Interaction of high-fat diet and brain trauma alters adipose tissue macrophages and brain microglia associated with exacerbated cognitive dysfunction.

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.

Bi-directional neuro-immune dysfunction after chronic experimental brain injury.

BACKGROUND: It is well established that traumatic brain injury (TBI) causes acute and chronic alterations in systemic immune function and that systemic immune changes contribute to posttraumatic neuroinflammation and neurodegeneration. However, how TBI affects bone marrow (BM) hematopoietic stem/progenitor cells chronically and to what extent such changes may negatively impact innate immunity and neurological function has not been examined. METHODS: To further understand the role of BM cell derivatives on TBI outcome, we generated BM chimeric mice by transplanting BM from chronically injured or sham (i.e., 90 days post-surgery) congenic donor mice into otherwise healthy, age-matched, irradiated CD45.2 C57BL/6 (WT) hosts. Immune changes were evaluated by flow cytometry, multiplex ELISA, and NanoString technology. Moderate-to-severe TBI was induced by controlled cortical impact injury and neurological function was measured using a battery of behavioral tests. RESULTS: TBI induced chronic alterations in the transcriptome of BM lineage-c-Kit+Sca1+ (LSK+) cells in C57BL/6 mice, including modified epigenetic and senescence pathways. After 8 weeks of reconstitution, peripheral myeloid cells from TBI→WT mice showed significantly higher oxidative stress levels and reduced phagocytic activity. At eight months after reconstitution, TBI→WT chimeric mice were leukopenic, with continued alterations in phagocytosis and oxidative stress responses, as well as persistent neurological deficits. Gene expression analysis revealed BM-driven changes in neuroinflammation and neuropathology after 8 weeks and 8 months of reconstitution, respectively. Chimeric mice subjected to TBI at 8 weeks and 8 months post-reconstitution showed that longer reconstitution periods (i.e., time post-injury) were associated with increased microgliosis and leukocyte infiltration. Pre-treatment with a senolytic agent, ABT-263, significantly improved behavioral performance of aged C57BL/6 mice at baseline, although it did not attenuate neuroinflammation in the acutely injured brain. CONCLUSIONS: TBI causes chronic activation and progressive dysfunction of the BM stem/progenitor cell pool, which drives long-term deficits in hematopoiesis, innate immunity, and neurological function, as well as altered sensitivity to subsequent brain injury.

The brain-bone marrow axis and its implications for chronic traumatic brain injury.

Traumatic brain injury (TBI) causes acute and chronic alterations in systemic immune function which contribute to posttraumatic neuroinflammation and neurodegeneration. However, how TBI affects bone marrow (BM) hematopoietic stem/progenitor cells chronically and to what extent such changes may negatively impact innate immunity and neurological function has not been examined. To further understand the role of BM cell derivatives on TBI outcome, we generated BM chimeric mice by transplanting BM from chronically injured or sham congenic donor mice into otherwise healthy, age-matched, irradiated hosts. After 8 weeks of reconstitution, peripheral myeloid cells from TBI→WT mice showed significantly higher oxidative stress levels and reduced phagocytic activity. At eight months after reconstitution, TBI→WT chimeric mice were leukopenic, with continued alterations in phagocytosis and oxidative stress responses, as well as persistent neurological deficits. Gene expression analysis revealed BM-driven changes in neuroinflammation and neuropathology after 8 weeks and 8 months of reconstitution, respectively. Chimeric mice subjected to TBI showed that longer reconstitution periods were associated with increased microgliosis and leukocyte infiltration. Thus, TBI causes chronic activation and progressive dysfunction of the BM stem/progenitor cell pool, which drives long-term deficits in innate immunity and neurological function, as well as altered sensitivity to subsequent brain injury.

Enhanced Akt/GSK-3ß/CREB signaling mediates the anti-inflammatory actions of mGluR5 positive allosteric modulators in microglia and following traumatic brain injury in male mice.

We have previously shown that treatment with a mGluR5 positive allosteric modulator (PAM) is neuroprotective after experimental traumatic brain injury (TBI), limiting post-traumatic neuroinflammation by reducing pro-inflammatory microglial activation and promoting anti-inflammatory and neuroprotective responses. However, the specific molecular mechanisms governing this anti-inflammatory shift in microglia remain unknown. Here we show that the mGluR5 PAM, VU0360172 (VuPAM), regulates microglial inflammatory responses through activation of Akt, resulting in the inhibition of GSK-3ß. GSK-3ß regulates the phosphorylation of CREB, thereby controlling the expression of inflammation-related genes and microglial plasticity. The anti-inflammatory action of VuPAM in microglia is reversed by inhibiting Akt/GSK-3ß/CREB signaling. Using a well-characterized TBI model and CX3CR1gfp/+ mice to visualize microglia in vivo, we demonstrate that VuPAM enhances Akt/GSK-3ß/CREB signaling in the injured cortex, as well as anti-inflammatory microglial markers. Furthermore, in situ analysis revealed that GFP + microglia in the cortex of VuPAM-treated TBI mice co-express pCREB and the anti-inflammatory microglial phenotype marker YM1. Taken together, our data show that VuPAM decreases pro-inflammatory microglial activation by modulating Akt/GSK-3ß/CREB signaling. These findings serve to clarify the potential neuroprotective mechanisms of mGluR5 PAM treatment after TBI, and suggest novel therapeutic targets for post-traumatic neuroinflammation. Cover Image for this issue: https://doi.org/10.1111/jnc.15048.

Mithramycin selectively attenuates DNA-damage-induced neuronal cell death.

DNA damage triggers cell death mechanisms contributing to neuronal loss and cognitive decline in neurological disorders, including traumatic brain injury (TBI), and as a side effect of chemotherapy. Mithramycin, which competitively targets chromatin-binding sites of specificity protein 1 (Sp1), was used to examine previously unexplored neuronal cell death regulatory mechanisms via rat primary neurons in vitro and after TBI in mice (males). In primary neurons exposed to DNA-damage-inducing chemotherapy drugs in vitro we showed that DNA breaks sequentially initiate DNA-damage responses, including phosphorylation of ATM, H2AX and tumor protein 53 (p53), transcriptional activation of pro-apoptotic BH3-only proteins, and mitochondrial outer membrane permeabilization (MOMP), activating caspase-dependent and caspase-independent intrinsic apoptosis. Mithramycin was highly neuroprotective in DNA-damage-dependent neuronal cell death, inhibiting chemotherapeutic-induced cell death cascades downstream of ATM and p53 phosphorylation/activation but upstream of p53-induced expression of pro-apoptotic molecules. Mithramycin reduced neuronal upregulation of BH3-only proteins and mitochondrial dysfunction, attenuated caspase-3/7 activation and caspase substrates' cleavage, and limited c-Jun activation. Chromatin immunoprecipitation indicated that mithramycin attenuates Sp1 binding to pro-apoptotic gene promoters without altering p53 binding suggesting it acts by removing cofactors required for p53 transactivation. In contrast, the DNA-damage-independent neuronal death models displayed caspase initiation in the absence of p53/BH3 activation and were not protected even when mithramycin reduced caspase activation. Interestingly, experimental TBI triggers a multiplicity of neuronal death mechanisms. Although markers of DNA-damage/p53-dependent intrinsic apoptosis are detected acutely in the injured cortex and are attenuated by mithramycin, these processes may play a reduced role in early neuronal death after TBI, as caspase-dependent mechanisms are repressed in mature neurons while other, mithramycin-resistant mechanisms are active. Our data suggest that Sp1 is required for p53-mediated transactivation of neuronal pro-apoptotic molecules and that mithramycin may attenuate neuronal cell death in conditions predominantly involving DNA-damage-induced p53-dependent intrinsic apoptosis.

Early or Late Bacterial Lung Infection Increases Mortality After Traumatic Brain Injury in Male Mice and Chronically Impairs Monocyte Innate Immune Function.

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.

Endocannabinoid modulation of inflammatory hyperalgesia in the IFN-α mouse model of depression.

Depression is a well-recognised effect of long-term treatment with interferon-alpha (IFN-α), a widely used treatment for chronic viral hepatitis and malignancy. In addition to the emotional disturbances, high incidences of painful symptoms such as headache and joint pain have also been reported following IFN-α treatment. The endocannabinoid system plays an important role in emotional and nociceptive processing, however it is unknown whether repeated IFN-α administration induces alterations in this system. The present study investigated nociceptive responding in the IFN-α-induced mouse model of depression and associated changes in the endocannabinoid system. Furthermore, the effects of modulating peripheral endocannabinoid tone on inflammatory pain-related behaviour in the IFN-α model was examined. Repeated IFN-α administration (8000 IU/g/day) to male C57/Bl6 mice increased immobility in the forced swim test and reduced sucrose preference, without altering body weight gain or locomotor activity, confirming development of the depressive-like phenotype. There was no effect of repeated IFN-α administration on latency to respond in the hot plate test on day 4 or 7 of treatment, however, formalin-evoked nociceptive behaviour was significantly increased in IFN-α treated mice following 8 days of IFN-α administration. 2-Arachidonoyl glycerol (2-AG) levels in the periaqueductal grey (PAG) and rostroventromedial medulla (RVM), and anandamide (AEA) levels in the RVM, were significantly increased in IFN-α-, but not saline-, treated mice following formalin administration. There was no change in endocannabinoid levels in the prefrontal cortex, spinal cord or paw tissue between saline- or IFNα-treated mice in the presence or absence of formalin. Furthermore, repeated IFN-α and/or formalin administration did not alter mRNA expression of genes encoding the endocannabinoid catabolic enzymes (fatty acid amide hydrolase or monoacylglycerol lipase) or endocannabinoid receptor targets (CB1, CB2 or PPARs) in the brain, spinal cord or paw tissue. Intra plantar administration of PF3845 (1 µg/10 µl) or MJN110 (1 µg/10 µl), inhibitors of AEA and 2-AG catabolism respectively, attenuated formalin-evoked hyperalgesia in IFN-α, but not saline-, treated mice. In summary, increasing peripheral endocannabinoid tone attenuates inflammatory hyperalgesia induced following repeated IFN-α administration. These data provide support for the endocannabinoid system in mediating and modulating heightened pain responding associated with IFNα-induced depression.

Sex Differences in Acute Neuroinflammation after Experimental Traumatic Brain Injury Are Mediated by Infiltrating Myeloid Cells.

The inflammatory response to moderate-severe controlled cortical impact (CCI) in adult male mice has been shown to exhibit greater glial activation compared with age-matched female mice. However, the relative contributions of resident microglia and infiltrating peripheral myeloid cells to this sexually dimorphic neuroinflammatory responses remains unclear. Here, 12-week-old male and female C57Bl/6 mice were subjected to sham or CCI, and brain samples were collected at 1, 3, or 7 days post-injury for flow cytometry analysis of cytokines, reactive oxygen species (ROS), and phagocytosis in resident microglia (CD45intCD11b+) versus infiltrating myeloid cells (CD45hiCD11b+). Motor (rotarod, cylinder test), affect (open field), and cognitive (Y-maze) function tests also were performed. We demonstrate that male microglia had increased phagocytic activity and higher ROS levels in the non-injured brain, whereas female microglia had increased production of tumor necrosis factor (TNF) α and interleukin (IL)-1ß. Following CCI, males showed a significant influx of peripheral myeloid cells by 1 day post-injury followed by proliferation of resident microglia at 3 days. In contrast, myeloid infiltration and microglial activation responses in female CCI mice were significantly reduced. No sex differences were observed for TNFα, IL-1ß, transforming growth factor ß, NOX2, ROS production, or phagocytic activity in resident microglia or infiltrating cells at any time. However, across these functions, infiltrating myeloid cells were significantly more reactive than resident microglia. Female CCI mice also had improved motor function at 1 day post-injury compared with male mice. Thus, we conclude that sexually dimorphic responses to moderate-severe CCI result from the rapid activation and infiltration of pro-inflammatory myeloid cells to brain in male, but not female, mice.

Chronic Alterations in Systemic Immune Function after Traumatic Brain Injury.

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.

FAAH, but not MAGL, inhibition modulates acute TLR3-induced neuroimmune signaling in the rat, independent of sex.

Toll-like receptor (TLR)3 is a key component of the innate immune response to viral infection. The present study firstly examined whether sex differences exist in TLR3-induced inflammatory, endocrine, and sickness responses. The data revealed that TLR3-induced expression of interferon- or NFkB-inducible genes (IFN-α/ß, IP-10, or TNF-α), either peripherally (spleen) or centrally (hypothalamus), did not differ between male and female rats, with the exception of TLR3-induced IFN-α expression in the spleen of female, but not male, rats 8 hr post TLR3 activation. Furthermore, TLR3 activation increased plasma corticosterone levels, induced fever, and reduced locomotor activity and body weight - effects independent of sex. Thus, the acute-phase inflammatory, endocrine, and sickness responses to TLR3 activation exhibit minimal sex-related differences. A further aim of this study was to examine whether enhancing endocannabinoid tone - namely, 2-arachidonylglycerol (2-AG) or N-arachidonoylethanolamine (AEA), exhibited similar effects on TLR3-induced inflammatory responses in male versus female rats. Systemic administration of the monoacylglycerol lipase (MAGL) inhibitor MJN110 and subsequent increases in 2-AG levels did not alter the TLR3-induced increase in IP-10, IRF7, or TNF-α expression in the spleen or the hypothalamus of male or female rats. In contrast, the fatty acid amide hydrolase (FAAH) inhibitor URB597 increased levels of AEA and related N-acylethanolamines, an effect associated with the attenuation of TLR3-induced inflammatory responses in the hypothalamus, but not the spleen, of male and female rats. These data support a role for FAAH, but not MAGL, substrates in the modulation of TLR3-induced neuroinflammatory responses, effects independent of sex.

NOX2 deficiency alters macrophage phenotype through an IL-10/STAT3 dependent mechanism: implications for traumatic brain injury.

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.