Mechanism of action of the tuberculosis and Crohn disease risk factor IRGM in autophagy

ABSTRACT

Polymorphisms in the IRGM gene, associated with Crohn disease (CD) and tuberculosis, are among the earliest identified examples documenting the role of autophagy in human disease. Functional studies have shown that IRGM protects against these diseases by modulating autophagy, yet the exact molecular mechanism of IRGM's activity has remained unknown. We have recently elucidated IRGM's mechanism of action. IRGM functions as a platform for assembling, stabilizing, and activating the core autophagic machinery, while at the same time physically coupling it to conventional innate immunity receptors. Exposure to microbial products or bacterial invasion increases IRGM expression, which leads to stabilization of AMPK. Specific protein-protein interactions and post-translational modifications such as ubiquitination of IRGM, lead to a co-assembly with IRGM of the key autophagy regulators ULK1 and BECN1 in their activated forms. IRGM physically interacts with 2 other CD risk factors, ATG16L1 and NOD2, placing these 3 principal players in CD within the same molecular complex. This explains how polymorphisms altering expression or function of any of the 3 factors individually can affect the same process—autophagy. Furthermore, IRGM's interaction with NOD2, and additional pattern recognition receptors such as NOD1, RIG-I, and select TLRs, transduces microbial signals to the core autophagy apparatus. This work solves the long-standing enigma of how IRGM controls autophagy.

IRGM is a protein encoded by a gene where polymorphisms predisposing to Crohn disease (CD) and tuberculosis occur in diverse human populations. The connection between IRGM, along with the core autophagy factor ATG16L1, with human disease in a 2007 GWAS study helped establish a strong connection between the autophagy pathway and human disease. Despite being previously connected to autophagy in 2006, the mechanism of IRGM action has hitherto proven difficult to resolve stemming from the lack of a true IRGM ortholog in model organisms. Although IRGM is distantly related to a class of immunity-related GTPases (IRGs) in mice, human IRGM is N- and C-terminally truncated and has other alterations relative to mouse IRGs. Thus, it has been difficult to predict IRGM's mode of action; IRGM has been assumed to act indirectly, with supporting evidence in favor of this model coming from studies in 2010 showing that IRGM affects mitochondrial function and the fission-fusion status of the mitochondrial network.

We have extended studies of the action of IRGM, finding that its expression promotes stabilization of total cellular AMPK including its activated (Ser172 phosphorylated) form. When IRGM is knocked down, we find a decrease in cellular levels of ULK1, ATG14, and ATG16L1. This effect is specific because levels of BECN1 and ATG12–ATG5 are unaffected. IRGM furthermore interacts with and controls the core autophagy machinery (Fig. 1) by associating with autophagy factors ULK1, BECN1, AMBRA1, ATG14, and ATG16L1. Through these interactions, IRGM promotes the assembly of active ULK1 into complexes with BECN1 and increases the abundance of the ULK1-dependent activating phosphorylation of BECN1 at Ser15. When we analyzed the constituents of the BECN1 complex in the presence or absence of IRGM, we found that more ATG14 (an autophagy initiation complex member) and less BCL2 and KIAA0226/Rubicon (BECN1 inhibitors) coimmunoprecipitate with BECN1 when IRGM is expressed. Thus, IRGM promotes autophagy initiation by increasing AMPK signaling and by driving the assembly of the autophagy machinery in its activated state.

Figure 1.

IRGM directly regulates the core autophagic apparatus. See text for details.

Given its known connections to inflammatory (CD) and infectious (tuberculosis) diseases, we tested IRGM's role in controlling antimicrobial autophagy and inflammatory responses to microbial products. We find that IRGM expression can be strongly induced by infection of monocytic cells with a CD-associated E. coli isolate or by treatment with the microbial products LPS or muramyl dipeptide (MDP). Whereas LPS or MDP treatment induce autophagy, IRGM knockdown abrogates this effect. Exposure of macrophage-like cells to microbes or microbial products (especially MDP) increases the abundance of ULK1 and ATG16L1 in complexes with IRGM. Together, these data demonstrate that IRGM promotes the formation of active autophagy complexes and couples them with sensors of microbial challenge.

MDP is detected by a pattern recognition receptor (PRR), NOD2, which, like IRGM and ATG16L1, is a CD risk factor. Based on the responsiveness of the IRGM-directed autophagy to the NOD2 agonist MDP, we tested whether NOD2 might interact with IRGM. These 2 proteins directly interact, and all 3 CD risk factors colocalize in cells. IRGM-NOD2 interactions are potentiated in the presence of MDP or by deletion of the auto-inhibitory LRR domain of NOD2, mirroring the effects of NOD disinhibition by MDP.

NOD2 has several effects on IRGM. NOD2 promotes IRGM oligomerization and enhances IRGM's interaction with the autophagy regulators ULK1 and BECN1. The presence of NOD2 elicits K63-linked polyubiquitination of IRGM. NOD2-dependent ubiquitination of IRGM is important for several of its biochemical properties and functions. We found that an IRGM mutant that can no longer be ubiquitinated in the presence of NOD2 (IRGM-K^mut) has a reduced capacity to oligomerize and to interact with ULK1, BECN1, and AMBRA1 as well as a diminished capacity to increase cellular ULK1 protein levels.

The above findings suggest that NOD2 interactions affect IRGM function. To test this, we reconstituted an IRGM-NOD2 system in HEK293T cells, which normally do not express either protein. We found that wild-type IRGM, but not IRGM-K^mut, promotes the autophagic degradation of NOD2. Furthermore, whereas inflammatory signaling, as measured by NFKB/NF-κB nuclear translocation, is strongly induced in cells expressing NOD2 alone upon bacterial infection, co-expression of wild-type IRGM but not that of IRGM-K^mut reverses this effect. Similarly, wild-type expression, but not that of IRGM-K^mut, reduces intracellular bacterial colony-forming units. Thus, IRGM's ability to affect the core autophagy status and assembly, in a manner requiring NOD2-dependent ubiquitination, correlates with protection against infection and inflammation.

In summary, our study shows how IRGM works (Fig. 1). This protein controls autophagy and exerts its antiinflammatory and antimicrobial activities by 4 discrete but convergent mechanisms: (i) IRGM indirectly activates AMPK and stabilizes cellular core autophagy regulators, such as ULK1 and ATG14. (ii) IRGM associates with and directs co-assembly of the core autophagic machinery, including ULK1, BECN1, ATG16L1, and additional components, leading to activation of autophagy. (iii) IRGM physically connects this core autophagy machinery with a subset of cytoplasmic PRRs reporting microbial presence. One of these PRRs is NOD2, which transduces microbial recognition signals to IRGM, enhancing its ubiquitination, a process that augments IRGM's ability to assemble the autophagy core machinery. We noticed similar relationships between IRGM and several other PRRs, including NOD1, DDX58/RIG-I, and TLR3, all of which interact with IRGM and promote its K63-linked polyubiquitination, indicating that IRGM is positioned at a nexus of various innate immunity sensory inputs. (iv) 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. This latter feature is also reflected in IRGM-dependent degradation of NOD2, thus establishing a negative feedback loop. In conclusion, our study places IRGM at the hub of the core autophagy machinery and connects it with innate immune sensors. This explains both the role and mechanism of human IRGM in its autophagic function and its known disease contexts.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.