Gaby Reichmann

Brain Dendritic Cells and Macrophages/Microglia in Central Nervous System Inflammation

Microglia subpopulations were studied in mouse experimental autoimmune encephalomyelitis and toxoplasmic encephalitis. CNS inflammation was associated with the proliferation of CD11b+ brain cells that exhibited the dendritic cell (DC) marker CD11c. These cells constituted up to 30% of the total CD11b+ brain cell population. In both diseases CD11c+ brain cells displayed the surface phenotype of myeloid DC and resided at perivascular and intraparenchymatic inflammatory sites. By lacking prominent phagocytic organelles, CD11c+ cells from inflamed brain proved distinct from other microglia, but strikingly resembled bone marrow-derived DC and thus were identified as DC. This brain DC population comprised cells strongly secreting IL-12p70, whereas coisolated CD11c− microglia/brain macrophages predominantly produced TNF-α, GM-CSF, and NO. In comparison, the DC were more potent stimulators of naive or allogeneic T cell proliferation. Both DC and CD11c− microglia/macrophages from inflamed brain primed naive T cells from DO11.10 TCR transgenic mice for production of Th1 cytokines IFN-γ and IL-2. Resting microglia that had been purified from normal adult brain generated immature DC upon exposure to GM-CSF, while CD40 ligation triggered terminal maturation. Consistently, a functional maturation of brain DC was observed to occur following the onset of encephalitis. In conclusion, these findings indicate that in addition to inflammatory macrophage-like brain cells, intraparenchymatical DC exist in autoimmune and infectious encephalitis. These DC functionally mature upon disease onset and can differentiate from resident microglia. Their emergence, maturation, and prolonged activity within the brain might contribute to the chronicity of intracerebral Th1 responses.

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.

In Vivo Monitoring of Inflammation After Cardiac and Cerebral Ischemia by Fluorine Magnetic Resonance Imaging

Background— In this study, we developed and validated a new approach for in vivo visualization of inflammatory processes by magnetic resonance imaging using biochemically inert nanoemulsions of perfluorocarbons (PFCs). Methods and Results— Local inflammation was provoked in 2 separate murine models of acute cardiac and cerebral ischemia, followed by intravenous injection of PFCs. Simultaneous acquisition of morphologically matching proton (1H) and fluorine (19F) images enabled an exact anatomic localization of PFCs after application. Repetitive 1H/19F magnetic resonance imaging at 9.4 T revealed a time-dependent infiltration of injected PFCs into the border zone of infarcted areas in both injury models, and histology demonstrated a colocalization of PFCs with cells of the monocyte/macrophage system. We regularly found the accumulation of PFCs in lymph nodes. Using rhodamine-labeled PFCs, we identified circulating monocytes/macrophages as the main cell fraction taking up injected nanoparticles. Conclusions— PFCs can serve as a “positive” contrast agent for the detection of inflammation by magnetic resonance imaging, permitting a spatial resolution close to the anatomic 1H image and an excellent degree of specificity resulting from the lack of any 19F background. Because PFCs are nontoxic, this approach may have a broad application in the imaging and diagnosis of numerous inflammatory disease states.

Phenotype and Functions of Brain Dendritic Cells Emerging During Chronic Infection of Mice with Toxoplasma gondii

During chronic infection of mice with Toxoplasma gondii, gene message for IL-12p40, CD86, and the potassium channel Kv1.3 was detected in brain mononuclear cells, suggesting the presence of dendritic cells (DC) in the CNS. Consistently, cells bearing the DC markers CD11c and 33D1 were localized at inflammatory sites in the infected brain. The number of isolated CD11c+ brain cells increased until peak inflammation. The cells exhibited the surface phenotype of myeloid DC by coexpressing 33D1 and F4/80, little DEC-205, and no CD8α. These brain DC were mature, as indicated by high-level expression of MHC class II, CD40, CD54, CD80, and CD86. They triggered Ag-specific and primary allogeneic T cell responses at very low APC/T cell ratios. Among mononuclear cells from encephalitic brain, DC were the main producers of IL-12. Evidence for a parasite-dependent development of DC from CNS progenitors was obtained in vitro: after inoculation of primary brain cell culture with T. gondii, IL-12-secreting dendriform cells emerged, and DC marker genes were expressed. Different stimuli elicited the generation and maturation of brain DC: neutralization of parasite-induced GM-CSF prevented outgrowth of dendriform cells and concomitant release of IL-12. IL-12 production was up-regulated by external IFN-γ but was stopped by inhibiting parasite replication. Consistently, DC isolated from GM-CSF-treated brain cell culture were activated to secrete IL-12 by exposure to parasite lysate. In sum, these results demonstrate T. gondii-induced expansion and functional maturation of DC in the CNS and, thus, highlight a mechanism that may contribute to the chronicity of the host response.

The CD40/CD40 Ligand Interaction Is Required for Resistance to Toxoplasmic Encephalitis

ABSTRACT Since the CD40/CD40 ligand (CD40L) interaction is involved in the regulation of macrophage production of interleukin 12 (IL-12) and T-cell production of gamma interferon (IFN-γ), effector cell functions associated with resistance to Toxoplasma gondii, the role of CD40L in immunity to this parasite was assessed. Infection of C57BL/6 mice with T. gondii results in an upregulation of CD40 expression on accessory cell populations at local sites of infection as well as in lymphoid tissues. Splenocytes from C57BL/6 mice infected with T. gondii for 5 days produced high levels of IL-12 and IFN-γ when stimulated with toxoplasma lysate antigen, and blocking CD40L did not significantly alter the production of IFN-γ or IL-12 by these cells. Similar results were observed with splenocytes and mononuclear cells isolated from the brains of chronically infected mice. Interestingly, although CD40L−/− mice infected withT. gondii produced less IL-12 than wild-type mice, they produced comparable levels of IFN-γ but succumbed to toxoplasmic encephalitis 4 to 5 weeks after infection. The inability of CD40L−/− mice to control parasite replication in the brain correlated with the ability of soluble CD40L, in combination with IFN-γ, to activate macrophages in vitro to control replication ofT. gondii. Together, these results identify an important role for the CD40/CD40L interaction in resistance to T. gondii. However, this interaction may be more important in the control of parasite replication in the brain rather than the generation of protective T-cell responses during toxoplasmosis.

GRA7, an excretory 29 kDa Toxoplasma gondii dense granule antigen released by infected host cells

Monoclonal antibody (mAb) TxE2, reactive with Toxoplasma gondii excretory products, detects an acidic 29 kDa protein (p29) which, in 2D gel electrophoresis, exhibits a migration pattern distinct from those of the toxoplasmic excretory proteins described so far. The sequence of seven peptides from tryptic digestion of isolated p29 allowed the design of primers to obtain the coding DNA sequence. The full-length gene was amplified from genomic DNA of T. gondii strain BK and the sequence was identical with that of the corresponding cDNA, providing evidence for an intron-free gene structure. A single mRNA transcript of 1.3 kb was detected by Northern blot analysis. The deduced 236 amino acid protein contains a putative N-terminal signal peptide, one site of potential N-linked glycosylation, and, close to the C-terminus, a further hydrophobic, putative transmembrane domain. With synthetic peptides spanning the sequence of p29, the epitope for mAb TxE2 was mapped adjacent to the putative signal sequence. The antigen, which represents almost 0.5% of T. gondii protein, is expressed in strains of all three intraspecies subgroups, and is associated with the parasite dense granules as demonstrated by immunoelectron microscopy. In tachyzoite-infected cells, p29 accumulates within the parasitophorous vacuole and co-localizes with its delimiting membrane. In bradyzoite-infected cells, p29 is present within the host cell cytoplasm as detected by immunofluorescence staining, and, furthermore, in the supernatant of cyst-bearing cell culture lacking extracellular parasites as shown by enzyme-linked immunosorbent assay (ELISA). Thus, p29 which is named dense granule protein (GRA)7 may indicate the presence of intracellular toxoplasma.

Dendritic cells and dendritic-like microglia in focal cortical ischemia of the mouse brain.

Intracerebral dendritic cells (DC) have recently been identified in neuroinflammation initiated peripherally by brain-targeted autoimmunity or infection. The present study detects DC in photochemically induced cortical ischemia of the mouse brain, a brain-intrinsic lesion model characterized by the lack of an overt T cell response. Concomitant to leukocyte infiltration of the infarcted area, cells expressing the pan-DC surface marker CD11c appeared at the lesion and persisted for weeks. These DC were located at the border zone of the infarct and remote from the lesion in degenerating corticothalamic fibre tracts and subcortical nuclei. All CD11c+ brain cells displayed a uniform CD11b+/CD8alpha-/CD205- surface phenotype, indicating a myeloid origin, and were immature DC based on their MHC class II+/CD40-/CD80+/CD86+/- profile. By expressing high levels of CD45, most DC from ischemic brain seemed to be blood-derived while a minority were CD45(low), thus corresponding to resident microglia. Consistently, round-shaped CD11c+ cells were found at the lesion whereas CD11c+ cells at subcortical sites were ramified like parenchymal microglia. These findings evidence a recruitment of myeloid DC to ischemic brain lesions and suggest that reactive microglia in remote areas transform into dendritic-like cells. Brain-infiltrating DC and their microglial counterparts may play a role in the inflammatory response to cerebral ischemia independently of T cells.

Host cells of Toxoplasma gondii encystation in infected primary culture from mouse brain.

Abstract In order to identify brain cell types that serve as host cells of Toxoplasma gondii encystation primary cultures from murine brain were infected and stained for neural and parasite stage-specific markers. In mixed culture inoculated with T. gondii tachyzoites, MAP2+ neurons, GFAP+ astrocytes, F4/80+ microglia, and O1+ oligodendrocytes proved to be infected as detected by parallel labeling of SAG1. At 4 days following infection with bradyzoites, cysts developed in neuronal, astroglial, and microglial host cells as clarified using bradyzoite-specific antibody 4F8. Additional staining of SAG1 revealed that astrocytes in bradyzoite-infected brain cell culture can also harbor tachyzoite-containing vacuoles. Stage conversion was observed shortly after inoculation and was accompanied by an increase in parasite proliferation. However, tachyzoites became rare in prolonged culture. By contrast, the numbers of cysts and of the bradyzoites isolated multiplied during long-term culture. These findings demonstrate that both glial and neuronal host cells allow T. gondii encystation in the absence of T cell-derived cytokines and imply that a brain-internal spreading of bradyzoites may sustain chronic infection.

Characterization of TgROP9 (p36), a novel rhoptry protein of Toxoplasma gondii tachyzoites identified by T cell clone ☆

T cell clone 3Tx19 detects a Toxoplasma gondii tachyzoite protein which, in high resolution 2D gel electrophoresis, runs at 36 kDa apparent MW with two spots of pI 5.9 and 6.5, thus exhibiting a migration pattern distinct from those of other known Toxoplasma antigens. The sequences of peptide fragments from tryptic digestion of the more prominent protein spot allowed the design of oligonucleotide primers to obtain the coding cDNA sequence. Sequence analysis of cDNA from strain BK revealed a 363 amino acid open reading frame, defined by all nine peptide sequences determined. The deduced protein sequence contains two hydrophobic segments, one near the N-terminus including a predicted signal peptide and a shorter second at the carboxy terminus, but homology to any other known protein is lacking. With synthetic peptides covering the complete primary structure, the epitope for clone 3Tx19 was mapped within the deduced partial sequence, which had remained unconfirmed by tryptic peptides. Antibodies raised against another, putative B cell epitope peptide detected the same two protein spots in 2D gel, indicating that they are antigenically related isoforms. The protein p36 is expressed by T. gondii isolates of all three intraspecies subgroups, but not in the bradyzoite stage. In intracellular tachyzoites, p36 colocalizes with rhoptry proteins and has a distribution pattern disparate from that of dense granule and microneme proteins. Subcellular fractionation indicated that p36 is a soluble constituent of tachyzoites. We suggest that this T cell-stimulatory novel rhoptry protein of T. gondii be named ROP9. It represents a marker of the tachyzoite stage.

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.