Previous studies had implicated the IFN-γ transcription factor signal transducer and activator of transcription 1 (STAT1) as a tumor suppressor. However, accumulating evidence has correlated increased STAT1 activation with increased tumor progression in multiple types of cancer, including breast cancer. Indeed, we present evidence that tumor up-regulation of STAT1 activity in human and mouse mammary tumors correlates with increasing disease progression to invasive carcinoma. A microarray analysis comparing low aggressive TM40D and highly aggressive TM40D-MB mouse mammary carcinoma cells revealed significantly higher STAT1 activity in the TM40D-MB cells. Ectopic overexpression of constitutively active STAT1 in TM40D cells promoted mobilization of myeloid-derived suppressor cells (MDSCs) and inhibition of antitumor T cells, resulting in aggressive tumor growth in tumor-transplanted, immunocompetent mice. Conversely, gene knockdown of STAT1 in the metastatic TM40D-MB cells reversed these events and attenuated tumor progression. Importantly, we demonstrate that in human breast cancer, the presence of tumor STAT1 activity and tumor-recruited CD33(+) myeloid cells correlates with increasing disease progression from ductal carcinoma in situ to invasive carcinoma. We conclude that STAT1 activity in breast cancer cells is responsible for shaping an immunosuppressive tumor microenvironment, and inhibiting STAT1 activity is a promising immune therapeutic approach.

High expression levels of cyclooxygenase 2 expression and infiltration by regulatory T cells (Tregs) are often associated with tumor progression. We have recently reported a prostaglandin E2 (PGE2)-dependent recruitment of Tregs to the tumor, suggesting that targeting specific PGE2 receptors may constitute a valuable approach to ablate the immuno-editing that occurs along with disease progression.

Extensive evidence indicates that alcohol (ethanol) consumption affects human health by altering normal physiological functions of the immune system. This study investigated the effect of a single in vivo exposure of macrophages to clinically relevant levels of ethanol (1.2 and 2.2 g/kg). Following 3 hour exposure, both doses of ethanol decreased ex vivo TNFα production by splenic and alveolar macrophages (AMs). Interestingly, the higher dose of ethanol resulted in sustained suppression of LPS-induced TNFα production at 3 and 6 hours post ethanol administration, as well as decreased IL-6 and IL-12 production after 6 hours, compared control treated groups. LPS or E. coli-stimulated production of TNFα and IL-6 by AMs were reduced at 3 hours after ethanol administration, compared to the saline treated animals. Additionally, acute in vivo (2.2 g/kg) or in vitro (50 mM) ethanol exposure transiently decreased AMs and RAW264.7 murine macrophage phagocytosis of Pseudomonas aeruginosa as early as 3 hours after initial exposure, with functional recovery by 24 hours. To investigate a single receptor pathway, we focused on Fcgamma-receptor (FcgammaR) mediated phagocytosis. In ethanol treated murine AMs and the RAW264.7 cells, phagocytosis of IgG-coated beads demonstrated a 40 and 50% reduction in actin recruitment to the site of the phagosome compared control conditions. Prior to actin polymerization, the phagocytic response requires recruitment of the adhesion molecules paxillin and vinculin, forming a structure we term "phagosomal adhesion". Ethanol did not alter paxillin phosphorylation during phagocytosis via the FcgammaR-pathway but suppressed vinculin phosphorylation, suggesting a defect in phagosomal adhesion maturation. Small GTPases are known to regulate normal actin polymerization. RAW264.7 cells exposed to NSC23766 (a Rac1 inhibitor) show 45% suppression in actin polymerization to the phagosome, regardless of ethanol exposure. This underscores the importance of normal Rac activity during FcgammaR-mediated phagocytosis. Following 15 minutes of IgG-coated bead phagocytosis, Rac activation increases by only 50% in ethanol exposed cells, compared to 175% in control conditions. Taken together, we conclude that the suppressive effects of clinically observed levels of ethanol are dose dependent, have effects that last after clearance of the ethanol from circulation, and can affect multiple macrophage functions.

BACKGROUND:The targeting of the immune system through immunotherapies to prevent tumor tolerance and immune suppression are at the front lines of breast cancer treatment and research. Human and laboratory studies have attributed breast cancer progression and metastasis to secondary organs such as the bone, to a number of factors, including elevated levels of prostaglandin E2 (PGE2) and the enzyme responsible for its production, cyclooxygenase 2 (COX2). Due to the strong connection of COX2 with immune function, we focused on understanding how variance in COX2 expression manipulates the immune profile in a syngeneic, and immune-competent, mouse model of breast cancer. Though there have been correlative findings linking elevated levels of COX2 and Tregs in other cancer models, we sought to elucidate the mechanisms by which these immuno-suppressive cells are recruited to breast tumor and the means by which they promote tumor tolerance.METHODOLOGY/PRINCIPAL FINDINGS:To elucidate the mechanisms by which exacerbated COX2 expression potentiates metastasis we genetically manipulated non-metastatic mammary tumor cells (TM40D) to over-express COX2 (TM40D-COX2). Over-expression of COX2 in this mouse breast cancer model resulted in an increase in bone metastasis (an observation that was ablated following suppression of COX2 expression) in addition to an exacerbated Treg recruitment in the primary tumor. Interestingly, other immune-suppressive leukocytes, such as myeloid derived suppressor cells, were not altered in the primary tumor or the circulation. Elevated levels of PGE2 by tumor cells can directly recruit CD4+CD25+ cells through interactions with their EP2 and/or EP4 receptors, an effect that was blocked using anti-PGE2 antibody. Furthermore, increased Treg recruitment to the primary tumor contributed to the greater levels of apoptotic CD8+ T cells in the TM40D-COX2 tumors.CONCLUSION/SIGNIFICANCE:Due to the systemic effects of COX2 inhibitors, we propose targeting specific EP receptors as therapeutic interventions to breast cancer progression.

Clinical and laboratory investigations have provided evidence that ethanol suppresses normal lung immunity. Our initial studies revealed that acute ethanol exposure results in transient suppression of phagocytosis of Pseudomonas aeruginosa by macrophages as early as 3h after initial exposure. Focusing on mechanisms by which ethanol decreases macrophage Fcγ-receptor (FcγR) phagocytosis we targeted the study on the focal adhesion and cytoskeletal elements that are necessary for phagosome progression. Ethanol inhibited macrophage phagocytosis of IgG-coated bead recruitment of actin to the site of the phagosome, dampened the phosphorylation of vinculin, but had no effect on paxillin phosphorylation suggesting a loss in “phagosomal adhesion” maturation. Moreover, our observations revealed that FcγR-phagocytosis induced Rac activation, which was increased by only 50% in ethanol exposed cells, compared to 175% in the absence of ethanol. This work is the first to show evidence of the cellular mechanisms involved in the ethanol-induced suppression of FcγR-mediated phagocytosis.

Pulmonary infections are a major cause of mortality in the critically ill burn patient. Alcohol consumption before burn increases the risk of pulmonary infection. Previously, we have shown an elevated mortality and lung pathology in mice given ethanol before burn and intratracheal infection relative to controls. Here we examine the cellular composition at 24 and 48 h in the circulation and the alveoli of infected mice given alcohol and burn. At 24 h after injury, blood neutrophils obtained from mice exposed to ethanol before burn and infection were 2-fold above those of the experimental controls (P < 0.05). By 48 h, the number of circulating neutrophils decreased and was comparable to levels found in untreated animals. Moreover, at 24 h, bronchoalveolar lavage cells obtained from all treatment groups had similar frequencies and contained 80% neutrophils regardless of treatment. In contrast, the following day, neutrophils were elevated 2-fold only in the alveoli of infected burn animals and 5-fold when ethanol preceded the injury (P < 0.05). These data were confirmed by immunofluorescence microscopy using a neutrophil-specific marker (P < 0.05). Levels of neutrophil chemoattractants, KC and macrophage inflammatory protein 2, and the cytokine, IL-1β, were 2-fold greater in the lungs of infected mice given burn, regardless of ethanol exposure, relative to infected sham injured animals (P < 0.05). Like the number of neutrophils, by the second day after injury, KC and macrophage inflammatory protein 2 remained 5-fold higher in the animals given ethanol, burn, and infection, when compared with other groups (P < 0.05). A similar pattern was seen for pulmonary levels of IL-1β (P < 0.05). Additionally, a reduction in neutrophil apoptosis was observed at the 24-h time point in infected mice exposed to ethanol and burn (P < 0.05). Targeting proinflammatory mediators in mice exposed to ethanol before burn and infection may help alleviate prolonged neutrophil accumulation in the lungs.

ABSTRACT The ability of a pathogen to evade host immunity successfully, in contrast to the host's capacity to defend itself against a foreign invader, is a complex struggle, in which eradication of infection is dictated by a robust immunologic response. Often, there are external factors that can alter the outcome by tipping the scale to benefit pathogen establishment rather than resolution by the host's defense system. These external sources, such a cigarettes, alcohol, or environmental pollutants, can negatively influence the effectiveness of the immune system's response to a pathogen. The observed suppression of immune function can be attributed to dysregulated cytokine and chemokine production, the loss of migratory potential, or the inability to phagocytose pathogens by immune cells. This review will focus on the mechanisms involved during the toxin-induced suppression of phagocytosis. The accumulated data support the importance of studying the mechanisms of phagocytosis following exposure to these factors, in that this effect alone cannot only leave the host susceptible to infection but also promote alterations in many other macrophage functions necessary for pathogen clearance and restoration of homeostasis.

Here, we studied in vitro cytokine production by splenic macrophages obtained from young and aged BALB/c wild type (WT) and IL-6 knockout (IL-6 KO) mice. Relative to macrophages obtained from young WT mice given lipopolysaccharide (LPS), those from aged WT mice had decreased production of proinflammatory cytokines. In contrast, when compared to macrophages from young IL-6 KO mice, LPS stimulation yielded higher levels of these cytokines by cells from aged IL-6 KO mice. Aging or IL-6 deficiency did not affected the percentage of F4/80+ macrophages, or the surface expression of Toll-like receptor 4 (TLR4) and components of the IL-6 receptor. Overall, our results indicate that IL-6 plays a role in regulating the age-related defects in macrophages through alteration of proinflammatory cytokines, adding to the complexity of IL-6-mediated impairment of immune cell function with increasing age.

Myosin light chain kinase and pulmonary responses after acute ethanol exposure and burn injury Anita Zahs, Luis Ramirez, Melanie D. Bird, Jerrold R. Turner and Elizabeth J. Kovacs, Cellular and Molecular Biochemistry Program, Department of Surgery, Cellular and Molecular Biochemistry Program, Loyola University Medical Center, Maywood, IL USA, Department of Surgery, Loyola University Medical Center, Maywood, IL USA, Alcohol Research Program, Loyola University Medical Center, Maywood, IL USA, Department of Pathology, University of Chicago, Chicago, IL USA.

Antigen-specific CD8D T cell response are reduced by chronic ethanol and by TLR agonists: CD8D T cell alterations Prajwal Gurung, Jodi McGill, Betty M. Young, Ruth A. Coleman, Kevin L. Legge and Robert T. Cook, Department of Pathology, Carver College of Medicine, University of Iowa, Iowa City, IA USA, Interdisciplinary Graduate Program in Immunology, Carver College of Medicine, University of Iowa, Iowa City, IA USA.

Cell-mediated immunity is extremely important for resolution of infection and for proper healing from injury. However, the cellular immune response is dysregulated following injuries such as burn and hemorrhage. Sex hormones are known to regulate immunity, and a well-documented dichotomy exists in the immune response to injury between the sexes. This disparity is caused by differences in immune cell activation, infiltration, and cytokine production during and after injury. Estrogen and testosterone can positively or negatively regulate the cellular immune response either by aiding in resolution or by compounding the morbidity and mortality. It is apparent that the hormonal dysregulation is dependent not only on the type of injury sustained but also the amount of circulating hormones. Therefore, it may be possible to design sex-specific therapies to improve immunological function and patient outcome. (C) 2008 Elsevier Inc. All rights reserved.

Extensive evidence indicates that ethanol (alcohol) has immunomodulatory properties. Many of its effects on innate immune response are dose dependent, with acute or moderate use associated with attenuated inflammatory responses, and heavy ethanol consumption linked with augmentation of inflammation. Ethanol may modify innate immunity via functional alterations of the cells of the innate immune system. Mounting evidence indicates that ethanol can diversely affect antigen recognition and intracellular signaling events, which include activation of mitogen activated protein kinases, and NFκB, mediated by Toll-like receptors, leading to altered inflammatory responses. The mechanism(s) underlying these changes may involve dose-dependent effects of ethanol on the fluidity of cell membrane, resulting in interference with the timely assembly or disassembly of lipid rafts. Ethanol could also modify cell activation by specific interactions with cell membrane molecules.

Both clinical and experimental data have linked acute ethanol exposure to increased susceptibility to infection as well as increased morbidity and mortality after injury. Macrophages play an integral role in the innate immune system and are important in priming the adaptive immune system. In this study, we investigated the effect of a single in vivo exposure of macrophages to physiologically relevant levels of ethanol (1.2 and 2.9 g/kg) followed by ex vivo stimulation with lipopolysaccharide (LPS) or bacteria. Our study confirms the work of others showing that a single administration of ethanol suppresses the production of tumor necrosis factor-alpha(TNF-alpha), interleukin-6 (IL-6), and IL-12 in response to LPS. There was no effect of ethanol on LPS induction of cytokine production at 30 min after treatment. In contrast, at 3 h, both doses of ethanol exposure decreased ex vivo TNF-alpha production by splenic and alveolar macrophages. Interestingly, the higher dose of ethanol resulted in sustained suppression of LPS-induced TNF-alpha production at 3 and 6 h after ethanol administration, as well as decreased IL-6 and IL-12 production after 6 h, as compared to control (saline-treated groups). Alveolar macrophages behaved similarly at 3 h after ethanol treatment. LPS-stimulated production of TNF-alpha and IL-6 was reduced at 3 h after ethanol administration, when compared with the saline-treated animals. Alveolar macrophages stimulated for 3 h with bacteria also showed decreased TNF-alpha and IL-6 production after harvested from mice given 2.9 g/kg ethanol for 3 h. This time point and high dose of ethanol also resulted in decreased Pseudomonas aeruginosa phagocytosis by alveolar macrophages. Taken together, we conclude that the effects of physiological levels of ethanol are dose dependent, have effects that last after ethanol is cleared from the circulation, and can affect multiple macrophage functions.

Development of biochips containing living cells for biodetection, drug screening and tissue engineering applications is limited by a lack of reconfigurable material interfaces and actuators. Here we describe a new class of nanostructured magnetizable materials created with a femtosecond laser surface etching technique that function as multiplexed magnetic field gradient concentrators. When combined with magnetic microbeads coated with cell adhesion ligands, these materials form microarrays of 'virtual' adhesive islands that can support cell attachment, resist cell traction forces and maintain cell viability. A cell death (apoptosis) response can then be actuated on command by removing the applied magnetic field, thereby causing cell retraction, rounding and detachment. This simple technology may be used to create reconfigurable interfaces that allow users to selectively discard contaminated or exhausted cellular sensor elements, and to replace them with new living cellular components for continued operation in future biomedical microdevices and biodetectors.