Cytotoxic T lymphocytes raised against Japanese encephalitis virus: effector cell phenotype, target specificity and in vitro virus clearance.

Several H-2 defined cell lines were examined for their ability to support infection and replication of Japanese encephalitis virus (JEV) before their use in in vitro and in vivo stimulation protocols for generating cytotoxic T lymphocytes (CTLs) against JEV. Among II different cell lines tested, two H-2(d) macrophage tumour lines (P388D1, RAW 264.7), an H-2(d) hybridoma (Sp2/0), an H-2K(k)D(d) neuroblastoma (Neuro 2a), and H-2(k) fibroblast cell line (L929) were found to support JEV infection and replication. These cell lines were used to generate anti-JEV CTLs by using in vivo immunization followed by in vitro stimulation of BALB/c mice. We observed that not only syngeneic and allogeneic infected cells but also JEV-infected xenogeneic cells could prime BALB/c mice for the generation of JEV-specific CTLs upon subsequent in vitro stimulation of splenocytes with JEV-infected syngeneic cells. Although infected xenogeneic cells were used for immunization, the anti-JEV effecters that were generated lysed infected syngeneic targets but not JEV-infected xenogeneic or allogeneic target cells in a 5h Cr-51 release assay. These anti-JEV effecters recognized syngeneic target cells infected with West Nile virus to a lesser extent and were shown to be Lyt-2.2(+) T cells. The results of unlabelled cold target competition studies suggested alterations in the cell surface expression of viral antigenic determinants recognized by these CTLs. We further demonstrate that the JEV-specific CTLs generated could virtually block the release of infectious virus particles from infected P388D1 and Neuro 2a cells in vitro.

Context dependent non canonical WNT signaling mediates activation of fibroblasts by transforming growth factor-beta

Actions of transforming growth factor-beta are largely context dependent. For instance, TGF-beta is growth inhibitory to epithelial cells and many tumor cell-lines while it stimulates the growth of mesenchymal cells. TGF-beta also activates fibroblast cells to a myofibroblastic phenotype. In order to understand how the responsiveness of fibroblasts to TGF-beta would change in the context of transformation, we have compared the differential gene regulation by TGF-beta in immortal fibroblasts (hFhTERT), transformed fibroblasts (hFhTERT-LTgRAS) and a human fibrosarcoma cell-line (HT1080). The analysis revealed regulation of 6735, 4163, and 3478 probe-sets by TGF-beta in hFhTERT, hFhTERT-LTgRAS and HT1080 cells respectively. Intriguingly, 5291 probe-sets were found to be either regulated in hFhTERT or hFhTERT-LTgRAS cells while 2274 probe-sets were regulated either in hFhTERT or HT1080 cells suggesting that the response of immortal hFhTERT cells to TGF-beta is vastly different compared to the response of both the transformed cells hFhTERT-LTgRAS and HT1080 to TGF-beta. Strikingly, WNT pathway showed enrichment in the hFhTERT cells in Gene Set Enrichment Analysis. Functional studies showed induction of WNT4 by TGF-beta in hFhTERT cells and TGF-beta conferred action of these cells was mediated by WNT4. While TGF-beta activated both canonical and non-canonical WNT pathways in hFhTERT cells, Erk1/2 and p38 Mitogen Activated Protein Kinase pathways were activated in hFhTERT-LTgRAS and HT1080 cells. This suggests that transformation of immortal hFhTERT cells by SV40 large T antigen and activated RAS caused a switch in their response to TGF-beta which matched with the response of HT1080 cells to TGF-beta. These data suggest context dependent activation of non-canonical signaling by TGF-beta. (C) 2015 Published by Elsevier Inc.

Glioblastoma-derived Macrophage Colony-stimulating Factor (MCSF) Induces Microglial Release of Insulin-like Growth Factor-binding Protein 1 (IGFBP1) to Promote Angiogenesis

Glioblastoma (grade IV glioma/GBM) is the most common primary adult malignant brain tumor with poor prognosis. To characterize molecular determinants of tumor-stroma interaction in GBM, we profiled 48 serum cytokines and identified macrophage colony-stimulating factor (MCSF) as one of the elevated cytokines in sera from GBM patients. Both MCSF transcript and protein were up-regulated in GBM tissue samples through a spleen tyrosine kinase (SYK)-dependent activation of the PI3K-NF kappa B pathway. Ectopic overexpression and silencing experiments revealed that glioma-secreted MCSF has no role in autocrine functions and M2 polarization of macrophages. In contrast, silencing expression of MCSF in glioma cells prevented tube formation of human umbilical vein endothelial cells elicited by the supernatant from monocytes/microglial cells treated with conditioned medium from glioma cells. Quantitative proteomics based on stable isotope labeling by amino acids in cell culture showed that glioma-derived MCSF induces changes in microglial secretome and identified insulin-like growth factor-binding protein 1 (IGFBP1) as one of the MCSF-regulated proteins secreted by microglia. Silencing IGFBP1 expression in microglial cells or its neutralization by an antibody reduced the ability of supernatants derived from microglial cells treated with glioma cell-conditioned medium to induce angiogenesis. In conclusion, this study shows up-regulation of MCSF in GBM via a SYK-PI3K-NF kappa B-dependent mechanism and identifies IGFBP1 released by microglial cells as a novel mediator of MCSF-induced angiogenesis, of potential interest for developing targeted therapy to prevent GBM progression.

Three-dimensional growth as multicellular spheroid activates the proangiogenic phenotype of colorectal carcinoma cells via LFA-1-dependent VEGF: implications on hepatic micrometastasis

Background: The recruitment of vascular stromal and endothelial cells is an early event occurring during cancer cell growth at premetastatic niches, but how the microenvironment created by the initial three-dimensional (3D) growth of cancer cells affects their angiogenesis-stimulating potential is unclear. Methods: The proangiogenic profile of CT26 murine colorectal carcinoma cells was studied in seven-day cultured 3D-spheroids of <300 mu m in diameter, produced by the hanging-drop method to mimic the microenvironment of avascular micrometastases prior to hypoxia occurrence. Results: Spheroid-derived CT26 cells increased vascular endothelial growth factor (VEGF) secretion by 70%, which in turn increased the in vitro migration of primary cultured hepatic sinusoidal endothelium (HSE) cells by 2-fold. More importantly, spheroid-derived CT26 cells increased lymphocyte function associated antigen (LFA)-1-expressing cell fraction by 3-fold; and soluble intercellular adhesion molecule (ICAM)-1, given to spheroid-cultured CT26 cells, further increased VEGF secretion by 90%, via cyclooxygenase (COX)-2-dependent mechanism. Consistent with these findings, CT26 cancer cells significantly increased LFA-1 expression in non-hypoxic avascular micrometastases at their earliest inception within hepatic lobules in vivo; and angiogenesis also markedly increased in both subcutaneous tumors and hepatic metastases produced by spheroid-derived CT26 cells. Conclusion: 3D-growth per se enriched the proangiogenic phenotype of cancer cells growing as multicellular spheroids or as subclinical hepatic micrometastases. The contribution of integrin LFA-1 to VEGF secretion via COX-2 was a micro environmental-related mechanism leading to the pro-angiogenic activation of soluble ICAM-1-activated colorectal carcinoma cells. This mechanism may represent a new target for specific therapeutic strategies designed to block colorectal cancer cell growth at a subclinical micrometastatic stage within the liver.

Endothelial cells release phenotypically and quantitatively distinct microparticles in activation and apoptosis

Background: Endothelial cells (EC) shed endothelial microparticles (EMP) in activation and apoptosis. Objectives: We compared the antigenic expression of EMP species released during activation as compared to apoptosis, in three cell lines. Methods: EC from renal and brain microvascular (MiVEC) and coronary macrovascular (MaVEC) origin were incubated with TNF-alpha to induce activation, or deprived of growth factors to induce apoptosis. Antigens expressed on EMP and EC were assayed flow cytometrically and included constitutive markers (CD31, CD51/61, CD105), inducible markers (CD54, CD62E and CD106), and annexin V binding. Results: It was found that in apoptosis, constitutive markers in EMP were markedly increased (CD31>CD105), with a concomitant decrease in expression in EC. Annexin V EC surface binding and annexin V+ EMP were more sharply increased in apoptosis than in activation. In contrast, in activation, inducible markers in EMP were markedly increased in both EMP and EC (CD62E>CD54>CD 106). Coronary MaVEC released significantly less EMP than MiVEC. Conclusion: EC release qualitatively and quantitatively distinct EMP during activation compared to apoptosis. Analysis of EMP phenotypic signatures may provide clinically useful information on the status of the endothelium. (C) 2003 Elsevier Science Ltd. All rights reserved.

B Cell Activation in Insulin Resistance and Obesity

Our group has demonstrated that inflammatory diseases such as type 2 diabetes (DM), inflammatory bowel disease (IBD), and periodontal disease (PD) are associated with altered B cell function that may contribute to disease pathogenesis. B cells were found to be highly activated with characteristics of inflammatory cells. Obesity is a pre-disease state for cardiovascular disease and type 2 diabetes and is considered a state of chronic inflammation. Therefore, we sought to better characterize B cell function and phenotype in obese patients. We demonstrate that (Toll-like receptor) TLR4 and CD36 expression by B cells is elevated in obese subjects, suggesting increased sensing of lipopolysaccharide (LPS) and other TLR ligands. These ligands may be of microbial, from translocation from a leaky gut, or host origin. To better assess microbial ligand burden and host response in the bloodstream, we measured LPS binding protein (LBP), bacterial/permeability increasing protein (BPI), and high mobility group box 1 (HMGB1). Thus far, our data demonstrate an increase in LBP in DM and obesity indicating increased responses to TLR ligands in the blood. Interestingly, B cells responded to certain types of LPS by phosphorylating extracellular-signal-regulated kinases (ERK) 1/2. A better understanding of the immunological state of obesity and the microbial and endogenous TLR ligands that may be activating B cells will help identify novel therapeutics to reduce the risk of more dangerous conditions, such as cardiovascular disease.

ROLE OF MULTIPLE TOLL-LIKE RECEPTOR ACTIVATION IN REGULATING CYTOTOXIC T CELL PRIMING TO LYMPOCYTIC CHORIOMENINGITIS VIRUS INFECTIONS

Foreign pathogens are recognized by toll-like receptors (TLR), present on various immune cells such as professional antigen-presenting cells (pAPCs). On recognition of its ligand, these receptors activate pAPCs, which may in turn influence naïve CD8+ T cell activation and affect their abilities to clear viral infection. However, how TLR ligands (TLR-L) can regulate CD8+ T cell responses have not been fully elucidated. This thesis will focus on examining how the presence of components from foreign pathogens, e.g. viral or bacterial infection, can contribute to shaping host immunity during concurrent viral infections. Since nitric oxide (NO), an innate effector immune molecule, was recently suggested to regulate proteasome activity; we sought to examine if NO can influence MHC-I antigen presentation during viral infections. The data in this section of the thesis provides evidence that combined TLR engagement can alter the presentation of certain CD8+ epitopes due to NO-induced inhibition in proteasome activity. Taken together, the data demonstrate that TLR ligation can influence the adaptive immune response due to induction of specific innate effector molecules such as NO. Next, the influence of combined TLR engagement on CD8+ T cell immunodominance hierarchies during viral infections was examined. In this section, we established that dual TLR2 and TLR3 stimulation alters immunodominance hierarchies of LCMV epitopes as a result of reduced uptake of cell-associated antigens and reduced cross-presentation of NP396 consequently suppressing NP396-specific CD8+ T cell responses. These findings are significant as they highlight a new role for TLR ligands in regulating anti-viral CD8+ T cell responses through impairing cross-presentation of cell-associated antigens depending on the type of TLR present in the environment during infections. Finally, we addressed TLR ligand induced type I interferon production and the signalling pathways that regulate them in two different mouse macrophage populations – those derived from the spleen or bone marrow. In this study, we observed that concomitant TLR2 stimulation blocked the induction of type I IFN induced by TLR4 in bone marrow-derived macrophages, but not spleen-derived macrophages in SOCS3-dependent manner. Taken together, the data presented in this thesis have defined new facets of how anti-viral responses are regulated by TLR activation, especially if multiple receptors are engaged simultaneously.

Vitamin D Receptor Modulates the Neoplastic Phenotype Through Antagonistic Growth Regulatory Signals

Vitamin D receptor (VDR) can modulate functionally antagonistic growth regulatory pathways, involving beta-catenin/E-cadherin on one hand and osteopontin (OPN) on the other. This study investigates effects of VDR ligand treatment on the balance of these discordant signals and on associated cell behavior. Treatment of Rama 37 or SW480 cells by 1 alpha,25-(OH)(2) D-3 or analogs suppressed beta-catenin/Lef-1/Tcf signaling and upregulated E-cadherin, consistent with a cancer-inhibitory action. Conversely, treatment also increased transcription of OPN that may be implicated in tumor progression. Molecular crosstalk was observed between the antagonistic VDR-dependent signals, in that beta-catenin/Lef-1/Tcf molecules modulated VDR activation of OPN. Treatment effects on cell growth were related to a constitutive balance of OPN and E-cadherin expression. No growth effects were observed in Rama 37 cells that have low OPN and high E-cadherin expression. Conversely, treatment of Rama 37 stably transfected subclones that had high OPN and/or low level E-cadherin induced small but significant increases of cell attachment to fibronectin, anchorage-independent growth or invasion. This study shows that relative expression levels of key VDR downstream genes may influence growth regulation by 1 alpha,25-(OH)(2) D-3 or analogs. These findings may be relevant to the cell- or tissue-specificity of vitamin D growth regulation. (C) 2009 Wiley-Liss, Inc.

Langerhans cells are critical in the development of atopic dermatitis-like inflammation and symptoms in mice

Genetic or vitamin D3-induced overexpression of thymic stromal lymphopoietin (TSLP) by keratinocytes results in an atopic dermatitis (AD)-like inflammatory phenotype in mice echoing the discovery of high TSLP expression in epidermis from AD patients. Although skin dendritic cells (DC) are suspected to be involved in AD, direct evidence of a pathogenetic role for skin DC in TSLP-induced skin inflammation has not yet been demonstrated. In a mouse model of AD, i.e. mice treated with the low-calcemic vitamin D3 analogue, MC903, we show that epidermal Langerhans cells (LC)-depleted mice treated with MC903 do neither develop AD-like inflammation nor increased serum IgE as compared to vitamin D3 analogue-treated control mice. Accordingly, we show that, in mice treated with MC903 or in K14-TSLP transgenic mice, expression of maturation markers by LC is increased whereas maturation of dermal DC is not altered. Moreover, only LC are responsible for the polarization of naive CD4+ T cells to a Th2 phenotype, i.e. decrease in interferon-gamma and increase in interleukin (IL)-13 production by CD4+ T cells. This effect of LC on T-lymphocytes does not require OX40-L/CD134 and is mediated by a concomitant down-regulation of IL-12 and CD70. Although it was previously stated that TSLP up-regulates the production of thymus and activation-regulated chemokine (TARC)/chemokine (C-C motif) ligand 17 (CCL17) and macrophage-derived chemokine (MDC)/CCL22 by human LC in vitro, our work shows that production of these Th2- cell attracting chemokines is increased only in keratinocytes in response to TSLP overexpression. These results demonstrate that LC are required for the development of AD in mouse models of AD involving epidermal TSLP overexpression.

CD18-dependent activation of the neutrophil NADPH oxidase during phagocytosis of Escherichia coli or Staphylococcus aureus is regulated by class III but not class I or II PI3Ks

Phagocytosis and activation of the NADPH oxidase are important mechanisms by which neutrophils and macrophages engulf and kill microbial pathogens. We investigated the role of PI3K signaling pathways in the regulation of the oxidase during phagocytosis of Staphylococcus aureus and Escherichia coli by mouse and human neutrophils, a mouse macrophage-like cell line and a human myeloid-like cell line. Phagocytosis of these bacteria was promoted by serum, independent of serum-derived antibodies, and effectively abolished in mouse neutrophils lacking the beta(2)-integrin common chain, CD18. A combination of PI3K isoform-selective inhibitors, mouse knock-outs, and RNA-interference indicated CD18-dependent activation of the oxidase was independent of class I and II PI3Ks, but substantially dependent on the single class III isoform (Vps34). Class III PI3K was responsible for the synthesis of PtdIns( 3) P on phagosomes containing either bacteria. The use of mouse neutrophils carrying an appropriate knock-in mutation indicated that PtdIns(3) P binding to the PX domain of their p40(phox) oxidase subunit is important for oxidase activation in response to both S aureus and E coli. This interaction does not, however, account for all the PI3K sensitivity of these responses, particularly the oxidase response to E coli, suggesting that additional mechanisms for PtdIns( 3) P-regulation of the oxidase must exist. ( Blood. 2008; 112: 5202-5211)

Helminth Cysteine Proteases Inhibit TRIF-dependent Activation of Macrophages via Degradation of TLR3

Helminth pathogens prepare a Th2 type immunological environment in their hosts to ensure their longevity. They achieve this by secreting molecules that not only actively drive type 2 responses but also suppress type 1 responses. Here, we show that the major cysteine proteases secreted from the helminth pathogens Fasciola hepatica (FheCL1) and Schistosoma mansoni (SmCB1) protect mice from the lethal effects of lipopolysaccharide by preventing the release of inflammatory mediators, nitric oxide, interleukin-6, tumor necrosis factor alpha, and interleukin-12, from macrophages. The proteases specifically block the MyD88-independent TRIF-dependent signaling pathway of Toll-like receptor (TLR) 4 and TLR3. Microscopical and flow cytometric studies, however, show that alteration of macrophage function by cysteine protease is not mediated by cleavage of components of the TLR4 complex on the cell surface but occurs by degradation of TLR3 within the endosome. This is the first study to describe a parasite molecule that degrades this receptor and pinpoints a novel mechanism by which helminth parasites modulate the innate immune responses of their hosts to suppress the development of Th1 responses.

Inactivation of macrophage Rab7 by Burkholderia cenocepacia

Strains of the Burkholderia cepacia complex can survive within macrophages by arresting the maturation of phagocytic vacuoles. The bacteria preclude fusion of the phagosome with lysosomes by a process that is poorly understood. Using murine macrophages, we investigated the stage at which maturation is arrested and analyzed the underlying mechanism. Vacuoles containing B. cenocepacia strain J2315, an isolate of the transmissible ET12 clone, recruited Rab5 and synthesized phosphatidylinositol-3-phosphate, indicating progression to the early phagosomal stage. Despite the fact that the B. cenocepacia-containing vacuoles rarely fused with lysosomes, they could nevertheless acquire the late phagosomal markers CD63 and Rab7. Fluorescence recovery after photobleaching and use of a probe that detects Rab7-guanosine triphosphate indicated that activation of Rab7 was impaired by B. cenocepacia, accounting at least in part for the inability of the vacuole to merge with lysosomes. The Rab7 defect was not due to excessive cholesterol accumulation and was confined to the infected vacuoles. Jointly, these experiments indicate that B. cenocepacia express virulence factors capable of interfering with Rab7 function and thereby with membrane traffic.

Elevated TRIB2 with NOTCH1 activation in paediatric/adult T-ALL

TRIB2 is a potent oncogene, elevated in a subset of human acute myeloid leukaemias (AML) with a mixed myeloid/lymphoid phenotype and NOTCH1 mutations. Although rare in AML, activating NOTCH1 mutations occur in 50% of all T cell acute lymphoblastic leukaemias (T-ALL). TRIB2 is a NOTCH1 target gene that functions in the degradation of key proteins and modulation of MAPK signalling pathways, implicated in haematopoietic cell survival and proliferation. This study showed that TRIB2 expression level is highest in the lymphoid compartment of normal haematopoietic cells, specifically in T cells. Analysis of TRIB2 expression across 16 different subtypes of human leukaemia demonstrated that TRIB2 expression was higher in ALL phenotypes versus all other phenotypes including AML, chronic lymphocytic leukaemia (CLL), myelodysplastic syndrome (MDS) and chronic myeloid leukaemia (CML). A T cell profile was distinguished by high TRIB2 expression in normal and malignant haematopoiesis. High TRIB2 expression was seen in T-ALL with normal karyotype and correlated with NOTCH signalling pathways. High TRIB2 expression correlated with NOTCH1/FBXW7 mutations in a paediatric T-ALL cohort, strongly linking NOTCH1 activation and high TRIB2 expression in paediatric T-ALL. The relationship between TRIB2 and T cell signalling pathways uniquely identifies leukaemia subtypes and will be useful in the advancement of our understanding of T cell and ALL biology.

TGFβ and CCN2/CTGF mediate actin related gene expression by differential E2F1/CREB activation

CCN2/CTGF is an established effector of TGFß driven responses in diabetic nephropathy. We have identified an interaction between CCN2 and TGFß leading to altered phenotypic differentiation and inhibited cellular migration. Here we determine the gene expression profile associated with this phenotype and define a transcriptional basis for differential actin related gene expression and cytoskeletal function.

Macrophages Control Vascular Stem/Progenitor Cell Plasticity Through Tumor Necrosis Factor-α-Mediated Nuclear Factor-κB Activation

Objective: Vascular lineage differentiation of stem/progenitor cells can contribute to both tissue repair and exacerbation of vascular diseases such as in vein grafts. The role of macrophages in controlling vascular progenitor differentiation is largely unknown and may play an important role in graft development. This study aims to identify the role of macrophages in vascular stem/progenitor cell differentiation and thereafter elucidate the mechanisms that are involved in the macrophage- mediated process.

Approach and Results: We provide in vitro evidence that macrophages can induce endothelial cell (EC) differentiation of the stem/progenitor cells while simultaneously inhibiting their smooth muscle cell differentiation. Mechanistically, both effects were mediated by macrophage-derived tumor necrosis factor-α (TNF-α) via TNF-α receptor 1 and canonical nuclear factor-κB activation. Although the overexpression of p65 enhanced EC (or attenuated smooth muscle cell) differentiation, p65 or TNF-α receptor 1 knockdown using lentiviral short hairpin RNA inhibited EC (or rescued smooth muscle cell) differentiation in response to TNF-α. Furthermore, TNF-α–mediated EC differentiation was driven by direct binding of nuclear factor-κB (p65) to specific VE-cadherin promoter sequences. Subsequent experiments using an ex vivo decellularized vessel scaffold confirmed an increase in the number of ECs and reduction in smooth muscle cell marker expression in the presence of TNF-α. The lack of TNF-α in a knockout mouse model of vein graft decreased endothelialization and significantly increased thrombosis formation.

Conclusions: Our study highlights the role of macrophages in directing vascular stem/progenitor cell lineage commitment through TNF-α–mediated TNF-α receptor 1 and nuclear factor-κB activation that is likely required for endothelial repair in vascular diseases such as vein graft.