Gregory A. Taylor

Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages.

Mycobacterium tuberculosis is an intracellular pathogen persisting within phagosomes through interference with phagolysosome biogenesis. Here we show that stimulation of autophagic pathways in macrophages causes mycobacterial phagosomes to mature into phagolysosomes. Physiological induction of autophagy or its pharmacological stimulation by rapamycin resulted in mycobacterial phagosome colocalization with the autophagy effector LC3, an elongation factor in autophagosome formation. Autophagy stimulation increased phagosomal colocalization with Beclin-1, a subunit of the phosphatidylinositol 3-kinase hVPS34, necessary for autophagy and a target for mycobacterial phagosome maturation arrest. Induction of autophagy suppressed intracellular survival of mycobacteria. IFN-gamma induced autophagy in macrophages, and so did transfection with LRG-47, an effector of IFN-gamma required for antimycobacterial action. These findings demonstrate that autophagic pathways can overcome the trafficking block imposed by M. tuberculosis. Autophagy, which is a hormonally, developmentally, and, as shown here, immunologically regulated process, represents an underappreciated innate defense mechanism for control of intracellular pathogens.

Human IRGM Induces Autophagy to Eliminate Intracellular Mycobacteria

Immunity-related p47 guanosine triphosphatases (IRG) play a role in defense against intracellular pathogens. We found that the murine Irgm1 (LRG-47) guanosine triphosphatase induced autophagy and generated large autolysosomal organelles as a mechanism for the elimination of intracellular Mycobacterium tuberculosis. We also identified a function for a human IRG protein in the control of intracellular pathogens and report that the human Irgm1 ortholog, IRGM, plays a role in autophagy and in the reduction of intracellular bacillary load.

A Pathogenetic Role for TNFα in the Syndrome of Cachexia, Arthritis, and Autoimmunity Resulting from Tristetraprolin (TTP) Deficiency

Tristetraprolin (TTP) is a widely expressed potential transcription factor that contains two unusual CCCH zinc fingers and is encoded by the immediate-early response gene, Zfp-36. Mice made deficient in TTP by gene targeting appeared normal at birth, but soon manifested marked medullary and extramedullary myeloid hyperplasia associated with cachexia, erosive arthritis, dermatitis, conjunctivitis, glomerular mesangial thickening, and high titers of anti-DNA and antinuclear antibodies. Myeloid progenitors from these mice showed no increase in sensitivity to growth factors. Treatment of young TTP-deficient mice with antibodies to tumor necrosis factor alpha (TNF alpha) prevented the development of essentially all aspects of the phenotype. These results indicate a role for TTP in regulating TNF alpha synthesis, secretion, turnover, or action. TTP-deficient mice may serve as useful models of the autoimmune inflammatory state resulting from chronic effective TNF alpha excess.

P47 GTPASES: REGULATORS OF IMMUNITY TO INTRACELLULAR PATHOGENS

Activation of the innate immune system by interferon-γ(IFN-γ) is crucial for host resistance to infection. IFN-γ induces the expression of a wide range of mediators that undermine the ability of pathogens to survive in host cells, including a newly discovered family of 47-kDa GTPases. Elimination of different p47 GTPases in mice by gene targeting severely cripples IFN-γ-regulated defence against Toxoplasma gondii, Listeria monocytogenes, Mycobacterium spp. and other pathogens. In this article, we review our understanding of the role of p47 GTPases in resistance to intracellular infection and discuss the present evidence concerning their mode of action.

Degradation of the Met tyrosine kinase receptor by the ubiquitin-proteasome pathway.

The Met tyrosine kinase receptor is a widely expressed molecule which mediates pleiotropic cellular responses following activation by its ligand, hepatocyte growth factor/scatter factor (HGF/SF). In this communication we demonstrate that significant Met degradation is induced by HGF/SF and that this degradation can be blocked by lactacystin, an inhibitor of proteasome activity. We also show that Met is rapidly polyubiquitinated in response to ligand and that polyubiquitinated Met molecules, which are normally unstable, are stabilized by lactacystin. Both HGF/SF-induced degradation and polyubiquitination of Met were shown to be dependent on the receptor possessing intact tyrosine kinase activity. Finally, we found that a normally highly labile 55-kDa fragment of the Met receptor is stabilized by lactacystin and demonstrate that it represents a cell-associated remnant that is generated following the ligand-independent proteolytic cleavage of the Met receptor in its extracellular domain. This truncated Met molecule encompasses the kinase domain of the receptor and is itself tyrosine phosphorylated. We conclude that the ubiquitin-proteasome pathway plays a significant role in the degradation of the Met tyrosine kinase receptor as directed by ligand-dependent and -independent signals. We propose that this proteolytic pathway may be important for averting cellular transformation by desensitizing Met signaling following ligand stimulation and by eliminating potentially oncogenic fragments generated via extracellular cleavage of the Met receptor.

The von Hippel-Lindau Tumor Suppressor Gene Inhibits Hepatocyte Growth Factor/Scatter Factor-Induced Invasion and Branching Morphogenesis in Renal Carcinoma Cells

ABSTRACT Loss of function in the von Hippel-Lindau (VHL) tumor suppressor gene occurs in familial and most sporadic renal cell carcinomas (RCCs). VHL has been linked to the regulation of cell cycle cessation (G0) and to control of expression of various mRNAs such as for vascular endothelial growth factor. RCC cells express the Met receptor tyrosine kinase, and Met mediates invasion and branching morphogenesis in many cell types in response to hepatocyte growth factor/scatter factor (HGF/SF). We examined the HGF/SF responsiveness of RCC cells containing endogenous mutated (mut) forms of the VHL protein (VHL-negative RCC) with that of isogenic cells expressing exogenous wild-type (wt) VHL (VHL-positive RCC). We found that VHL-negative 786-0 and UOK-101 RCC cells were highly invasive through growth factor-reduced (GFR) Matrigel-coated filters and exhibited an extensive branching morphogenesis phenotype in response to HGF/SF in the three-dimensional (3D) GFR Matrigel cultures. In contrast, the phenotypes of A498 VHL-negative RCC cells were weaker, and isogenic RCC cells ectopically expressing wt VHL did not respond at all. We found that all VHL-negative RCC cells expressed reduced levels of tissue inhibitor of metalloproteinase 2 (TIMP-2) relative to the wt VHL-positive cells, implicating VHL in the regulation of this molecule. However, consistent with the more invasive phenotype of the 786-0 and UOK-101 VHL-negative RCC cells, the levels of TIMP-1 and TIMP-2 were reduced and levels of the matrix metalloproteinases 2 and 9 were elevated compared to the noninvasive VHL-positive RCC cells. Moreover, recombinant TIMPs completely blocked HGF/SF-mediated branching morphogenesis, while neutralizing antibodies to the TIMPs stimulated HGF/SF-mediated invasion in vitro. Thus, the loss of the VHL tumor suppressor gene is central to changes that control tissue invasiveness, and a more invasive phenotype requires additional genetic changes seen in some but not all RCC lines. These studies also demonstrate a synergy between the loss of VHL function and Met signaling.

Gamma interferon-induced inhibition of Toxoplasma gondii in astrocytes is mediated by IGTP.

ABSTRACT Toxoplasma gondii is an important pathogen in the central nervous system, causing a severe and often fatal encephalitis in patients with AIDS. Gamma interferon (IFN-γ) is the main cytokine preventing reactivation of Toxoplasma encephalitis in the brain. Microglia are important IFN-γ-activated effector cells controlling the growth of T. gondii in the brain via a nitric oxide (NO)-mediated mechanism. IFN-γ can also activate astrocytes to inhibit the growth of T. gondii. Previous studies found that the mechanism in murine astrocytes is independent of NO and all other known anti-Toxoplasma mechanisms. In this study we investigated the role of IGTP, a recently identified IFN-γ-regulated gene, in IFN-γ inhibition of T. gondii in murine astrocytes. Primary astrocytes were cultivated from IGTP-deficient mice, treated with IFN-γ, and then tested for anti-Toxoplasma activity. In wild-type astrocytesT. gondii growth was significantly inhibited by IFN-γ, whereas in astrocytes from IGTP-deficient mice IFN-γ did not cause a significant inhibition of growth. Immunoblot analysis confirmed that IFN-γ induced significant levels of IGTP in wild-type murine astrocytes within 24 h. These results indicate that IGTP plays a central role in the IFN-γ-induced inhibition of T. gondii in murine astrocytes.

p47 GTPases regulate Toxoplasma gondii survival in activated macrophages.

ABSTRACT The cytokine gamma interferon (IFN-γ) is critical for resistance to Toxoplasma gondii. IFN-γ strongly activates macrophages and nonphagocytic host cells to limit intracellular growth of T. gondii; however, the cellular factors that are required for this effect are largely unknown. We have shown previously that IGTP and LRG-47, members of the IFN-γ-regulated family of p47 GTPases, are required for resistance to acute T. gondii infections in vivo. In contrast, IRG-47, another member of this family, is not required. In the present work, we addressed whether these GTPases are required for IFN-γ-induced suppression of T. gondii growth in macrophages in vitro. Bone marrow macrophages that lacked IGTP or LRG-47 displayed greatly attenuated IFN-γ-induced inhibition of T. gondii growth, while macrophages that lacked IRG-47 displayed normal inhibition. Thus, the ability of the p47 GTPases to limit acute infection in vivo correlated with their ability to suppress intracellular growth in macrophages in vitro. Using confocal microscopy and sucrose density fractionation, we demonstrated that IGTP largely colocalizes with endoplasmic reticulum markers, while LRG-47 was mainly restricted to the Golgi. Although both IGTP and LRG-47 localized to vacuoles containing latex beads, neither protein localized to vacuoles containing live T. gondii. These results suggest that IGTP and LRG-47 are able to regulate host resistance to acute T. gondii infections through their ability to inhibit parasite growth within the macrophage.

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

Although IFN-γ 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-γ-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-γ-dependent manner. LRG-47−/− mice were unable to control bacterial replication, but survived the acute phase, succumbing 11–16 wk postinfection. IFN-γ-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-γ 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-γ response to persistent infection, LRG-47 can have downstream regulatory effects on lymphocyte survival.

Identification of a Novel GTPase, the Inducibly Expressed GTPase, That Accumulates in Response to Interferon γ

Interferon γ is a pleiotropic cytokine that regulates many immune functions. We have identified a novel protein, inducibly expressed GTPase (IGTP), whose expression was regulated by interferon γ in macrophages. In mouse RAW 264.7 macrophages, IGTP mRNA levels were almost undetectable but increased within 1 h of exposure to interferon γ, peaked at very high levels within 3 h, and remained at high levels to at least 48 h; pretreatment of the cells with cycloheximide blocked the majority of mRNA accumulation. In the mouse, the mRNA was highly expressed in thymus, spleen, lung, and small intestine. Using interspecific backcross analysis, the Igtp gene was mapped to mouse chromosome 11. The IGTP cDNA encoded a putative polypeptide of Mr 48,507 and pI 7.79 that contained three consensus GTP binding motifs, GXXXXGK(S/T), DXXG, and NTKXD. Both IGTP that had been immunoprecipitated from RAW cells and a glutathione S-transferase IGTP fusion protein were able to convert GTP to GDP in vitro. Subcellular protein fractionation and Western blotting localized IGTP to the cytosol of RAW cells. In addition, the protein was homologous to proteins encoded by three previously cloned cDNAs, IRG-47, TGTP/Mg21, and LRG-47, and thus may be representative of a new family of interferon γ-regulated GTPases.