Gustavo Salguero

Toll-like receptor 2/6 stimulation promotes angiogenesis via GM-CSF as a potential strategy for immune defense and tissue regeneration

Toll-like receptors (TLRs) are known primarily as pathogen recognition receptors of the innate immunity, initiating inflammatory pathways to organize the immune defense. More recently, an involvement of TLRs in various physiologic and pathologic processes has been reported. Because many of these processes implicate angiogenesis, we here elucidated the role of a TLR2/6-dependent pathway on angiogenesis using the TLR2/6 agonist macrophage-activating lipopeptide of 2 kDa (MALP-2), a common bacterial lipopeptide. In vivo and in vitro Matrigel assays demonstrated that MALP-2 promoted angiogenesis in a TLR2/6-dependent manner. Moreover, MALP-2 induced endothelial cell proliferation and migration and a strong secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF release in response to MALP-2 from isolated vascular segments was completely prevented when the endothelium was removed. MALP-2 containing Matrigel implants exhibited vascular structures as well as CD45(+) cells. MALP-2 induced migration of leukocytes and likewise GM-CSF release, particularly from the monocyte population. Inhibition of GM-CSF by siRNA or antibodies suppressed MALP-2-induced angiogenesis in vitro and in vivo. These results clearly identified a TLR2/6-dependent induction of angiogenesis by the bacterial lipopeptide MALP-2, which is mediated by GM-CSF. This might represent a general mechanism to enhance or restore blood flow and recruit immune cells for pathogen defense and tissue regeneration.

Ex vivo expanded hematopoietic progenitor cells improve cardiac function after myocardial infarction: Role of β-catenin transduction and cell dose

Cell-based therapy after myocardial infarction (MI) is a promising therapeutic option but the relevant cell subsets and dosage requirements are poorly defined. We hypothesized that cell therapy for myocardial infarction is improved by ex vivo expansion and high-dose transplantation of defined hematopoietic progenitor cells (HPCs). Since beta-catenin promotes self-renewal of stem cells we evaluated the therapeutic efficacy of beta-catenin-mediated ex vivo expansion of mouse HPCs in a mouse model of myocardial ischemia/reperfusion followed by intraarterial cell delivery. The impact of cell dose was determined by comparing a low-dose (LD, 5 x 10(5) cells) vs. a high-dose (HD, 1 x 10(7) cells) cell transplantation regimen of beta-catenin-HPCs. The impact of beta-catenin modification of HPCs was determined by comparing control-transduced HPCs (GFP-HPCs) vs. transgenic beta-catenin-HPCs. HD beta-catenin-HPCs significantly improved LV function and end-systolic and end-diastolic dimensions as compared to saline and LD beta-catenin-HPCs. Furthermore, while treatment with HD GFP-HPC resulted in a modest cardiac improvement the application of beta-catenin-HPCs was superior, resulting in a significant improvement in EF, FS and LVESD over saline and control GFP-HPC treatment. Although myocardial engraftment of HPCs was only transient, as determined by cell quantification after dye labeling, beta-catenin-HPC treatment significantly decreased infarct size, reduced cardiomyocyte apoptosis and increased capillary angiogenesis in vitro and in vivo. Ex vivo expanded HPCs improve cardiac function and remodeling post MI in a cell number- and beta-catenin-dependent manner.

Renovascular hypertension by two-kidney one-clip enhances endothelial progenitor cell mobilization in a p47phox-dependent manner.

Background Enhanced mechanical forces, e.g. in arterial hypertension, stimulate the formation of reactive oxygen species (ROS) by the NAD(P)H oxidase. Since bone marrow derived endothelial progenitor cells (EPCs) contribute to vascular remodeling and repair, we investigated whether renovascular hypertension stimulates EPC mobilization in a NAD(P)H oxidase-dependent manner. Methods Renovascular hypertension was induced by two-kidney one-clip (2K1C) in C57BL/6 (WT) and in mice lacking the p47phox subunit of the NAD(P)H oxidase (p47phox−/−). Results In WT, 2K1C increased blood pressure levels by 32.4 ± 4 mmHg, which was associated with a four-fold increase in circulating EPCs (Sca-1+;Flk-1+). In p47phox−/− mice, the increase in blood pressure was significantly reduced (15.1 ± 1.8 mmHg, P < 0.05) and not associated with increased EPCs. Inhibitors of the renin–angiotensin system (RAS) and nonspecific vasodilators normalized blood pressure and inhibited EPC mobilization in WT mice after 2K1C. In addition, p47phox deficiency and pharmacological ROS blockage abrogated 2K1C-induced blood pressure elevation and EPC mobilization. Stromal cell derived factor (SDF)-1 and matrix metalloproteinase (MMP)-9 activity in the bone marrow, required for EPC mobilization, were modulated in WT mice after 2K1C. In contrast, no alterations in SDF-1 and MMP-9 were observed in p47phox−/− mice. Moreover, enhanced migration of Lin− bone marrow mononuclear cells was observed when stimulated with plasma from 2K1C WT mice but not when stimulated with plasma from 2K1C p47phox−/− mice. Conclusion Enhanced mechanical stretch in renovascular hypertension induces EPC mobilization in a p47phox−/− -dependent manner, involving bone marrow SDF-1 and MMP-9 which may contribute to compensatory vascular adaptation in renovascular hypertension.

Dendritic Cell–Mediated Immune Humanization of Mice: Implications for Allogeneic and Xenogeneic Stem Cell Transplantation

De novo regeneration of immunity is a major problem after allogeneic hematopoietic stem cell transplantation (HCT). HCT modeling in severely compromised immune-deficient animals transplanted with human stem cells is currently limited because of incomplete maturation of lymphocytes and scarce adaptive responses. Dendritic cells (DC) are pivotal for the organization of lymph nodes and activation of naive T and B cells. Human DC function after HCT could be augmented with adoptively transferred donor-derived DC. In this study, we demonstrate that adoptive transfer of long-lived human DC coexpressing high levels of human IFN-α, human GM-CSF, and a clinically relevant Ag (CMV pp65 protein) promoted human lymphatic remodeling in immune-deficient NOD.Rag1−/−.IL-2rγ−/− mice transplanted with human CD34+ cells. After immunization, draining lymph nodes became replenished with terminally differentiated human follicular Th cells, plasma B cells, and memory helper and cytotoxic T cells. Human Igs against pp65 were detectable in plasma, demonstrating IgG class-switch recombination. Human T cells recovered from mice showed functional reactivity against pp65. Adoptive immunotherapy with engineered DC provides a novel strategy for de novo immune reconstitution after human HCT and a practical and effective tool for studying human lymphatic regeneration in vivo in immune deficient xenograft hosts.

Interleukin-1 Assembles a Proangiogenic Signaling Module Consisting of Caveolin-1, Tumor Necrosis Factor Receptor–Associated Factor 6, p38–Mitogen-Activated Protein Kinase (MAPK), and MAPK-Activated Protein Kinase 2 in Endothelial Cells

Objective—Interleukin-1&bgr; (IL-1&bgr;) is a major cytokine linking inflammation and angiogenesis in pathological vascular processes, such as atherosclerosis and tumor neoangiogenesis. However, signaling pathways mediating IL-1&bgr;-induced proangiogenic processes in endothelial cells (ECs) have barely been elucidated yet. Therefore, the present study investigated IL-1&bgr;-induced proangiogenic signaling in ECs. Methods and Results—IL-1&bgr; potently induced tube formation and migration of ECs. This was associated with and dependent on activation of p38–mitogen-activated protein kinase (MAPK) and MAPK-activated protein kinase 2 (MK2) as determined by pharmacological inhibition and gene silencing. Furthermore, silencing of the adaptor protein tumor necrosis factor receptor–associated factor 6 (TRAF6) (lentiviral short hairpin RNA) inhibited these IL-1&bgr;-induced processes. Moreover, IL-1&bgr; promoted translocation of TRAF6 to insoluble cellular fractions (containing membrane rafts/caveolae) and interaction of TRAF6 with caveolin-1. Accordingly, cellular cholesterol depletion (cyclodextrin) and silencing of caveolin-1 (small interfering RNA) inhibited IL-1&bgr;-induced activation of p38-MAPK and MK2, as well as IL-1&bgr;-induced tube formation and migration. Finally, silencing of TRAF6 and MK2 deficiency inhibited IL-1&bgr;-induced microvessel outgrowth in murine aortic rings ex vivo, and deficiency of MK2 or caveolin-1 significantly reduced IL-1&bgr;-induced angiogenesis in mice in vivo (Matrigel plug assay). Conclusion—IL-1&bgr; assembles a proangiogenic signaling module consisting of caveolin-1, TRAF6, p38-MAPK, and MK2 in ECs, representing a potential target to intervene into angiogenesis-dependent processes and diseases.

Elevated frequencies of leukemic myeloid and plasmacytoid dendritic cells in acute myeloid leukemia with the FLT3 internal tandem duplication

Some 30% of acute myeloid leukemia (AML) patients display an internal tandem duplication (ITD) mutation in the FMS-like tyrosine kinase 3 (FLT3) gene. FLT3-ITDs are known to drive hematopoietic stem cells towards FLT3 ligand independent growth, but the effects on dendritic cell (DC) differentiation during leukemogenesis are not clear. We compared the frequency of cells with immunophenotype of myeloid DC (mDC: Lin−, HLA-DR+, CD11c+, CD86+) and plasmacytoid DC (pDC: Lin−, HLA-DR+, CD123+, CD86+) in diagnostic samples of 47 FLT3-ITD− and 40 FLT3-ITD+ AML patients. The majority of ITD+ AML samples showed high frequencies of mDCs or pDCs, with significantly decreased HLA-DR expression compared with DCs detectable in ITD− AML samples. Interestingly, mDCs and pDCs sorted out from ITD+ AML samples contained the ITD insert revealing their leukemic origin and, upon ex vivo culture with cytokines, they acquired DC morphology. Notably, mDC/pDCs were detectable concurrently with single lineage mDCs and pDCs in all ITD+ AML (n = 11) and ITD− AML (n = 12) samples analyzed for mixed lineage DCs (Lin−, HLA-DR+, CD11c+, CD123+). ITD+ AML mDCs/pDCs could be only partially activated with CD40L and CpG for production of IFN-α, TNF-α, and IL-1α, which may affect the anti-leukemia immune surveillance in the course of disease progression.

Digitoxin elicits anti-inflammatory and vasoprotective properties in endothelial cells: Therapeutic implications for the treatment of atherosclerosis?

Pro-inflammatory processes initiated in the endothelium represent a crucial step in the pathogenesis of inflammatory cardiovascular disease, such as atherosclerosis. Recent observations pointed to potential anti-inflammatory properties of the cardiac glycoside digitoxin. Therefore, the present study investigated potential anti-inflammatory and vasoprotective properties of digitoxin as well as the underlying signaling pathways affected in endothelial cells (EC). Digitoxin, employing therapeutical concentrations used in patients (3-30nM), potently inhibited the IL-1beta-induced expression of MCP-1 and VCAM-1 in EC and the capacity of corresponding cell culture supernatants on monocyte migration as well as monocyte adhesion to endothelial monolayers, respectively. Furthermore, digitoxin prevented the IL-1beta-induced activation of p44/42-MAPK and NF-kappaB without affecting activation of JNK and p38-MAPK. Inhibition of NF-kappaB signaling but not p44/42-MAPK mimicked the observed inhibitory effects of digitoxin on MCP-1 expression and monocyte migration. Moreover, digitoxin inhibited NF-kappaB signaling at the level of TAK-1/IKK. Additionally, digitoxin prevented TNF-alpha-induced apoptosis in EC accompanied by activation of Akt. Blockade of PI-3-kinase, activator of Akt, prevented the anti-apoptotic properties of digitoxin and impaired its inhibitory action on NF-kappaB signaling and MCP-1 expression. Finally, digitoxin activated endothelial NO-synthase, which was blocked by inhibition of PI-3-kinase, Ca(2+)/Calmodulin-dependent-proteinkinase-II and chelation of intracellular calcium. Digitoxin elicits anti-inflammatory and vasoprotective properties by blocking NF-kappaB and activating PI-3-kinase/Akt signaling as well as Ca(2+)/Calmodulin-dependent-proteinkinase-II in EC. These observations indicate a potential therapeutical application of digitoxin in the treatment of cardiovascular diseases, such as atherosclerosis.

Identity, potency, in vivo viability, and scaling up production of lentiviral vector-induced dendritic cells for melanoma immunotherapy

SmartDCs (Self-differentiated Myeloid-derived Antigen-presenting-cells Reactive against Tumors) consist of highly viable dendritic cells (DCs) induced to differentiate with lentiviral vectors (LVs) after an overnight ex vivo transduction. Tricistronic vectors co-expressing cytokines (granulocyte-macrophage-colony stimulating factor [GM-CSF], interleukin [IL]-4) and a melanoma antigen (tyrosine related protein 2 [TRP2]) were used to transduce mouse bone marrow cells or human monocytes. Sixteen hours after transduction, the cells were dispensed in aliquots and cryopreserved for identity, potency, and safety analyses. Thawed SmartDCs readily differentiated into highly viable cells with a DC immunophenotype. Prime/boost subcutaneous administration of 1×10(6) thawed murine SmartDCs into C57BL/6 mice resulted into TRP2-specific CD8(+) T-cell responses and protection against lethal melanoma challenge. Human SmartDC-TRP2 generated with monocytes obtained from melanoma patients secreted endogenous cytokines associated with DC activation and stimulated TRP2-specific autologous T-cell expansion in vitro. Thawed human SmartDCs injected subcutaneously in NOD.Rag1(-/-).IL2rγ(-/-) mice maintained DC characteristics and viability for 1 month in vivo and did not cause any signs of pathology. For development of good manufacturing practices, CD14(+) monocytes selected by magnetic-activated cell separation were transduced in a closed bag system (multiplicity of infection of 5), washed, and cryopreserved. Fifty percent of the monocytes used for transduction were recovered for cryopreservation. Thawed SmartDCs produced in two independent runs expressed the endogenous cytokines GM-CSF and IL-4, and the resulting homogeneous SmartDCs that self-differentiated in vitro contained approximately 1.5-3.0 copies of integrated LVs per cell. Thus, this method facilitates logistics, standardization, and high recovery for the generation of viable genetically reprogrammed DCs for clinical applications.

Lentiviral vectors for induction of self-differentiation and conditional ablation of dendritic cells

Development of lentiviral vectors (LVs) in the field of immunotherapy and immune regeneration will strongly rely on biosafety of the gene transfer. We demonstrated previously the feasibility of ex vivo genetic programming of mouse bone marrow precursors with LVs encoding granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), which induced autonomous differentiation of long-lived dendritic cells (DCs), referred to as self-differentiated myeloid-derived antigen-presenting-cells reactive against tumors (SMART-DCs). Here, LV biosafety was enhanced by using a DC-restricted and physiological promoter, the major histocompatibility complex (MHC) II promoter, and including co-expression of the herpes simplex virus-thymidine kinase (sr39HSV-TK) conditional suicide gene. Tricistronic vectors co-expressing sr39HSV-TK, GM-CSF and IL-4 transcriptionally regulated by the MHCII promoter or the ubiquitous cytomegalovirus (CMV) promoter were compared. Despite the different gene transfer effects, such as the kinetics, levels of transgene expression and persistency of integrated vector copies, both vectors induced highly viable SMART-DCs, which persisted for at least 70 days in vivo and could be ablated with the pro-drug Ganciclovir (GCV). SMART-DCs co-expressing the tyrosine-related protein 2 melanoma antigen administered subcutaneously generated antigen-specific, anti-melanoma protective and therapeutic responses in the mouse B16 melanoma model. GCV administration after immunotherapy did not abrogate DC vaccination efficacy. This demonstrates proof-of-principle of genetically programmed DCs that can be ablated pharmacologically.

Engineered dendritic cells from cord blood and adult blood accelerate effector T cell immune reconstitution against HCMV

Human cytomegalovirus (HCMV) harmfully impacts survival after peripheral blood hematopoietic stem cell transplantation (PB-HSCT). Delayed immune reconstitution after cord blood (CB)-HSCT leads to even higher HCMV-related morbidity and mortality. Towards a feasible dendritic cell therapy to accelerate de novo immunity against HCMV, we validated a tricistronic integrase-defective lentiviral vector (coexpressing GM-CSF, IFN-α, and HCMV pp65 antigen) capable to directly induce self-differentiation of PB and CB monocytes into dendritic cells processing pp65 (“SmyleDCpp65”). In vitro, SmyleDCpp65 resisted HCMV infection, activated CD4+ and CD8+ T cells and expanded functional pp65-specific memory cytotoxic T lymphocytes (CTLs). CD34+ cells obtained from PB and CB were transplanted into irradiated NOD.Rag1−/−.IL2γc−/− mice. Donor-derived SmyleDCpp65 administration after PB-HSCT stimulated peripheral immune effects: lymph node remodeling, expansion of polyclonal effector memory CD8+ T cells in blood, spleen and bone marrow, and pp65-reactive CTL and IgG responses. SmyleDCpp65 administration after CB-HSCT significantly stimulated thymopoiesis. Expanded frequencies of CD4+/CD8+ T cell precursors containing increased levels of T-cell receptor excision circles in thymus correlated with peripheral expansion of effector memory CTL responses against pp65. The comparative in vivo modeling for PB and CB-HSCT provided dynamic and spatial information regarding human T and B cell reconstitution. In vivo potency supports future clinical development of SmyleDCpp65.