ARTICLE IN PRESS

Immunobiology 213 (2008) 367–375

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REVIEW

The IRG proteins: A function in search of a mechanism

Jonathan Howard�

Institute for Genetics, University of Cologne, Zuelpicher Strasse 47, 50674 Cologne, Germany

Received 25 September 2007; received in revised form 24 October 2007; accepted 19 November 2007

Abstract

The IRG proteins (p47 GTPases) constitute one of the strongest resistance systems known to be active againstintracellular pathogens in mice. The proteins are induced by interferons and assemble on phagosomes andparasitophorous vacuoles of a number of different micro-organisms in all cell types assayed. There are presently threeexperimentally based views as to how they exert their cell-autonomous activity against intracellular pathogens:blocking of interferon-mediated acceleration of phagosome maturation, induction of autophagic membranes, anddirect destruction of the parasitophorous vacuole membrane. Failure of hemopoietic stem cells during infection isassociated with targeted deletion of one IRG protein, Irgm1. The significance of this non-cell-autonomous phenotypeis discussed.r 2007 Elsevier GmbH. All rights reserved.

Keywords: IRGM; LRG-47; Irgm1; Irga6; Toxoplasma gondii; Parasitophorous vacuole; Cell-autonomous immunity

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367

A cell-autonomous resistance function. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 368

IRG proteins localise to pathogen-containing vacuoles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369

What do IRG proteins do when they reach a pathogen-containing vacuole? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369

Regulation of IRG proteins in vivo: the Irgm1 problem. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372

Summary: the human factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373

Introduction

It is astonishing how long it took for some of the mostimportant innate resistance mechanisms of mammals tobe discovered. The hegemony of the adaptive immune

e front matter r 2007 Elsevier GmbH. All rights reserved.

bio.2007.11.005

1 470 4864; fax: +49 221 470 6749.

ess: j.howard@uni-koeln.de.

system over the field probably suppressed research intonon-adaptive mechanisms for decades. It was said thatNiels Jerne would not allow seminars on macrophagesto be held at the Basel Institute for Immunology. Thefirst mammalian Toll-like receptor was documentedonly in 1997 (Medzhitov et al., 1997), shortly after thediscovery that a molecule of this structural type, Tollitself, was required for fungal resistance in Drosophila

ARTICLE IN PRESSJ. Howard / Immunobiology 213 (2008) 367–375368

(Lemaitre et al., 1996). The first demonstration that aToll-like receptor, TLR4, was indeed a mammalianresistance factor, had to wait yet another year (Poltoraket al., 1998).

IRG proteins had a less explosive but also surpris-ingly late start considering their abundance in cellstreated with gamma-interferon (IFNg). The first one,IRG-47, was cloned from an interferon-induced mousepre-B cell line in 1992 (Gilly and Wall, 1992). Theyslowly crept onto the map after that, derived fromvarious cell types, as a novel family of IFNg-inducibleGTPases without a function. LRG-47 (Sorace et al.,1995) and TGTP (Carlow et al., 1995; Lafuse et al.,1995) were identified in 1995, IGTP in 1996 (Tayloret al., 1996) and both IIGP and GTPI in 1998 (Boehmet al., 1998). The complete IRG gene family was finallydescribed in mouse and man by us in 2005 (Bekpenet al., 2005). At that time, we also introduced a newnomenclature for the IRG gene family which tookaccount of its considerable complexity, both in themouse and human genomes, and also across phylogeny.The new nomenclature for the six genes mentionedabove, which had been named in haphazard fashion aseach was discovered, is given in Table 1. For the rest ofthis text I will use only the new nomenclature except atthe first introduction of each gene or protein.

The role of IRG proteins as resistance factors couldhave been predicted from their striking induction byIFNg, but the first experimental evidence came fromGreg Taylor, George van der Woude and colleagues,who generated a genomic knock-out mouse of Irgm3(IGTP) (Taylor et al., 2000). The mouse was morpho-logically normal and normally resistant to two viruses,mouse cytomegalovirus and Ebola virus (!), as well as tothe intracellular Gram-positive bacterium, Listeria

monocytogenes. It was, however, extraordinarily suscep-tible to infection with the ME49 avirulent strain of

Table 1. New and old nomenclatures for IRG genes and

proteins (adapted from Martens and Howard, 2006)

Common

names/gene

names

New name

(gene/

protein)

Date and original

reference

IRG-47,

IRG47, Irg47

Irgd/Irgd 1992 (Gilly and Wall,

1992)

LRG-47,

LRG47, Lrg47

Irgm1/Irgm1 1995 (Sorace et al., 1995)

TGTP, Mg21,

Tgtp

Irgb6/Irgb6 1995 (Carlow et al., 1995;

Lafuse et al., 1995)

IGTP, Igtp Irgm3/Irgm3 1996 (Taylor et al., 1996)

IIGP, IIGP1,

Iigp1

Irga6/Irga6 1998 (Boehm et al., 1998)

GTPI, IIGP2 Irgm2/Irgm2 1998 (Boehm et al., 1998)

CINEMA Irgc/Irgc 2005 (Bekpen et al., 2005)

Toxoplasma gondii, the mice all dying in the acute phaseof the disease, before day 12. This first striking resultyielded several important conclusions: while the familyof IRG proteins in the mouse may be large, neverthelessloss of a single member resulted in a fatal diseasesusceptibility, thus the multiple members of the, at thattime still growing, family were not redundant to oneanother. Secondly, the loss of resistance showed a degreeof specificity, making it unlikely that Irgm3 belonged toone of the universal signal transduction pathways of theinnate immune system, and more likely that it partici-pated in a relatively specific step in immunity close to thepathogen itself. Taylor and colleagues added importantnew information to the story when they published twofurther genomic knock-outs of IRG genes, in this caseIrgm1 (LRG-47) and Irgd (IRG-47) (Collazo et al.,2001). Firstly, both these two knock-outs were suscep-tible to T. gondii infection, in the case of Irgm1, highlysusceptible, in the case of Irgd, moderately so, theinfected animals dying over the 2 months after infection.Now three IRG genes had been shown to be essentialand non-redundant for immunity to a single pathogen.Secondly, the Irgm1-deficient mice were also extremelysusceptible to L. monocytogenes, unlike the Irgm3-andthe Irgd-deficient mice. Thus each IRG protein wasshown to have its distinct characteristics, while theparticipation of all three IRG proteins in a non-redundant resistance to T. gondii raised the possibilitythat IRG proteins function as a multi-componentmachine. In the meantime, several further pathogenshave been added to the list of those for which IRGdeficiency results in whole-animal susceptibility. Thesehave been extensively reviewed (MacMicking, 2004,2005; Taylor, 2004; Martens and Howard, 2006; Tayloret al., 2007). However, none of these experiments,although critical in drawing attention to the IRGproteins as novel resistance factors, was able to provideany clues as to the mechanism of the resistance itself.

A cell-autonomous resistance function

A critical step in the analysis of the functionalmechanism of resistance was taken by Halonen et al.(2001) in collaboration with Greg Taylor. These showedthat the susceptibility of Irgm3-deficient mice wasreflected in susceptibility of single astrocytes to T. gondii

infection. Primary mouse astrocytes in culture arenormally susceptible to T. gondii infection, but becomeresistant after treatment with IFNg (Halonen et al.,1998). However astrocytes from Irgm3-deficient micedid not become resistant after IFNg treatment. Thus itappeared that the susceptibility of the IRG protein-deficient mouse was secondary to the susceptibility ofindividual infected cells, which were permissive foruncontrolled pathogen replication. Cell autonomous

ARTICLE IN PRESSJ. Howard / Immunobiology 213 (2008) 367–375 369

loss of resistance has been seen in other in vitro cellularsystems with Toxoplasma and other pathogens andinvolving genomic knockouts of several IRG proteins(MacMicking et al., 2003; Butcher et al., 2005; Martenset al., 2005; Santiago et al., 2005; Bernstein-Hanleyet al., 2006), transfection of functionally dominant-negative mutant IRG sequences (Martens et al., 2005)and a genetic polymorphism resulting in defectiveexpression of Irgb10 associated with susceptibility toChlamydia trachomatis (Bernstein-Hanley et al., 2006).It is certain that IRG proteins can mediate resistance ina cell-autonomous manner. A series of observationshave, however, been made in the case of Irgm1 that seemto speak for an additional non-cell-autonomous prop-erty. Mice deficient in Irgm1 are susceptible to a widerange of different organisms, and in some cases a cell-autonomous defect has also been documented (T. gondii

(Butcher et al., 2005), Trypanosoma cruzi (Santiagoet al., 2005), Mycobacterium tuberculosis (MacMickinget al., 2003), C. trachomatis (Joern Coers, personalcommunication)). Thus Irgm1 is not an exception inprinciple to the rule of cell autonomy. NeverthelessIrgm1-deficient mice infected with either Mycobacterium

avium or T. cruzi, to both of which they are highlysusceptible, show a profound leucocytopenia that hasbeen proposed to result from a failure of stem cellproliferation and/or differentiation during haemopoieticstress following infection with these highly immunosti-mulatory pathogens (Taylor, 2007). The responsibilityof the leucocytopenia, as distinct from the IFNg-dependent cell-autonomous immunity, for susceptibilityto these two pathogens has not yet been fully resolved. Itwas reported that Irgm1-deficient macrophages have acell-autonomous defect in control of T. cruzi replication(Santiago et al., 2005), but no reduction in pathogennumber has been reported in IFNg-stimulated macro-phages infected with M. avium. I discuss a possibleexplanation for the haemopoietic defect in infectedIrgm1-deficient mice below.

IRG proteins localise to pathogen-containing

vacuoles

We showed by immunofluorescence with specificantisera that five (Irga6, Irgb6, Irgd, Irgm2, Irgm3)out of the six IFNg-induced IRG proteins investigatedlocalised strongly to parasitophorous vacuoles inT. gondii-infected astrocytes (Martens et al., 2005) andhave since generalised this further to a number of celltypes including primary hepatocytes, L929 fibroblasts,mouse bone marrow-derived macrophages, mouse 3T3cells of various origins, primary mouse embryonicfibroblasts, and a variety of mouse transformed cellsderived from various tissues (unpublished results). Theexception was Irgm1, which could not be found on the

vacuole. Irgb10, which we did not investigate at thattime, has since also been shown to localise to theT. gondii vacuole (unpublished results with JoernCoers), and Joern Coers and colleagues have shownthat Irgb10, which is a resistance factor forC. trachomatis (see above), also localises to C. tracho-

matis vacuoles in IFNg-stimulated mouse MEFs(submitted for publication). Taylor and colleaguesfailed to detect Irgm1 or Irgm3 on T. gondii vacuolesin IFNg-stimulated BMM (Butcher et al., 2005), but atleast in the case of Irgm3 the negative was probably atechnical problem since this IRG protein was subse-quently detected on the vacuole both at the light(Taylor, personal communication) and electron micro-scopic (Ling et al., 2006) levels.

The case of Irgm1 is clearly exceptional. The proteinis well expressed after IFNg induction in all cell typestested, and has been shown to be a powerful resistancefactor for many infectious agents including T. gondii.Yet it fails to localise to the T. gondii vacuole at anydetectable level. Indeed, we were able to show in an earlyreport (Martens et al., 2004) in cells phagocytosing latexbeads that Irgm1 localises to a quite distinct vacuolarcompartment, the phagosomal–lysosomal compartment,co-localising with filamentous actin at phagocytic cupsand subsequently in an intracellular LAMP1-positivecompartment consistent with lysosomes. In this locationIrgm1 would have no access to the vacuoles of T. gondii

or C. trachomatis (though its localisation to the latterclass of vacuoles has not been reported). This is,however, the entry compartment of many bacteria,including Mycobacteria, and Irgm1 was indeedidentified by Western blot to be associated withmycobacterial phagosomes in IFNg-induced macro-phages (MacMicking et al., 2003). This localisationhas subsequently been confirmed by immunofluores-cence (John MacMicking, personal communication). Atpresent there is little clear information about thepropensity of other IRG proteins to locate to phagocyticvacuoles of any kind. We have been unable to localiseany other IRG protein except Irgm1 to latex beadphagosomes (unpublished results). However, Taylorand colleagues did detect Irgm3 on these structures(Butcher et al., 2005).

What do IRG proteins do when they reach a

pathogen-containing vacuole?

This is now the heart of the problem. IRG proteinsare undoubtedly potent cell-autonomous resistancefactors for a number of diseases, even if cell-autono-mous resistance has not been demonstrated in every casewhere a whole-animal susceptibility has been found inthe knock-out. There have been three proposals basedon experimental investigations for the mechanism of

ARTICLE IN PRESSJ. Howard / Immunobiology 213 (2008) 367–375370

action of vacuolar IRG proteins (see also a recent reviewby Taylor et al., 2007).

MacMicking et al. (2003) observed reduced acidifica-tion of M. tuberculosis-containing phagosomes fromIFNg-induced Irgm1-deficient macrophages, correlatingwith reduced expression of the vacuolar proton pump atthe phagosomal membrane. It was unlikely that thisphenotype was due to direct interference with the protonpump. It seemed, rather, that the phenotype reflectedarrested maturation of the phagosome. Without specify-ing a mechanism, the conclusion from these experimentsis that the presence of Irgm1 on the phagosomalmembrane defeats the complex array of processes bywhich Mycobacteria seek to delay phagosomal matura-tion (Mueller and Pieters, 2006).

Subsequently, Deretic and colleagues proposed anentirely distinct mechanism, namely induced autophagy,for control of mycobacterial infection in mouse macro-phages (Gutierrez et al., 2004). They observed anincrease in markers associated with autophagy inRAW264.7 macrophages transfected 12 h earlier withan EGFP-tagged construct of Irgm1. This phenotyperesembled the phenotype of cells stimulated for 2 h withIFNg, but not apparently Type I, interferon, and wasassociated with large apparently autophagic vacuolescontaining BCG. In a later paper the same groupreiterated this finding with RAW264.7 macrophagesstimulated for 24 h with IFNg (Singh et al., 2006). Theassociation between Irgm1 transfection and formationof autophagic structures is intriguing and certainlyjustifies further work. However it is difficult to attributethe effects of stimulating cells with IFNg for only 2 h tothe action of IRG proteins, since these are not detectablein the cell for at least 8 h after induction. The timing ismore realistic in the second report, but it is difficult toescape the conclusion that RAW264.7 cells may developlarge autophagic structures as a result of severaldifferent inducers. The distinctive role of Irgm1 remainsto be shown: in this context it is surprising that nogeneral or specific autophagic defect has been reportedin Irgm1-deficient macrophages. It is perhaps worth

Fig. 1. Vesiculation and disruption of the T. gondii parasitophorous

infection with ME49 strain tachyzoites (A, B) and in vivo mouse ‘‘pr

strain of T. gondii carrying a GFP transgene (C). Panel A is modifi

(2005). Both are reprinted courtesy of PLO. Panel C is modified from

Rockefeller University Press. Further details about the figures can b

mouse astrocytes stained with antiserum against Irga6 (Martens et

white arrows (filled white arrowhead, plasma membrane; open whit

vacuolar membrane is indicated with a black filled arrowhead. Immu

the neighbourhood of the parasitophorous vacuole membrane. In som

membrane vesicles heavily stained with Irga6 are apparent (insert, b

against the T. gondii granule protein, GRA7, which is secreted and t

positive material is still seen in the dense granules inside the T. gondii

PV membrane. (C) Paraformaldehyde-fixed section of T. gondii in p

indicate ruffling and blebbing of the PV membrane very similar to

noting that an explanation in terms of autophagy wouldalso subsume MacMicking’s observations. If Irgm1promotes autophagy of the mycobacterial phagosome,this would override mycobacterial mechanisms ofresistance to maturation at the level of the phagosomeitself.

We have proposed a third activity for IRG proteinsassociated with the T. gondii vacuole in IFNg-inducedastrocytes and fibroblasts, distinct from either of theabove. We observed at light and EM levels dramaticmodifications to the IRG protein-coated parasitopho-rous membrane consistent with vesiculation (Martenset al., 2005) (Fig. 1A, B). In regions of intensevesiculation the PVM was disrupted (Fig. 1A, inset).We further observed that T. gondii organisms within thedisrupted vacuoles showed gross morphological changesconsistent with death (Martens et al., 2005). An IRGprotein activity associated with vesiculation of the PVMseems perfectly reasonable, if, as sometimes said, theseproteins have a functional relationship to the dynamins.Vesiculation may serve to extract membrane from thePVM, leading to tension and ultimately disruption.However, while disruption of the PVM may expose theenclosed organism to the cytosol, it is unknown whetherthe cytosol is an inimical compartment for T. gondii

survival. Another closely related Apicomplexan,Sarcocystis, lives and replicates in the cytosol (Jakelet al., 2001), while Plasmodium sporozoites penetratethe cytosol of several hepatocytes before forming astable parasitophorous vacuole (Mota et al., 2001). Welooked for but failed to find a consistent association ofLC3-positive, putatively autophagic, membranesaround disrupting vacuoles, although undoubtedlyLC3 was activated in many infected cells (Martenset al., 2005). Furthermore, we have observed some, butnot complete, reduction in restriction of T. gondii ininterferon-treated fibroblasts taken from mice deficientin the autophagy protein, atg5 (Konen-Waisman andHoward, 2007). Our observations on vesiculation of theT. gondii PVM were, however, confirmed by work fromthe laboratory of George Yap, working with peritoneal

vacuole membrane in IFNg-induced mouse astrocytes 6 h after

imed’’ peritoneal macrophages 2 h after infection with the PTG

ed from Fig. 3A and panel B from Fig. 3D of Martens et al.

Fig. 3D3 of Ling et al. (2006) and reprinted by courtesy of the

e found in the source references. (A) Ultrathin cryosection of

al., 2005). The two membranes of T. gondii are indicated with

e arrowhead, inner membrane complex). The parasitophorous

nogold particles indicating the presence of Irga6 are located in

e regions the PV membrane appears to be disrupted and small

lack arrows). (B) Ultrathin cryosection stained with antiserum

ransferred to the PV membrane shortly after infection. GRA7-

(Martens et al., 2005). The image shows massive ruffling of the

rimed peritoneal macrophage (Ling et al., 2006). Black arrows

the images in panels A and B.

ARTICLE IN PRESSJ. Howard / Immunobiology 213 (2008) 367–375 371

macrophages from Toxoplasma-primed mice (Linget al., 2006) (Fig. 1C). These workers further observeddisruption of the T. gondii plasma membrane itself, and

association of the disrupting organisms with largeputatively autophagic membrane bounded structures.The Martens and Yap studies both suggest an early role

ARTICLE IN PRESSJ. Howard / Immunobiology 213 (2008) 367–375372

for IRG proteins at the PVM, but in Yap’s study therole of IRG proteins at later stages, the killing of thepathogen, the stripping of the pathogen plasma mem-brane and the engulfment in autophagic membranes,remains to be tested. There were several differencesbetween the Yap study and our own, including the celltype examined, and the method of induction, which wasby IFNg treatment in vitro in our experiments and by‘‘priming’’ of peritoneal macrophages in vivo by priorimmunisation with two injections of a different T. gondii

strain in the other (Ling et al., 2006). Peritoneal cellsfrom unprimed mice were not rendered resistant toT. gondii by treatment with IFNg in vitro, an anomalythat deserves further investigation.

In conclusion, the field is very far from establishing adefinitive resistance mechanism for the IRG proteins inany infection. Several possibilities have been floated,each with some experimental support, but it is difficultto discern a common thread at present. A key issue willbe whether there are distinctive mechanisms operatingfor different pathogens.

Regulation of IRG proteins in vivo: the Irgm1

problem

We have presented preliminary evidence that correctIRG protein function in the IFNg-stimulated cell isregulated by the presence of other IRG proteins(Martens and Howard, 2006). In that analysis we wereable to show that the three GMS (Boehm et al., 1998)proteins, Irgm1, Irgm2 and Irgm3, distinguished fromall other GTPases by their unique use in the G1nucleotide-binding motif of the large hydrophobicresidue, methionine, in place of the usual lysine, arerequired for normal function of Irga6. We have sincebeen able to analyse these phenomena in considerabledetail (Hunn et al., manuscript in preparation). Theoriginal observation was that certain IRG proteins,namely Irga6 and Irgb6, were grossly mislocalised andaggregated when transfected or otherwise expressedalone in cells not induced with IFNg. This mislocalisa-tion could be corrected by co-expression of all threeGMS proteins. There is also evidence that the assemblyof IRG proteins on the T. gondii parasitophorousvacuole is also to some degree cooperative (Hunnet al., manuscript in preparation). These observationsseem to provide an explanatory basis for the surprisingnon-redundancy between IRG genes that has beenobserved especially in T. gondii and C. trachomatis

immunity.If regulatory or cooperative interactions are generally

true for members of the IRG protein family, this mayalso throw some light on the anomalous properties ofIrgm1. Irgm1 is one of the three GMS subfamilyproteins that are required for correct function of the

GKS proteins Irga6, Irgb6 and Irgd. Absence of Irgm1on the T. gondii vacuole would be consistent with aregulatory, rather than executive role of this GTPase.Absence of Irgm1 has also been reported to lead tolymphomyeloid failure during infection with Mycobac-teria and Trypanosoma (Feng et al., 2004; Santiagoet al., 2005). We can put these observations together. InIrgm1-deficient mice, induction with IFNg will lead toexpression of mislocalised GKS IRG proteins in all cells.The consequences of such mislocalisation are unpredict-able. We have observed growth impairment in 3T3 cellsexpressing Irgb6 under the control of an induciblepromoter, but not in all clones (unpublished results),results which echo earlier findings of growth failure andunstable expression in cells transfected with a constitu-tively expressed construct of Irgb6 (Carlow et al., 1998).It may also be of interest that Irgb6 has been reported tobe expressed at a high level in activated haemopoieticstem cells (HSC) (Terskikh et al., 2001; Venezia et al.,2004). It is not clear whether this is ‘‘autochthonous’’transcription, or whether it is the result of a burst ofIFNg expression during the activation of HSC inmarrow. Other IRG proteins were reported to beoverrepresented in this population, but Irgb6 is thepredominant transcript (see references above). IndeedIrgb6 (TGTP) may be preferentially expressed in cells ofthe lymphomyeloid system: it was initially described inimmunologically activated T cells (Carlow et al., 1995),although there has been no systematic study of IRGgene expression in individual lymphomyeloid compart-ments. Nevertheless, it is interesting to consider thepossibility that the marked lymphomyeloid failurereported in Irgm1-deficient mice may result, not fromsome essential function of LRG-47 mediated on stemcell development, but rather from a cytopathic conse-quence in lymphomyeloid stem cells of mislocalisation,possibly aggregation, of other IRG proteins in theabsence of an essential regulator of their behaviour. Oneobvious problem with this explanation for the infection-associated marrow failure of Irgm1-deficient mice is thatmice deficient in Irgm3, which is also required fornormal behaviour of GKS proteins, should also showan immunological defect, but in fact these mice arenormally resistant to both Mycobacteria and Trypano-

soma and no lymphomyeloid defect has been reported.A plausible response could be that Irgm1 may bespecifically required for, for example, Irgb6 regulation,while Irgm3 may favour Irga6 or another GKS protein,and perhaps de-regulated Irgb6 is the main cause of theproblem. Whether correct or not, the hypothesis thatlymphomyeloid failure in Irgm1-deficient animals is dueto failure to regulate the GKS IRG proteins in sensitivestem cell compartments at least deserves a hearing.

If induction of IRG proteins in lymphomyeloid stemcells leads rapidly to a systemic immunodeficiency, wemay also have here an explanation for why Irgm1

ARTICLE IN PRESSJ. Howard / Immunobiology 213 (2008) 367–375 373

deficiency is associated with susceptibility to so manypathogens, unlike the rest of the IRG family. It will beimportant for the future to test each case specifically fora cell-autonomous component as well as a lymphomye-loid deficiency. For instance, Irgm1 deficiency leads toextreme vulnerability to L. monocytogenes (Taylor et al.,2000), an organism which resides only briefly in acytoplasmic vacuole before escaping to the cytosol.There is as yet no report of a cell-autonomous model forListeria susceptibility in Irgm1 deficiency, and equallythere has been no description of the lymphomyeloidpicture in Listeria-infected Irgm1-deficient mice. Listeria

does, however, stimulate a massive, systemic IFNgresponse which in turn stimulates induction of all theknown IRG proteins except IRGC (Boehm et al., 1998;Bekpen et al., 2005).

Summary: the human factor

The IRG resistance system is fascinating in its powerand its complexity. It is also fascinating that the entiresystem is absent in man. While IRG genes can be tracedthrough all of vertebrate ancestry (Bekpen et al., 2005)and even further back (Hunn et al., in preparation),their representation is extremely erratic. They are, forexample, still unknown in birds, though it is of coursepossible that they are all there, hiding in some smallunsequenced fragment of the chicken genome. It seems,nevertheless, that it is possible to handle all theintracellular pathogens that IRG proteins target withoutthe help of IRG proteins, at least in man. The singlefragment of a GMS gene, known as IRGM, that is allthat remains of the entire immune IRG system in thehuman genome, is highly unlikely to play any role inIFNg-mediated immunity (Bekpen et al., 2005).A strong case can be made that other resistance factors,and especially perhaps, tryptophan depletion viaIFNg-inducible indoleamine dioxygenase (IDO), maycover for the lack of IRG proteins in our species(Konen-Waisman and Howard, 2007). Surprisingly,however, the IRGM fragment has been implicated inbasal resistance to Mycobacterium bovis BCG in humanmacrophages, with a possible connection to an autop-hagic response (Singh et al., 2006), and more recentlyIRGM has emerged as a major player in susceptibility toCrohn’s Disease through whole-genome screening in alarge-scale case–control study (Parkes et al., 2007).Again, though indirectly, a connection with autophagyhas surfaced, since a second high-risk factor in Crohn’sis also a component of the autophagic mechanism(Hampe et al., 2007). We shall have to see. So far, thereis only a single-experimental claim on behalf of IRGMthat it plays any role in human immunity.

If such a powerful resistance mechanism as the IRGsystem gets lost again and again (I include the birds

here) it seems certain that possession of this mechanismbrings costs as well as benefits. What are the costs?Right now, one can only speculate that the closeintegration of the IRG mechanism renders its hostvulnerable to single mutations, perhaps especially in theregulatory GMS genes. Is it significant that the humanIRGM fragment appears to have an integrated retro-viral element directly upstream which controls itstranscription? Was this the event that precipitated theloss of the whole IRG system? An appropriatephylogenetic study of the genomes of the higherprimates should help us to find out.

Acknowledgements

The author is indebted to all the members of his lab,and especially to Sascha Martens, Steffi Konen-Waisman, Julia Hunn, Natasa Papic, Yang Xhao andSasha Khaminets who worked at various times on theT. gondii project. Julia Hunn also provided helpfulcomments on the manuscript. Thanks also to Dr. GabyReichmann of the Institute for Medical Microbiology,University of Dusseldorf, who first introduced us toT. gondii. Dr. George Yap of the University of Medicineand Dentistry, New Jersey Medical School in Newark,generously sent a high-resolution image of his publishedelectron micrograph, shown in Fig. 1C. Work from theauthor’s laboratory has been generously supported bythe Schwerpunktprogramm SPP1110 ‘‘Innate Immu-nity’’, as well as by the following special researchprojects (SFBs): SFB670 ‘‘Cell-autonomous Immunity’’,SFB635 ‘‘Post-translational control of protein function’’and SFB680 ‘‘Molecular basis of evolutionary innovation’’.

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