Toxoplasma gondii is a pathogen of global importance. Innate immunity is critical for control of acute infection. In vivo mouse models have shown that monocytes are rapidly recruited to sites of T. gondii infection and MCP-1 and CCR2 knock-out mice that fail to do so succumb to infection, underscoring the importance of monocytes in host defense. However, the role of human monocytes in immunity to T. gondii and their specific interactions with T. gondii are not well understood.

Human monocytes are actively infected by T. gondii and permissive to parasite replication. Infected human monocytes release interleukin-1beta (IL-1beta), a "master regulator" of inflammation that has been shown to be protective against T. gondii in vivo. However, the host and parasite factors regulating T. gondii-induced IL-1beta production in human monocytes were unknown. We found that T. gondii induces IL-1beta transcript, post-translational processing, and release from human monocytes. We demonstrate a role for host inflammasome components caspase-1 and ASC and the parasite protein GRA15 in the induction and release of IL-1beta. This work provides important mechanistic insight into the regulation of a key mediator of inflammation and is the first to identify a T. gondii factor that drives innate immune responses in human cells.

Recently, there has been increasing appreciation for the heterogeneity of circulating monocytes. Three phenotypically and functionally distinct subpopulations have been defined in humans based on CD14 and CD16 expression. However, the interactions of specific monocyte subsets with T. gondii and their functional outcomes have not been elucidated. We found that T. gondii preferentially invades classical (CD14+CD16-) and intermediate (CD14+CD16+) monocytes and that these subsets are more permissive to intracellular parasite survival. All monocyte subsets were capable of phagocytosing and degrading Mycalolide B-treated parasites. T. gondii infection induces IL-1beta in all three monocyte subsets, but the resident monocytes (CD14loCD16+) showed an enhanced IL-1beta response. These data suggest that resident monocytes are less permissive to infection and may play a critical role in parasite control.

Collectively, our work defines host and parasite pathways driving innate immunity and contributes to a better understanding of the role of human monocytes in parasite control.

Toxoplasma gondii is an obligate intracellular eukaryotic parasite that is estimated to infect one-third of the global population and is especially life-threatening in developing fetuses and immunocompromised individuals. Innate immune cells contribute to host defense against T. gondii infection. Specifically, monocytes are rapidly recruited to sites of infection and CCR2 or CCL2 KO mice are more susceptible to T. gondii infection. Monocytes protect against infection by initiating a robust inflammatory response which is in part mediated by release of IL-1β during activation of the NLRP3 inflammasome. IL-1β is also implicated in the development of many autoimmune disorders like rheumatoid arthritis and atherosclerosis, but there is still much that is unknown about how human monocytes produce, process and release IL-1β. We have identified that T. gondii induces IL-1β production via a Syk-CARD9-NF-κB signaling axis in primary human peripheral blood monocytes. Syk was rapidly phosphorylated during T. gondii infection of primary monocytes, and inhibiting Syk with the pharmacological inhibitors R406 or entospletinib, or genetic ablation of Syk in THP-1 cells, reduced IL-1β release. Inhibition of Syk in primary cells or deletion of Syk in THP-1 cells decreased parasite-induced transcription and production of pro-IL-1β protein. Inhibition of PKCδ, CARD9/MALT-1 and IKK also reduced p65 phosphorylation and pro-IL-1β production in T. gondii-infected primary monocytes, and genetic knock-out (KO) of PKCδ or CARD9 in THP-1 cells also reduced pro-IL-1β protein levels and IL-1β release during T. gondii infection, indicating that Syk functions upstream of this NF-κB-dependent signaling pathway for IL-1β transcriptional activation. We have also found that primary human monocytes treated with a caspase-8 inhibitor released less IL-1β after T. gondii infection than control cells. Similarly, caspase-1 and caspase-8 KO human monocytic THP-1 cells, but not caspase-4 or -5 KO cells were impaired in their release of IL-1β after infection compared to empty vector (EV) THP-1 cells. We found caspase-8 deficiency did not significantly affect the pro-IL-1β transcripts or production of pro-IL-1β protein. Similarly, caspase-8 had no significant affect on NLRP3 inflammasome activation or cleavage of pro-IL-1β to mature IL-1β during T. gondii infection. Instead, caspase-8 deficiency appeared to stunt the release mechanism of IL-1β from infected cells as mature-IL-1β would accumulate intracellularly in these KO cells. IL-1β release from T. gondii-infected primary human monocytes did require an NLRP3-ASC-caspase-1 inflammasome. While the release mechanism of IL-1β during NLRP3 inflammasome activation often requires cleavage of gasdermin D (GSDMD), formation of pores in the cell membrane and induction of an inflammatory form of cell death known as pyroptosis, human monocytes released IL-1β independent of these factors. Taken together, our data indicate that T. gondii induces a Syk-CARD9/MALT-1-NF-κB signaling pathway and activation of the NLRP3 inflammasome for the release of IL-1β in a cell death- and GSDMD-independent manner. This research also describes a novel role for caspase-8 in IL-1β release from T. gondii-infected monocytes and contributes to the growing notion that IL-1β can be released from human myeloid cells independent of cell death. Collectively, this research expands our understanding of the molecular basis for human innate immune regulation of inflammation and host defense during parasite infection.

Toxoplasma gondii is a foodborne parasite that infects virtually all warm-blooded animals and can cause severe disease in individuals with compromised or weakened immune systems. During dissemination in its infected hosts, T. gondii traverses endothelial barriers to enter tissues and establish chronic infections that underlie the most severe manifestations of toxoplasmosis. Despite a growing appreciation for the importance of endothelial infection in T. gondii pathogenesis, the molecular interactions occurring at this host-pathogen interface remain poorly defined. The research presented here examines T. gondii infection in the context of human primary endothelial cells in static conditions and, for the first time, under the physiological conditions of shear stress found in the bloodstream. We demonstrate that T. gondii infection of primary human umbilical vein endothelial cells (HUVEC) altered cell morphology and dysregulated barrier function, increasing permeability to low molecular weight polymers. Altered cell morphology coincided with parasite-induced remodeling of the three main components of the cytoskeleton: actin microfilaments, intermediate filaments, and microtubules. By conducting a global transcriptome analysis of infected endothelial cells, we identified gene expression changes associated with mechanotransduction and show that T. gondii infection activated Hippo signaling. Infected endothelial cells exposed to shear forces exhibit disrupted planar cell polarity and fragmented Golgi. RNA-Seq analysis on infected endothelial cells exposed to shear suggests that infection alters expression of genes associated with cell proliferation and lipid metabolism. Collectively, this work reveals novel interactions occurring at the interface between endothelial cells and T. gondii to provide insights into processes linked to parasite dissemination and pathogenesis.

Alzheimer’s disease is estimated to affect more than 6 million people in the United States, and this number is expected to grow in the next 20 years. As a result, development of therapeutics that alleviate or prevent this disease is becoming increasingly important. Existing therapies approved by the FDA target amyloid aggregates, one of the defining pathological signs of Alzheimer's disease, via anti-amyloid antibodies. These anti-amyloid antibodies rely on galvanizing an immune system response in the brain to reduce amyloid, with the hope of also improving or restoring cognition. While these therapies have offered some hope and reprieve to those with Alzheimer's disease, concerns over limited efficacy and dangerous side effects have highlighted the importance of developing newer treatments that carefully control immune responses in the brain. To do this, a greater understanding of the neuroimmune environment that contributes to and prevents Alzheimer’s pathology is needed.

Toxoplasma gondii is an obligate intracellular eukaryotic parasite that can be used as a tool to study immune mediated responses to amyloid plaques in Alzheimer's disease. T. gondii is a widely prevalent parasite, estimated to infect up to 80% of humans, depending on geographic location. In Alzheimer's model mice, infection with T. gondii has been shown to reduce amyloid plaques, and this effect has been attributed to an increase in phagocytic monocytes recruited to the brain, and increased proliferation of homeostatic microglia. However, many questions remain on how these cells, as well as other neuroimmune cells, could contribute to a reduction in amyloid during infection.

Herein, we describe both regional and global reductions in amyloid within the brains of 5xFAD Alzheimer’s model mice following infection, providing evidence that amyloid reduction may be both a focal and systemic response to T. gondii infection. We also identify and describe the temporal recruitment of immune cells to the brain over the course of infection in 5xFAD model mice, showing that T cells and monocytes are recruited to the brain as early as acute infection, and these cell populations remain elevated into chronic infection. Generation and infection of bone marrow chimeric 5xFAD mice allowed us to determine that during chronic infection, recruited monocytes originate in both the skull and more peripheral bone marrow, and can be found closely associated with amyloid plaques in the brain. Additionally, myeloid cells in the brain may be more phagocytic of amyloid, as amyloid colocalization with phagolysosomes is increased following infection. Amyloid morphology at this chronic time point is also more diffuse around cores, which may further imply that surrounding myeloid cells are phagocytosing the edges of plaques.

Finally, we identify interesting trends in Alzheimer’s pathology in the brains of a small cohort of patients with Alzheimer's disease and controls who have been serologically tested for T. gondii infection. Future studies in a larger cohort may allow us to determine whether these trends in the pathology become significant. This research thus builds on existing literature, using the parasite T. gondii as a tool to expand our understanding of how neuroimmune responses can contribute to changes in Alzheimer's pathology in both mice and humans, and may enable advances in therapeutic development for Alzheimer’s disease.

Immune defense against the ubiquitous brain-resident parasite Toxoplasma gondii requires a balanced immune response that ultimately results in chronic infection of the host. Monocytes play a key role in host defense and are critical for controlling T. gondii in the periphery. Monocytes also enter the brain and protect against Toxoplasmic encephalitis. However, the dynamic and regional relationship between brain infiltration of the parasite and host monocytes is unknown. In addition, the ability of T. gondii to disseminate through the brain is poorly understood. In this dissertation, we report the mobilization of inflammatory and patrolling monocytes into the blood and brain during T. gondii infection of mice and define their regional localization. We also describe previously unappreciated effects of T. gondii infection on the blood-brain barrier (BBB) and the novel observation that infected cells can shuttle T. gondii at the BBB and in the brain parenchyma.

Many of these questions were addressed using longitudinal analysis of mice using two-photon, intravital imaging of the brain microvasculature through cranial windows. This imaging approach revealed that CCR2-RFP monocytes were recruited to the blood-brain barrier (BBB) within two weeks of T. gondii infection, exhibited distinct rolling and crawling behavior at the BBB, and accumulated within the vessel lumen before entering the parenchyma. Optical clearing of intact T. gondii-infected brains and light sheet microscopy coupled with computational alignment of brains to the Allen annotated reference atlas indicated consistent patterns of monocyte infiltration during T. gondii infection, specifically in the olfactory tubercle and pontine areas. These data provide novel insights into the localization of monocytes in the brain during CNS infection and raise the provocative hypothesis that regional neuroinflammation may contribute to the behavioral phenotypes observed during T. gondii infection of mice.

At the BBB, we observed profound vascular remodeling in T. gondii-infected mice expressing the tight junction protein claudin-5 fused to eGFP in endothelial cells. We detected increased tortuosity of individual endothelial cells and reorganization of the vascular network. In addition, two-photon imaging revealed T. gondii within highly motile cells travelling along cerebral blood vessels. These parasite-infected cells were highly dynamic and traveled at speeds greater than 5 µm/min. As a result, infected cell motility resulted in significantly greater displacement of T. gondii in the brain than individual parasites migrating using their own motility. These data revealed that the infection of motile cells enhanced the dissemination of T. gondii in the brain.

Collectively, this work highlights inherent trade-offs in T. gondii immunity, revealing the delicate balance between mounting a protective monocyte immune response while suggesting it may also contribute to parasite-induced pathology and spread.

Mammalian circulation relies on a closed high-pressure system to deliver oxygen to tissues via red blood cells within blood vessels lined by endothelial cells. However, this high-pressure system makes us vulnerable to hemorrhage if a blood vessel is damaged. Therefore, we have a mechanism to rapidly seal a damaged vessel with a platelet-fibrin clot in a physiological process called hemostasis. However, clots can form in an intact blood vessel and obstruct blood flow, in a pathological process called thrombosis. Such clots can be life threatening if they interfere with blood flow in critical organs, such as the brain (i.e., ischemic stroke). However, recent research has revealed that thrombosis can be involved in augmenting and supporting the innate immune response to control infection through immunothrombosis.

As recent work has demonstrated that Toxoplasma gondii infects and lyses central nervous system endothelial cells that form the blood-brain barrier (BBB), T. gondii-infection is a good model to study the interplay between coagulation, immunity, and infection. However, little is known about the effect of T. gondii infection on the BBB and the functional consequences of infection on cerebral blood flow (CBF) during the different stages of infection. To study CBF changes during T. gondii infection longitudinally we developed a surgical method to achieve chronic optical access for >100 days. Then, using an in vivo infection model in mice, we demonstrated that brain endothelial upregulate the adhesion molecules ICAM-1 and VCAM-1 and become morphologically more tortuous during T. gondii infection. Longitudinal two-photon imaging of cerebral blood vessels during infection revealed vascular occlusion in the brain, prompting an analysis of the coagulation cascade. We detected platelet-fibrin clots within the cerebral vasculature during acute infection. Longitudinal analysis of CBF using laser-speckle imaging during T. gondii infection demonstrated that CBF decreased during acute infection, recovered during stable chronic infection, and decreased again during reactivation of the infection. Finally, we demonstrate that treatment of mice with an anticoagulant, during infection partially rescued CBF in T. gondii-infected mice. These data provide insight into the host-pathogen interactions of a CNS pathogen within the brain vasculature and suggest that thrombosis and changes in cerebral hemodynamics may be an unappreciated aspect of infection with T. gondii.

Protective immunity to Toxoplasma gondii depends on innate immune activation and the establishment of a robust Th1 response. Notably, however, T. gondii can subvert host immunity by altering the transcriptional program and the proteome of infected cells. The mechanisms by which the parasite alters the expression and function of cell surface receptors, which play a key role in immune activation, are not well understood. As critical regulators of T cell activation, the expression of the costimulatory receptors B7-1 and B7-2 are tightly controlled. We examined the molecular mechanisms governing their induction in macrophages. We found that all three strains of T. gondii (types I, II, and III) up-regulated the expression of B7-2. Transcriptional profiling by microarray analysis revealed nodes of regulation in infected cells, in particular the induction of MAPK signaling. Using specific inhibitors against MAPKs, we determined that parasite-induced B7-2 is dependent on JNK. We demonstrate that B7-2 induced by T. gondii is capable of activating T cells and promoting their proliferation.

We also examined the regulation of the CD40 receptor on the surface of T. gondii-infected macrophages. CD40 expression was dramatically increased only in type II T. gondii-infected cells. Forward genetic analysis suggested that GRA15 from type II strains (GRA15_II) was the parasite factor responsible for the CD40-inducing phenotype. Using type I parasites and THP-1 cells stably expressing GRA15_II, we confirmed that GRA15_II was sufficient to induce CD40. We also found that engagement of CD40 in type II-infected cells amplified the IL-12 response. These data indicate that GRA15_II may promote parasite immunity through CD40-mediated production of IL-12.

Using a proteomic approach, we sought to determine how T. gondii infection globally alters the expression of cell surface proteins. Using this approach we found that the expression of Fc&gammaR is altered on infected cells, which may represent a previously undescribed immune evasion strategy by T. gondii. The dataset generated using this protocol may lead to the identification of novel surface markers specific to infected cells that can serve as therapeutic targets.

Collectively, our data suggest that changes in the expression of surface proteins on infected cells may profoundly shape T. gondii immunity.

Inflammation is a tightly regulated process necessary to protect the body against infection but, in excess, can be dangerous and lead to damage. Toxoplasma gondii is an obligate intracellular foodborne pathogen with the unique ability to cross the blood-brain barrier and form lifelong parasite cysts in neurons. This infection drives a protective immune response in the brain that can control parasitic growth but not clear the infection. CCR2 chemokine receptor-expressing monocytes play a necessary role in controlling T. gondii infection both in the periphery and the brain. Once in the brain, monocytes are specifically recruited to areas containing clusters of T. gondii. However, the drivers of monocyte recruitment to sites of infection, specifically to areas containing T. gondii, are not well understood. This research aimed to understand the signals that drive protective immunity against T. gondii infection. In the brain, we did determine a critical role for chemokine production in neuroinflammation. In the brain, we determined that the cells producing the potent CCR2-binding chemokine, CCL2, changed over the course of infection: microglia were the main CCL2 producers during acute infection, whereas astrocytes became the dominant CCL2 producers during chronic infection. Interestingly, the ablation of CCL2 production from astrocytes reduced immune cell recruitment to the brain during chronic infection and decreased control of the parasitic infection. We also found that activation of the transcription factor, NF-B, and CCL2 production are increased near parasites in the brain. However, the parasite effector protein GRA15 does not drive immune cell recruitment to parasites. Furthermore, T. gondii replication and host cell lysis play significant roles in driving immune cells to parasites in the brain. Additionally, we found that Piezo1 is upregulated during infection and Piezo1 expression in myeloid cells aids in parasite dissemination to the liver during early acute infection. However, Piezo1 expression in myeloid cells does not affect dissemination to the brain during late acute infection nor control of parasitic burden throughout chronic infection. Collectively, this work highlights the role of host cell responses to T. gondii in driving immune cell recruitment to control parasitic infection.

Toxoplasma gondii is a highly successful parasite that infects approximately one-third of the human population and can cause fatal disease in immunocompromised individuals. Systemic parasite dissemination to organs such as the brain and eye is critical to disease pathogenesis. T. gondii can disseminate via the circulation, and both intracellular and extracellular modes of transport have been proposed. To examine the dynamics of both of these dissemination mechanisms we have developed a fluidic system combined with time-lapse fluorescence microscopy. Using this approach, we showed that T. gondii-infected primary human monocytes and THP-1 cells exhibited altered adhesion dynamics compared to uninfected monocytes: infected cells rolled at significantly higher velocities (2.5 to 4.6-fold) and over greater distances (2.6 to 4.8-fold) than uninfected monocytes before firmly adhering. Since infected monocytes appeared delayed in their transition to firm adhesion, we examined the effects of infection on integrin expression and function. T. gondii did not affect the expression of LFA-1, VLA-4, or MAC-1 or the ability of Mn²⁺ to activate these integrins. However, T. gondii infection impaired LFA-1 and VLA-4 clustering and pseudopod extension in response to integrin ligands.

We then applied this same fluidic system to questions of extracellular parasite adhesion to endothelium and showed that shear force influenced parasite adhesion and motility dynamics and the outcome of parasite interactions with endothelium. Extracellular parasites were capable of adhesion to primary human endothelium in shear stress conditions, and interestingly, shear stress enhanced T. gondii helical gliding, resulting in a significantly greater displacement. In addition, shear stress increased the percentage of tachyzoites that invaded or migrated across the endothelium. By examining T. gondii deficient in the adhesion protein MIC2, we found that MIC2 contributed to initial adhesion but was not required for adhesion strengthening. These data suggest that in fluidic conditions, T. gondii adhesion to endothelium may be mediated by a multistep cascade of interactions that is governed by unique combinations of adhesion molecules.

This dissertation work has led to a better understanding of the mechanisms by which T. gondii interacts with and migrates across endothelium into tissues, where the parasites ultimately cause disease.