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In a murine model for neurocysticercosis (NCC), intracranial inoculation of the helminth parasite Mesocestoides corti induces multiple Toll-like receptors (TLRs), among which TLR2 is upregulated first and to a relatively high extent. Here, we report that TLR2(-/-) mice displayed significantly increased susceptibility to parasite infection accompanied by increased numbers of parasites in the brain parenchyma compared to infection in wild-type (WT) mice. This coincided with an increased display of microglial nodule formations and greater neuropathology than in the WT. Parasite-infected TLR2(-/-) brains exhibited a scarcity of lymphocytic cuffing and displayed reduced numbers of infiltrating leukocytes. Fluorescence-activated cell sorter (FACS) analyses revealed significantly lower numbers of CD11b(+) myeloid cells, γδ T cells, αβ T cells, and B cells in the brains of parasite-infected TLR2(-/-) mice. This correlated with significantly reduced levels of inflammatory mediators, including tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), CCL2, CCL3, and interleukin-6 (IL-6) in the central nervous system (CNS) of TLR2(-/-) mice. As TLR2 has been implicated in immune regulation of helminth infections and as alternatively activated macrophages (AAMs) are thought to play a profound regulatory role in such infections, induction of AAMs in infected TLR2(-/-) mice was compared with that in WT mice. Parasite-infected WT brains showed larger numbers of macrophages/microglia (CD11b(+) cells) expressing AAM-associated molecules such as YM1, Fizz1 (found in inflammatory zone-1 antigen), and arginase 1 than TLR2(-/-) brains, consistent with a protective role of AAMs during infection. Importantly, these results demonstrate that TLR2-associated responses modulate the disease severity of murine NCC.

Murine neurocysticercosis is a parasitic infection transmitted through the direct ingestion of Taenia solium eggs, which differentially disrupts the barriers that protect the microenvironment of the central nervous system. Among the host factors that are involved in this response, matrix metalloproteinases (MMPs) have been recently described as important players. Doxycycline is a commonly prescribed antimicrobial drug that acts as an anti-inflammatory agent with broad inhibitory properties against MMPs. In this study, we examined the effects of doxycycline treatment in a murine model of neurocysticercosis. Animals treated with doxycycline exhibited reduced morbidity and mortality throughout the course of infection. Although similar levels of leukocyte infiltration were observed with both treatment regimens, doxycycline appeared to provide improved conditions for host survival, as reduced levels of apoptosis were detected among infiltrates as well as in neurons. As an established MMP blocker, doxycycline reduced the degradation of junctional complex proteins in parenchymal vessels. In addition, doxycycline treatment was associated with an overall reduction in the expression and activity of MMPs, particularly in areas of leukocyte infiltration. These results indicate that a broad-range inhibitor of MMPs promotes host survival and suggest the potential of doxycycline as a therapeutic agent for the control of inflammatory responses associated with neurocysticercosis.

The functions of Toll-like receptors (TLRs) 11-13 in central nervous system (CNS) infections are currently unknown. Using a murine model of neurocysticercosis, we investigated the expression and distribution of TLRs 11-13 by using both gene specific real-time PCR analysis and in situ immunofluorescence microscopy in both control and neurocysticercosis brains. In the mock infected brain, mRNAs of TLRs 11-13 were constitutively expressed. Parasite infection caused an increase of both mRNAs and protein levels of all three TLRs by several fold. All three TLR proteins were present in both CNS and immune cell types. Among them TLR13 was expressed the most in terms of number of positive cells and brain areas expressing it, followed by TLR11 and TLR12 respectively. Among the nervous tissue cells, TLRs 11-13 protein levels appeared highest in neurons. However, TLR13 expression was also present in ependymal cells, endothelial cells of pial blood vessels, and astrocytes. In contrast, infiltrating CD11b and CD11c positive myeloid cells predominantly produced TLR11 protein, particularly early during infection at 1 wk post infection (approximately 50% cells). TLRs 12 and 13 proteins were present on approximately 5% of infiltrating immune cells. The infiltrating cells positive for TLRs 11-13 were mostly of myeloid origin, CD11b+ cells. This report provides a comprehensive analysis of the expression of TLRs 11-13 in normal and parasite infected mouse brains and suggests a role for them in CNS infections.

During the course of murine neurocysticercosis (NCC), disruption of the unique protective barriers in the central nervous system (CNS) is evidenced by extravasation of leukocytes. This process varies according to the anatomical sites and diverse vascular beds analyzed. To examine mechanisms involved in the observed differences, the expression and activity of eight matrix metalloproteinases (MMPs) were analyzed in a murine model of NCC. The mRNA expression of the MMPs studied was upregulated as a result of infection, and active MMPs were mainly detected in leukocytes migrating into the brain. Polarized expression and gelatinolytic activity of several MMPs were identified in immune cells extravasating pial vessels as early as 1 day post infection. In contrast, leukocytes expressing active MMPs and extravasating parenchymal vessels were not observed until 5 weeks post infection. In ventricular areas, most of the MMP activity was detected in leukocytes traversing the ependyma from leptomeningeal infiltrates. In addition, immune cells continued to express active MMPs after exiting vessels suggesting that enzymatic activity of MMPs is not just required for diapedesis. These results correlate with our previous studies showing differential kinetics in the disruption of the CNS barriers upon infection and help document the important role of MMPs during leukocyte infiltration and inflammation.

In a mouse model of neurocysticercosis, the expression and distribution of Toll-like receptors (TLRs) was investigated by using both gene array analyses and in situ immunofluorescence microscopy (IF). In the normal uninfected brain, mRNA of all the TLRs are constitutively expressed albeit TLR5, TLR7, TLR8 and TLR9 to a lesser extent. In these animals, however, expression of TLR1, TLR3, TLR4 and TLR9 proteins was not detected. In contrast, parasite infection increased both gene and protein level expression of all the TLRs several fold except TLR5 where only the mRNA was upregulated. Importantly, TLRs were differentially distributed among various central nervous system (CNS) cell types and infiltrating leukocytes. TLR2 was almost exclusively localized to nervous tissue cells, particularly astrocytes, while TLR1 and TLR9 proteins were essentially limited to infiltrating leukocytes. All other TLRs tested were detected in both CNS and immune cell types. Interestingly, ependymal cells and neurofilaments of the cerebellar white matter of infected mice exhibited a substantial upregulation of TLR7 and TLR8 proteins respectively. These data provide a comprehensive analysis of TLR expression in the normal and parasite infected brain and suggest a role for TLRs in the interplay of immune cells and CNS cells during infection.

Brain homeostasis is normally protected by the blood brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCB), barriers that function in distinct CNS compartments and consist of different types of blood vessels including pial (subarachnoid spaces, leptomeninges), parenchymal (cerebral cortex) and ventricular vessels. In this study, a mouse model of neurocysticercosis was used to distinguish between changes in the permeability of the BBB and the BCB and determine the association of such alterations on leukocyte infiltration. Mice were intracranially infected with the parasite Mesocestoides corti and sacrificed at various times post infection. Different anatomical areas of infected brain were analyzed by three color immunofluoresence utilizing antibodies against serum proteins to assess brain barrier permeability, glial fibrillary acidic protein (GFAP) to detect astrocytes, and specific cell surface markers to determine the subpopulations of leukocytes infiltrating the CNS at particular sites. The results indicate increased permeability of all three types of vessels/structural sites as a result of infection evidenced by serum proteins and leukocyte extravasation but with considerable differences in the timing and extent of these permeability changes. Parenchymal vessels were the most resilient to changes in permeability whereas pial vessels were the least. Choroid plexus vessels of the ventricles also appeared less susceptible to increased permeability compared with pial vessels. In addition, parenchymal vessels appeared impermeable to particular types of immune cells even after extended periods of infection. Additionally, alterations in reactive astrocytes juxtaposed to blood vessels that exhibited increased permeability displayed increased expression of cytokines known to regulate brain barrier function. The results suggest that access of leukocytes and serum derived factors into the infected brain depend on several parameters including the anatomical area, type of vascular bed, cell phenotype and cytokine microenvironment.

In a recently developed mouse model for neurocysticercosis, the immune response was characterized by a massive influx of gammadelta T cells and a type 1 pathway of cytokine expression. To understand the role of gammadelta T cells during this infection, the cellular and cytokine response was analyzed in mice that lack gammadelta T cells (TCRdelta(-/-)). In TCRdelta(-/-) mice, Mesocestoides corti metacestodes preferentially invaded the extraparenchymal areas of the brain. Furthermore, parasites were able to escape from the brain and establish a systemic infection with liver and peritoneal involvement. Immunopathological studies indicated that TCRdelta(-/-) mice develop little inflammatory response and less neurological symptomatology. Significantly reduced numbers of T cells, macrophages, dendritic cells, and mast cells were present in the brain. The cytokine response in the brain of TCRdelta(-/-) mice appears to be a mixed type1/type 2 response with low levels of IL-2, IL-4, IL-10, IL-12, IL-13, IL-15, and IFN-gamma. To further investigate the immunological significance of this cell population, gammadelta T cells were adoptively transferred into intracranially infected TCRdelta(-/-) mice. gammadelta T cells were specifically recruited into the CNS in response to this parasitic infection, and they were able to target the infected brain within 12 h after transfer. These results suggest that gammadelta T cells are key players in the immune response elicited during this CNS infection and direct a type 1 response in wild-type mice upon infection.

Neurocysticercosis is the most common parasitic disease of the central nervous system worldwide. It is caused by the metacestode form of the helminth Taenia solium. Study of the immune response in the human brain has been limited by the chronic progression of the disease, the influence of corticosteroid treatment, and the scarcity of patients who undergo surgical intervention. To better understand the immune response and associated pathology in neurocysticercosis, a mouse model was developed by intracranial infection of BALB/c mice with Mesocestoides corti, a cestode organism related to T. solium. The immune response reveals the presence of abundant inflammatory infiltrates appearing as early as 2 days postinfection in extraparenchymal regions. In contrast, infiltration of immune cells into parenchymal tissue is significantly delayed. There is a natural progression of innate (neutrophils and macrophages), early induced (NK cells and gamma delta T cells), and adaptive immune responses (alpha beta T cells and B cells) in infected mice. Gamma delta T cells are the predominant T cell population. A cell-mediated Th1 pathway of cytokine expression is evident in contrast to the previously described Th2 phenotype induced in the periphery.