Immunity-related GTPase IRGM at the intersection of autophagy, inflammation, and tumorigenesis

The human immunity-related GTPase M (IRGM) is a GTP-binding protein that regulates selective autophagy including xenophagy and mitophagy. IRGM impacts autophagy by (1) affecting mitochondrial fusion and fission, (2) promoting the co-assembly of ULK1 and Beclin 1, (3) enhancing Beclin 1 interacting partners (AMBRA1, ATG14L1, and UVRAG), (4) interacting with other key proteins (ATG16L1, p62, NOD2, cGAS, TLR3, and RIG-I), and (5) regulating lysosomal biogenesis. IRGM also negatively regulates NLRP3 inflammasome formation and therefore, maturation of the important pro-inflammatory cytokine IL-1β, impacting inflammation and pyroptosis. Ultimately, this affords protection against chronic inflammatory diseases. Importantly, ten IRGM polymorphisms (rs4859843, rs4859846, rs4958842, rs4958847, rs1000113, rs10051924, rs10065172, rs11747270, rs13361189, and rs72553867) have been associated with human inflammatory disorders including cancer, which suggests that these genetic variants are functionally relevant to the autophagic and inflammatory responses. The current review contextualizes IRGM, its modulation of autophagy, and inflammation, and emphasizes the role of IRGM as a cross point of immunity and tumorigenesis.


What is IRGM?
The immunity-related-GTPases (IRGs), also known as p47 GTPases, perform a pivotal function in innate immunity. The murine IRG gene family comprises 23 genes as tandem clusters on three chromosomes [5]. Murine models demonstrate that one of these genes, Irgm1, influences autophagic flux at the maturation phase when localised to the lysosomal compartment [6]. Murine Irgm1 modulates autophagy by assisting with autophagosome formation [7] and preventing lysosomal deacidification [7]. Irgm1 lysosomal localisation is IFN-γ-induced [8] during bacterial infections such as Salmonella enterica serovar typhimurium infection [9].
Interestingly, this family is reduced to only three genes in humans. This is possibly the consequence of host-pathogen coevolution driven by competition between IRG resistance proteins and pathogen virulence factors as it has been suggested by seminal studies conducted on Chlamydia muridarum [10] and C. trachomatis [11].
The three identifiable IRG genes in humans are IRGC , IRGQ, and IRGM. IRGC and IRGQ are located in chromosome 19 and are not involved in human immunity [5]. IRGM, which is located in chromosome 5q33.1, is the mammalian ortholog of murine Irgm1 and has a role in immunity, providing protection against intracellular pathogens [12]. Bekpen et al. [13] identified the process of ancestral Irgm1 pseudogenation and subsequent reactivation via insertion of the endogenous retroviral element 9 (EVR9) in human lineages. Some murine Irgm1 autophagy-related functions are performed similarly by IRGM. Of note, IRGM, in association with ATG8, translocates Stx17, an SNARE component, to the autophagosome for lysosomal fusion [14]. In addition, human IRGM functions upstream of autophagic initiation and throughout the autophagic process. Importantly, human IRGM is not IFN-γ-dependant, lacking a γ-activated sequence (GAS) [5]; however, recent evidence suggests it does act as a master negative regulator of cellular interferon responses [13]. There are four IRGM isoforms (IRGMa, IRGMb, IRGMc, and IRGMd) with distinct functions. Isoforms IRGMa and IRGMc lack the C-terminal G5 (SAK) motif, which is present in IRGMb and IRGMd [12]. IRGMd isoform becomes embedded within the mitochondrial membrane via cardiolipin, depolarising the membrane, inducing Bax-Bak-dependant cell death by increasing mitochondrial fission [12,15]. Isoforms IRGMa and IRGMc also exert the same effect at high concentrations, albeit with different kinetic profiles [15]. Tian et al. [16] demonstrated that concentrations of IRGMb, IRGMc, and IRGMd were higher in cancerous tissue compared to paracancerous tissue in melanoma patients, while IRGMa concentrations were relatively lower. In addition, IRGMb was shown to increase melanoma cell survival via increasing autophagic flux, in an HMGB1dependant manner [16]. This study also established IRGMb as a notable independent risk factor for melanoma progression [16]. Given that generation of alternatively spliced isoforms is frequently associated with drug resistance in cancer therapy, further studies are required to determine the role of IRGM isoforms in diverse tumours.

IRGM in cancer
The role of autophagy in cancer is context-dependent, acting as both a promoter and suppressor of tumorigenesis [24]. Evidence that mutations in ATG genes are associated with cancer was first provided when a monoallelic deletion in BECN1, an essential autophagy gene encoding Beclin 1, was shown to result in tumorigenesis in breast cancer [25]. This was later observed in 40-75% cases of breast, ovary, and prostate cancers [26,27]. Studies on other important autophagy genes (ATG2B, ATG3, ATG5, ATG9, ATG12, and ATG16L1) have also been conducted and suggest that autophagy plays a key role in tumorigenesis [28,29].
IRGM appears to be a fulcrum between immunity and tumorigenesis. Studies on melanoma [16,30], hepatocellular carcinoma [31], glioma [32], and gastric cancer [33] have shown that IRGM can promote carcinogenesis. In human glioma cell lines, overexpression of IRGM was linked to increased cell colony formation, cell proliferation, and Akt activation [32]. Xu et al. [34] also established that highly expressed IRGM in glioma cells promoted IL-8 production and M2 macrophage polarization via p62/TRAF6/NK-κB signalling. In gastric cancer, mRNA and protein levels of IRGM were shown to be significantly upregulated in the peripheral blood of cancer patients compared to healthy controls, and these levels were higher in stage IV than in stage I cancer patients [33]. Furthermore, there were significant differences in IRGM expression in patients presenting with melanoma, wherein the highest expression occurred in metastatic tumours, followed by primary tumours, and finally, benign adjacent nevus tissue [16]. A similar pattern was observed according to disease stage, where patients in stages III-IV of melanoma showed significantly higher expression of IRGM mRNA and protein levels, compared to patients in stages I and II [16]. In hepatocellular carcinoma (HCC), overexpression of the metallocarboxypeptidase AGBL2, an independent prognostic biomarker that promotes HCC cell survival and proliferation, enhanced autophagy and inhibited apoptosis via IRGM up-regulation [31].
Given the plethora of IRGM-related mechanisms that impact autophagy, a pathway that plays a complex role in cancer per se, it is pivotal to establish the precise mechanisms by which IRGM can promote carcinogenesis.

IRGM in other inflammatory conditions
Inflammation is a hallmark of cancer. In fact, many chronic inflammatory disorders including inflammatory bowel diseases (IBD) (e.g., Crohn's disease (CD) and ulcerative colitis) [35] and autoimmune diseases (e.g., primary Sjogren syndrome, rheumatoid arthritis, systemic lupus erythematosus, and systemic sclerosis) [36] are well known to significantly increase the risk of cancer. Recent studies demonstrate that IRGM modulates anti-inflammatory processes, particularly in the context of IBD [37]. Irgm1 deficient murine models have exhibited functional abnormalities in intestinal Paneth cells and hyper-inflammation in colon and ileum, upon dextran sodium sulphate exposure [38]. Mehto et al. [39] have revealed that IRGM is a negative regulator of the NLRP3 inflammasome, suppressing inflammation and providing protection against inflammatory diseases including CD. IRGM has been shown to control inflammation by interacting with SQSTM1/p62 and mediating p62-dependent selective autophagy of NLRP3 and ASC [39]. In addition, IRGM appears to block NLPR3 and ASC oligomerization, hindering inflammasome assembly [39]. Thus, IRGM restricts inflammasome activity and protects from pyroptosis (Fig. 3) [39]. Furthermore, by inhibiting inflammasome activation, Irgm1 has been shown to negatively regulate cellular inflammation in immune and intestinal epithelial cells in a CD murine model [39]. Knockdown of IRGM has resulted in morphology changes of dendritic cells, leading to hyperstability of the immunologic synapse as well as increased T-cell activation [37]. This mechanism might explain the loss of immune tolerance in the intestine and increased adaptive immunity in CD patients who carry ATG16L1 and IRGM risk alleles [40].
However, IRGM has also been shown to support immunopathogenesis. In mouse and human intestinal epithelial cell lines, it was found that IRGM can modulate necroptosis and release damage associated molecular patterns to induce gastrointestinal inflammation [43]. Furthermore, Fang et al. [44] have demonstrated that Irgm1 deficient murine models lead to macrophage apoptosis rescue by preventing ROS accumulation and phosphorylation of JNK/p38/ERK in the MAPK pathway. Irgm1 imparts a pro-inflammatory M1 macrophage phenotype by stabilising M1-associated transcription factors Irf5 and Irf8 [45]. In addition, Irgm1haplodeficient mice demonstrated reduced iNOS activity in M1 macrophages and reduced M1 polarization [45].

IRGM during microbial insult
Infectious diseases represent the third leading cause of cancer worldwide. In fact, 15.4% of cancers are attributable to carcinogenic infections [46]. Helicobacter pylori, high-risk human papillomavirus (HPV), hepatitis B virus (HBV), and hepatitis C virus (HCV) account for 90% of infection-related cancers worldwide [46].
H. pylori-infected patients harbouring IRGM rs13361189 demonstrated a remarkably increased risk of gastric cancer development [49]. Considering that H. pylori induces IRGM downregulation in a strain-dependant manner, IRGM polymorphisms and microbial suppression act synergistically to prevent xenophagic clearance [49]. The deletion or knockdown of the murine ortholog Irgm1 results in increased susceptibility to both intracellular and extracellular bacteria, including Citrobacter rodentium [50], S. typhimurium [9], Mycobacterium tuberculosis [12], Listeria monocytogenes [51], and C. trachomatis [52]. Numerous mechanisms for increased susceptibility in murine Irgm1 −/− models have been proposed: failure of monocyte maturation upon lamina propia infiltration and apoptosis [50], abolishment of macrophage movement and adhesion [9], and loss of intracellular bacterial restriction mechanisms [52]. In vitro models using M. leprae and M. tuberculosis corroborate murine models, identifying increased IRGM expression upon infection [12,53]. Interestingly, impaired IRGM expression results in persistent replication of adherent-invasive Escherichia coli (AIEC) in epithelial cells and macrophages, which has been implicated in CD pathogenesis, with further increased production of IL-6 and TNF-α [47]. The inability of macrophage-mediated AIEC clearance has been further demonstrated in CD patient-derived macrophages harbouring IRGM rs10065172 [54].
IRGM is also a fundamental negative regulator of type-1 IFN response against viral pathogens [21,55]. IRGM plays a key role in replication of HCV, an important risk factor for hepatocellular carcinoma, by regulating Golgi fragmentation and leading to co-localization of Golgi vesicles with replicating HCV [48]. Furthermore, autophagosome formation is stimulated in HIV-infected and HCV-infected HeLa cells, via IRGM interaction with HIV-NEF and HCV-NS3 [56]. This may aid in viral survival by autophagic degradation of nucleic acid-sensing proteins RIG-I and cGAS [21]. Epithelial and monocytic cells lines with abolished IRGM demonstrate enhanced antiviral properties, including MHC-I presentation and PKR stress granule formation, and are resistant to ZIKV and SARS-CoV-2 infection [55].
While the interaction of IRGM and fungi remains understudied, Rosentul et al. [57] investigated the effects of HIV+patient-derived peripheral blood mononuclear cells cytokine stimulation by Candida albicans blastoconidia, demonstrating patients harbouring IRGM rs13361189 had increased IL-8 levels compared to patients not harbouring this variant. IRGM also plays a pivotal role in limiting parasitic protozoan proliferation, including Trypanosoma cruzi and Toxoplasma Gondii, by ensuring macrophage maturation and secluding protozoan vacuoles to the lysosome [51,58].
In the era of the microbiome, the limited number of studies investigating the potential impact of IRGM on whole microbial communities needs to be addressed. This would be particularly pertinent in gastrointestinal disorders. To date, the most relevant study investigating the impact of IRGM genetic variants on gut dysbiosis [59] demonstrated that several IBD risk alleles, including IRGM rs11741861, are associated with a decreased abundance of the genus Roseburia in healthy individuals (FDR = 0.017).

IRGM germline variants associated with disease
IRGM polymorphisms have been investigated in relation to cancer including gastric cancer, renal cell carcinoma, and glioma (Table 1) [49,[60][61][62]. Of these, consistent associations have been found between IRGM rs4958847 and rs13361189 and gastric cancer; we [49] showed that rs4958847 decreases the risk of gastric cancer in ethnic Han Chinese populations, while Burada et al. [60] showed comparable results in Caucasian populations. Interestingly, both studies reported borderline associations between IRGM rs13361189 and gastric carcinogenesis. IRGM rs13361189 is in perfect linkage disequilibrium with a 20-kb deletion located immediately upstream of the IRGM promoter gene [63]. This deletion is replaced with seven nucleotides, causing IRGM segregation in the population with two distinct upstream sequences and alters IRGM regulation, which subsequently affects autophagy [63]. Importantly, IRGM rs13361189 appears to increase the risk of other types of cancer in Chinese populations, as evidenced by Ge et al. [61] who demonstrated an increased risk of glioma in subjects harbouring this polymorphism.
Several studies establishing links between IRGM polymorphisms and other human inflammatory diseases have been conducted (Table 1) The most established associations have been with CD and tuberculosis. An early meta-analysis by Li et al. [64], including 5183 CD patients and 5571 healthy controls, showed a significant association between rs13361189 and CD, but not rs4958847 and rs10065172. However, a meta-analysis by Lu et al. [65], comprising a much larger study sample size (20590 IBD cases and 27670 controls), has demonstrated that these three IRGM polymorphisms (rs13361189, rs4958847, and rs10065172) significantly increase the risk of CD. In addition, subgroup analyses by ethnicity conducted in both meta-analyses [64,65] revealed significant associations between these IRGM polymorphisms and an increased risk of CD among Caucasians but not among Asian populations. Recently, Ajayi et al. [66] demonstrated that subjects harbouring IRGM polymorphisms (rs13361189 and rs10065172) present with reduced IRGM expression in their serum and terminal ileum, indicating that these disease-associated SNP also affect IRGM expression, not only protein activity. Importantly, IRGM polymorphisms also appear to exacerbate symptoms (rs4958847) [67] and complicate prognoses (rs4958847 and rs13361189) [68,69] following treatment of CD.
A meta-analysis by Xie et al. [70], investigating the association between IRGM polymorphisms status and the risk of tuberculosis, comprising 3780 patients with tuberculosis and 4835 controls, reported a decreased risk of this disease in the presence of IRGM rs10065172, rs4958842, rs4859843, and rs4859846. The capacity for IRGM polymorphisms to affect tuberculosis development appears to be species-dependant, as the variant rs9637876 (−261TT) results in significant protection from M. tuberculosis, but not M. africanum or M. bovis [71].

Conclusions
Over the past decade, autophagy has evolved from a purely homeostatic tool to a complex axis of immunological, inflammatory, and carcinogenic processes, spurred by

Conflict of interests
The authors have no conflict of interests to declare that are relevant to the content of this article.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. No association Song et al. [87] Korean rs10065172 Decreases risk rs72553867 and rs12654043 No association Bahari et al. [88] Iranian rs4958843 and rs4958846 Decreases risk rs4958842 No association King et al. [89] African-American rs10065172 Increases risk rs11747270 Increases