is warranted to elucidate the specific roles of these genes in Crohn's disease and provide additional clues for treatment and prevention.

5 Autophagy modulation for the treatment of Crohn's disease

Patients with Crohn's disease may require surgical or pharmacologic treatment, which treatment they receive depends on the severity of their disease or other clinical features, such as age of onset and major comorbidities (Cushing and Higgins 2021). Further studies of several drugs currently used to treat Crohn's disease have shown that they affect autophagy, such as anti-TNF drugs and thiopurines (Nys et al. 2013). In addition, there is a growing recognition of the potential therapeutic benefits of small-molecule autophagy modulators, including autophagy inducers and inhibitors, for the treatment of Crohn's disease. Currently, relevant studies on small-molecule autophagy inhibitors are very limited, and therefore, they are not discussed in this section.

5.1 Therapeutic potential of small-molecule autophagy inducers in Crohn's disease

As mentioned previously, since autophagy-related genes are linked to Crohn's disease susceptibility, autophagy modulation could be an effective therapeutic intervention strategy. Therefore, it is necessary to summarize the current state of research on autophagy inducers in Crohn's disease (Table 2).

As a major regulator of cellular autophagy, mTOR is an attractive pharmacological target for the manipulation of autophagy, and it was originally identified as a target for rapamycin (Sabers et al. 1995). Rapamycin is a Food and Drug Administration (FDA) -approved drug and has shown some potential in the treatment of Crohn's disease in several small studies. For example, Massey et al. described the use of rapamycin in the treatment of perianal Crohn's disease in a 37-year-old woman, which resulted in significant symptom improvement (Massey et al. 2008). Celastrol, another autophagy inducer, was identified to induce autophagy in colonic tissues by blocking the PI3K/Akt/mTOR signaling pathway (Zhao et al. 2015). One week's administration of celastrol improves colitis in IL10-deficient mice, a murine model of Crohn's disease. IL-10, as an anti-inflammatory cytokine, plays an important role in maintaining intestinal immune homeostasis and preventing inflammatory responses, and the use of an IL10-deficient mouse model can mimic the disease of Crohn's disease. Subsequently, it was found that pre-treatment of peritoneal meacrophages with the selective cannabinoid receptor 2 (CB2R) agonist HU 308 promotes the phosphorylation of the AMPK-mTOR-P70S6K signaling cascade, which in turn enhances autophagic processes in peritoneal macrophages. This enhanced autophagy contributes to the inhibition of the initiation and activation of NLRP3 inflammatory vesicles and reduces the inflammatory cascade response, which ultimately mitigates DSS-induced colitis (Ke et al. 2016). In addition, docosahexaenoic acid (DHA) reduces the severity of chronic colitis in IL10-deficient mice by enhancing autophagy via mTOR pathway inhibition (Zhao et al. 2017).

Autophagy inducers that function on other pathways are as interesting as those that act on the mTOR signaling pathway. Kuo et al. identified a new small molecule autophagy regulator BRD5631 through high-throughput screening (Kuo et al. 2015). BRD5631 enhanced autophagy through an mTOR-independent pathway to clear invading bacteria and inhibit IL-1β production in HeLa cells containing the Crohn's disease-associated allele ATG16L1 (T300A). Chlorpromazine has also been used as an autophagy inducer and can restore autophagic flux defects. It was found that chlorpromazine treatment of human monocyte-derived macrophages (MDM) restored autophagic flux and overcame the bacterial killing deficiency in Niemann-Pick disease type C1 (NPC1). Early-onset IBD with granuloma formation and a Crohn's disease phenotype is predisposed by mutations in the NPC1 gene (Schwerd et al. 2017). In contrast to other autophagy inducers that have primarily been investigated in experimental studies, chlorpromazine has been approved by the FDA to treat psychosis (Plaze et al. 2021). However, there are still no clinical studies of chlorpromazine in treating Crohn's disease. Therefore, chlorpromazine is still classified as an autophagy inducer with therapeutic potential. Intestinal fibrosis-induced obstruction stands as a frequent complication in Crohn's disease, and Holvoet et al. evaluated the effect of locally acting Rho kinases (ROCK) inhibitors (AMA0825) on intestinal fibrosis. AMA0825-treated mice showed enhanced autophagy in intestinal fibroblasts, a significant decrease in p62 levels, and a rise in the number of autophagosomes, thereby preventing and reversing intestinal fibrosis (Holvoet et al. 2017). In addition to ROCK, another potential target for avoiding intestinal fibrosis is CD147, and the inhibitor of this protein, AC-73, may offer a promising new anti-fibrotic treatment option for Crohn's disease. Mice with chronic TNBS colitis exhibit higher levels of CD147 protein than control mice during the development of colon tissue fibrosis. By promoting autophagy and preventing the activation of the CD147/ERK1/2 and STAT3 signaling pathways, AC-73 treatment lowers intestinal fibrosis (Butera et al. 2022). Intestinal epithelial cells have an important barrier role in Crohn's disease. Rizzo et al. found that baicalin extracted from Scutellaria baicalensis root enhanced intestinal barrier function by inducing autophagy to counteract LPS-stimulation-induced down-regulation of LC3 and ATG5 gene expression in HT-29 cells (a human colonic epithelial cell line) (Rizzo et al. 2021). These findings highlight potential therapeutic implications for autophagy modulators in Crohn's disease, though further evidence is needed to establish their conventional treatment status.

5.2 Modulation of autophagy by current small-molecule drug for the treatment of Crohn's disease

Current drug treatment options for Crohn's disease include traditional "step-up" therapy and newer "step-down" therapy. Traditional "step-up" therapy uses 5-ASA medications as first-line treatment, then corticosteroids as second-line treatment, and finally immunomodulators and anti-TNF antibodies as third-line treatment. "Step-down" therapy, on the other hand, allows for the use of immunomodulators and anti-TNF antibodies as initial treatment options for patients with severe Crohn's disease (Buchner et al. 2011). In this section, we will review the modulation of autophagy involved in small-molecule drugs currently used to treat Crohn's disease, as anti-TNF antibodies are not small-molecule drugs and will be excluded from this discussion (Table 3). Although a direct correlation between drug modulation of autophagy and disease improvement in patients with Crohn's disease has not been definitively established by current research, gaining a more in-depth understanding of the mechanisms of action of these drugs is of significant value. This is particularly crucial for the targeted and personalized application of these drugs within the Crohn's disease patient population.

5-ASA medications, such as sulfasalazine, mesalazine, olsalazine, and balsalazide, are among the most frequently prescribed treatments for Crohn's disease and exhibit a wide range of anti-inflammatory and immunomodulatory effects (Noureldin et al. 2021). A qualitative interview study of physicians with at least 4 years of independent experience treating patients with Crohn's disease showed that almost all physicians (93.3%) had prescribed 5-ASA medications for Crohn's disease patients and endorsed 5-ASA medications as the first-line treatment for Crohn's disease (Ma et al. 2018). One study in which mesalazine was able to act on the autophagy pathway caught our attention. Notably, mesalazine ameliorated dextran sulfate sodium (DSS)-induced colitis by activating the macrophage AMPK β1 subunit, enhancing autophagy, and causing a decrease in the production of macrophage proinflammatory factors (Banskota et al. 2021). In addition, sulfasalazine can induce autophagy via the NF-κB/mTOR pathway, thereby inhibiting neoplastic intimal hyperplasia after carotid artery injury in mice (Zhang et al. 2023).

Since many years ago, corticosteroids have been used to treat active Crohn's disease and continue to be the cornerstone treatment for recurring mild to severe Crohn's disease (Vavricka et al. 2014). Corticosteroids inhibit immune cell migration and the expression of adhesion molecules in inflamed tissues, as well as affecting the stability of messenger mRNA and down-regulating the transcription of genes involved in the production of pro-inflammatory cytokines (Yang and Lichtenstein 2002). Traditional corticosteroids like prednisone, prednisolone, and methylprednisolone are thought to be the most efficacious treatment for producing mild to moderate Crohn's disease remission (Malchow et al. 1984; Sandborn et al. 2007). Newer corticosteroids, such as budesonide, have the potential to minimize systemic medication and adverse effects while retaining efficacy (Vavricka et al. 2014). The FDA has approved delayed-release budesonide to treat mild to moderately active Crohn's disease (Chopra et al. 2006). Although corticosteroids have a limited effect on autophagy in Crohn's disease, studies on the relationship between corticosteroids and autophagy have never ceased. Recently, methylprednisolone and budesonide's modulatory effects on autophagy have been researched. Jang et al. showed that methylprednisolone could inhibit autophagy in rat femoral head vascular endothelial cells via the PI3K/Akt/mTOR pathway (Jang et al. 2022). By increasing autophagic flux, methylprednisolone, on the other hand, can encourage autophagy in osteoblasts (Zhao et al. 2020b). Additionally, in asthmatic patients, inhaling budesonide can inhibit the autophagy of airway macrophages by reducing the expression of LC3 and Beclin 1, and the inhibition of autophagy can increase the expression of IL-10, thereby controlling asthma inflammation (Maneechotesuwan et al. 2021).

The primary mechanism of action for immunomodulators in treating Crohn's disease involves the inhibition of immune system activity and the reduction of inflammation as well as the immune response, thereby effectively managing disease symptoms. Immunomodulators used to maintain Crohn's disease remission include thiopurines (6-mercaptopurine and azathioprine) and methotrexate (MTX) (Djuric et al. 2018). MTX is commonly considered an alternative therapeutic choice for individuals with Crohn's disease who have shown inadequate response to conventional thiopurine treatments (Savelkoul et al. 2022). Interestingly, these immunomodulators for maintaining remission in Crohn's disease also seem to affect the autophagic process. Morgan et al. investigated the effects of azathioprine on bone using a DSS model of colitis in mice and discovered that LC3 labeling was enhanced in the azathioprine-treated mice, implying autophagy induction (Morgan et al. 2019). In addition, by increasing the conversion of LC3-I to LC3-II, MTX was shown to activate autophagy in spermatocyte cells (**ong et al. 2020). Other immunomodulators such as the calcineurin inhibitors cyclosporine and tacrolimus may help induce remission in severe Crohn's disease (Venner and Bernstein 2022). Tacrolimus and cyclosporine are both classified as calcineurin inhibitors, and tacrolimus has a more stable oral bioavailability and fewer side effects than cyclosporine for Crohn's disease (Chow and Leong 2007). Moreover, Hinterleitner et al. showed that the combination of cyclosporine, prednisolone, and azathioprine improved perianal fistulae in Crohn's disease (Hinterleitner et al. 1997). Our attention has also been drawn to the induction of autophagy by tacrolimus and cyclosporine. The induction of autophagy by cyclosporine was first described by Pallet et al. in 2008, who found that autophagosome production and LC3-II expression could be seen in primary cultured human renal tubular cells (Pallet et al. 2008). Tacrolimus induces autophagy by upregulating LC3 expression, which inhibits puromycin-induced podocyte injury in mice (Yang et al. 2020) and may protect vascular endothelial cells from low-density lipoprotein damage (Xu et al. 2018).

6 Conclusion and perspective

Autophagy is essential for preserving intestinal homeostasis, regulating the relationship between immune responses and gut microbiota, and assisting the host in defending against intestinal pathogens (Mizushima 2018). Modulation of autophagy is an exciting therapeutic approach to treat Crohn's disease, and it has been shown that small-molecule drugs currently used for Crohn's disease can affect the autophagic pathway. However, it remains to be proven whether the autophagy regulation involved in these drugs is directly related to disease improvement. Given that these drugs have been clinically validated over a long period with proven safety and efficacy, it will become critical to investigate their direct impact on disease improvement in terms of autophagy regulation. In addition, a variety of small-molecule autophagy inducers have been recognized as potential therapeutic agents for Crohn's disease and have shown some therapeutic effects. However, the identified small-molecule autophagy inducers are still in the experimental stage, and further in-depth studies are still needed to determine whether they can enter into clinical applications, and the number of autophagy inducers with potential therapeutic effects is relatively limited, so it is imperative to search for more autophagy inducers. Notably, we discovered that starting from traditional Chinese medicine can give us fresh perspectives on how to discover small molecule autophagy inducers. A recent study found that the traditional Chinese herbal combination Xue-Jie-San (XJS) is beneficial in relieving clinical symptoms and aiding the repair of intestinal ulcers in Crohn's disease patients. Following therapy, XJS induces autophagy by blocking the mTOR/ULK1 signaling pathway. XJS also inhibits Notch1 and FGL1 signaling, which both affect mTOR signaling (Gao et al. 2023). In addition, Sanguisorba officinalis L. (SO) was able to suppress intestinal inflammation by promoting ATG7-dependent autophagy in colorectal macrophages (Yasueda et al. 2020). In the future, we can focus on extracting effective small-molecule components from traditional Chinese medicine, so as to better find small-molecule autophagy inducers with potential therapeutic effects. Although autophagy modulators show promise in the treatment of Crohn's disease, several challenges remain. First, we need a deeper understanding of the specific role and regulatory mechanisms of the autophagy pathway in the development of Crohn's disease. Second, since autophagy involves multiple signaling pathways and molecules, modulators targeting autophagy may have complex effects and side effects, and therefore more targeted and selective autophagy modulators need to be developed. In addition, the long-term safety and efficacy of autophagy modulators need to be further investigated.

In conclusion, this review provides an in-depth analysis of the impact of mutations in autophagy-related genes on Crohn's disease, offers a synthesis of the current understanding of how small-molecule drugs modulate autophagy in the treatment of this condition, and investigates the therapeutic promise of small-molecule autophagy inducers for Crohn's disease. We aim to highlight the significance of autophagy modulation in Crohn's disease, with the aspiration of contributing to the development of more efficacious treatments that can alleviate their suffering, and improve their quality of life.