Divergent co-transcriptomes of different host cells infected with Toxoplasma gondii reveal cell type-specific host-parasite interactions.

The apicomplexan parasite Toxoplasma gondii infects various cell types in avian and mammalian hosts including humans. Infection of immunocompetent hosts is mostly asymptomatic or benign, but leads to development of largely dormant bradyzoites that persist predominantly within neurons and muscle cells. Here we have analyzed the impact of the host cell type on the co-transcriptomes of host and parasite using high-throughput RNA sequencing. Murine cortical neurons and astrocytes, skeletal muscle cells (SkMCs) and fibroblasts differed by more than 16,200 differentially expressed genes (DEGs) before and after infection with T. gondii. However, only a few hundred of them were regulated by infection and these largely diverged in neurons, SkMCs, astrocytes and fibroblasts indicating host cell type-specific transcriptional responses after infection. The heterogeneous transcriptomes of host cells before and during infection coincided with ~5,400 DEGs in T. gondii residing in different cell types. Finally, we identified gene clusters in both T. gondii and its host, which correlated with the predominant parasite persistence in neurons or SkMCs as compared to astrocytes or fibroblasts. Thus, heterogeneous expression profiles of different host cell types and the parasites' ability to adapting to them may govern the parasite-host cell interaction during toxoplasmosis.

Transcriptional Responses in the Murine Spleen after Toxoplasma gondii Infection: Inflammasome and Mucus-Associated Genes.

The spleen plays an important role in coordinating both adaptive and innate immune responses. Here, the transcriptional response to T. gondii infection in the murine spleen was characterized concerning inflammasome sensors (two different models: seven days after oral or four weeks after intraperitoneal infection). Additionally, Tff1KO and Tff3KO mice were investigated because TFF genes are often upregulated during inflammation. The expression of the pattern-recognition receptors Nlrp3, Nlrp12, and Nlrp1a was significantly increased after infection. This increase was diminished in Tff1KO and Tff3KO mice pointing towards a positive regulation of the inflammatory response by Tff1 and Tff3. Furthermore, the transcription of Tff1 (encoding a motogenic lectin) and other secretory genes was analyzed, i.e., gastrokines (Gkn), IgG Fc binding protein (Fcgbp), and the mucin Muc2. The corresponding gene products belong to an interactome protecting mucous epithelia. Tff1 was significantly induced after infection, which might increase the motility of immune cells. In contrast, Gkn3, Fcgbp, and Muc2 were downregulated seven days after oral infection; whereas four weeks after i.p. infection only Gkn3 remained downregulated. This might be an indication that Gkn3, Fcgbp, and Muc2 are involved in the transient disruption of the splenic architecture and its reorganization, which is characteristic after T. gondii infection.

Increased Cerebral Tff1 Expression in Two Murine Models of Neuroinflammation.

BACKGROUND/AIMS: The trefoil factor family (TFF) peptide TFF1 is a typical secretory product of the gastric mucosa and a very low level of expression occurs in nearly all regions of the murine brain. TFF1 possesses a lectin activity and binding to a plethora of transmembrane glycoproteins could explain the diverse biological effects of TFF1 (e.g., anti-apoptotic effect). It was the aim to test whether TFF expression is changed during neuroinflammation. METHODS: Expression profiling was performed using semi-quantitative RT-PCR analyses in two murine models of neuroinflammation, i.e. Toxoplasma gondii-induced encephalitis and experimental autoimmune encephalomyelitis (EAE), the latter being the most common animal model of multiple sclerosis. Tff1 expression was also localized using RNA in situ hybridization histochemistry. RESULTS: We report for the first time on a significant transcriptional induction in cerebral Tff1 expression in both T. gondii-induced encephalitis and EAE. In contrast, Tff2 and Tff3 expression were not altered. Tff1 transcripts were predominantly localized in the internal granular layer of the cerebellum indicating neuronal expression. Furthermore, also glial cells are expected to express Tff1. Characterization of both experimental models by expression profiling (e.g., inflammasome sensors, inflammatory cytokines, microglial marker Iba1, ependymin related protein 1) revealed differences concerning the expression of the inflammasome sensor Nlrp1 and interleukin 17a. CONCLUSION: The up-regulated expression of Tff1 is probably the result of a complex inflammatory process as its expression is induced by tumor necrosis factor α as well as interleukins 1ß and 17. However on the transcript level, Tff1KO mice did not show any significant signs of an altered immune response after infection with T. gondii in comparison with the wild type animals.

Neuronal gp130 expression is crucial to prevent neuronal loss, hyperinflammation, and lethal course of murine Toxoplasma encephalitis.

The obligate intracellular parasite Toxoplasma gondii infects and persists within neurons of approximately one-third of the human population. Intracerebral control of T. gondii largely depends on interferon (IFN)-γ-producing T cells, which induce antiparasitic effector mechanisms in infected cells, as well as immunosuppressive cytokines, which prevent immunopathology. To gain further insight into the role of neurons in Toxoplasma encephalitis (TE), we generated C57BL/6 synapsin-I (Syn)-Cre gp130(fl/fl) mice, which lack gp130, the signal-transducing receptor for the IL-6 family of cytokines, in their neurons. On infection with T. gondii, Syn-Cre gp130(fl/fl) mice failed to control T. gondii infection and died of necrotizing TE before day 77. In contrast, gp130(fl/fl) control mice efficiently restricted parasite replication and survived the infection. TE in Syn-Cre gp130(fl/fl) mice was characterized by a hyperinflammatory immune response with increased numbers of IL-17- and IFN-γ-producing CD4 and CD8 T cells but reduced intracerebral production of immunosuppressive transforming growth factor (TGF)-ß and IL-27. Additional in vitro experiments found that IL-6 stimulation of neurons induced gp130-dependent TGF-ß1, TGF-ß2, and IL-27 production. Importantly, gp130 expression and stimulation with IL-6 cytokine family members also reduced death and apoptosis of infected cultured neurons. Correspondingly, TE in Syn-Cre gp130(fl/fl) but not gp130(fl/fl) mice was characterized by progressive neuronal loss. Collectively, these findings indicate a crucial protective function of gp130-expressing neurons in a model of chronic encephalitis.

Toxoplasma gondii actively inhibits neuronal function in chronically infected mice.

Upon infection with the obligate intracellular parasite Toxoplasma gondii, fast replicating tachyzoites infect a broad spectrum of host cells including neurons. Under the pressure of the immune response, tachyzoites convert into slow-replicating bradyzoites, which persist as cysts in neurons. Currently, it is unclear whether T. gondii alters the functional activity of neurons, which may contribute to altered behaviour of T. gondii-infected mice and men. In the present study we demonstrate that upon oral infection with T. gondii cysts, chronically infected BALB/c mice lost over time their natural fear against cat urine which was paralleled by the persistence of the parasite in brain regions affecting behaviour and odor perception. Detailed immunohistochemistry showed that in infected neurons not only parasitic cysts but also the host cell cytoplasm and some axons stained positive for Toxoplasma antigen suggesting that parasitic proteins might directly interfere with neuronal function. In fact, in vitro live cell calcium (Ca(2+)) imaging studies revealed that tachyzoites actively manipulated Ca(2+) signalling upon glutamate stimulation leading either to hyper- or hypo-responsive neurons. Experiments with the endoplasmatic reticulum Ca(2+) uptake inhibitor thapsigargin indicate that tachyzoites deplete Ca(2+) stores in the endoplasmatic reticulum. Furthermore in vivo studies revealed that the activity-dependent uptake of the potassium analogue thallium was reduced in cyst harbouring neurons indicating their functional impairment. The percentage of non-functional neurons increased over time In conclusion, both bradyzoites and tachyzoites functionally silence infected neurons, which may significantly contribute to the altered behaviour of the host.

Gp130-dependent astrocytic survival is critical for the control of autoimmune central nervous system inflammation.

Astrocytes are activated in experimental autoimmune encephalomyelitis (EAE) and have been suggested to either aggravate or ameliorate EAE. However, the mechanisms leading to an adverse or protective effect of astrocytes on the course of EAE are incompletely understood. To gain insight into the astrocyte-specific function of gp130 in EAE, we immunized mice lacking cell surface expression of gp130, the signal-transducing receptor for cytokines of the IL-6 family, with myelin oligodendrocyte glycoprotein(35-55) peptide. These glial fibrillary acid protein (GFAP)-Cre gp130(fl/fl) mice developed clinically a significantly more severe EAE than control mice and succumbed to chronic EAE. Loss of astrocytic gp130 expression resulted in apoptosis of astrocytes in inflammatory lesions of GFAP-Cre gp130(fl/fl) mice, whereas gp130(fl/fl) control mice developed astrogliosis. Astrocyte loss of GFAP-Cre gp130(fl/fl) mice was paralleled by significantly larger areas of demyelination and significantly increased numbers of CD4 T cells in the CNS. Additionally, loss of astrocytes in GFAP-Cre gp130(fl/fl) mice resulted in a reduction of CNS regulatory Foxp3(+) CD4 T cells and an increase of IL-17-, IFN-γ-, and TNF-producing CD4 as well as IFN-γ- and TNF-producing CD8 T cells, illustrating that astrocytes regulate the phenotypic composition of T cells. An analysis of mice deficient in either astrocytic gp130- Src homology region 2 domain-containing phosphatase 2/Ras/ERK or gp130-STAT1/3 signaling revealed that prevention of astrocyte apoptosis, restriction of demyelination, and T cell infiltration were dependent on the astrocytic gp130-Src homology region 2 domain-containing phosphatase 2/Ras/ERK, but not on the gp130-STAT1/3 pathway, further demonstrating that gp130-dependent astrocyte activation is crucial to ameliorate EAE.

Protective Toxoplasma gondii-specific T-cell responses require T-cell-specific expression of protein kinase C-theta.

Protein kinase C-theta (PKC-theta) is important for the activation of autoreactive T cells but is thought to be of minor importance for T-cell responses in infectious diseases, suggesting that PKC-theta may be a target for the treatment of T-cell-mediated autoimmune diseases. To explore the function of PKC-theta in a chronic persisting infection in which T cells are crucial for pathogen control, we infected BALB/c PKC-theta(-/-) and PKC-theta(+/+) wild-type mice with Toxoplasma gondii. The PKC-theta(-/-) mice succumbed to necrotizing Toxoplasma encephalitis due to an insufficient parasite control up to day 40, whereas the wild-type mice survived. The number of T. gondii-specific CD4 and CD8 T cells was significantly reduced in the PKC-theta(-/-) mice, resulting in the impaired production of protective cytokines (gamma interferon, tumor necrosis factor) and antiparasitic effector molecules (inducible nitric oxide, gamma interferon-induced GTPase) in the spleen and brain. In addition, Th2-cell numbers were reduced in infected the PKC-theta(-/-) mice, paralleled by the diminished GATA3 expression of PKC-theta(-/-) CD4 T cells and reduced T. gondii-specific IgG production in serum and cerebrospinal fluid. Western blot analysis of splenic CD4 and CD8 T cells revealed an impaired activation of the NF-kappaB, AP-1, and MAPK pathways in T. gondii-infected PKC-theta(-/-) mice. Adoptive transfer of wild-type CD4 plus CD8 T cells significantly protected PKC-theta(-/-) mice from death by increasing the numbers of gamma interferon-producing T. gondii-specific CD4 and CD8 T cells, illustrating a cell-autonomous, protective function of PKC-theta in T cells. These findings imply that PKC-theta inhibition drastically impairs T. gondii-specific T-cell responses with fatal consequences for intracerebral parasite control and survival.