Immunity-Related GTPases (IRG)
The IRGs are a family of large GTP-binding proteins that play roles in immune and inflammatory processes (Pilla-Moffett et al. 2016; Kim et al. 2012; Hunn et al. 2011). They are closely related to three other families of GTP-binding proteins: the Mx proteins, guanylate-binding proteins (GBPs), and very large IFN-inducible GTPases. The IRGs were discovered in the 1990s by scientists who were conducting screens for mouse genes that were upregulated by IFN-γ and/or lipopolysaccharide (LPS). Those IRGs were named and described piecemeal, but later, a standardized nomenclature (Bekpen et al. 2005) placed the IRGs into subfamilies based on amino acid homology within the GTP-binding regions. Most subfamilies contain a canonical GKS sequence within the G1 motif in the GTP-binding region (IRGA, IRGB, IRGC, IRGD, IRGE, and IRGF), while a noncanonical GMS sequence is found only in the IRGM proteins, which is thought to lead to the distinct functional roles of this subfamily. The IRG genes have been found across vertebrates, yet their distribution is uneven with C57Bl/6 mice, for instance, having 23 members and humans only two. This expansion of the IRGs in some species may reflect relatively recent evolutionary events to combat unique pressures from endemic pathogens. Indeed, knock-out mouse studies have established that IRGs are important for immunity against a variety of protozoan and bacterial pathogens. More recently, human GWAS studies have associated variants in the human IRGM gene with Crohn’s disease, mycobacterial resistance, and sepsis outcomes.
Expression of most mouse IRG genes is highly induced by type I and type II interferons (IFN), as a consequence of multiple interferon-stimulated response elements (ISRE) and γ-activated sequences (GAS) found in the promoters of the genes (Bekpen et al. 2005). It is notable, however, that the positions of these elements vary from gene to gene in the mouse, implying that there has been evolutionary pressure to maintain IFN-regulated expression. The transcriptional response to IFNγ is rapid; for Irgm3, for instance, mRNA accumulates within 1 h of stimulation, reaching maximal levels within 3 h and having a half-life of about 4.5 h (Taylor et al. 1996). Irgm3 protein accumulates within 3 h of induction and reaches maximal levels within 8 h that do not decrease in the continued presence of IFN-. Expression of IRGs has been noted in a wide variety of tissues and cell types, both hematopoietic and nonhematopoietic. Expression of some mouse IRGs (Sorace et al. 1995), and of human IRGM (Chauhan et al. 2015), is induced by LPS. However, a variety of other cytokines do not induce expression of IFN-regulated IRG genes, underscoring the specificity of IFN and LPS in controlling expression (Sorace et al. 1995).
IRGs are members of the dynamin protein superfamily (Martens and Howard 2006). Although IRGs share little sequence homology with the dynamins outside of the GTP-binding region, they do share the GTPase function, a high affinity for lipid membranes, the ability to assemble into dimers and oligomers, and the ability to mechanically alter membranes. As a consequence of these biochemical properties, the IRGs are thought to be involved in membrane remodeling and trafficking events within cells. The related guanylate-binding proteins (GBP) and the Mx proteins are similarly related to the dynamins (Martens and Howard 2006).
The abilities to bind and hydrolyze GTP to GDP have been confirmed for Irgm1, Irgm3, and Irga6 (Taylor et al. 1997; Uthaiah et al. 2003). Extensive biochemical and structural studies of Irga6 (Uthaiah et al. 2003; Schulte et al. 2016; Ghosh et al. 2004) have served as models that may be representative of the majority of IRG proteins.
IRG proteins are found in many different membrane compartments within the cell depending on the particular IRG, including the endoplasmic reticulum, the Golgi, lysosomes, mitochondria, peroxisomes, and the plasma membrane, with the exact localization pattern varying with the particular IRG (Taylor et al. 1997; Martens et al. 2004; Henry et al. 2014; Haldar et al. 2013). The degree to which the proteins associate with membranes is also variable, ranging from >90% (for the IRGMs) to <10% (e.g., for IRGC) (Martens et al. 2004). Diverse mechanisms are involved in membrane localization including myristoylation, palmitoylation, and amphipathic helices (Martens et al. 2004; Henry et al. 2014). Targeting to most of these membranes does not involve GTP hydrolysis. However, many IRGs relocalize to vacuoles/phagosomes in cells infected with pathogens (most notably Toxoplasma gondii) with GTP hydrolysis triggering protein dimerization on the membrane. IRG complexes that assemble on pathogen-containing vacuoles are heterotypic, and the assembly is cooperative and ordered (Hunn et al. 2008; Khaminets et al. 2010).
The creation and analysis of mice lacking IRG proteins – Irgm1, Irgm3, Irgd, and Irga6 – have established the prominent role that the GTPases play in innate immunity to multiple intracellular pathogens (Taylor et al. 2000; Collazo et al. 2001; MacMicking et al. 2003; Henry et al. 2007; Al-Zeer et al. 2009; Taylor 2007; Liesenfeld et al. 2011). The clear but distinct phenotypes of the IRG-deficient mouse strains have also emphasized that the genes are nonredundant and of varying importance for immune resistance depending on the pathogen.
IRG proteins are extensively involved in resistance to Toxoplasma gondii in mice, which may have predicated the expansion of the gene family in that species (Gazzinelli et al. 2014). Mice lacking Irgm1 or Irgm3 demonstrate acute susceptibility to this protozoan parasite on par with the susceptibility in IFNγ-deficient mice, underscoring the essential role for the IRGM proteins in IFNγ-induced resistance (Taylor et al. 2000; Collazo et al. 2001). In contrast, Irgd and Irga6-deficient mice demonstrate weak susceptibility that becomes manifest later during the infection (Collazo et al. 2001; Liesenfeld et al. 2011). The role of the IRGs in resistance is tied to their ability to provide cell autonomous resistance to T. gondii: Macrophages or astrocytes that lack IRG proteins demonstrate varying degrees of impaired IFNγ-induced T. gondii killing activity (Butcher et al. 2005; Halonen et al. 2001). Data from several studies have suggested a model in which GKS IRG proteins load onto the T. gondii vacuole, where they drive vesiculation of that vacuole, releasing the parasite into the cytosol of the cell where it is destroyed (Ling et al. 2006; Zhao et al. 2009). GMS IRGM proteins, in contrast, do not load to the same extent on the vacuole; rather, they regulate the GKS proteins, such that in absence of the GMS IRG proteins, the GKS IRG proteins inappropriately activate and form aberrant aggregates, leaving them unavailable to load as efficiently onto T. gondii vacuoles (Hunn et al. 2008). The pivotal function of IRG protein family in eradicating T. gondii is underscored by the fact that virulent strains of the parasite have acquired the ability to phosphorylate IRGs, which prevents their loading onto the parasitophorous vacuole (Fentress et al. 2008; Steinfeldt et al. 2015). IRG-deficient mice also have altered resistance to other parasites including Leishmania major, Trypanosoma cruzi, and Plasmodium berghei (Santiago et al. 2005; de Souza et al. 2003; Murray et al. 2015; Guo et al. 2015), but the underlying mechanism(s) in those cases have not been elucidated and may well be distinct from the vacuole attack mechanism seen with T. gondii.
Irgm1-deficient mice also display increased susceptibility to bacterial pathogens including Listeria monocytogenes, Salmonella typhimurium, Mycobacterium tuberculosis, Mycobacterium avium, and Chlamydia trachomatis (Collazo et al. 2001; MacMicking et al. 2003; Henry et al. 2007; Taylor 2007; Feng et al. 2004; Bernstein-Hanley et al. 2006). Irgm1 is unique among the mouse IRGs in this respect, as other IRG-deficient mice have shown little or no susceptibility to bacteria, with the exception of C. trachomatis (Collazo et al. 2001; Al-Zeer et al. 2009; Coers et al. 2008). Multiple changes in the immune response of Irgm1-deficient mice have been noted that may contribute to susceptibility to bacterial infection; these include decreased bacterial processing/killing in IFN-activated macrophages (MacMicking et al. 2003; Henry et al. 2007; Gutierrez et al. 2004) and decreased T cell responses (Feng et al. 2004, 2008a, b). Both may result from the decreased autophagic functioning (Traver et al. 2011) and/or altered mitochondrial dynamics (Henry et al. 2014) that have been documented in Irgm1-deficient cells. Alterations in these processes may result from a direct role of Irgm1 in autophagy and/or mitochondrial dynamics, or indirectly as a result of the GKS IRG protein aggregates that accumulate in cells deficient in IRGM proteins such as Irgm1 (Maric-Biresev et al. 2016). A role for human IRGM in bacterial resistance has also been suggested by GWAS studies (King et al. 2011; Intemann et al. 2009), as well as by in vitro studies with model bacterial pathogens showing decreased bacterial killing in host cells treated with IRGM siRNA (McCarroll et al. 2008; Singh et al. 2006; Brest et al. 2011; Lapaquette et al. 2010). This role for IRGM in antibacterial immunity is likely also driven by the role(s) it plays in autophagy (Chauhan et al. 2015; Singh et al. 2006) and/or mitochondrial functioning (Singh et al. 2010).
IRGM proteins have been linked to inflammatory disease in two settings: Crohn’s disease and sepsis. In mice, Irgm1-deficiency leads to enhanced intestinal inflammation when mice are exposed to dextran sodium sulfate, a standard chemical inducer of experimental colitis (Wirtz et al. 2007). The mice display increases in both ileal and colonic inflammation, while accompanying the inflammation are enhanced weight loss, colonic shortening, intestinal bleeding, and loss of stool consistency. This syndrome is accompanied by altered autophagic processing of secretory granules in Paneth cells and altered production of defensins and inflammatory cytokines. In humans, multiple IRGM gene variants have been identified that lend susceptibility to Crohn’s Disease (Wellcome Trust Case Consortium 2007; Parkes et al. 2007). These not only increase the risk of developing CD but also increase the severity of disease, including fistulating behavior (Latiano et al. 2009), ileal involvement (Roberts et al. 2008), and the need for surgery (Sehgal et al. 2012). Most of the IRGM variants occur in noncoding regions of the gene and are thought to affect expression rather than protein function. Regarding sepsis, Irgm1-deficient mice display enhanced production of the proinflammatory cytokine, tumor necrosis factor, when exposed to LPS, as well as enhanced mortality to LPS-induced shock (Bafica et al. 2007). GWAS studies in humans have associated IRGM gene variants with poor outcomes in sepsis patients (Kimura et al. 2014). The underlying mechanism(s) have not been determined, but the aforementioned impairments in autophagy may drive overly robust cytokine responses to bacterial products such as LPS.
Interferons trigger expression of a diverse range of proteins that provide mechanisms of immune resistance to pathogens. The immunity-related GTPases (IRG) are a family of interferon-induced, membrane-binding proteins that mediate membrane remodeling and membrane trafficking events, which enhance immune functions that contribute to eradication of pathogenic protozoa and bacteria. The best characterized of these mechanisms is the IRG-driven vesiculation of the parasitophorous vacuole in T. gondii-infected cells. Other functions of IRG proteins are continuing to emerge, with this research being driven by GWAS studies associating variants of the human IRG gene with Crohn’s disease, mycobacterial resistance, and sepsis outcomes. Current models suggest that these associations relate to the ability of IRGM proteins to modulate the membrane-associated processes of autophagy and/or mitochondrial fission. This, in turn, impacts relevant immune processes including intracellular processing of bacteria in host cells, the generation of secretory granules in intestinal Paneth cells, and inflammatory cytokine production.
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