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Spinal cord injury (SCI) produces a toxic inflammatory microenvironment that negatively affects plasticity and recovery. Recently, we showed glial activation and peripheral myeloid cell infiltration extending beyond the epicenter through the remote lumbar cord after thoracic SCI. The presence and role of infiltrating monocytes is important, especially in the lumbar cord where locomotor central pattern generators are housed. Therefore, we compared the inflammatory profile of resident microglia and peripheral myeloid cells after SCI. Bone marrow chimeras received midthoracic contusive SCI, and trafficking was determined 1-7 days later. Fluorescence-activated cell (FAC) sorting showed similar infiltration timing of both neutrophils and macrophages in epicenter and lumbar regions. While neutrophil numbers were attenuated by day 3, macrophages remained unchanged at day 7, suggesting that macrophages have important long-term influence on the microenvironment. Nanostring gene array identified a strong proinflammatory profile of infiltrating macrophages relative to microglia at both epicenter and lumbar sites. Macrophages had elevated expression of inflammatory cytokines (IL-1β, IFNγ), chemokines (CCL2, CXCL2), mediators (COX-1, MMP-9), and receptors (CCR2, Ly6C), and decreased expression of growth promoting genes (GDNF, BDNF). Importantly, lumbar macrophages had elevated expression of active trafficking genes (CCR2, l-selectin, MMP-9) compared with epicenter macrophages. Further, acute rehabilitation exacerbated the inflammatory profile of infiltrated macrophages in the lumbar cord. Such high inflammatory potential and negative response to rehabilitation of infiltrating macrophages within lumbar locomotor central pattern generators likely impedes activity-dependent recovery. Therefore, limiting active trafficking of macrophages into the lumbar cord identifies a novel target for SCI therapies to improve locomotion.

A novel Gram-stain-negative, rod-shaped, motile, non-spore-forming, facultatively anaerobic marine bacterium was isolated from the gastrointestinal tract of the sandworm Alitta succinea collected from Grice Cove, South Carolina, USA. The strain was arginine dihydrolase-positive, and oxidase- and catalase-positive. Growth occurred between 10 and 37 °C, with optimal growth occurring between 30 and 32 °C. Comparative 16S rRNA gene sequence analysis showed its nearest neighbours are members of the genus Kistimonas of the family Hahellaceae , which is found in the order Oceanospirillales , class Gammaproteobacteria . The closest related species was Kistimonas asteriae KMD 001T with 16S rRNA gene sequence similarity of 99.0 %. However, DNA–DNA hybridization between these strains revealed less than 70 % DNA–DNA relatedness, supporting the novel species status of the strain. The major fatty acids were C16 : 0, C18 : 0, C18 : 1ω7c and a summed feature that contained C16 : 1ω6c/C16 : 1ω7c. The major respiratory quinone was ubiquinone-9 and the predominant polar lipids were phosphatidylserine, phosphoethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The genomic DNA G+C content was 52.5 mol%. Based on the data presented, strain BGP-2T is considered to represent a novel member of the genus Kistimonas , for which the name Kistimonas alittae sp. nov. is proposed. The type strain is BGP-2T (=CCUG 65711T=JCM 30010T).

Spinal cord injury (SCI) promotes inflammation along the neuroaxis that jeopardizes plasticity, intrinsic repair and recovery. While inflammation at the injury site is well-established, less is known within remote spinal networks. The presence of bone marrow-derived immune (myeloid) cells in these areas may further impede functional recovery. Previously, high levels of the gelatinase, matrix metalloproteinase-9 (MMP-9) occurred within the lumbar enlargement after thoracic SCI and impeded activity-dependent recovery. Since SCI-induced MMP-9 potentially increases vascular permeability, myeloid cell infiltration may drive inflammatory toxicity in locomotor networks. Therefore, we examined neurovascular reactivity and myeloid cell infiltration in the lumbar cord after thoracic SCI. We show evidence of region-specific recruitment of myeloid cells into the lumbar but not cervical region. Myeloid infiltration occurred with concomitant increases in chemoattractants (CCL2) and cell adhesion molecules (ICAM-1) around lumbar vasculature 24 h and 7 days post injury. Bone marrow GFP chimeric mice established robust infiltration of bone marrow-derived myeloid cells into the lumbar gray matter 24 h after SCI. This cell infiltration occurred when the blood-spinal cord barrier was intact, suggesting active recruitment across the endothelium. Myeloid cells persisted as ramified macrophages at 7 days post injury in parallel with increased inhibitory GAD67 labeling. Importantly, macrophage infiltration required MMP-9.

Immune-activated microglia from aged mice produce exaggerated levels of cytokines. Despite high levels of microglial interleukin (IL)-10 in the aged brain, neuroinflammation was prolonged and associated with depressive-like deficits. Because astrocytes respond to IL-10 and, in turn, attenuate microglial activation, we investigated if astrocyte-mediated resolution of microglial activation was impaired with age. Here, aged astrocytes had a dysfunctional profile with higher glial fibrillary acidic protein, lower glutamate transporter expression, and significant cytoskeletal re-arrangement. Moreover, aged astrocytes had reduced expression of growth factors and IL-10 receptor-1 (IL-10R1). After in vivo lipopolysaccharide immune challenge, aged astrocytes had a molecular signature associated with reduced responsiveness to IL-10. This IL-10 insensitivity of aged astrocytes resulted in a failure to induce IL-10R1 and transforming growth factor β and resolve microglial activation. In addition, adult astrocytes reduced microglial activation when co-cultured ex vivo, whereas aged astrocytes did not. Consistent with the aging studies, IL-10R(KO) astrocytes did not augment transforming growth factor β after immune challenge and failed to resolve microglial activation. Collectively, a major cytokine-regulatory loop between activated microglia and astrocytes is impaired in the aged brain.

Amplified neuroinflammatory responses following an immune challenge occur with normal aging and can elicit or exacerbate neuropathology. The mechanisms mediating this sensitized or “primed” immune response in the aged brain are not fully understood. The alarmin high mobility group box 1 (HMGB1) can be released under chronic pathological conditions and initiate inflammatory cascades. This led us to investigate whether HMGB1 regulates age-related priming of the neuroinflammatory response. Here, we show that HMGB1 protein and mRNA were elevated in the hippocampus of unmanipulated aged rats (24-month-old F344XBN rats). Furthermore, aged rats had increased HMGB1 in the CSF, suggesting increased HMGB1 release. We demonstrate that blocking HMGB1 signaling with an intracisterna magna (ICM) injection of the competitive antagonist to HMGB1, Box-A, downregulates basal expression of several inflammatory pathway genes in the hippocampus of aged rats. This indicates that blocking the actions of HMGB1 might reduce age-associated inflammatory priming. To test this hypothesis, we evaluated whether HMGB1 antagonism blocks the protracted neuroinflammatory and sickness response to peripheral Escherichia coli ( E. coli ) infection in aged rats. ICM pretreatment of aged rats with Box-A 24 h before E. coli infection prevented the extended hippocampal cytokine response and associated cognitive and affective behavioral changes. ICM pretreatment with Box-A also inhibited aging-induced potentiation of the microglial proinflammatory response to lipopolysaccharide ex vivo . Together, these results suggest that HMGB1 mediates neuroinflammatory priming in the aged brain. Blocking the actions of HMGB1 appears to “desensitize” aged microglia to an immune challenge, thereby preventing exaggerated behavioral and neuroinflammatory responses following infection. SIGNIFICANCE STATEMENT The world9s population is aging, highlighting a need to develop treatments that promote quality of life in aged individuals. Normal aging is associated with precipitous drops in cognition, typically following events that induce peripheral inflammation (e.g., infection, surgery, heart attack). Peripheral immune stimuli cause exaggerated immune responses in the aged brain, which likely underlie these behavioral deficits. Here, we investigated whether the alarmin high mobility group box 1 (HMGB1) mediates age-associated “priming” of the neuroinflammatory response. HMGB1 is elevated in aged rodent brain and CSF. Blocking HMGB1 signaling downregulated expression of inflammatory pathway genes in aged rat brain. Further, HMGB1 antagonism prevented prolonged infection-induced neuroinflammatory and sickness responses in aged rats. Overall, blocking HMGB1 “desensitized” microglia in the aged brain, thereby preventing pathological infection-elicited neuroinflammatory responses.

Antiviral immunity is severely compromised following trauma to the central nervous system. In mice with chronic spinal cord injury (SCI), primary infection with influenza virus leads to high mortality rates due to impaired expansion of virus-specific CD8 T cells. One strategy to increase resistance to viral infections is to generate memory immune cells that protect from recurrent infections. However, it is unknown if chronic SCI also impairs secondary immune responses to influenza challenge as it does primary responses. Here, we used a mouse model of chronic SCI and a clinically relevant influenza A infection to investigate CD8 T cell response. As shown previously, chronic SCI mice had impaired primary antiviral responses with high mortality rates and decreased expansion of virus-specific CD8 T cells following intranasal infection. To investigate CD8 T cell memory, we used two strains of influenza A virus [PR8(H1N1) and X31(H3N2)] that share internal proteins but differ in surface antigens. Chronic SCI mice immunized with live X31 were able to generate memory CD8 T cells that secreted IFNγ upon stimulation with viral peptides ex vivo, which was comparable to immunized uninjured mice. Importantly, immunization prior to challenge with a lethal dose of PR8 resulted in no mortality and significant CD8 T cell recall responses in both uninjured and chronic SCI mice. In addition, memory CD8 T cells generated before SCI remained functional up to 8 weeks after injury. These pre-existing memory CD8 T cells provided full protection from lethal PR8 challenge given at the chronic timepoint following injury. Overall, this study shows that memory CD8 T cells generated either before or after chronic SCI still remain functional. These results highlight the need for proper immunization of SCI patients and show the potential of memory T cells to confer protection against not only influenza, but other viral infections as well.

Activation of the peripheral immune system elicits a coordinated response from the central nervous system. Key to this immune to brain communication is that glia, microglia, and astrocytes, interpret and propagate inflammatory signals in the brain that influence physiological and behavioral responses. One issue in glial biology is that morphological analysis alone is used to report on glial activation state. Therefore, our objective was to compare behavioral responses after in vivo immune (lipopolysaccharide, LPS) challenge to glial specific mRNA and morphological profiles. Here, LPS challenge induced an immediate but transient sickness response with decreased locomotion and social interaction. Corresponding with active sickness behavior (2-12 h), inflammatory cytokine mRNA expression was elevated in enriched microglia and astrocytes. Although proinflammatory cytokine expression in microglia peaked 2-4 h after LPS, astrocyte cytokine, and chemokine induction was delayed and peaked at 12 h. Morphological alterations in microglia (Iba-1(+)) and astrocytes (GFAP(+)), however, were undetected during this 2-12 h timeframe. Increased Iba-1 immunoreactivity and de-ramified microglia were evident 24 and 48 h after LPS but corresponded to the resolution phase of activation. Morphological alterations in astrocytes were undetected after LPS. Additionally, glial cytokine expression did not correlate with morphology after four repeated LPS injections. In fact, repeated LPS challenge was associated with immune and behavioral tolerance and a less inflammatory microglial profile compared with acute LPS challenge. Overall, induction of glial cytokine expression was sequential, aligned with active sickness behavior, and preceded increased Iba-1 or GFAP immunoreactivity after LPS challenge.

Cancer patients frequently suffer from fatigue, a complex syndrome associated with tiredness and depressed mood. Cancer-related fatigue (CRF) can be present at the time of diagnosis, escalates during treatment, and can persist for years after treatment. CRF negatively influences quality of life, limits functional independence, and is associated with decreased survival in patients with incurable disease. We have previously shown that increased pro-inflammatory cytokine expression in the brain contributes to depressive- and fatigue-like behaviors in a mouse model of CRF. Inflammatory cytokines increase the activity of indoleamine 2,3-dioxygenase (IDO) and kynurenine 3-monooxygenase (KMO), which competitively reduce serotonin synthesis. Reduced serotonin availability in the brain and increased production of alternative neuroactive metabolites of tryptophan are thought to contribute to the development of depression and fatigue. The purpose of this study was to determine the effects of fluoxetine, a selective serotonin reuptake inhibitor (SSRI), on brain cytokines and behavioral measures of fatigue and depression in tumor-bearing mice. Here we show that tumor growth increased brain expression of pro-inflammatory cytokines and KMO. Treatment with fluoxetine had no effect on tumor growth, muscle wasting, fatigue behavior, or cytokine expression in the brain. Fluoxetine, however, reduced depressive-like behaviors in tumor bearing mice. In conclusion, our data confirm that increased brain expression of pro-inflammatory cytokines is associated with tumor-induced fatigue- and depressive-like behaviors. However, it is possible to separate the effects of tumor growth on mood and fatigue-like behaviors using SSRIs such as fluoxetine.

While there clearly is an intimate relationship between astrocytes and microglia, few studies have examined these potentially dynamic interactions. In this study, cytokine-mediated communication between microglia and astrocytes under inflammatory conditions was investigated. We have previously shown that activated microglia produce Interleukin (IL)-10, a regulatory cytokine that plays an important role in resolving neuroinflammation. Nonetheless, the mechanism by which IL-10 attenuates pro-inflammatory cytokine expression in the brain is unclear. Here, we show that IL-10 redirected astrocytes regulate the activation of microglia in a transforming growth factor (TGF)-β dependent manner. In support of this concept, astrocytes in the brain maintained higher IL-10 receptor (IL-10R1) expression and primary astrocytes in culture were markedly more sensitive to the anti-inflammatory effects of IL-10 compared with microglia. Moreover, studies using primary cultures and an astrocyte-microglia coculture system revealed that astrocytes mediated the anti-inflammatory effects of IL-10 on microglia through the production of TGFβ. For instance, only when astrocytes were present did IL-10 stimulation reduce the expression of IL-1β and increase expression of anti-inflammatory mediators fractalkine receptor (CX3CR1) and interleukin 4 receptor-α (IL-4Rα) in microglia. Importantly, these IL-10-astrocyte dependent effects on microglia were blocked by a TGFβ inhibitor. Furthermore, inhibition of TGFβ signaling in the brain resulted in prolonged sickness behavior and amplified pro-inflammatory cytokine expression in mice challenged with lipopolysaccharide. Taken together, IL-10 stimulated the production of TGFβ by astrocytes, which in turn, attenuated microglial activation. Overall, these findings provide novel insight into the mechanisms by which astrocytes modulate microglia under inflammatory conditions. GLIA 2014;62:881–895

The potential use of geologic salt beds as terminal repositories for nuclear waste has necessitated research on the interaction of the waste with indigenous microbiota. Microorganisms may affect actinide solubility by degrading organic complexing agents present in the waste. A halophilic bacterium and Archaea indigenous to a salt formation in New Mexico were examined for their ability to degrade acetate, oxalate, citrate, and ethylenediamine tetraacetate under aerobic conditions in low and high-magnesium brines. All complexing agents, except EDTA, were utilized, suggesting that microorganisms indigenous to such repositories can potentially play a beneficial role in mitigating actinide mobility.

Innate immunity within the central nervous system (CNS) is primarily provided by resident microglia. Microglia are pivotal in immune surveillance and also facilitate the co-ordinated responses between the immune system and the brain. For example, microglia interpret and propagate inflammatory signals that are initiated in the periphery. This transient microglial activation helps mount the appropriate physiological and behavioural response following peripheral infection. With normal ageing, however, microglia develop a more inflammatory phenotype. For instance, in several models of ageing there are increased pro-inflammatory cytokines in the brain and increased expression of inflammatory receptors on microglia. This increased inflammatory status of microglia with ageing is referred to as primed, reactive or sensitized. A modest increase in the inflammatory profile of the CNS and altered microglial function in ageing has behavioural and cognitive consequences. Nonetheless, there are major differences in microglial biology between young and old age when the immune system is challenged and microglia are activated. In this context, microglial activation is amplified and prolonged in the aged brain compared with adults. The cause of this amplified microglial activation may be related to impairments in several key regulatory systems with age that make it more difficult to resolve microglial activation. The consequences of impaired regulation and microglial hyper-activation following immune challenge are exaggerated neuroinflammation, sickness behaviour, depressive-like behaviour and cognitive deficits. Therefore the purpose of this review is to discuss the current understanding of age-associated microglial priming, consequences of priming and reactivity, and the impairments in regulatory systems that may underlie these age-related deficits.

Aims Cancer-related fatigue (CRF) is often accompanied by depressed mood, both of which reduce functional status and quality of life. Research suggests that increased expression of pro-inflammatory cytokines is associated with skeletal muscle wasting and depressive- and fatigue-like behaviors in rodents and cancer patients. We have previously shown that treatment with ibuprofen, a nonsteroidal anti-inflammatory drug, preserved muscle mass in tumor-bearing mice. Therefore, the purpose of the present study was to determine the behavioral effects of ibuprofen in a mouse model of CRF. Main methods Mice were injected with colon-26 adenocarcinoma cells and treated with ibuprofen (10 mg/kg) in the drinking water. Depressive-like behavior was determined using the forced swim test (FST). Fatigue-like behaviors were determined using voluntary wheel running activity (VWRA) and grip strength. The hippocampus, gastrocnemius muscle, and serum were collected for cytokine analysis. Key findings Tumor-bearing mice showed depressive-like behavior in the FST, which was not observed in mice treated with ibuprofen. VWRA and grip strength declined in tumor-bearing mice, and ibuprofen attenuated this decline. Tumor-bearing mice had decreased gastrocnemius muscle mass and increased expression of IL-6, MAFBx and MuRF mRNA, biomarkers of protein degradation, in the muscle. Expression of IL-1β and IL-6 was also increased in the hippocampus. Treatment with ibuprofen improved muscle mass and reduced cytokine expression in both the muscle and hippocampus of tumor-bearing mice. Significance Ibuprofen treatment reduced skeletal muscle wasting, inflammation in the brain, and fatigue- and depressive-like behavior in tumor-bearing mice. Therefore, ibuprofen warrants evaluation as an adjuvant treatment for CRF.

Activation of the peripheral immune system elicits a coordinated response from the central nervous system. Key to this immune to brain communication is that glia, microglia, and astrocytes, interpret and propagate inflammatory signals in the brain that influence physiological and behavioral responses. One issue in glial biology is that morphological analysis alone is used to report on glial activation state. Therefore, our objective was to compare behavioral responses after in vivo immune (lipopolysaccharide, LPS) challenge to glial specific mRNA and morphological profiles. Here, LPS challenge induced an immediate but transient sickness response with decreased locomotion and social interaction. Corresponding with active sickness behavior (2-12 h), inflammatory cytokine mRNA expression was elevated in enriched microglia and astrocytes. Although proinflammatory cytokine expression in microglia peaked 2-4 h after LPS, astrocyte cytokine, and chemokine induction was delayed and peaked at 12 h. Morphological alterations in microglia (Iba-1(+)) and astrocytes (GFAP(+)), however, were undetected during this 2-12 h timeframe. Increased Iba-1 immunoreactivity and de-ramified microglia were evident 24 and 48 h after LPS but corresponded to the resolution phase of activation. Morphological alterations in astrocytes were undetected after LPS. Additionally, glial cytokine expression did not correlate with morphology after four repeated LPS injections. In fact, repeated LPS challenge was associated with immune and behavioral tolerance and a less inflammatory microglial profile compared with acute LPS challenge. Overall, induction of glial cytokine expression was sequential, aligned with active sickness behavior, and preceded increased Iba-1 or GFAP immunoreactivity after LPS challenge.

Glia of the central nervous system (CNS) help to maintain homeostasis in the brain and support efficient neuronal function. Microglia are innate immune cells of the brain that mediate responses to pathogens and injury. They have key roles in phagocytic clearing, surveying the local microenvironment and propagating inflammatory signals. An interruption in homeostasis induces a cascade of conserved adaptive responses in glia. This response involves biochemical, physiological and morphological changes and is associated with the production of cytokines and secondary mediators that influence synaptic plasticity, cognition and behavior. This reorganization of host priorities represents a beneficial response that is normally adaptive but may become maladaptive when the profile of microglia is compromised. For instance, microglia can develop a primed or pro-inflammatory mRNA, protein and morphological profile with aging, traumatic brain injury and neurodegenerative disease. As a result, primed microglia exhibit an exaggerated inflammatory response to secondary and sub-threshold challenges. Consequences of exaggerated inflammatory responses by microglia include the development of cognitive deficits, impaired synaptic plasticity and accelerated neurodegeneration. Moreover, impairments in regulatory systems in these circumstances may make microglia more resistant to negative feedback and important functions of glia can become compromised and dysfunctional. Overall, the purpose of this review is to discuss key concepts of microglial priming and immune-reactivity in the context of aging, traumatic CNS injury and neurodegenerative disease. This article is part of a Special Issue entitled 'Neuroimmunology and Synaptic Function'.

Cancer patients frequently suffer from fatigue, a complex syndrome associated with loss of muscle mass, weakness, and depressed mood. Cancer-related fatigue (CRF) can be present at the time of diagnosis, during treatment, and persists for years after treatment. CRF negatively influences quality of life, limits functional independence, and is associated with decreased survival in patients with incurable disease. Currently there are no effective treatments to reduce CRF. The aim of this study was to use a mouse model of tumor growth and discriminate between two main components of fatigue: loss of muscle mass/function and altered mood/motivation. Here we show that tumor growth increased fatigue- and depressive-like behaviors, and reduced body and muscle mass. Decreased voluntary wheel running activity (VWRA) and increased depressive-like behavior in the forced swim and sucrose preference tests were evident in tumor-bearing mice within the first two weeks of tumor growth and preceded the loss of body and muscle mass. At three weeks, tumor-bearing mice had reduced grip strength but this was not associated with altered expression of myosin isoforms or impaired contractile properties of muscles. These increases in fatigue and depressive-like behaviors were paralleled by increased expression of IL-1β mRNA in the cortex and hippocampus. Minocycline administration reduced tumor-induced expression of IL-1β in the brain, reduced depressive-like behavior, and improved grip strength without altering muscle mass. Taken together, these results indicate that neuroinflammation and depressed mood, rather than muscle wasting, contribute to decreased voluntary activity and precede major changes in muscle contractile properties with tumor growth.

Repeated social defeat (RSD) in mice causes myeloid cell trafficking to the brain that contributes to the development of prolonged anxiety-like behavior. Myeloid cell recruitment following RSD occurs in regions where neuronal and microglia activation is observed. Thus, we hypothesized that crosstalk between neurons, microglia, and endothelial cells contributes to brain myeloid cell trafficking via chemokine signaling and vascular adhesion molecules. Here we show that social defeat caused an exposure- and brain region-dependent increase in several key adhesion molecules and chemokines involved in the recruitment of myeloid cells. For example, RSD induced distinct patterns of adhesion molecule expression that may explain brain region-dependent myeloid cell trafficking. VCAM-1 and ICAM-1 mRNA expression were increased in an exposure-dependent manner. Furthermore, RSD-induced VCAM-1 and ICAM-1 protein expression were localized to the vasculature of brain regions implicated in fear and anxiety responses, which spatially corresponded to previously reported patterns of myeloid cell trafficking. Next, mRNA expression of additional adhesion molecules (E- and P-selectin, PECAM-1) and chemokines (CXCL1, CXCL2, CXCL12, CCL2) were determined in the brain. Social defeat induced an exposure-dependent increase in mRNA levels of E-selectin, CXCL1, and CXCL2 that increased with additional days of social defeat. While CXCL12 was unaffected by RSD, CCL2 expression was increased by six days of social defeat. Last, comparison between enriched CD11b(+) cells (microglia/macrophages) and enriched GLAST-1(+)/CD11b(-) cells (astrocytes) revealed RSD increased mRNA expression of IL-1β, CCL2, and CXCL2 in microglia/macrophages but not in astrocytes. Collectively, these data indicate that key mediators of leukocyte recruitment were increased in the brain vasculature following RSD in an exposure- and brain region-dependent manner.

Activated microglia from aged mice produce exaggerated levels of both pro-and anti-inflammatory cytokines, including interleukin (IL)-10. Despite higher levels of IL-10, microglial activation is prolonged in the aged brain following systemic immune challenge. Recently we showed that astrocytes of adult mice express the IL-10 receptor (IL-10R) and that IL-10 re-directs active astrocytes to produce TGFbeta, which in turn, attenuates the activation of microglia. Therefore, the purpose of this study was to investigate the degree to which these key cytokine interactions between glia are impaired in the aged brain. Here we report that along with microglia, astrocytes also had a primed morphological profile in aged mice. In addition, aged astrocytes had increased GFAP and vimentin expression, and decreased IL-10R surface expression compared to adults. Following acute immune challenge in vivo, adult astrocytes up-regulated IL-10R and TGFbeta mRNA. Aged astrocytes, however, failed to increase expression of these mediators. This lack of regulation by TGFbeta was associated with decreased TGFbeta signaling and exaggerated expression of pro-inflammatory mediators in aged microglia. Additionally, active microglia cultured ex vivo with adult astrocytes reduced inflammatory markers while those cultured with aged astrocytes did not. In summary, these novel data indicate that astrocytes have an important role in regulating microglia via TGFbeta signaling and that an impaired IL-10 response in aged astrocytes contributes to age-related deficits in the regulation of active microglia.

While there clearly is an intimate relationship between astrocytes and microglia, few studies have examined these potentially dynamic interactions. In this study, cytokine-mediated communication between microglia and astrocytes under inflammatory conditions was investigated. We have previously shown that activated microglia produce Interleukin (IL)-10, a regulatory cytokine that plays an important role in resolving neuroinflammation. Nonetheless, the mechanism by which IL-10 attenuates pro-inflammatory cytokine expression in the brain is unclear. Here we show that IL-10 re-directed astrocytes regulate the activation of microglia in a Transforming growth factor (TGF)-β dependent manner. In support of this concept, astrocytes in the brain maintained higher IL-10 receptor (IL-10R1) expression and primary astrocytes in culture were markedly more sensitive to the anti-inflammatory effects of IL-10 compared to microglia. Moreover, studies using primary cultures and an astrocyte-microglia co-culture system revealed that astrocytes mediated the anti-inflammatory effects of IL-10 on microglia through the production of TGFβ. For instance, only when astrocytes were present did IL-10 stimulation reduce the expression of IL-1β and increase expression of anti-inflammatory mediators fractalkine receptor (CX3CR1) and interleukin 4 receptor-α (IL-4Rα) in microglia. Importantly, these IL-10-astrocyte dependent effects on microglia were blocked by a TGFβ inhibitor. Furthermore, inhibition of TGFβ signaling in the brain resulted in prolonged sickness behavior and amplified pro-inflammatory cytokine expression in mice challenged with lipopolysaccharide (LPS). Taken together, IL-10 stimulated the production of TGFβ by astrocytes, which in turn, attenuated microglial activation. Overall, these findings provide novel insight into the mechanisms by which astrocytes modulate microglia under inflammatory conditions.

We previously reported that systemic injection of AAV9 in aged mice (P550) results in significantly fewer transduced CNS cells compared to adult mice (P49). This was not due to the presence of neutralizing antibodies in the aged mice. To determine whether aging in the brain and/or the periphery is responsible for this reduction, we quantified CNS transduction following stereotaxic brain injection in aged or adult mice. Aged male Balb/c mice (n = 6, P550) and adult mice (n = 7, P50) received direct injections into the striatum and ipsilateral hippocampus with scAAV9-CB expressing green fluorescent protein or scAAV9-CB expressing glial cell line derived neurotrophic factor (GFP or GDNF). All mice were euthanized three weeks post-injection. Brains of aged and adult mice injected with scAAV9-CB-GFP were sliced and processed for near-infrared imaging to quantify vector spread and GFP intensity. GFP expression was similar in directly injected aged and adult brains. Vector spread along the rostrocaudal axis was approximately 2 mm from either the striatal or hippocampal injection site and did not vary with age. Brains of mice directly injected with scAAV9-CB-GDNF were hemisected and processed for ELISA to quantify GDNF protein levels. GDNF levels in the striatum and hippocampus reached 2x those of uninjected controls and no differences in GDNF expression were detected between aged and adult injected mice. Together, we found that AAV9 CNS transduction is similar in aged and adult mouse brains following direct injection. These results suggest that reduced CNS transduction in aged mice following AAV9 systemic injection is due to alterations in the periphery. Future studies include investigation of altered viral blood brain barrier penetration and viral retention within peripheral tissues of aged mice.

This report summarizes the progress made in the ongoing task of characterizing the microbial community structures within the WIPP repository and in surrounding groundwaters. Through cultivation and DNA-based identification, the potential activity of these organisms is being inferred, thus leading to a better understanding of their impact on WIPP performance. Members of the three biological domains - Bacteria, Archaea, and Eukarya (in this case, Fungi) - that are associated with WIPP halite have been identified. Thus far, their activity has been limited to aerobic respiration; anaerobic incubations are underway. WIPP halite constitutes the near-field microbial environment. We expect that microbial activity in this setting will proceed from aerobic respiration, through nitrate reduction to focus on sulfate reduction. This is also the current WIPP performance assessment (PA) position. Sulfate reduction can occur at extremely high ionic strengths, and sulfate is available in WIPP brines and in the anhydrite interbeds. The role of methanogenesis in the WIPP remains unclear, due to both energetic constraints imposed by a high-salt environment and substrate selectivity, and it is no longer considered in PA. Archaea identified in WIPP halite thus far fall exclusively within the family Halobacteriaceae. These include Halobacterium noricense, cultivated from both low- andmore » high-salt media, and a Halorubrum-like species. The former has also been detected in other salt mines worldwide; the latter likely constitutes a new species. Little is known of its function, but it was prevalent in experiments investigating the biodegradation of organic complexing agents in WIPP brines. Bacterial signatures associated with WIPP halite include members of the phylum Proteobacteria - Halomonas, Pelomonas, Limnobacter, and Chromohalobacter - but only the latter has been isolated. Also detected and cultivated were Salinicoccus and Nesterenkonia spp. Fungi were also isolated from halite. Although these were most likely introduced into the WIPP as contaminants from above-ground, their survival and potential role in the WIPP (e.g., cellulose degradation) is under investigation. WIPP groundwaters comprise the far-field microbial environment. Bacteria cultivated and identified from the overlying Culebra and nearby borehole groundwater are capable of aerobic respiration, denitrification, fermentation, metal reduction, and sulfate reduction and are distributed across many different phyla. Two of the Bacteria found in groundwater were also found in WIPP halite (Chromohalobacter sp. and Virgibacillus sp.). Archaea identified in groundwater include Halococcus saccharolyticus, Haloferax sp., and Natrinema sp. The differences in the microbial communities detected thus far in halite and groundwater suggest that there will be significant differences in the associated metabolic potential of the near- and far-field environments. Whereas the near-field is dominated by Archaea with more limited metabolic capabilities, the far-field is dominated by Bacteria with extremely broad capabilities. Because the majority of the repository's lifetime will be anoxic, ongoing and future work focuses on the presence and role of anaerobic organisms in WIPP. Further tasks on biosorption, cellulose degradation, and bioreduction are being performed using organisms obtained from this characterization work.« less

An impaired ability to regulate the activation of microglia by fractalkine (CX3CL1) leads to persistent neuroinflammation and behavioral alterations following lipopolysaccharide (LPS) challenge. While these responses are usually transient, LPS injection caused prolonged depressive-like behavior in fractalkine receptor deficient mice (CX3CR1(-/-)) that was associated with exaggerated microglial activation and induction of the tryptophan (TRP) degrading enzyme indoleamine 2,3-dioxygenase (IDO). IDO activation and subsequent generation of neuroactive kynurenine metabolites may have a pivotal role in the development of depression. Therefore, the purpose of this study was to determine the extent to which LPS-induced depressive-like behavior in CX3CR1(-/-) mice was dependent on IDO activation. CX3CR1(-/-) mice were implanted prior to LPS challenge with a slow release pellet of 1-methyl-tryptophan (1-MT), a competitive inhibitor of IDO. Here we show that the depressive-like behavior evident in CX3CR1(-/-) mice 72 h after LPS injection was abrogated by inhibition of IDO. LPS also decreased body weight and locomotor activity in CX3CR1(-/-) mice, but these effects were independent of 1-MT. Consistent with the increased metabolism of TRP by IDO, the ratio of 3-hydroxykynurenine (3-HK) to TRP was increased in the brain 72 h after LPS. Increased serotonin (5-HT) turnover was also evident in the brain. The LPS-associated increases in both 3-HK:TRP and 5-HIAA:5-HT ratios were prevented by the inhibition of IDO. Last, IDO blockade attenuated microglial activation in the prefrontal cortex and hippocampus 72 h after LPS. Collectively these data indicate that LPS-induced IDO activation contributes to persistent microglial activation and depressive-like behavior in CX3CR1(-/-) mice.

Cancer patients frequently suffer concomitantly from loss of muscle mass, fatigue and depression. Understanding cancer-related fatigue (CRF) is critical because it negatively influences quality of life, functional independence, and survival. In the present study, we used a mouse model of tumor progression to discriminate between two main components of fatigue: loss of muscle mass/function and altered mood/motivation. Here we show that tumor progression over time was associated with increased fatigue, reduced body and muscle mass, and increased depressive-like behavior. Tumor induced fatigue, however, was independent of the functional capabilities of skeletal muscle. Moreover, fatigue, depressive-like behavior and increased cytokine expression in the brain were evident in tumor bearing mice within the first 2 weeks and preceded the loss of body and muscle mass. For example, within 2 weeks of tumor progression there was reduced voluntary wheel running activity and increased depressive-like behavior (e.g., resignation and anhedonia) that was not associated with general malaise or reduced muscle mass and contractile function. In addition, these changes in motivation and mood were paralleled by increased expression of IL-1β in the brain. Last, administration of minocycline, an anti-inflammatory agent and microglial inhibitor, ameliorated the tumor-induced increase in brain IL-1β expression, depressive-like behavior and fatigue. Taken together, these results indicate that inflammation and depressed mood are important early contributors to decreased physical activity and weakness associated with CRF.

Psychosocial stress is associated with increased inflammation and higher prevalence of mental health disorders like anxiety and depression. Through the activation of several neuroendocrine pathways, psychological stress leads to significant physiological, immunological, and behavioral changes. Repeated social defeat (RSD), a murine model of psychosocial stress, recapitulates many of the behavioral and immunological effects observed in humans, including increased circulating cytokines, immune cell recruitment, and prolonged anxiety-like behavior. The aim of this study was to elucidate the mechanisms underlying stress-induced immune cell trafficking to the brain that leads to the development of a reactive endothelium and behavioral changes. We show that RSD caused an exposure-dependent increase in the gene expression of ICAM1, VCAM1, E-selectin, CXCL1, and CXCL2 in the brain that increased with additional days of stress. RSD-induced ICAM1 and VCAM1 protein expression were localized to the vasculature of brain regions implicated in fear and anxiety responses, which spatially corresponded to previously reported patterns of myeloid cell trafficking. Comparison between enriched CD11b+ cells (microglia/macrophages) and enriched GLAST-1+/CD11b- cells (astrocytes) revealed that RSD increased the gene expression of IL-1 beta, CCL2, and CXCL2 in microglia/macrophages, but not astrocytes. Collectively, these data indicate that critical adhesion mediators are increased in the brain vasculature following RSD. This study begins to establish a mechanism by which the brain facilitates stress-induced immune cell recruitment that may underlie anxiety and mood disorders.

Activated microglia from aged mice produce exaggerated levels of both pro-and anti-inflammatory cytokines, including interleukin (IL)-10. Despite higher levels of IL-10, microglial activation is prolonged in the aged brain. Astrocytes express the IL-10 receptor and produce cytokines, including TGFβ, that attenuate microglial activation. Therefore, to examine the failure of IL-10 to resolve inflammation within the aged brain, we evaluated the sensitivity of microglia and astrocytes to IL-10. We found that in vivo, astrocytes have higher surface expression of IL-10 receptor compared to microglia. In vitro, IL-10 lowered IL-1β expression in activated astrocytes, and not microglia. Furthermore, IL-10 increased TGFβ production in activated astrocytes. TGFβ, in turn increased expression of the regulatory receptors CX3CR1 and IL-4R on microglia. In astrocyte and microglia co-cultures, IL-10 decreased IL-1β expression in microglia and increased IL-4R expression. Blockade of TGFβ signaling decreased IL-4R induction and also decreased CX3CR1 expression in microglia co-cultured with astrocytes. Thus, astrocyte production of TGFβ plays an important role in regulating immune activated microglia. Next, we examined the inflammatory status of astrocytes in adult and aged mice and found that astrocytes in aged mice had increased GFAP and vimentin expression, and decreased IL-10 receptor surface expression compared to adults. Overall, astrocytes from aged mice are more inflammatory and less sensitive to IL-10, which may negatively affect TGFβ production and regulation of microglia in the aged brain.