Cell-autonomous immunity to Toxoplasma gondii in mouse and man

Microbes and Infection 9 (2007) 1652e1661 www.elsevier.com/locate/micinf

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Cell-autonomous immunity to Toxoplasma gondii in mouse and man Stephanie Ko¨nen-Waisman, Jonathan C. Howard* Institute for Genetics, University of Cologne, Zu¨lpicher Strasse 47, 50674 Cologne, Germany Available online 14 September 2007

Abstract The protozoan, Toxoplasma gondii, is a natural pathogen of mouse and a zoonosis of man. Immunity against the pathogen is largely mediated by interferon-stimulated cell-autonomous mechanisms that are strikingly different between man and mouse. There are many poorly understood host and pathogen variables that affect the outcome of infection. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Interferon; Innate immunity; Apicomplexa

1. Introduction Toxoplasma gondii is a close relative of the malaria parasite, Plasmodium falciparum and its congeners. As an experimental model it has enormous technical advantages over Plasmodium and has frequently been used as a convenient alternative on the principle that difficult things can be found out and researched in the easy model, then transferred to the more demanding Plasmodium situation. The advantages are these: the domestic cat is a primary host, mice and other small rodents are natural secondary hosts. Oocysts and subsequently sporozoites can be obtained relatively easily from cat faeces and the asexual cycle can be indefinitely extended at convenience by continuous passaging in vitro of the fast replicating tachyzoites which are also infectious both in vitro and in vivo. In recent years sophisticated genetic procedures have been developed for T. gondii which enormously extend the range of experiments. The normal mature human immune system is remarkably resistant to acute toxoplasmosis and as a result the

Abbreviations: IRG, immunity-related GTPase; IFNg, interferon gamma; IFNb, interferon beta; IDO, indoleamine 2,3-dioxygenase; NVDV, Newcastle Virus Disease Virus; INOS2, inducible nitric oxide synthase 2; TNFa/TNFR, tumor necrosis factor alpha/TNF receptor; MoPn, mouse pneumonitis variant of Chlamydia trachomatis; Nramp1, natural resistance-associated macrophage protein 1. * Corresponding author. Tel.: þ49 221 470 4864; fax: þ49 221 470 6749. E-mail address: [email protected] (J.C. Howard). 1286-4579/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.micinf.2007.09.005

organism has been of relatively modest interest as a human pathogen, but doubly valuable as an experimental tool because of the low risk. Increasingly, however, toxoplasmosis is becoming a clinical problem because of iatrogenic and AIDS-related immunodeficiency. Disease processes in T. gondii infection have been studied most intensively in man and mouse but, surprisingly, experimental findings from one species have often not been generalised to the other. This is surely due, on the one hand, to a degree of ‘‘brand loyalty’’ in the research communities that focus on host responses, while on the other hand the community that approaches toxoplasmosis from the pathogen side moves freely, but somewhat insouciantly, between material from a variety of different host species, taking advantage of the organism’s remarkable promiscuity and ability to thrive in essentially any mammalian cell. Nevertheless it has been apparent for many years that important components of host immunity to T. gondii are indeed radically different between man and mouse. The biology of the situation is still far from clear, but it is important to recognize that mouse is a natural secondary host for the organism, locked into a complex dynamic and evolving relationship with both pathogen and primary host, while for humans Toxoplasmosis is a zoonosis with no evolutionary consequences. The purpose of this brief review is to highlight one aspect of immunity against T. gondii that has been intensively worked on in both man and mouse, and that is the cell-autonomous resistance that both species can generate when cells of either species are stimulated with interferons.

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Dubremetz and Joiner correctly observed in 1993 [1] how deficient the then available battery of tools was for really critical molecular work with T. gondii. Nevertheless one of the most valuable and interesting tools was in fact already in their hands, namely the great ease with which T. gondii infections in the tachyzoite stage can be conducted in cultivated cells in vitro. This provided and continues to provide a window on the complex interactions between the pathogen and the individual host cell as convenient as for any bacterial species. Not only this, but the ease of in vitro infection had already been used as early as 1968 [2] to demonstrate the phenomenon that is the subject of this review, namely the ability of individual cells, normally permissive to infection by T. gondii, to be stimulated by interferons to develop an intrinsic state of resistance which we call cell-autonomous immunity. 2. Acute mortality in the mouse We focus on the earliest immune events following infection, namely the consequences of the induction of natural killer cell-dependent gamma interferon before the engagement of the adaptive immune system [3] (reviewed in ref. [4]). These early processes determine the outcome of infection in the mouse. If they fail, then infection even with an avirulent strain such as ME49 is followed by death from day 8 to 11 after infection. Acute mortality in mouse T. gondii infection is based largely on hyperactivation of Th1 inflammatory cytokines, especially IL-12 [5], IL-1, and gamma interferon itself [6,7]. Clearly the earliest immune events serve to control the size of the early stimulus to cytokine production. The primary IL-12 source in T. gondii infection is the plasmacytoid dendritic cell and not the macrophage [8]. Indeed there is very little evidence that macrophages normally play a significant role in the acute response to T. gondii infection although their cellautonomous ability to restrain infecting T. gondii is often studied (e.g. ref. [9]). Sher and colleagues have shown that components of the lymphomyeloid system and non-lymphomyeloid compartments are both required for normal interferon-dependent resistance to T. gondii [10]. Which non-lymphomyeloid cells participate in this resistance is an interesting unanswered question. With the discovery of numerous cell-autonomous, interferon-dependent immune processes that can be mediated by any cell type (see Reviews in this edition of Microbes and Cells), it has become a reasonable working hypothesis that acute mortality to T. gondii infection in the mouse is due to a failure of these processes, whether in lymphomyeloid or nonlymphomyeloid cells. In experimental settings these failures can be generated by inactivation of components of the interferon signalling pathway [11], of the interferons themselves, and of the interferon-inducible effector mechanisms that are directly responsible for cell-autonomous resistance. With these results in mind it is also possible to formulate a view of virulence differences between natural T. gondii strains that would certainly bear testing. It has been apparent for some time that the population genetics of T. gondii are consistent with a recent world-wide expansion of two highly similar genotypes and their recombinants arguably derived

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from a single mating [12e14]. Although >98% identical overall at the sequence level the two genotypes are strikingly distinct from each other in their virulence in mice, by up to 4 orders of magnitude. Much research has been devoted to the analysis of this profound functional dichotomy and several parameters plausibly contributing to virulence have been noted [12,15e18]. It seems likely, however, that the key distinction between virulent and avirulent T. gondii strains will turn out to be their ability to interfere with cell-autonomous resistance mechanisms, whether with the interferon-signalling pathway which initiates effector processes, or with the effector processes themselves. Furthermore, bearing in mind the fundamental differences in the nature of cell-autonomous resistance between man and mouse (see below) it is highly unlikely that T. gondii virulence mechanisms that counter cell-autonomous resistance in the mouse will have much relevance to the infection process in man. Indeed intensive study of virulent and avirulent T. gondii infection in man has failed to reveal any significant differences between them in terms of invasiveness or morbidity, although there are significant differences in the incidence of the two virulence types in different clinical categories of patient at risk [19,20] and extensive referencing therein. The relevance of these subtleties in the pattern of toxoplasmosis due to different virulence types in man to the dramatic virulence difference in mice remains completely unclear. The absence of a clear distinction in terms of virulence in the human certainly supports the proposition that the genetic distinctions between virulent and avirulent T. gondii are to do with the relationship of this organism with mice and not with man. 3. Cell-autonomous immunity in non-lymphomyeloid cells Although non-lymphomyeloid cells are implicated in interferon-dependent resistance to T. gondii, their identity remains unknown. However interferon-inducible, cell-autonomous immunity to T. gondii has been demonstrated in several cell types including macrophages and astrocytes. Because of the ease with which mouse primary embryonic fibroblasts and human foreskin fibroblasts can be obtained, they are the cell type of choice for genetic and cell-biological investigations of nonlymphomyeloid cell-autonomous immunity. It is, however, a surprising fact that no systematic analysis of cellular resistance in fibroblasts has been reported, in particular taking account of genetic factors. We here focus mostly on the question whether human and mouse fibroblasts, representing ordinary non-lymphomyeloid cells, can display interferon-dependent cell-autonomous immunity against T. gondii and if so what mechanisms are at their disposal; we discuss to a minor extent whether macrophages can perform the same role and if so whether they employ the same or different or additional mechanisms. The previously cited pioneering experiments of Remington [2] no longer seem technically ideal, but their form is still routine for examining cell-autonomous immunity. In Remington’s experiment L929 mouse fibroblasts were stimulated with interferon and infected with virulent RH strain T. gondii. After 12

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and 24 h the monolayers were examined visually for cell destruction and scored on an arbitrary scale. The L cells were rendered resistant to T. gondii cytopathic effects by pre-treatment with interferon. There are, however, three curious aspects to these results that deserve further analysis. Firstly, it is unclear what the cytopathic effects of the RH strain T. gondii were in Remington’s experiment that were so well advanced by 24 h after infection with a multiplicity of 2.5. Cytopathic effects would not normally be apparent under such conditions for at least 3 days with this T. gondii strain. Secondly, the interferon used was Type I viral interferon, presumably IFNb, produced from L cells themselves by infection with Newcastle Virus Disease Virus (NVDV). Essentially all subsequent work on interferon effects in T. gondii infection has been based on IFNg. Thirdly, when the topic of interferon-induced cell-autonomous resistance to T. gondii was taken up again by Pfefferkorn [21] and L929 cells were again the host cell of interest, no interferon-induced resistance to T. gondii RH strain was detectable. Pfefferkorn and colleagues generalized this failure of IFNg-mediated resistance to mouse fibroblasts in general, claiming similar results from 3T3 fibroblasts as well [21]. In a completely independent analysis, however, Shirahata and Shimizu [22] showed clear control of T. gondii in L cells treated with supernatants from immune spleen cells. These effects were later shown by the same group to be due to IFNg [23]. These inconsistent results provoked us to look more closely into the status of mouse fibroblasts as mediators of cell-autonomous immunity to T. gondii. We infected a number of different mouse fibroblasts in vitro with the non-virulent T. gondii strain, ME49, and measured T. gondii replication on the third day after infection by incorporation of 3H-uracil. The data in Fig. 1aec show that we were able to confirm completely Pfefferkorn’s result [24] for one population of L-929 cells, which showed no IFNg-inducible resistance to infection with ME49 strain T. gondii. Another batch of L-929 cells, known as P2 (gift from Joern Coers, Department of Microbiology, Harvard University), showed weak but significant IFNg-induced resistance (Fig. 1d). Diverging from the results of Pfefferkorn [21], however, a subline of NIH-3T3 fibroblasts generated substantial resistance to infection under identical experimental conditions (Fig. 1e). In our hands, C57BL/6 primary mouse embryonic fibroblasts are completely resistant to T. gondii after induction by IFNg (Fig. 1f,g). Thus under our assay conditions mouse L929 cells do indeed seem to show rather a striking defect in their IFNg-inducible cell-autonomous resistance to T. gondii. We asked whether the basis for this defect lay in the genetics of the strain of origin of L929 cells, C3H/An, a little-used, ancient substrain of C3H. However a liver tumor cell, NCTC1469, originating from the same strain at the same time as L929, was as competent to resist T. gondii after IFNg stimulation as primary embryonic fibroblasts of the C57BL/6 strain. That no genetic defect associated with fibroblasts of C3H origin is responsible for the defect in L929 cells was shown by the complete control of T. gondii replication by primary embryonic fibroblasts derived from another C3H subline, C3H/HeJ (Fig. 1i). It therefore seems

that L929 cells suffer from a specific if not always absolute deficit in a cell-autonomous resistance function that is not general to other fibroblasts or fibroblast-derived cell lines. Most mouse fibroblasts can control T. gondii replication completely after induction with IFNg. We will come later to the likely mechanisms responsible for this resistance. The failure of mouse L929 cells to control T. gondii after IFNg induction seemed to Pfefferkorn [21] to be consistent with the behaviour of human fibroblasts [24]. These cells have been shown many times to control T. gondii after IFNg treatment, and to a first approximation the mechanism also seems established, namely the induction of an active catabolic enzyme of tryptophan, indoleamine 2,3-dioxygenase, which opens the indole ring generating kynurenine as a degradation product [24]. The depletion of tryptophan in cell culture medium by IDO is clearly a necessary component of the IFNg-induced resistance mechanism in human cells since the addition of excess tryptophan completely blocks the T. gondii inhibitory effect of IFNg. Furthermore T. gondii furnished with a tryptophan-synthesizing enzyme, and thereby rendered autotrophic for this amino acid, cannot be inhibited by IFNg treatment of infected cells [25]. The consistency of Pfefferkorn’s findings in L929 cells with these results from human cells arises from his observation that L929 cells are unable to synthesize IDO following IFNg induction, although very low levels of IDO were detected in IFNg-induced L929 cells in another study [26]. Thus the failure of L929 cells in both IDO production and IFNg-mediated T. gondii control seemed to generalise the importance of IDO as a resistance mechanism, a conclusion supported by the resistance achieved in mouse cells transfected with an inducible construct expressing mouse IDO [27]. Unfortunately for this simple conclusion, however, failure to synthesize IDO following IFNg is general for mouse fibroblasts and indeed for several other mouse cell types including macrophages [28]. An extended analysis of 25 mouse cell lines documented IDO production in only 7, and significant levels in only 3 [26]. More recently, 6 out of 7 human cell lines were shown to produce IDO after IFNg stimulation while only 1 of 10 mouse cell lines did so [29]. The situation is thus complex. Following IFNg induction, human fibroblasts can arrest T. gondii growth through expression of IDO and subsequent tryptophan depletion while mouse fibroblasts cannot, but most mouse fibroblasts, whether primary or derived from cell lines, can nevertheless resist T. gondii effectively. L929 cells appear to be the exception in being completely or largely unable to control T. gondii after IFNg induction. The first question is, what other interferon-dependent mechanism do mouse fibroblasts normally use to control T. gondii? And the answer seems clearly to be the recently described IFNg-inducible multigene family of p47 or IRG GTPases [30,31] and described elsewhere in this Forum [32]. While little is yet known about the mechanism of control, failure of this system results in loss of control of T. gondii growth. There are relatively few published data from fibroblasts, but the results from Taylor and colleagues show clear effects of gene disruption of Irgm3 (IGTP) in mouse astrocytes

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Fig. 1. L929 fibroblasts show no IFNg-inducible resistance to T. gondii growth. Cultured cells were stimulated with 100 U of IFNg for 24 h (black bars) or left unstimulated (white bars) before infection with different multiplicities of T. gondii strain ME49. Forty eight hours post-infection cells 3H-uracil was added to each well. After a further 24 h incubation cultures were harvested and radioactivity incorporated into T. gondii DNA was measured in a beta scintillation spectrometer. To compare different experiments the counts per minute were normalized to % of the highest value. L929 cells (AeC) show a striking inability to control T. gondii infection compared with primary mouse embryonic fibroblasts (F, G, I), 3T3 fibroblasts (E) or NCTC1649 (H), a liver cell line derived from the same C3H substrain (C3H/An) as L929 cells. Another subline of L929 (P2, a gift from Joern Coers, Harvard University Department of Microbiology) showed some weak control of T. gondii replication.

[33] and we have shown a clear but weaker effect from disruption of Irga6 (IIGP1) [34]. Stronger effects on interferondependent control of T. gondii growth were however seen when interferon-treated cells were transfected with a dominant

negative form of Irga6 (K92A) [34] and likewise with a dominant negative mutant of Irgb6 (TGTP) (unpublished results). The second question provoked from experimental T. gondii infection in mouse fibroblasts is then, what is wrong with

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mouse L929 cells? Is the IRG system defective in this cell line? We have analysed the IFNg-inducible expression of 6 IRG proteins in L929 cells [35] and it is not different in any obvious way from that of other cells, for instance 3T3 cells, which are competent to control T. gondii (see Fig. 1E). Nevertheless the importance of the IRG GTPases as cell-autonomous resistance molecules in the mouse is such that a further detailed study of the L929 cell situation would be justified, including accurate quantitation and kinetics of induction of the known proteins. The failure of L929 cells to control T. gondii may reveal a component of the mechanism of IRG protein function that has so far escaped attention. This discussion has so far not commented on the original observations of Remington [2], in which good control of T. gondii infection was induced in L929 cells by Type I interferon. Although it was early days in the interferon business it is quite clear from the nature of the preparation that this was indeed type I interferon, probably largely IFNb. This result now stands in contrast to the situation observed repeatedly with IFNg on this cell line. Indeed there is a second report of control of T. gondii in L929 cells [23]. In this case, IFNg was produced from mouse spleen cells by antigen or lectin stimulation, and was undoubtedly contaminated with many other cytokines, including almost certainly also type I interferon. In this system, however, unlike Remington, the authors were unable to show the same effect for NVDV-induced Type I IFN alone. It has been reported elsewhere that Type I IFN is not such a strong stimulator of the IRG system as Type II [36], but Remington’s and possibly also Shirata’s results may suggest that Type I interferon can contribute a significant component of the IFN-mediated resistance of mouse fibroblasts and presumably other non-professional APC. We therefore recently examined the possibility that our refractory L929

cells could be made resistant to T. gondii by treatment with IFNb. The clear-cut result was that they could not (Fig. 2a), any more than they could by IFNg. In parallel, mouse embryonic fibroblasts showed their characteristic IFNg-induced resistance to T. gondii growth, while treatment with IFNb induced a significantly weaker effect (Fig. 2b), consistent with the weaker induction of IRG proteins by type I IFN. For the time being, therefore, Remington’s experiment [2] remains un-reproduced. A more extensive and professional screen of available L929 lines and sublines would seem appropriate. Between mouse and man there have been two major mechanisms clearly identified, even if not fully worked out, which enable normal non-phagocytic tissue cells to resist T. gondii following IFNg induction. These are IDO and tryptophan depletion for human and IRG GTPases in mouse, where the mechanism probably includes but is probably not limited to destruction of the parasitophorous vacuole membrane [34,37]. There have, however, been strong hints that further cell-autonomous mechanisms can be induced by IFNg. A strong IFNg-induced resistance was described in human vascular endothelial cells [38] that was demonstrably not due to tryptophan depletion, nor could be explained either by reactive oxygen intermediates or by nitric oxide mechanisms characteristic of both mouse and human macrophages. This interesting result could not be generalized to human brain microvascular endothelial cells, where a clear IDO-dependent, IFNg-inducible resistance was documented [39]. Whether these divergent results represent technical issues or a genuine divergence in mechanism is not clear. A possible role for iron depletion in T. gondii resistance is also suggested by an experiment on rat enterocytes stimulated with IFNg [40]. These cells developed a resistance to T. gondii that could not be

Fig. 2. Beta-interferon is unable to induce resistance to T. gondii in L929 cells. L929 cells and C57BL/6 embryonic fibroblasts were left untreated (white bars) or stimulated for 24 h with 500 U IFNb (dark grey bars), 100 U IFNg (black bars), 500 U IFNb þ100 U IFNg (light grey bars) before infection with T. gondii strain ME49 at different multiplicities. Toxoplasma replication was measured at 72 h after infection as in Fig. 1.

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attributed to NO, active oxygen or L-tryptophan depletion. Rats are well-endowed with IRG genes, and IFNg-inducible IRG proteins would be good candidates for this resistance. T. gondii inhibition was, however, relieved by providing excess iron either as ferrous salt, deferoxamine or Fe-transferrin. The same experiment was attempted in mouse astrocytes [41], and here the resistance to T. gondii could not be restored by the provision of iron. Indeed this resistance appears to be exclusively due to the induction of IRG proteins {Halonen SK, 2001 #2907}. It would indeed be surprising if IRG proteins are not involved in T. gondii resistance in rat cells, but this remains to be demonstrated. 4. Autophagy and apoptosis It has recently been made clear that mouse macrophages can develop a cell-autonomous resistance to T. gondii that is independent of IFNg, of INOS2 and of at least three members of the IRG family of resistance GTPases [42]. This resistance is stimulated through CD40, requires TNFa signalling through the TNFR2 and is probably mediated via the induction of autophagy [43,44]. Whether this pathway is also active after IFNg stimulation of cells is not known. It is also not known whether this pathway is available to non-professional cells infected by T. gondii, although CD40 has been shown to be expressed in several primary cell types including fibroblasts in both mouse and man [45e47]. In a parallel development, IRG GTPases have been implicated in the initiation of autophagy-dependent resistance mechanisms in both mouse and, surprisingly, man [48]. We [34] reported the formation of LC3-positive membranes in the vicinity of T. gondii parasitophorous vesicles already ringed by IRG proteins in IFNg-stimulated astrocytes, while Yap and colleagues showed autophagic-like forms

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surrounding T. gondii vacuoles in IFNg-induced bone marrow-derived macrophages where the vacuolar membranes had already been disrupted [37]. In both these cases it is unclear whether the IRG proteins are directly involved in the autophagic process, as has been claimed for mycobacterial resistance and Irgm1 (LRG-47) [49]. More research is needed to establish whether the autophagic process is a downstream mediator of the action of IRG proteins or an independent effector mechanism in its own right. Until now, no evidence has been adduced for an involvement of autophagy in the IFNg-induced resistance of mouse fibroblasts to T. gondii. We therefore examined the resistance to T. gondii of IFNg-induced transformed embryonic fibroblasts from mice deficient in the autophagic mediator, Atg5. Fig. 3 shows the results of an experiment of this type. The IFNg-induced resistance of the Atg5-deficient cells was diminished relative to the wild type, suggesting that autophagy may play a significant role in the IFNg-mediated control of T. gondii in fibroblasts. This result would further implicate IRG proteins in the regulation of autophagy during infection of IFNg-induced cells. A powerful inhibition by T. gondii infection of cellular apoptotic pathways has been documented in many systems (reviewed in ref. [50]). It would be of considerable interest to initiate a focused investigation of the role played by apoptosis and its inhibition in a primary mouse fibroblast system of T. gondii infection. In view of the strenuous efforts made by T. gondii to inhibit apoptosis of the infected cell it seems likely that apoptosis is indeed a potential defense mechanism of some power. The question revolves around the role of apoptosis as a cell-autonomous resistance mechanism, but the field is confused by evidence that T. gondii can also induce noncell-autonomous apoptotic events occurring in lymphocytes and macrophages in environments containing T. gondii-infected

Fig. 3. Autophagy may contribute to IFNg-induced resistance of mouse fibroblasts to T. gondii. SV40-transformed embryonic fibroblasts from wild-type and atg5deficient mice were stimulated for 24 h with 100 U IFNg (grey bars), 200 U IFNg (black bars) or left untreated before infection with T. gondii strain ME49 at different multiplicities. Toxoplasma replication was measured at 72 h after infection as in Fig. 1. The atg5-deficient and control fibroblasts were a generous gift from Dr Nobura Mizushima, Tokyo Medical and Dental University.

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cells (reviewed in ref. [51]). A recent study implicated IFNg, acting through nitric oxide production, in the bystander apoptosis of macrophages during T. gondii infection [52]. 5. Chlamydia and T. gondii: common themes While human resistance to T. gondii may be of medical importance, it is unlikely to play any role in the evolution of hostparasite relations for T. gondii. The situation in the mouse is, however, quite different, where T. gondii lives in a continuous cat-and-mouse game between its main primary and secondary hosts. Unfortunately little account has been taken in the past of the fine structure of co-adaptation between T. gondii and either cat or mouse. Recently, however, an interesting analysis has begun to develop of the complex species-specific relations that have evolved between Chlamydial species and the human and mouse host species [53]. In several respects Chlamydial resistance seems to mimic T. gondii resistance in man and mouse. Just as with T. gondii, the critical medical issue is the differential relation between the human and mouse Chlamydial species and the interferon response of the two hosts. Practically speaking, the mouse is a poor clinical model for human C. trachomatis because its resistance response, as for T. gondii, is based on the cell-autonomous, IFNg-dependent expression of IRG proteins, three of which have already been implicated in the resistance [54,55]. The natural Chlamydia of mouse, C. muridarum MoPn, extremely closely related to human C. trachomatis and responsible for murine pneumonia, is controlled in the C57BL/6 mouse, but is relatively insensitive to IFNg in vivo [56] although there are in vitro data showing that C. muridarum is sensitive to IFNg treatment of C57BL/6 macrophages [57]. The relative protection of C. muridarum from the IFNg response has recently been attributed to the possession by this murine Chlamydia strain of a resistance molecule, a Yersinia YopT homologue, which may target the IRG proteins and render them ineffective [29,54]. In contrast, the human variant, C. trachomatis lacks Yopt, consistent with the lack of IFNg-inducible IRG genes in man, but possesses a tryptophan synthase which is argued to allow C. trachomatis to evade the human IFNg-dependent IDO response. This curious but provocative proposal remains to be substantiated experimentally, specifically, that C. muridarum YopT homologues have a destructive enzymatic effect on IRG proteins. The argument is based on two points, firstly that Yersinia YopT proteins target Rho GTPases and IRG gene products are GTPases; secondly, that one IRG protein, Irga6, which in a knock-down analysis has been implicated in cellautonomous immunity to C. trachomatis in mouse epithelial cells [54], possesses a C-terminal sequence, -CLRN, reminiscent of the C-terminal CaaX sequence which, following isoprenylation, is the target for the yersinial YopT toxin in isoprenylated Rho GTPases. It should however be observed that Irga6 is myristoylated at the N-terminus [58], while the arginine in the penultimate position should anyway prevent the -CLRN sequence from functioning as an isoprenylation signal. It has not, however been experimentally excluded that Irga6 may be isoprenylated as well.

There have been a few systematic attempts to analyse polymorphic variation associated with T. gondii resistance in the mouse (e.g. ref. [59]) and these have led only to the identification of the MHC as a determinant of differential natural resistance of inbred strains [60]. Some important groundwork was, however, laid in a linkage analysis based on the A/J  C57BL/ 6 recombinant inbred lines developed at the Jackson laboratory [60,61]. In addition to confirming a significant role for the MHC, this study identified several genomic regions showing significant associations with T. gondii sensitivity. Not unexpectedly, different genomic regions showed associations with overall mortality and brain cysts as separate traits. No specific genes stand out except the expected strong association of cyst number with the MHC. Regions on chromosome 18 and chromosome 11 hint at linkage to the two main clusters of IRG genes, but this would require confirmation through a detailed analysis, like that performed by the Dietrich group in their demonstration that mouse Irgb10 is a resistance gene for Chlamydia trachomatis [55]. While the chromosome 11 associations could be consistent with a role for polymorphism in the IRG genes in controlling resistance, the highest association is telomeric to the IRG region of the chromosome. Interestingly, a major dominant locus controlling resistance to T. gondii has recently been mapped to a small region telomeric to the IRG genes on rat chromosome 12 which is syntenic to mouse chromosome 11 [62]. The list of known genes in that region does not immediately suggest an obvious candidate. The only locus in the relevant region that suggests a possible connection with T. gondii infection is that encoding the actinbinding protein, profilin. The profilin homologue of T. gondii is an important immune activator through TLR11 [63], but there is no obvious argument connecting this fact with the sequence or expression of the profilin of the host. Not only have there been few attempts to dissect the genetic variation in response to T. gondii in the mouse, but surprisingly these attempts have not been soundly focused. Even when a potential resistance gene is identified, this has not, except in one case, been followed up in a search for polymorphic variation correlating with susceptibility and resistance. The exception was the study cited above which was stimulated by the idea that the bcg gene (Nramp1), identified as responsible for differential resistance to Mycobacteria in mice (see review in this Forum [64]), might also play a part in T. gondii resistance [61]. In the event, the analysis excluded a role for bcg in differential resistance to T. gondii. However considering the complexity of expression of IDO and its important role in T. gondii resistance in human cells, it would seem natural to look for polymorphic variation governing expression levels in different cell types. Even stronger would be the expectation that the IRG proteins will display genetic variation in expression and in sequence which will have an impact on T. gondii resistance. To our knowledge, no such study has been completed. Several years ago my laboratory sought and failed to find polymorphic variation in Irga6 (IIGP1) from a number of inbred mouse strains (Eva Glowalla and J.C. Howard, unpublished results). The Dietrich group, in their study of genetic control of Chlamydia resistance, were able to

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identify a promoter variation resulting in very low expression of Irgb10 and susceptibility to Chlamydia trachomatis [55]. We found no effect of the reduced expression of Irgb10 derived from the C3H/HeJ allele on T. gondii survival in IFNg-treated mouse embryonic fibroblasts derived from that analysis (unpublished results with Dr Joern Coers). But these preliminary negative results are only scratching the surface of the problem. It is now a straightforward matter to screen many individuals for polymorphic variation in the genes of interest: in the case of the IRG genes, perhaps 20 genes would be worth analyzing in 20 inbred strains and a similar number from wild mice. The IRG genes are evolving rapidly, as is already apparent from their complexity within the mouse and other mammalian orders [65], and from further preliminary analysis of IRG genes from two other Mus musculus subspecies, musculus and castaneus (J.C. Howard and Jing-Tao Li, unpublished). A systematic analysis of the pattern of evolution of these powerful resistance genes would be of considerable interest. 6. Summary: human versus mouse cell-autonomous immunity to T. gondii By essentially every test the immune resistance of mouse and man to T. gondii infection differs. A normal healthy mouse can be killed by a single live individual of a virulent strain of T. gondii, while the infection presents essentially no threat to a normal healthy human, except a baby in utero. Indeed the distinction between virulent (Type I) and avirulent (Type II and III) T. gondii strains, which dominates the literature on T. gondii genetics, plays little role in human infection. In cellular responses in vitro, cell-autonomous resistance in mouse cells is strongly associated with the IFNg-inducible IRG GTPases while in man the main cell-autonomous resistance mechanism is IDO. This is not just a matter of emphasis. The IFNg-inducible IRG GTPases have all been lost from the genome of all the higher primates (Bekpen and unpublished results) and there is no evidence that the residual fragment IRGM, is involved in T. gondii immunity. Similarly, cells showing IFNg-inducible resistance to T. gondii in human all produce high levels of IDO, while almost no mouse cell line studied so far produces any. Activation of TLR11 by T. gondii plays an important role in the development of early resistance in mice, but there is no expressed homologue to TLR11 in man [66]. Just as is being asked in the Chlamydia system, one must ask to what extent one can still consider the mouse as a meaningful model for human T. gondii infection. Nevertheless, the study of the host-pathogen relationship between T. gondii and one of its natural mammalian hosts remains a fascinating study in its own right that will throw new light on the evolution of immune mechanisms active against intracellular pathogens. Furthermore, the properties of T. gondii as a human pathogen are determined by its interactions with its natural hosts, yet we still know embarrassingly little about these. As a striking example, the ROP2 family kinases that were recently identified as key factors determining differential virulence between T. gondii types in the mouse [15,67], are unknown in the closely-related Plasmodium genome [68].

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It seems certain that these proteins evolved as part of the intimate antagonistic relationship between T. gondii and the immune resistance repertoire of the mouse and other small mammals that are prey for cats.

Acknowledgments We are indebted to many members of this laboratory for stimulating discussions of many of the issues raised here, to Joern Coers (Microbiology, Harvard) for the gift of several genotypes of mouse embryonic fibroblasts and the P2 subline of L929 cells, as well as for many valuable insights, and to Annabelle Schnaith, Martin Kro¨nke and Nobura Mizushima for access to Atg5-deficient and control fibroblasts. We are particularly grateful to Julia Hunn for help in preparing the figures and for her critical reading of the manuscript. We acknowledge funding support from Collaborative Research Centres SFB635, SFB670 and SFB680 from the German Research Foundation (DFG).

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