N-VEGF, the Autoregulatory Arm of VEGF-A.

Vascular endothelial growth factor A (VEGF-A) is a secreted protein that stimulates angiogenesis in response to hypoxia. Under hypoxic conditions, a non-canonical long isoform called L-VEGF is concomitantly expressed with VEGF-A. Once translated, L-VEGF is proteolytically cleaved to generate N-VEGF and VEGF-A. Interestingly, while VEGF-A is secreted and affects the surrounding cells, N-VEGF is mobilized to the nucleus. This suggests that N-VEGF participates in transcriptional response to hypoxia. In this study, we performed a series of complementary experiments to examine the functional role of N-VEGF. Strikingly, we found that the mere expression of N-VEGF followed by its hypoxia-independent mobilization to the nucleus was sufficient to induce key genes associated with angiogenesis, such as Hif1α,VEGF-A isoforms, as well as genes associated with cell survival under hypoxia. Complementarily, when N-VEGF was genetically depleted, key hypoxia-induced genes were downregulated and cells were significantly susceptible to hypoxia-mediated apoptosis. This is the first report of N-VEGF serving as an autoregulatory arm of VEGF-A. Further experiments will be needed to determine the role of N-VEGF in cancer and embryogenesis.

MafK Mediates Chromatin Remodeling to Silence IRF8 Expression in Non-immune Cells in a Cell Type-SpecificManner.

The regulation of gene expression is a result of a complex interplay between chromatin remodeling, transcription factors, and signaling molecules. Cell differentiation is accompanied by chromatin remodeling of specific loci to permanently silence genes that are not essential for the differentiated cell activity. The molecular cues that recruit the chromatin remodeling machinery are not well characterized. IRF8 is an immune-cell specific transcription factor and its expression is augmented by interferon-γ. Therefore, it serves as a model gene to elucidate the molecular mechanisms governing its silencing in non-immune cells. Ahigh-throughput shRNA library screen in IRF8 expression-restrictive cells enabled the identification of MafK as modulator of IRF8 silencing, affecting chromatin architecture. ChIP-Seq analysis revealed three MafK binding regions (-25 kb, -20 kb, and IRF8 6th intron) within the IRF8 locus. These MafK binding sites are sufficient to repress a reporter gene when cloned in genome-integrated lentiviral reporter constructs in only expression-restrictive cells. Conversely, plasmid-based constructs do not demonstrate such repressive effect. These results highlight the role of these MafK binding sites in mediating repressed chromatin assembly. Finally, a more thorough genomic analysis was performed, using CRISPR-Cas9 to delete MafK-int6 binding region in IRF8 expression-restrictive cells. Deleted clones exhibited an accessible chromatin conformation within the IRF8 locus that was accompanied by a significant increase in basal expression of IRF8 that was further induced by interferon-γ. Taken together, we identified and characterized several MafK binding elements within the IRF8 locus that mediate repressive chromatin conformation resulting in the silencing of IRF8 expression in a celltype-specific manner.

Prevention of liver metastases through perioperative acute CpG-C immune stimulation.

Following excision of colorectal tumors, metastatic disease is prevalent, primarily occurs in the liver, and is highly predictive of poor prognosis. The perioperative period is now recognized as critical in determining the incidence of postoperative metastases and long-term cancer outcomes. Thus, various perioperative prophylactic interventions are currently studied during this time frame. However, immune stimulation during the perioperative period has rarely been attempted due to specific contraindications to surgery and various adverse effects. Here, to prevent liver metastases, we perioperatively employed a TLR-9 agonist, CpG-C, which exhibits minimal pyrogenic and other adverse effects in patients. We found that marginating-hepatic (MH) cells in BALB/c mice contained high percentage of NK cells, but exhibited negligible NK cytotoxicity, as previously reported in humans. However, a single CpG-C administration (25-100 µg/mouse) doubled MH-NK cell numbers, increased NK cell activation and maturation markers (NKp46, CD11b), decreased the inhibitory NKG2A ligand, and dramatically increased MH-NK-cell cytotoxicity against the syngeneic CT26 colon cancer line. Moreover, in operated mice, this innocuous intervention also markedly improved resistance to CT26 and MC38 hepatic metastases in BALB/c and C57BL/6 mice, respectively. Beneficial effects of CpG-C were mediated through activation of MH-NK cells, as indicated by an in vivo NK depletion study. Last, CpG-C protected against surgery-induced suppression of MH-NK cytotoxicity and improved their activation indices. Thus, we suggest that systemic perioperative CpG-C treatment should be considered and studied as a novel therapeutic approach to improve long-term cancer outcomes in colorectal cancer patients.

The Third Intron of IRF8 Is a Cell-Type-Specific Chromatin Priming Element during Mouse Embryonal Stem Cell Differentiation.

Interferon regulatory factor 8 (IRF8) is a nuclear transcription factor that plays a key role in the hierarchical differentiation of hematopoietic stem cells toward monocyte/dendritic cell lineages. Therefore, its expression is mainly limited to bone marrow-derived cells. The molecular mechanisms governing this cell-type-restricted expression have been described. However, the molecular mechanisms that are responsible for its silencing in non-hematopoietic cells are elusive. Recently, we demonstrated a role for IRF8 third intron in restricting its expression in non-hematopoietic cells. Furthermore, we showed that this intron alone is sufficient to promote repressed chromatin a cell-type-specific manner. Here we demonstrate the effect of the IRF8 third intron on chromatin conformation during murine embryonal stem cell differentiation. Using genome editing, we provide data showing that the third intron plays a key role in priming the chromatin state of the IRF8 locus during cell differentiation. It mediates dual regulatory effects in a cell-type-specific mode. It acts as a repressor element governing chromatin state of the IRF8 locus during embryonal stem cell differentiation to cardiomyocytes that are expression-restrictive cells. Conversely, it functions as an activator element that is essential for open chromatin structure during the differentiation of these cells to dendritic cells that are expression-permissive cells. Together, these results point to the role of IRF8 third intron as a cell-type-specific chromatin priming element during embryonal stem cell differentiation. These data add another layer to our understanding of the molecular mechanisms governing misexpression of a cell-type-specific gene such as IRF8.

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.

The Third Intron of the Interferon Regulatory Factor-8 Is an Initiator of Repressed Chromatin Restricting Its Expression in Non-Immune Cells.

Interferon Regulatory Factor-8 (IRF-8) serves as a key factor in the hierarchical differentiation towards monocyte/dendritic cell lineages. While much insight has been accumulated into the mechanisms essential for its hematopoietic specific expression, the mode of restricting IRF-8 expression in non-hematopoietic cells is still unknown. Here we show that the repression of IRF-8 expression in restrictive cells is mediated by its 3rd intron. Removal of this intron alleviates the repression of Bacterial Artificial Chromosome (BAC) IRF-8 reporter gene in these cells. Fine deletion analysis points to conserved regions within this intron mediating its restricted expression. Further, the intron alone selectively initiates gene silencing only in expression-restrictive cells. Characterization of this intron's properties points to its role as an initiator of sustainable gene silencing inducing chromatin condensation with suppressive histone modifications. This intronic element cannot silence episomal transgene expression underlining a strict chromatin-dependent silencing mechanism. We validated this chromatin-state specificity of IRF-8 intron upon in-vitro differentiation of induced pluripotent stem cells (iPSCs) into cardiomyocytes. Taken together, the IRF-8 3rd intron is sufficient and necessary to initiate gene silencing in non-hematopoietic cells, highlighting its role as a nucleation core for repressed chromatin during differentiation.

HDAC3 Is a Master Regulator of mTEC Development.

The thymus provides a unique microenvironment enabling development and selection of T lymphocytes. Medullary thymic epithelial cells (mTECs) play a pivotal role in this process by facilitating negative selection of self-reactive thymocytes and the generation of Foxp3(+) regulatory T cells. Although studies have highlighted the non-canonical nuclear factor κB (NF-κB) pathway as the key regulator of mTEC development, comprehensive understanding of the molecular pathways regulating this process still remains incomplete. Here, we demonstrate that the development of functionally competent mTECs is regulated by the histone deacetylase 3 (Hdac3). Although histone deacetylases are global transcriptional regulators, this effect is highly specific only to Hdac3, as neither Hdac1 nor Hdac2 inactivation caused mTEC ablation. Interestingly, Hdac3 induces an mTEC-specific transcriptional program independently of the previously recognized RANK-NFκB signaling pathway. Thus, our findings uncover yet another layer of complexity of TEC lineage divergence and highlight Hdac3 as a major and specific molecular switch crucial for mTEC differentiation.

Requirement of Stat3 Signaling in the Postnatal Development of Thymic Medullary Epithelial Cells.

Thymic medullary regions are formed in neonatal mice as islet-like structures, which increase in size over time and eventually fuse a few weeks after birth into a continuous structure. The development of medullary thymic epithelial cells (TEC) is dependent on NF-κB associated signaling though other signaling pathways may contribute. Here, we demonstrate that Stat3-mediated signals determine medullary TEC cellularity, architectural organization and hence the size of the medulla. Deleting Stat3 expression selectively in thymic epithelia precludes the postnatal enlargement of the medulla retaining a neonatal architecture of small separate medullary islets. In contrast, loss of Stat3 expression in cortical TEC neither affects the cellularity or organization of the epithelia. Activation of Stat3 is mainly positioned downstream of EGF-R as its ablation in TEC phenocopies the loss of Stat3 expression in these cells. These results indicate that Stat3 meditated signal via EGF-R is required for the postnatal development of thymic medullary regions.

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.

Impact of surgical extent and sex on the hepatic metastasis of colon cancer.

PURPOSE: Extensive oncological surgeries were previously suggested to increase cancer recurrence rates. We herein studied the impact of different surgical procedures and sex on colorectal cancer liver metastasis, employing several tumor inoculation approaches in BALB/c mice. METHODS: Experimental hepatic metastases of the syngeneic CT26 colorectal cancer line were induced either by intra-portal inoculation or intra-splenic inoculation, employing different tumor loads. Following intra-splenic inoculation, the entire spleen or an injected hemi-spleen was removed. Additionally, the magnitude of the surgical trauma accompanying the injection procedure was manipulated. RESULTS: Increasing the surgical trauma by adding laparotomy or extending the length of the surgery and hypothermia did not significantly affect the number of liver metastases or liver weight for any of the injection methods and tumor loads. The development of metastasis was significantly greater in males than in females under all conditions studied--a difference not explained by the direct effects of sex hormones on in vitro CT26 proliferation or vitality. CONCLUSION: Concurring with less controlled clinical observations, the surgical extensiveness did not significantly affect CT26 hepatic metastasis, potentially due to a ceiling effect of the surgical trauma on the metastatic process. The sexual dimorphism observed for the CT26 metastasis should be investigated in the context of surgical stress and considering anti-CT26 immunoreactivity.

The marginating-pulmonary immune compartment in mice exhibits increased NK cytotoxicity and unique cellular characteristics.

To test whether marginating-pulmonary (MP) leukocytes in mice have a unique potential to identify and destroy aberrant circulating cells, we compared MP to circulating leukocytes with respect to natural killer (NK) cytotoxicity, proinflammatory characteristics, molecular determinants of activation, and response to IL-12 immunostimulation. Cytotoxicity was assessed employing the YAC-1, B16F10, and 3LL target lines. C57BL/6 mice were injected with either saline or murine IL-12 (0.1 or 0.5 µg/mouse), either once or three times 48-h apart. Twenty-four hours after last injection, cardiac blood was withdrawn and MP leukocytes were collected by forced lung perfusion. NK cytotoxicity, cellular composition, and surface molecular markers were studied. MP leukocytes exhibited greater NK cytotoxicity than circulating leukocytes against the syngeneic B16F10 and 3LL tumor lines, but not against the allogeneic YAC-1 line. NKG2D and IL-12 receptor expression predicted NK cytotoxicity in circulating leukocytes, but not in MP leukocytes. IFNγ-receptor, IL-12-receptor, CD69, CD11a, and CD11b showed different patterns of expression in the two leukocyte populations, suggesting pro-inflammatory characteristics of the MP compartment. IL-12 stimulation caused differential effects on these markers and also elevated cytotoxicity in both compartments, but in different effector: target ratio-dependent patterns. MP leukocytes may play a critical role in eliminating aberrant circulating cells due to their enhanced NK cytotoxicity and given their strategic location in the lungs vasculature, which forces physical interactions with all circulating aberrant cells. MP-NK cells are unique in their cytotoxic mechanisms against syngeneic targets and in their activation profile and response to immunostimulatory agents.

In vivo suppression of NK cell cytotoxicity by stress and surgery: glucocorticoids have a minor role compared to catecholamines and prostaglandins.

Most in vitro and ex-vivo studies indicate a profound suppression of NK cell cytotoxicity (NKCC) by glucocorticoids; while catecholamines and prostaglandins were reported both to suppress and to enhance NKCC. However, methodological considerations hinder our ability to deduce from these findings to the impact of endogenous release of these factors on in vivo levels of NKCC and their implications to NK-dependent resistance to pathologies in living humans or animals. Here we used an in vivo approach that sensitively and specifically reflects NKCC in living F344 rats, based on lung clearance of NK-sensitive tumor cells (MADB106), and based on comparing effects between NK-intact and NK-depleted rats. To study the role of corticosterone, epinephrine, and prostaglandins, we administered these factors to rats, or antagonized their endogenous release following different stress paradigms or surgery. The results indicated that endogenous or exogenous elevated corticosterone levels can suppress in vivo NKCC levels, but only under some conditions, and mostly secondarily to the NK-suppressing impact of epinephrine. Specifically, corticosterone-induced NKCC suppression occurred (i) only under prolonged, but not short exposure to stress, and mainly in males; (ii) was smaller than the prominent impact of epinephrine; (iii) was mostly ascribed to corticosterone-induced potentiation of the effects of epinephrine or/and prostaglandins; and (iv) was completely abolished through antagonizing epinephrine or/and prostaglandins. Overall, these findings markedly limit the significance of stress/surgery-induced corticosterone release in the in vivo suppression of NKCC, and highlight the blockade of epinephrine or/and prostaglandins as effective and clinically feasible approaches to overcome such immuno-suppressive effects.

Resilience of the immune system in healthy young students to 30-hour sleep deprivation with psychological stress.

OBJECTIVE: Young adults often encounter sleep deprivation and stressful events. Both have been separately reported to modulate immunity, and occasionally they occur simultaneously. We assessed the combined effects of these conditions on immune competence in healthy students. METHODS: Twenty-three participants (mean age 24 years; SD 1.86; 14 females) were exposed to 30 h of sleep deprivation during which they conducted physiological, social and cognitive tasks. The control group consisted of 18 participants (mean age 23.67 years; SD 1.46; 11 females). All participants underwent cognitive and psychological evaluations at 10:00 AM, followed by blood and saliva collection, 3 days before sleep deprivation induction and on the morning following it. Immune/endocrine measures included blood counts of lymphocytes, granulocytes, monocytes and natural killer (NK) cells; levels of several cell surface markers; NK cytotoxicity; plasma levels of interleukin (IL)-6, IL-10, dehydroepiandrosterone and neuropeptide Y, and plasma and salivary cortisol levels. RESULTS: Although the experimental protocol significantly elevated state anxiety and psychological dissociation levels, no effects were evident in any of the immunological/endocrine indices. In contrast, expected sex differences in immune measures were found, including significantly higher NK cytotoxicity and monocyte counts in males, validating the integrity of the measurements. CONCLUSIONS: The findings suggest resilience of the immune system to a combined sleep deprivation and stressful exposure in young adults, while previous studies reported immune perturbations following either of these conditions separately. These apparent contradictions might reflect differences in the study design or in the methodology used for immunological assessments, including the time of sample collection, the combination of sleep deprivation with stress and our in vivo assessment of cytokine levels.

Correction of hypertelorbitism: evaluation of relapse on long-term follow-up.

BACKGROUND: Hypertelorbitism has been associated with a variety of congenital deformities. Appropriate timing for surgical correction remains controversial. We present our long-term experience of 33 patients with hypertelorbitism undergoing facial bipartition or orbital box osteotomy. METHODS: Patients with hypertelorbitism treated with either facial bipartition or orbital box osteotomy and repositioning who had long-term follow-up were studied (n=33). Age at the time of first surgery, preoperative interdacryon distance, and immediate postoperative interdacryon distance were recorded. Relapse was determined on postoperative follow-up, and the need for secondary correction was noted. Physician satisfaction score (range, 0-4) was also assessed. RESULTS: Patients had a mean total follow-up of 14.0 years. With regard to age at the time of initial procedure, patients younger than 6 years were all noted to have relapse, and 83% underwent revision surgery. In patients 6 years or older, only 11% had relapse and required a second operation. Yet, satisfaction scores were similar (3.2 versus 3.5). With regard to the severity of hypertelorbitism, there was no relapse noted among patients with mild hypertelorbitism (interorbital distance [IOD], 30-34 mm). Among those with moderate hypertelorbitism (IOD, 35-40 mm), 29.4% developed relapse. By contrast, all patients with severe hypertelorbitism (IOD, >40 mm) were noted to have relapse requiring repeat correction. Satisfaction scores were similar (3.4 versus 3.3 versus 3.1). CONCLUSIONS: Relapse after surgery for hypertelorbitism is related to the age of the patient at correction and the preoperative severity. When possible, surgical repositioning of the orbits should be delayed until later childhood.

Fish oil attenuates surgery-induced immunosuppression, limits post-operative metastatic dissemination and increases long-term recurrence-free survival in rodents inoculated with cancer cells.

BACKGROUND & AIMS: Omega-3 fatty acids (ω-3FA) attenuate postoperative immunosuppression vis-à-vis infection. Since immune-surveillance targets metastasizing cancer cells, we assessed the effect of ω-3FA consumption on 1) early post-operative Natural Killer cell (NK) cytotoxicity and metastases and 2) long-term recurrence-free survival, in two rodent models of surgery-promoted metastases. METHODS: C57BL/6J mice were fed standard, ω-3FA-enriched, or ω-6FA-enriched chow, beginning one week before subcutaneous footpad implantation of syngeneic melanoma cells. When tumors reached the volume of 110 µl, the tumor-bearing footpad was amputated, and long-term recurrence-free survival was assessed. Also, F344 rats were fed ω-3FA or ω-6FA for a month before undergoing or not undergoing laparotomy, and were intravenously inoculated with radio-labeled syngeneic adenocarcinoma cells. Marginating-pulmonary (MP)-leukocytes were harvested, and lung tumor retention (LTR) of metastases was assessed. RESULTS: ω-3FA consumption did not affect the growth of footpad tumors, but significantly enhanced post-amputation recurrence-free survival in mice. Surgery had a deleterious effect on NK cell activity and LTR whereas ω-3FA had large beneficial effects in non-operated rats and an even greater impact in operated rats. CONCLUSIONS: ω-3FA feeding attenuates or even overcomes postoperative NK cell suppression, increases resistance to experimental and spontaneous metastasis, and enhances recurrence-free survival following excision of metastasizing primary tumors. These findings warrant clinical studies of ω-3FA-based nutrition in patients undergoing resection of a primary tumor.

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 mm, 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.

PML is a key component for the differentiation of myeloid progenitor cells to macrophages.

IFN regulatory factor-8 (IRF-8, previously known as ICSBP) is a key transcription factor driving the differentiation of granulocyte\monocyte progenitor (GMP) cells toward monocyte\macrophage lineage. The promyelocytic leukemia (PML) gene is an immediate target gene regulated by IRF-8 in response to IFN-γ activation. PML is a multifunctional protein that has many isoforms serving as the scaffold components for nuclear bodies (NBs) engaged in numerous proteins interactions. The role of PML in the retinoic acid pathway that drives GMPs to granulopoiesis is documented in the literature. Here, we show that PML is also involved in monopoiesis by mediating some of the IRF-8 activities during the differentiation of murine-derived bone marrow macrophages (BMMs). PML silencing resulted in altered expression level of key transcription factors essential for monopoiesis that was accompanied by silencing of typical myeloid-specific genes. Interestingly, this altered expression resembled that of the GMPs and that of BMMs derived from IRF-8(-/-) mice altogether supporting the role of PML in monopoiesis. Further, PML silencing led to reduced colony-forming capacity of bone marrow cells highlighting the dual function of PML in myelopoiesis. Last, PML overexpression only partially rescued the phenotype of IRF-8(-/-) BMMs. Together, our data show that PML is an important factor for monopoiesis and not solely for granulopoiesis. This suggests that PML-NBs respond to an incoming signal that affects the fate of GMP driving cell differentiation to granulocytes or monocytes.

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 ß-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.

CpG-C immunotherapeutic efficacy is jeopardized by ongoing exposure to stress: potential implications for clinical use.

Bi-directional influences between stress hormones and immune responses have been repeatedly documented, however, in the clinical setting they are rarely considered when immunotherapeutic approaches are used or studied in patients. As some immunotherapeutic treatments have shown great potential in animal models but have had limited success in patients, we hypothesize that ongoing psychological and physiological stress responses in patients, which do not characterize the setting of animal studies, contribute to this discrepancy. In the current study we examined the interaction between ongoing water stress and CpG-C immunotherapy to determine whether stress that precedes immunotherapy can modulate the efficacy of CpG-C immunostimulation. C57BL/6 mice were exposed to water stress or served as controls. Two hours following the commencement of the stress protocol animals were injected with CpG-C, non-CpG, or PBS, and sacrificed 1, 4 or 12h thereafter. We found that in CpG-C-treated animals stress eliminated the elevation of plasma IL-12, and synergistically elevated corticosterone levels. Furthermore, stress markedly reduced the total number of myeloid (33D1(+)), plasmacytoid (mPDCA-1(+)) and plasmacytoid-derived (33D1(+)mPDCA-1(+)) dendritic cells in CpG-C-treated animals, as well as the numbers of these cell sub-types expressing CD11b, CD80 and CD69. These changes were more dramatic in the blood than in the spleen. Overall, these findings indicate that under no-stress conditions CpG-C induces a robust immune response, which is significantly diminished when immunostimulation is attempted during ongoing stress. If these findings hold in humans, potential prophylactic treatments should be found to limit the deleterious effects of ongoing stress on the efficacy of immunotherapy.

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 (3x0.2 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-gamma); 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.