Yael Goldfarb

Perioperative Use of β-blockers and COX-2 Inhibitors May Improve Immune Competence and Reduce the Risk of Tumor Metastasis

BackgroundCOX inhibitors and β-blockers were recently suggested to reduce cancer progression through inhibition of tumor proliferation and growth factor secretion, induction of tumor apoptosis, and prevention of cellular immune suppression during the critical perioperative period. Here we evaluated the perioperative impact of clinically applicable drugs from these categories in the context of surgery, studying natural killer (NK) cell activity and resistance to experimental metastases.MethodsF344 rats were treated with COX-1 inhibitors (SC560), COX-2 inhibitors (indomethacin, etodolac, or celecoxib), a β-blocker (propranolol), or a combination of a COX-2 inhibitor and a β-blocker (etodolac and propranolol). Rats underwent laparotomy, and were inoculated intravenously with syngeneic MADB106 tumor cells for the assessment of lung tumor retention (LTR). Additionally, the impact of these drug regimens on postoperative levels of NK cytotoxicity was studied in peripheral blood and marginating-pulmonary leukocytes.ResultsSurgery increased MADB106 LTR. COX-2 inhibition, but not COX-1 inhibition, reduced postoperative LTR. Etodolac and propranolol both attenuated the deleterious impact of surgery, and their combined use abolished it. Surgery decreased NK cytotoxicity per NK cell in both immune compartments, and only the combination of etodolac and propranolol significantly attenuated these effects. Lastly, the initiation of drug treatment three days prior to surgery yielded the same beneficial effects as a single pre-operative administration, but, as discussed, prolonged treatment may be more advantageous clinically.ConclusionsExcess prostaglandin and catecholamine release contributes to postoperative immune-suppression. Treatment combining perioperative COX-2 inhibition and β-blockade is practical in operated cancer patients, and our study suggests potential immunological and clinical benefits.

Improving postoperative immune status and resistance to cancer metastasis: a combined perioperative approach of immunostimulation and prevention of excessive surgical stress responses.

Background:Surgical procedures, including primary tumor resection, have been suggested to suppress immune competence and to promote postoperative infections and cancer metastasis. Catecholamines and prostaglandins were recently implicated in these processes, and in directly promoting tumor angiogenesis and invasion. Objective:To examine the integration of 2 complementary approaches to reduce postoperative immunosuppression and metastatic progression: (1) perioperative immunostimulation with CpG-C and (2) pharmacological blockade of the tumor-promoting and immunosuppressing effects of catecholamines and prostaglandins, using propranolol (P) and etodolac (E), respectively. Methods:F344 rats were treated before surgery with CpG-C, P+E, both interventions, or vehicles, and were intravenously inoculated with syngeneic MADB106 mammary adenocarcinoma cells. Blood was withdrawn, marginating-pulmonary leukocytes were harvested, and NK activity and lung MADB106 tumor retention were assessed. In addition, C57BL/6 mice were implanted with syngeneic B16F10.9 melanoma cells. When tumors reached 100 mm3, mice were treated with CpG-C/vehicle, and 24 hours later the tumor was excised along with P+E/vehicle treatment. Recurrence-free survival was monitored thereafter. Results:Each of the regimens alone, CpG-C or P+E, showed improvement in most indices examined, including improved long-term recurrence-free survival rates. Most importantly, the combined treatment yielded additive or synergistic effects, further improving tumor clearance from the lungs and enhancing NK numbers and cytotoxicity via different, but complimentary, mechanisms. Conclusions:Treatment aimed at perioperative enhancement of CMI and simultaneous inhibition of excessive catecholamine and prostaglandin responses, employing CpG-C, propranolol, and etodolac, could be successful in limiting postoperative immunosuppression and metastatic progression, more so than each treatment alone.

The deacetylase Sirt1 is an essential regulator of Aire-mediated induction of central immunological tolerance

Aire is a transcriptional regulator that induces the promiscuous expression of thousands of tissue-restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs), a step critical for the induction of immunological self-tolerance. Studies have offered molecular insights into how Aire operates, but more comprehensive understanding of this process still remains elusive. Here we found abundant expression of the protein deacetylase Sirtuin-1 (Sirt1) in mature Aire+ mTECs, wherein it was required for the expression of Aire-dependent TRA-encoding genes and the subsequent induction of immunological self-tolerance. Our study elucidates a previously unknown molecular mechanism for Aire-mediated transcriptional regulation and identifies a unique function for Sirt1 in preventing organ-specific autoimmunity.

Neuronal, Endocrine, and Anorexic Responses to the T-Cell Superantigen Staphylococcal Enterotoxin A: Dependence on Tumor Necrosis Factor-α

Staphylococcal enterotoxin A (SEA) is a microbial superantigen that activates T-lymphocytes and induces production of various cytokines, including tumor necrosis factor-α (TNFα). Previously, it was shown that SEA activates the hypothalamic-pituitary-adrenal axis and augments gustatory neophobic behaviors. In the present study, it was hypothesized that these effects involve neuronal activation in forebrain regions mediating fear and/or anxiety and are dependent on the production of TNFα. Male C57BL/6J mice were given intraperitoneal injections of 10 μg of SEA and 5 μg of lipopolysaccharide (LPS) or saline and perfused 2 h later for histochemical determination of brain c-Fos immunoreactivity (IR). The results showed increased c-Fos IR in the paraventricular nucleus, arcuate nucleus, central nucleus of the amygdala, bed nucleus of the stria terminalis, and lateral septum. Challenge of TNF-/- mice with SEA did not produce a significant increase in brain c-Fos IR, although c-Fos was increased after exposure to a psychogenic stressor (i.e., open field). In additional experiments, the elevated corticosterone response to SEA was abrogated in TNF-/- mice and was shown to be corticotropin-releasing hormone dependent. Finally, the augmented reduction in novel food intake after SEA challenge was attenuated in TNF-/- mice as well as in wild-type mice administered antibody to TNFα. In conclusion, challenge with SEA recruits brain regions mediating stress and anxiety responses, an effect that requires endogenous TNFα. Whether this is indicative of all T-cell superantigens remains to be determined, although it stands in contrast to other models of neuroimmunomodulation (e.g., LPS) that involve multiple cytokine influences.

Neural and behavioral responses to systemic immunologic stimuli: a consideration of bacterial T cell superantigens.

Immune responses represent a source of systemic stress which impacts the brain and modifies various neuroendocrine and behavioral functions. Therefore, the immune system has been conceived of as a potential contributor to stress-related behavioral abnormalities, such as depression. Much of this knowledge has been gained through research focused largely on the administration of cytokines and/or bacterial endotoxin (eg., LPS), which targets innate immune cells, such as macrophages. However, fewer studies have addressed the effects of T cell activation on central nervous system (CNS) function. The discovery and characterization of bacterial superantigens (SAgs) has introduced an important opportunity for studying how T cell activation influences CNS function. Superantigens target unique variable regions of the beta chain of the mouse and human T cell receptor. This is restricted by the class II molecule of the major histocompatibility complex (MHC), and results in the production of a cytokine cascade that includes interleukin-2 (IL-2), interferon-gamma (IFNgamma), tumor necrosis factor (TNF) and many other cytokines, including IL-6. The best studied SAgs are the staphylococcal enterotoxins, of which staphylococcal enterotoxins A and B (SEA and SEB), have been shown to produce significant changes in behavior and activation of the hypothalamic-pituitary-adrenal (HPA) axis. Importantly, a T cell requirement was necessary to produce these changes. Furthermore, the anorexic or hypophagic effects of SAg challenge in mice appears to be related to anxiety-like processes, since challenge with both SEA or SEB reduces consumption of mainly novel food or food presented in a novel context. In the present paper, these studies are reviewed and related to known alterations in both anxiogenic and anxiolytic neuropeptides. It is suggested that immunologically-induced changes in the brain activate both categories of neuropeptides, thereby sustaining an adaptive state of arousal that promotes appropriate behavioral adjustments during infectious illness.

CpG-C oligodeoxynucleotides limit the deleterious effects of β-adrenoceptor stimulation on NK cytotoxicity and metastatic dissemination

Suppression of natural killer (NK) cell activity is common after stress, has been reported to predict malignant recurrence in cancer patients, and was shown to underlie metastatic dissemination in animal models. We have previously reported that catecholamines play a major role in NK cell suppression, particularly in the context of physiologic stress and surgery. In the current study using Fisher 344 rats, we examined the prophylactic use of different regimens of type-C CpG oligodeoxynucleotides (CpG-C ODN) on NK activity and metastatic dissemination in the context of pharmacologic stress (using metaproterenol for β-adrenoceptor stimulation). Our results indicated that the beneficial effects of CpG-C ODN were more profound under pharmacologic stress than under baseline conditions. A bolus of CpG-C ODN (330 μg/kg, intraperitoneally) 24 hours before metaproterenol-challenge was most effective at reducing lung tumor retention of an experimental syngeneic mammary adenocarcinoma (MADB106), although having no observable side effects. Depletion of NK cells revealed their key role in improving baseline levels of resistance to metastatic dissemination after CpG-C ODN administration. When NK cell cytotoxicity was assessed in the circulation and the marginating-pulmonary immune compartments, we found that CpG-C ODN protected individual NK cells from metaproterenol-induced suppression in both compartments. Moreover, in the critical marginating-pulmonary compartment, CpG-C ODN also elevated baseline cytotoxicity per NK cell against MADB106 tumor cells, and increased NK cell numbers in nonstressed rats. Overall, prophylactic CpG-C ODN treatment can improve immunocompetence and potentially reduce metastatic dissemination, especially in clinical settings characterized by enhanced sympathetic stress responses.

Continuous stress disrupts immunostimulatory effects of IL-12

Immune stimulation by biological response modifiers is a common approach in tumor immunotherapy. IL-12 was found effective in various animal studies, but clinical trials showed limited success. However, among other differences, animal models do not simulate psychological or physiological stress while employing IL-12, whereas cancer patients often experience distress while treated with immunostimulants. Thus, in the current study we assessed the impact of continuous stress on the efficacy of IL-12 immunostimulation. F344 rats were subjected to a pharmacological stress paradigm (continuous administration of a β-adrenergic agonist) or to a 20 h behavioral stress paradigm (wet cage exposure) commencing 2h before IL-12 administration. Twenty-six hours after stress initiation, we studied indices known to reflect IL-12 immunostimulatory impacts, including NK cell numbers and activity in different immune compartments, and in vivo resistance to MADB106 lung tumor colonization. The results indicated that both the pharmacological and behavioral stress paradigms significantly reduced the increase in the number and activity of marginating-pulmonary NK cells evident in non-stressed IL-12 treated animals. Additionally, stressed animals exhibited a lower IL-12-induced improvement of MADB106 lung clearance, an in vivo index that markedly depends on total marginating-pulmonary NK activity. These deleterious effects of stress were more prominent in males than in females. Overall, the findings demonstrate that prolonged stress exposure can disrupt the efficacy of simultaneous immunostimulatory treatments, irrespective of stress effects on baseline immune measures. Neuroendocrine and cellular mediating mechanisms are yet unknown, but the potential clinical ramifications of these findings warrant consideration in clinical trials employing immunostimulatory agents.

AIRE: From promiscuous molecular partnerships to promiscuous gene expression.

Autoimmune regulator (AIRE) is a unique transcriptional regulator that induces promiscuous expression of thousands of tissue‐restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs), a step critical for the induction of immunological self‐tolerance. The past 15 years have seen dramatic progress in our understanding of how AIRE induces immunological self‐tolerance on a molecular level. This major advancement can be greatly attributed to the identification of a large variety of proteins that physically associate with AIRE, supporting and regulating its transcription‐transactivation capacity. These diverse molecular partnerships have been shown to play roles in shuttling AIRE to the nucleus, securing AIREs interaction with nuclear matrix and chromatin, releasing RNA polymerase‐II from its stalled state and potentiating AIRE‐mediated gene expression, among others. In this review we discuss the relationship of AIRE with its vast and rather diverse repertoire of partners and highlight how such “promiscuous partnerships” contribute to the phenomenon of “promiscuous gene expression” in the thymus.

The Marginating-Pulmonary Immune Compartment in rats: Characteristics of continuous inflammation and activated NK cells

A significant role has been indicated for cellular immunity in controlling circulating cancer cells, but most autologous tumor cells seem resistant, in vitro, to natural killer cell (NKC) and cytotoxic T lymphocytes cytotoxicity. Addressing this apparent contradiction, we recently identified a unique leukocyte population, marginating-pulmonary (MP)-leukocytes, which exhibit potent natural killer (NK) cytotoxicity. Here, we characterize the MP-compartment in naive and immunostimulated rats, and assessed its cytotoxicity against “NK-resistant” tumors cells. Animals were treated with poly I-C (3×0.2 mg/kg) or saline, and circulating-leukocytes and MP-leukocytes were collected and analyzed in terms of cellular composition, cellular activation markers, and NK cytotoxicity of leukocytes and purified NKCs. Compared with circulating-leukocytes, MP-leukocytes showed greater proportion of granulocytes, monocytes, NKCs, and large NKCs; higher expression of activation and adhesion markers (CD25, CD11a, CD11b, and NKR-P1, IFN-γ); and elevated NK cytotoxicity of leukocytes and purified NKCs against several syngeneic and xenogeneic NK-resistant target cells (from both F344 and BDX inbred rats). In immunostimulated animals (treated with poly I-C), but not in naive animals, purified NKCs from the MP-compartment showed markedly superior cytotoxicity, suggesting that poly I-C immunostimulation uniquely affect MP-NKCs, and that in naive animals other MP-leukocytes support NK cytotoxicity. Overall, the results suggest that the MP-compartment is characterized by a continuous activated inflammatory microenvironment uniquely affected by immunostimulation. If similarly potent MP-NKCs exist in patients, then circulating autologous tumor cells that are considered “NK-resistant” could actually be controlled by MP-NKCs. Innate immunity may assume greater role in controlling malignant spread, especially after immunostimulation.

Transcriptional programs that control expression of the autoimmune regulator gene Aire

Aire is a transcriptional regulator that induces promiscuous expression of thousands of genes encoding tissue-restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs). While the target genes of Aire are well characterized, the transcriptional programs that regulate its own expression have remained elusive. Here we comprehensively analyzed both cis-acting and trans-acting regulatory mechanisms and found that the Aire locus was insulated by the global chromatin organizer CTCF and was hypermethylated in cells and tissues that did not express Aire. In mTECs, however, Aire expression was facilitated by concurrent eviction of CTCF, specific demethylation of exon 2 and the proximal promoter, and the coordinated action of several transcription activators, including Irf4, Irf8, Tbx21, Tcf7 and Ctcfl, which acted on mTEC-specific accessible regions in the Aire locus.