Acute Effect of Caffeine on the Synthesis of Pro-Inflammatory Cytokines in the Hypothalamus and Choroid Plexus during Endotoxin-Induced Inflammation in a Female Sheep Model.

This study was designed to determine the effect of acute caffeine (CAF) administration, which exerts a broad spectrum of anti-inflammatory activity, on the synthesis of pro-inflammatory cytokines and their receptors in the hypothalamus and choroid plexus (ChP) during acute inflammation caused by the injection of bacterial endotoxin-lipopolysaccharide (LPS). The experiment was performed on 24 female sheep randomly divided into four groups: control; LPS treated (iv.; 400 ng/kg of body mass (bm.)); CAF treated (iv.; 30 mg/kg of bm.); and LPS and CAF treated. The animals were euthanized 3 h after the treatment. It was found that acute administration of CAF suppressed the synthesis of interleukin (IL-1ß) and tumor necrosis factor (TNF)α, but did not influence IL-6, in the hypothalamus during LPS-induced inflammation. The injection of CAF reduced the LPS-induced expression of TNF mRNA in the ChP. CAF lowered the gene expression of IL-6 cytokine family signal transducer (IL6ST) and TNF receptor superfamily member 1A (TNFRSF1) in the hypothalamus and IL-1 type II receptor (IL1R2) in the ChP. Our study on the sheep model suggests that CAF may attenuate the inflammatory response at the hypothalamic level and partly influence the inflammatory signal generated by the ChP cells. This suggests the potential of CAF to suppress neuroinflammatory processes induced by peripheral immune/inflammatory challenges.

Metformin Treatment Attenuates Brain Inflammation and Rescues PACAP/VIP Neuropeptide Alterations in Mice Fed a High-Fat Diet.

High-fat diet (HFD)-induced comorbid cognitive and behavioural impairments are thought to be the result of persistent low-grade neuroinflammation. Metformin, a first-line medication for the treatment of type-2 diabetes, seems to ameliorate these comorbidities, but the underlying mechanism(s) are not clear. Pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP) are neuroprotective peptides endowed with anti-inflammatory properties. Alterations to the PACAP/VIP system could be pivotal during the development of HFD-induced neuroinflammation. To unveil the pathogenic mechanisms underlying HFD-induced neuroinflammation and assess metformin's therapeutic activities, (1) we determined if HFD-induced proinflammatory activity was present in vulnerable brain regions associated with the development of comorbid behaviors, (2) investigated if the PACAP/VIP system is altered by HFD, and (3) assessed if metformin rescues such diet-induced neurochemical alterations. C57BL/6J male mice were divided into two groups to receive either standard chow (SC) or HFD for 16 weeks. A further HFD group received metformin (HFD + M) (300 mg/kg BW daily for 5 weeks) via oral gavage. Body weight, fasting glucose, and insulin levels were measured. After 16 weeks, the proinflammatory profile, glial activation markers, and changes within the PI3K/AKT intracellular pathway and the PACAP/VIP system were evaluated by real-time qPCR and/or Western blot in the hypothalamus, hippocampus, prefrontal cortex, and amygdala. Our data showed that HFD causes widespread low-grade neuroinflammation and gliosis, with regional-specific differences across brain regions. HFD also diminished phospho-AKT(Ser473) expression and caused significant disruptions to the PACAP/VIP system. Treatment with metformin attenuated these neuroinflammatory signatures and reversed PI3K/AKT and PACAP/VIP alterations caused by HFD. Altogether, our findings demonstrate that metformin treatment rescues HFD-induced neuroinflammation in vulnerable brain regions, most likely by a mechanism involving the reinstatement of PACAP/VIP system homeostasis. Data also suggests that the PI3K/AKT pathway, at least in part, mediates some of metformin's beneficial effects.

Andrographolide ameliorates neuroinflammation in APP/PS1 transgenic mice.

Alzheimer's disease is a devastating neurodegenerative disorder, with no disease-modifying treatment available yet. There is increasing evidence that neuroinflammation plays a critical role in the pathogenesis of AD. Andrographolide (Andro), a labdane diterpene extracted from the herb Andrographis paniculata, has been reported to exhibit neuroprotective property in central nervous system diseases. However, its effects on Aß and Aß-induced neuroinflammation have not yet been studied. In the present study, we found that Andro administration significantly alleviated cognitive impairments, reduced amyloid-ß deposition, inhibited microglial activation, and decreased the secretion of proinflammatory factors in APP/PS1 mice. Furthermore, transcriptome sequencing analysis revealed that Andro could significantly decrease the expression of Itgax, TLR2, CD14, CCL3, CCL4, TLR1, and C3ar1 in APP/PS1 mice, which was further validated by qRT-PCR. Our results suggest that Andro might be a potential therapeutic drug for AD by regulating neuroinflammation.

Gut Hormone GIP Induces Inflammation and Insulin Resistance in the Hypothalamus.

The hypothalamus plays a critical role in controlling energy balance. High-fat diet (HFD) feeding increases the gene expression of proinflammatory mediators and decreases insulin actions in the hypothalamus. Here, we show that a gut-derived hormone, glucose-dependent insulinotropic polypeptide (GIP), whose levels are elevated during diet-induced obesity, promotes and mediates hypothalamic inflammation and insulin resistance during HFD-induced obesity. Unbiased ribonucleic acid sequencing of GIP-stimulated hypothalami revealed that hypothalamic pathways most affected by intracerebroventricular (ICV) GIP stimulation were related to inflammatory-related responses. Subsequent analysis demonstrated that GIP administered either peripherally or centrally, increased proinflammatory-related factors such as Il-6 and Socs3 in the hypothalamus, but not in the cortex of C57BL/6J male mice. Consistently, hypothalamic activation of IκB kinase-ß inflammatory signaling was induced by ICV GIP. Further, hypothalamic levels of proinflammatory cytokines and Socs3 were significantly reduced by an antagonistic GIP receptor (GIPR) antibody and by GIPR deficiency. Additionally, centrally administered GIP reduced anorectic actions of insulin in the brain and diminished insulin-induced phosphorylation of Protein kinase B and Glycogen synthase kinase 3ß in the hypothalamus. Collectively, these findings reveal a previously unrecognized role for brain GIP signaling in diet-induced inflammation and insulin resistance in the hypothalamus.

From the Cover: Lung-Specific Overexpression of Constitutively Active IKK2 Induces Pulmonary and Systemic Inflammations but Not Hypothalamic Inflammation and Glucose Intolerance.

The lung is constantly exposed to ambient pollutants such as ambient fine particulate matter (PM2.5), making it one of the most frequent locations of inflammation in the body. Given the establishment of crucial role of inflammation in the pathogenesis of cardiometabolic diseases, pulmonary inflammation is thus widely believed to be an important risk factor for cardiometabolic diseases. However, the causality between them has not yet been well established. To determine if pulmonary inflammation is sufficient to cause adverse cardiometabolic effects, SFTPC-rtTA+/-tetO-cre+/-pROSA-inhibitor κB kinase 2(IKK2)ca+/- (LungIKK2ca) and littermate SFTPC-rtTA+/-tetO-cre-/-pROSA-IKK2ca+/- wildtype (WT) mice were fed with doxycycline diet to induce constitutively active Ikk2 (Ikk2ca) overexpression in the lung and their pulmonary, systemic, adipose, and hypothalamic inflammations, vascular function, and glucose homeostasis were assessed. Feeding with doxycycline diet resulted in IKK2ca overexpression in the lungs of LungIKK2ca but not WT mice. This induction of IKK2ca was accompanied by marked pulmonary inflammation as evidenced by significant increases in bronchoalveolar lavage fluid leukocytes, pulmonary macrophage infiltration, and pulmonary mRNA expression of tumor necrosis factor α (Tnfα) and interleukin-6 (Il-6). This pulmonary inflammation due to lung-specific overexpression of IKK2ca was sufficient to increase circulating TNFα and IL-6 levels, adipose expression of Tnfα and Il-6 mRNA, aortic endothelial dysfunction, and systemic insulin resistance. Unexpectedly, no significant alteration in hypothalamic expression of Tnfα and Il-6 mRNA and glucose intolerance were observed in these mice. Pulmonary inflammation is sufficient to induce systemic inflammation, endothelial dysfunction, and insulin resistance, but not hypothalamic inflammation and glucose intolerance.

Specific suppression of microgliosis cannot circumvent the severe neuropathology in peroxisomal ß-oxidation-deficient mice.

An important hallmark of various neurodegenerative disorders is the proliferation and activation of microglial cells, the resident immune cells of the central nervous system (CNS). Mice that lack multifunctional protein-2 (MFP2), the key enzyme in peroxisomal ß-oxidation, develop excessive microgliosis that positively correlates with behavioral deficits whereas no neuronal loss occurs. However, the precise contribution of neuroinflammation to the fatal neuropathology of MFP2 deficiency remains largely unknown. Here, we first attempted to suppress the inflammatory response by administering various anti-inflammatory drugs but they failed to reduce microgliosis. Subsequently, Mfp2-/- mice were treated with the selective colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX5622 as microglial proliferation and survival is dependent on CSF1R signaling. This resulted in the elimination of >95% of microglia from control mice but only 70% of the expanded microglial population from Mfp2-/- mice. Despite microglial diminution in Mfp2-/- brain, inflammatory markers remained unaltered and residual microglia persisted in a reactive state. CSF1R inhibition did not prevent neuronal dysfunction, cognitive decline and clinical deterioration of Mfp2-/- mice. Collectively, the unaltered inflammatory profile despite suppressed microgliosis concurrent with persevering clinical decline strengthens our hypothesis that neuroinflammation importantly contributes to the Mfp2-/- phenotype.

Curcumin attenuates acute inflammatory injury by inhibiting the TLR4/MyD88/NF-κB signaling pathway in experimental traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) initiates a neuroinflammatory cascade that contributes to substantial neuronal damage and behavioral impairment, and Toll-like receptor 4 (TLR4) is an important mediator of thiscascade. In the current study, we tested the hypothesis that curcumin, a phytochemical compound with potent anti-inflammatory properties that is extracted from the rhizome Curcuma longa, alleviates acute inflammatory injury mediated by TLR4 following TBI. METHODS: Neurological function, brain water content and cytokine levels were tested in TLR4⁻/⁻ mice subjected to weight-drop contusion injury. Wild-type (WT) mice were injected intraperitoneally with different concentrations of curcumin or vehicle 15 minutes after TBI. At 24 hours post-injury, the activation of microglia/macrophages and TLR4 was detected by immunohistochemistry; neuronal apoptosis was measured by FJB and TUNEL staining; cytokines were assayed by ELISA; and TLR4, MyD88 and NF-κB levels were measured by Western blotting. In vitro, a co-culture system comprised of microglia and neurons was treated with curcumin following lipopolysaccharide (LPS) stimulation. TLR4 expression and morphological activation in microglia and morphological damage to neurons were detected by immunohistochemistry 24 hours post-stimulation. RESULTS: The protein expression of TLR4 in pericontusional tissue reached a maximum at 24 hours post-TBI. Compared with WT mice, TLR4⁻/⁻ mice showed attenuated functional impairment, brain edema and cytokine release post-TBI. In addition to improvement in the above aspects, 100 mg/kg curcumin treatment post-TBI significantly reduced the number of TLR4-positive microglia/macrophages as well as inflammatory mediator release and neuronal apoptosis in WT mice. Furthermore, Western blot analysis indicated that the levels of TLR4 and its known downstream effectors (MyD88, and NF-κB) were also decreased after curcumin treatment. Similar outcomes were observed in the microglia and neuron co-culture following treatment with curcumin after LPS stimulation. LPS increased TLR4 immunoreactivity and morphological activation in microglia and increased neuronal apoptosis, whereas curcumin normalized this upregulation. The increased protein levels of TLR4, MyD88 and NF-κB in microglia were attenuated by curcumin treatment. CONCLUSIONS: Our results suggest that post-injury, curcumin administration may improve patient outcome by reducing acute activation of microglia/macrophages and neuronal apoptosis through a mechanism involving the TLR4/MyD88/NF-κB signaling pathway in microglia/macrophages in TBI.

A role for orexin in cytotoxic chemotherapy-induced fatigue.

Fatigue is the most common symptom related to cytotoxic chemotherapeutic treatment of cancer. Peripheral inflammation associated with cytotoxic chemotherapy is likely a causal factor of fatigue. The neural mechanisms by which cytotoxic chemotherapy associated inflammation induces fatigue behavior are not known. This lack of knowledge hinders development of interventions to reduce or prevent this disabling symptom. Infection induced fatigue/lethargy in rodents is mediated by suppression of hypothalamic orexin activity. Orexin is critical for maintaining wakefulness and motivated behavior. Though there are differences between infection and cytotoxic chemotherapy in some symptoms, both induce peripheral inflammation and fatigue. Based on these similarities we hypothesized that cytotoxic chemotherapy induces fatigue by disrupting orexin neuron activity. We found that a single dose of a cytotoxic chemotherapy cocktail (cyclophosphamide, adriamycin, 5-fluorouracil - CAF) induced fatigue/lethargy in mice and rats as evidenced by a significant decline in voluntary locomotor activity measured by telemetry. CAF induced inflammatory gene expression - IL-1R1 (p<0.001), IL-6 (p<0.01), TNFα (p<0.01), and MCP-1 (p<0.05) - in the rodent hypothalamus 6-24h after treatment during maximum fatigue/lethargy. CAF decreased orexin neuron activity as reflected by decreased nuclear cFos localization in orexin neurons 24h after treatment (p<0.05) and by decreased orexin-A in cerebrospinal fluid 16 h after treatment (p<0.001). Most importantly, we found that central administration of 1 µg orexin-A restored activity in CAF-treated rats (p<0.05). These results demonstrate that cytotoxic chemotherapy induces hypothalamic inflammation and that suppression of hypothalamic orexin neuron activity has a causal role in cytotoxic chemotherapy-induced fatigue in rodents.

Purinergic receptors modulate MAP kinases and transcription factors that control microglial inflammatory gene expression.

Following many types of brain injury, microglial cell hyperactivation, and the subsequent release of neurotoxic mediators into the CNS contributes to inflammation and neuronal death. Among the proteins important for modulating the inflammatory function of microglia are the P2 purinergic receptors for which extracellular adenine nucleotides, such as ATP, are ligands. Because adenine nucleotides are abundant in the extracellular fluid following brain injury, ATP may represent an important component of the inflammatory microenvironment controlling microglial cell function. Although much work has been done examining the mechanisms whereby adenine nucleotides stimulate inflammatory mediator production, little is known concerning their complementary inhibitory effects. In this review we will focus on what is currently known about the microglial inhibitory effects of adenine nucleotides in the context of inflammation and summarize the current knowledge of their effects via purinergic receptors on microglial signal transduction pathways including transcription factors important for controlling inflammatory gene expression. The relevance of these mechanisms to microglial inflammatory function and physiology will be discussed. Further, we present data here illustrating that MAP kinase signal transduction pathways are altered in activated microglia that have been primed with or co-exposed to adenine nucleotides; effects that are stimulus- and MAPK pathway-specific. We also demonstrate the ability of P2X7 receptors to stimulate the phosphorylation of CREB, a putative inhibitory transcription factor in microglia. Together, these data indicate that ATP may be an endogenous inhibitor or neuroprotective molecule decreasing the inflammatory capacity of microglia.

MCP-1 and murine prion disease: separation of early behavioural dysfunction from overt clinical disease.

Prion diseases are chronic, fatal neurodegenerative conditions of the CNS. We have investigated the role of monocyte chemoattractant protein-1 (MCP-1) in the ME7 model of murine prion disease. MCP-1 expression increased in the CNS throughout disease progression and was positively correlated with microglial activation. We subsequently compared the inflammatory response, pathology and behavioural changes in wild-type (wt) mice and MCP-1 knockout mice (MCP-1-/-) inoculated with ME7. Late-stage clinical signs were delayed by 4 weeks in MCP-1-/- mice, and survival time increased by 2-3 weeks. By contrast, early changes in affective behaviours and locomotor activity were not delayed in onset. There was also no difference in microglial activation or neuronal death in the hippocampus and thalamus of wt mice and MCP-1-/- mice. These results highlight an important dissociation between prolonged survival, early behavioural dysfunction and hippocampal/thalamic pathology when considering therapeutic intervention for human prion diseases and other chronic neurodegenerative conditions.

COX-3: a splice variant of cyclooxygenase-1 in mouse neural tissue and cells.

We have detected an expressed mRNA encoding a splice variant of COX-1 in the mouse central nervous system. This isoform, referred to as COX-3, is identical in sequence to COX-1 except for the in-frame retention of intron 1. Like its counterpart COX-1, COX-3 does not generally appear to be induced by acute inflammatory stimulation.

Gene expression profiling of 12633 genes in Alzheimer hippocampal CA1: transcription and neurotrophic factor down-regulation and up-regulation of apoptotic and pro-inflammatory signaling.

Alterations in transcription, RNA editing, translation, protein processing, and clearance are a consistent feature of Alzheimer's disease (AD) brain. To extend our initial study (Alzheimer Reports [2000] 3:161-167), RNA samples isolated from control and AD hippocampal cornu ammonis 1 (CA1) were analyzed for 12633 gene and expressed sequence tag (EST) expression levels using DNA microarrays (HG-U95Av2 Genechips; Affymetrix, Santa Clara, CA). Hippocampal CA1 tissues were carefully selected from several hundred potential specimens obtained from domestic and international brain banks. To minimize the effects of individual differences in gene expression, RNA of high spectral quality (A(260/280) > or= 1.9) was pooled from CA1 of six control or six AD subjects. Results were compared as a group; individual gene expression patterns for the most-changed RNA message levels were also profiled. There were no significant differences in age, postmortem interval (mean < or = 2.1 hr) nor tissue pH (range 6.6-6.9) between the two brain groups. AD tissues were derived from subjects clinically classified as CDR 2-3 (CERAD/NIA). Expression data were analyzed using GeneSpring (Silicon Genetics, Redwood City, CA) and Microarray Data Mining Tool (Affymetrix) software. Compared to controls and 354 background/alignment markers, AD brain showed a generalized depression in brain gene transcription, including decreases in RNA encoding transcription factors (TFs), neurotrophic factors, signaling elements involved in synaptic plasticity such as synaptophysin, metallothionein III, and metal regulatory factor-1. Three- or morefold increases in RNAs encoding DAXX, cPLA(2), CDP5, NF-kappaBp52/p100, FAS, betaAPP, DPP1, NFIL6, IL precursor, B94, HB15, COX-2, and CEX-1 signals were strikingly apparent. These data support the hypothesis of widespread transcriptional alterations, misregulation of RNAs involved in metal ion homeostasis, TF signaling deficits, decreases in neurotrophic support and activated apoptotic and neuroinflammatory signaling in moderately affected AD hippocampal CA1.