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1.
J Neuroinflammation ; 21(1): 113, 2024 Apr 29.
Article in English | MEDLINE | ID: mdl-38685031

ABSTRACT

Obesity increases the morbidity and mortality of traumatic brain injury (TBI). Detailed analyses of transcriptomic changes in the brain and adipose tissue were performed to elucidate the interactive effects between high-fat diet-induced obesity (DIO) and TBI. Adult male mice were fed a high-fat diet (HFD) for 12 weeks prior to experimental TBI and continuing after injury. High-throughput transcriptomic analysis using Nanostring panels of the total visceral adipose tissue (VAT) and cellular components in the brain, followed by unsupervised clustering, principal component analysis, and IPA pathway analysis were used to determine shifts in gene expression patterns and molecular pathway activity. Cellular populations in the cortex and hippocampus, as well as in VAT, during the chronic phase after combined TBI-HFD showed amplification of central and peripheral microglia/macrophage responses, including superadditive changes in selected gene expression signatures and pathways. Furthermore, combined TBI and HFD caused additive dysfunction in Y-Maze, Novel Object Recognition (NOR), and Morris water maze (MWM) cognitive function tests. These novel data suggest that HFD-induced obesity and TBI can independently prime and support the development of altered states in brain microglia and VAT, including the disease-associated microglia/macrophage (DAM) phenotype observed in neurodegenerative disorders. The interaction between HFD and TBI promotes a shift toward chronic reactive microglia/macrophage transcriptomic signatures and associated pro-inflammatory disease-altered states that may, in part, underlie the exacerbation of cognitive deficits. Thus, targeting of HFD-induced reactive cellular phenotypes, including in peripheral adipose tissue immune cell populations, may serve to reduce microglial maladaptive states after TBI, attenuating post-traumatic neurodegeneration and neurological dysfunction.


Subject(s)
Brain Injuries, Traumatic , Brain , Cognitive Dysfunction , Diet, High-Fat , Macrophages , Mice, Inbred C57BL , Microglia , Animals , Diet, High-Fat/adverse effects , Microglia/metabolism , Microglia/pathology , Male , Mice , Cognitive Dysfunction/etiology , Cognitive Dysfunction/pathology , Cognitive Dysfunction/metabolism , Macrophages/metabolism , Macrophages/pathology , Brain Injuries, Traumatic/pathology , Brain Injuries, Traumatic/metabolism , Brain/pathology , Brain/metabolism , Adipose Tissue/metabolism , Adipose Tissue/pathology , Recognition, Psychology/physiology , Obesity/pathology , Obesity/complications , Maze Learning/physiology
2.
bioRxiv ; 2023 Jul 29.
Article in English | MEDLINE | ID: mdl-37546932

ABSTRACT

Obesity increases the morbidity and mortality of traumatic brain injury (TBI). We performed a detailed analysis of transcriptomic changes in the brain and adipose tissue to examine the interactive effects between high-fat diet-induced obesity (DIO) and TBI in relation to central and peripheral inflammatory pathways, as well as neurological function. Adult male mice were fed a high-fat diet (HFD) for 12 weeks prior to experimental TBI and continuing after injury. Combined TBI and HFD resulted in additive dysfunction in the Y-Maze, novel object recognition (NOR), and Morris water maze (MWM) cognitive function tests. We also performed high-throughput transcriptomic analysis using Nanostring panels of cellular compartments in the brain and total visceral adipose tissue (VAT), followed by unsupervised clustering, principal component analysis, and IPA pathway analysis to determine shifts in gene expression programs and molecular pathway activity. Analysis of cellular populations in the cortex and hippocampus as well as in visceral adipose tissue during the chronic phase after combined TBI-HFD showed amplification of central and peripheral microglia/macrophage responses, including superadditive changes in select gene expression signatures and pathways. These data suggest that HFD-induced obesity and TBI can independently prime and support the development of altered states in brain microglia and visceral adipose tissue macrophages, including the disease-associated microglia/macrophage (DAM) phenotype observed in neurodegenerative disorders. The interaction between HFD and TBI promotes a shift toward chronic reactive microglia/macrophage transcriptomic signatures and associated pro-inflammatory disease-altered states that may, in part, underlie the exacerbation of cognitive deficits. Targeting of HFD-induced reactive cellular phenotypes, including in peripheral adipose tissue macrophages, may serve to reduce microglial maladaptive states after TBI, attenuating post-traumatic neurodegeneration and neurological dysfunction.

3.
Front Immunol ; 12: 710608, 2021.
Article in English | MEDLINE | ID: mdl-34504493

ABSTRACT

Aging adversely affects inflammatory processes in the brain, which has important implications in the progression of neurodegenerative disease. Following traumatic brain injury (TBI), aged animals exhibit worsened neurological function and exacerbated microglial-associated neuroinflammation. Type I Interferons (IFN-I) contribute to the development of TBI neuropathology. Further, the Cyclic GMP-AMP Synthase (cGAS) and Stimulator of Interferon Genes (STING) pathway, a key inducer of IFN-I responses, has been implicated in neuroinflammatory activity in several age-related neurodegenerative diseases. Here, we set out to investigate the effects of TBI on cGAS/STING activation, IFN-I signaling and neuroinflammation in young and aged C57Bl/6 male mice. Using a controlled cortical impact model, we evaluated transcriptomic changes in the injured cortex at 24 hours post-injury, and confirmed activation of key neuroinflammatory pathways in biochemical studies. TBI induced changes were highly enriched for transcripts that were involved in inflammatory responses to stress and host defense. Deeper analysis revealed that TBI increased expression of IFN-I related genes (e.g. Ifnb1, Irf7, Ifi204, Isg15) and IFN-I signaling in the injured cortex of aged compared to young mice. There was also a significant age-related increase in the activation of the DNA-recognition pathway, cGAS, which is a key mechanism to propagate IFN-I responses. Finally, enhanced IFN-I signaling in the aged TBI brain was confirmed by increased phosphorylation of STAT1, an important IFN-I effector molecule. This age-related activation of cGAS and IFN-I signaling may prove to be a mechanistic link between microglial-associated neuroinflammation and neurodegeneration in the aged TBI brain.


Subject(s)
Aging/immunology , Brain Injuries, Traumatic/immunology , Interferon Type I/physiology , Nucleotidyltransferases/metabolism , Aging/metabolism , Animals , Enzyme Activation , Interferon Type I/genetics , Male , Membrane Proteins/physiology , Mice , Mice, Inbred C57BL , Microglia/physiology , Neurodegenerative Diseases/etiology , Neuroinflammatory Diseases/etiology , Signal Transduction/physiology
4.
Trends Neurosci ; 44(5): 406-418, 2021 05.
Article in English | MEDLINE | ID: mdl-33495023

ABSTRACT

Traumatic brain injury (TBI) is a debilitating disorder associated with chronic progressive neurodegeneration and long-term neurological decline. Importantly, there is now substantial and increasing evidence that TBI can negatively impact systemic organs, including the pulmonary, gastrointestinal (GI), cardiovascular, renal, and immune system. Less well appreciated, until recently, is that such functional changes can affect both the response to subsequent insults or diseases, as well as contribute to chronic neurodegenerative processes and long-term neurological outcomes. In this review, we summarize evidence showing bidirectional interactions between the brain and systemic organs following TBI and critically assess potential underlying mechanisms.


Subject(s)
Brain Injuries, Traumatic , Cognitive Dysfunction , Animals , Brain , Humans , Mice , Mice, Inbred C57BL
5.
Int J Mol Sci ; 21(15)2020 Jul 23.
Article in English | MEDLINE | ID: mdl-32718090

ABSTRACT

Radiotherapy for brain tumors induces neuronal DNA damage and may lead to neurodegeneration and cognitive deficits. We investigated the mechanisms of radiation-induced neuronal cell death and the role of miR-711 in the regulation of these pathways. We used in vitro and in vivo models of radiation-induced neuronal cell death. We showed that X-ray exposure in primary cortical neurons induced activation of p53-mediated mechanisms including intrinsic apoptotic pathways with sequential upregulation of BH3-only molecules, mitochondrial release of cytochrome c and AIF-1, as well as senescence pathways including upregulation of p21WAF1/Cip1. These pathways of irradiation-induced neuronal apoptosis may involve miR-711-dependent downregulation of pro-survival genes Akt and Ang-1. Accordingly, we demonstrated that inhibition of miR-711 attenuated degradation of Akt and Ang-1 mRNAs and reduced intrinsic apoptosis after neuronal irradiation; likewise, administration of Ang-1 was neuroprotective. Importantly, irradiation also downregulated two novel miR-711 targets, DNA-repair genes Rad50 and Rad54l2, which may impair DNA damage responses, amplifying the stimulation of apoptotic and senescence pathways and contributing to neurodegeneration. Inhibition of miR-711 rescued Rad50 and Rad54l2 expression after neuronal irradiation, enhancing DNA repair and reducing p53-dependent apoptotic and senescence pathways. Significantly, we showed that brain irradiation in vivo persistently elevated miR-711, downregulated its targets, including pro-survival and DNA-repair molecules, and is associated with markers of neurodegeneration, not only across the cortex and hippocampus but also specifically in neurons isolated from the irradiated brain. Our data suggest that irradiation-induced miR-711 negatively modulates multiple pro-survival and DNA-repair mechanisms that converge to activate neuronal intrinsic apoptosis and senescence. Using miR-711 inhibitors to block the development of these regulated neurodegenerative pathways, thus increasing neuronal survival, may be an effective neuroprotective strategy.


Subject(s)
DNA Repair/radiation effects , MicroRNAs/biosynthesis , Neurodegenerative Diseases/metabolism , Neurons/metabolism , Radiation Injuries, Experimental/metabolism , Up-Regulation/radiation effects , X-Rays/adverse effects , Animals , Cell Death/radiation effects , DNA Damage , Male , Mice , Neurodegenerative Diseases/etiology , Neurodegenerative Diseases/pathology , Neurons/pathology , Radiation Injuries, Experimental/pathology
6.
Crit Care Med ; 48(5): e418-e428, 2020 05.
Article in English | MEDLINE | ID: mdl-32149839

ABSTRACT

OBJECTIVES: Respiratory infections in the postacute phase of traumatic brain injury impede optimal recovery and contribute substantially to overall morbidity and mortality. This study investigated bidirectional innate immune responses between the injured brain and lung, using a controlled cortical impact model followed by secondary Streptococcus pneumoniae infection in mice. DESIGN: Experimental study. SETTING: Research laboratory. SUBJECTS: Adult male C57BL/6J mice. INTERVENTIONS: C57BL/6J mice were subjected to sham surgery or moderate-level controlled cortical impact and infected intranasally with S. pneumoniae (1,500 colony-forming units) or vehicle (phosphate-buffered saline) at 3 or 60 days post-injury. MAIN RESULTS: At 3 days post-injury, S. pneumoniae-infected traumatic brain injury mice (TBI + Sp) had a 25% mortality rate, in contrast to no mortality in S. pneumoniae-infected sham (Sham + Sp) animals. TBI + Sp mice infected 60 days post-injury had a 60% mortality compared with 5% mortality in Sham + Sp mice. In both studies, TBI + Sp mice had poorer motor function recovery compared with TBI + PBS mice. There was increased expression of pro-inflammatory markers in cortex of TBI + Sp compared with TBI + PBS mice after both early and late infection, indicating enhanced post-traumatic neuroinflammation. In addition, monocytes from lungs of TBI + Sp mice were immunosuppressed acutely after traumatic brain injury and could not produce interleukin-1ß, tumor necrosis factor-α, or reactive oxygen species. In contrast, after delayed infection monocytes from TBI + Sp mice had higher levels of interleukin-1ß, tumor necrosis factor-α, and reactive oxygen species when compared with Sham + Sp mice. Increased bacterial burden and pathology was also found in lungs of TBI + Sp mice. CONCLUSIONS: Traumatic brain injury causes monocyte functional impairments that may affect the host's susceptibility to respiratory infections. Chronically injured mice had greater mortality following S. pneumoniae infection, which suggests that respiratory infections even late after traumatic brain injury may pose a more serious threat than is currently appreciated.


Subject(s)
Brain Injuries, Traumatic/epidemiology , Monocytes/metabolism , Respiratory Tract Infections/epidemiology , Staphylococcal Infections/epidemiology , Animals , Brain Injuries, Traumatic/physiopathology , Disease Models, Animal , Inflammation Mediators/metabolism , Male , Mice , Mice, Inbred C57BL , Pneumonia, Staphylococcal , Respiratory Tract Infections/mortality , Staphylococcal Infections/mortality
7.
J Neurosci ; 40(14): 2960-2974, 2020 04 01.
Article in English | MEDLINE | ID: mdl-32094203

ABSTRACT

Chronic neuroinflammation with sustained microglial activation occurs following severe traumatic brain injury (TBI) and is believed to contribute to subsequent neurodegeneration and neurological deficits. Microglia, the primary innate immune cells in brain, are dependent on colony stimulating factor 1 receptor (CSF1R) signaling for their survival. In this preclinical study, we examined the effects of delayed depletion of chronically activated microglia on functional recovery and neurodegeneration up to 3 months postinjury. A CSF1R inhibitor, Plexxikon (PLX) 5622, was administered to adult male C57BL/6J mice at 1 month after controlled cortical impact to remove chronically activated microglia, and the inhibitor was withdrawn 1-week later to allow for microglial repopulation. Following TBI, the repopulated microglia displayed a ramified morphology similar to that of Sham uninjured mice, whereas microglia in vehicle-treated TBI mice showed the typical chronic posttraumatic hypertrophic morphology. PLX5622 treatment limited TBI-associated neuropathological changes at 3 months postinjury; these included a smaller cortical lesion, reduced hippocampal neuron cell death, and decreased NOX2- and NLRP3 inflammasome-associated neuroinflammation. Furthermore, delayed depletion of chronically activated microglia after TBI led to widespread changes in the cortical transcriptome and altered gene pathways involved in neuroinflammation, oxidative stress, and neuroplasticity. Using a variety of complementary neurobehavioral tests, PLX5622-treated TBI mice also had improved long-term motor and cognitive function recovery through 3 months postinjury. Together, these studies demonstrate that chronic phase removal of neurotoxic microglia after TBI using CSF1R inhibitors markedly reduce chronic neuroinflammation and associated neurodegeneration, as well as related motor and cognitive deficits.SIGNIFICANCE STATEMENT Traumatic brain injury (TBI) is a debilitating neurological disorder that can seriously impact the patient's quality of life. Microglial-mediated neuroinflammation is induced after severe TBI and contributes to neurological deficits and on-going neurodegenerative processes. Here, we investigated the effect of breaking the neurotoxic neuroinflammatory loop at 1-month after controlled cortical impact in mice by pharmacological removal of chronically activated microglia using a colony stimulating factor 1 receptor (CSF1R) inhibitor, Plexxikon 5622. Overall, we show that short-term elimination of microglia during the chronic phase of TBI followed by repopulation results in long-term improvements in neurological function, suppression of neuroinflammatory and oxidative stress pathways, and a reduction in persistent neurodegenerative processes. These studies are clinically relevant and support new concepts that the therapeutic window for TBI may be far longer than traditionally believed if chronic and evolving microglial-mediated neuroinflammation can be inhibited or regulated in a precise manner.


Subject(s)
Brain Injuries, Traumatic/pathology , Microglia/drug effects , Nerve Degeneration/pathology , Neuroprotective Agents/pharmacology , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/antagonists & inhibitors , Animals , Brain Injuries, Traumatic/metabolism , Brain Injuries, Traumatic/physiopathology , Disease Models, Animal , Male , Mice , Mice, Inbred C57BL , Microglia/metabolism , Nerve Degeneration/metabolism , Nerve Degeneration/physiopathology
8.
J Neurosci ; 40(11): 2357-2370, 2020 03 11.
Article in English | MEDLINE | ID: mdl-32029532

ABSTRACT

DNA damage and type I interferons (IFNs) contribute to inflammatory responses after traumatic brain injury (TBI). TBI-induced activation of microglia and peripherally-derived inflammatory macrophages may lead to tissue damage and neurological deficits. Here, we investigated the role of IFN-ß in secondary injury after TBI using a controlled cortical impact model in adult male IFN-ß-deficient (IFN-ß-/-) mice and assessed post-traumatic neuroinflammatory responses, neuropathology, and long-term functional recovery. TBI increased expression of DNA sensors cyclic GMP-AMP synthase and stimulator of interferon genes in wild-type (WT) mice. IFN-ß and other IFN-related and neuroinflammatory genes were also upregulated early and persistently after TBI. TBI increased expression of proinflammatory mediators in the cortex and hippocampus of WT mice, whereas levels were mitigated in IFN-ß-/- mice. Moreover, long-term microglia activation, motor, and cognitive function impairments were decreased in IFN-ß-/- TBI mice compared with their injured WT counterparts; improved neurological recovery was associated with reduced lesion volume and hippocampal neurodegeneration in IFN-ß-/- mice. Continuous central administration of a neutralizing antibody to the IFN-α/ß receptor (IFNAR) for 3 d, beginning 30 min post-injury, reversed early cognitive impairments in TBI mice and led to transient improvements in motor function. However, anti-IFNAR treatment did not improve long-term functional recovery or decrease TBI neuropathology at 28 d post-injury. In summary, TBI induces a robust neuroinflammatory response that is associated with increased expression of IFN-ß and other IFN-related genes. Inhibition of IFN-ß reduces post-traumatic neuroinflammation and neurodegeneration, resulting in improved neurological recovery. Thus, IFN-ß may be a potential therapeutic target for TBI.SIGNIFICANCE STATEMENT TBI frequently causes long-term neurological and psychiatric changes in head injury patients. TBI-induced secondary injury processes including persistent neuroinflammation evolve over time and can contribute to chronic neurological impairments. The present study demonstrates that TBI is followed by robust activation of type I IFN pathways, which have been implicated in microglial-associated neuroinflammation and chronic neurodegeneration. We examined the effects of genetic or pharmacological inhibition of IFN-ß, a key component of type I IFN mechanisms to address its role in TBI pathophysiology. Inhibition of IFN-ß signaling resulted in reduced neuroinflammation, attenuated neurobehavioral deficits, and limited tissue loss long after TBI. These preclinical findings suggest that IFN-ß may be a potential therapeutic target for TBI.


Subject(s)
Brain Damage, Chronic/physiopathology , Brain Injuries, Traumatic/physiopathology , Interferon-beta/physiology , Nerve Degeneration/etiology , Animals , Brain Damage, Chronic/etiology , Brain Injuries, Traumatic/complications , Cerebral Cortex/metabolism , Exploratory Behavior/physiology , Gene Expression Regulation , Hippocampus/metabolism , Inflammation , Interferon-beta/biosynthesis , Interferon-beta/deficiency , Interferon-beta/genetics , Male , Maze Learning/physiology , Memory Disorders/etiology , Memory Disorders/physiopathology , Mice , Mice, Inbred C57BL , Microglia/physiology , Movement Disorders/etiology , Movement Disorders/physiopathology , Random Allocation , Receptor, Interferon alpha-beta/immunology , Signal Transduction , Up-Regulation
9.
Neurotherapeutics ; 16(1): 216-230, 2019 01.
Article in English | MEDLINE | ID: mdl-30225790

ABSTRACT

Micro-RNAs (miRs) are short, noncoding RNAs that negatively regulate gene expression at the post-transcriptional level and have been implicated in the pathophysiology of secondary damage after traumatic brain injury (TBI). Among miRs linked to inflammation, miR-155 has been implicated as a pro-inflammatory factor in a variety of organ systems. We examined the expression profile of miR-155, following experimental TBI (controlled cortical impact) in adult male C57Bl/6 mice, as well as the effects of acute or delayed administration of a miR-155 antagomir on post-traumatic neuroinflammatory responses and neurological recovery. Trauma robustly increased miR-155 expression in the injured cortex over 7 days. Similar TBI-induced miR-155 expression changes were also found in microglia/macrophages isolated from the injured cortex at 7 days post-injury. A miR-155 hairpin inhibitor (antagomir; 0.5 nmol), administered intracerebroventricularly (ICV) immediately after injury, attenuated neuroinflammatory markers at both 1 day and 7 days post-injury and reduced impairments in spatial working memory. Delayed ICV infusion of the miR-155 antagomir (0.5 nmol/day), beginning 24 h post-injury and continuing for 6 days, attenuated neuroinflammatory markers at 7 days post-injury and improved motor, but not cognitive, function through 28 days. The latter treatment limited NADPH oxidase 2 expression changes in microglia/macrophages in the injured cortex and reduced cortical lesion volume. In summary, TBI causes a robust and persistent neuroinflammatory response that is associated with increased miR-155 expression in microglia/macrophages, and miR-155 inhibition reduces post-traumatic neuroinflammatory responses and improves neurological recovery. Thus, miR-155 may be a therapeutic target for TBI-related neuroinflammation.


Subject(s)
Antagomirs/administration & dosage , Brain Injuries, Traumatic , MicroRNAs/antagonists & inhibitors , Neurogenic Inflammation , Animals , Brain Injuries, Traumatic/genetics , Brain Injuries, Traumatic/physiopathology , Male , Mice , Mice, Inbred C57BL , Neurogenic Inflammation/genetics , Recovery of Function/drug effects , Recovery of Function/genetics
10.
Sci Rep ; 8(1): 11326, 2018 07 27.
Article in English | MEDLINE | ID: mdl-30054538

ABSTRACT

Neuroinflammation is recognized as one of the obligatory pathogenic features of neurodegenerative diseases including Alzheimer's, Parkinson's or prion diseases. In prion diseases, space and time correlations between deposition of disease-associated, pathogenic form of the prion protein or PrPSc and microglial-mediated neuroinflammation has been established. Yet, it remains unclear whether activation of microglia is triggered directly by a contact with PrPSc, and what molecular features of PrPSc microglia sense and respond to that drive microglia to inflammatory states. The current study asked the questions whether PrPSc can directly trigger activation of microglia and whether the degree of microglia response depends on the nature of terminal carbohydrate groups on the surface of PrPSc particles. PrPSc was purified from brains of mice infected with mouse-adapted prion strain 22L or neuroblastoma N2a cells stably infected with 22L. BV2 microglial cells or primary microglia were cultured in the presence of purified 22L. We found that exposure of BV2 cells or primary microglia to purified PrPSc triggered proinflammatory responses characterized by an increase in the levels of TNFα, IL6, nitric oxide (NO) and expression of inducible Nitric Oxide Synthase (iNOS). Very similar patterns of inflammatory response were induced by PrPSc purified from mouse brains and neuroblastoma cells arguing that microglia response is independent of the source of PrPSc. To test whether the microglial response is mediated by carbohydrate epitopes on PrPSc surface, the levels of sialylation of PrPSc N-linked glycans was altered by treatment of purified PrPSc with neuraminidase. Partial cleavage of sialic acid residues was found to boost the inflammatory response of microglia to PrPSc. Moreover, transient degradation of Iκßα observed upon treatment with partially desialylated PrPSc suggests that canonical NFκB activation pathway is involved in inflammatory response. The current study is the first to demonstrate that PrPSc can directly trigger inflammatory response in microglia. In addition, this work provides direct evidence that the chemical nature of the carbohydrate groups on PrPSc surface is important for microglial activation.


Subject(s)
Inflammation/immunology , Microglia/immunology , PrPSc Proteins/immunology , Prion Diseases/immunology , Animals , Brain/immunology , Brain/metabolism , Brain/pathology , Carbohydrates/immunology , Epitopes/immunology , Gene Expression Regulation , Humans , Inflammation/genetics , Inflammation/pathology , Interleukin-6/genetics , Mice , Microglia/metabolism , Microglia/pathology , N-Acetylneuraminic Acid/immunology , Nitric Oxide/genetics , Nitric Oxide Synthase Type II/genetics , PrPSc Proteins/genetics , PrPSc Proteins/metabolism , Primary Cell Culture , Prion Diseases/genetics , Prion Diseases/pathology , Tumor Necrosis Factor-alpha/genetics
11.
J Neurotrauma ; 35(13): 1419-1436, 2018 07 01.
Article in English | MEDLINE | ID: mdl-29421977

ABSTRACT

There is a compelling link between severe brain trauma and immunosuppression in patients with traumatic brain injury (TBI). Although acute changes in the systemic immune compartment have been linked to outcome severity, the long-term consequences of TBI on systemic immune function are unknown. Here, adult male C57Bl/6 mice underwent moderate-level controlled cortical impact (CCI) or sham surgery, and systemic immune function was evaluated at 1, 3, 7, 14, and 60 days post-injury. Bone marrow, blood, thymus, and spleen were examined by flow cytometry to assess changes in immune composition, reactive oxygen species (ROS) production, phagocytic activity, and cytokine production. Bone marrow derived macrophages (BMDMs) from sham and 60-day CCI mice were cultured for immune challenge studies using lipopolysaccharide (LPS) and interleukin-4 (IL-4) models. Acutely, TBI caused robust bone marrow activation and neutrophilia. Neutrophils and monocytes exhibited impairments in respiratory burst, cytokine production, and phagocytosis; in contrast, ROS levels and pro-inflammatory cytokine production were chronically elevated at 60 days post-injury. Cultures of BMDMs from chronic CCI mice demonstrated defects in LPS- and IL-4-induced polarization when compared with stimulated BMDMs from sham mice. TBI also caused thymic involution, inverted CD4:CD8 ratios, chronic T lymphopenia, greater memory conversion, increased T cell activation, impaired interferon γ induction, and chronically elevated Th1 cytokine and ROS production. Collectively, our in-depth phenotypic and functional analyses demonstrate that TBI induces widespread suppression of innate and adaptive immune responses after TBI. Moreover, at chronic time points, TBI mice exhibit hallmarks of accelerated immune aging, displaying chronic deficits in systemic immune function.


Subject(s)
Adaptive Immunity/immunology , Brain Injuries, Traumatic/immunology , Immunity, Innate/immunology , Animals , Male , Mice , Mice, Inbred C57BL
12.
J Neuroinflammation ; 14(1): 65, 2017 03 24.
Article in English | MEDLINE | ID: mdl-28340575

ABSTRACT

BACKGROUND: NADPH oxidase (NOX2) is an enzyme system that generates reactive oxygen species (ROS) in microglia and macrophages. Excessive ROS production is linked with neuroinflammation and chronic neurodegeneration following traumatic brain injury (TBI). Redox signaling regulates macrophage/microglial phenotypic responses (pro-inflammatory versus anti-inflammatory), and NOX2 inhibition following moderate-to-severe TBI markedly reduces pro-inflammatory activation of macrophages/microglia resulting in concomitant increases in anti-inflammatory responses. Here, we report the signaling pathways that regulate NOX2-dependent macrophage/microglial phenotype switching in the TBI brain. METHODS: Bone marrow-derived macrophages (BMDMs) prepared from wildtype (C57Bl/6) and NOX2 deficient (NOX2-/-) mice were treated with lipopolysaccharide (LPS; 10 ng/ml), interleukin-4 (IL-4; 10 ng/ml), or combined LPS/IL-4 to investigate signal transduction pathways associated with macrophage activation using western immunoblotting and qPCR analyses. Signaling pathways and activation markers were evaluated in ipsilateral cortical tissue obtained from adult male wildtype and NOX2-/- mice that received moderate-level controlled cortical impact (CCI). A neutralizing anti-IL-10 approach was used to determine the effects of IL-10 on NOX2-dependent transitions from pro- to anti-inflammatory activation states. RESULTS: Using an LPS/IL-4-stimulated BMDM model that mimics the mixed pro- and anti-inflammatory responses observed in the injured cortex, we show that NOX2-/- significantly reduces STAT1 signaling and markers of pro-inflammatory activation. In addition, NOX2-/- BMDMs significantly increase anti-inflammatory marker expression; IL-10-mediated STAT3 signaling, but not STAT6 signaling, appears to be critical in regulating this anti-inflammatory response. Following moderate-level CCI, IL-10 is significantly increased in microglia/macrophages in the injured cortex of NOX2-/- mice. These changes are associated with increased STAT3 activation, but not STAT6 activation, and a robust anti-inflammatory response. Neutralization of IL-10 in NOX2-/- BMDMs or CCI mice blocks STAT3 activation and the anti-inflammatory response, thereby demonstrating a critical role for IL-10 in regulating NOX2-dependent transitions between pro- and anti-inflammatory activation states. CONCLUSIONS: These studies indicate that following TBI NOX2 inhibition promotes a robust anti-inflammatory response in macrophages/microglia that is mediated by the IL-10/STAT3 signaling pathway. Thus, therapeutic interventions that inhibit macrophage/microglial NOX2 activity may improve TBI outcomes by not only limiting pro-inflammatory neurotoxic responses, but also enhancing IL-10-mediated anti-inflammatory responses that are neuroprotective.


Subject(s)
Brain Injuries, Traumatic/immunology , Inflammation/pathology , Macrophage Activation/immunology , Macrophages/immunology , NADPH Oxidase 2/deficiency , Animals , Brain Injuries, Traumatic/metabolism , Brain Injuries, Traumatic/pathology , Inflammation/immunology , Inflammation/metabolism , Interleukin-10/immunology , Interleukin-10/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Phenotype , STAT3 Transcription Factor/immunology , STAT3 Transcription Factor/metabolism
13.
Brain Behav Immun ; 58: 291-309, 2016 Nov.
Article in English | MEDLINE | ID: mdl-27477920

ABSTRACT

Following traumatic brain injury (TBI), activation of microglia and peripherally derived inflammatory macrophages occurs in association with tissue damage. This neuroinflammatory response may have beneficial or detrimental effects on neuronal survival, depending on the functional polarization of these cells along a continuum from M1-like to M2-like activation states. The mechanisms that regulate M1-like and M2-like activation after TBI are not well understood, but appear in part to reflect the redox state of the lesion microenvironment. NADPH oxidase (NOX2) is a critical enzyme system that generates reactive oxygen species in microglia/macrophages. After TBI, NOX2 is strongly up-regulated in M1-like, but not in M2-like polarized cells. Therefore, we hypothesized that NOX2 drives M1-like neuroinflammation and contributes to neurodegeneration and loss of neurological function after TBI. In the present studies we inhibited NOX2 activity using NOX2-knockout mice or the selective peptide inhibitor gp91ds-tat. We show that NOX2 is highly up-regulated in infiltrating macrophages after injury, and that NOX2 deficiency reduces markers of M1-like activation, limits tissue loss and neurodegeneration, and improves motor recovery after moderate-level control cortical injury (CCI). NOX2 deficiency also promotes M2-like activation after CCI, through increased IL-4Rα signaling in infiltrating macrophages, suggesting that NOX2 acts as a critical switch between M1- and M2-like activation states after TBI. Administration of gp91ds-tat to wild-type CCI mice starting at 24h post-injury reduces deficits in cognitive function and increased M2-like activation in the hippocampus. Collectively, our data indicate that increased NOX2 activity after TBI drives M1-like activation that contributes to inflammatory-mediated neurodegeneration, and that inhibiting this pathway provides neuroprotection, in part by altering M1-/M2-like balance towards the M2-like neuroinflammatory response.


Subject(s)
Brain Injuries, Traumatic/metabolism , Brain Injuries, Traumatic/pathology , Encephalitis/metabolism , Macrophages/metabolism , Microglia/metabolism , NADPH Oxidase 2/metabolism , Animals , Cerebral Cortex/metabolism , Cerebral Cortex/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , NADPH Oxidase 2/genetics , Spatial Memory
14.
Exp Cell Res ; 335(2): 258-68, 2015 Jul 15.
Article in English | MEDLINE | ID: mdl-26022664

ABSTRACT

Macrophages can be polarised to adopt the M1 or M2 phenotype and functional outcomes of activation include altered secretion of immune molecules such as insulin-like growth factor (IGF)-1 as well as upregulation of cell surface molecules specifically associated with each state. Interleukin (IL)-4 mediates its effects through two receptors, the type I and II receptors and activation of these receptors results in phosphorylation of signal transducers and activators of transcription (STAT)6. JAK3 is activated as a consequence of ligation of the type I IL-4R, which participates in Akt activation. We set out to investigate the impact of perturbation of IGF-1 tone on IL-4- and interferon (IFN)γ-induced activation, the mechanisms by which this may occur and the contribution of type I IL-4R activation to adoption of the M2 state. The data presented here indicate that IL-4-induced activation of Akt is JAK3-dependent, enhanced by release of IGF-1 and necessary for full adoption of the M2 phenotype, since blocking IGF-1 activity blunts the ability of IL-4 to induce activation of Akt and to upregulate expression of some M2-associated molecules. In addition, differential control of the expression of mannose receptor (MRC1), arginase-1 (Arg-1), chitinase-3 like 3 (Chi3l3) and found in inflammatory zone 1 (FIZZ1) was observed. The IFNγ-induced decrease in IGF-1 was exacerbated by inhibition of phosphatidylinositol-3 (PI3) kinase, indicating that Akt may regulate its own activation via IGF-1. Overall, a deficit in IGF-1/Akt signalling is associated with decreased capacity to induce the M2 state and an increased responsiveness to IFNγ.


Subject(s)
Insulin-Like Growth Factor I/physiology , Macrophages/enzymology , Proto-Oncogene Proteins c-akt/physiology , Animals , Cells, Cultured , Interleukin-4/physiology , Janus Kinase 3/metabolism , Macrophage Activation , Macrophages/immunology , Mice , Signal Transduction , Transcriptome
15.
J Neuroinflammation ; 12: 67, 2015 Apr 09.
Article in English | MEDLINE | ID: mdl-25890218

ABSTRACT

BACKGROUND: Lipopolysaccharide (LPS) and interferon-γ (IFNγ) increase expression of tumour necrosis factor-α (TNFα) that characterizes the M1 activation state of macrophages. Whereas it is accepted that the immune system undergoes changes with age, there is inconsistency in the literature with respect to the impact of age on the response of macrophages to inflammatory stimuli. Here, we investigate the effect of age on the responsiveness of bone marrow-derived macrophages (BMDMs) to LPS and IFNγ. The context for addressing this question is that macrophages, which infiltrate the brain of aged animals, will encounter the neuroinflammatory environment that has been described with age. METHODS: Brain tissue, prepared from young and aged rats, was assessed for expression of inflammatory markers by PCR and for evidence of infiltration of macrophages by flow cytometry. BMDMs were prepared from the long bones of young and aged rats, maintained in culture for 8 days and incubated in the presence or absence of LPS (100 ng/ml) or IFNγ (50 ng/ml). Cells were harvested and assessed for mRNA expression of markers of M1 activation including TNFα and NOS2, or for expression of IFNγR1 and TLR4 by western immunoblotting. To assess whether BMDMs induced glial activation, mixed glial cultures were incubated in the presence of conditioned media obtained from unstimulated BMDMs of young and aged rats and evaluated for expression of inflammatory markers. RESULTS: Markers associated with M1 activation were expressed to a greater extent in BMDMs from aged rats in response to LPS and IFNγ, compared with cells from young rats. The increased responsiveness was associated with increases in IFNγ receptor (IFNγR) and Toll-like receptor 4 (TLR4). The data show that conditioned media from BMDMs of aged rats increased the expression of pro-inflammatory mediators in glial cells. Significantly, there was an age-related increase in macrophage infiltration into the brain, and this was combined with increased expression of IFNγ and the Toll-like receptor 4 agonist, high-mobility group protein B1 (HMGB1). CONCLUSION: Exposure of infiltrating macrophages to the inflammatory microenvironment that develops in the brain with age is likely to contribute to a damaging cascade that negatively impacts neuronal function.


Subject(s)
Aging , Cytokines/metabolism , Gene Expression Regulation/drug effects , Interferon-gamma/pharmacology , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Age Factors , Analysis of Variance , Animals , Animals, Newborn , Cells, Cultured , Cytokines/genetics , Enzyme-Linked Immunosorbent Assay , Male , Nitric Oxide Synthase Type II/metabolism , RNA, Messenger , Rats , Rats, Wistar , Tumor Necrosis Factor-alpha/metabolism
16.
J Alzheimers Dis ; 44(3): 949-62, 2015.
Article in English | MEDLINE | ID: mdl-25374107

ABSTRACT

Macrophages are key cells in tissue defense in the periphery and, under certain circumstances, infiltrate the central nervous system, where they may play a similar role in the brain, perhaps supporting the function of microglia. Macrophages have been shown to adopt different activation states in response to various stimuli. Specifically, when exposed to inflammatory stimuli such as interferon (IFN)γ, the cells adopt the M1 phenotype, whereas when exposed to anti-inflammatory cytokines such as interleukin (IL)-4 or IL-13, the M2 phenotype is adopted. While M1 macrophages are associated with tissue defense and destruction of invading pathogens, M2 macrophages are involved in tissue repair and in terminating inflammation. It is well known that an inflammatory microenvironment exists in the brain of aged animals and also in the brain of mice that overexpress amyloid-ß protein precursor (AßPP) and presenilin 1 (PS1; AßPP/PS1 mice), a commonly-used model of Alzheimer's disease (AD). Recent studies have revealed that immune cells, including macrophages, infiltrate the brain in both circumstances raising the possibility that these cells adopt the M1 activation state and contribute to the already-existing neuroinflammation. We set out to examine the responses of bone marrow-derived macrophages prepared from wildtype and AßPP/PS1 mice and demonstrate that cells from AßPP/PS1 mice, even after several days in culture, respond more profoundly to IFNγ than those from wildtype mice. We suggest that this propensity to respond to M1-polarizing stimuli, together with the described changes in the brain of AßPP/PS1 mice, contribute to the development of chronic neuroinflammation.


Subject(s)
Alzheimer Disease/pathology , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Interferon-gamma/pharmacology , Macrophages/drug effects , Alzheimer Disease/genetics , Amyloid beta-Protein Precursor/genetics , Animals , Brain/drug effects , Brain/metabolism , Cells, Cultured , Claudin-5/metabolism , Cytokines/metabolism , Disease Models, Animal , Endothelium/drug effects , Female , Humans , Male , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neutrophil Infiltration/drug effects , Neutrophil Infiltration/genetics , Presenilin-1/genetics , Receptors, Cell Surface/metabolism , Receptors, Immunologic , Zonula Occludens-1 Protein/metabolism
17.
Neurotherapeutics ; 11(4): 857-69, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25096154

ABSTRACT

Traumatic brain injury (TBI) causes microglial activation and related neurotoxicity that contributes to chronic neurodegeneration and loss of neurological function. Selective activation of metabotropic glutamate receptor 5 (mGluR5) by the orthosteric agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), is neuroprotective in experimental models of TBI, and has potent anti-inflammatory effects in vitro. However, the therapeutic potential of CHPG is limited due to its relatively weak potency and brain permeability. Highly potent, selective and brain penetrant mGluR5 positive allosteric modulators (PAMs) have been developed and show promise as therapeutic agents. We evaluated the therapeutic potential of a novel mGluR5 PAM, VU0360172, after controlled cortical impact (CCI) in mice. Vehicle, VU0360172, or VU0360172 plus mGluR5 antagonist (MTEP), were administered systemically to CCI mice at 3 h post-injury; lesion volume, hippocampal neurodegeneration, microglial activation, and functional recovery were assessed through 28 days post-injury. Anti-inflammatory effects of VU0360172 were also examined in vitro using BV2 and primary microglia. VU0360172 treatment significantly reduced the lesion, attenuated hippocampal neurodegeneration, and improved motor function recovery after CCI. Effects were mediated by mGluR5 as co-administration of MTEP blocked the protective effects of VU0360172. VU0360172 significantly reduced CD68 and NOX2 expression in activated microglia in the cortex at 28 days post-injury, and also suppressed pro-inflammatory signaling pathways in BV2 and primary microglia. In addition, VU0360172 treatment shifted the balance between M1/M2 microglial activation states towards an M2 pro-repair phenotype. This study demonstrates that VU0360172 confers neuroprotection after experimental TBI, and suggests that mGluR5 PAMs may be promising therapeutic agents for head injury.


Subject(s)
Brain Injuries/prevention & control , Cerebral Cortex/drug effects , Glycine/analogs & derivatives , Neuroprotective Agents/pharmacology , Phenylacetates/pharmacology , Receptor, Metabotropic Glutamate 5/agonists , Allosteric Regulation , Animals , Brain Injuries/metabolism , Cell Count , Cerebral Cortex/metabolism , Cerebral Cortex/pathology , Disease Models, Animal , Glycine/pharmacology , Hippocampus/drug effects , Hippocampus/pathology , Male , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred C57BL , Microglia/drug effects , Microglia/metabolism , Motor Activity/drug effects , NADPH Oxidase 2 , NADPH Oxidases/metabolism , Nitric Oxide Synthase Type II/metabolism , Pyridines/pharmacology , Receptor, Metabotropic Glutamate 5/antagonists & inhibitors , Recovery of Function , Thiazoles/pharmacology
18.
J Neuroinflammation ; 9: 126, 2012 Jun 14.
Article in English | MEDLINE | ID: mdl-22697788

ABSTRACT

BACKGROUND: Compelling evidence has implicated neuroinflammation in the pathogenesis of a number of neurodegenerative conditions. Chronic activation of both astrocytes and microglia leads to excessive secretion of proinflammatory molecules such as TNF α, IL-6 and IL-1 ß with potentially deleterious consequences for neuronal viability. Many signaling pathways involving the mitogen-activated protein kinases (MAPKs), nuclear factor κ B (NF κ B) complex and the Janus kinases (JAKs)/signal transducers and activators of transcription (STAT)-1 have been implicated in the secretion of proinflammatory cytokines from glia. We sought to identify signaling kinases responsible for cytokine production and to delineate the complex interactions which govern time-related responses to lipopolysaccharide (LPS). METHODS: We examined the time-related changes in certain signaling events and the release of proinflammatory cytokines from LPS-stimulated co-cultures of astrocytes and microglia isolated from neonatal rats. RESULTS: TNF α was detected in the supernatant approximately 1 to 2 hours after LPS treatment while IL-1 ß and IL-6 were detected after 2 to 3 and 4 to 6 hours, respectively. Interestingly, activation of NF κ B signaling preceded release of all cytokines while phosphorylation of STAT1 was evident only after 2 hours, indicating that activation of JAK/STAT may be important in the up-regulation of IL-6 production. Additionally, incubation of glia with TNF α induced both phosphorylation of JAK2 and STAT1 and the interaction of JAK2 with the TNF α receptor (TNFR1). Co-treatment of glia with LPS and recombinant IL-6 protein attenuated the LPS-induced release of both TNF α and IL-1 ß while potentiating the effect of LPS on suppressor of cytokine signaling (SOCS)3 expression and IL-10 release. CONCLUSIONS: These data indicate that TNF α may regulate IL-6 production through activation of JAK/STAT signaling and that the subsequent production of IL-6 may impact on the release of TNF α, IL-1 ß and IL-10.


Subject(s)
Cytokine Receptor gp130/metabolism , Interleukin-1beta/biosynthesis , Janus Kinase 2/physiology , Lipopolysaccharides/physiology , Neuroglia/metabolism , Animals , Animals, Newborn , Cells, Cultured , Coculture Techniques , Cytokine Receptor gp130/physiology , MAP Kinase Signaling System/physiology , Neuroglia/immunology , Rats , Rats, Wistar , Recombinant Proteins/pharmacology , Signal Transduction/physiology
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