No abstract

To examine the ability of dendritic cells (DC) to discriminate between helminth and microbial Ag and induce appropriately polarized Th responses, mouse DC were copulsed with the helminth Ag, schistosome egg Ag (SEA), along with the bacterium Proprionebacterium acnes, Pa, and transferred into wild-type mice. Strikingly, SEA/Pa-copulsed DC induced concurrent Pa-specific Th1 (but not Th2) responses and SEA-specific Th2 (but not Th1) responses. Although DC exposed to both Ag undergo many of the maturation-associated changes that accompany exposure to Pa alone, Pa-induced IL-12 production was inhibited by SEA. Examination of Ag uptake revealed that SEA and Pa are acquired via discrete pathways and enter nonoverlapping intracellular compartments. Data suggest that segregation of SEA and Pa into distinct compartments, coupled with SEA-induced modifications of the DC maturation pathway, are significant components of the ability of DC to interpret signals inherent to SEA and Pa and induce appropriately polarized Th responses.

To better understand the initiation of CD8+ T cell responses during infection, the primary response to the intracellular parasite Toxoplasma gondii was characterized using 2-photon microscopy combined with an experimental system that allowed visualization of dendritic cells (DCs) and parasite specific CD8+ T cells. Infection with T. gondii induced localization of both these populations to the sub-capsular/interfollicular region of the draining lymph node and DCs were required for the expansion of the T cells. Consistent with current models, in the presence of cognate antigen, the average velocity of CD8+ T cells decreased. Unexpectedly, infection also resulted in modulation of the behavior of non-parasite specific T cells. This TCR-independent process correlated with the re-modeling of the lymph node micro-architecture and changes in expression of CCL21 and CCL3. Infection also resulted in sustained interactions between the DCs and CD8+ T cells that were visualized only in the presence of cognate antigen and were limited to an early phase in the response. Infected DCs were rare within the lymph node during this time frame; however, DCs presenting the cognate antigen were detected. Together, these data provide novel insights into the earliest interaction between DCs and CD8+ T cells and suggest that cross presentation by bystander DCs rather than infected DCs is an important route of antigen presentation during toxoplasmosis.

Toxoplasma gondii is a leading cause of congenital birth defects, as well as a cause for ocular and neurological diseases in humans. Its cytoskeleton is essential for parasite replication and invasion and contains many unique structures that are potential drug targets. Therefore, the biogenesis of the cytoskeletal structure of T. gondii is not only important for its pathogenesis, but also of interest to cell biology in general. Previously, we and others identified a new T. gondii cytoskeletal protein, TgMORN1, which is recruited to the basal complex at the very beginning of daughter formation. However, its function remained largely unknown. In this study, we generated a knock-out mutant of TgMORN1 (ΔTgMORN1) using a Cre-LoxP based approach. We found that the structure of the basal complex was grossly affected in ΔTgMORN1 parasites, which also displayed defects in cytokinesis. Moreover, ΔTgMORN1 parasites showed significant growth impairment in vitro, and this translated into greatly attenuated virulence in mice. Therefore, our results demonstrate that TgMORN1 is required for maintaining the structural integrity of the parasite posterior end, and provide direct evidence that cytoskeleton integrity is essential for parasite virulence and pathogenesis.

Parasite differentiation is commonly associated with transitions between complex life cycle stages and with long-term persistence in the host, and it is therefore critical for pathogenesis. In the protozoan parasite Toxoplasma gondii, interconversion between rapidly growing tachyzoites and latent encysted bradyzoites is accompanied by numerous morphological and metabolic adaptations. In order to explore early cell biological events associated with this differentiation process, we have exploited fluorescent reporter proteins targeted to various subcellular locations. Combining these markers with efficient in vitro differentiation and time-lapse video microscopy provides a dynamic view of bradyzoite development in living cultures, demonstrating subcellular reorganization, maintenance of the mitochondrion, and missegregation of the apicoplast. Bradyzoites divide asynchronously, using both endodyogeny and endopolygeny, and are highly motile both within and between host cells. Cysts are able to proliferate without passing through an intermediate tachyzoite stage, via both the migration of free bradyzoites and the fission of bradyzoite cysts, suggesting a mechanism for dissemination during chronic infection.

Infection with the parasite Toxoplasma gondii leads to the induction of a Th1-type response dominated by IFN-γ production and control of this pathogen. Cells of the innate immune system are essential in initiating this response both through the production of IL-12 as well as the presentation of parasite-derived Ags to MHC-restricted T cells. Although dendritic cells (DCs) have been implicated in these events, the contribution of individual DC populations remains unclear. Therefore, multiparameter flow cytometry was used to identify and characterize subsets of murine DCs during acute toxoplasmosis. This approach confirmed that infection leads to the expansion and activation of conventional DC (cDC) subsets. Unexpectedly, however, this analysis further revealed that plasmacytoid DCs are also expanded and that these cells up-regulate MHC class II and costimulatory molecules associated with their acquired ability to prime naive CD4+ T cells. Furthermore, T. gondii-activated plasmacytoid DCs produce high levels of IL-12 and both plasmacytoid DC maturation and cytokine production are dependent on TLR11. Together these studies suggest that pDCs are a prominent DC subset involved in the initial stages of T. gondii infection, presenting parasite Ags and producing cytokines that are important for controlling infection.

No abstract

Despite its noted ability to induce strong cellular immunity, and its known susceptibility to IFN-γ-dependent immune effector mechanisms, the protozoan Toxoplasma gondii is a highly successful parasite, able to replicate, disseminate, and either kill the host or, more commonly, establish resistant encysted life forms before the emergence of protective immune responses. We sought to understand how the parasite gains the advantage. Using transgenic clonal parasite lines engineered to express fluorescent markers in combination with dendritic cells (DC) grown from the bone marrow of wild-type mice or transgenic mice expressing fluorescent protein-tagged MHC class II molecules, we used flow cytometry and fluorescence microscopy to analyze the responses of infected DC to both invasion by the parasite and subsequent DC maturation signals. We found that T. gondii preferentially invades immature dendritic cells but fails to activate them in the process, and renders them resistant to subsequent activation by TLR ligands or the immune-system-intrinsic maturation signal CD40L. The functional consequences of T. gondii-mediated suppression of DC activation are manifested in a relative inability of infected immature DC to activate naive CD4+ Th lymphocytes, or to secrete cytokines, such IL-12 and TNF-α, that play important roles in innate and/or adaptive immunity. The findings reveal that T. gondii suppresses the ability of immature DC to participate in innate immunity and to induce adaptive immune responses. The ability of T. gondii to temporarily evade recognition could provide a selective advantage that permits dissemination and establishment before adaptive immune response initiation.