Felicita Baratelli

Prostaglandin E2 Induces FOXP3 Gene Expression and T Regulatory Cell Function in Human CD4+ T Cells

Naturally occurring CD4+CD25+ regulatory T cells (T reg) are pivotal in suppressing immune responses and maintaining tolerance. The identification of molecules controlling T reg differentiation and function is important in understanding host immune responses in malignancy and autoimmunity. In this study we show that PGE2 enhances the in vitro inhibitory function of human purified CD4+CD25+ T reg cells. Moreover, PGE2 induces a regulatory phenotype in CD4+CD25− T cells. PGE2-treated T cell-mediated inhibition of anti-CD3-stimulated lymphocyte proliferation did not require cell contact. Phenotypic analysis revealed that PGE2 diminished CD25 expression in both CD4+CD25dim T cells and CD4+CD25bright T reg cells. PGE2 exposure induced the T reg cell-specific transcription factor forkhead/winged helix transcription factor gene (FOXP3) in CD4+CD25− T cells and significantly up-regulated its expression in CD4+CD25+ T reg cells. Similarly, 24-h incubation with supernatants from cyclooxygenase-2-overexpressing lung cancer cells that secrete high levels of PGE2 significantly induced FOXP3 in CD4+CD25− T cells. Finally, PGE2 up-regulated FOXP3 at both mRNA and protein levels and enhanced FOXP3 promoter activity. This is the first report indicating that PGE2 can modulate FOXP3 expression and T reg function in human lymphocytes.

Tumor Cyclooxygenase-2/Prostaglandin E2–Dependent Promotion of FOXP3 Expression and CD4+CD25+ T Regulatory Cell Activities in Lung Cancer

Cyclooxygenase (COX)-2 and its product prostaglandin (PG) E2 underlie an immunosuppressive network that is important in the pathogenesis of non-small cell lung cancer. CD4+ CD25+ T regulatory (Treg) cells play an important role in maintenance of immunologic self-tolerance. CD4+ CD25+ Treg cell activities increase in lung cancer and appear to play a role in suppressing antitumor immune responses. Definition of the pathways controlling Treg cell activities will enhance our understanding of limitation of the host antitumor immune responses. Tumor-derived COX-2/PGE2 induced expression of the Treg cell-specific transcription factor, Foxp3, and increased Treg cell activity. Assessment of E-prostanoid (EP) receptor requirements revealed that PGE2-mediated induction of Treg cell Foxp3 gene expression was significantly reduced in the absence of the EP4 receptor and ablated in the absence of the EP2 receptor expression. In vivo, COX-2 inhibition reduced Treg cell frequency and activity, attenuated Foxp3 expression in tumor-infiltrating lymphocytes, and decreased tumor burden. Transfer of Treg cells or administration of PGE2 to mice receiving COX-2 inhibitors reversed these effects. We conclude that inhibition of COX-2/PGE2 suppresses Treg cell activity and enhances antitumor responses.

Inflammation and lung carcinogenesis: applying findings in prevention and treatment

Lung carcinogenesis is a complex process requiring the acquisition of genetic mutations that confer the malignant phenotype as well as epigenetic alterations that may be manipulated in the course of therapy. Inflammatory signals in the lung cancer microenvironment can promote apoptosis resistance, proliferation, invasion, metastasis, and secretion of proangiogenic and immunosuppressive factors. Here, we discuss several prototypical inflammatory mediators controlling the malignant phenotype in lung cancer. Investigation into the detailed molecular mechanisms underlying the tumor-promoting effects of inflammation in lung cancer has revealed novel potential drug targets. Cytokines, growth factors and small-molecule inflammatory mediators released in the developing tumor microenvironment pave the way for epithelial–mesenchymal transition, the shift from a polarized, epithelial phenotype to a highly motile mesenchymal phenotype that becomes dysregulated during tumor invasion. Inflammatory mediators within the tumor microenvironment are derived from neoplastic cells as well as stromal and inflammatory cells; thus, lung cancer develops in a host environment in which the deregulated inflammatory response promotes tumor progression. Inflammation-related metabolic and catabolic enzymes (prostaglandin E2 synthase, prostaglandin I2 synthase and 15-hydroxyprostaglandin dehydrogenase), cell-surface receptors (E-type prostaglandin receptors) and transcription factors (ZEB1, SNAIL, PPARs, STATs and NF-κB) are differentially expressed in lung cancer cells compared with normal lung epithelial cells and, thus, may contribute to tumor initiation and progression. These newly discovered molecular mechanisms in the pathogenesis of lung cancer provide novel opportunities for targeted therapy and prevention in lung cancer.

Prostaglandin E2-Dependent Enhancement of Tissue Inhibitors of Metalloproteinases-1 Production Limits Dendritic Cell Migration through Extracellular Matrix

Dendritic cell (DC) migration is crucial for the initiation of immune responses. The balance between metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) has been shown to modulate DC migration. PGE2, which is overproduced in a wide variety of human malignancies, has been implicated in MMP and TIMP regulation in various cells, including monocytes. In the present study, we hypothesized that tumor-derived PGE2 would affect DC migratory capacity through the extracellular matrix (ECM) by altering MMP and TIMP balance. Treatment of monocyte-derived immature DC with exogenous PGE2 induced TIMP-1 secretion but not MMP-9 production and was correlated with reduced DC migration through ECM. Because recombinant TIMP-1 replicated PGE2 inhibition of DC migration while anti-TIMP-1 neutralizing Ab reversed it, we conclude that PGE2-mediated induction of TIMP-1 was responsible for the reduced migration of PGE2-treated DC. Similarly, DC cultured for 48 h in supernatants from cyclooxygenase-2 overexpressing lung cancer cells that secrete high levels of PGE2, exhibited decreased migration through ECM. Finally, analysis of E prostanoid receptor expression and their selective inhibition revealed that the enhanced TIMP-1 secretion in PGE2-treated DC was mediated predominantly by the E prostanoid receptor 2. These findings indicate that PGE2-dependent enhancement of TIMP-1 production causes reduced migration of DC through ECM.

Novel CCL21-Vault Nanocapsule Intratumoral Delivery Inhibits Lung Cancer Growth

Background Based on our preclinical findings, we are assessing the efficacy of intratumoral injection of dendritic cells (DC) transduced with an adenoviral vector expressing the secondary lymphoid chemokine (CCL21) gene (Ad-CCL21-DC) in a phase I trial in advanced non-small cell lung cancer (NSCLC). While this approach shows immune enhancement, the preparation of autologous DC for CCL21 genetic modification is cumbersome, expensive and time consuming. We are evaluating a non-DC based approach which utilizes vault nanoparticles for intratumoral CCL21 delivery to mediate antitumor activity in lung cancer. Principal Findings Here we describe that vault nanocapsule platform for CCL21 delivery elicits antitumor activity with inhibition of lung cancer growth. Vault nanocapsule packaged CCL21 (CCL21-vaults) demonstrated functional activity in chemotactic and antigen presenting activity assays. Recombinant vaults impacted chemotactic migration of T cells and this effect was predominantly CCL21 dependent as CCL21 neutralization abrogated the CCL21 mediated enhancement in chemotaxis. Intratumoral administration of CCL21-vaults in mice bearing lung cancer enhanced leukocytic infiltrates (CXCR3+T, CCR7+T, IFNγ+T lymphocytes, DEC205+ DC), inhibited lung cancer tumor growth and reduced the frequencies of immune suppressive cells [myeloid derived suppressor cells (MDSC), T regulatory cells (Treg), IL-10 T cells]. CCL21-vaults induced systemic antitumor responses by augmenting splenic T cell lytic activity against parental tumor cells. Significance This study demonstrates that the vault nanocapsule can efficiently deliver CCL21 to sustain antitumor activity and inhibit lung cancer growth. The vault nanocapsule can serve as an “off the shelf” approach to deliver antitumor cytokines to treat a broad range of malignancies.

Modulation of Pulmonary Leukotriene B4 Production by Cyclooxygenase-2 Inhibitors and Lipopolysaccharide

Purpose: Emerging data continue to link carcinogenesis to inflammatory events involving the eicosanoid metabolic pathways. We therefore evaluated the effects of cyclooxygenase (COX)-2 inhibition on leukotriene (LT) B4 synthesis in the lungs of active smokers, as part of a pilot lung cancer chemoprevention study with celecoxib (Celebrex), an oral COX-2 inhibitor. Experimental Design: Bronchoalveolar lavage was performed before celecoxib treatment and after 1 month of celecoxib treatment to recover alveolar macrophages (AMs) and lining fluid for study. After harvest, AMs were immediately stimulated in vitro with the calcium ionophore A23187. AMs obtained from smokers before treatment and from ex-smoker control subjects were also cultured overnight with SC58236, a selective COX-2 inhibitor, with or without lipopolysaccharide stimulation. Results: Treatment with oral celecoxib only modestly increased LTB4 levels in bronchoalveolar lavage, without increasing the mRNA transcription of 5-lipoxygenase (5-LOX) or 5-LOX-activating protein in AMs, whereas the acute calcium ionophore-stimulated LTB4 production from smokers’ AMs was markedly increased by 10.6-fold. In addition, smokers’ AMs were twice as responsive in producing LTB4 when exposed to lipopolysaccharide compared with ex-smokers’ AMs. Concomitant COX-2 inhibition with SC58236, however, did not significantly impact these changes, whereas the 5-LOX inhibitor Zileuton blocked the generation of LTB4 in a dose-responsive manner. Finally, cycloheximide increased the production of LTB4 under all conditions, suggesting a shunting phenomenon and/or the presence of pathway inhibitors. Conclusions: Our findings suggest that whereas oral celecoxib is capable of modulating LTB4 production in the lung microenvironment, under physiologic conditions, this effect is probably not functionally significant.

PGE2 confers survivin‐dependent apoptosis resistance in human monocyte‐derived dendritic cells

Control of apoptosis is fundamental for dendritic cell (DC) homeostasis. Numerous factors maintain DC viability throughout their lifespan, including inhibitor of apoptosis proteins. Among them, survivin is overexpressed in many human malignancies, but its physiological function in normal cells has not been fully delineated. Prostaglandin E2 (PGE2), also overproduced in several malignancies, has shown to induce proapoptotic and antiapoptotic effects in different cell types, including immune cells. In DC, PGE2 predominantly affects maturation and modulates immune functions. Here, we show that exposure of monocyte‐derived DC to PGE2 (10−5 M) for 72 h significantly increased DC survivin mRNA and protein expression. In contrast, DC, matured with lipopolysaccharide or tumor necrosis factor α, did not reveal survivin induction in response to PGE2. Following exposure to apoptotic stimuli, DC treated with PGE2 exhibited an overall increased viability compared with control DC, and this effect was correlated inversely with caspase‐3 activation. Moreover, PGE2‐treated, survivin‐deficient DC demonstrated reduced viability in response to apoptotic stimuli. Further analysis indicated that PGE2 induced DC survivin expression in an E prostanoid (EP)2/EP4 receptor and phosphatidylinositol‐3 kinase‐dependent manner. These findings suggest that PGE2‐dependent regulation of survivin is important in modulating apoptosis resistance in human DC.

IL-27 inhibits epithelial-mesenchymal transition and angiogenic factor production in a STAT1-dominant pathway in human non-small cell lung cancer

BackgroundInterleukin-27 signaling is mediated by the JAK-STAT pathway via activation of STAT1 and STAT3, which have tumor suppressive and oncogenic activities, respectively. Epithelial–mesenchymal transition (EMT) and angiogenesis are key processes in carcinogenesis. Although IL-27 has been shown to have potent anti-tumor activity in various cancer models, the role of IL-27 in EMT and angiogenesis is poorly understood. In this study, we investigated the role of IL-27 in regulating EMT and angiogenesis through modulation of the STAT pathways in human non-small cell lung carcinoma (NSCLC) cells.MethodsSTAT activation following IL-27 exposure was measured in human NSCLC cell lines. Expression of epithelial (E-cadherin, γ-catenin) and mesenchymal (N-cadherin, vimentin) markers were assessed by Western blot analysis. Production of pro-angiogenic factors (VEGF, IL-8/CXCL8, CXCL5) were examined by ELISA. Cell motility was examined by an in vitro scratch and transwell migration assays. Selective inhibitors of STAT1 (STAT1 siRNAs) and STAT3 (Stattic) were used to determine whether both STAT1 and STAT3 are required for IL-27 mediated inhibition of EMT and secretion of angiogenic factors.ResultsOur results demonstrate that IL-27 stimulation in NSCLC resulted in 1) STAT1 and STAT3 activation in a JAK-dependent manner, 2) development of epithelial phenotypes, including a decrease in the expression of a transcriptional repressor for E-cadherin (SNAIL), and mesenchymal marker (vimentin) with a reciprocal increase in the expression of epithelial markers, 3) inhibition of cell migration, and 4) reduced production of pro-angiogenic factors. STAT1 inhibition in IL-27–treated cells reversed the IL-27 effect with resultant increased expression of Snail, vimentin and the pro-angiogenic factors. The inhibition of STAT3 activation had no effect on the development of the epithelial phenotype.ConclusionIL-27 induces mesenchymal to epithelial transition and inhibits the production of pro-angiogenic factors in a STAT1–dominant pathway. These findings highlight the importance of STAT1 in repressing lung carcinogenesis and describe a new anti-tumor mechanism of IL-27.

Pre-clinical characterization of GMP grade CCL21-gene modified dendritic cells for application in a phase I trial in Non-Small Cell Lung Cancer

BackgroundOur previous studies have demonstrated that transduction of human dendritic cells (DC) with adenovirus encoding secondary lymphoid chemokine, CCL21, led to secretion of biologically active CCL21 without altering DC phenotype or viability. In addition, intratumoral injections of CCL21-transduced DC into established murine lung tumors resulted in complete regression and protective anti-tumor immunity. These results have provided the rationale to generate a clinical grade adenoviral vector encoding CCL-21 for ex vivo transduction of human DC in order to assess intratumoral administration in late stage human lung cancer.MethodsIn the current study, human monocyte-derived DC were differentiated by exposure to GM-CSF and IL-4 from cryopreserved mononuclear cells obtained from healthy volunteers. Transduction with clinical grade adenoviral vector encoding CCL21 (1167 viral particles per cell) resulted in secretion of CCL21 protein.ResultsCCL21 protein production from transduced DC was detected in supernatants (24–72 hours, 3.5–6.7 ng/4–5 × 106 cells). DC transduced with the clinical grade adenoviral vector were > 88% viable (n = 16), conserved their phenotype and maintained integral biological activities including dextran uptake, production of immunostimulatory cytokines/chemokines and antigen presentation. Furthermore, supernatant from CCL21-DC induced the chemotaxis of T2 cells in vitro.ConclusionViable and biologically active clinical grade CCL21 gene-modified DC can be generated from cryopreserved PBMC.

Overexpression of CCL-21/Secondary Lymphoid Tissue Chemokine in Human Dendritic Cells Augments Chemotactic Activities for Lymphocytes and Antigen Presenting Cells

BackgroundEx vivo generated dendritic cells (DC) genetically modified to express secondary lymphoid tissue chemokine (CCL-21/SLC) have been shown to stimulate potent antitumor responses in murine models. When injected intratumorally, CCL-21 colocalizes DC and lymphocyte effector cells at the tumor site. This may improve tumor antigen presentation and T cell activation by utilizing the tumor as an in vivo source of antigen for DC. In order to develop DC-based cancer therapies for intratumoral injection that could promote tumor antigen uptake and presentation in situ, we constructed and characterized an adenoviral vector that expresses human CCL-21 (AdCCL-21).ResultsHuman monocyte derived DC were cultured in GM-CSF and IL-4 for 6 days. Following AdCCL-21 transduction, CCL-21 protein production was assessed by ELISA on day 8. DC transduced with AdCCL-21 at multiplicities of infection (MOIs) of 50:1 or 100:1 produced up to 210 ± 9 ng/ml and 278 ± 6.5 ng/ml /106 cells/48 hours, respectively. Following transduction, an immature DC phenotype was maintained and an upregulation of the costimulatory molecule, CD86 was noted. In addition, supernatant from AdCCL-21-DC caused significant chemotaxis of peripheral blood lymphocytes and mature DC.ConclusionsThese studies demonstrate that AdCCL-21-DC generate functional levels of CCL-21 without adversely altering DC phenotype. These findings strengthen the rationale for further investigation of AdCCL-21-DC as a DC-based therapy in cancer treatment.