Ultrastructure and pathology of prion protein amyloid accumulation and cellular damage in extraneural tissues of scrapie-infected transgenic mice expressing anchorless prion protein

In most human and animal prion diseases the abnormal disease-associated prion protein (PrPSc) is deposited as non-amyloid aggregates in CNS, spleen and lymphoid organs. In contrast, in humans and transgenic mice with PrP mutations which cause expression of PrP lacking a glycosylphosphatidylinositol (GPI)-anchor, most PrPSc is in the amyloid form. In transgenic mice expressing only anchorless PrP (tg anchorless), PrPSc is deposited not only in CNS and lymphoid tissues, but also in extraneural tissues including heart, brown fat, white fat, and colon. In the present paper, we report ultrastructural studies of amyloid PrPSc deposition in extraneural tissues of scrapie-infected tg anchorless mice. Amyloid PrPSc fibrils identified by immunogold-labeling were visible at high magnification in interstitial regions and around blood vessels of heart, brown fat, white fat, colon, and lymphoid tissues. PrPSc amyloid was located on and outside the plasma membranes of adipocytes in brown fat and cardiomyocytes, and appeared to invaginate and disrupt the plasma membranes of these cell types, suggesting cellular damage. In contrast, no cellular damage was apparent near PrPSc associated with macrophages in lymphoid tissues and colon, with enteric neuronal ganglion cells in colon or with adipocytes in white fat. PrPSc localized in macrophage phagolysosomes lacked discernable fibrils and might be undergoing degradation. Furthermore, in contrast to wild-type mice expressing GPI-anchored PrP, in lymphoid tissues of tg anchorless mice, PrPSc was not associated with follicular dendritic cells (FDC), and FDC did not display typical prion-associated pathogenic changes.     

Systemic inflammation and delirium: important co-factors in the progression of dementia

It is widely accepted that inflammation plays some role in the progression of chronic neurodegenerative diseases such as AD (Alzheimer's disease), but its precise role remains elusive. It has been known for many years that systemic inflammatory insults can signal to the brain to induce changes in CNS (central nervous system) function, typically grouped under the syndrome of sickness behaviour. These changes are mediated via systemic and CNS cytokine and prostaglandin synthesis. When patients with dementia suffer similar systemic inflammatory insults, delirium is a frequent consequence. This profound and acute exacerbation of cognitive dysfunction is associated with poor prognosis: accelerating cognitive decline and shortening time to permanent institutionalization and death. Therefore a better understanding of how delirium occurs during dementia and how these episodes impact on existing neurodegeneration are now important priorities. The current review summarizes the relationship between dementia, systemic inflammation and episodes of delirium and addresses the basic scientific approaches currently being pursued with respect to understanding acute cognitive dysfunction during aging and dementia. In addition, despite there being limited studies on this subject, it is becoming increasingly clear that infections and other systemic inflammatory conditions do increase the risk of AD and accelerate the progression of established dementia. These data suggest that systemic inflammation is a major contributor to the progression of dementia and constitutes an important clinical target.     

Prions and the proteasome

Prion diseases are fatal neurodegenerative disorders that include Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy in animals. They are unique in terms of their biology because they are caused by the conformational re-arrangement of a normal host-encoded prion protein, PrPC, to an abnormal infectious isoform, PrPSc. Currently the precise mechanism behind prion-mediated neurodegeneration remains unclear. It is hypothesised than an unknown toxic gain of function of PrPSc, or an intermediate oligomeric form, underlies neuronal death. Increasing evidence suggests a role for the ubiquitin proteasome system (UPS) in prion disease. Both wild-type PrPC and disease-associated PrP isoforms accumulate in cells after proteasome inhibition leading to increased cell death, and abnormal beta-sheet-rich PrP isoforms have been shown to inhibit the catalytic activity of the proteasome. Here we review potential interactions between prions and the proteasome outlining how the UPS may be implicated in prion-mediated neurodegeneration.     

Prion Strain Differences in Accumulation of PrPSc on Neurons and Glia Are Associated with Similar Expression Profiles of Neuroinflammatory Genes: Comparison of Three Prion Strains

Misfolding and aggregation of host proteins are important features of the pathogenesis of neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, frontotemporal dementia and prion diseases. In all these diseases, the misfolded protein increases in amount by a mechanism involving seeded polymerization. In prion diseases, host prion protein is misfolded to form a pathogenic protease-resistant form, PrPSc, which accumulates in neurons, astroglia and microglia in the CNS. Here using dual-staining immunohistochemistry, we compared the cell specificity of PrPSc accumulation at early preclinical times post-infection using three mouse scrapie strains that differ in brain regional pathology. PrPSc from each strain had a different pattern of cell specificity. Strain 22L was mainly associated with astroglia, whereas strain ME7 was mainly associated with neurons and neuropil. In thalamus and cortex, strain RML was similar to 22L, but in substantia nigra, RML was similar to ME7. Expression of 90 genes involved in neuroinflammation was studied quantitatively using mRNA from thalamus at preclinical times. Surprisingly, despite the cellular differences in PrPSc accumulation, the pattern of upregulated genes was similar for all three strains, and the small differences observed correlated with variations in the early disease tempo. Gene upregulation correlated with activation of both astroglia and microglia detected in early disease prior to vacuolar pathology or clinical signs. Interestingly, the profile of upregulated genes in scrapie differed markedly from that seen in two acute viral CNS diseases (LaCrosse virus and BE polytropic Friend retrovirus) that had reactive gliosis at levels similar to our prion-infected mice.     

Different therapeutic outcomes in experimental allergic encephalomyelitis dependent upon the mode of delivery of IL-10: a comparison of the effects of protein, adenoviral or retroviral IL-10 delivery into the central nervous system

Experimental allergic encephalomyelitis (EAE) is a CNS autoimmune disease mediated by the action of CD4(+) T cells, macrophages, and proinflammatory cytokines. IL-10 is a cytokine shown to have many anti-inflammatory properties. Studies have shown both inhibition and exacerbation of EAE after systemic IL-10 protein administration. We have compared the inhibitory effect in EAE of Il10 gene delivery in the CNS. Fibroblasts transduced with retroviral vectors expressing IL-10 could inhibit EAE. This was not associated with a prevention of cellular recruitment but an alteration in their phenotype, notably an increase in the numbers of CD8(+) T and B cells. In marked contrast, CNS delivery of adenovirus coding for mouse IL-10 or IL-10 protein performed over a wide dose range failed to inhibit disease, despite producing similar or greater amounts of IL-10 protein. Thus the action of IL-10 may differ depending on the local cytokine microenvironment produced by the gene-secreting cell types.     

Autocrine Regulation of Macrophage Activation via Exocytosis of ATP and Activation of P2Y11 Receptor

It is important to understand the mechanisms that regulate macrophage activation to establish novel therapies for inflammatory diseases, such as sepsis; a systemic inflammatory response syndrome generally caused by bacterial lipopolysaccharide (LPS). In this study, we investigated the involvement of extracellular ATP-mediated autocrine signaling in LPS-induced macrophage activation. We show here that ATP release via exocytosis, followed by activation of P2Y11 receptor, is a major pathway of the macrophage activation, leading to release of cytokines. Treatment of human monocyte THP-1 cells with LPS induced rapid ATP release from cells, and this release was blocked by knockdown of SLC17A9 (vesicular nucleotide transporter, VNUT), which is responsible for exocytosis of ATP. ATP-enriched vesicles were found in cytosol of THP-1 cells. These data suggest the involvement of vesicular exocytosis in the release of ATP. Knockdown of SLC17A9, the P2Y11 antagonist NF157 or knockdown of P2Y11 receptor significantly suppressed both M1-type polarization and IL-6 production in THP-1 cells, indicating an important role of activation of P2Y11 receptor by released ATP in macrophage activation. Next, the effect of NF157 on LPS-induced immune activation was examined in vivo. Administration of LPS to mice caused increase of serum IL-1ß, IL-6, IL-12 and TNF-alpha levels at 3–24 h after the administration. Pre-treatment of LPS-treated mice with NF157 suppressed both elevation of proinflammatory cytokines in serum and M1 polarization of peritoneal/spleen macrophages. Moreover, post-treatment with NF157 at 30 min after administration of LPS also suppressed the elevation of serum cytokines levels. We conclude that vesicular exocytosis of ATP and autocrine, positive feedback through P2Y11 receptors is required for the effective activation of macrophages. Consequently, P2Y11 receptor antagonists may be drug candidates for treatment of inflammatory diseases such as sepsis.     

Prion Peptide Uptake in Microglial Cells – The Effect of Naturally Occurring Autoantibodies against Prion Protein

In prion disease, a profound microglial activation that precedes neurodegeneration has been observed in the CNS. It is still not fully elucidated whether microglial activation has beneficial effects in terms of prion clearance or whether microglial cells have a mainly detrimental function through the release of pro-inflammatory cytokines. To date, no disease-modifying therapy exists. Several immunization attempts have been performed as one therapeutic approach. Recently, naturally occurring autoantibodies against the prion protein (nAbs-PrP) have been detected. These autoantibodies are able to break down fibrils of the most commonly used mutant prion variant PrP106-126 A117V and prevent PrP106-126 A117V-induced toxicity in primary neurons. In this study, we examined the phagocytosis of the prion peptide PrP106-126 A117V by primary microglial cells and the effect of nAbs-PrP on microglia. nAbs-PrP considerably enhanced the uptake of PrP106-126 A117V without inducing an inflammatory response in microglial cells. PrP106-126 A117V uptake was at least partially mediated through scavenger receptors. Phagocytosis of PrP106-126 A117V with nAbs-PrP was inhibited by wortmannin, a potent phosphatidylinositol 3-kinase inhibitor, indicating a separate uptake mechanism for nAbs-PrP mediated phagocytosis. These data suggest the possible mechanisms of action of nAbs-PrP in prion disease.     

Serum amyloid A, cytokines, and corticosterone responses in germfree and conventional mice after lipopolysaccharide injection.

To determine why germfree mice are less susceptible to lipopolysaccharide (LPS) than conventional mice, we studied serum levels of serum amyloid A (SAA), tumor necrosis factor (TNF), interleukin 1 (IL-1), IL-6, and corticosterone in mice after treatment with LPS. A single injection of LPS caused an elevation of SAA, an acute-phase protein in the mouse, in both conventional and germfree IQI mice, and the response was significantly less in germfree mice. LPS-induced elevations of serum TNF, IL-1, and IL-6 levels were also significantly less in germfree mice, while serum corticosterone levels were greater in germfree mice than in conventional mice. These results suggest that the lower susceptibility to LPS and a smaller response of SAA elevation by LPS in germfree mice may result from less elevation in serum of these cytokines in these mice, which are known to mediate the acute phase response of SAA. High levels of serum corticosterone in germfree mice may be partly responsible for the lower responsiveness of these inflammatory cytokines to LPS in these mice.     

Spleen-Derived Interleukin-10 Downregulates the Severity of High-Fat Diet-Induced Non-Alcoholic Fatty Pancreas Disease

Obesity is associated with systemic low-grade inflammation and is a risk factor for non-alcoholic fatty pancreas disease (NAFPD), but the molecular mechanisms of these associations are not clear. Interleukin (IL)-10, a potent anti-inflammatory cytokine, is released during acute pancreatitis and is known to limit inflammatory responses by downregulating the release of proinflammatory mediators. The origin of IL-10 that suppresses pancreatitis has not been investigated. Since obesity is known to reduce expression of proinflammatory cytokines in the spleen, we examined whether spleen-derived IL-10 regulates NAFPD caused by high-fat (HF) diet-induced obesity. The following investigations were performed: 1) IL-10 induction from spleen was examined in male mice fed a HF diet; 2) triglyceride content, expression of pro- and anti-inflammatory cytokines and infiltration of M1 and M2 macrophages were determined to evaluate ectopic fat accumulation and inflammatory responses in the pancreas of splenectomy (SPX)-treated mice fed HF diet; 3) exogenous IL-10 was systemically administered to SPX-treated obese mice and the resulting pathogenesis caused by SPX was assessed; and 4) IL-10 knockout (IL-10KO) mice were treated with SPX and ectopic fat deposition and inflammatory conditions in the pancreas were investigated. Obesity impaired the ability of the spleen to synthesize cytokines, including IL-10. SPX aggravated fat accumulation and inflammatory responses in the pancreas of HF diet-induced obese mice and these effects were inhibited by systemic administration of IL-10. Moreover, SPX had little effect on fat deposition and inflammatory responses in the pancreas of IL-10KO mice. Our findings indicate that obesity reduces IL-10 production by the spleen and that spleen-derived IL-10 may protect against the development of NAFPD.     

Inhibiting adenosine deaminase modulates the systemic inflammatory response syndrome in endotoxemia and sepsis

By pharmacological manipulation of endogenous adenosine, using chemically distinct methods, we tested the hypothesis that endogenous adenosine tempers proinflammatory cytokine responses and oxyradical-mediated tissue damage during endotoxemia and sepsis. Rats were pretreated with varying doses of pentostatin (PNT; adenosine deaminase inhibitor) or 8-sulfophenyltheophylline (8-SPT; adenosine receptor antagonist) and then received either E. coli endotoxin (lipopolysaccharide; 0.01 or 2.0 mg/kg) or a slurry of cecal matter in 5% dextrose in water (200 mg/kg). Resultant levels of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-10 were measured in serum and in liver and spleen. Untreated, 2 mg/kg lipopolysaccharide elevated serum TNF-alpha, IL-1beta, and IL-10. PNT dose dependently attenuated, without ablating, the elevation in serum TNF-alpha and IL-1beta and raised liver and spleen IL-10. PNT also attenuated elevation of TNF-alpha in serum, liver, and spleen at 4 and 24 h after sepsis induction, and 8-SPT resulted in higher proinflammatory cytokines. Modulating endogenous adenosine was also effective in exacerbated (8-SPT) or diminished (PNT) tissue peroxidation. Survival from sepsis was also improved when PNT was used as a posttreatment. These data indicate that endogenous adenosine is an important modulatory component of systemic inflammatory response syndromes. These data also indicate that inhibition of adenosine deaminase may be a novel and viable therapeutic approach to managing the systemic inflammatory response syndrome without ablating important physiological functions.     

Splenectomy exacerbates lung injury after ischemic acute kidney injury in mice

Patients with acute kidney injury (AKI) have increased serum proinflammatory cytokines and an increased occurrence of respiratory complications. The aim of the present study was to examine the effect of renal and extrarenal cytokine production on AKI-mediated lung injury in mice. C57Bl/6 mice underwent sham surgery, splenectomy, ischemic AKI, or ischemic AKI with splenectomy and kidney, spleen, and liver cytokine mRNA, serum cytokines, and lung injury were examined. The proinflammatory cytokines IL-6, CXCL1, IL-1β, and TNF-α were increased in the kidney, spleen, and liver within 6 h of ischemic AKI. Since splenic proinflammatory cytokines were increased, we hypothesized that splenectomy would protect against AKI-mediated lung injury. On the contrary, splenectomy with AKI resulted in increased serum IL-6 and worse lung injury as judged by increased lung capillary leak, higher lung myeloperoxidase activity, and higher lung CXCL1 vs. AKI alone. Splenectomy itself was not associated with increased serum IL-6 or lung injury vs. sham. To investigate the mechanism of the increased proinflammatory response, splenic production of the anti-inflammatory cytokine IL-10 was determined and was markedly upregulated. To confirm that splenic IL-10 downregulates the proinflammatory response of AKI, IL-10 was administered to splenectomized mice with AKI, which reduced serum IL-6 and improved lung injury. Our data demonstrate that AKI in the absence of a counter anti-inflammatory response by splenic IL-10 production results in an exuberant proinflammatory response and lung injury.     

High prion and PrPSc levels but delayed onset of disease in scrapie-inoculated mice heterozygous for a disrupted PrP gene.

BACKGROUND: It has been proposed that the prion, the infectious agent of transmissible spongiform encephalopathies, is PrPSc, a post-translationally modified form of the normal host protein PrPC. We showed previously that mice devoid of PrPC (Prn-p0/0) are completely resistant to scrapie. We now report on the unexpected response of heterozygous (Prn-p0/+) mice to scrapie infection. MATERIALS AND METHODS: Prn-p0/+, Prn-p0/0 and Prn-p+/+ mice were obtained from crosses of Prn-p0/+ mice. Mice were inoculated intracerebrally with mouse-adapted scrapie agent and the clinical progression of the disease recorded. Mice were sacrificed at intervals, PrPSc was determined as protease-resistant PrP and the prion titer by the incubation time assay. RESULTS: Prn-p0/+ mice, which have about half the normal level of PrPC in their brains, show enhanced resistance to scrapie, as manifested by a significant delay in onset and progression of clinical disease. However, while in wild type animals an increase in prion titer and PrPSc levels is followed within weeks by scrapie symptoms and death, heterozygous Prn-p0/+ mice remain free of symptoms for many months despite similar levels of scrapie infectivity and PrPSc. CONCLUSIONS: Our findings extend previous reports showing an inverse relationship between PrP expression level and incubation time for scrapie. However, contrary to expectation, overall accumulation of PrPSc and prions to a high level do not necessarily lead to clinical disease. These findings raise the question whether high titers of prion infectivity could also persist for long periods under natural circumstances in the absence of clinical symptoms.     

Prognostic value of serial serum interleukin-6 level estimation in patients with lung cancer: a preliminary report.

In the present report, we serially measured the levels of interleukin-6 (IL-6) and some acute-phase proteins (APP) in 61 lung cancer patients undergoing radiotherapy in order to investigate the relationship between the response to the treatment and the changes in parameters of systemic inflammatory response. The patients were divided into two groups depending on the response to the treatment. The first group (referred to as responders) comprised 32 patients with stable disease, partial remission or total remission. Twenty-nine patients with progression of the disease were included to the second group (referred to as non-responders). Six patients died due to the lung cancer during the study. We showed a decrease in IL-6 serum level and C-reactive protein (CRP) level in responders but not in non-responders. However, the most interesting results were obtained after retrospective analysis of the data of six deceased patients. In these patients we observed an elevation of IL-6 and CRP before the patients'' deaths. Following the changes in acute-phase response and interleukin-6 serum levels in lung cancer patients seems to be helpful in prognosis of the outcome of the disease. Based on our data, we conclude that an elevation in IL-6 and/or CRP level in patients with lung cancer may serve as an adverse prognostic factor.     

Dimethyl fumarate, an immune modulator and inducer of the antioxidant response, suppresses HIV replication and macrophage-mediated neurotoxicity: a novel candidate for HIV neuroprotection

Despite antiretroviral therapy (ART), HIV infection promotes cognitive dysfunction and neurodegeneration through persistent inflammation and neurotoxin release from infected and/or activated macrophages/microglia. Furthermore, inflammation and immune activation within both the CNS and periphery correlate with disease progression and morbidity in ART-treated individuals. Accordingly, drugs targeting these pathological processes in the CNS and systemic compartments are needed for effective, adjunctive therapy. Using our in vitro model of HIV-mediated neurotoxicity, in which HIV-infected monocyte-derived macrophages release excitatory neurotoxins, we show that HIV infection dysregulates the macrophage antioxidant response and reduces levels of heme oxygenase-1 (HO-1). Furthermore, restoration of HO-1 expression in HIV-infected monocyte-derived macrophages reduces neurotoxin release without altering HIV replication. Given these novel observations, we have identified dimethyl fumarate (DMF), used to treat psoriasis and showing promising results in clinical trials for multiple sclerosis, as a potential neuroprotectant and HIV disease-modifying agent. DMF, an immune modulator and inducer of the antioxidant response, suppresses HIV replication and neurotoxin release. Two distinct mechanisms are proposed: inhibition of NF-κB nuclear translocation and signaling, which could contribute to the suppression of HIV replication, and induction of HO-1, which is associated with decreased neurotoxin release. Finally, we found that DMF attenuates CCL2-induced monocyte chemotaxis, suggesting that DMF could decrease recruitment of activated monocytes to the CNS in response to inflammatory mediators. We propose that dysregulation of the antioxidant response during HIV infection drives macrophage-mediated neurotoxicity and that DMF could serve as an adjunctive neuroprotectant and HIV disease modifier in ART-treated individuals.     

Natural and experimental prion diseases of humans and animals.

Prions cause transmissible and genetic neurodegenerative diseases. Infectious prion particles are composed largely, if not entirely, of an abnormal isoform of the prion protein (PrPSc), which is encoded by a chromosomal gene. Although the PrP gene is single copy, transgenic mice with both alleles of the PrP gene ablated develop normally. A post-translational process, as yet unidentified, converts the cellular prion protein (PrPC) into PrPSc. Scrapie incubation times, neuropathology and prion synthesis in transgenic mice are controlled by the PrP gene. Mutations in this gene are genetically linked to the development of neurodegeneration. Transgenic mice expressing mutant PrP spontaneously develop neurological dysfunction and spongiform neuropathology. Future investigations of prion diseases using molecular biological and genetic approaches promise to yield much new information about these once enigmatic disorders.     

Phosphatidic acid regulates systemic inflammatory responses by modulating the Akt-mammalian target of rapamycin-p70 S6 kinase 1 pathway

Macrophages are pivotal effector cells in the innate immune system. When microbial products bind to pathogen recognition receptors, macrophages are activated and release a broad array of mediators, such as cytokines, that orchestrate the inflammatory responses of the host. Phosphatidic acid (PA) has been implicated as an important metabolite of phospholipid biosynthesis and in membrane remodeling and has been further suggested to be a crucial second messenger in various cellular signaling events. Here we show that PA is an essential regulator of inflammatory response. Deleterious effects of PA are associated with the secretion of proinflammatory cytokines, such as tumor necrosis factor-alpha, interleukin-1beta, interleukin-6, and the production of nitric oxide, prostaglandin E2, which are predominantly released by macrophage Raw264.7 cells. Furthermore, the administration of PA to mice increased the serum cytokine level. Moreover, direct or lipopolysaccharide-induced PA accumulation by macrophages led to the Akt-dependent activation of the mammalian target of rapamycin-p70 S6 kinase 1, a process required for the induction of inflammatory mediators. These findings demonstrate the importance of the role of PA in systemic inflammatory responses, and provide a potential usefulness as specific targets for the development of therapies.     

Roquinimex-mediated protection effect on the development of chronic graft-versus-host disease in mice is associated with induction of Th1 cytokine production and inhibition of proinflammatory cytokine production

Roquinimex is an immunomodulator that can effectively inhibit the development of several autoimmune diseases in animal models, but the mechanism is still unknown. In this study, we investigated the effect of roquinimex on chronic graft-versus-host disease (GVHD) in mice, a well-established model for human systemic lupus erythematosus (SLE). Oral administration of roquinimex significantly suppressed the development of proteinuria and ameliorated nephritis symptoms in chronic GVHD mice. In addition, renal histopathology and immunohistochemistry studies revealed reduced glomerulonephritis and decreased IgG deposition in chronic GVHD mice treated with roquinimex. Chronic GVHD is characterized by a predominance of Th2 cytokines, and proinflammatory cytokines that also play an important role in the pathology of tissue damage. Therefore, we focused on the effect of roquinimex on cytokine production. Chronic GVHD mouse splenocytes exhibited severely reduced interferon (IFN)-gamma production in response to Concanavalin (Con A) stimulation and an overt Th2 skewness. Roquinimex treatment, however, induced IFN-gamma production and restored the Th1/Th2 cytokine balance, although only a minimal effect of roquinimex on interleukin (IL)-4 secretion was observed. The production of the proinflammatory cytokines TNF-alpha and IL-1 beta by peritoneal macrophages from lipopolysaccharide (LPS)-treated GVHD mice was significantly inhibited by roquinimex treatment. These data suggested that the beneficial effect of roquinimex on lupus might, at least in part, result from a restoration of Th1/Th2 cytokine balance and inhibition of inflammatory cytokine production.