Changes of the enteric nervous system in amyloid-ß protein precursor transgenic mice correlate with disease progression.

In Alzheimer's disease (AD), fatal neuronal cell loss occurs long before relevant evidence can lead to a reliable diagnosis. If characteristic pathological alterations take place in the enteric nervous system (ENS), it could be one of the most promising targets for an early diagnosis, using submucosal biopsies from the gut. We therefore investigated time- and spatial-dependent changes in an amyloid-ß protein precursor (AßPP) overexpressing transgenic mouse model to examine early changes within the ENS. Wholemount preparations and paraffin sections were analyzed for the expression of neuronal, glial, and innate immunity markers. Isolated myenteric networks were screened for differences in overall protein expression, and a motility analysis delivered functional data. The level of AßPP in the gut was significantly higher in the AD mouse model than in wild-type mice and also higher in the gut than in the brain at all ages investigated. The transcriptional level of Nestin, GFAP, and TLR4 increased with age with a peak at 3 months. At the protein level, human amyloid-ß was located in myenteric neurons. Myenteric networks showed a reduction of the neuronal density in AßPP compared to wild-type mice, which was functionally relevant as revealed by motility analysis. The ENS undergoes significant changes during the early onset of AßPP expression in AD mouse models that appear before those seen in the brain as demonstrated in this study. Thus, there is a chance of determining similar alterations in the human gut of AD patients, which could be used to develop early diagnostic approaches.

Screening of innate immune receptors in neurodegenerative diseases: a similar pattern.

In Alzheimer's disease (AD), Parkinson's disease (PD), dementia with Lewy bodies (DLB) and amyotrophic lateral sclerosis (ALS), neuroinflammatory responses are considered to contribute to neuronal injury. Recently, the innate immune receptors, toll-like receptors (TLRs) and the LPS receptor (CD14) have been related to neurodegeneration. In this study, we systematically assessed the expression of most TLRs and CD14 in AD, PD/DLB and ALS using murine models of these diseases and human post-mortem brain tissues. A common upregulation of TLR2 and CD14 was found in all three animal models. While these two receptors could also be detected in AD patient tissues, they were absent from DLB and ALS tissues. This uniform pattern of innate immune response in animal models of neurodegenerative diseases clearly indicates that this response is part of a non-specific neuroinflammatory effector phase rather than a disease-specific event. The less dynamic disease progression in humans and the location (extracellular versus intracellular) of the aggregated proteins deposits might explain the divergent results seen between animal models and human tissues.

Moderately elevated plant sterol levels are associated with reduced cardiovascular risk--the LASA study.

Functional foods with supplementation of plant sterols are already used by millions of people. However, at the same time it is current scientific thinking that elevation of plant sterols in the circulation causes coronary heart disease. Therefore, this study aimed to define the risk for coronary heart disease associated with moderately high plant sterol plasma levels in a cohort of elderly. In this study, we evaluated the association between plant sterols and coronary heart disease in a cohort of 1242 subjects older than 65 years, participating at the Longitudinal Aging Study Amsterdam (LASA). Concentrations of sitosterol, campesterol, brassicasterol and stigmasterol were assessed using highly sensitive and specific gas chromatography-mass spectrometry-selected ion-monitoring. Plant sterol concentrations (and their ratios to cholesterol) were slightly, however, significantly lower in patients with coronary heart disease. Moreover, high plasma concentrations of a marker plant sterol, sitosterol, were associated with a markedly reduced risk for coronary heart disease (OR 0.78, CI 0.62-0.98, p<0.05). In contrast neither plant stanols (sitostanol or campestanol) nor the cholesterol synthesis markers (lathosterol, lanosterol and desmosterol) nor their ratios to cholesterol were significantly different in the study groups. These data suggest that plant sterols could have neutral or even protective effects on development of coronary heart disease, which have to be confirmed in interventional trials.

Role of the toll-like receptor 4 in neuroinflammation in Alzheimer's disease.

Microglial activation is a key feature in Alzheimer's disease and is considered to contribute to progressive neuronal injury by release of neurotoxic products. The innate immune receptor Toll-like-receptor 4 (TLR4), localized on the surface of microglia, is a first-line host defense receptor against invading microorganisms. Here, we show that a spontaneous loss-of-function mutation in the Tlr4 gene strongly inhibits microglial and monocytic activation by aggregated Alzheimer amyloid peptide resulting in a significantly lower release of the inflammatory products IL-6, TNFalpha and nitric oxide. Treatment of primary murine neuronal cells with supernatant of amyloid peptide-stimulated microglia demonstrates that Tlr4 contributes to amyloid peptide-induced microglial neurotoxicity. In addition, stimulation experiments in transfected HEK293 cells allowed to define a tri-molecular receptor complex consisting of TLR4, MD-2 and CD14 necessary for full cellular activation by aggregated amyloid peptide. A clinical relevance of these findings is supported by a marked upregulation of Tlr4 mRNA in APP transgenic mice and by an increased expression of TLR4 in Alzheimer's disease brain tissue associated with amyloid plaque deposition. Together, these observations provide the first evidence for a role of the key innate immune receptor, TLR4, in neuroinflammation in Alzheimer's disease.

Suppression of microglial inflammatory activity by myelin phagocytosis: role of p47-PHOX-mediated generation of reactive oxygen species.

Multiple sclerosis (MS) is pathologically characterized by inflammatory demyelination and neuronal injury. Although phagocytosis of myelin debris by microglia and macrophages in acute MS lesions is well documented, its pathophysiological significance is unclear. Using real-time quantitative PCR, flow cytometry, ELISA, and reactive oxygen species (ROS) measurement assays, we demonstrated that phagocytosis of myelin modulates activation of microglial cells prestimulated by interferon-gamma (IFN-gamma) or a combination of IFN-gamma and lipopolysaccharide with a biphasic temporal pattern, i.e., enhanced production of proinflammatory mediators during the first phase (< or = 6 h), followed by suppression during the second (6-24 h) phase. In this second phase, myelin phagocytosis leads to an enhanced release of prostaglandin E2 and ROS in microglia, whereas the production of anti-inflammatory cytokines (particularly interleukin-10) remains unchanged. Suppression of inflammatory microglial activation by myelin phagocytosis was reversed by treatment with superoxide dismutase and catalase, by inhibition of the NADPH-oxidase complex, or by specific knockdown of the NADPH-oxidase-required adaptor p47-phagocyte oxidase (PHOX). Furthermore, we observed that myelin phagocytosis destabilized tumor necrosis factor-alpha and interferon-induced protein-10 mRNA through an adenine-uridine-rich elements-involved mechanism, which was reversed by blocking the function of NADPH-oxidase complex. We conclude that phagocytosis of myelin suppresses microglial inflammatory activities via enhancement of p47-PHOX-mediated ROS generation. These results suggest that intervention in ROS generation could represent a novel therapeutic strategy to reduce neuroinflammation in MS.

The LPS receptor, CD14, in experimental autoimmune encephalomyelitis and multiple sclerosis.

Innate immune receptors are crucial for defense against microorganisms. Recently, a cross-talk between innate and adaptive immunity has been considered. Here, we provide first evidence for a role of the key innate immune receptor, LPS receptor (CD14) in pathophysiology of experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis. Indicating a functional importance in vivo, we show that CD14 deficiency increased clinical symptoms in active experimental autoimmune encephalomyelitis. Consistent with these observations, CD14 deficient mice exhibited a markedly enhanced infiltration of monocytes and neutrophils in brain and spinal cord. Moreover, we observed an increased immunoreactivity of CD14 in biopsy and post mortem brain tissues of multiple sclerosis patients compared to age-matched controls. Thus, the key innate immune receptor, CD14, may be of pathophysiological relevance in experimental autoimmune encephalomyelitis and multiple sclerosis.

Toll-like receptor 2 deficiency delays pneumococcal phagocytosis and impairs oxidative killing by granulocytes.

Phagocytosis and killing of Streptococcus pneumoniae was compared in blood-derived wild-type (WT) and Toll-like receptor 2 (TLR2)-deficient (TLR2-/-) polymorphonuclear leukocytes (PMN). Phagocytosis of green fluorescent protein-transformed pneumococci was delayed in TLR2-/- PMN. These cells exhibited also a lower oxidative bactericidal activity against S. pneumoniae than WT PMN, suggesting that TLR2 modulates bacterial clearance in PMN.

Regulation of Streptococcus pneumoniae distribution by Toll-like receptor 2 in vivo.

The phagocyte pattern recognition receptor Toll-like receptor 2 (TLR2) and the multi-receptor adaptor MyD88 contribute to the reduction of bacterial load in infections with intra- and extra-cellular Gram-positive bacteria. Their mechanism of antibacterial action is mostly unresolved but evident in vivo by an increased pathogen burden in infected TLR2-/- and MyD88-/- compared to C57BL/6 wild type (wt) mice. We had previously observed higher bacterial numbers in brains of TLR2-/- than of wt mice with meningitis. Here we study bacteria-phagocyte interaction by comparing S. pneumoniae distribution and localization in wt and TLR2-/- brain by confocal microscopy using a green fluorescent protein-transformed encapsulated S. pneumoniae (C5017). Colony-forming units were similarly distributed in TLR2-/- and wt mice and exclusively localized in meninges and ventricles. Bacteria were more abundant in ventricles, in and around TLR2-/- than wt GLT1v+ plexus choroideus epithelial cells. S. pneumoniae were also found in and around Gr-1+ granulocytes, but never in F4/80+ macrophages, Iba1+ microglia, GFAP+ astrocytes, Meca-31+ endothelial cells or Neun+ neurons of either mouse strain. The results indicate that TLR2 does not change bacterial distribution, but may contribute to antibacterial defense by modulating S. pneumoniae adherence and uptake in plexus epithelia.

Toll-like receptor-2 deficiency is associated with enhanced brain TNF gene expression during pneumococcal meningitis.

TNF is a marker of disease activity in bacterial meningitis. To investigate TNF modulation by Toll-like receptor-2 (TLR2), we studied temporal and anatomical expression patterns of TLR2 and TNF in a pneumococcal meningitis model in wild type (wt) and TLR2(-/-) mice. We show by in situ hybridization that transcripts of TLR2 and of the comolecules CD14, MD-2, TLR1/6 strongly increased and colocalized with TNF in CD45-positive infiltrating cells in the ventricles, corpus callosum and the meninges. TNF gene and protein expression was stronger in TLR2(-/-) than wt brains and associated with increased IkappaB expression suggesting that TLR2 is controlling inflammation via TNF regulation.

CD14 deficiency leads to increased MIP-2 production, CXCR2 expression, neutrophil transmigration, and early death in pneumococcal infection.

CD14 is a myeloid receptor for bacterial cell membrane/wall components, for which we previously showed a strong induction in cerebrospinal fluid (CSF) during meningitis. Here, we studied CD14 function in murine Streptococcus pneumoniae meningitis by using wild-type (WT), CD14(-/-) mice, and WT mice pretreated with neutralizing anti-CD14 antibodies. Early polymorphonuclear leukocytes (PMN) immigration was more pronounced in CSF of CD14(-/-) than of WT mice. This was not a result of altered adherence molecule expression in blood and CSF PMN or brain endothelial cells. Macrophage inflammatory protein-2 (MIP-2) and keratinocyte-derived chemokine levels were similar in CSF in both strains, but MIP-2 was higher in infected brain and in brain-derived endothelial cells infected in vitro in CD14(-/-) than in WT mice. CD14(-/-) PMN demonstrated increased expression of CXC chemokine receptor 2 (CXCR2) after infection and stronger in vitro chemotaxis than WT PMN toward CSF from WT or CD14(-/-) mice and toward MIP-2. Excess PMN migration in CD14(-/-) mice did not result in improved bacterial clearing but in increased tumor necrosis factor in CSF, higher disease severity, and earlier death. Pretreatment with anti-CXCR2 reduced PMN infiltration into CSF and brain MIP-2 production and abolished earlier mortality in CD14(-/-) mice. In conclusion, CD14 plays a protective role in pneumococcal meningitis by slowing PMN migration via MIP-2 and CXCR2 modulation.

LPS receptor (CD14): a receptor for phagocytosis of Alzheimer's amyloid peptide.

The amyloid beta peptide 42 (Abeta(42)) plays a key role in neurotoxicity in Alzheimer's disease. Mononuclear phagocytes, i.e. microglia, have the potential to clear Abeta by phagocytosis. Recently, the lipopolysaccharide (LPS) receptor CD14 was shown to mediate phagocytosis of bacterial components and furthermore to contribute to neuroinflammation in Alzheimer's disease. Here, we investigated whether this key innate immunity receptor can interact with Abeta(42) and mediate phagocytosis of this peptide. Using flow cytometry, confocal microscopy and two-photon fluorescence lifetime imaging (FLIM) combined with fluorescence resonance energy transfer (FRET), we demonstrated a direct molecular interaction in the range of a few nanometers between Abeta(42) and CD14 in human CD14-transfected Chinese hamster ovary cells. Investigations using cells that were genetically deficient for this receptor showed that in <30 minutes exogenous Abeta(42) added to cultured primary microglial cells was phagocytosed into the cytoplasmic compartment in a CD14-dependent manner. This phagocytosis occurred at Abeta(42) concentration ranges that were considerably lower than the threshold to activate a cellular inflammatory reaction. In contrast, there was no association of CD14 to microglial internalization of microbeads. In complementary clinical experiments, we detected a pronounced CD14 immunoreactivity on parenchymal microglia spatially correlated to characteristic Alzheimer's disease lesion sites in brain sections of Alzheimer's disease patients but not in brain sections of control subjects. By showing a close interaction between CD14 and Abeta(42), demonstrating a direct role of CD14 in Abeta(42) phagocytosis, and detecting CD14-specific staining in brains of Alzheimer's disease patients, our results indicate a role of the LPS receptor in the pathophysiology of Alzheimer's disease, which could be of therapeutic relevance.

Generation and functional characterization of a clonal murine periportal Kupffer cell line from H-2Kb -tsA58 mice.

Murine Kupffer cells (KCs) are heterogeneous and survive only for a short time in vitro. Here, a clonal, murine KC line was generated from transgenic mice, expressing the thermolabile mutant tsA58 of the Simian virus 40 large T antigen under the control of the H-2K(b) promoter. Thirty-three degrees Celsius and 37 degrees C but not 39 degrees C have been permissive for growth of the clone; it required conditioned media from hepatocytes and endothelial cells for proliferation. In contrast to primary cells, the cells of the clone were uniform, survived detachment, and could therefore be analyzed by cytofluorimetry. The clone, as primary KCs, constitutively expressed nonspecific esterase, peroxidase, MOMA-2, BM8, scavenger receptor A, CD14, and Toll-like receptor 4 (TLR4); the antigen-presenting molecules CD40, CD80, and CD1d; and endocytosed dextran-fluorescein isothiocyanate. It lacked complement, Fc receptors, F4/80 marker, and the phagosomal coat protein tryptophan aspartate-containing coat protein (TACO). The clone exhibited CD14- and TLR4/MD2-independent, plasma-dependent lipopolysaccharide (LPS) binding, Escherichia coli and Streptococcus pneumoniae phagocytosis, and LPS- and interferon-gamma-induced NO production but no tumor necrosis factor alpha, interleukin (IL)-6, or IL-10 release. The large size, surface-marker expression, and capacity to clear gram-negative and -positive bacteria indicate that the clone was derived from the periportal, large KC subpopulation. The clone allows molecular studies of anti-infective and immune functions of KCs.