Anjali Chauhan

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 CD45high 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 TCRvβ 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.

Ischemic stroke induces gut permeability and enhances bacterial translocation leading to sepsis in aged mice.

Aging is an important risk factor for post-stroke infection, which accounts for a large proportion of stroke-associated mortality. Despite this, studies evaluating post-stroke infection rates in aged animal models are limited. In addition, few studies have assessed gut microbes as a potential source of infection following stroke. Therefore we investigated the effects of age and the role of bacterial translocation from the gut in post-stroke infection in young (8-12 weeks) and aged (18-20 months) C57Bl/6 male mice following transient middle cerebral artery occlusion (MCAO) or sham surgery. Gut permeability was examined and peripheral organs were assessed for the presence of gut-derived bacteria following stroke. Furthermore, sickness parameters and components of innate and adaptive immunity were examined. We found that while stroke induced gut permeability and bacterial translocation in both young and aged mice, only young mice were able to resolve infection. Bacterial species seeding peripheral organs also differed between young (Escherichia) and aged (Enterobacter) mice. Consequently, aged mice developed a septic response marked by persistent and exacerbated hypothermia, weight loss, and immune dysfunction compared to young mice following stroke.

Interferon regulatory factor 4/5 signaling impacts on microglial activation after ischemic stroke in mice

Microglial activation is a key element in initiating and perpetuating inflammatory responses to stroke. Interferon regulatory factor 5 (IRF5) and IRF4 signaling have been found critical in mediating macrophage pro‐inflammatory (M1) and anti‐inflammatory (M2) phenotypes, respectively, in peripheral inflammation. We hypothesize that the IRF5/4 regulatory axis also mediates microglial activation after stroke. C57BL6 mice of 8–12 weeks were subject to a 90‐min middle cerebral artery occlusion, and the brains evaluated at 24 h, 3, 10 and 30 days after reperfusion. Flow cytometry was utilized to examine microglial activation and cytokine expression. RT‐PCR was performed for mRNA levels of IRF5/4 in sorted microglia. Microglial expression of IRF5/4 was examined by immunohistochemistry, and brain cytokine levels were determined by ELISA. Our results revealed that the IRF5 mRNA level in sorted microglia increased at 3 days of stroke; whereas IRF4 mRNA level exhibited biphasic increases, with a transient rise at 24 h and a peak at 10 days. The same pattern was seen in IRF5/4 protein colocalization with Iba‐1+ cells by IHC. Intracellular levels of TNF‐α and IL‐1β in microglia peaked at 3 days of stroke, and IL‐4+IL‐10+ double‐positive microglia significantly increased at day 10. Brain levels of these cytokines were consistent with microglial cytokine changes. Worse behavior test results were seen at 3 days vs. 10 days of stroke. We conclude that microglia phenotypes are dynamic to ischemic stroke, and IRF5/4 signaling may regulate microglial M1/M2 activation and impact on stroke outcomes.

Splenectomy protects aged mice from injury after experimental stroke

Elderly stroke patients and aged animals subjected to experimental stroke have significantly worse functional recovery and higher mortality compared to younger subjects. Activation of the peripheral immune system is known to influence stroke outcome. Prior studies have shown that splenectomy reduces ischemic brain injury in young mice. As immune function changes with aging, it is unclear whether splenectomy will confer similar benefits in aged animals. We investigated the contribution of spleen to brain injury after cerebral ischemia in aged male mice. Splenic architecture and immune cell composition were altered in aged mice. Splenectomy 2 weeks before stroke resulted in improved neurobehavioral and infarct outcomes in aged male mice. In addition, there was a reduction in peripheral immune cell infiltration into the brain and decreased levels of peripheral inflammatory cytokines after stroke in aged splenectomized mice. Splenectomy immediately after reperfusion also improved behavioral and infarct outcomes. This study suggests that inhibition of the splenic immune response is a translationally relevant target to pursue for stroke treatment in aged individuals.

Sex differences in adipose tissue CD8+ T cells and regulatory T cells in middle-aged mice

The prevalence of cardiovascular disease has increased among middle-aged women in the United States, yet has declined in middle-aged men. In experimental stroke, middle-aged females have larger strokes and greater inflammation than age-matched males or younger females. The mechanism underlying this shift from an “ischemia-protected” to an “ischemia-sensitive” phenotype in aging females is unknown. One potential factor is an age-related increase in systemic factors that induce inflammation. Increased abdominal fat deposition is seen in women during middle age. Adipose tissue plays a key role in obesity-induced systemic inflammation, including increased pro-inflammatory cytokines. We hypothesized that age and sex differences in adipose immune cells promote an augmented pro-inflammatory milieu in middle-aged females driven by a balance shift between pro-inflammatory and anti-inflammatory T cells. Abdominal adipose tissue immune cells from young (3–4 months) and middle-aged (15–16 months) male and female C57BL/6J mice were analyzed by flow cytometry. Plasma triglyceride (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) levels were determined with colorimetric assays. Middle-aged mice had higher adipose tissue mass compared to young mice. Lipid profiling showed no sex differences in TG and LDL, but middle-aged females had lower HDL (0.84 ± 0.07 μg/μl) than middle-aged males (1.35 ± 0.06 μg/μl). Flow cytometry data demonstrated an age-associated increase in adipose tissue CD8+ T cells that was augmented by female sex, with middle-aged females having a higher percentage of CD8+ cells (34.4 ± 3.2% of CD3+ T cells) than middle-aged males (24.4 ± 2.2%). This increase in CD8+ T-cell proportion was adipose tissue-specific, as this change was not observed in blood. Middle-aged females had higher numbers of activated (CD69+) CD8+ T cells than males. In addition, female CD8+ T cells produced higher levels of IFN-γ, TNF-α, and granzyme B ex vivo, and females had higher adipose levels of IFN-γ, RANTES and MIP-1β than middle-aged males. In parallel, females had lower levels of regulatory T cells (Tregs), an anti-inflammatory T-cell subtype, compared to age-matched males. In conclusion, middle-aged females have a detrimental combination of elevated pro-inflammatory T cells and decreased anti-inflammatory Tregs in adipose tissue, which may promote a pro-inflammatory milieu and contribute to increased cardiovascular disease burden in aging females.

Genetic deletion of the Rho GEF Net1 impairs mouse macrophage motility and actin cytoskeletal organization

ABSTRACT Macrophages are innate immune cells that constantly patrol an organism to fulfill protective and homeostatic roles. Previous studies have shown that Rho GTPase activity is required for macrophage mobility, yet the roles of upstream regulatory proteins controlling Rho GTPase function in these cells are not well defined. Previously we have shown that the RhoA GEF Net1 is required for human breast cancer cell motility and extracellular matrix invasion. To assess the role of Net1 in macrophage motility, we isolated bone marrow macrophage (BMM) precursors from wild type and Net1 knockout mice. Loss of Net1 did not affect the ability of BMM precursors to differentiate into mature macrophages in vitro, as measured by CD68 and F4/80 staining. However, Net1 deletion significantly reduced RhoA activation, F-actin accumulation, adhesion, and motility in these cells. Nevertheless, similar to RhoA/RhoB double knockout macrophages, Net1 deletion did not impair macrophage recruitment to the peritoneum in a mouse model of sterile inflammation. These data demonstrate that Net1 is an important regulator of RhoA signaling and motility in mouse macrophages in vitro, but that its function may be dispensable for macrophage recruitment to inflammatory sites in vivo.

Sex-Specific Factors in Stroke

Stroke is a sexually dimorphic disease, identified through evidence derived from preclinical and clinical trials. Efficacy of prevention strategies, recognition of symptoms, acute stroke treatment options, and stroke recovery are influenced by sex-specific factors. The terms, “sex” and “gender” are often used interchangeably. By definition, sex is a biological variable, typically characterized as male, female, or intersex and includes indicators of biological sex, including sex chromosomes (XX and XY) and reproductive organs. Gender refers to attitudes, feelings, and behaviors that a culture associates with a person’s biological sex. This chapter will focus on sex differences in ischemic stroke and will provide an update of emerging pre-clinical and clinical studies that confirm that sex is an important biological variable in ischemic brain injury.