Jens Zerrahn

Disruption of Toxoplasma gondii Parasitophorous Vacuoles by the Mouse p47-Resistance GTPases

The p47 GTPases are essential for interferon-γ-induced cell-autonomous immunity against the protozoan parasite, Toxoplasma gondii, in mice, but the mechanism of resistance is poorly understood. We show that the p47 GTPases, including IIGP1, accumulate at vacuoles containing T. gondii. The accumulation is GTP-dependent and requires live parasites. Vacuolar IIGP1 accumulations undergo a maturation-like process accompanied by vesiculation of the parasitophorous vacuole membrane. This culminates in disruption of the parasitophorous vacuole and finally of the parasite itself. Over-expression of IIGP1 leads to accelerated vacuolar disruption whereas a dominant negative form of IIGP1 interferes with interferon-γ-mediated killing of intracellular parasites. Targeted deletion of the IIGP1 gene results in partial loss of the IFN-γ-mediated T. gondii growth restriction in mouse astrocytes.

Streptococcus pneumoniae-induced p38 MAPK-dependent Phosphorylation of RelA at the Interleukin-8 Promotor

Streptococcus pneumoniae is the major cause of community-acquired pneumonia and one of the most common causes of death by infectious disease in industrialized countries. Little is known concerning the mechanisms of target cell activation in this disease. The present study shows that NF-κB and p38 MAPK signaling pathways contribute to chemokine synthesis by lung epithelial cells in response to pneumococci. In infected lungs of mice pneumococci stimulate expression of the interleukin (IL)-8 homolog keratinocyte-derived chemokine and granulocyte-macrophage colony-stimulating factor, as well as activate p38 MAPK. Human bronchial epithelium was chosen as a cellular model, because it establishes the first barrier against pathogens, and little is known about its function in innate immunity. Pneumococci infection induces expression of IL-8 and granulocyte-macrophage colony-stimulating factor as well as activation of p38 MAPK in human bronchial epithelial cells (BEAS-2B). Inhibition of p38 MAPK activity by SB202190 and SB203580 blocks pneumococci-induced cytokine release. In mouse lungs in vivo as well as in cultured cells, pneumococci activate NF-κBinanIκB kinase-dependent manner. Inhibition of p38 MAPK by chemical inhibitors or by RNA interference targeting p38α reduces pneumococci-induced NF-κB-dependent gene transcription. Blockade of p38 activity did not affect inducible nuclear translocation and recruitment of NF-κB/RelA to the IL-8 promotor but did reduce the level of phosphorylated RelA (serine 536) at IL-8 promotor and inhibited pneumococci-mediated recruitment of RNA polymerase II to IL-8 promotor. Thus, p38 MAPK contributes to pneumococci-induced chemokine transcription by modulating p65 NF-κB-mediated transactivation.

Regulatory interactions between IRG resistance GTPases in the cellular response to Toxoplasma gondii

Members of the immunity‐related GTPase (IRG) family are interferon‐inducible resistance factors against a broad spectrum of intracellular pathogens including Toxoplasma gondii. The molecular mechanisms governing the function and regulation of the IRG resistance system are largely unknown. We find that IRG proteins function in a system of direct, nucleotide‐dependent regulatory interactions between family members. After interferon induction but before infection, the three members of the GMS subfamily of IRG proteins, Irgm1, Irgm2 and Irgm3, which possess an atypical nucleotide‐binding site, regulate the intracellular positioning of the conventional GKS subfamily members, Irga6 and Irgb6. Following infection, the normal accumulation of Irga6 protein at the parasitophorous vacuole membrane (PVM) is nucleotide dependent and also depends on the presence of all three GMS proteins. We present evidence that an essential role of the GMS proteins in this response is control of the nucleotide‐bound state of the GKS proteins, preventing their GTP‐dependent activation before infection. Accumulation of IRG proteins at the PVM has previously been shown to be associated with a block in pathogen replication: our results relate for the first time the enzymatic properties of IRG proteins to their role in pathogen resistance.

CXCL5-secreting pulmonary epithelial cells drive destructive neutrophilic inflammation in tuberculosis

Successful host defense against numerous pulmonary infections depends on bacterial clearance by polymorphonuclear leukocytes (PMNs); however, excessive PMN accumulation can result in life-threatening lung injury. Local expression of CXC chemokines is critical for PMN recruitment. The impact of chemokine-dependent PMN recruitment during pulmonary Mycobacterium tuberculosis infection is not fully understood. Here, we analyzed expression of genes encoding CXC chemokines in M. tuberculosis-infected murine lung tissue and found that M. tuberculosis infection promotes upregulation of Cxcr2 and its ligand Cxcl5. To determine the contribution of CXCL5 in pulmonary PMN recruitment, we generated Cxcl5(-/-) mice and analyzed their immune response against M. tuberculosis. Both Cxcr2(-/-) mice and Cxcl5(-/-) mice, which are deficient for only one of numerous CXCR2 ligands, exhibited enhanced survival compared with that of WT mice following high-dose M. tuberculosis infection. The resistance of Cxcl5(-/-) mice to M. tuberculosis infection was not due to heightened M. tuberculosis clearance but was the result of impaired PMN recruitment, which reduced pulmonary inflammation. Lung epithelial cells were the main source of CXCL5 upon M. tuberculosis infection, and secretion of CXCL5 was reduced by blocking TLR2 signaling. Together, our data indicate that TLR2-induced epithelial-derived CXCL5 is critical for PMN-driven destructive inflammation in pulmonary tuberculosis.

IFN-γ-Inducible Irga6 Mediates Host Resistance against Chlamydia trachomatis via Autophagy

Chlamydial infection of the host cell induces Gamma interferon (IFNγ), a central immunoprotector for humans and mice. The primary defense against Chlamydia infection in the mouse involves the IFNγ-inducible family of IRG proteins; however, the precise mechanisms mediating the pathogens elimination are unknown. In this study, we identify Irga6 as an important resistance factor against C. trachomatis, but not C. muridarum, infection in IFNγ-stimulated mouse embryonic fibroblasts (MEFs). We show that Irga6, Irgd, Irgm2 and Irgm3 accumulate at bacterial inclusions in MEFs upon stimulation with IFNγ, whereas Irgb6 colocalized in the presence or absence of the cytokine. This accumulation triggers a rerouting of bacterial inclusions to autophagosomes that subsequently fuse to lysosomes for elimination. Autophagy-deficient Atg5−/− MEFs and lysosomal acidification impaired cells surrender to infection. Irgm2, Irgm3 and Irgd still localize to inclusions in IFNγ-induced Atg5−/− cells, but Irga6 localization is disrupted indicating its pivotal role in pathogen resistance. Irga6-deficient (Irga6−/−) MEFs, in which chlamydial growth is enhanced, do not respond to IFNγ even though Irgb6, Irgd, Irgm2 and Irgm3 still localize to inclusions. Taken together, we identify Irga6 as a necessary factor in conferring host resistance by remodelling a classically nonfusogenic intracellular pathogen to stimulate fusion with autophagosomes, thereby rerouting the intruder to the lysosomal compartment for destruction.

Phenotypic characterization of CD8(+)NKT cells.

We describe a novel CD8+NKT cell population expressing TCRα /β or TCRγ /δ . These CD8+NKT cells were prominent in the liver, and except for the thymus, virtually absent in other lymphoid organs. CD8+NKT cells expressed activation markers and comprised a high proportion of Ly49+ cells. The development of the majority of CD8+NKT cells expressing TCRα /β, but not TCRγ /δ, depended on classical MHC class I. No CD8+NKT cells were detectable in young athymic mice, whereas the cells expressing TCRγ /δ, but not TCRα /β, appeared randomly in aged athymic mice. CD8+NK1+ TCRα /β cells showed polyclonal TCRVβ usage and were virtually devoid of TCRVα14. CD8+NK1+ TCRγ /δ cells predominantly expressed TCRVγ1, 2 and 4, and Vδ4, 5, 6 and 7. CD8+NKT cells, in particular those expressing TCRγ /δ, were a major population in early life. IFN‐γ, but not IL‐4, was induced in CD8+NKT cells by in vitro stimulation, independent of the TCRα /β or TCRγ /δ lineage. Hence, these cells represent a unique, though heterogeneous T cell population that shares markers with, but is distinct from, both conventional NKT cells and conventional CD8+ T cells, and that may play a role in immune regulation.

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-γ-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-γ 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 IFN-γ-Inducible GTPase, Irga6, Protects Mice against Toxoplasma gondii but Not against Plasmodium berghei and Some Other Intracellular Pathogens

Clearance of infection with intracellular pathogens in mice involves interferon-regulated GTPases of the IRG protein family. Experiments with mice genetically deficient in members of this family such as Irgm1(LRG-47), Irgm3(IGTP), and Irgd(IRG-47) has revealed a critical role in microbial clearance, especially for Toxoplasma gondii. The in vivo role of another member of this family, Irga6 (IIGP, IIGP1) has been studied in less detail. We investigated the susceptibility of two independently generated mouse strains deficient in Irga6 to in vivo infection with T. gondii, Mycobacterium tuberculosis, Leishmania mexicana, L. major, Listeria monocytogenes, Anaplasma phagocytophilum and Plasmodium berghei. Compared with wild-type mice, mice deficient in Irga6 showed increased susceptibility to oral and intraperitoneal infection with T. gondii but not to infection with the other organisms. Surprisingly, infection of Irga6-deficient mice with the related apicomplexan parasite, P. berghei, did not result in increased replication in the liver stage and no Irga6 (or any other IRG protein) was detected at the parasitophorous vacuole membrane in IFN-γ-induced wild-type cells infected with P. berghei in vitro. Susceptibility to infection with T. gondii was associated with increased mortality and reduced time to death, increased numbers of inflammatory foci in the brains and elevated parasite loads in brains of infected Irga6-deficient mice. In vitro, Irga6-deficient macrophages and fibroblasts stimulated with IFN-γ were defective in controlling parasite replication. Taken together, our results implicate Irga6 in the control of infection with T. gondii and further highlight the importance of the IRG system for resistance to this pathogen.

Inactive and Active States of the Interferon-inducible Resistance GTPase, Irga6, in Vivo

Irga6, a myristoylated, interferon-inducible member of the immunity-related GTPase family, contributes to disease resistance against Toxoplasma gondii in mice. Accumulation of Irga6 on the T. gondii parasitophorous vacuole membrane is associated with vesiculation and ultimately disruption of the vacuolar membrane in a process that requires an intact GTP-binding domain. The role of the GTP-binding domain of Irga6 in pathogen resistance is, however, unclear. We provide evidence that Irga6 in interferon-induced, uninfected cells is predominantly in a GDP-bound state that is maintained by other interferon-induced proteins. However, Irga6 that accumulates on the parasitophorous vacuole membrane after Toxoplasma infection is in the GTP-bound form. We demonstrate that a monoclonal antibody, 10D7, specifically detects GTP-bound Irga6, and we show that the formation of the 10D7 epitope follows from a GTP-dependent conformational transition of the N terminus of Irga6, anticipating an important role of the myristoyl group on Irga6 function in vivo.

IIGP, a member of the IFN inducible and microbial defense mediating 47 kDa GTPase family, interacts with the microtubule binding protein hook3

Innate immunity against intracellular pathogens is critically determined by an as yet unknown interferon (IFN)-inducible mechanism exerted by members of the 47 kDa GTPase family. The association of IGTP and IIGP with membranous compartments, the endoplasmic reticulum and, in addition in case of IIGP, the Golgi, implicate these GTPases in intracellular membrane trafficking or processing. We identified the cytoplasmic linker molecule hook3 as an interactor for IIGP by yeast two-hybrid screening. The physical complex between these molecules was present in lysates of IFNγ-stimulated macrophages as demonstrated by co-immunoprecipitation. Only a minor subfraction of total cellular IIGP or hook3 was co-purified, indicating that this interaction is either transient and/or involves distinct subpopulations of the total cellular pools of these molecules. Binding of IIGP to hook3 depends on a GTP-bound conformation. Hook3 is a microtubule-binding protein which participates in the organization of the cis-Golgi compartment. Both proteins were detected in the Golgi-membrane-enriched fraction upon subcellular fractionation. Apart from the Golgi localization of both proteins, hook3 was detected in perinuclear regions in close spatial proximity to IIGP, associated with the endoplasmic reticulum. Our experiments identify hook3 as the first cooperation partner of a member of the 47 kDa GTPase protein family and indicate that hook3 links in an IFNγ-inducible fashion to cytoskeleton-based membrane trafficking.