Control of IFN-gamma-mediated host resistance to intracellular pathogens by immunity-related GTPases (p47 GTPases)

IRG proteins (also known as p47 GTPases) are key mediators of interferon-gamma-induced resistance to pathogens. Absence of certain IRG proteins leads to profound susceptibility to protozoa and bacteria in mice. Underlying their roles in host resistance, IRG proteins regulate the processing of pathogen-containing vacuoles in host cells, and regulate hematopoiesis following infection.     

Control of IFN-γ-mediated host resistance to intracellular pathogens by immunity-related GTPases (p47 GTPases)

IRG proteins (also known as p47 GTPases) are key mediators of interferon-γ-induced resistance to pathogens. Absence of certain IRG proteins leads to profound susceptibility to protozoa and bacteria in mice. Underlying their roles in host resistance, IRG proteins regulate the processing of pathogen-containing vacuoles in host cells, and regulate hematopoiesis following infection.     

Impaired macrophage function underscores susceptibility to Salmonella in mice lacking Irgm1 (LRG-47)

IRG proteins, or immunity-related GTPases (also known as p47 GTPases), are a group of IFN-regulated proteins that are highly expressed in response to infection. The proteins localize to intracellular membranes including vacuoles that contain pathogens in infected macrophages and other host cells. Current data indicate that the IRG protein Irgm1 (LRG-47) is critical for resistance to intracellular bacteria. This function is thought to be a consequence of regulating the survival of vacuolar bacteria in host cells. In the current work, the role of Irgm1 in controlling resistance to Salmonella typhimurium was explored to further define the mechanism through which the protein regulates host resistance. Irgm1-deficient mice displayed increased susceptibility to this bacterium that was reflected in increased bacterial loads in spleen and liver and decreased maturation of S. typhimurium granulomas. The mice also displayed an inability to concentrate macrophages at sites of bacterial deposition. In vitro, the ability of Irgm1-deficient macrophages to suppress intracellular growth of S. typhimurium was impaired. Furthermore, adhesion and motility of Irgm1-deficient macrophages after activation with IFN-gamma was markedly decreased. Altered adhesion/motility of those cells was accompanied by changes in cell morphology, density of adhesion-associated proteins, and actin staining. Together, these data suggest that in addition to regulating the maturation of pathogen-containing vacuoles, Irgm1 plays a key role in regulating the adhesion and motility of activated macrophages.     

Mice deficient in LRG-47 display enhanced susceptibility to Trypanosoma cruzi infection associated with defective hemopoiesis and intracellular control of parasite growth

IFN-gamma is known to be required for host control of intracellular Trypanosoma cruzi infection in mice, although the basis of its protective function is poorly understood. LRG-47 is an IFN-inducible p47GTPase that has been shown to regulate host resistance to intracellular pathogens. To investigate the possible role of LRG-47 in IFN-gamma-dependent control of T. cruzi infection, LRG-47 knockout (KO) and wild-type (WT) mice were infected with the Y strain of this parasite, and host responses were analyzed. When assayed on day 12 after parasite inoculation, LRG-47 KO mice, in contrast to IFN-gamma KO mice, controlled early parasitemia almost as effectively as WT animals. However, the infected LRG-47 KO mice displayed a rebound in parasite growth on day 15, and all succumbed to the infection by day 19. Additional analysis indicated that LRG-47-deficient mice exhibit unimpaired proinflammatory responses throughout the infection. Instead, reactivated disease in the KO animals was associated with severe splenic and thymic atrophy, anemia, and thrombocytopenia not observed in their WT counterparts. In addition, in vitro studies revealed that IFN-gamma-stimulated LRG-47 KO macrophages display defective intracellular killing of amastigotes despite normal expression of TNF and NO synthetase type 2 and that both NO synthetase type 2 and LRG-47 are required for optimum IFN-gamma-dependent restriction of parasite growth. Together, these data establish that LRG-47 can influence pathogen control by simultaneously regulating macrophage-microbicidal activity and hemopoietic function.     

The interferon-inducible p47 (IRG) GTPases in vertebrates: loss of the cell autonomous resistance mechanism in the human lineage

Background  

Members of the p47 (immunity-related GTPases (IRG) family) GTPases are essential, interferon-inducible resistance factors in mice that are active against a broad spectrum of important intracellular pathogens. Surprisingly, there are no reports of p47 function in humans.     

Mice deficient in LRG-47 display increased susceptibility to mycobacterial infection associated with the induction of lymphopenia

Although IFN-gamma is essential for host control of mycobacterial infection, the mechanisms by which the cytokine restricts pathogen growth are only partially understood. LRG-47 is an IFN-inducible GTP-binding protein previously shown to be required for IFN-gamma-dependent host resistance to acute Listeria monocytogenes and Toxoplasma gondii infections. To examine the role of LRG-47 in control of mycobacterial infection, LRG-47(-/-) and wild-type mice were infected with Mycobacterium avium, and host responses were analyzed. LRG-47 protein was strongly induced in livers of infected wild-type animals in an IFN-gamma-dependent manner. LRG-47(-/-) mice were unable to control bacterial replication, but survived the acute phase, succumbing 11-16 wk postinfection. IFN-gamma-primed, bone marrow-derived macrophages from LRG-47(-/-) and wild-type animals produced equivalent levels of TNF and NO upon M. avium infection in vitro and developed similar intracellular bacterial loads. In addition, priming for IFN-gamma production was observed in T cells isolated from infected LRG-47(-/-) mice. Importantly, however, mycobacterial granulomas in LRG-47(-/-) mice showed a marked lymphocyte deficiency. Further examination of these animals revealed a profound systemic lymphopenia and anemia triggered by infection. As LRG47(-/-) T lymphocytes were found to both survive and confer resistance to M. avium in recipient recombinase-activating gene-2(-/-) mice, the defect in cellular response and bacterial control in LRG-47(-/-) mice may also depend on a factor(s) expressed in a nonlymphocyte compartment. These findings establish a role for LRG-47 in host control of mycobacteria and demonstrate that in the context of the IFN-gamma response to persistent infection, LRG-47 can have downstream regulatory effects on lymphocyte survival.     

IL-18 contributes to host resistance against infection with Cryptococcus neoformans in mice with defective IL-12 synthesis through induction of IFN-gamma production by NK cells

The aim of this study was to examine the contribution of IL-18 in host defense against infection caused by Cryptococcus neoformans in mice with defective IL-12 production. Experiments were conducted in mice with a targeted disruption of the gene for IL-12p40 subunit (IL-12p40-/- mice). In these mice, host resistance was impaired, as shown by increased number of organisms in both lungs and brains, compared with control mice. Serum IFN-gamma was still detected in these mice at a considerable level (20-30% of that in control mice). The host resistance was moderately impaired in IL-12p40-/- mice compared with IFN-gamma-/- mice. Neutralizing anti-IFN-gamma mAb further increased the lung burdens of organisms. In addition, treatment with neutralizing anti-IL-18 Ab almost completely abrogated the production of IFN-gamma and also impaired the host resistance. Host resistance in IL-12p40-/- IL-18-/- mice was more profoundly impaired than in IL-12p40-/- mice. Administration of IL-12 as well as IL-18 increased the serum levels of IFN-gamma and significantly restored the reduced host resistance. Spleen cells obtained from infected IL-12p40-/- mice did not produce any IFN-gamma upon restimulation with the same organisms, while those from infected and IL-12-treated mice produced IFN-gamma. In contrast, IL-18 did not show such effect. Finally, depletion of NK cells by anti-asialo GM1 Ab mostly abrogated the residual production of IFN-gamma in IL-12p40-/- mice. Our results indicate that IL-18 contributes to host resistance to cryptococcal infection through the induction of IFN-gamma production by NK cells, but not through the development of Th1 cells, under the condition in which IL-12 synthesis is deficient.     

Chlamydia muridarum evades growth restriction by the IFN-gamma-inducible host resistance factor Irgb10

Chlamydiae are obligate intracellular bacterial pathogens that exhibit a broad range of host tropism. Differences in host tropism between Chlamydia species have been linked to host variations in IFN-gamma-mediated immune responses. In mouse cells, IFN-gamma can effectively restrict growth of the human pathogen Chlamydia trachomatis but fails to control growth of the closely related mouse pathogen Chlamydia muridarum. The ability of mouse cells to resist C. trachomatis replication is largely dependent on the induction of a family of IFN-gamma-inducible GTPases called immunity-related GTPases or IRGs. In this study we demonstrate that C. muridarum can specifically evade IRG-mediated host resistance. It has previously been suggested that C. muridarum inactivates the IRG protein Irga6 (Iigp1) to dampen the murine immune response. However, we show that Irga6 is dispensable for the control of C. trachomatis replication. Instead, an effective IFN-gamma response to C. trachomatis requires the IRG proteins Irgm1 (Lrg47), Irgm3 (Igtp), and Irgb10. Ectopic expression of Irgb10 in the absence of IFN-gamma is sufficient to reduce intracellular growth of C. trachomatis but fails to restrict growth of C. muridarum, indicating that C. muridarum can specifically evade Irgb10-driven host responses. Importantly, we find that Irgb10 protein intimately associates with inclusions harboring C. trachomatis but is absent from inclusions formed by C. muridarum. These data suggest that C. muridarum has evolved a mechanism to escape the murine IFN-gamma response by restricting access of Irgb10 and possibly other IRG proteins to the inclusion.     

Autophagic elimination of intracellular parasites: convergent induction by IFN-gamma and CD40 ligation?

Autophagy has recently been implicated in the immune elimination of the intracellular protozoan parasite, Toxoplasma gondii. Toxoplasma and other apicomplexan parasites actively invade host cells and form nonfusogenic parasitophorous vacuoles. Nevertheless, following entry into IFN-gamma-activated effector macrophages, vesiculation of the parasite vacuole or PV membrane ensues, in a process dependent upon the activity of p47 GTPases induced by IFN-gamma signaling. Subsequent disruption of the plasma membrane of the stripped parasites precedes autophagolysosomal elimination of T. gondii. In contrast, ligation of the CD40 receptor and autocrine signaling by TNF activate a seemingly distinct, p47 GTPase-independent mechanism leading to autophagic elimination of intracellular T. gondii, without prior disruption of the pathogen vacuole. Thus, two key pathways of the cell-mediated immune response, namely IFN-gamma and CD40/CD40L, trigger a common autophagolysosomal endpoint of parasite elimination, via distinct intermediary mechanisms.     

Mechanisms regulating the positioning of mouse p47 resistance GTPases LRG-47 and IIGP1 on cellular membranes: retargeting to plasma membrane induced by phagocytosis

The recently identified p47 GTPases are one of the most effective cell-autonomous resistance systems known against intracellular pathogens in the mouse. One member of the family, LRG-47, has been shown to be essential for immune control in vivo of Listeria monocytogenes, Toxoplasma gondii, Mycobacterium tuberculosis, and Mycobacterium avium, possibly by promoting acidification of the phagosome. However, the intracellular localization of LRG-47, and the nature of its association with the phagosomal or any other membrane system is unknown. In this study, we show that LRG-47 is a Golgi-associated protein in the IFN-stimulated cell, which is rapidly recruited to active plasma membrane upon phagocytosis and remains associated with phagosomes as they mature. We show that the Golgi localization of LRG-47 is dependent on the integrity of an amphipathic helix near the C terminus, whereas the plasma membrane localization depends on an unidentified signal associated with the G domain. Unlike LRG-47, but like the published p47 resistance GTPase, IGTP, a further p47 GTPase, IIGP1, is associated with the endoplasmic reticulum. However, unlike IGTP, IIGP1 is associated with the endoplasmic reticulum by an N-terminal myristoylation modification. Thus, the p47 GTPases are a diverse battery of intracellular defense factors dynamically associated with different membrane systems.     

NK cells respond to pulmonary infection with Mycobacterium tuberculosis, but play a minimal role in protection

Both innate and adaptive immune systems contribute to host defense against infection with Mycobacterium tuberculosis. NK cells have been associated with early resistance against intracellular pathogens and are known to be potent producers of the cytokine IFN-gamma. In C57BL/6 mice infected by aerosol exposure with M. tuberculosis, NK cells increased in the lungs over the first 21 days of infection. Expansion of the NK cell subset was associated with increased expression of activation and maturation markers. In addition, NK cells isolated from the infected lungs were capable of producing IFN-gamma and became positive for perforin. In vivo depletion of NK cells using a lytic Ab had no influence on bacterial load within the lungs. These findings indicate that NK cells can become activated during the early response to pulmonary tuberculosis in the mouse model and are a source of IFN-gamma, but their removal does not substantially alter the expression of host resistance.     

Crystal structure of IIGP1: a paradigm for interferon-inducible p47 resistance GTPases

Interferon-inducible p47 GTPases are critical mediators of cell-autonomous resistance against several intracellular pathogens. Here we present the first crystal structure of a member of this novel GTPase family, IIGP1, in its nucleotide-free, GDP-, and GppNHp-bound form. The structure shows a Ras-like G domain between an N-terminal three-helix bundle and a complex system of C-terminal helices and loops. Sequence comparison and secondary structure prediction suggest the IIGP1 structure to be a valid model for the p47 GTPase family. The IIGP1 crystals contain a noncrystallographic dimer. We show that the dimer is required for cooperative GTP hydrolysis and GTP-dependent oligomerization of IIGP1. We also present the GDP- and GppNHp-bound monomeric structures of two dimer interface mutants. Our structures direct approaches to the analysis of the catalytic mechanism of IIGP1 and provide a coherent basis for structure-function studies aimed at elucidating the mechanistic basis of pathogen resistance caused by these enigmatic GTPases.     

TLR adaptor MyD88 is essential for pathogen control during oral toxoplasma gondii infection but not adaptive immunity induced by a vaccine strain of the parasite

TLR adaptor MyD88 activation is important in host resistance to Toxoplasma gondii during i.p. infection, but the function of this signaling pathway during oral infection, in which mucosal immunity assumes a predominant role, has not been examined. In this study, we show that MyD88(-/-) mice fail to control the parasite and succumb within 2 wk of oral infection. Early during infection, T cell IFN-gamma production, recruitment of neutrophils and induction of p47 GTPase IGTP (Irgm3) in the intestinal mucosa were dependent upon functional MyD88. Unexpectedly, these responses were MyD88-independent later during acute infection. In particular, CD4(+) T cell IFN-gamma reached normal levels independently of MyD88, despite continued absence of IL-12 in these animals. The i.p. vaccination of MyD88(-/-) mice with an avirulent T. gondii uracil auxotroph elicited robust IFN-gamma responses and protective immunity to challenge with a high virulence T. gondii strain. Our results demonstrate that MyD88 is required to control Toxoplasma infection, but that the parasite can trigger adaptive immunity without the need for this TLR adaptor molecule.     

CD40-TRAF6 and autophagy-dependent anti-microbial activity in macrophages

A fundamental question in host-pathogen interaction is to determine if the immune system activates fusion with the lysosomes to eradicate pathogens. We recently reported that this task is accomplished by the interaction between CD40 expressed on macrophages and CD154 expressed on activated CD4+ T cells. CD40 stimulation of macrophages induces vacuole-lysosome fusion through autophagy and results in killing of the obligate intracellular pathogen Toxoplasma gondii. This response is independent of IFN-gamma, STAT1 and p47 GTPases. We now report that vacuole-lysosome fusion is dependent on synergy between TRAF6 signaling downstream of CD40 and TNF-alpha. These studies identified a new paradigm by which T cells eradicate an intracellular pathogen within macrophages.     

Manipulation of rab GTPase function by intracellular bacterial pathogens.

Intracellular bacterial pathogens have evolved highly specialized mechanisms to enter and survive within their eukaryotic hosts. In order to do this, bacterial pathogens need to avoid host cell degradation and obtain nutrients and biosynthetic precursors, as well as evade detection by the host immune system. To create an intracellular niche that is favorable for replication, some intracellular pathogens inhibit the maturation of the phagosome or exit the endocytic pathway by modifying the identity of their phagosome through the exploitation of host cell trafficking pathways. In eukaryotic cells, organelle identity is determined, in part, by the composition of active Rab GTPases on the membranes of each organelle. This review describes our current understanding of how selected bacterial pathogens regulate host trafficking pathways by the selective inclusion or retention of Rab GTPases on membranes of the vacuoles that they occupy in host cells during infection.     

The IFN-inducible Golgi- and endoplasmic reticulum- associated 47-kDa GTPase IIGP is transiently expressed during listeriosis

Members of the 47-kDa GTPase family are implicated in an IFN-gamma-induced, as yet unclear, mechanism that confers innate resistance against infection with intracellular pathogens. Overt immunological parameters are apparently uncompromised in mice deficient for individual members and the prototype of this family, IGTP, localizes to the endoplasmic reticulum. This suggests that these GTPases are involved in intracellular defense. We analyzed the expression of the 47-kDa GTPase cognate, IIGP, in splenic sections from mice infected with the intracellular pathogen Listeria monocytogenes by immunohistochemistry. An early transient IIGP induction was observed revealing the IFN-gamma responsiveness of cellular subcompartments within the spleen in early listeriosis. Marginal metallophilic macrophages and endothelial cells within the red and white pulp strongly expressed IIGP, while other splenocytes remained negative. In vitro analyses show that both type I and type II IFNs are prime stimuli for IIGP induction in various cells, including L. monocytogenes-infected or LPS-stimulated macrophages, endothelial cells, and activated T cells. Contrary to the subcellular localization of IGTP, IIGP was predominantly associated with the Golgi apparatus and also localizes to the endoplasmic reticulum. We conclude that IIGP exerts a distinct role in IFN-induced intracellular membrane trafficking or processing.     

The role of gamma interferon in antimicrobial immunity

Gamma interferon (IFN-gamma) is an important cytokine in the host defense against infection by viral and microbial pathogens. IFN-gamma induces a variety of physiologically significant responses that contribute to immunity. Treatment of animal cells with IFN-gamma or infection with viral or microbial pathogens leads to changes in the level of expression of several target genes as revealed by DNA microarray analyses. The signaling pathways leading to the induction of IFN-gamma-regulated gene products and, in some cases, their biochemical functions have been defined in exquisite detail. Studies of transgenic mutant mice deficient in proteins of the IFN-gamma response pathway firmly establish the importance of IFN-gamma in immunity.     

The host resistance locus sst1 controls innate immunity to Listeria monocytogenes infection in immunodeficient mice

Epidemiological, clinical, and experimental approaches have convincingly demonstrated that host resistance to infection with intracellular pathogens is significantly influenced by genetic polymorphisms. Using a mouse model of infection with virulent Mycobacterium tuberculosis (MTB), we have previously identified the sst1 locus as a genetic determinant of host resistance to tuberculosis. In this study we demonstrate that susceptibility to another intracellular pathogen, Listeria monocytogenes, is also influenced by the sst1 locus. The contribution of sst1 to anti-listerial immunity is much greater in immunodeficient scid mice, indicating that this locus controls innate immunity and becomes particularly important when adaptive immunity is significantly depressed. Similar to our previous observations using infection with MTB, the resistant allele of sst1 prevents formation of necrotic infectious lesions in vivo. We have shown that macrophages obtained from sst1-resistant congenic mice possess superior ability to kill L. monocytogenes in vitro. The bactericidal effect of sst1 is dependent on IFN-gamma activation and reactive oxygen radical production by activated macrophages after infection, but is independent of NO production. It is possible that there is a single gene that controls common IFN-dependent macrophage function, which is important in the pathogenesis of infections caused by both MTB and L. monocytogenes. However, host resistance to the two pathogens may be controlled by two different polymorphic genes encoded within the sst1 locus. The polymorphic gene(s) encoded within the sst1 locus that controls macrophage interactions with the two intracellular pathogens remains to be elucidated.