J.L. Eddy

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.

The syp Enhancer Sequence Plays a Key Role in Transcriptional Activation by the σ54-Dependent Response Regulator SypG and in Biofilm Formation and Host Colonization by Vibrio fischeri

Biofilm formation by Vibrio fischeri is a complex process that requires multiple regulators. One such regulator, the NtrC-like response regulator SypG, controls biofilm formation and host colonization by V. fischeri via its impact on transcription of the symbiosis polysaccharide (syp) locus. SypG is predicted to activate syp transcription by binding to the syp enhancer (SE), a conserved sequence located upstream of four syp promoters. In this study, we performed an in-depth analysis of the sequences necessary for SypG to promote syp transcription and biofilm formation. We found that the SE sequence is necessary for SypG-mediated syp transcription, identified individual bases necessary for efficient activation, and determined that SypG is able to bind to syp promoter regions. We also identified SE sequences outside the syp locus and established that SypG recognizes these sequences as well. Finally, deletion of the SE sequence upstream of sypA led to defects in both biofilm formation and host colonization that could be restored by reintroducing the SE sequence into its native location in the chromosome. This work thus fills in critical gaps in knowledge of the Syp regulatory circuit by demonstrating a role for the SE sequence in SypG-dependent control of biofilm formation and host colonization and by identifying new putative regulon members. It may also provide useful insights into other bacteria, such as Vibrio vulnificus and Vibrio parahaemolyticus, that have syp-like loci and conserved SE sequences.

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.

28. Chronic exposure to glucocorticoids primes an epigenetic proinflammatory signature in lymphocytes

Although glucocorticoids (GC) are well described as anti-inflammatory agents, GC can also be proinflammatory. The effect of GC on cytokine production, either pro or anti-inflammatory, is dependent on both the dose and duration of GC exposure. The purpose of this study was to determine whether the proinflammatory effect of GC arise from epigenetic pattern modifications in the regulatory regions of proinflammatory genes. Chronic GC treatment of natural killer cells in vitro resulted in a marked increase in stimulated proinflammatory cytokine (IFN-gamma and IL-6) production. qPCR revealed these changes to be transcriptionally regulated and the result of altered epigenetic patterns at proinflammatory cytokine regulatory regions. Chronic GC treatment increased the histone(H) 4 lysine(K) 8 acetylation at the IFN-gamma enhancer and IL-6 promoter, 2- and 4-fold, respectively. Additionally, the IL-6 promoter exhibited a 2-fold increase in H3K9 acetylation. Chromatin structure analysis confirmed a 25% increase in accessibility at the IFN-gamma locus and a 40% increase in accessibility at the IL-6 promoter. Increased histone acetylation resulted from the displacement of HDAC1 and HDAC3 at both regulatory regions. Chronic GC significantly reduced cellular HDAC3 protein levels by 50% while HDAC1 relocalized to a non-nuclear position. Thus chronic GC alters the epigenetic landscape, increasing chromatin accessibility and priming these proinflammatory cytokine genes for rapid expression. These data provide an epigenetic basis for the observed proinflammatory effects of GC.

68. Relationships between global epigenetic modifications and NK cell function in women with breast cancer

A diagnosis of breast cancer results in psychosocial distress measured by increased perceived stress (PSS) and depressive mood (CES-D). This psychosocial distress is accompanied by reduced NK cell activity and elevated proinflammatory cytokine production (i.e., immune dysregulation). The effect of psychosocial distress on immune dysregulation has been attributed to elevations in glucocorticoids, such as cortisol. Glucocorticoids can impact gene transcription and alter epigenetic modifications. The purpose of this study was to identify epigenetic modifications hypothesized to underlie this immune dysregulation during periods of psychosocial distress. Global epigenetic modifications were analyzed in woman diagnosed with breast cancer and a matched cancer free group. Relationships among psychosocial distress and global acetylation of histone 4-lysine −8 (H4-K8-Ac) and global phosphorylation of histone 3-serine 10 (H3-S10-P) were measured in NK cells. A significant negative correlation was measured between PSS and global H4-K8-Ac. In contrast, a positive significant correlation was observed among global H4-K8-Ac with NKCA and intracellular perforin levels. Further analysis found the effect of cortisol on perforin to be significantly mediated by global H4-K8-Ac. Conversely, a strong positive relationship was measured between PSS, H3-S10-P and with intracellular interferon gamma levels These data suggest global H4-H8-Ac and H3-S10-P to be important epigenetic modifications associated with both the immune suppressive and immune enhancing effects demonstrable in women experiencing this naturalistic stressor.

174. Proinflammatory cytokine promoter priming of natural killer (NK) cells following chronic glucocorticoid treatment

viral pathogens such as herpes simplex virus. While stress is thought to have a detrimental effect on the immune system’s ability to resist infection, studies have shown certain stressors (i.e., social disruption; SDR) can enhance innate immunity in the presence or absence of infection. Natural killer (NK) cells are bone marrow-derived innate lymphocytes known to be potent mediators in the early defense against viral challenges. Therefore, initial experiments exploring mechanisms by which SDR potentially enhances NK cell function in the absence of a pathogenic challenge were conducted. The objectives of these experiments were to determine the effect of an SDRinduced altered microenvironment on NK cell activation receptors (i.e., CD69 and CD16/32), MHC class 1-like inhibitory receptors (i.e., CD94/NKG2a and Ly49a), and cytolytic potential (i.e., CD107a). Flow cytometric analyses were used to examine the possible effects of SDR on NK cell receptor surface expression. Results indicate that SDR alone is capable of increasing surface expression of activating receptors, differentially reducing inhibitory receptor expression, and increasing cytolytic potential. Mechanisms found in these studies point to potential therapeutic targets in the prevention of viral infection, recurrence, severity, and transmission in susceptible patients.

Epigenetic effects of glucocorticoids on NK cells

335 Odor cues from tumor-bearing mice induces neuroimmune changes J. Palermo-Neto, L. Vismari, G.J. Alves Neuroimmunomodulation Research Group, School of Veterinary Medicine, University of São Paulo, Av Prof Orlando Marques de Paiva, 87, Sao paulo, Sao Paulo 05508-270, Brazil The Central Nervous System and the Immune System are intimately connected and the bidirectional communication between these systems is the subject of study of the psychoneuroimmunology field. We have already shown that cohabitation with tumor-bearing partner for 14 days induces several psychoneuroimmunological changes, such as increased locomotor activity, increased hypothalamic noradrenaline levels and turnover which in turn are responsible for decreasing the innate immune responses and animal resistance to tumor growth. The present experiment was designed to access the relevance of tactile, chemical, olfactory, and visual communication to the neuroimmune changes induced by cohabitation with a tumor-bearing partner. Mice that were not allowed to perceive odor cues from their sick partners presented no alterations in neutrophil activity (p > .05), a fact not observed after visual deprivation (p < .05) and physical isolation (p < .05). Mice use scents for intraspecies communication in many social contexts. Tumors produce volatile organic compounds released into the atmosphere through breath, sweat, and urine. The present results strongly suggest that volatile compounds released by Ehrlich tumor-injected mice are perceived by their conspecifics, inducing the neuroimmune changes reported for cohabitation with a sick companion. doi:10.1016/j.bbi.2010.07.104 Abstract # 337 Epigenetic effects of glucocorticoids on NK cells J.L. Eddy , K.N. Krukowski , L. Janusek , H.L. Mathews a a Loyola University Graduate School Chicago, Microbiology and Immunology, 2160 S. 1st Ave., Building 105, Rm 2890, Maywood, IL 60153, United States b Niehoff School of Nursing, Loyola University Chicago, United States The purpose of this study was to evaluate epigenetic mechanisms that underlie the effect of glucocorticoids on natural killer (NK) cells. Glucocorticoid treatment resulted in decreased natural killer cell activity (NKCA) as judged by K562 lysis which correlated with a reduction in both effector molecules (i.e. granzyme B) and cytokine production (i.e. IFN-gamma and TNF-alpha). At the molecular level, promoter specific epigenetic patterns were assessed by chromatin immunoprecipitation demonstrating a reduction in histone(H) 4 lysine(K) 8 acetylation at the proximal promoters of IFN-gamma, granzyme B, and perforin. Glucocorticoid receptor was demonstrable at these proximal promoters with concomitant nuclear translocation of histone deacetylase 3. Compared to untreated cells, glucocorticoid treatment inhibited the activation-induced upregulation of IFNgamma mRNA as well as IFN-gamma protein secretion, suggesting promoter region inaccessibility to multiple transcription factors. Acetylation status was fully recovered by treatment of NK cells with a histone deacetylase inhibitor, TSA, which also restored NKCA. These data demonstrate glucocorticoids to inhibit NKCA through glucocorticoid receptor recruitment of histone deacetylase (HDAC) 3 to the promoter regions of NK cell effector genes culminating in nucleosomal deacetylation. This results in a compacted chromatin landscape with down regulation of transcription through decreased promoter region accessibility. These data provide mechanistic insight into the effects of glucocorticoids on immune cell function, demonstrating a role for epigenetic pattern modifications by glucocorticoids. doi:10.1016/j.bbi.2010.07.105

70. Global acetylation patterns and immune dysregulation associated with psychological stress

brain parenchyma and to affect astroglial activation. Immune deficient mice possess higher levels of activated astrocytes along with a reduced expression of the synaptogenic protein, thrombospondin1 (TSP1), as compared to wild type mice. Moreover, immune deficient mice have fewer hippocampal synapses when compared to wild type counterparts. We show that while anti-inflammatory cytokine, interleukin (IL)-4, increases TSP1 expression in astrocytes, pro-inflammatory cytokines, such as IL-1beta and TNF-alpha inhibit its expression, which is, supposedly, responsible for reduced synapses and for cognitive impairment observed in immune deficient mice. Our results shed a light on a role of meningeal immunity in synaptogenesis, in particular, and in cognitive function, in general.