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 emerging importance of skull-brain interactions in traumatic brain injury.

The recent identification of skull bone marrow as a reactive hematopoietic niche that can contribute to and direct leukocyte trafficking into the meninges and brain has transformed our view of this bone structure from a solid, protective casing to a living, dynamic tissue poised to modulate brain homeostasis and neuroinflammation. This emerging concept may be highly relevant to injuries that directly impact the skull such as in traumatic brain injury (TBI). From mild concussion to severe contusion with skull fracturing, the bone marrow response of this local myeloid cell reservoir has the potential to impact not just the acute inflammatory response in the brain, but also the remodeling of the calvarium itself, influencing its response to future head impacts. If we borrow understanding from recent discoveries in other CNS immunological niches and extend them to this nascent, but growing, subfield of neuroimmunology, it is not unreasonable to consider the hematopoietic compartment in the skull may similarly play an important role in health, aging, and neurodegenerative disease following TBI. This literature review briefly summarizes the traditional role of the skull in TBI and offers some additional insights into skull-brain interactions and their potential role in affecting secondary neuroinflammation and injury outcomes.

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.

Azithromycin reduces hemoglobin-induced innate neuroimmune activation.

Neonatal intraventricular hemorrhage (IVH) releases blood products into the lateral ventricles and brain parenchyma. There are currently no medical treatments for IVH and surgery is used to treat a delayed effect of IVH, post-hemorrhagic hydrocephalus. However, surgery is not a cure for intrinsic brain injury from IVH, and is performed in a subacute time frame. Like many neurological diseases and injuries, innate immune activation is implicated in the pathogenesis of IVH. Innate immune activation is a pharmaceutically targetable mechanism to reduce brain injury and post-hemorrhagic hydrocephalus after IVH. Here, we tested the macrolide antibiotic azithromycin, which has immunomodulatory properties, to reduce innate immune activation in an in vitro model of microglial activation using the blood product hemoglobin (Hgb). We then utilized azithromycin in our in vivo model of IVH, using intraventricular blood injection into the lateral ventricle of post-natal day 5 rat pups. In both models, azithromycin modulated innate immune activation by several outcome measures including mitochondrial bioenergetic analysis, cytokine expression and flow cytometric analysis. This suggests that azithromycin, which is safe for neonates, could hold promise for modulating innate immune activation after IVH.

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.

Inhibition of autophagy in microglia and macrophages exacerbates innate immune responses and worsens brain injury outcomes.

Excessive and prolonged neuroinflammation following traumatic brain injury (TBI) contributes to long-term tissue damage and poor functional outcomes. However, the mechanisms contributing to exacerbated inflammatory responses after brain injury remain poorly understood. Our previous work showed that macroautophagy/autophagy flux is inhibited in neurons following TBI in mice and contributes to neuronal cell death. In the present study, we demonstrate that autophagy is also inhibited in activated microglia and infiltrating macrophages, and that this potentiates injury-induced neuroinflammatory responses. Macrophage/microglia-specific knockout of the essential autophagy gene Becn1 led to overall increase in neuroinflammation after TBI. In particular, we observed excessive activation of the innate immune responses, including both the type-I interferon and inflammasome pathways. Defects in microglial and macrophage autophagy following injury were associated with decreased phagocytic clearance of danger/damage-associated molecular patterns (DAMP) responsible for activation of the cellular innate immune responses. Our data also demonstrated a role for precision autophagy in targeting and degradation of innate immune pathways components, such as the NLRP3 inflammasome. Finally, inhibition of microglial/macrophage autophagy led to increased neurodegeneration and worse long-term cognitive outcomes after TBI. Conversely, increasing autophagy by treatment with rapamycin decreased inflammation and improved outcomes in wild-type mice after TBI. Overall, our work demonstrates that inhibition of autophagy in microglia and infiltrating macrophages contributes to excessive neuroinflammation following brain injury and in the long term may prevent resolution of inflammation and tissue regeneration.Abbreviations: Becn1/BECN1, beclin 1, autophagy related; CCI, controlled cortical impact; Cybb/CYBB/NOX2: cytochrome b-245, beta polypeptide; DAMP, danger/damage-associated molecular patterns; Il1b/IL1B/Il-1ß, interleukin 1 beta; LAP, LC3-associated phagocytosis; Map1lc3b/MAP1LC3/LC3, microtubule-associated protein 1 light chain 3 beta; Mefv/MEFV/TRIM20: Mediterranean fever; Nos2/NOS2/iNOS: nitric oxide synthase 2, inducible; Nlrp3/NLRP3, NLR family, pyrin domain containing 3; Sqstm1/SQSTM1/p62, sequestosome 1; TBI, traumatic brain injury; Tnf/TNF/TNF-α, tumor necrosis factor; Ulk1/ULK1, unc-51 like kinase 1.

Fn14-Directed DART Nanoparticles Selectively Target Neoplastic Cells in Preclinical Models of Triple-Negative Breast Cancer Brain Metastasis.

Triple-negative breast cancer (TNBC) patients with brain metastasis (BM) face dismal prognosis due to the limited therapeutic efficacy of the currently available treatment options. We previously demonstrated that paclitaxel-loaded PLGA-PEG nanoparticles (NPs) directed to the Fn14 receptor, termed "DARTs", are more efficacious than Abraxane─an FDA-approved paclitaxel nanoformulation─following intravenous delivery in a mouse model of TNBC BM. However, the precise basis for this difference was not investigated. Here, we further examine the utility of the DART drug delivery platform in complementary xenograft and syngeneic TNBC BM models. First, we demonstrated that, in comparison to nontargeted NPs, DART NPs exhibit preferential association with Fn14-positive human and murine TNBC cell lines cultured in vitro. We next identified tumor cells as the predominant source of Fn14 expression in the TNBC BM-immune microenvironment with minimal expression by microglia, infiltrating macrophages, monocytes, or lymphocytes. We then show that despite similar accumulation in brains harboring TNBC tumors, Fn14-targeted DARTs exhibit significant and specific association with Fn14-positive TNBC cells compared to nontargeted NPs or Abraxane. Together, these results indicate that Fn14 expression primarily by tumor cells in TNBC BMs enables selective DART NP delivery to these cells, likely driving the significantly improved therapeutic efficacy observed in our prior work.

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.

Depletion of CD4 T cells provides therapeutic benefits in aged mice after ischemic stroke.

T-lymphocytes have a multifaceted role in ischemic stroke, but the majority of studies have been conducted in young mice, which may limit the translational value of these findings. Previous studies have shown that aging results in T cell dysfunction, leading to enhanced production of pro-inflammatory cytokines and chemokines, including interferon gamma (IFN-γ) and interferon-gamma-inducible protein (IP-10). This study assessed the role of T cells and pro-inflammatory factors on histologic and functional outcomes in an aged mouse model. Levels of IP-10 were measured in the brain and serum of young and aged male mice following middle cerebral artery occlusion (MCAo) or sham surgery. Additionally, IP-10 levels were evaluated in stroke patients. To directly determine the role of brain-infiltrating T cells after stroke, a separate cohort of aged male and female animals received either an anti-CD4 depletion antibody or IgG isotype control at 72 and 96 h following experimental stroke. Behavioral assessments were performed on day 7 post-MCAo. CD4 T cell depletion resulted in improved behavioral outcomes, despite the lack of differences in infarct size between the isotype control and anti-CD4 antibody treated stroke groups. Circulating IP-10 levels were increased in both humans and mice with age and stroke, and depletion of CD4 T cells led to a reduction in IFN-γ and IP-10 levels in mice. Since anti-CD4 treatment was administered three days after stroke onset, targeting this inflammatory pathway may be beneficial to aged stroke patients who present outside of the current time window for thrombolysis and thrombectomy.

Old age increases microglial senescence, exacerbates secondary neuroinflammation, and worsens neurological outcomes after acute traumatic brain injury in mice.

After traumatic brain injury (TBI), individuals aged over 65 years show increased mortality and worse functional outcomes compared with younger persons. As neuroinflammation is a key pathobiological mechanism of secondary injury after TBI, we examined how aging affects post-traumatic microglial responses and functional outcomes. Young (3-month-old) and aged (18-month-old) male C57Bl/6 mice were subjected to moderate-level controlled cortical impact or sham surgery, and neurological function was evaluated. At 72 hours after injury, brain, blood, and spleen leukocyte counts were assessed ex vivo using flow cytometry. Aged mice demonstrated more severe deficits in forelimb grip strength, balance and motor coordination, spontaneous locomotor activity, and anxiety-like behavior. These animals also exhibited more robust microglial proliferation and significantly higher numbers of brain-infiltrating leukocytes. Microglia in aged mice showed impairments in phagocytic activity and higher production of interleukin-1ß (IL-1ß). Infiltrating myeloid cells in aged TBI mice also had deficits in phagocytosis but showed diminished proinflammatory cytokine production and greater reactive oxygen species production. Expression of several senescence markers (Bcl-2, p16ink4a, p21cip1a, lipofuscin, and H2AX [pS139]) was increased with age and/or TBI in both microglia and injured cortex. Although there was no difference in the number of circulating blood neutrophils as a function of age, young mice exhibited more pronounced TBI-induced splenomegaly and splenic myeloid cell expansion. Thus, worse post-traumatic behavioral outcomes in aged animals are associated with exaggerated microglial responses, increased leukocyte invasion, and upregulation of senescence markers.

Evaluation of the Neuroprotective Effect of Sirt3 in Experimental Stroke.

Sirtuins (Sirt) are a family of NAD+ dependent histone deacetylase (HDAC) proteins implicated in aging, cell cycle regulation, and metabolism. These proteins are involved in the epigenetic modification of neuromodulatory proteins after strokevia acetylation/deacetylation. The specific role of Sirt3, a mitochondrial sirtuin, in post-stroke injury has been relatively unexplored. Using male Sirt3 knockout (KO) mice and wild-type littermates (WT), we show that Sirt3 KO mice show significant neuroprotection at 3 days after ischemia/reperfusion (I/R) or stroke injury. The deacetylation activity of Sirt3, measured as the amount of reduced acetylated lysine, was increased after stroke. Stroke-induced increases in liver kinase 1 (LKB1) activity were also reduced in KO mice at 3 days after stroke. On further investigation, we found that the levels of Sirt1, another important member of the Sirtuin family, were increased in the brains of Sirt3 KO mice after stroke. To determine the translational relevance of these findings, we then tested the effects of pharmacological inhibition of Sirt3. We found no benefit of Sirt3 inhibition despite clear evidence of deacetylation. Overall, these data suggest that Sirt3 KO mice show neuroprotection by a compensatory rise in Sirt1 rather than the loss of Sirt3 after stroke. Further analysis reveals that the beneficial effects of Sirt1 might be mediated by a decrease in LKB1 activity after stroke. Finally, our data clearly demonstrate the importance of using both pharmacological and genetic methods in pre-clinical stroke studies.

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.

Inhibition of miR-141-3p Ameliorates the Negative Effects of Poststroke Social Isolation in Aged Mice.

BACKGROUND AND PURPOSE: Social isolation increases mortality and impairs recovery after stroke in clinical populations. These detrimental effects have been recapitulated in animal models, although the exact mechanism mediating these effects remains unclear. Dysregulation of microRNAs (miRNAs) occurs in both strokes as well as after social isolation, which trigger changes in many downstream genes. We hypothesized that miRNA regulation is involved in the detrimental effects of poststroke social isolation in aged animals. METHODS: We pair-housed 18-month-old C57BL/6 male mice for 2 weeks before a 60-minute right middle cerebral artery occlusion or sham surgery and then randomly assigned mice to isolation or continued pair housing immediately after surgery. We euthanized mice either at 3, 7, or 15 days after surgery and isolated the perilesional frontal cortex for whole microRNAome analysis. In an additional cohort, we treated mice 1 day after stroke onset with an in vivo-ready antagomiR-141 for 3 days. RESULTS: Using whole microRNAome analysis of 752 miRNAs, we identified miR-141-3p as a unique miRNA that was significantly upregulated in isolated mice in a time-dependent manner up to 2 weeks after stroke. Posttreatment with an antagomiR-141-3p reduced the postisolation-induced increase in miR-141-3p to levels almost equal to those of pair-housed stroke controls. This treatment significantly reduced mortality (by 21%) and normalized infarct volume and neurological scores in poststroke-isolated mice. Quantitative PCR analysis revealed a significant upregulation of Tgfßr1 (transforming growth factor beta receptor 1, a direct target of miR-141-3p) and Igf-1 (insulin-like growth factor 1) mRNA after treatment with antagomiR. Treatment also increased the expression of other pleiotropic cytokines such as Il-6 (interleukin 6) and Tnf-α (tumor necrosis factor-α), an indirect or secondary target) in brain tissue. CONCLUSIONS: miR-141-3p is increased with poststroke isolation. Inhibition of miR-141-3p improved mortality, neurological deficits, and decreased infarct volumes. Importantly, these therapeutic effects occurred in aged animals, the population most at risk for stroke and poststroke isolation.

Aging alters the immunological response to ischemic stroke.

The peripheral immune system plays a critical role in aging and in the response to brain injury. Emerging data suggest inflammatory responses are exacerbated in older animals following ischemic stroke; however, our understanding of these age-related changes is poor. In this work, we demonstrate marked differences in the composition of circulating and infiltrating leukocytes recruited to the ischemic brain of old male mice after stroke compared to young male mice. Blood neutrophilia and neutrophil invasion into the brain were increased in aged animals. Relative to infiltrating monocyte populations, brain-invading neutrophils had reduced phagocytic potential, and produced higher levels of reactive oxygen species and extracellular matrix-degrading enzymes (i.e., MMP-9), which were further exacerbated with age. Hemorrhagic transformation was more pronounced in aged versus young mice relative to infarct size. High numbers of myeloperoxidase-positive neutrophils were found in postmortem human brain samples of old (> 71 years) acute ischemic stroke subjects compared to non-ischemic controls. Many of these neutrophils were found in the brain parenchyma. A large proportion of these neutrophils expressed MMP-9 and positively correlated with hemorrhage and hyperemia. MMP-9 expression and hemorrhagic transformation after stroke increased with age. These changes in the myeloid response to stroke with age led us to hypothesize that the bone marrow response to stroke is altered with age, which could be important for the development of effective therapies targeting the immune response. We generated heterochronic bone marrow chimeras as a tool to determine the contribution of peripheral immune senescence to age- and stroke-induced inflammation. Old hosts that received young bone marrow (i.e., Young → Old) had attenuation of age-related reductions in bFGF and VEGF and showed improved locomotor activity and gait dynamics compared to isochronic (Old → Old) controls. Microglia in young heterochronic mice (Old → Young) developed a senescent-like phenotype. After stroke, aged animals reconstituted with young marrow had reduced behavioral deficits compared to isochronic controls, and had significantly fewer brain-infiltrating neutrophils. Increased rates of hemorrhagic transformation were seen in young mice reconstituted with aged bone marrow. This work suggests that age alters the immunological response to stroke, and that this can be reversed by manipulation of the peripheral immune cells in the bone marrow.

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.

Multiparity improves outcomes after cerebral ischemia in female mice despite features of increased metabovascular risk.

Females show a varying degree of ischemic sensitivity throughout their lifespan, which is not fully explained by hormonal or genetic factors. Epidemiological data suggest that sex-specific life experiences such as pregnancy increase stroke risk. This work evaluated the role of parity on stroke outcome. Age-matched virgin (i.e., nulliparous) and multiparous mice were subjected to 60 min of reversible middle cerebral artery occlusion and evaluated for infarct volume, behavioral recovery, and inflammation. Using an established mating paradigm, fetal microchimeric cells present in maternal mice were also tracked after parturition and stroke. Parity was associated with sedentary behavior, weight gain, and higher triglyceride and cholesterol levels. The multiparous brain exhibited features of immune suppression, with dampened baseline microglial activity. After acute stroke, multiparous mice had smaller infarcts, less glial activation, and less behavioral impairment in the critical recovery window of 72 h. Behavioral recovery was significantly better in multiparous females compared with nulliparous mice 1 mo after stroke. This recovery was accompanied by an increase in poststroke angiogenesis that was correlated with improved performance on sensorimotor and cognitive tests. Multiparous mice had higher levels of VEGF, both at baseline and after stroke. GFP+ fetal cells were detected in the blood and migrated to areas of tissue injury where they adopted endothelial morphology 30 d after injury. Reproductive experience has profound and complex effects on neurovascular health and disease. Inclusion of female mice with reproductive experience in preclinical studies may better reflect the life-long patterning of ischemic stroke risk in women.

Age-Associated Resident Memory CD8 T Cells in the Central Nervous System Are Primed To Potentiate Inflammation after Ischemic Brain Injury.

Aging is associated with an increase in basal inflammation in the CNS and an overall decline in cognitive function and poorer recovery following injury. Growing evidence suggests that leukocyte recruitment to the CNS is also increased with normal aging, but, to date, no systematic evaluation of these age-associated leukocytes has been performed. In this work, the effect of aging on CNS leukocyte recruitment was examined. Aging was associated with more CD45(high) leukocytes, primarily composed of conventional CD8(+) T cells. These results were strain independent and seen in both sexes. Intravascular labeling and immunohistology revealed the presence of parenchymal CD8(+) T cells in several regions of the brain, including the choroid plexus and meninges. These cells had effector memory (CD44(+)CD62L(-)) and tissue-resident phenotypes and expressed markers associated with TCR activation. Analysis of TCRvß repertoire usage suggested that entry into the CNS is most likely stochastic rather than Ag driven. Correlational analyses revealed a positive association between CD8 T cell numbers and decreased proinflammatory function of microglia. However, the effects of cerebral ischemia and ex vivo stimulation of these cells dramatically increased production of TNF, IFN-γ, and MCP-1/CCL2. Taken together, we identified a novel population of resident memory, immunosurveillant CD8 T cells that represent a hallmark of CNS aging and appear to modify microglia homeostasis under normal conditions, but are primed to potentiate inflammation and leukocyte recruitment following ischemic injury.

Functional differences between microglia and monocytes after ischemic stroke.

BACKGROUND: The brain's initial innate response to stroke is primarily mediated by microglia, the resident macrophage of the CNS. However, as early as 4 h after stroke, the blood-brain barrier is compromised and monocyte infiltration occurs. The lack of discriminating markers between these two myeloid populations has led many studies to generate conclusions based on the grouping of these two populations. A growing body of evidence now supports the distinct roles played by microglia and monocytes in many disease models. METHODS: Using a flow cytometry approach, combined with ex-vivo functional assays, we were able to distinguish microglia from monocytes using the relative expression of CD45 and assess the function of each cell type following stroke over the course of 7 days. RESULTS: We found that at 72 h after a 90-min middle cerebral artery occlusion (MCAO), microglia populations decrease whereas monocytes significantly increase in the stroke brain compared to sham. After stroke, BRDU incorporation into monocytes in the bone marrow increased. After recruitment to the ischemic brain, these monocytes accounted for nearly all BRDU-positive macrophages. Inflammatory activity peaked at 72 h. Microglia produced relatively higher reactive oxygen species and TNF, whereas monocytes were the predominant IL-1ß producer. Although microglia showed enhanced phagocytic activity after stroke, monocytes had significantly higher phagocytic capacity at 72 h. Interestingly, we found a positive correlation between TNF expression levels and phagocytic activity of microglia after stroke. CONCLUSIONS: In summary, the resident microglia population is vulnerable to the effects of severe ischemia, show compromised cell cycle progression, and adopt a largely pro-inflammatory phenotype after stroke. Infiltrating monocytes are primarily involved with early debris clearance of dying cells. These findings suggest that the early wave of infiltrating monocytes may be beneficial to stroke repair and future therapies aimed at mitigating microglia cell death may prove more effective than attempting to elicit targeted anti-inflammatory responses from damaged cells.

Sex, stroke, and inflammation: the potential for estrogen-mediated immunoprotection in stroke.

Stroke is the third leading cause of death and the primary cause of disability in the developed world. Experimental and clinical data indicate that stroke is a sexually dimorphic disease, with males demonstrating an enhanced intrinsic sensitivity to ischemic damage throughout most of their lifespan. The neuroprotective role of estrogen in the female brain is well established, however, estrogen exposure can also be deleterious, especially in older women. The mechanisms for this remain unclear. Our current understanding is based on studies examining estrogen as it relates to neuronal injury, yet cerebral ischemia also induces a robust sterile inflammatory response involving local and systemic immune cells. Despite the potent anti-inflammatory effects of estrogen, few studies have investigated the contribution of estrogen to sex differences in the inflammatory response to stroke. This review examines the potential role for estrogen-mediated immunoprotection in ischemic injury.