Karen Krukowski

Glucocorticoid Dysregulation of Natural Killer Cell Function through Epigenetic Modification

It is well-established that psychological distress reduces natural killer cell activity (NKCA) and dysregulates cytokine balance. This may be mediated by stress-induced release of glucocorticoids, which have broad effects on the immune system, including the suppression of NKCA and alteration of cytokine production. The purpose of this study was to evaluate epigenetic mechanisms that may underlie the effect of glucocorticoids on NK cells, using the human NK cell line, NK92. Treatment of NK92 cells with the synthetic glucocorticoid, dexamethasone, at a concentration of 10⁻⁷M, produced a significant reduction in NKCA. Glucocorticoid inhibition was a consequence of not only a reduced capacity of the NK cells to bind to tumor targets but also a reduced production of granule constituents (perforin and granzyme B) with no detectable effect on granule exocytosis. Glucocorticoids also reduced the constitutive and the stimulated production of the cytokines, IL-6, TNF alpha and IFN gamma, and reduced the surface expression of LFA-1. Glucocorticoid treatment also reduced global histone acetylation, the acetylation of histone 4 lysine position 8, and the accessibility of the proximal promoters of perforin, interferon gamma and granzyme B. Histone acetylation was recovered by treatment of the NK cells with a histone deacetylase inhibitor, which also restored NKCA and IFN gamma production. These results demonstrate glucocorticoids to dysregulate NK cell function at least in part through an epigenetic mechanism, which reduces promoter accessibility through modification of histone acetylation status. This epigenetic modification decreases the expression of effector proteins necessary to the full functional activity of NK cells.

Glucocorticoid receptor mediated suppression of natural killer cell activity: identification of associated deacetylase and corepressor molecules.

Physical and psychological stressors reduce natural killer cell function. This reduction in cellular function results from stress-induced release of glucocorticoids. Glucocorticoids act upon natural killer cells to deacetylate and transrepress immune response genes through epigenetic processes. However, other than the glucocorticoid receptor, the proteins that participate in this process are not well described in natural killer cells. The purpose of this study was to identify the proteins associated with the glucocorticoid receptor that are likely epigenetic participants in this process. Treatment of natural killer cells with the synthetic glucocorticoid, dexamethasone, produced a significant time dependent reduction in natural killer cell activity as early as 8h post treatment. This reduction in natural killer cell activity was preceded by nuclear localization of the glucocorticoid receptor with histone deacetylase 1 and the corepressor, SMRT. Other class I histone deacetylases were not associated with the glucocorticoid receptor nor was the corepressor NCoR. These results demonstrate histone deacetylase 1 and SMRT to associate with the ligand activated glucocorticoid receptor within the nuclei of natural killer cells and to be the likely participants in the histone deacetylation and transrepression that accompanies glucocorticoid mediated reductions in natural killer cell function.

Glucocorticoids regulate natural killer cell function epigenetically.

Although glucocorticoids are well known for their capacity to suppress the immune response, glucocorticoids can also promote immune responsiveness. It was the purpose of this investigation to evaluate the molecular basis for this apparent dichotomous immunologic effect. Glucocorticoid treatment of natural killer cells (NK) was shown to reduce NK cell cytolytic activity by reduction of histone promoter acetylation for perforin and granzyme B, which corresponded with reduced mRNA and protein for each. In contrast, glucocorticoid treatment increased histone acetylation at regulatory regions for interferon gamma and IL-6, as well as chromatin accessibility for each. This increase in histone acetylation was associated with increased proinflammatory cytokine mRNA and protein production upon cellular stimulation. These immunologic effects were evident at the level of the individual cell and demonstrate glucocorticoids to epigenetically reduce NK cell cytolytic activity while at the same time to prime NK cells for proinflammatory cytokine production.

Inhibition of the integrated stress response reverses cognitive deficits after traumatic brain injury

Significance Traumatic brain injury (TBI) is a leading cause of long-term neurological disability and affects an ever-growing population. Currently, there are no effective treatments for patients suffering from chronic TBI-induced cognitive impairments. Here, we found that suppression of the integrated stress response (ISR) with a drug-like small-molecule inhibitor, ISRIB, rescued cognition in two TBI mouse models, even when administered weeks after injury. Consistent with the behavioral results, ISRIB restored long-term potentiation deficits observed in TBI mice. Our data suggest that targeting ISR activation could serve as a promising approach for the treatment of chronic cognitive dysfunction after TBI. Traumatic brain injury (TBI) is a leading cause of long-term neurological disability, yet the mechanisms underlying the chronic cognitive deficits associated with TBI remain unknown. Consequently, there are no effective treatments for patients suffering from the long-lasting symptoms of TBI. Here, we show that TBI persistently activates the integrated stress response (ISR), a universal intracellular signaling pathway that responds to a variety of cellular conditions and regulates protein translation via phosphorylation of the translation initiation factor eIF2α. Treatment with ISRIB, a potent drug-like small-molecule inhibitor of the ISR, reversed the hippocampal-dependent cognitive deficits induced by TBI in two different injury mouse models—focal contusion and diffuse concussive injury. Surprisingly, ISRIB corrected TBI-induced memory deficits when administered weeks after the initial injury and maintained cognitive improvement after treatment was terminated. At the physiological level, TBI suppressed long-term potentiation in the hippocampus, which was fully restored with ISRIB treatment. Our results indicate that ISR inhibition at time points late after injury can reverse memory deficits associated with TBI. As such, pharmacological inhibition of the ISR emerges as a promising avenue to combat head trauma-induced chronic cognitive deficits.

Epigenetic patterns associated with the immune dysregulation that accompanies psychosocial distress

The molecular basis for psychosocial-distress mediated immune-dysregulation is not well understood. The purpose of this study was to determine whether peripheral blood mononuclear cell (PBMC) epigenetic pattern associates with this form of immune dysregulation. Women newly diagnosed with early stage breast cancer were enrolled into the study and psychosocial, immunological and epigenetic assessments were made at diagnosis and four months later, after completion of cancer treatment. At diagnosis women reported increased perceived stress, anxiety, and mood disturbance and the PBMC of these women exhibited reduced natural killer cell activity and reduced production of interferon gamma, which contrasted with results, obtained after completion of treatment. At the epigenetic level, a PBMC subset derived from women at diagnosis exhibited a distinct epigenetic pattern, with reduced nuclear acetylation of histone residues H4-K8 and H4-K12, as well as reduced phosphorylation of H3-S10, when compared to similar cells derived after the completion of treatment. Natural killer cell activity and interferon-gamma production were associated with nuclear acetylation and phosphorylation status of these histone residues. These findings demonstrate associations among nuclear epigenetic pattern and the immune dysregulation that accompanies psychosocial distress.

Repeated mild head injury leads to wide ranging deficits in higher order cognitive functions associated with the prefrontal cortex.

Traumatic brain injury (TBI) has long been identified as a precipitating risk factor for higher-order cognitive deficits associated with the frontal and prefrontal cortices (PFC). In addition, mild repetitive TBI (rTBI), in particular, is being steadily recognized to increase the risk of neurodegenerative disease. Thus, further understanding of how mild rTBI changes the pathophysiology of the brain to lead to cognitive impairment is warranted. The current models of rTBI lack knowledge regarding chronic higher-order cognitive functions and the underlying neuronal physiology, especially functions involving the PFC. Here, we establish that five repeated mild hits, allowing rotational acceleration of the head, lead to chronic deficits in PFC-dependent functions such as social behavior, spatial working memory, and environmental response with concomitant microgliosis and a small decrease in the adaptation rate of layer V pyramidal neurons in the medial PFC (mPFC). However, structural damage is not seen on in vivo T2-weighted magnetic resonance imaging (MRI), and extensive intrinsic excitability changes in layer V pyramidal neurons of the mPFC are not observed. Thus, this rTBI animal model can recapitulate chronic higher-order cognitive impairments without structural damage on MR imaging as observed in humans.

In vivo metabolic imaging of Traumatic Brain Injury

Complex alterations in cerebral energetic metabolism arise after traumatic brain injury (TBI). To date, methods allowing for metabolic evaluation are highly invasive, limiting our understanding of metabolic impairments associated with TBI pathogenesis. We investigated whether 13C MRSI of hyperpolarized (HP) [1-13C] pyruvate, a non-invasive metabolic imaging method, could detect metabolic changes in controlled cortical injury (CCI) mice (nu2009=u200957). Our results show that HP [1-13C] lactate-to-pyruvate ratios were increased in the injured cortex at acute (12/24u2009hours) and sub-acute (7 days) time points after injury, in line with decreased pyruvate dehydrogenase (PDH) activity, suggesting impairment of the oxidative phosphorylation pathway. We then used the colony-stimulating factor-1 receptor inhibitor PLX5622 to deplete brain resident microglia prior to and after CCI, in order to confirm that modulations of HP [1-13C] lactate-to-pyruvate ratios were linked to microglial activation. Despite CCI, the HP [1-13C] lactate-to-pyruvate ratio at the injury cortex of microglia-depleted animals at 7 days post-injury remained unchanged compared to contralateral hemisphere, and PDH activity was not affected. Altogether, our results demonstrate that HP [1-13C] pyruvate has great potential for in vivo non-invasive detection of cerebral metabolism post-TBI, providing a new tool to monitor the effect of therapies targeting microglia/macrophages activation after TBI.

Peripheral T Cells as a Biomarker for Oxygen-Ion-Radiation-Induced Social Impairments

Exposure to galactic cosmic rays (GCR) poses an obstacle to successful deep space missions, including missions to the Moon or Mars. Previously, we and others have identified chronic cognitive impairments associated with GCR in rodent model systems. The persistent cognitive loss previously reported is indicative of global changes in different regions of the brain, including the prefrontal cortex and the hippocampus. It has been shown that both of these brain regions are involved in social functions. Here we demonstrate that four months after a single exposure to oxygen ionizing radiation, which is a component of GCR, adult male mice have social memory deficits. Importantly, we identified circulating levels of CD8 T cells as predictors of social behavioral changes. Thus, CD8 T cells could be used as a potential peripheral biomarker. To the best of our knowledge we demonstrate for the first time that GCR-induced impairments in social behavior are directly linked to peripheral immune changes. These results further advance our understanding of the challenges encountered during space exploration.

Temporary microglia-depletion after cosmic radiation modifies phagocytic activity and prevents cognitive deficits

Microglia are the main immune component in the brain that can regulate neuronal health and synapse function. Exposure to cosmic radiation can cause long-term cognitive impairments in rodent models thereby presenting potential obstacles for astronauts engaged in deep space travel. The mechanism/s for how cosmic radiation induces cognitive deficits are currently unknown. We find that temporary microglia depletion, one week after cosmic radiation, prevents the development of long-term memory deficits. Gene array profiling reveals that acute microglia depletion alters the late neuroinflammatory response to cosmic radiation. The repopulated microglia present a modified functional phenotype with reduced expression of scavenger receptors, lysosome membrane protein and complement receptor, all shown to be involved in microglia-synapses interaction. The lower phagocytic activity observed in the repopulated microglia is paralleled by improved synaptic protein expression. Our data provide mechanistic evidence for the role of microglia in the development of cognitive deficits after cosmic radiation exposure.

Author Correction: Temporary microglia-depletion after cosmic radiation modifies phagocytic activity and prevents cognitive deficits

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