Myeloid caspase-8 restricts RIPK3-dependent proinflammatory IL-1ß production and CD4 T cell activation in autoimmune demyelination.

Caspase-8 functions at the crossroad of programmed cell death and inflammation. Here, using genetic approaches and the experimental autoimmune encephalomyelitis model of inflammatory demyelination, we identified a negative regulatory pathway for caspase-8 in infiltrated macrophages whereby it functions to restrain interleukin (IL)-1ß-driven autoimmune inflammation. Caspase-8 is partially activated in macrophages/microglia in active lesions of multiple sclerosis. Selective ablation of Casp8 in myeloid cells, but not microglia, exacerbated autoimmune demyelination. Heightened IL-1ß production by caspase-8-deficient macrophages underlies exacerbated activation of encephalitogenic T cells and production of GM-CSF and interferon-γ. Mechanistically, IL-1ß overproduction by primed caspase-8-deficient macrophages was mediated by RIPK1/RIPK3 through the engagement of NLRP3 inflammasome and was independent of cell death. When instructed by autoreactive CD4 T cells in the presence of antigen, caspase-8-deficient macrophages, but not their wild-type counterparts, released significant amount of IL-1ß that in turn acted through IL-1R to amplify T cell activation. Moreover, the worsened experimental autoimmune encephalomyelitis progression in myeloid Casp8 mutant mice was completely reversed when Ripk3 was simultaneously deleted. Together, these data reveal a functional link between T cell-driven autoimmunity and inflammatory IL-1ß that is negatively regulated by caspase-8, and suggest that dysregulation of the pathway may contribute to inflammatory autoimmune diseases, such as multiple sclerosis.

Role of omega-3 endocannabinoids in the modulation of T-cell activity in a multiple sclerosis experimental autoimmune encephalomyelitis (EAE) model.

Epidemiological studies show that omega-3 fatty acid consumption is associated with improved conditions in neurodegenerative diseases such as multiple sclerosis (MS). However, the mechanism of this association is not well understood. Emerging evidence suggests that parent molecules such as docosahexaenoic acid are converted into downstream metabolites that are capable of directly modulating immune responses. In vitro, we found that docosahexaenoyl ethanolamide (DHEA), another dietary component and its epoxide metabolite, reduced the polarization of naïve T-cells toward proinflammatory Th1 and Th17 phenotypes. Furthermore, we identified that DHEA and related endocannabinoids are changing during the disease progression in mice undergoing relapse-remitting experimental autoimmune encephalomyelitis (RR-EAE). In addition, daily administration of DHEA to mice delayed the onset of disease, the rate of relapse, and the severity of clinical scores at relapse in RR-EAE, an animal model of MS. Collectively, these data indicate that DHEA and their downstream metabolites reduce the disease severity in the RR-EAE model of MS and can be potential dietary adjuvants to existing MS therapeutics.

Mononuclear phagocyte morphological response to chemoattractants is dependent on geranylgeranyl pyrophosphate.

Statins are used to treat hypercholesterolemia and function by inhibiting the production of the rate-limiting metabolite mevalonate. As such, statin treatment not only inhibits de novo synthesis of cholesterol but also isoprenoids that are involved in prenylation, the posttranslational lipid modification of proteins. The immunomodulatory effects of statins are broad and often conflicting. Previous work demonstrated that statins increased survival and inhibited myeloid cell trafficking in a murine model of sepsis, but the exact mechanisms underlying this phenomenon were unclear. Herein, we investigated the role of prenylation in chemoattractant responses. We found that simvastatin treatment abolished chemoattractant responses induced by stimulation by C5a and FMLP. The inhibitory effect of simvastatin treatment was unaffected by the addition of either farnesyl pyrophosphate (FPP) or squalene but was reversed by restoring geranylgeranyl pyrophosphate (GGPP). Treatment with prenyltransferase inhibitors showed that the chemoattractant response to both chemoattractants was dependent on geranylgeranylation. Proteomic analysis of C15AlkOPP-prenylated proteins identified several geranylgeranylated proteins involved in chemoattractant responses, including RHOA, RAC1, CDC42, and GNG2. Chemoattractant responses in THP-1 human macrophages were also geranylgeranylation dependent. These studies provide data that help clarify paradoxical findings on the immunomodulatory effects of statins. Furthermore, they establish the role of geranylgeranylation in mediating the morphological response to chemoattractant C5a.NEW & NOTEWORTHY The immunomodulatory effect of prenylation is ill-defined. We investigated the role of prenylation on the chemoattractant response to C5a. Simvastatin treatment inhibits the cytoskeletal remodeling associated with a chemotactic response. We showed that the chemoattractant response to C5a was dependent on geranylgeranylation, and proteomic analysis identified several geranylgeranylated proteins that are involved in C5a receptor signaling and cytoskeletal remodeling. Furthermore, they establish the role of geranylgeranylation in mediating the response to chemoattractant C5a.

α-Tocopherol Depletion Exacerbates Lipopolysaccharide-Induced Reduction of Grip Strength.

BACKGROUND: α-Tocopherol (αT) deficiency causes several neurologic disorders, such as spinocerebellar ataxia, peripheral neuropathy, and myopathy. Furthermore, decreased antibody production, impaired ex vivo T cell function, and elevated cytokine production are observed in humans and mice with αT deficiency. Although modeling αT deficiency in animals is challenging, αT depletion can be more readily achieved in α-tocopherol transfer protein-null (Ttpa-/-) mice than wild-type (WT) mice. Thus, the Ttpa-/- mouse model is a useful tool for studying metabolic consequences of low αT status. Optimizing this mouse model and selecting the reliable indicators/markers of deficiency are still needed. OBJECTIVE: Our objective was to assess whether αT depletion alters lipopolysaccharide (LPS)-induced inflammatory response in the brain and/or grip strength used as a proxy for fatigue. METHODS: WT and Ttpa-/- weanling littermates (n = 37-40/genotype) were fed an αT deficient diet ad libitum for 9 wk. Mice were then injected with LPS (10 µg/mouse) or saline (control) intraperitoneally and killed 4 h later. Concentrations of αT in diet and tissues were measured via high-pressure liquid chromatography. Grip strength was evaluated via a grip strength meter apparatus 2 d before and 3.5 h after LPS injection. Cerebellar and serum interleukin-6 (IL-6) concentrations were measured via enzyme-linked immunosorbent assay. RESULTS: αT concentrations in the liver, heart, and adipose tissue of WT mice were higher than Ttpa-/- mice. Although αT was detected in the brain, muscle, and serum of WT mice, it was undetectable in these tissues of Ttpa-/- mice. Cerebellar and serum concentrations of IL-6 were increased in LPS-treated groups but were not significantly affected by genotype. Grip strength was reduced in LPS-treated groups, an effect that was more pronounced in Ttpa-/- mice. CONCLUSIONS: Systemic LPS administration caused an acute inflammatory response with a concomitant decline in grip strength, especially in Ttpa-/- mice. αT depletion appears to exacerbate reductions in grip strength brought on by systemic inflammation.

The emergence of inflammatory microglia during gut inflammation is not affected by FFAR2 expression in intestinal epithelial cells or peripheral myeloid cells.

Gut inflammation can trigger neuroinflammation and is linked to mood disorders. Microbiota-derived short-chain fatty acids (SCFAs) can modulate microglia, yet the mechanism remains elusive. Since microglia do not express free-fatty acid receptor (FFAR)2, but intestinal epithelial cells (IEC) and peripheral myeloid cells do, we hypothesized that SCFA-mediated FFAR2 activation within the gut or peripheral myeloid cells may impact microglia inflammation. To test this hypothesis, we developed a tamoxifen-inducible conditional knockout mouse model targeting FFAR2 exclusively on IEC and induced intestinal inflammation with dextran sodium sulfate (DSS), a well-established colitis model. Given FFAR2's high expression in myeloid cells, we also investigated its role by selectively deleting it in these populations of cells. In an initial study, male and female wild-type mice received 0 or 2% DSS for 5d and microglia were isolated 3d later to assess inflammatory status. DSS induced intestinal inflammation and upregulated inflammatory gene expression in microglia, indicating inflammatory signaling via the gut-brain axis. Despite the lack of significant effects of sex in the intestinal phenotype, male mice showed higher microglial inflammatory response than females. Subsequent studies using FFAR2 knockout models revealed that FFAR2 expression in IECs or immune myeloid cells did not affect DSS-induced colonic pathology (i.e. clinical and histological scores and colon length), or colonic expression of inflammatory genes. However, FFAR2 knockout led to an upregulation of several microglial inflammatory genes in control mice and downregulation in DSS-treated mice, suggesting that FFAR2 may constrain neuroinflammatory gene expression under healthy homeostatic conditions but may permit it during intestinal inflammation. No interactions with sex were observed, suggesting sex does not play a role on FFAR2 potential function in gut-brain communication in the context of colitis. To evaluate the role of FFAR2 activated by microbiota-derived SCFAs, we employed the same knockout and DSS models adding fermentable dietary fiber (0 or 2.5% inulin for 8 wks). Despite no genotype or fiber main effects, contrary to our hypothesis, inulin feeding augmented DSS-induced inflammation and signs of colitis, suggesting context-dependent effects of fiber. These findings highlight microglial involvement in colitis-associated neuroinflammation and advance our understanding of FFAR2's role in the gut-brain axis. Although not integral, we observed that the role of FFAR2 differs between homeostatic and inflammatory conditions, underscoring the need to consider different inflammatory conditions and disease contexts when investigating the role of FFAR2 and SCFAs in the gut-brain axis.

Dietary Cholesterol Causes Inflammatory Imbalance and Exacerbates Morbidity in Mice Infected with Influenza A Virus.

Influenza is a common cause of pneumonia-induced hospitalization and death, but how host factors function to influence disease susceptibility or severity has not been fully elucidated. Cellular cholesterol levels may affect the pathogenesis of influenza infection, as cholesterol is crucial for viral entry and replication, as well as immune cell proliferation and function. However, there is still conflicting evidence on the extent to which dietary cholesterol influences cholesterol metabolism. In this study, we examined the effects of a high-cholesterol diet in modulating the immune response to influenza A virus (IAV) infection in mice. Mice were fed a standard or a high-cholesterol diet for 5 wk before inoculation with mouse-adapted human IAV (Puerto Rico/8/1934), and tissues were collected at days 0, 4, 8, and 16 postinfection. Cholesterol-fed mice exhibited dyslipidemia characterized by increased levels of total serum cholesterol prior to infection and decreased triglycerides postinfection. Cholesterol-fed mice also displayed increased morbidity compared with control-fed mice, which was neither a result of immunosuppression nor changes in viral load. Instead, transcriptomic analysis of the lungs revealed that dietary cholesterol caused upregulation of genes involved in viral-response pathways and leukocyte trafficking, which coincided with increased numbers of cytokine-producing CD4+ and CD8+ T cells and infiltrating dendritic cells. Morbidity as determined by percent weight loss was highly correlated with numbers of cytokine-producing CD4+ and CD8+ T cells as well as granulocytes. Taken together, dietary cholesterol promoted IAV morbidity via exaggerated cellular immune responses that were independent of viral load.

Astrocytes lure CXCR2-expressing CD4+ T cells to gray matter via TAK1-mediated chemokine production in a mouse model of multiple sclerosis.

Multiple sclerosis (MS) is a chronic neurological disease of the central nervous system driven by peripheral immune cell infiltration and glial activation. The pathological hallmark of MS is demyelination, and mounting evidence suggests neuronal damage in gray matter is a major contributor to disease irreversibility. While T cells are found in both gray and white matter of MS tissue, they are typically confined to the white matter of the most commonly used mouse model of MS, experimental autoimmune encephalomyelitis (EAE). Here, we used a modified EAE mouse model (Type-B EAE) that displays severe neuronal damage to investigate the interplay between peripheral immune cells and glial cells in the event of neuronal damage. We show that CD4+ T cells migrate to the spinal cord gray matter, preferentially to ventral horns. Compared to CD4+ T cells in white matter, gray matter-infiltrated CD4+ T cells were mostly immobilized and interacted with neurons, which are behaviors associated with detrimental effects to normal neuronal function. T cell-specific deletion of CXCR2 significantly decreased CD4+ T cell infiltration into gray matter in Type-B EAE mice. Further, astrocyte-targeted deletion of TAK1 inhibited production of CXCR2 ligands such as CXCL1 in gray matter, successfully prevented T cell migration into spinal cord gray matter, and averted neuronal damage and motor dysfunction in Type-B EAE mice. This study identifies astrocyte chemokine production as a requisite for the invasion of CD4+T cell into the gray matter to induce neuronal damage.

Endometriosis leads to central nervous system-wide glial activation in a mouse model of endometriosis.

BACKGROUND: Chronic pelvic pain (CPP) is a common symptom of endometriosis. Women with endometriosis are also at a high risk of suffering from anxiety, depression, and other psychological disorders. Recent studies indicate that endometriosis can affect the central nervous system (CNS). Changes in the functional activity of neurons, functional magnetic resonance imaging signals, and gene expression have been reported in the brains of rat and mouse models of endometriosis. The majority of the studies thus far have focused on neuronal changes, whereas changes in the glial cells in different brain regions have not been studied. METHODS: Endometriosis was induced in female mice (45-day-old; n = 6-11/timepoint) by syngeneic transfer of donor uterine tissue into the peritoneal cavity of recipient animals. Brains, spines, and endometriotic lesions were collected for analysis at 4, 8, 16, and 32 days post-induction. Sham surgery mice were used as controls (n = 6/timepoint). The pain was assessed using behavioral tests. Using immunohistochemistry for microglia marker ionized calcium-binding adapter molecule-1 (IBA1) and machine learning "Weka trainable segmentation" plugin in Fiji, we evaluated the morphological changes in microglia in different brain regions. Changes in glial fibrillary acidic protein (GFAP) for astrocytes, tumor necrosis factor (TNF), and interleukin-6 (IL6) were also evaluated. RESULTS: We observed an increase in microglial soma size in the cortex, hippocampus, thalamus, and hypothalamus of mice with endometriosis compared to sham controls on days 8, 16, and 32. The percentage of IBA1 and GFAP-positive area was increased in the cortex, hippocampus, thalamus, and hypothalamus in mice with endometriosis compared to sham controls on day 16. The number of microglia and astrocytes did not differ between endometriosis and sham control groups. We observed increased TNF and IL6 expression when expression levels from all brain regions were combined. Mice with endometriosis displayed reduced burrowing behavior and hyperalgesia in the abdomen and hind-paw. CONCLUSION: We believe this is the first report of central nervous system-wide glial activation in a mouse model of endometriosis. These results have significant implications for understanding chronic pain associated with endometriosis and other issues such as anxiety and depression in women with endometriosis.

Influenza A virus infection disrupts oligodendrocyte homeostasis and alters the myelin lipidome in the adult mouse.

BACKGROUND: Recent data suggest that myelin may be altered by physiological events occurring outside of the central nervous system, which may cause changes to cognition and behavior. Similarly, peripheral infection by non-neurotropic viruses is also known to evoke changes to cognition and behavior. METHODS: Mice were inoculated with saline or influenza A virus. Bulk RNA-seq, lipidomics, RT-qPCR, flow cytometry, immunostaining, and western blots were used to determine the effect of infection on OL viability, protein expression and changes to the lipidome. To determine if microglia mediated infection-induced changes to OL homeostasis, mice were treated with GW2580, an inhibitor of microglia activation. Additionally, conditioned medium experiments using primary glial cell cultures were also used to test whether secreted factors from microglia could suppress OL gene expression. RESULTS: Transcriptomic and RT-qPCR analyses revealed temporal downregulation of OL-specific transcripts with concurrent upregulation of markers characteristic of cellular stress. OLs isolated from infected mice had reduced cellular expression of myelin proteins compared with those from saline-inoculated controls. In contrast, the expression of these proteins within myelin was not different between groups. Similarly, histological and immunoblotting analysis performed on various brain regions indicated that infection did not alter OL viability, but increased expression of a cellular stress marker. Shot-gun lipidomic analysis revealed that infection altered the lipid profile within the prefrontal cortex as well as in purified brain myelin and that these changes persisted after recovery from infection. Treatment with GW2580 during infection suppressed the expression of genes associated with glial activation and partially restored OL-specific transcripts to baseline levels. Finally, conditioned medium from activated microglia reduced OL-gene expression in primary OLs without altering their viability. CONCLUSIONS: These findings show that peripheral respiratory viral infection with IAV is capable of altering OL homeostasis and indicate that microglia activation is likely involved in the process.

A hydrolyzed lipid blend diet promotes myelination in neonatal piglets in a region and concentration-dependent manner.

The impact of early life nutrition on myelin development is of interest given that cognitive and behavioral function depends on proper myelination. Evidence shows that myelination can be altered by dietary lipid, but most of these studies have been performed in the context of disease or impairment. Here, we assessed the effects of lipid blends containing various levels of a hydrolyzed fat (HF) system on myelination in healthy piglets. Piglets were sow-reared, fed a control diet, or a diet containing 12%, 25%, or 53% HF consisting of cholesterol, fatty acids, monoglycerides, and phospholipid from lecithin. At postnatal day 28/29, magnetic resonance imaging (MRI) was performed to assess changes to brain development, followed by brain collection for microscopic analyses of myelin in targeted regions using CLARITY tissue clearing, immunohistochemistry, and electron microscopy techniques. Sow-reared piglets exhibited the highest overall brain white matter volume by MRI. However, a 25% HF diet resulted in the greatest total myelin density in the prefrontal cortex based on 3D modeling analysis of myelinated filaments. Nodal gap length and g-ratio were inversely correlated with percentage of HF in the corpus callosum, as well as in the PFC and internal capsule for g-ratio, indicating that a 53% HF diet resulted in the thickest myelin per axon and a 0% HF control diet the thinnest in specific brain regions. These findings indicate that HF promoted myelination in the neonatal piglet in a region- and concentration-dependent manner.

STAT3 signaling in myeloid cells promotes pathogenic myelin-specific T cell differentiation and autoimmune demyelination.

Multiple sclerosis (MS) is an autoimmune inflammatory demyelinating disease of the central nervous system. Dysregulation of STAT3, a transcription factor pivotal to various cellular processes including Th17 cell differentiation, has been implicated in MS. Here, we report that STAT3 is activated in infiltrating monocytic cells near active MS lesions and that activation of STAT3 in myeloid cells is essential for leukocyte infiltration, neuroinflammation, and demyelination in experimental autoimmune encephalomyelitis (EAE). Genetic disruption of Stat3 in peripheral myeloid lineage cells abrogated EAE, which was associated with decreased antigen-specific T helper cell responses. Myeloid cells from immunized Stat3 mutant mice exhibited impaired antigen-presenting functions and were ineffective in driving encephalitogenic T cell differentiation. Single-cell transcriptome analyses of myeloid lineage cells from preclinical wild-type and mutant mice revealed that loss of myeloid STAT3 signaling disrupted antigen-dependent cross-activation of myeloid cells and T helper cells. This study identifies a previously unrecognized requisite for myeloid cell STAT3 in the activation of myelin-reactive T cells and suggests myeloid STAT3 as a potential therapeutic target for autoimmune demyelinating disease.

Transient Receptor Potential Vanilloid 4-Dependent Microglial Function in Myelin Injury and Repair.

Microglia are found pathologically at all stages of multiple sclerosis (MS) lesion development and are hypothesized to contribute to both inflammatory injury and neuroprotection in the MS brain. Transient receptor potential vanilloid 4 (TRPV4) channels are widely expressed, play an important role as environmental sensors, and are involved in calcium homeostasis for a variety of cells. TRPV4 modulates myeloid cell phagocytosis in the periphery and microglial motility in the central nervous system. We hypothesized that TRPV4 deletion would alter microglia phagocytosis in vitro and lessen disease activity and demyelination in experimental autoimmune encephalitis (EAE) and cuprizone-induced demyelination. We found that genetic deletion of TRPV4 led to increased microglial phagocytosis in vitro but did not alter the degree of demyelination or remyelination in the cuprizone mouse model of MS. We also found no difference in disease in EAE following global or microglia-specific deletion of Trpv4. Additionally, lesioned and normal appearing white matter from MS brains exhibited similar TRPV4 expression compared to healthy brain tissue. Taken together, these findings indicate that TRPV4 modulates microglial activity but does not impact disease activity in mouse models of MS, suggesting a muted and/or redundant role in MS pathogenesis.

TAK1 inhibition in mouse astrocyte cultures ameliorates cytokine-induced chemokine production and neutrophil migration.

Systemic inflammation can exacerbate symptoms of many neurological diseases. This effect may be facilitated by glial cells of the central nervous system (CNS) that alter their transcriptional responses and up-regulate cytokine and chemokine expression which can, in turn trigger immune surveillance. In this study, we sought to determine the effects of pro-inflammatory cytokine stimulation (TNF, IL-1α, IL-1ß) on astrocyte and microglia chemokine secretion. Primary cultures of astrocytes or microglia were stimulated with the recombinant cytokines and the levels of secreted chemokines were semi-quantitatively determined using a chemokine-specific proteome profiler array and densitometry. Pharmacological inhibitors were used to determine the effects of p38 MAPK, JNK, ERK1/2, NFkB, and transforming growth factor beta-associated kinase 1 (TAK1) in controlling chemokine production. Finally, neutrophil migration assays were performed to demonstrate functionality. Our data show that stimulated astrocytes secrete at least eight chemokines as a response to cytokine stimulation. These include those involved in neutrophil chemo-attraction and proved capable of promoting neutrophil migration in vitro. In contrast, microglia up-regulated few chemokines in response to cytokine stimulation and did not promote neutrophil migration. However, microglia readily secreted chemokines following stimulation with the toll-like receptor agonists. Finally, we show that both the production of chemokines and neutrophil migration resulting from cytokine stimulation of astrocytes was dependent on TAK1 signaling. Collectively, this study adds to the understanding of how astrocytes and microglia respond to stimuli and their role in promoting neutrophil migration to the CNS during inflammatory conditions.

Crosstalk between microglia and patient-derived glioblastoma cells inhibit invasion in a three-dimensional gelatin hydrogel model.

BACKGROUND: Glioblastoma is the most common and deadly form of primary brain cancer, accounting for more than 13,000 new diagnoses annually in the USA alone. Microglia are the innate immune cells within the central nervous system, acting as a front-line defense against injuries and inflammation via a process that involves transformation from a quiescent to an activated phenotype. Crosstalk between GBM cells and microglia represents an important axis to consider in the development of tissue engineering platforms to examine pathophysiological processes underlying GBM progression and therapy. METHODS: This work used a brain-mimetic hydrogel system to study patient-derived glioblastoma specimens and their interactions with microglia. Here, glioblastoma cells were either cultured alone in 3D hydrogels or in co-culture with microglia in a manner that allowed secretome-based signaling but prevented direct GBM-microglia contact. Patterns of GBM cell invasion were quantified using a three-dimensional spheroid assay. Secretome and transcriptome (via RNAseq) were used to profile the consequences of GBM-microglia interactions. RESULTS: Microglia displayed an activated phenotype as a result of GBM crosstalk. Three-dimensional migration patterns of patient-derived glioblastoma cells showed invasion was significantly decreased in response to microglia paracrine signaling. Potential molecular mechanisms underlying with this phenotype were identified from bioinformatic analysis of secretome and RNAseq data. CONCLUSION: The data demonstrate a tissue engineered hydrogel platform can be used to investigate crosstalk between immune cells of the tumor microenvironment related to GBM progression. Such multi-dimensional models may provide valuable insight to inform therapeutic innovations to improve GBM treatment.

Influenza infection triggers disease in a genetic model of experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system. Most MS patients experience periods of symptom exacerbation (relapses) followed by periods of partial recovery (remission). Interestingly, upper-respiratory viral infections increase the risk for relapse. Here, we used an autoimmune-prone T-cell receptor transgenic mouse (2D2) and a mouse-adapted human influenza virus to test the hypothesis that upper-respiratory viral infection can cause glial activation, promote immune cell trafficking to the CNS, and trigger disease. Specifically, we inoculated 2D2 mice with influenza A virus (Puerto Rico/8/34; PR8) and then monitored them for symptoms of inflammatory demyelination. Clinical and histological experimental autoimmune encephalomyelitis was observed in ∼29% of infected 2D2 mice. To further understand how peripheral infection could contribute to disease onset, we inoculated wild-type C57BL/6 mice and measured transcriptomic alterations occurring in the cerebellum and spinal cord and monitored immune cell surveillance of the CNS by flow cytometry. Infection caused temporal alterations in the transcriptome of both the cerebellum and spinal cord that was consistent with glial activation and increased T-cell, monocyte, and neutrophil trafficking to the brain at day 8 post infection. Finally, Cxcl5 expression was up-regulated in the brains of influenza-infected mice and was elevated in cerebrospinal fluid of MS patients during relapse compared with specimens acquired during remission. Collectively, these data identify a mechanism by which peripheral infection may exacerbate MS as well as other neurological diseases.

Indoleamine 2,3-dioxygenase 1 deletion promotes Theiler's virus-induced seizures in C57BL/6J mice.

OBJECTIVE: Viral encephalitis increases the risk for developing seizures and epilepsy. Indoleamine 2,3-dioxygenase 1 (Ido1) is induced by inflammatory cytokines and functions to metabolize tryptophan to kynurenine. Kynurenine can be further metabolized to produce kynurenic acid and the N-methyl-d-aspartate receptor agonist quinolinic acid (QuinA). In the present study, we sought to determine the role of Ido1 in promoting seizures in an animal model of viral encephalitis. METHODS: C57BL/6J and Ido1 knockout mice (Ido1-KO) were infected with Theiler's murine encephalomyelitis virus (TMEV). Quantitative real-time polymerase chain reaction was used to evaluate hippocampal expression of proinflammatory cytokines, Ido1, and viral RNA. Body weights and seizure scores were recorded daily. Elevated zero maze was used to assess differences in behavior, and hippocampal pathology was determined by immunohistochemistry. RESULTS: Infected C57BL/6J mice up-regulated proinflammatory cytokines, Ido1, and genes encoding the enzymatic cascade responsible for QuinA production in the kynurenine pathway prior to the onset of seizures. Seizure incidence was elevated in Ido1-KO compared to C57BL/6J mice. Infection increased locomotor activity in Ido1-KO compared to C57BL/6J mice. Furthermore, the occurrence of seizures was associated with hyperexcitability. Neither expression of proinflammatory cytokines nor viral RNA was altered as a result of genotype. Immunohistochemical analysis revealed increased hippocampal pathology in Ido1-KO mice. SIGNIFICANCE: Our findings suggest that Ido1 deletion promotes seizures and neuropathogenesis during acute TMEV encephalitis.

Activation of oligodendroglial Stat3 is required for efficient remyelination.

Multiple sclerosis is the most prevalent demyelinating disease of the central nervous system (CNS) and is histologically characterized by perivascular demyelination as well as neurodegeneration. While the degree of axonal damage is correlated with clinical disability, it is believed that remyelination can protect axons from degeneration and slow disease progression. Therefore, understanding the intricacies associated with myelination and remyelination may lead to therapeutics that can enhance the remyelination process and slow axon degeneration and loss of function. Ciliary neurotrophic factor (CNTF) family cytokines such as leukemia inhibitory factor (LIF) and interleukin 11 (IL-11) are known to promote oligodendrocyte maturation and remyelination in experimental models of demyelination. Because CNTF family member binding to the gp130 receptor results in activation of the JAK2/Stat3 pathway we investigated the necessity of oligodendroglial Stat3 in transducing the signal required for myelination and remyelination. We found that Stat3 activation in the CNS coincides with myelination during development. Stimulation of oligodendrocyte precursor cells (OPCs) with CNTF or LIF promoted OPC survival and final differentiation, which was completely abolished by pharmacologic blockade of Stat3 activation with JAK2 inhibitor. Similarly, genetic ablation of Stat3 in oligodendrocyte lineage cells prevented CNTF-induced OPC differentiation in culture. In vivo, while oligodendroglial Stat3 signaling appears to be dispensable for developmental CNS myelination, it is required for oligodendrocyte regeneration and efficient remyelination after toxin-induced focal demyelination in the adult brain. Our data suggest a critical function for oligodendroglial Stat3 signaling in myelin repair.

Effects of exercise in a relapsing-remitting model of experimental autoimmune encephalomyelitis.

Previous research has examined the effects of exercise in experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis. However, all previous studies have utilized a chronic model of EAE, with exercise delivered prior to or immediately after induction of EAE. To our knowledge, no study has examined the effects of exercise delivered during a remission period after initial disease onset in a relapsing-remitting model of EAE (RR-EAE). The current study examines the effects of both voluntary wheel running and forced treadmill exercise on clinical disability and hippocampal brain-derived neurotrophic factor (BDNF) in SJL mice with RR-EAE. The results demonstrate no significant effects of exercise delivered during remission after initial disease onset on clinical disability scores or levels of hippocampal BDNF in mice with RR-EAE. Furthermore, our results demonstrate no significant increase in citrate synthase activity in the gastrocnemius and soleus muscles of mice in the running wheel or treadmill conditions compared with the sedentary condition. These results suggest that the exercise stimuli might have been insufficient to elicit differences in clinical disability or hippocampal BDNF among treatment conditions. © 2016 Wiley Periodicals, Inc.

Postnatal Iron Deficiency Alters Brain Development in Piglets.

BACKGROUND: Cognitive deficits associated with postnatal iron deficiency (ID) suggest abnormal brain development, but little is known about animals with gyrencephalic brains. OBJECTIVE: The objective was to assess the impact of ID on brain development in piglets. METHODS: Male and female Yorkshire piglets were reared from postnatal day (PD) 2 until PD 29 or 30 by using milk replacer adequate [control (CON)] or deficient (100 compared with 10 mg/kg) in iron and subjected to MRI to assess brain macrostructure, microstructure, and metabolites in the dorsal hippocampi and intervening space. After MRI, brains were collected for histology. Hematocrit, hemoglobin, and liver iron were measured to determine iron status. RESULTS: Hematocrit and hemoglobin in ID piglets were less than CON after PD 14 (P < 0.001), and at the study end liver iron in ID piglets was less than CON (P < 0.001). Brain region volumes were not affected by ID, but changes in brain composition were evident. ID piglets had less white matter in 78,305 voxels, with large clusters in the hippocampus and cortex. ID piglets had less gray matter in 13,625 voxels primarily in cortical areas and more gray matter in 28,017 voxels, most notably in olfactory bulbs and hippocampus. The major effect of ID on white matter was supported by lower fractional anisotropy values in the corpus callosum (0.300 compared with 0.284, P = 0.006) and in whole brain white matter (0.313 compared with 0.307, P = 0.002) in ID piglets. In coronal brain sections, corpus callosum width was less (P = 0.043) in ID piglets. Inositol was lower (P = 0.01) and phosphocholine was higher (P = 0.03) in hippocampus of ID piglets. CONCLUSIONS: Postnatal ID in piglets affects brain development, especially white matter. If the effects of ID persist, it might explain the lasting detrimental effects on cognition.

Galectin-9 protein is up-regulated in astrocytes by tumor necrosis factor and promotes encephalitogenic T-cell apoptosis.

Demyelination and axonal damage in multiple sclerosis (MS) are thought to be a consequence of inflammatory processes that are perpetuated by activated glia and infiltrating leukocytes. Galectin-9 is a ß-galactoside binding lectin capable of modulating immune responses and appears to be up-regulated in MS. However, its role in the pathogenesis of MS has yet to be determined. Here, we report that proinflammatory cytokines induce galectin-9 (Gal-9) expression in primary astrocytes and the mechanism by which TNF up-regulates Gal-9. Astrocytes did not express Gal-9 under basal conditions nor did IL-6, IL-10, or IL-13 trigger Gal-9 expression. In contrast, IL-1ß, IFN-γ, and particularly TNF up-regulated Gal-9 in astrocytes. TNF-induced Gal-9 expression was dependent on TNF receptor 1 (TNFR1) as TNF failed to induce Gal-9 in TNFR1(-/-) astrocytes. Blockade of the JNK MAP kinase pathway with the JNK inhibitor SP600125 abrogated TNF-induced Gal-9, whereas p38 and MEK inhibitors had minimal effects. Furthermore, specific knockdown of c-Jun via siRNA in astrocytes before TNF treatment greatly suppressed Gal-9 transcription, suggesting that TNF induces astroglial Gal-9 through the TNF/TNFR1/JNK/cJun signaling pathway. Finally, utilizing astrocytes from Lgals9 mutant (Gal-9(-/-)) mice as well as a myelin basic protein-specific Tim-3(+) encephalitogenic T-cell clone (LCN-8), we found that conditioned medium from TNF-stimulated Gal-9(+/+) but not Gal-9(-/-) astrocytes increased the percentage of apoptotic encephalitogenic T-cells. Together, our results suggest that Gal-9 is induced in astrocytes by TNF via the JNK/c-Jun pathway and that astrocyte-derived Gal-9 may function as an immunoregulatory protein in response to ongoing neuroinflammation.