Nucleotide-Oligomerization-Domain-2 Affects Commensal Gut Microbiota Composition and Intracerebral Immunopathology in Acute Toxoplasma gondii Induced Murine Ileitis

Background Within one week following peroral high dose infection with Toxoplasma (T.) gondii, susceptible mice develop non-selflimiting acute ileitis due to an underlying Th1-type immunopathology. The role of the innate immune receptor nucleotide-oligomerization-domain-2 (NOD2) in mediating potential extra-intestinal inflammatory sequelae including the brain, however, has not been investigated so far. Methodology/Principal Findings Following peroral infection with 100 cysts of T. gondii strain ME49, NOD2-/- mice displayed more severe ileitis and higher small intestinal parasitic loads as compared to wildtype (WT) mice. However, systemic (i.e. splenic) levels of pro-inflammatory cytokines such as TNF-α and IFN-γ were lower in NOD2-/- mice versus WT controls at day 7 p.i. Given that the immunopathological outcome might be influenced by the intestinal microbiota composition, which is shaped by NOD2, we performed a quantitative survey of main intestinal bacterial groups by 16S rRNA analysis. Interestingly, Bifidobacteria were virtually absent in NOD2-/- but not WT mice, whereas differences in remaining bacterial species were rather subtle. Interestingly, more distinct intestinal inflammation was accompanied by higher bacterial translocation rates to extra- intestinal tissue sites such as liver, spleen, and kidneys in T. gondii infected NOD2-/- mice. Strikingly, intracerebral inflammatory foci could be observed as early as seven days following T. gondii infection irrespective of the genotype of animals, whereas NOD2-/- mice exhibited higher intracerebral parasitic loads, higher F4/80 positive macrophage and microglia numbers as well as higher IFN-γ mRNA expression levels as compared to WT control animals. Conclusion/Significance NOD2 signaling is involved in protection of mice from T. gondii induced acute ileitis. The parasite-induced Th1-type immunopathology at intestinal as well as extra-intestinal sites including the brain is modulated in a NOD2-dependent manner

Impaired Chromatin Remodelling at STAT1-Regulated Promoters Leads to Global Unresponsiveness of Toxoplasma gondii-Infected Macrophages to IFN-γ

Intracellular pathogens including the apicomplexan and opportunistic parasite Toxoplasma gondii profoundly modify their host cells in order to establish infection. We have shown previously that intracellular T. gondii inhibit up-regulation of regulatory and effector functions in murine macrophages (MΦ) stimulated with interferon (IFN)-γ, which is the cytokine crucial for controlling the parasites' replication. Using genome-wide transcriptome analysis we show herein that infection with T. gondii leads to global unresponsiveness of murine macrophages to IFN-γ. More than 61% and 89% of the transcripts, which were induced or repressed by IFN-γ in non-infected MΦ, respectively, were not altered after stimulation of T. gondii-infected cells with IFN-γ. These genes are involved in a variety of biological processes, which are mostly but not exclusively related to immune responses. Analyses of the underlying mechanisms revealed that IFN-γ-triggered nuclear translocation of STAT1 still occurred in Toxoplasma-infected MΦ. However, STAT1 bound aberrantly to oligonucleotides containing the IFN-γ-responsive gamma-activated site (GAS) consensus sequence. Conversely, IFN-γ did not induce formation of active GAS-STAT1 complexes in nuclear extracts from infected MΦ. Mass spectrometry of protein complexes bound to GAS oligonucleotides showed that T. gondii-infected MΦ are unable to recruit non-muscle actin to IFN-γ-responsive DNA sequences, which appeared to be independent of stimulation with IFN-γ and of STAT1 binding. IFN-γ-induced recruitment of BRG-1 and acetylation of core histones at the IFN-γ-regulated CIITA promoter IV, but not β-actin was diminished by >90% in Toxoplasma-infected MΦ as compared to non-infected control cells. Remarkably, treatment with histone deacetylase inhibitors restored the ability of infected macrophages to express the IFN-γ regulated genes H2-A/E and CIITA. Taken together, these results indicate that Toxoplasma-infected MΦ are unable to respond to IFN-γ due to disturbed chromatin remodelling, but can be rescued using histone deacetylase inhibitors

MyD88 Is Required for Protection from Lethal Infection with a Mouse-Adapted SARS-CoV

A novel human coronavirus, SARS-CoV, emerged suddenly in 2003, causing approximately 8000 human cases and more than 700 deaths worldwide. Since most animal models fail to faithfully recapitulate the clinical course of SARS-CoV in humans, the virus and host factors that mediate disease pathogenesis remain unclear. Recently, our laboratory and others developed a recombinant mouse-adapted SARS-CoV (rMA15) that was lethal in BALB/c mice. In contrast, intranasal infection of young 10-week-old C57BL/6 mice with rMA15 results in a nonlethal infection characterized by high titer replication within the lungs, lung inflammation, destruction of lung tissue, and loss of body weight, thus providing a useful model to identify host mediators of protection. Here, we report that mice deficient in MyD88 (MyD88−/−), an adapter protein that mediates Toll-like receptor (TLR), IL-1R, and IL-18R signaling, are far more susceptible to rMA15 infection. The genetic absence of MyD88 resulted in enhanced pulmonary pathology and greater than 90% mortality by day 6 post-infection. MyD88−/− mice had significantly higher viral loads in lung tissue throughout the course of infection. Despite increased viral loads, the expression of multiple proinflammatory cytokines and chemokines within lung tissue and recruitment of inflammatory monocytes/macrophages to the lung was severely impaired in MyD88−/− mice compared to wild-type mice. Furthermore, mice deficient in chemokine receptors that contribute to monocyte recruitment to the lung were more susceptible to rMA15-induced disease and exhibited severe lung pathology similar to that seen in MyD88−/−mice. These data suggest that MyD88-mediated innate immune signaling and inflammatory cell recruitment to the lung are required for protection from lethal rMA15 infection

CD11b+, Ly6G+ Cells Produce Type I Interferon and Exhibit Tissue Protective Properties Following Peripheral Virus Infection

The goal of the innate immune system is containment of a pathogen at the site of infection prior to the initiation of an effective adaptive immune response. However, effector mechanisms must be kept in check to combat the pathogen while simultaneously limiting undesirable destruction of tissue resulting from these actions. Here we demonstrate that innate immune effector cells contain a peripheral poxvirus infection, preventing systemic spread of the virus. These innate immune effector cells are comprised primarily of CD11b+Ly6C+Ly6G- monocytes that accumulate initially at the site of infection, and are then supplemented and eventually replaced by CD11b+Ly6C+Ly6G+ cells. The phenotype of the CD11b+Ly6C+Ly6G+ cells resembles neutrophils, but the infiltration of neutrophils typically occurs prior to, rather than following, accumulation of monocytes. Indeed, it appears that the CD11b+Ly6C+Ly6G+ cells that infiltrated the site of VACV infection in the ear are phenotypically distinct from the classical description of both neutrophils and monocyte/macrophages. We found that CD11b+Ly6C+Ly6G+ cells produce Type I interferons and large quantities of reactive oxygen species. We also observed that depletion of Ly6G+ cells results in a dramatic increase in tissue damage at the site of infection. Tissue damage is also increased in the absence of reactive oxygen species, although reactive oxygen species are typically thought to be damaging to tissue rather than protective. These data indicate the existence of a specialized population of CD11b+Ly6C+Ly6G+ cells that infiltrates a site of virus infection late and protects the infected tissue from immune-mediated damage via production of reactive oxygen species. Regulation of the action of this population of cells may provide an intervention to prevent innate immune-mediated tissue destruction

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

Parasite fate and involvement of infected cells in the induction of CD4+ and CD8+ T cell responses to Toxoplasma gondii

During infection with the intracellular parasite Toxoplasma gondii, the presentation of parasite-derived antigens to CD4+ and CD8+ T cells is essential for long-term resistance to this pathogen. Fundamental questions remain regarding the roles of phagocytosis and active invasion in the events that lead to the processing and presentation of parasite antigens. To understand the most proximal events in this process, an attenuated non-replicating strain of T. gondii (the cpsII strain) was combined with a cytometry-based approach to distinguish active invasion from phagocytic uptake. In vivo studies revealed that T. gondii disproportionately infected dendritic cells and macrophages, and that infected dendritic cells and macrophages displayed an activated phenotype characterized by enhanced levels of CD86 compared to cells that had phagocytosed the parasite, thus suggesting a role for these cells in priming naïve T cells. Indeed, dendritic cells were required for optimal CD4+ and CD8+ T cell responses, and the phagocytosis of heat-killed or invasion-blocked parasites was not sufficient to induce T cell responses. Rather, the selective transfer of cpsII-infected dendritic cells or macrophages (but not those that had phagocytosed the parasite) to naïve mice potently induced CD4+ and CD8+ T cell responses, and conferred protection against challenge with virulent T. gondii. Collectively, these results point toward a critical role for actively infected host cells in initiating T. gondii-specific CD4+ and CD8+ T cell responses

Barrier Tissue Macrophages: Functional Adaptation to Environmental Challenges

Macrophages are found throughout the body, where they have crucial roles in tissue development, homeostasis and remodeling, as well as being sentinels of the innate immune system that can contribute to protective immunity and inflammation. Barrier tissues, such as the intestine, lung, skin and liver, are exposed constantly to the outside world, which places special demands on resident cell populations such as macrophages. Here we review the mounting evidence that although macrophages in different barrier tissues may be derived from distinct progenitors, their highly specific properties are shaped by the local environment, which allows them to adapt precisely to the needs of their anatomical niche. We discuss the properties of macrophages in steady-state barrier tissues, outline the factors that shape their differentiation and behavior and describe how macrophages change during protective immunity and inflammation

Extramedullary Myelopoiesis in Malaria Depends on Mobilization of Myeloid-Restricted Progenitors by IFN-γ Induced Chemokines

Resolution of a variety of acute bacterial and parasitic infections critically relies on the stimulation of myelopoiesis leading in cases to extramedullary hematopoiesis. Here, we report the isolation of the earliest myeloid-restricted progenitors in acute infection with the rodent malaria parasite, Plasmodium chabaudi. The rapid disappearance of these infection-induced myeloid progenitors from the bone marrow (BM) equated with contraction of the functional myeloid potential in that organ. The loss of BM myelopoiesis was not affected by the complete genetic inactivation of toll-like receptor signaling. De-activation of IFN-γ signaling completely abrogated the contraction of BM myeloid progenitors. Radiation chimeras of Ifngr1-null and control BM revealed that IFN-γ signaling in an irradiation-resistant stromal compartment was crucial for the loss of early myeloid progenitors. Systemic IFN-γ triggered the secretion of C-C motif ligand chemokines CCL2 and CCL7 leading to the egress of early, myeloid-committed progenitors from the bone marrow mediated by their common receptor CCR2. The mobilization of myeloid progenitors initiated extramedullary myelopoiesis in the spleen in a CCR2-dependent manner resulting in augmented myelopoiesis during acute malaria. Consistent with the lack of splenic myelopoiesis in the absence of CCR2 we observed a significant persistence of parasitemia in malaria infected CCR2-deficient hosts. Our findings reveal how the activated immune system mobilizes early myeloid progenitors out of the BM thereby transiently establishing myelopoiesis in the spleen in order to contain and resolve the infection locally