Immunity-related GTPase Family M Research Articles

Race-specific association of an IRGM risk allele with cytokine expression in human subjects

Immunity-related GTPase family M (IRGM), located on human chromosome 5q33.1, encodes a protein that promotes autophagy and suppresses the innate immune response. The minor allele of rs13361189 (−4299T>C), a single nucleotide polymorphism in the IRGM promoter, has been associated with several diseases, including Crohn’s disease and tuberculosis. Although patterns of linkage disequilibrium and minor allele frequency for this polymorphism differ dramatically between subjects of European and African descent, studies of rs13361189 have predominantly been conducted in Europeans and the mechanism of association is poorly understood. We recruited a cohort of 68 individuals (30 White, 34 African American, 4 other race) with varying rs13361189 genotypes and assessed a panel of immune response measures including whole blood cytokine induction following ex vivo stimulation with Toll-like Receptor ligands. Minor allele carriers were found to have increased serum immunoglobulin M, C-reactive protein, and circulating CD8+ T cells. No differences in whole blood cytokines were observed between minor allele carriers and non-carriers in the overall study population; however, minor allele status was associated with increased induction of a subset of cytokines among African American subjects, and decreased induction among White subjects. These findings underline the importance of broad racial inclusion in genetic studies of immunity.

The role of SIRT1 in the process of Toxoplasma gondii infection of RAW 264.7 macrophages.

Toxoplasma gondii is an opportunistic pathogenic protozoan that can infect almost all kinds of warm-blooded animals, including humans. T. gondii can evade the host's immune response, a process known as immune evasion. Our main objective was to evaluate the role played by Sirtuin1 (SIRT1) [one of the sirtuins (SIRTs) that are a family of nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylases (HDACs)] in the T. gondii infection of RAW264.7 macrophages. In this study, we evaluated and observed alterations in the activity, expression, and localization of SIRT1 and assessed its involvement in the CD154/IFN-γ (CD40 ligand/interferon gamma) killing pathway and in autophagy during T. gondii infection. The inhibition of SIRT1 in host cells effectively reduced the number of intracellular tachyzoites, and the mechanism behind this effect might be the upregulation of IRGM1 [murine ortholog of IRGM (immunity-related GTPase family M)] and the initiation of autophagy. To the best of our knowledge, our study is the first to prove that T. gondii infection upregulates SIRT1 in RAW264.7 cells and that the inhibition of SIRT1 reduces the number of intracellular tachyzoites. Moreover, the upregulation of IRGM1 and the activation of autophagy may contribute to the intracellular inhibition of T. gondii caused by SIRT1 inhibition.

Crohn's disease IRGM risk alleles are associated with altered gene expression in human tissues.

Crohn's disease (CD) is a chronic inflammatory gastrointestinal disorder. Genetic association studies have implicated dysregulated autophagy in CD. Among risk loci identified are a promoter single nucleotide polymorphism (SNP)( rs13361189 ) and two intragenic SNPs ( rs9637876 , rs10065172 ) in immunity-related GTPase family M ( IRGM) a gene that encodes a protein of the autophagy initiation complex. All three SNPs have been proposed to modify IRGM expression, but reports have been divergent and largely derived from cell lines. Here, analyzing RNA-Sequencing data of human tissues from the Genotype-Tissue Expression Project, we found that rs13361189 minor allele carriers had reduced IRGM expression in whole blood and terminal ileum, and upregulation in ileum of ZNF300P1, a locus adjacent to IRGM on chromosome 5q33.1 that encodes a long noncoding RNA. Whole blood and ileum from minor allele carriers had altered expression of multiple additional genes that have previously been linked to colitis and/or autophagy. Notable among these was an increase in ileum of LTF (lactoferrin), an established fecal inflammatory biomarker of CD, and in whole blood of TNF, a key cytokine in CD pathogenesis. Last, we confirmed that risk alleles at all three loci associated with increased risk for CD but not ulcerative colitis in a case-control study. Taken together, our findings suggest that genetically encoded IRGM deficiency may predispose to CD through dysregulation of inflammatory gene networks. Gene expression profiling of disease target tissues in genetically susceptible populations is a promising strategy for revealing new leads for the study of molecular pathogenesis and, potentially, for precision medicine. NEW & NOTEWORTHY Single nucleotide polymorphisms in immunity-related GTPase family M ( IRGM), a gene that encodes an autophagy initiation protein, have been linked epidemiologically to increased risk for Crohn's disease (CD). Here, we show for the first time that subjects with risk alleles at two such loci, rs13361189 and rs10065172 , have reduced IRGM expression in whole blood and terminal ileum, as well as dysregulated expression of a wide array of additional genes that regulate inflammation and autophagy.

Variants in the autophagy-related gene IRGM confer susceptibility to non-alcoholic fatty liver disease by modulating lipophagy

Autophagy has been shown to be crucial in the regulation of the intracellular lipid stores in hepatocytes. We hypothesize that immunity-related GTPase family M (IRGM) gene (an autophagy-related gene) variants confer the susceptibility to non-alcoholic fatty liver disease (NAFLD) development. 832 obese children and adolescents aged 6-18years were recruited. NAFLD was determined by liver ultrasonography. We genotyped PNPLA3 rs738409, GCKR rs780094, TM6SF2 rs58542926, six IRGM single nucleotide polymorphisms (rs13361189, rs9637876, rs72553867, rs10065172, rs1000113, and rs11747270). To understand the molecular mechanism, we examined the effects of IRGM knockdown and overexpression on autophagic flux and lipid droplet metabolism in human hepatoma cells. 22.8% of recruited obese children and adolescents had NAFLD. Multiple logistic regression analysis revealed that after controlling for the effects of age- and gender-adjusted body mass index, gender, PNPLA3, GCKR, and TM6SF2 polymorphisms, variant IRGM rs10065172 TT genotype independently increased the odds ratio of NAFLD by 2.04 (95% confidence interval 1.22-3.42; p=0.007), as compared to the CC genotype. The predictive model was validated by means of 10-fold cross validation. Functional assay revealed that IRGM knockdown inhibited autophagic flux and increased lipid droplet content in HepG2 and PLC/PRF/5 cells, which were reversed by the autophagy inducer rapamycin administration. Similarly, wortmannin (an autophagy inhibitor) increased intracellular lipid droplet content. In contrast, overexpression of IRGM caused decreased lipid droplet content in HepG2 cells. Our findings suggest that IRGM may contribute to the development of human NAFLD by altering hepatic lipid metabolism through the autophagy pathway. Autophagy is involved in the process of lipid metabolism in hepatocytes. The mechanism of autophagy regulation by IRGM has just been unveiled. This study demonstrates that genetic variants in IRGM confer risk of human non-alcoholic fatty liver disease. The functional studies reveal how IRGM regulates hepatic lipid droplet content.

Coxsackievirus A16 elicits incomplete autophagy involving the mTOR and ERK pathways.

Autophagy is an important homeostatic process for the degradation of cytosolic proteins and organelles and has been reported to play an important role in cellular responses to pathogens and virus replication. However, the role of autophagy in Coxsackievirus A16 (CA16) infection and pathogenesis remains unknown. Here, we demonstrated that CA16 infection enhanced autophagosome formation, resulting in increased extracellular virus production. Moreover, expression of CA16 nonstructural proteins 2C and 3C was sufficient to trigger autophagosome accumulation by blocking the fusion of autophagosomes with lysosomes. Interestingly, we found that Immunity-related GTPase family M (IRGM) was crucial for the activation of CA16 infection-induced autophagy; in turn, reducing IRGM expression suppressed autophagy. Expression of viral protein 2C enhanced IRGM promoter activation, thereby increasing IRGM expression and inducing autophagy. CA16 infection inhibited Akt/mTOR signaling and activated extracellular signal-regulated kinase (ERK) signaling, both of which are necessary for autophagy induction. In summary, CA16 can use autophagy to enhance its own replication. These results raise the possibility of targeting the autophagic pathway for the treatment of hand, foot, and mouth disease (HFMD).

Tu1849 Defining and Targeting IRGM's Mechanism of Action in Crohn's Disease

Crohn's disease (CD) is a chronic, relapsing inflammatory disease, which affects nearly 1.4 million Americans. Genome-wide association studies (GWAS) have identified several genetic risk loci for CD including IRGM (immunity-related GTPase family M). IRGM functions in the innate immune control of intracellular pathogens. IRGM localizes to the mitochondria and induces mitochondrial fission which is linked to the autophagic elimination of intracellular bacteria. Surprisingly, IRGM expression is not lost but stronger in the epithelium of CD patients carrying the IRGM risk allele and more unexpectedly it is associated with an increase in intracellular bacteria. So how IRGM signaling contributes to the pathogenesis of CD remains unresolved. Our laboratory has generated a unique mouse model (DKO) of CD with a high mucosal expression of IRGM which duplicates the functional, histological and clinical features of human CD. The DKO mice were generated by genetic deletion of two anti-apoptotic proteins (villin/gelsolin) that regulate mitochondrial homeostasis and mucosal healing. Our studies with DKO mice and human CD patients demonstrate that by acting on the mitochondria, IRGM confers autophagic protection when expressed in moderation but induces cell death when over-expressed, explaining IRGM's action both in defense against bacteria and in damaging inflammation in CD. Furthermore, IRGM induced cell death is accompanied by the release of HMGB1 (high mobility group protein B1), a major proinflammatory alarmin/DAMP (danger-associated molecular pattern). This we demonstrate is the molecular basis of IRGM's role in the pathogenesis of CD. Our innovative study uses a novel mouse model of CD, provides a novel paradigm about the role of IRGM in CD and includes translational studies to diagnose, treat and monitor CD patients.

Tu1852 Resveratrol Ameliorates Oxidative Stress-Induced Intestinal Epithelial Barrier Dysfunction by Upregulating Heme Oxygenase-1 Expression

Crohn's disease (CD) is a chronic, relapsing inflammatory disease, which affects nearly 1.4 million Americans. Genome-wide association studies (GWAS) have identified several genetic risk loci for CD including IRGM (immunity-related GTPase family M). IRGM functions in the innate immune control of intracellular pathogens. IRGM localizes to the mitochondria and induces mitochondrial fission which is linked to the autophagic elimination of intracellular bacteria. Surprisingly, IRGM expression is not lost but stronger in the epithelium of CD patients carrying the IRGM risk allele and more unexpectedly it is associated with an increase in intracellular bacteria. So how IRGM signaling contributes to the pathogenesis of CD remains unresolved. Our laboratory has generated a unique mouse model (DKO) of CD with a high mucosal expression of IRGM which duplicates the functional, histological and clinical features of human CD. The DKO mice were generated by genetic deletion of two anti-apoptotic proteins (villin/gelsolin) that regulate mitochondrial homeostasis and mucosal healing. Our studies with DKO mice and human CD patients demonstrate that by acting on the mitochondria, IRGM confers autophagic protection when expressed in moderation but induces cell death when over-expressed, explaining IRGM's action both in defense against bacteria and in damaging inflammation in CD. Furthermore, IRGM induced cell death is accompanied by the release of HMGB1 (high mobility group protein B1), a major proinflammatory alarmin/DAMP (danger-associated molecular pattern). This we demonstrate is the molecular basis of IRGM's role in the pathogenesis of CD. Our innovative study uses a novel mouse model of CD, provides a novel paradigm about the role of IRGM in CD and includes translational studies to diagnose, treat and monitor CD patients.

Elevated expression of immunity-related GTPase family M in gastric cancer.

Recent researches have suggested that autophagy may play critical roles in tumorigenesis. Immunity-related GTPase family M (IRGM) is a human protein highlighted for its contribution to autophagy upon inflammation and infections. Studies have shown that IRGM is involved in the development of several cancers. In the current study, we investigated expression of IRGM and gastric cancer. Levels of messenger RNA (mRNA) and protein were examined by real-time reverse transcription PCR (RT-PCR) and Western blot, respectively. Data showed that mRNA level of IRGM was significantly increased in peripheral blood mononuclear cells (PBMCs) of gastric cancer patients than in PBMCs from healthy controls (p > 0.05). Moreover, both mRNA and protein levels were significantly higher in cancer tissues compared to adjacent noncancerous stomach tissues (1.28-fold, p < 0.001; 1.19-fold, p < 0.01, respectively). However, the level of IRGM seemed not to be affected by Helicobacter pylori infection. In addition, we investigated the correlation between IRGM expression and cancer stages and identified that stage IV patients had upregulated mRNA level and protein level of IRGM in cancer tissues than those stage I patients. Our findings suggest that expression of IRGM is dysregulated in gastric cancer and that the molecule may affect progression of the disease.

THE EMERGING ROLES OF HUMAN IMMUNITY-RELATED GTPASE M (IRGM) GENE

Article history: st 2014 Immunity-related GTPase family M (IRGM) protein, discovered for the first time in 1990, belongs to IRG family of proteins and is divided into five subfamilies (IRGA, IRGB, IRGC, IRGD and IRGM). These are responsive to interferon- and play a pivotal role in immune response to pathogens in mice. Owing to lack of interferon response element in the promoter region, human IRGM does not respond to interferon- stimulation, which is why it was earlier thought to be non-functional gene. Moreover, evolutionary history suggests that this gene was non-functional for ~25 million years ago. Bioinformatics has been instrumental in elucidating its evolutionary history as well as functions, especially in its interactions with the proteins of autophagy pathway. Recently, several studies have demonstrated the various functions of IRGM in limiting different pathogens in humans. This review discusses how and various roles played by IRGM unraveled in the recent years.

Structural Characterization of Human Immunity-Related GTPase Family M (IRGM)

The Immunity-Related GTPase Family M protein (IRGM) is involved in regulating cellular autophagy. Cellular knockdown of IRGM was shown to allow RNA viruses to hijack the autophagic immune response. Additionally, recent genetic studies have shown that underexpression of IRGM is associated with the incidence of Crohn's disease and infection by Mycobacterium tuberculosis. IRGM is an interferon-induced GTPase with an evolutionary conserved P-loop. It is an effector of the interferon-gamma pathway, but, unlike its protein family members, is not directly activated by the pathway. Its mechanism of action has been proposed to occur by translocation of IRGM to the mitochondria through recognition of cardiolipin, and affecting mitochondrial fission to induce autophagy. This potential interaction with cardiolipin might indicate the presence of a unique GTPase recognition and activation fold within IRGM. Our goal is to determine the X-ray crystal structure of IRGM in an effort to understand its molecular role in normal and diseased states. Additionally, we seek to test its interaction with and mechanism of recognition to mitochondrial cardiolipin as well as other autophagy-inducing binding partners. Currently, we have managed to express human IRGM in bacterial cells and have purified it to homogeneity using affinity and size-exclusion chromatography. These findings will serve to elucidate the mechanism of action of IRGM. Crucially, we hope to gain an understanding of its contributing role to Crohn's disease and tuberculosis infection at the molecular level, potentially paving the way to structure-based drug design and therapeutic opportunities.

Association between Variants of the Autophagy Related Gene – IRGM and Susceptibility to Crohn’s Disease and Ulcerative Colitis: A Meta-Analysis

BackgroundPolymorphisms in immunity-related GTPase family M (IRGM) gene may be associated with inflammatory bowel disease (IBD) by affecting autophagy. However, the genetic association studies on three common variants in IRGM gene (rs13361189, rs4958847 and rs10065172) have shown inconsistent results.Methodology/ Principal FindingsThe PubMed and Embase were searched up to June 5, 2013 for studies on the association between three IRGM polymorphisms and IBD risk. Data were extracted and the odd ratios (ORs) and 95% confidence intervals (95% CIs) were calculated. Finally, we performed a meta-analysis of 25 eligible studies in 3 SNPs located at IRGM gene by using a total of 20590 IBD cases and 27670 controls. The analysis showed modest significant association for the rs13361189, rs4958847 and rs10065172 variants in Crohn’s disease (CD): the risk estimates for the allele contrast were OR=1.306 (1.200-1.420), p=5.2×10-10, OR=1.182 (1.082-1.290), p=0.0002, and OR=1.248 (1.057-1.473), p=0.009 respectively (still significant when the p value was Bonferroni adjusted to 0.017). When stratified by ethnicity, significantly increased CD risk was observed in Europeans, but not in Asians. Conversely, there was no association of rs13361189 or rs4958847 variant with risk of ulcerative colitis (UC).Conclusions/ SignificanceThese results indicated that autophagy gene-IRGM polymorphisms appear to confer susceptibility to CD but not UC, especially in Europeans. Our data may provide further understanding of the role of autophagy in the pathogenesis of CD.

IRGM in autophagy and viral infections

Autophagy is a cell autonomous process allowing each individual cell to fight intracellular pathogens. Autophagy can destroy pathogens within the cytosol, and can elicit innate and adaptive immune responses against microorganisms. Nevertheless, numerous pathogens have developed molecular strategies enabling them to avoid or even exploit autophagy for their own benefit. IRGM (immunity-related GTPase family M) is a human protein recently highlighted for its contribution to autophagy upon infections. The physical association of IRGM with mitochondria and different autophagy-regulating proteins, ATG5, ATG10, SH3GLB1, and LC3, contribute to explain how IRGM could regulate autophagy. Whereas IRGM is involved in autophagy-mediated immunity against bacteria, certain viruses seem to have developed strategies to manipulate autophagy through the selective targeting of this protein. Furthermore, irgm variants are linked to infection-associated human pathologies such as the inflammatory Crohn’s disease. Here, we discuss how IRGM might contribute to human autophagy upon viral infection, and why its targeting might be beneficial to virus replication.

Ginsenoside Re enhances survival of human CD4 + T cells through regulation of autophagy

In the present study, we examined the effects of ginsenoside Re (Re) on cytokine expression, cytokine-dependent autophagy and cell survival in human CD4 + T cells. When CD4 + T cells isolated from human peripheral blood were treated with Re, LC3 and monodansylcadaverine (MDC), representative markers of autophagy, were decreased in a dose-dependent manner. Interestingly, Re suppressed the production of interferon-gamma (IFN-γ) and immunity-related GTPase family M (IRGM) in CD4 + T cells whereas no changes in other autophagy-related signaling molecules (ERK, p38 and AKT-mTOR-p70S6k) were found. Concomitantly, we observed that Re increased the proliferation of CD4 + T cells with decreased cell death. Our results demonstrate that ginsenoside Re enhanced viability of CD4 + T cells through the regulation of IFN-γ-dependent autophagy activity.

NKX2-3 and IRGM variants are associated with disease susceptibility to IBD in Eastern European patients

To investigate variants of immunity-related GTPase family M (IRGM) and NKX2-3 genes and genotype-phenotype in Eastern European patients with inflammatory bowel disease (IBD). We analyzed 1707 Hungarian and Czech subjects with Crohn's disease (CD) (n = 810, age: 37.1 ± 12.6 years, duration: 10.7 ± 8.4 years) and ulcerative colitis (UC) (n = 428, age: 43.7 ± 15.0 years, duration: 12.6 ± 9.9 years), as well as 469 healthy controls. IRGM rs13361189, NKX2-3 rs10883365 and ECM1 rs13294 polymorphisms were tested by LightCycler allele discrimination. Detailed clinical phenotypes were determined by reviewing the medical charts. NKX2-3 rs10883365 variant allele was associated with increased risk for CD (P = 0.009, OR = 1.24, 95% CI = 1.06-1.48) and UC (P = 0.001, OR = 1.36, 95% CI = 1.13-1.63), whereas variant IRGM allele increased risk for CD (P = 0.029, OR = 1.36, 95% CI = 1.03-1.79). In contrast, ECM1 rs13294 was not associated with either CD or UC. In CD, the variant IRGM allele was associated with a colon-only location (P = 0.02, OR = 1.62, 95% CI = 1.07-2.44), whereas in UC, the ECM1 variant was associated with cutaneous manifestations (P = 0.002, OR = 3.36, 95% CI = 1.48-7.63). Variant alleles did not predict resistance to steroids or azathioprine, efficacy of infliximab, or need for surgery. NKX2-3 and IRGM are susceptibility loci for IBD in Eastern European patients. Further studies are needed to confirm the reported phenotype-genotype associations.

Germline variants of IRGM in childhood-onset Crohn’s disease

Genetic association of the autophagy genes, autophagy-related 16-like 1 (ATG16L1) and immunity-related GTPase family, M (IRGM), has been confirmed by meta-analysis of three index genome-wide association studies (GWASs), and in...

Genotyping of IRGM Tetranucleotide Promoter Oligorepeats by Fluorescence Resonance Energy Transfer

Fluorescence resonance energy transfer (FRET) genotyping has been well established for the rapid assessment of single nucleotide polymorphisms (SNPs) and deletions. A design is presented that allows the typing of short tandem oligo repeat sequences using the LightTyper/LightCycler system. The protocol was evaluated and applied to the typing of a tetranucleotide promoter repeat of the human gene encoding the immunity-related GTPase family, M (IRGM) molecule in >2000 individuals from Ghana, West Africa.

Insufficient Evidence for Association of NOD2/CARD15 or Other Inflammatory Bowel Disease-Associated Markers with Gvhd or Other Outcomes in T-Replete, Unrelated Donor Transplantation Facilitated by the NMDP

Genome wide association studies have identified more than 30 distinct loci linked with susceptibility to Crohn's disease (CD), a common idiopathic inflammatory bowel disease (IBD). Among these candidate genes, NOD2/CARD15 encodes a cytosolic protein that recognizes bacterial cell wall components and results in downstream activation of innate pathways of host defense. NOD2/CARD15 Variants are associated with increased susceptibility to CD. IRGM (immunity-related GTPase family, M) is involved in autophagy and intracellular pathogen clearance. Variations in or near the IRGM locus have been associated with increased susceptibility to CD. IL23R (interleukin 23 receptor) variants appear to have a protective role in the development of IBD although the mechanism for this is unknown. Initial studies in European cohorts described an association in which NOD2/CARD15 donor or recipient variants increased severe acute GvHD/transplant-related mortality and reduced survival in HLA-matched sibling transplants. In T cell depleted unrelated donor transplants for acute leukemia, an increased risk of relapse and death was observed in pairs with recipient NOD2/CARD15 variants. However, this association was not confirmed in a recent independent study in 101 unrelated Swedish subjects. No studies have yet evaluated IRGM or IL23R variants in GvHD or other transplant outcomes.Methods: Pre-transplant samples from 390 patients and their 10/10 HLA-A, B, C, DRB1 and DQB1-matched unrelated donors were provided by the National Marrow Donor Program Research Repository. Patients received T-replete transplants for early stage AML, ALL, CML, MDS using myeloablative conditioning and accrued between 1995–2004. Gut decontamination was not routinely used. Patients and donors were of predominately European American descent 92.2% and 94.5%, respectively with 6.7% racial/ethnic minorities. DNA was genotyped for three NOD2/CARD15 SNPs (rs2066844, rs2066845 and rs2066847), two IL23R SNPs (rs11209026 and rs11465804), and two IRGM SNPs (rs4958847 and rs13361189) using Sequenom and/or Taqman assays. Multivariate analyses were performed for overall (OS) and disease free survival (DFS), transplant-related mortality (TRM), relapse, and acute and chronic GvHD, adjusting for clinical variables.Results: Out of 390 donor recipient pairs, NOD2/CARD15 variant SNPs were found in 56 donors (14%) and 67 recipients (17%), with an overall frequency of 16% in this cohort. In 12 pairs (3%), both donor and recipient had a mutant SNP. These frequencies are lower than reported in the earlier European cohorts but our study population included 6.7% racial/ethnic minorities. For IL23R, 13% of donors and 16% of recipients had variant SNPs (n=353 pairs analyzed), with 11 pairs (3%) having both donor and recipient variants. For IRGM, variant SNPs were seen in 23% of both donors and recipients (n=353 pairs analyzed). The overall incidence of 100 day acute grade III–IV (21%) and 1-year chronic (53%) GvHD as well as OS and DFS at 100 days (85 and 84%), 6 months (77 and 74%) and 1 year (67 and 63%) were similar to published outcomes. For IRGM, there was a trend (0.01<p<0.05) for recipient mutations to be associated with higher OS, LFS, and lower TRM. However, none of the 3 IBD-associated alleles showed evidence of a statistically significant association with any of the aforementioned outcomes at p <0.01. In particular there was no association between the three NOD2/CARD15 SNPs (rs2066844, rs2066845 and rs2066847) and the incidence of acute or chronic GvHD.Conclusions: Our study finds insufficient evidence to support an association between the 3 examined IBD-associated SNPs and adverse outcomes following matched unrelated donor HCTs in a (predominately Caucasian) cohort of US patients. Several factors may contribute to this discrepancy from prior studies, including our larger cohort size, differences in patient characteristics, exclusion of sibling matched donor HCTs and T cell depletion procedures. Nevertheless, our findings do not support the use of IBD-associated allele mutation status in the donor/recipient selection process.