ABSTRACT
Platelet factor 4 (PF4) is a pleiotropic inflammatory chemokine, which has been implicated in various inflammatory disorders including liver fibrosis. However, its role in acute liver diseases has not yet been elucidated. Here we describe an unexpected, anti-inflammatory role of PF4. Serum concentrations of PF4 were measured in patients and mice with acute liver diseases. Acute liver injury in mice was induced either by carbon tetrachloride or by D-galactosamine hydrochloride and lipopolysaccharide. Serum levels of PF4 were decreased in patients and mice with acute liver diseases. PF4-/- mice displayed increased liver damage in both models compared to control which was associated with increased apoptosis of hepatocytes and an enhanced pro-inflammatory response of liver macrophages. In this experimental setting, PF4-/- mice were unable to generate activated Protein C (APC), a protein with anti-inflammatory activities on monocytes/macrophages. In vitro, PF4 limited the activation of liver resident macrophages. Hence, the systemic application of PF4 led to a strong amelioration of experimental liver injury. Along with reduced liver injury, PF4 improved the severity of the pro-inflammatory response of liver macrophages and induced increased levels of APC. PF4 has a yet unidentified direct anti-inflammatory effect in two models of acute liver injury. Thus, attenuation of acute liver injury by systemic administration of PF4 might offer a novel therapeutic approach for acute liver diseases.
ABSTRACT
Herpes Simplex Virus type 1 (HSV-1), a neurotropic pathogen widespread in human population, infects the enteric nervous system (ENS) in humans and rodents and causes intestinal neuromuscular dysfunction in rats. Although infiltration of inflammatory cells in the myenteric plexus and neurodegeneration of enteric nerves are common features of patients suffering from functional intestinal disorders, the proof of a pathogenic link with HSV-1 is still unsettled mainly because the underlying mechanisms are largely unknown. In this study we demonstrated that following intragastrical administration HSV-1 infects neurons within the myenteric plexus resulting in functional and structural alterations of the ENS. By infecting mice with HSV-1 replication-defective strain we revealed that gastrointestinal neuromuscular anomalies were however independent of viral replication. Indeed, enteric neurons exposed to UV-inactivated HSV-1 produced monocyte chemoattractant protein-1 (MCP-1/CCL2) to recruit activated macrophages in the longitudinal muscle myenteric plexus. Infiltrating macrophages produced reactive oxygen and nitrogen species and directly harmed enteric neurons resulting in gastrointestinal dysmotility. In HSV-1 infected mice intestinal neuromuscular dysfunctions were ameliorated by in vivo administration of (i) liposomes containing dichloromethylene bisphosphonic acid (clodronate) to deplete tissue macrophages, (ii) CCR2 chemokine receptor antagonist RS504393 to block the CCL2/CCR2 pathway, (iii) Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) and AR-C 102222 to quench production of nitrogen reactive species produced via iNOS. Overall these data demonstrate that HSV-1 infection makes enteric neurons recruit macrophages via production of a specific chemoattractant factor. The resulting inflammatory reaction is mandatory for intestinal dysmotility. These findings provide insights into the neuro-immune communication that occurs in the ENS following HSV-1 infection and allow recognition of an original pathophysiologic mechanism underlying gastrointestinal diseases as well as identification of novel therapeutic targets.
Subject(s)
Enteric Nervous System/drug effects , Enteric Nervous System/virology , Gastrointestinal Motility/drug effects , Herpes Simplex/metabolism , Herpesvirus 1, Human/pathogenicity , Macrophages/metabolism , Neurons/drug effects , Adaptive Immunity , Animals , Arginine/analogs & derivatives , Arginine/metabolism , Chemokine CCL2/metabolism , Clodronic Acid , Disease Models, Animal , Enteric Nervous System/metabolism , Enteric Nervous System/pathology , Herpes Simplex/immunology , Herpes Simplex/pathology , Herpes Simplex/virology , Ileum/immunology , Ileum/pathology , Ileum/virology , Inflammation/metabolism , Liposomes/metabolism , Male , Mice , Mice, Inbred C57BL , Myenteric Plexus/drug effects , Myenteric Plexus/metabolism , Myenteric Plexus/pathology , Myenteric Plexus/virology , NG-Nitroarginine Methyl Ester/metabolism , Neurons/virology , Rats , Reactive Nitrogen Species/metabolism , Reactive Nitrogen Species/toxicity , Reactive Oxygen Species/metabolism , Reactive Oxygen Species/toxicity , Receptors, Chemokine , Virus Internalization , Virus ReplicationABSTRACT
Here we report methods of preparation for liposome formulations containing lipophilic drugs. In contrast to the encapsulation of water soluble compounds into the entrapped aqueous volume of a liposome, drugs with lipophilic properties are incorporated into the phospholipid bilayer membrane. Water-soluble molecules, for example cytotoxic or antiviral nucleosides can be transformed into lipophilic compounds by attachment of long alkyl chains, allowing their stable incorporation into liposome membranes and taking advantage of the high loading capacity lipid bilayers provide for lipophilic molecules. We created a new class of cytotoxic drugs by chemical transformation of the hydrophilic drugs cytosine-arabinoside (ara-C), 5-fluoro-deoxyuridine (5-FdU), and ethinylcytidine (ETC) into lipophilic compounds and their formulation in liposomes.The concept of chemical modification of water-soluble molecules by attachment of long alkyl chains and their stable incorporation into liposome bilayer membranes represent a very promising method for the development of new drugs not only for the treatment of tumors or infections but also for many other diseases.
Subject(s)
Hydrophobic and Hydrophilic Interactions , Liposomes/chemistry , Pharmaceutical Preparations/chemistry , Dialysis , Filtration , PressureABSTRACT
Respiratory immunization is an attractive way to generate systemic and mucosal protective memory responses that are required for preventing mucosally transmitted infections. However, the molecular and cellular mechanisms for controlling memory T cell responses remain incompletely understood. In this study, we investigated the role of respiratory macrophage (MΦ) in regulating CD4 T cell responses to recombinant adenovirus-based (rAd) vaccines. We demonstrated that rAd intranasal (i.n.) vaccination induced migration and accumulation of respiratory MΦ and circulatory monocytes in the mediastinal lymph nodes and lung parenchyma. Under the influence of respiratory MΦ CD4 T cells exhibited slow proliferation kinetics and an increased tendency of generating central memory, as opposed to effector memory, CD4 T cell responses in vitro and in vivo. Correspondingly, depletion of MΦ using clodronate-containing liposome prior to i.n. immunization significantly enhanced CD4 T cell proliferation and increased the frequency of CD4 memory T cells in the airway lumen, demonstrating that MΦ initially serve as a negative regulator in limiting generation of mucosal tissue-resident memory CD4 T cells. However, clodronate-containing liposome delivery following i.n. immunization markedly reduced the frequencies of memory CD4 T cells in the airway lumen and spleen, indicating that respiratory MΦ and potentially circulating monocytes are critically required for maintaining long-term memory CD4 T cells. Collectively, our data demonstrate that rAd-induced mucosal CD4 T memory responses are regulated by respiratory MΦ and/or monocytes at multiple stages.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Lung/immunology , Macrophages, Alveolar/immunology , Vaccines, Synthetic/immunology , Adenoviridae/genetics , Animals , Cell Communication , Cell Movement , Cell Proliferation , Cells, Cultured , Clodronic Acid/administration & dosage , Female , Immunity, Mucosal , Immunologic Memory , Macrophages, Alveolar/drug effects , Mice , Mice, Inbred BALB C , Mice, Transgenic , VaccinationABSTRACT
Advanced stages of cancer often involve multiple tumors in different locations in the body. These tumors are associated with a microenvironment that can influence tumor responses to immunotherapy. Whether tumors and their disparate microenvironment can interact together at distance in a multiple tumor setting, through a form of cross-talk, and affect their responses to immunotherapy has never been described. Our study investigated the cross-talk between two tumors with disparate microenvironments in a mouse model. We demonstrated that immunosuppressive visceral tumors could influence distant subcutaneous (SC) tumors to render them resistant to immunotherapy. We observed distinct modifications in the SC tumor microenvironment following cross-talk with kidney tumors that exhibit a type-2 macrophage-related immunosuppressive microenvironment. Indeed, when a concomitant kidney tumor was present in the mouse, the SC tumors were highly infiltrated with M2 macrophages and had a reduced T cell and NK cell effector immune profile. Finally, blocking the M2-associated chemokine CCL2 or depleting macrophages, significantly improved the effect of immunotherapy on growth of SC tumors in the presence of concomitant kidney tumors. This work emphasizes the potential negative influence that a tumor, with a strong immunosuppressive microenvironment, can exert on distant tumors that would normally be treatment-responsive. This report may lead to a new vision of the prioritization in the treatment of advanced metastatic cancer.
ABSTRACT
Liposomes and liposome-derived nanovesicles such as archaeosomes and virosomes have become important carrier systems in vaccine development and the interest for liposome-based vaccines has markedly increased. A key advantage of liposomes, archaeosomes and virosomes in general, and liposome-based vaccine delivery systems in particular, is their versatility and plasticity. Liposome composition and preparation can be chosen to achieve desired features such as selection of lipid, charge, size, size distribution, entrapment and location of antigens or adjuvants. Depending on the chemical properties, water-soluble antigens (proteins, peptides, nucleic acids, carbohydrates, haptens) are entrapped within the aqueous inner space of liposomes, whereas lipophilic compounds (lipopeptides, antigens, adjuvants, linker molecules) are intercalated into the lipid bilayer and antigens or adjuvants can be attached to the liposome surface either by adsorption or stable chemical linking. Coformulations containing different types of antigens or adjuvants can be combined with the parameters mentioned to tailor liposomal vaccines for individual applications. Special emphasis is given in this review to cationic adjuvant liposome vaccine formulations. Examples of vaccines made with CAF01, an adjuvant composed of the synthetic immune-stimulating mycobacterial cordfactor glycolipid trehalose dibehenate as immunomodulator and the cationic membrane forming molecule dimethyl dioctadecylammonium are presented. Other vaccines such as cationic liposome-DNA complexes (CLDCs) and other adjuvants like muramyl dipeptide, monophosphoryl lipid A and listeriolysin O are mentioned as well. The field of liposomes and liposome-based vaccines is vast. Therefore, this review concentrates on recent and relevant studies emphasizing current reports dealing with the most studied antigens and adjuvants, and pertinent examples of vaccines. Studies on liposome-based veterinary vaccines and experimental therapeutic cancer vaccines are also summarized.
ABSTRACT
The vascular disrupting agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a murine agonist of the stimulator of interferon genes (STING), appears to target the tumor vasculature primarily as a result of stimulating pro-inflammatory cytokine production from tumor-associated macrophages (TAMs). Since there were relatively few reports of DMXAA effects in genetically-engineered mutant mice (GEMM), and models of non-small cell lung cancer (NSCLC) in particular, we examined both the effectiveness and macrophage dependence of DMXAA in various NSCLC models. The DMXAA responses of primary adenocarcinomas in K-rasLA1/+ transgenic mice, as well as syngeneic subcutaneous and metastatic tumors, generated by a p53R172HΔg/+; K-rasLA1/+ NSCLC line (344SQ-ELuc), were assessed both by in vivo bioluminescence imaging as well as by histopathology. Macrophage-dependence of DMXAA effects was explored by clodronate liposome-mediated TAM depletion. Furthermore, a comparison of the vascular structure between subcutaneous tumors and metastases was carried out using micro-computed tomography (micro-CT). Interestingly, in contrast to the characteristic hemorrhagic necrosis produced by DMXAA in 344SQ-ELuc subcutaneous tumors, this agent failed to cause hemorrhagic necrosis of either 344SQ-ELuc-derived metastases or autochthonous K-rasLA1/+ NSCLCs. In addition, we found that clodronate liposome-mediated depletion of TAMs in 344SQ-ELuc subcutaneous tumors led to non-hemorrhagic necrosis due to tumor feeding-vessel occlusion. Since NSCLC were comprised exclusively of TAMs with anti-inflammatory M2-like phenotype, the ability of DMXAA to re-educate M2-polarized macrophages was examined. Using various macrophage phenotypic markers, we found that the STING agonists, DMXAA and the non-canonical endogenous cyclic dinucleotide, 2'3'-cGAMP, were both capable of re-educating M2 cells towards an M1 phenotype. Our findings demonstrate that the choice of preclinical model and the anatomical site of a tumor can determine the vascular disrupting effectiveness of DMXAA, and they also support the idea of STING agonists having therapeutic utility as TAM repolarizing agents.
Subject(s)
Carcinoma, Non-Small-Cell Lung/blood supply , Carcinoma, Non-Small-Cell Lung/drug therapy , Cell Polarity/drug effects , Macrophages/pathology , Membrane Proteins/agonists , Neovascularization, Pathologic/drug therapy , Nucleotides, Cyclic/therapeutic use , Xanthones/therapeutic use , Adenocarcinoma/blood supply , Adenocarcinoma/drug therapy , Animals , Carcinoma, Non-Small-Cell Lung/pathology , Cell Membrane Permeability/drug effects , Clodronic Acid/pharmacology , Clodronic Acid/therapeutic use , Humans , Inflammation/pathology , Liposomes/chemistry , Lung Neoplasms/blood supply , Lung Neoplasms/drug therapy , Macrophages/drug effects , Male , Mice, Transgenic , Necrosis , Neovascularization, Pathologic/pathology , Nucleotides, Cyclic/pharmacology , Phenotype , Subcutaneous Tissue/drug effects , Tumor Suppressor Protein p53/metabolism , Xanthones/pharmacology , Xenograft Model Antitumor Assays , ras Proteins/metabolismABSTRACT
The spread of multidrug-resistant microorganisms globally has created an urgent need for novel therapeutic strategies to combat urinary tract infections (UTIs). Immunomodulatory therapy may provide benefit, as treatment of mice with dexamethasone during acute UTI improved outcome by reducing the development of chronic cystitis, which predisposes to recurrent infection. Here we discovered soluble biomarkers engaged in myeloid cell development and chemotaxis that were predictive of future UTI recurrence when elevated in the sera of young women with UTI. Translation of these findings revealed that temperance of the neutrophil response early during UTI, and specifically disruption of bladder epithelial transmigration of neutrophils by inhibition of cyclooxygenase-2, protected mice against chronic and recurrent cystitis. Further, proteomics identified bladder epithelial remodeling consequent to chronic infection that enhances sensitivity to neutrophil damage. Thus, cyclooxygenase-2 expression during acute UTI is a critical molecular trigger determining disease outcome and drugs targeting cyclooxygenase-2 could prevent recurrent UTI.
ABSTRACT
Chronic viral infections lead to CD8(+) T cell exhaustion, characterized by impaired cytokine secretion. Presence of the immune-regulatory cytokine IL-10 promotes chronicity of Lymphocytic Choriomeningitis Virus (LCMV) Clone 13 infection, while absence of IL-10/IL-10R signaling early during infection results in viral clearance and higher percentages and numbers of antiviral, cytokine producing T cells. IL-10 is produced by several cell types during LCMV infection but it is currently unclear which cellular sources are responsible for induction of viral chronicity. Here, we demonstrate that although dendritic cells produce IL-10 and overall IL-10 mRNA levels decrease significantly in absence of CD11c(+) cells, absence of IL-10 produced by CD11c(+) cells failed to improve the LCMV-specific T cell response and control of LCMV infection. Similarly, NK cell specific IL-10 deficiency had no positive impact on the LCMV-specific T cell response or viral control, even though high percentages of NK cells produced IL-10 at early time points after infection. Interestingly, we found markedly improved T cell responses and clearance of normally chronic LCMV Clone 13 infection when either myeloid cells or T cells lacked IL-10 production and mice depleted of monocytes/macrophages or CD4(+) T cells exhibited reduced overall levels of IL-10 mRNA. These data suggest that the decision whether LCMV infection becomes chronic or can be cleared critically depends on early CD4(+) T cell and monocyte/macrophage produced IL-10.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Interleukin-10/immunology , Lymphocytic Choriomeningitis/immunology , Lymphocytic choriomeningitis virus/immunology , Macrophages/immunology , Animals , CD4-Positive T-Lymphocytes/pathology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/pathology , Chronic Disease , Interleukin-10/genetics , Lymphocytic Choriomeningitis/genetics , Lymphocytic Choriomeningitis/pathology , Lymphocytic choriomeningitis virus/genetics , Macrophages/pathology , Mice , Mice, Knockout , Receptors, Interleukin-10/genetics , Receptors, Interleukin-10/immunologyABSTRACT
Macrophages are the primary effector cells of the innate immune system and are also activated in response to tissue injury. The avian cochlea contains a population of resident macrophages, but the precise function of those cells is not known. The present study characterized the behavior of cochlear macrophages after aminoglycoside ototoxicity and also examined the possible role of macrophages in sensory regeneration. We found that the undamaged chick cochlea contains a large resting population of macrophages that reside in the hyaline cell region, immediately outside the abneural (inferior) border of the sensory epithelium. Following ototoxic injury, macrophages appear to migrate out of the hyaline cell region and towards the basilar membrane, congregating immediately below the lesioned sensory epithelium. In order to determine whether recruited macrophages contribute to the regeneration of sensory receptors, we quantified supporting cell proliferation and hair cell recovery after the elimination of most resident macrophages via application of liposomally-encapsulated clodronate. Examination of macrophage-depleted specimens at two days following ototoxic injury revealed no deficits in hair cell clearance, when compared to normal controls. In addition, we found that elimination of macrophages did not affect either regenerative proliferation of supporting cells or the production of replacement hair cells. However, we did find that macrophage-depleted cochleae contained reduced numbers of proliferative mesothelial cells below the basilar membrane. Our data suggest that macrophages are not required for normal debris clearance and regeneration, but that they may play a role in the maintenance of the basilar membrane.
Subject(s)
Basilar Membrane , Cochlea , Hair Cells, Auditory , Macrophages , Aminoglycosides , Animals , Basilar Membrane/injuries , Basilar Membrane/physiology , Chickens/physiology , Cochlea/cytology , Cochlea/physiology , Hair Cells, Auditory/cytology , Hair Cells, Auditory/physiology , Immunity, Innate/physiology , Macrophages/cytology , Macrophages/physiology , Nerve Regeneration/physiologyABSTRACT
Interplay between macrophages and dendritic cells in the processing and presentation of bacterial antigens for T-cell immune responses remains poorly understood. Using a Listeria monocytogenes (Lm) infection model, we demonstrate that dendritic cells (DCs) require the support of macrophages to elicit protective immunity against Lm infection. DCs themselves were inefficient at taking up Lm but capable of taking up microparticles (MPs) released by Lm-infected macrophages. These MPs transferred Lm antigens to DCs, allowing DCs to present Lm antigen to effector T cells. MP-mediated Lm antigen transfer required MHC class I participation, since MHC class I deficiency in macrophages resulted in a significant reduction of T-cell activation. Moreover, the vaccination of mice with MPs from Lm-infected macrophages produced strong protective immunity against Lm infection. We here identify an intrinsic antigen transfer program between macrophages and DCs during Lm infection, and emphasize that macrophages also play an essential role in DC-elicited Lm-specific T-cell responses.
Subject(s)
Cell-Derived Microparticles/metabolism , Dendritic Cells/immunology , Immunity/immunology , Listeria monocytogenes/immunology , Listeriosis/immunology , Macrophages/metabolism , Macrophages/microbiology , Actin Cytoskeleton/metabolism , Animals , Antigen Presentation/immunology , Antigens, Bacterial/immunology , Cell Differentiation/immunology , Cell Proliferation , Cells, Cultured , Dendritic Cells/cytology , Listeriosis/microbiology , Listeriosis/prevention & control , Macrophages/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Subcellular Fractions/metabolism , T-Lymphocytes/cytology , T-Lymphocytes/immunologyABSTRACT
Tumor necrosis factor receptor (TNFR) signaling may result in survival, apoptosis or programmed necrosis. The latter is called necroptosis if the receptor-interacting protein 1 (RIP1) inhibitor necrostatin-1 (Nec-1) or genetic knockout of RIP3 prevents it. In the lethal mouse model of TNFα-mediated shock, addition of the pan-caspase inhibitor zVAD-fmk (zVAD) accelerates time to death. Here, we demonstrate that RIP3-deficient mice are protected markedly from TNFα-mediated shock in the presence and absence of caspase inhibition. We further show that the fusion protein TAT-crmA, previously demonstrated to inhibit apoptosis, also prevents necroptosis in L929, HT29 and FADD-deficient Jurkat cells. In contrast to RIP3-deficient mice, blocking necroptosis by Nec-1 or TAT-crmA did not protect from TNFα/zVAD-mediated shock, but further accelerated time to death. Even in the absence of caspase inhibition, Nec-1 application led to similar kinetics. Depletion of macrophages, natural killer (NK) cells, granulocytes or genetic deficiency for T lymphocytes did not influence this model. Because RIP3-deficient mice are known to be protected from cerulein-induced pancreatitis (CIP), we applied Nec-1 and TAT-crmA in this model and demonstrated the deterioration of pancreatic damage upon addition of these substances. These data highlight the importance of separating genetic RIP3 deficiency from RIP1 inhibition by Nec-1 application in vivo and challenge the current definition of necroptosis.
Subject(s)
Apoptosis , Receptor-Interacting Protein Serine-Threonine Kinases/antagonists & inhibitors , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Shock/genetics , Shock/pathology , Tumor Necrosis Factor-alpha/toxicity , Animals , Apoptosis/drug effects , Caspase Inhibitors , Caspases/metabolism , Cell Line, Tumor , Female , Gene Products, tat/genetics , Humans , Imidazoles/pharmacology , Indoles/pharmacology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Mice, Nude , Necrosis , Pancreatitis/chemically induced , Pancreatitis/genetics , Pancreatitis/pathology , Recombinant Fusion Proteins/pharmacology , Serpins/genetics , Shock/chemically induced , Shock/mortality , Viral Proteins/geneticsABSTRACT
BACKGROUND: Several stromal cell subtypes including macrophages contribute to tumor progression by inducing epithelial-mesenchymal transition (EMT) at the invasive front, a mechanism also linked to metastasis. Tumor associated macrophages (TAM) reside mainly at the invasive front but they also infiltrate tumors and in this process they mainly assume a tumor promoting phenotype. In this study, we asked if TAMs also regulate EMT intratumorally. We found that TAMs through TGF-ß signaling and activation of the ß-catenin pathway can induce EMT in intratumoral cancer cells. METHODS: We depleted macrophages in F9-teratocarcinoma bearing mice using clodronate-liposomes and analyzed the tumors for correlations between gene and protein expression of EMT-associated and macrophage markers. The functional relationship between TAMs and EMT was characterized in vitro in the murine F9 and mammary gland NMuMG cells, using a conditioned medium culture approach. The clinical relevance of our findings was evaluated on a tissue microarray cohort representing 491 patients with non-small cell lung cancer (NSCLC). RESULTS: Gene expression analysis of F9-teratocarcinomas revealed a positive correlation between TAM-densities and mesenchymal marker expression. Moreover, immunohistochemistry showed that TAMs cluster with EMT phenotype cells in the tumors. In vitro, long term exposure of F9-and NMuMG-cells to macrophage-conditioned medium led to decreased expression of the epithelial adhesion protein E-cadherin, activation of the EMT-mediating ß-catenin pathway, increased expression of mesenchymal markers and an invasive phenotype. In a candidate based screen, macrophage-derived TGF-ß was identified as the main inducer of this EMT-associated phenotype. Lastly, immunohistochemical analysis of NSCLC patient samples identified a positive correlation between intratumoral macrophage densities, EMT markers, intraepithelial TGF-ß levels and tumor grade. CONCLUSIONS: Data presented here identify a novel role for macrophages in EMT-promoted tumor progression. The observation that TAMs cluster with intra-epithelial fibroblastoid cells suggests that the role of macrophages in tumor-EMT extends beyond the invasive front. As macrophage infiltration and pronounced EMT tumor phenotype correlate with increased grade in NSCLC patients, we propose that TAMs also promote tumor progression by inducing EMT locally in tumors.
Subject(s)
Epithelial-Mesenchymal Transition , Macrophages/pathology , Teratocarcinoma/pathology , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Animals , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Disease Progression , Epithelial-Mesenchymal Transition/drug effects , Humans , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Macrophages/physiology , Mice , Neoplasm Invasiveness/pathology , Teratocarcinoma/genetics , Teratocarcinoma/metabolism , Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta/pharmacologyABSTRACT
Epoxyeicosatrienoic acids (EETs) are small molecules produced by cytochrome P450 epoxygenases. They are lipid mediators that act as autocrine or paracrine factors to regulate inflammation and vascular tone. As a result, drugs that raise EET levels are in clinical trials for the treatment of hypertension and many other diseases. However, despite their pleiotropic effects on cells, little is known about the role of these epoxyeicosanoids in cancer. Here, using genetic and pharmacological manipulation of endogenous EET levels, we demonstrate that EETs are critical for primary tumor growth and metastasis in a variety of mouse models of cancer. Remarkably, we found that EETs stimulated extensive multiorgan metastasis and escape from tumor dormancy in several tumor models. This systemic metastasis was not caused by excessive primary tumor growth but depended on endothelium-derived EETs at the site of metastasis. Administration of synthetic EETs recapitulated these results, while EET antagonists suppressed tumor growth and metastasis, demonstrating in vivo that pharmacological modulation of EETs can affect cancer growth. Furthermore, inhibitors of soluble epoxide hydrolase (sEH), the enzyme that metabolizes EETs, elevated endogenous EET levels and promoted primary tumor growth and metastasis. Thus, our data indicate a central role for EETs in tumorigenesis, offering a mechanistic link between lipid signaling and cancer and emphasizing the critical importance of considering possible effects of EET-modulating drugs on cancer.
Subject(s)
Eicosanoids/metabolism , Neoplasm Metastasis/physiopathology , Neoplasms, Experimental/metabolism , Animals , Aryl Hydrocarbon Hydroxylases/genetics , Aryl Hydrocarbon Hydroxylases/metabolism , Cytochrome P-450 CYP2C8 , Cytochrome P-450 CYP2J2 , Cytochrome P-450 Enzyme Inhibitors , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Endothelium, Vascular/metabolism , Epoxy Compounds/metabolism , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Neoplasm Metastasis/pathology , Neoplasms, Experimental/blood supply , Neoplasms, Experimental/pathology , Neovascularization, Pathologic , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Vascular Endothelial Growth Factor A/metabolismABSTRACT
S100A7/psoriasin, a member of the epidermal differentiation complex, is widely overexpressed in invasive estrogen receptor (ER)α-negative breast cancers. However, it has not been established whether S100A7 contributes to breast cancer growth or metastasis. Here, we report the consequences of its expression on inflammatory pathways that impact breast cancer growth. Overexpression of human S100A7 or its murine homologue mS100a7a15 enhanced cell proliferation and upregulated various proinflammatory molecules in ERα-negative breast cancer cells. To examine in vivo effects, we generated mice with an inducible form of mS100a7a15 (MMTV-mS100a7a15 mice). Orthotopic implantation of MVT-1 breast tumor cells into the mammary glands of these mice enhanced tumor growth and metastasis. Compared with uninduced transgenic control mice, the mammary glands of mice where mS100a7a15 was induced exhibited increased ductal hyperplasia and expression of molecules involved in proliferation, signaling, tissue remodeling, and macrophage recruitment. Furthermore, tumors and lung tissues obtained from these mice showed further increases in prometastatic gene expression and recruitment of tumor-associated macrophages (TAM). Notably, in vivo depletion of TAM inhibited the effects of mS100a7a15 induction on tumor growth and angiogenesis. Furthermore, introduction of soluble hS100A7 or mS100a7a15 enhanced chemotaxis of macrophages via activation of RAGE receptors. In summary, our work used a powerful new model system to show that S100A7 enhances breast tumor growth and metastasis by activating proinflammatory and metastatic pathways.
Subject(s)
Inflammation/genetics , Mammary Neoplasms, Experimental/genetics , S100 Proteins/genetics , Signal Transduction/genetics , Animals , Blotting, Western , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation , Chemokines/genetics , Chemokines/metabolism , Chemotaxis/genetics , Cytokines/genetics , Cytokines/metabolism , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Inflammation/metabolism , Inflammation/pathology , Macrophages/metabolism , Macrophages/pathology , Male , Mammary Neoplasms, Experimental/metabolism , Mammary Neoplasms, Experimental/pathology , Mice , Mice, Transgenic , Neoplasm Metastasis , Oligonucleotide Array Sequence Analysis , Receptor for Advanced Glycation End Products , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolism , Reverse Transcriptase Polymerase Chain Reaction , S100 Calcium Binding Protein A7 , S100 Proteins/metabolism , Up-Regulation , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Amino-bisphosphonates (alendronate, pamidronate) were covalently linked in a three step synthesis, with protected and triazolylated derivatives of therapeutically used nucleoside analogs (5-FdU, araC, AZT) by substitution of their triazolyl residue. From the deprotected and chromatographically purified reaction mixtures N4-[alkyl-(hydroxyphosphono) phosphonate]-cytidine combining two differently cytotoxic functions were obtained. This new family of bisphosphonates (BPs) contains as novelty an alkyl side chain with a cytotoxic nucleoside. The BPs moiety allows for a high binding to hydroxyapatite which is a prerequisite for bone targeting of the drugs. In vitro binding of 5-FdU-alendronate (5-FdU-ale) to hydroxyapatite showed a sixfold increased binding of these BPs as compared to 5-FdU. Exploratory cytotoxic properties of 5-FdU-ale were tested on a panel of human tumor cell lines resulting in growth inhibition ranging between 5% and 38%. The determination of IC50-concentrations of the conjugate in Lewis lung carcinoma and murine macrophages showed an incubation time dependent growth inhibition with higher sensitivity towards the tumor cells. We assume that the antimetabolite-BPs can be cleaved into different active metabolites that may exert cytotoxic and other therapeutic effects. However, the underlying mechanisms of these promising new antimetabolite-BPs conjugates remain to be evaluated in future experiments.
Subject(s)
Alendronate/analogs & derivatives , Antimetabolites, Antineoplastic/chemistry , Bone Density Conservation Agents/chemistry , Cytidine/chemistry , Diphosphonates/chemistry , Fluorouracil/analogs & derivatives , Alendronate/chemical synthesis , Alendronate/chemistry , Alendronate/toxicity , Animals , Antimetabolites, Antineoplastic/chemical synthesis , Antimetabolites, Antineoplastic/toxicity , Arabinofuranosyluracil/chemistry , Bone and Bones/drug effects , Cell Line, Tumor , Drug Screening Assays, Antitumor , Durapatite/chemistry , Fluorouracil/chemical synthesis , Fluorouracil/chemistry , Fluorouracil/toxicity , Humans , Macrophages/drug effects , Mice , Pamidronate , Uridine/analogs & derivatives , Uridine/chemistry , Zidovudine/chemistryABSTRACT
BACKGROUND: Most bladder cancer patients experience lymphatic metastasis in the course of disease progression, yet the relationship between lymphangiogenesis and lymphatic metastasis is not well known. The aim of this study is to elucidate underlying mechanisms of how expanded lymphatic vessels and tumor microenvironment interacts each other and to find effective therapeutic options to inhibit lymphatic metastasis. RESULTS: The orthotopic urinary bladder cancer (OUBC) model was generated by intravesical injection of MBT-2 cell lines. We investigated the angiogenesis, lymphangiogenesis, and CD11b+/CD68+ tumor-associated macrophages (TAM) by using immunofluorescence staining. OUBC displayed a profound lymphangiogenesis and massive infiltration of TAM in primary tumor and lymphatic metastasis in lymph nodes. TAM flocked near lymphatic vessels and express higher levels of VEGF-C/D than CD11b- cells. Because VEGFR-3 was highly expressed in lymphatic vascular endothelial cells, TAM could assist lymphangiogenesis by paracrine manner in bladder tumor. VEGFR-3 expressing adenovirus was administered to block VEGF-C/D signaling pathway and clodronate liposome was used to deplete TAM. The blockade of VEGF-C/D with soluble VEGF receptor-3 markedly inhibited lymphangiogenesis and lymphatic metastasis in OUBC. In addition, the depletion of TAM with clodronate liposome exerted similar effects on OUBC. CONCLUSION: VEGF-C/D are the main factors of lymphangiogenesis and lymphatic metastasis in bladder cancer. Moreover, TAM plays an important role in these processes by producing VEGF-C/D. The inhibition of lymphangiogenesis could provide another therapeutic target to inhibit lymphatic metastasis and recurrence in patients with invasive bladder cancer.
Subject(s)
Lymphangiogenesis , Macrophages/metabolism , Urinary Bladder Neoplasms/metabolism , Vascular Endothelial Growth Factor Receptor-3/metabolism , Animals , Antigens, CD/genetics , Antigens, CD/metabolism , Antigens, Differentiation, Myelomonocytic/genetics , Antigens, Differentiation, Myelomonocytic/metabolism , CD11b Antigen/genetics , CD11b Antigen/metabolism , Cell Line, Tumor , Female , Flow Cytometry , Gene Expression Regulation, Neoplastic , Immunohistochemistry , Lymph Nodes/metabolism , Lymph Nodes/pathology , Lymphatic Metastasis , Macrophages/pathology , Mice , Mice, Inbred C3H , Neovascularization, Pathologic , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Solubility , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/pathology , Vascular Endothelial Growth Factor C/genetics , Vascular Endothelial Growth Factor C/metabolism , Vascular Endothelial Growth Factor D/genetics , Vascular Endothelial Growth Factor D/metabolism , Vascular Endothelial Growth Factor Receptor-3/geneticsABSTRACT
Interleukin-12 (IL-12) can promote tumor regression via activation of multiple lymphocytic and myelocytic effectors. Whereas the cytotoxic mechanisms employed by T/NK/NKT cells in IL-12-mediated tumor kill are well defined, the antitumor role of macrophage-produced cytotoxic metabolites has been more controversial. To this end, we investigated the specific role of nitric oxide (NO), a major macrophage effector molecule, in post-IL-12 tumor regression. Analysis of tumors following a single intratumoral injection of slow-release IL-12 microspheres showed an IFNγ-dependent sevenfold increase in inducible nitric oxide synthase (iNOS) expression within 48 h. Flow cytometric analysis of tumor-resident leukocytes and in vivo depletion studies identified CD11b(+) F4/80(+) Gr1(lo) macrophages as the primary source of iNOS. Blocking of post-therapy iNOS activity with N-nitro-L: -arginine methyl ester (L-NAME) dramatically enhanced tumor suppression revealing the inhibitory effect of NO on IL-12-driven antitumor immunity. Superior tumor regression in mice receiving combination treatment was associated with enhanced survival and proliferation of activated tumor-resident CD8+ T-effector/memory cells (Tem). These findings demonstrate that macrophage-produced NO negatively regulates the antitumor activity of IL-12 via its detrimental effects on CD8+ T cells and identify L-NAME as a potent adjuvant in IL-12 therapy of cancer.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Interleukin-12/immunology , Interleukin-12/pharmacology , Nitric Oxide/immunology , Animals , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/pathology , Female , Flow Cytometry , Humans , Injections, Intralesional , Interleukin-12/antagonists & inhibitors , Lymphocyte Activation/drug effects , Lymphocytes, Tumor-Infiltrating/drug effects , Lymphocytes, Tumor-Infiltrating/immunology , Macrophages/drug effects , Macrophages/enzymology , Macrophages/immunology , Male , Mice , Mice, Inbred BALB C , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide/metabolism , Nitric Oxide Synthase Type II/immunology , Nitric Oxide Synthase Type II/metabolism , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Clinical and experimental evidence indicates that inflammatory processes contribute to the pathophysiology of epilepsy, but underlying mechanisms remain mostly unknown. Using immunohistochemistry for CD45 (common leukocyte antigen) and CD3 (T-lymphocytes), we show here microglial activation and infiltration of leukocytes in sclerotic tissue from patients with mesial temporal lobe epilepsy (TLE), as well as in a model of TLE (intrahippocampal kainic acid injection), characterized by spontaneous, nonconvulsive focal seizures. Using specific markers of lymphocytes, microglia, macrophages, and neutrophils in kainate-treated mice, we investigated with pharmacological and genetic approaches the contribution of innate and adaptive immunity to kainate-induced inflammation and neurodegeneration. Furthermore, we used EEG analysis in mutant mice lacking specific subsets of lymphocytes to explore the significance of inflammatory processes for epileptogenesis. Blood-brain barrier disruption and neurodegeneration in the kainate-lesioned hippocampus were accompanied by sustained ICAM-1 upregulation, microglial cell activation, and infiltration of CD3(+) T-cells. Moreover, macrophage infiltration was observed, selectively in the dentate gyrus where prominent granule cell dispersion was evident. Unexpectedly, depletion of peripheral macrophages by systemic clodronate liposome administration affected granule cell survival. Neurodegeneration was aggravated in kainate-lesioned mice lacking T- and B-cells (RAG1-knock-out), because of delayed invasion by Gr-1(+) neutrophils. Most strikingly, these mutant mice exhibited early onset of spontaneous recurrent seizures, suggesting a strong impact of immune-mediated responses on network excitability. Together, the concerted action of adaptive and innate immunity triggered locally by intrahippocampal kainate injection contributes seizure-suppressant and neuroprotective effects, shedding new light on neuroimmune interactions in temporal lobe epilepsy.
Subject(s)
Brain/pathology , Epilepsy, Temporal Lobe/etiology , Leukocytes/pathology , Microglia/pathology , Adaptive Immunity , Analysis of Variance , Animals , Biomarkers/metabolism , Brain/metabolism , Disease Models, Animal , Electroencephalography , Epilepsy, Temporal Lobe/chemically induced , Epilepsy, Temporal Lobe/metabolism , Epilepsy, Temporal Lobe/pathology , Humans , Immunity, Innate , Immunohistochemistry , Intercellular Adhesion Molecule-1/metabolism , Kainic Acid , Leukocyte Common Antigens/metabolism , Leukocyte Count , Leukocytes/metabolism , Male , Mice , Mice, Knockout , Microglia/metabolism , Neurons/metabolism , Neurons/pathology , Sclerosis/metabolism , Sclerosis/pathologyABSTRACT
Prodrugs can have the advantage over parent drugs in increased activation and cellular uptake. The multidrug ETC-L-FdUrd and the duplex drug ETC-FdUrd are composed of two different monophosphate-nucleosides, 5-fluoro-2'deoxyuridine (FdUrd) and ethynylcytidine (ETC), coupled via a glycerolipid or phosphodiester, respectively. The aim of the study was to determine cytotoxicity levels and mode of drug cleavage. Moreover, we determined whether a liposomal formulation of ETC-L-FdUrd would improve cytotoxic activity and/or cleavage. Drug effects/cleavage were studied with standard radioactivity assays, HPLC and LC-MS/MS in FM3A/0 mammary cancer cells and their FdUrd resistant variants FM3A/TK(-). ETC-FdUrd was active (IC(50) of 2.2 and 79 nM) in FM3A/0 and TK(-) cells, respectively. ETC-L-FdUrd was less active (IC(50): 7 nM in FM3A/0 vs 4500 nM in FM3A/TK(-)). Although the liposomal formulation was less active than ETC-L-FdUrd in FM3A/0 cells (IC(50):19.3 nM), resistance due to thymidine kinase (TK) deficiency was greatly reduced. The prodrugs inhibited thymidylate synthase (TS) in FM3A/0 cells (80-90%), but to a lower extent in FM3A/TK(-) (10-50%). FdUMP was hardly detected in FM3A/TK(-) cells. Inhibition of the transporters and nucleotidases/phosphatases resulted in a reduction of cytotoxicity of ETC-FdUrd, indicating that this drug was cleaved outside the cells to the monophosphates, which was verified by the presence of FdUrd and ETC in the medium. ETC-L-FdUrd and the liposomal formulation were neither affected by transporter nor nucleotidase/phosphatase inhibition, indicating circumvention of active transporters. In vivo, ETC-FdUrd and ETC-L-FdURd were orally active. ETC nucleotides accumulated in both tumor and liver tissues. These formulations seem to be effective when a lipophilic linker is used combined with a liposomal formulation.