Lymph node formation and B cell homeostasis require IKK-α in distinct endothelial cell-derived compartments.

Global inactivation of IκB kinase (IKK)-α results in defective lymph node (LN) formation and B cell maturation, and loss of IKK-α-dependent noncanonical NF-κB signaling in stromal organizer and hematopoietic cells is thought to underlie these distinct defects. We previously demonstrated that this pathway is also activated in vascular endothelial cells (ECs). To determine the physiologic function of EC-intrinsic IKK-α, we crossed IkkαF/F mice with Tie2-cre or Cdh5-cre mice to ablate IKK-α in ECs. Notably, the compound defects of global IKK-α inactivation were recapitulated in IkkαTie2 and IkkαCdh5 mice, as both lacked all LNs and mature follicular and marginal zone B cell numbers were markedly reduced. However, as Tie2-cre and Cdh5-cre are expressed in all ECs, including blood forming hemogenic ECs, IKK-α was also absent in hematopoietic cells (HC). To determine if loss of HC-intrinsic IKK-α affected LN development, we generated IkkαVav mice lacking IKK-α in only the hematopoietic compartment. While mature B cell numbers were significantly reduced in IkkαVav mice, LN formation was intact. As lymphatic vessels also arise during development from blood ECs, we generated IkkαLyve1 mice lacking IKK-α in lymphatic ECs (LECs) to determine if IKK-α in lymphatic vessels impacts LN development. Strikingly, while mature B cell numbers were normal, LNs were completely absent in IkkαLyve1 mice. Thus, our findings reveal that IKK-α in distinct EC-derived compartments is uniquely required to promote B cell homeostasis and LN development, and we establish that LEC-intrinsic IKK-α is absolutely essential for LN formation.

Meningeal B Cell Clusters Correlate with Submeningeal Pathology in a Natural Model of Multiple Sclerosis.

Multiple sclerosis (MS) is an idiopathic demyelinating disease in which meningeal inflammation correlates with accelerated disease progression. The study of meningeal inflammation in MS has been limited because of constrained access to MS brain/spinal cord specimens and the lack of experimental models recapitulating progressive MS. Unlike induced models, a spontaneously occurring model would offer a unique opportunity to understand MS immunopathogenesis and provide a compelling framework for translational research. We propose granulomatous meningoencephalomyelitis (GME) as a natural model to study neuropathological aspects of MS. GME is an idiopathic, progressive neuroinflammatory disease of young dogs with a female bias. In the GME cases examined in this study, the meninges displayed focal and disseminated leptomeningeal enhancement on magnetic resonance imaging, which correlated with heavy leptomeningeal lymphocytic infiltration. These leptomeningeal infiltrates resembled tertiary lymphoid organs containing large B cell clusters that included few proliferating Ki67+ cells, plasma cells, follicular dendritic/reticular cells, and germinal center B cell-like cells. These B cell collections were confined in a specialized network of collagen fibers associated with the expression of the lympho-organogenic chemokines CXCL13 and CCL21. Although neuroparenchymal perivascular infiltrates contained B cells, they lacked the immune signature of aggregates in the meningeal compartment. Finally, meningeal B cell accumulation correlated significantly with cortical demyelination reflecting neuropathological similarities to MS. Hence, during chronic neuroinflammation, the meningeal microenvironment sustains B cell accumulation that is accompanied by underlying neuroparenchymal injury, indicating GME as a novel, naturally occurring model to study compartmentalized neuroinflammation and the associated pathology thought to contribute to progressive MS.

Netrin 1 regulates blood-brain barrier function and neuroinflammation.

Blood-brain barrier function is driven by the influence of astrocyte-secreted factors. During neuroinflammatory responses the blood-brain barrier is compromised resulting in central nervous system damage and exacerbated pathology. Here, we identified endothelial netrin 1 induction as a vascular response to astrocyte-derived sonic hedgehog that promotes autocrine barrier properties during homeostasis and increases with inflammation. Netrin 1 supports blood-brain barrier integrity by upregulating endothelial junctional protein expression, while netrin 1 knockout mice display disorganized tight junction protein expression and barrier breakdown. Upon inflammatory conditions, blood-brain barrier endothelial cells significantly upregulated netrin 1 levels in vitro and in situ, which prevented junctional breach and endothelial cell activation. Finally, netrin 1 treatment during experimental autoimmune encephalomyelitis significantly reduced blood-brain barrier disruption and decreased clinical and pathological indices of disease severity. Our results demonstrate that netrin 1 is an important regulator of blood-brain barrier maintenance that protects the central nervous system against inflammatory conditions such as multiple sclerosis and experimental autoimmune encephalomyelitis.

Immunologic privilege in the central nervous system and the blood-brain barrier.

The brain is in many ways an immunologically and pharmacologically privileged site. The blood-brain barrier (BBB) of the cerebrovascular endothelium and its participation in the complex structure of the neurovascular unit (NVU) restrict access of immune cells and immune mediators to the central nervous system (CNS). In pathologic conditions, very well-organized immunologic responses can develop within the CNS, raising important questions about the real nature and the intrinsic and extrinsic regulation of this immune privilege. We assess the interactions of immune cells and immune mediators with the BBB and NVU in neurologic disease, cerebrovascular disease, and intracerebral tumors. The goals of this review are to outline key scientific advances and the status of the science central to both the neuroinflammation and CNS barriers fields, and highlight the opportunities and priorities in advancing brain barriers research in the context of the larger immunology and neuroscience disciplines. This review article was developed from reports presented at the 2011 Annual Blood-Brain Barrier Consortium Meeting.

Mesocestoides corti intracranial infection as a murine model for neurocysticercosis.

Neurocysticercosis (NCC) is the most common parasitic disease of the central nervous system (CNS) caused by the larval form of the tapeworm Taenia solium. NCC has a long asymptomatic period with little or no inflammation, and the sequential progression to symptomatic NCC depends upon the intense inflammation associated with degeneration of larvae. The mechanisms involved in these progressive events are difficult to study in human patients. Thus it was necessary to develop an experimental model that replicated NCC. In this review, we describe studies of a murine model of NCC in terms of the release/secretion of parasite antigens, immune responses elicited within the CNS environment and subsequent pathogenesis. In particular, the kinetics of leukocyte subsets infiltrating into the brain are discussed in the context of disruption of the CNS barriers at distinct anatomical sites and the mechanisms contributing to these processes. In addition, production of various inflammatory mediators and the mechanisms involved in their induction by the Toll-like receptor signaling pathway are described. Overall, the knowledge gained from the mouse model of NCC has provided new insights for understanding the kinetics of events contributing to different stages of NCC and should aid in the formulation of more effective therapeutic approaches.

Doxycycline treatment decreases morbidity and mortality of murine neurocysticercosis: evidence for reduction of apoptosis and matrix metalloproteinase activity.

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.

Multiple expression of matrix metalloproteinases in murine neurocysticercosis: Implications for leukocyte migration through multiple central nervous system barriers.

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.

Differential release and phagocytosis of tegument glycoconjugates in neurocysticercosis: implications for immune evasion strategies.

Neurocysticercosis (NCC) is an infection of the central nervous system (CNS) by the metacestode of the helminth Taenia solium. The severity of the symptoms is associated with the intensity of the immune response. First, there is a long asymptomatic period where host immunity seems incapable of resolving the infection, followed by a chronic hypersensitivity reaction. Since little is known about the initial response to this infection, a murine model using the cestode Mesocestoides corti (syn. Mesocestoides vogae) was employed to analyze morphological changes in the parasite early in the infection. It was found that M. corti material is released from the tegument making close contact with the nervous tissue. These results were confirmed by infecting murine CNS with ex vivo-labeled parasites. Because more than 95% of NCC patients exhibit humoral responses against carbohydrate-based antigens, and the tegument is known to be rich in glycoconjugates (GCs), the expression of these types of molecules was analyzed in human, porcine, and murine NCC specimens. To determine the GCs present in the tegument, fluorochrome-labeled hydrazides as well as fluorochrome-labeled lectins with specificity to different carbohydrates were used. All the lectins utilized labeled the tegument. GCs bound by isolectinB4 were shed in the first days of infection and not resynthesized by the parasite, whereas GCs bound by wheat germ agglutinin and concavalinA were continuously released throughout the infectious process. GCs bound by these three lectins were taken up by host cells. Peanut lectin-binding GCs, in contrast, remained on the parasite and were not detected in host cells. The parasitic origin of the lectin-binding GCs found in host cells was confirmed using antibodies against T. solium and M. corti. We propose that both the rapid and persistent release of tegumental GCs plays a key role in the well-known immunomodulatory effects of helminths, including immune evasion and life-long inflammatory sequelae seen in many NCC patients.

Evidence for differential changes of junctional complex proteins in murine neurocysticercosis dependent upon CNS vasculature.

The delicate balance required to maintain homeostasis of the central nervous system (CNS) is controlled by the blood-brain barrier (BBB). Upon injury, the BBB is disrupted compromising the CNS. BBB disruption has been represented as a uniform event. However, our group has shown in a murine model of neurocysticercosis (NCC) that BBB disruption varies depending upon the anatomical site/vascular bed analyzed. In this study further understanding of the mechanisms of BBB disruption was explored in blood vessels located in leptomeninges (pial vessels) and brain parenchyma (parenchymal vessels) by examining the expression of junctional complex proteins in murine brain infected with Mesocestoides corti. Both pial and parenchymal vessels from mock infected animals showed significant colocalization of junctional proteins and displayed an organized architecture. Upon infection, the patterned organization was disrupted and in some cases, particular tight junction and adherens junction proteins were undetectable or appeared to be undergoing proteolysis. The extent and timing of these changes differed between both types of vessels (pial vessel disruption within days versus weeks for parenchymal vessels). To approach potential mechanisms, the expression and activity of matrix metalloproteinase-9 (MMP-9) were evaluated by in situ zymography. The results indicated an increase in MMP-9 activity at sites of BBB disruption exhibiting leukocyte infiltration. Moreover, the timing of MMP activity in pial and parenchymal vessels correlated with the timing of permeability disruption. Thus, breakdown of the BBB is a mutable process despite the similar structure of the junctional complex between pial and parenchymal vessels and involvement of MMP activity.

Differential changes in junctional complex proteins suggest the ependymal lining as the main source of leukocyte infiltration into ventricles in murine neurocysticercosis.

The blood brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCB) limit the influx of immune mediators and bloodstream compounds into the central nervous system (CNS). Upon injury or infection, the integrity of these barriers is compromised and leukocyte infiltration occurs. The BCB is located in the choroid plexuses (CPs) found within ventricles of the brain, and it is considered one of the main routes of cellular infiltration into the CNS into healthy individuals. Our group recently showed that in a murine model of neurocysticercosis (NCC), there is a moderate increase in infiltration of leukocytes into ventricles, but the BCB is hardly compromised. To elucidate the role played by CPs and surrounding ependyma in leukocyte infiltration at ventricular sites, we analyzed changes in the expression of junctional complex proteins in animals intracranially infected with Mesocestoides corti. The results indicate that infection does not change the expression pattern of junctional complex proteins in CPs, but structural alterations and disappearance of these proteins were evident in ependyma adjacent to the internal leptomeninges. The kinetics and magnitude of these changes directly correlated with the extent of leukocyte infiltration through ependyma and with the expression and activity of MMPs. The results of this study indicate that the anatomical elements of the BCB are minimally disrupted during the course of murine NCC. Thus, most of the leukocytes infiltrating ventricles appear to extravasate through pial vessels located in the internal leptomeninges juxtaposed to the ependyma layer and then traverse the ependyma cells. In addition, MMP activity seems to be involved in this process. These results provide evidence for a previously undescribed entry route for leukocytes into the CNS.

Breakdown of the blood brain barrier and blood-cerebrospinal fluid barrier is associated with differential leukocyte migration in distinct compartments of the CNS during the course of murine NCC.

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.

The inflammatory cell infiltrates in porcine cysticercosis: immunohistochemical analysis during various stages of infection.

Taenia solium metacestodes cause cysticercosis in both humans and pigs. In the former host species, the central nervous system involvement (neurocysticercosis (NCC)) may range from asymptomatic to life-threatening, but little is known about the corresponding variation in tissue response due to the difficulty in obtaining parasite-infected brain biopsies. The use of pigs as animal models for cysticercosis is ideal because the histological description of the animal's response around the parasites resembles the one recorded in human specimens. In this study the histological features of the immune response in swine were complemented by immunohistochemical analysis to determine the phenotype of the inflammatory cells. The presence of mononuclear cells and eosinophils, and the co-localization of MHC-II with B lymphocytes and monocytes/macrophages within the granulomas surrounding the parasites, were features that closely resembled the descriptions made in prior studies with human specimens. In addition, there were novel findings such as the upregulation of the adhesin CD44 in cells resembling monocytes/macrophages, eosinophils and in astrocytes from the central nervous system. The upregulation of CD44 may be important for the recruitment of inflammatory cells to the site of the lesion. Finally, the presence of null-gamma delta-T cells since stage I of the immune response was similar to the early detection of these cells in mouse models for cysticercosis.