Sensitization of primary cultures from rat dorsal root ganglia with lipopolysaccharide (LPS) requires a robust inflammatory response.

OBJECTIVE: We investigated whether it is possible to induce a state of "LPS-sensitization" in neurons of primary cultures from rat dorsal root ganglia by pre-treatment with ultra-low doses of LPS. METHODS: DRG primary cultures were pre-treated with low to ultra-low doses of LPS (0.001-0.1 µg/ml) for 18 h, followed by a short-term stimulation with a higher LPS-dose (10 µg/ml for 2 h). TNF-α in the supernatants was measured as a sensitive read out. Using the fura-2 340/380 nm ratio imaging technique, we further investigated the capsaicin-evoked Ca2+-signals in neurons from DRG, which were pre-treated with a wide range of LPS-doses. RESULTS: Release of TNF-α evoked by stimulation with 10 µg/ml LPS into the supernatant was not significantly modified by pre-exposure to low to ultra-low LPS-doses. Capsaicin-evoked Ca2+-signals were significantly enhanced by pre-treatment with LPS doses being above a certain threshold. CONCLUSION: Ultra-low doses of LPS, which per se do not evoke a detectable inflammatory response, are not sufficient to sensitize neurons (Ca2+-responses) and glial elements (TNF-α-responses) of the primary afferent somatosensory system.

Gabapentinoids Suppress Lipopolysaccharide-Induced Interleukin-6 Production in Primary Cell Cultures of the Rat Spinal Dorsal Horn.

INTRODUCTION: Gabapentin and pregabalin are drugs to treat neuropathic pain. Several studies highlighted effects on presynaptic terminals of nociceptors. Via binding to α2δ subunits of voltage-gated calcium channels, gabapentinoids modulate the synaptic transmission of nociceptive information. However, recent studies revealed further properties of these substances. Treatment with gabapentin or pregabalin in animal models of neuropathic pain resulted not only in reduced symptoms of hyperalgesia but also in an attenuated activation of glial cells and decreased production of pro-inflammatory mediators in the spinal dorsal horn. METHODS: In the present study, we aimed to investigate the impact of gabapentinoids on the inflammatory response of spinal dorsal horn cells, applying the established model of neuro-glial primary cell cultures of the superficial dorsal horn (SDH). We studied effects of gabapentin and pregabalin on lipopolysaccharide (LPS)-induced cytokine release (bioassays), expression of inflammatory marker genes (RT-qPCR), activation of transcription factors (immunocytochemistry), and Ca2+ responses of SDH neurons to stimulation with substance P and glutamate (Ca2+-imaging). RESULTS: We detected an attenuated LPS-induced expression and release of interleukin-6 by SDH cultures in the presence of gabapentinoids. In addition, a significant main effect of drug treatment was observed for mRNA expression of microsomal prostaglandin E synthase 1 and the inhibitor of nuclear factor kappa B. Nuclear translocation of inflammatory transcription factors in glial cells was not significantly affected by gabapentinoid treatment. Moreover, both substances did not modulate neuronal responses upon stimulation with substance P or glutamate. CONCLUSION: Our results provide evidence for anti-inflammatory capacities of gabapentinoids on the acute inflammatory response of SDH primary cultures upon LPS stimulation. Such effects may contribute to the pain-relieving effects of gabapentinoids.

Manifestation of lipopolysaccharide-induced tolerance in neuro-glial primary cultures of the rat afferent somatosensory system.

OBJECTIVE: Bacterial lipopolysaccharide (LPS) may contribute to the manifestation of inflammatory pain within structures of the afferent somatosensory system. LPS can induce a state of refractoriness to its own effects termed LPS tolerance. We employed primary neuro-glial cultures from rat dorsal root ganglia (DRG) and the superficial dorsal horn (SDH) of the spinal cord, mainly including the substantia gelatinosa to establish and characterize a model of LPS tolerance within these structures. METHODS: Tolerance was induced by pre-treatment of both cultures with 1 µg/ml LPS for 18 h, followed by a short-term stimulation with a higher LPS dose (10 µg/ml for 2 h). Cultures treated with solvent were used as controls. Cells from DRG or SDH were investigated by means of RT-PCR (expression of inflammatory genes) and immunocytochemistry (translocation of inflammatory transcription factors into nuclei of cells from both cultures). Supernatants from both cultures were assayed for tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) by highly sensitive bioassays. RESULTS: At the mRNA-level, pre-treatment with 1 µg/ml LPS caused reduced expression of TNF-α and enhanced IL-10/TNF-α expression ratios in both cultures upon subsequent stimulation with 10 µg/ml LPS, i.e. LPS tolerance. SDH cultures further showed reduced release of TNF-α into the supernatants and attenuated TNF-α immunoreactivity in microglial cells. In the state of LPS tolerance macrophages from DRG and microglial cells from SDH showed reduced LPS-induced nuclear translocation of the inflammatory transcription factors NFκB and NF-IL6. Nuclear immunoreactivity of the IL-6-activated transcription factor STAT3 was further reduced in neurons from DRG and astrocytes from SDH in LPS tolerant cultures. CONCLUSION: A state of LPS tolerance can be induced in primary cultures from the afferent somatosensory system, which is characterized by a down-regulation of pro-inflammatory mediators. Thus, this model can be applied to study the effects of LPS tolerance at the cellular level, for example possible modifications of neuronal reactivity patterns upon inflammatory stimulation.

Primary culture of the rat spinal dorsal horn: a tool to investigate the effects of inflammatory stimulation on the afferent somatosensory system.

One maladaptive consequence of inflammatory stimulation of the afferent somatosensory system is the manifestation of inflammatory pain. We established and characterized a neuroglial primary culture of the rat superficial dorsal horn (SDH) of the spinal cord to test responses of this structure to neurochemical, somatosensory, or inflammatory stimulation. Primary cultures of the rat SDH consist of neurons (43%), oligodendrocytes (35%), astrocytes (13%), and microglial cells (9%). Neurons of the SDH responded to cooling (7%), heating (18%), glutamate (80%), substance P (43%), prostaglandin E2 (8%), and KCl (100%) with transient increases in the intracellular calcium [Ca2+]i. Short-term stimulation of SDH primary cultures with LPS (10 µg/ml, 2 h) caused increased expression of pro-inflammatory cytokines, inflammatory transcription factors, and inducible enzymes responsible for inflammatory prostaglandin E2 synthesis. At the protein level, increased concentrations of tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6) were measured in the supernatants of LPS-stimulated SDH cultures and enhanced TNFα and IL-6 immunoreactivity was observed specifically in microglial cells. LPS-exposed microglial cells further showed increased nuclear immunoreactivity for the inflammatory transcription factors NFκB, NF-IL6, and pCREB, indicative of their activation. The short-term exposure to LPS further caused a reduction in the strength of substance P as opposed to glutamate-evoked Ca2+-signals in SDH neurons. However, long-term stimulation with a low dose of LPS (0.01 µg/ml, 24 h) resulted in a significant enhancement of glutamate-induced Ca2+ transients in SDH neurons, while substance P-evoked Ca2+ signals were not influenced. Our data suggest a critical role for microglial cells in the initiation of inflammatory processes within the SDH of the spinal cord, which are accompanied by a modulation of neuronal responses.

The transcription factor nuclear factor interleukin 6 mediates pro- and anti-inflammatory responses during LPS-induced systemic inflammation in mice.

The transcription factor nuclear factor interleukin 6 (NF-IL6) plays a pivotal role in neuroinflammation and, as we previously suggested, hypothalamus-pituitary-adrenal-axis-activation. Here, we investigated its contribution to immune-to-brain communication and brain controlled sickness symptoms during lipopolysaccharide (LPS)-induced (50 or 2500 µg/kg i.p.) systemic inflammation in NF-IL6-deficient (KO) or wildtype mice (WT). In WT LPS induced a dose-dependent febrile response and reduction of locomotor activity. While KO developed a normal fever after low-dose LPS-injection the febrile response was almost abolished 3-7 h after a high LPS-dose. High-dose LPS-stimulation was accompanied by decreased (8 h) followed by enhanced (24 h) inflammation in KO compared to WT e.g. hypothalamic mRNA-expression including microsomal prostaglandin E synthase, inducible nitric oxide synthase and further inflammatory mediators, neutrophil recruitment to the brain as well as plasma levels of inflammatory markers such as IL-6 and IL-10. Interestingly, KO showed reduced locomotor activity even under basal conditions, but enhanced locomotor activity to novel environment stress. Hypothalamic-pituitary-adrenal-axis-activity of KO was intact, but tryptophan-metabolizing enzymes were shifted to enhanced serotonin production and reuptake. Overall, we showed for the first time that NF-IL6 plays a dual role for sickness response and immune-to-brain communication: acting pro-inflammatory at 8h but anti-inflammatory at 24 h after onset of the inflammatory response reflecting active natural programming of inflammation. Moreover, reduced locomotor activity observed in KO might be due to altered tryptophan metabolism and serotonin reuptake suggesting some role for NF-IL6 as therapeutic target for depressive disorders.

Critical role for peripherally-derived interleukin-10 in mediating the thermoregulatory manifestations of fever and hypothermia in severe forms of lipopolysaccharide-induced inflammation.

Although peripherally released interleukin (IL)-10 has a critical regulatory role in limiting fever in mild-to-moderate forms of inflammation, its role in regulating the more complex thermoregulatory manifestations of hypothermia and fever noted during severe inflammation is less clear. Using cytokine antagonism, we therefore investigated the involvement of peripherally released IL-10 in mediating hypothermia, fever and inflammation induced by intraperitoneal (IP) administration of a large dose of lipopolysaccharide (LPS). Male Wistar rats (200-250 g) were anaesthetized and implanted intra-abdominally with temperature-sensitive radiotelemeters. Rats were randomly assigned to receive IL-10 antiserum (IL-10AS) or normal sheep serum IP, 4 h before receiving an IP injection of LPS (10 mg/kg) or phosphate-buffered saline (PBS). Inflammatory responses were measured in plasma and tissue samples (spleen, liver and brain) at 90 min and 6 h after the IP injection of LPS or PBS. Administration of LPS induced an initial period of hypothermia (~90 min) after which fever developed. Pre-treating rats with IL-10AS abolished the LPS-induced increase in plasma IL-10 levels, attenuated the hypothermia and increased the amplitude of the fever. Moreover, IL-10AS pre-treatment augmented the LPS-induced increase in plasma levels of tumor necrosis factor-alpha (90 min and 6 h), IL-1ß (90 min), prostaglandin E2 (90 min) and IL-6 (6 h), in the periphery, but not the hypothalamus, over the duration of hypothermia and fever. Via its action on the synthesis of inflammatory mediators in the spleen and liver, endogenous IL-10 plays a crucial regulatory role in mediating hypothermia and fever during severe aspectic (LPS-induced) systemic inflammation.

Leptin is involved in age-dependent changes in response to systemic inflammation in the rat.

Obesity contributes to a state of subclinical peripheral and central inflammation and is often associated with aging. Here we investigated the source and contribution of adipose tissue derived cytokines and the cytokine-like hormone leptin to age-related changes in lipopolysaccharide (LPS)-induced brain-controlled sickness-responses. Old (24 months) and young (2 months) rats were challenged with LPS or saline alone or in combination with a neutralizing leptin antiserum (LAS) or control serum. Changes in the sickness-response were monitored by biotelemetry. Additionally, ex vivo fat-explants from young and old rats were stimulated with LPS or saline and culture medium collected and analyzed by cytokine-specific bioassays/ELISAs. We found enhanced duration/degree of the sickness-symptoms, including delayed but prolonged fever in old rats. This response was accompanied by increased plasma-levels of interleukin (IL)-6 and IL-1ra and exaggerated expression of inflammatory markers in brain and liver analyzed by RT-PCR including inhibitor κBα, microsomal prostaglandin synthase and cyclooxygenase 2 (brain). Moreover, for the first time, we were able to show prolonged elevated plasma leptin-levels in LPS-treated old animals. Treatment with LAS in young rats tended to attenuate the early- and in old rats the prolonged febrile response. Fat-explants exhibited unchanged IL-6 but reduced IL-1ra and tumor necrosis factor (TNF)-α release from adipose tissue of aged compared to young animals. In addition, we found increased expression of the endogenous immune regulator microRNA146a in aged animals suggesting a role for these mediators in counteracting brain inflammation. Overall, our results indicate a role of adipose tissue and leptin in "aging-related-inflammation" and age-dependent modifications of febrile-responses.

The apicomplexan parasite Eimeria arloingi induces caprine neutrophil extracellular traps.

As a novel effector mechanism polymorphonuclear neutrophils (PMN) release neutrophil extracellular traps (NETs), which represent protein-labeled DNA matrices capable of extracellular trapping and killing of invasive pathogens. Here, we demonstrate for the first time NET formation performed by caprine PMN exposed to different stages (sporozoites and oocysts) of the goat apicomplexan protozoan parasite Eimeria arloingi. Scanning electron microscopy as well as fluorescence microscopy of sporozoites- and oocysts-PMN co-cultures revealed a fine network of DNA fibrils partially covering the parasites. Immunofluorescence analyses confirmed the co-localization of histones (H3), neutrophil elastase (NE), and myeloperoxidase (MPO) in extracellular traps released from caprine PMN. In addition, the enzymatic activity of NE was found significantly enhanced in sporozoite-exposed caprine PMN. The treatment of caprine NET structures with deoxyribonuclease (DNase) and the NADPH oxidase inhibitor diphenylene iodondium (DPI) significantly reduced NETosis confirming the classical characteristics of NETs. Caprine NETs efficiently trapped vital sporozoites of E. arloingi since 72% of these stages were immobilized-but not killed-in NET structures. As a consequence, early infection rates were significantly reduced when PMN-pre-exposed sporozoites were allowed to infect adequate host cells. These findings suggest that NETs may play an important role in the early innate host response to E. arloingi infection in goats.

Activation of the inflammatory transcription factor nuclear factor interleukin-6 during inflammatory and psychological stress in the brain.

BACKGROUND: The transcription factor nuclear factor interleukin 6 (NF-IL6) is known to be activated by various inflammatory stimuli in the brain. Interestingly, we recently detected NF-IL6-activation within the hypothalamus-pituitary-adrenal (HPA)-axis of rats after systemic lipopolysaccharide (LPS)-injection. Thus, the aim of the present study was to investigate whether NF-IL6 is activated during either, inflammatory, or psychological stress in the rat brain. METHODS: Rats were challenged with either the inflammatory stimulus LPS (100 µg/kg, i.p.) or exposed to a novel environment. Core body temperature (Tb) and motor activity were monitored using telemetry, animals were killed at different time points, brains and blood removed, and primary cell cultures of the anterior pituitary lobe (AL) were investigated. Analyses were performed using immunohistochemistry, RT-PCR, and cytokine-specific bioassays. RESULTS: Stress stimulation by a novel environment increased NF-IL6-immunoreactivity (IR) in the pituitary's perivascular macrophages and hypothalamic paraventricular cells and a rise in Tb lasting approximately 2 h. LPS stimulation lead to NF-IL6-IR in several additional cell types including ACTH-IR-positive corticotrope cells in vivo and in vitro. Two other proinflammatory transcription factors, namely signal transducer and activator of transcription (STAT)3 and NFκB, were significantly activated and partially colocalized with NF-IL6-IR in cells of the AL only after LPS-stimulation, but not following psychological stress. In vitro NF-IL6-activation was associated with induction and secretion of TNFα in folliculostellate cells, which could be antagonized by the JAK-STAT-inhibitor AG490. CONCLUSIONS: We revealed, for the first time, that NF-IL6 activation occurs not only during inflammatory LPS stimulation, but also during psychological stress, that is, a novel environment. Both stressors were associated with time-dependent activation of NF-IL6 in different cell types of the brain and the pituitary. Moreover, while NF-IL6-IR was partially linked to STAT3 and NFκB activation, TNFα production, and ACTH-IR after LPS stimulation; this was not the case after exposure to a novel environment, suggesting distinct underlying signaling pathways. Overall, NF-IL6 can be used as a broad activation marker in the brain and might be of interest for therapeutic approaches not only during inflammatory but also psychological stress.

Interleukin-10 modulates the synthesis of inflammatory mediators in the sensory circumventricular organs: implications for the regulation of fever and sickness behaviors.

BACKGROUND: Whereas the role played by interleukin (IL)-10 in modulating fever and sickness behavior has been linked to it targeting the production of pro-inflammatory cytokines in the circulation, liver and spleen, it is not known whether it could directly target the local production of pro-inflammatory cytokines within the sensory circumventricular organs (CVOs) situated within the brain, but outside the blood-brain barrier. Using inactivation of IL-10, we, therefore, investigated whether IL-10 could modulate the synthesis of pro-inflammatory cytokines within the sensory CVOs, in particular the organum vasculosum laminae terminalis (OVLT) and area postrema (AP). FINDINGS: Primary OVLT and AP microcultures were established from topographically excised rat pup brain tissue. The microcultures were pretreated with either IL-10 antibodies (AB) (10 µl/350 µl medium) or phosphate-buffered saline (PBS) (10 µl/350 µl medium) before being incubated with lipopolysaccharide (LPS) (100 µg/ml) or PBS in complete medium for 6 h. Supernatants were removed from the microcultures after 6 h of incubation with LPS and used for the determination of IL-6 and tumor necrosis factor (TNF)-α. Pre-treating the OVLT and AP microcultures with IL-10 antibodies significantly enhanced the LPS-induced increase in TNF-α and IL-6 in the supernatant obtained from the microcultures. CONCLUSIONS: Our results show for the first time that the LPS-induced release of pro-inflammatory cytokines in cells cultured from the AP and OVLT can be modulated in the presence of IL-10 antibodies. Thus, we have identified that the sensory CVOs may have a key role to play in both the initiation and modulation of neuroinflammation.

Decreased plasma levels of the brain-derived neurotrophic factor correlate with right heart congestion in pulmonary arterial hypertension.

Background: The brain-derived neurotrophic factor (BDNF) may promote development of pulmonary hypertension and right ventricular (RV) failure. However, BDNF plasma levels were decreased in patients with left ventricular (LV) failure. Therefore, we investigated BDNF plasma levels in pulmonary hypertension patients and the role of BDNF in mouse models of pulmonary hypertension and isolated RV failure. Methods: BDNF plasma levels were correlated to pulmonary hypertension in two patient cohorts, including either post- and pre-capillary pulmonary hypertension patients (first cohort) or only pre-capillary pulmonary hypertension patients (second cohort). In the second cohort, RV dimensions and load-independent function were determined by imaging and pressure-volume catheter measurements, respectively. For induction of isolated RV pressure overload, heterozygous Bdnf knockout (Bdnf+/- ) mice were subjected to pulmonary arterial banding (PAB). For induction of pulmonary hypertension, mice with inducible knockout of BDNF in smooth muscle cells (Bdnf/Smmhc knockout) were exposed to chronic hypoxia. Results: Plasma BDNF levels were decreased in patients with pulmonary hypertension. Following adjustment for covariables, BDNF levels negatively correlated in both cohorts with central venous pressure. In the second cohort, BDNF levels additionally negatively correlated with RV dilatation. In animal models, BDNF downregulation attenuated RV dilatation in Bdnf+ /- mice after PAB or hypoxic Bdnf/Smmhc knockout mice, although they developed pulmonary hypertension to a similar extent. Conclusions: Similar to LV failure, circulating levels of BDNF were decreased in pulmonary hypertension patients, and low BDNF levels were associated with right heart congestion. Decreased BDNF levels did not worsen RV dilatation in animal models, and thus, may be the consequence, but not the cause of RV dilatation.

SK-Channel Activation Alters Peripheral Metabolic Pathways in Mice, but Not Lipopolysaccharide-Induced Fever or Inflammation.

PURPOSE: Previously, we have shown that CyPPA (cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine), a pharmacological small-conductance calcium-activated potassium (SK)-channel positive modulator, antagonizes lipopolysaccharide (LPS)-induced cytokine expression in microglial cells. Here, we aimed to test its therapeutic potential for brain-controlled sickness symptoms, brain inflammatory response during LPS-induced systemic inflammation, and peripheral metabolic pathways in mice. METHODS: Mice were pretreated with CyPPA (15 mg/kg IP) 24 hours before and simultaneously with LPS stimulation (2.5 mg/kg IP), and the sickness response was recorded by a telemetric system for 24 hours. A second cohort of mice were euthanized 2 hours after CyPPA or solvent treatment to assess underlying CyPPA-induced mechanisms. Brain, blood, and liver samples were analyzed for inflammatory mediators or nucleotide concentrations using immunohistochemistry, real-time PCR and Western blot, or HPLC. Moreover, we investigated CyPPA-induced changes of UCP1 expression in brown adipose tissue (BAT)-explant cultures. RESULTS: CyPPA treatment did not affect LPS-induced fever, anorexia, adipsia, or expression profiles of inflammatory mediators in the hypothalamus or plasma or microglial reactivity to LPS (CD11b staining and CD68 mRNA expression). However, CyPPA alone induced a rise in core body temperature linked to heat production via altered metabolic pathways like reduced levels of adenosine, increased protein content, and increased UCP1 expression in BAT-explant cultures, but no alteration in ATP/ADP concentrations in the liver. CyPPA treatment was accompanied by altered pathways, including NFκB signaling, in the hypothalamus and cortex, while circulating cytokines remained unaltered. CONCLUSION: Overall, while CyPPA has promise as a treatment strategy, in particular according to results from in vitro experiments, we did not reveal anti-inflammatory effects during severe LPS-induced systemic inflammation. Interestingly, we found that CyPPA alters metabolic pathways inducing short hyperthermia, most likely due to increased energy turnover in the liver and heat production in BAT.

Neuroinflammation in Primary Cultures of the Rat Spinal Dorsal Horn Is Attenuated in the Presence of Adipose Tissue-Derived Medicinal Signalling Cells (AdMSCs) in a Co-cultivation Model.

Neuroinflammation within the superficial dorsal horn (SDH) of the spinal cord induces inflammatory pain with symptoms of hyperalgesia and allodynia. Glial activation and production of inflammatory mediators (e.g. cytokines) is associated with modulation of nociceptive signalling. In this context, medicinal signalling cells, e.g. obtained from adipose tissue (AdMSCs), gained attention due to their capacity to modulate the inflammatory response in several diseases, e.g. spinal cord injury. We applied the recently established mixed neuroglial primary cell culture of the rat SDH to investigate effects of AdMSCs on the inflammatory response of SDH cells. Following establishment of a co-cultivation system, we performed specific bioassays for tumour necrosis factor alpha (TNFα) and interleukin (IL)-6, RT-qPCR and immunocytochemistry to detect changes in cytokine production and glial activation upon inflammatory stimulation with lipopolysaccharide (LPS). LPS-induced expression and release of pro-inflammatory cytokines (TNFα, IL-6) by SDH cells was significantly attenuated in the presence of AdMSCs. Further evidence for anti-inflammatory capacities of AdMSCs derived from a blunted LPS-induced TNFα/IL-10 expression ratio and suppressed nuclear translocation of the inflammatory transcription factor nuclear factor kappa B (NFκB) in SDH microglial cells. Expression of IL-10, transforming growth factor beta (TGF-ß) and TNFα-stimulated gene-6 (TSG-6) was detected in AdMSCs, which are putative candidates for anti-inflammatory capacities of these cells. We present a novel co-cultivation system of AdMSCs with neuroglial primary cultures of the SDH to investigate immunomodulatory effects of AdMSCs at a cellular level.

Parthenolide attenuates LPS-induced fever, circulating cytokines and markers of brain inflammation in rats.

Parthenolide, a sesquiterpene lactone, has been reported to exhibit a variety of anti-inflammatory and immunomodulatory effects. To test the effect of parthenolide on brain inflammatory responses, brain oxidative stress and fever, we treated rats with parthenolide (1 mg/kg), simultaneously or 1 h prior to a systemic (i.p.) challenge with a moderate dose (100 µg/kg) of lipopolysaccharide (LPS). The initial hypothermia was exaggerated; the second phase of the biphasic LPS-induced fever and circulating interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) were significantly attenuated only in parthenolide-pretreated animals. In the hypothalamus, markers of NFκB/NF-IL6 pathway activation (inhibitor κBα, NF-IL6 and the serin/threonin kinase-like protein mRNA expression) and markers of oxidative stress (including nuclear respiratory factor 1) and NFκB immunoreactivity were significantly reduced while NF-IL6 immunoreactivity and suppressor of cytokine signaling 3 mRNA expression remained unaltered, 8 h after LPS-stimulation with parthenolide-pretreatment. Importantly, this response was accompanied by decreased mRNA expression of the rate limiting enzyme in prostaglandin synthesis, cyclooxygenase 2 (COX2), known for its critical role in fever induction pathways. A direct action of parthenolide on brain cells was also confirmed in a primary neuro-glial cell culture of the vascular organ of the lamina terminalis a pivotal brain structure for fever manifestation with a leaky blood-brain barrier. In summary, pretreatment with parthenolide attenuates the febrile response during LPS-induced systemic inflammation by reducing circulating IL-6 and TNFα and decreasing hypothalamic NFκB/NF-IL6 activation, oxidative stress and expression of COX2. Thus parthenolide appears to have the potential to reduce brain inflammation.

LPS Primes Brain Responsiveness to High Mobility Group Box-1 Protein.

High mobility group box (HMGB)1 action contributes to late phases of sepsis, but the effects of increased endogenous plasma HMGB1 levels on brain cells during inflammation are unclear. Here, we aimed to further investigate the role of HMGB1 in the brain during septic-like lipopolysaccharide-induced inflammation in rats (LPS, 10 mg/kg, i.p.). HMGB-1 mRNA expression and release were measured in the periphery/brain by RT-PCR, immunohistochemistry and ELISA. In vitro experiments with disulfide-HMGB1 in primary neuro-glial cell cultures of the area postrema (AP), a circumventricular organ with a leaky blood-brain barrier and direct access to circulating mediators like HMGB1 and LPS, were performed to determine the direct influence of HMGB1 on this pivotal brain structure for immune-to-brain communication. Indeed, HMGB1 plasma levels stayed elevated after LPS injection. Immunohistochemistry of brains and AP cultures confirmed LPS-stimulated cytoplasmatic translocation of HMGB1 indicative of local HMGB1 release. Moreover, disulfide-HMGB1 stimulation induced nuclear factor (NF)-κB activation and a significant release of interleukin-6, but not tumor necrosis factor α, into AP culture supernatants. However, only a few AP cells directly responded to HMGB1 with increased intracellular calcium concentration. Interestingly, priming with LPS induced a seven-fold higher percentage of responsive cells to HMGB1. We conclude that, as a humoral and local mediator, HMGB1 enhances brain inflammatory responses, after LPS priming, linked to sustained sepsis symptoms.

Neurons and astrocytes of the chicken hypothalamus directly respond to lipopolysaccharide and chicken interleukin-6.

In 4-5-month-old chicken, intravenous injections of bacterial lipopolysaccharide (LPS) induced a dose-dependent fever response and a pronounced increase of circulating interleukin-6 (IL-6). To assess a possible role for IL-6 in the brain of birds, a hypothalamic neuro-glial primary culture from 1-day-old chicken was established. Each well of cultured hypothalamic cells contained some 615 neurons, 1350 astrocytes, and 580 microglial cells on average. Incubation of chicken hypothalamic primary cultures with 10 or 100 µg/ml LPS induced a dose-dependent release of bioactive IL-6 into the supernatant. Populations of hypothalamic neurons (4%) and astrocytes (12%) directly responded to superfusion with buffer containing 10 µg/ml LPS with a transient increase of intracellular calcium, a sign of direct cellular activation. Stimulation of hypothalamic cultures with buffer containing 50 ng/ml chicken IL-6 induced calcium signaling in 11% of neurons and 22% of astrocytes investigated. These results demonstrate that IL-6 is produced in the periphery and in the hypothalamus in response to LPS in chicken. The observed cellular responses of hypothalamic cells to chicken IL-6 indicate that this cytokine may readily be involved in the manifestation of fever in the avian hypothalamus.

Age-Dependent Changes of Adipokine and Cytokine Secretion From Rat Adipose Tissue by Endogenous and Exogenous Toll-Like Receptor Agonists.

White adipose tissue but recently also brown adipose tissue have emerged as endocrine organs. Age-associated obesity is accompanied by prolonged and elevated lipopolysaccharide (LPS)-induced sickness symptoms and increased cytokine and adipokine levels in the circulation partially originating from adipose tissue. In the present study, ex vivo fat explants were used to investigate how the exogenous pathogen-associated molecular pattern (PAMP) LPS or the endogenous danger-associated molecular patterns (DAMPs) high mobility group box-1 protein (HMGB1) and biglycan modulate the release of cytokines and adipokines/batokines and, thus, could influence systemic and/or local inflammation. The response of adipose tissue (epididymal, retroperitoneal, subcutaneous, and brown) was compared between young lean and old obese rats (2 vs. 24 months old). LPS induced a strong interleukin (IL)-6 and tumor necrosis factor (TNF) alpha release into the supernatant of all adipose tissue types investigated. HMGB1 (subcutaneous) and biglycan (retroperitoneal) led to an increased release of IL-6 and TNFalpha (HMGB1) and decreased visfatin and adiponectin (biglycan) secretion from epididymal adipose tissue (young rats). Visfatin was also decreased by HMGB1 in retroperitoneal adipose tissue of old rats. We found significantly higher leptin (all fat pads) and adiponectin (subcutaneous) levels in supernatants of adipose tissue from old compared to young rats, whereas visfatin secretion showed the opposite. The expression of the biglycan receptor Toll-like receptor (TLR) 2 as well as the LPS and HMGB1 receptors TLR4 and receptor for advanced glycation end products (RAGE) were reduced with age (TLR4/RAGE) and by stimulation with their ligands (subcutaneous). Overall, we revealed that adipokines/adipose-tissue released cytokines show some modulation of their release caused by mediators of septic (batokines) and sterile inflammation with potential implication for acute and chronic disease. Moreover, aging may increase or decrease the release of fat-derived mediators. These data show that DAMPS and LPS locally modulate cytokine secretion while only DAMPS but not LPS can locally alter adipokine secretion during inflammation.

Effects of gabapentinoids on responses of primary cultures from rat dorsal root ganglia to inflammatory or somatosensory stimuli.

Background Gabapentinoids are known to reduce neuropathic pain. The aim of this experimental study was to investigate whether gabapentinoids exert anti-inflammatory and/or anti-nociceptive effects at the cellular level using primary cultures of rat dorsal root ganglia (DRG). Methods Cells from rat DRG were cultured in the presence of gabapentin or pregabalin, and we tested the effects of subsequent stimulation with lipopolysaccharide (LPS) on the expression of genes (real-time polymerase chain reaction) and production of tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6) by specific bioassays. Using Ca2+ imaging, we further investigated in neurons the effects of gabapentinoids upon stimulation with the TRPV-1 agonist capsaicin. Results There is a small influence of gabapentinoids on the inflammatory response to LPS stimulation, namely, a significantly reduced expression of IL-6. Pregabalin and gabapentin further seem to exert a moderate inhibitory influence on capsaicin-induced Ca2+ signals in DRG neurons. Conclusions Although the single inhibitory effects of gabapentinoids on inflammatory and nociceptive responses are moderate, a combination of both effects might provide an explanation for the proposed function of these substances as an adjuvant for the reduction of neuropathic pain.

Tumor necrosis factor-alpha, interleukin-1beta and nitric oxide induce calcium transients in distinct populations of cells cultured from the rat area postrema.

The area postrema (AP) represents the medullary sensory circumventricular organ lacking endothelial blood-brain barrier function at the base of the 4th cerebral ventricle. Administration of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) or the nitric oxide (NO) donor diethylamino-diazenolate-2-oxide (DEA) caused fast transient rises in intracellular calcium concentrations ([Ca(2+)]i) in distinct populations of cells investigated in a primary microculture of the rat AP. TNF-alpha caused rapid elevations of [Ca(2+)]i in 8% of all neurons and astrocytes investigated, with limited responses of microglial cells and no responses of oligodendrocytes. 15% of all neurons investigated responded to IL-1beta, while only 5-7% of the other cell types showed rises in [Ca(2+)]i. The most pronounced effects were caused by treatment with DEA with some 20% responsive astrocytes and oligodendrocytes, 15% neurons and 10% microglial cells. Evidently, the AP can act as a sensor for circulating TNF-alpha and IL-1beta, or for locally produced cytokines and NO during infection and inflammation.

Visualizing and Profiling Lipids in the OVLT of Fat-1 and Wild Type Mouse Brains during LPS-Induced Systemic Inflammation Using AP-SMALDI MSI.

Lipids, including omega-3 polyunsaturated fatty acids (n-3-PUFAs), modulate brain-intrinsic inflammation during systemic inflammation. The vascular organ of the lamina terminalis (OVLT) is a brain structure important for immune-to-brain communication. We, therefore, aimed to profile the distribution of several lipids (e.g., phosphatidyl-choline/ethanolamine, PC/PE), including n-3-PUFA-carrying lipids (esterified in phospholipids), in the OVLT during systemic lipopolysaccharide(LPS)-induced inflammation. We injected wild type and endogenously n-3-PUFA producing fat-1 transgenic mice with LPS (i.p., 2.5 mg/kg) or PBS. Brain samples were analyzed using immunohistochemistry and high-resolution atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization orbital trapping mass spectrometry imaging (AP-SMALDI-MSI) for spatial resolution of lipids. Depending on genotype and treatment, several distinct distribution patterns were observed for lipids [e.g., lyso(L)PC (16:0)/(18:0)] proposed to be involved in inflammation. The distribution patterns ranged from being homogeneously disseminated [LPC (18:1)], absent/reduced signaling within the OVLT relative to adjacent preoptic tissue [PE (38:6)], either treatment- and genotype-dependent or independent low signal intensities [LPC (18:0)], treatment- and genotype-dependent [PC 38:6)] or independent accumulation in the OVLT [PC (38:7)], and accumulation in commissures, e.g., nerve fibers like the optic nerve [LPE (18:1)]. Overall, screening of lipid distribution patterns revealed distinct inflammation-induced changes in the OVLT, highlighting the prominent role of lipid metabolism in brain inflammation. Moreover, known and novel candidates for brain inflammation and immune-to-brain communication were detected specifically within this pivotal brain structure, a window between the periphery and the brain. The biological significance of these newly identified lipids abundant in the OVLT and the adjacent preoptic area remains to be further analyzed.