Regulation Of Cell Trafficking Research Articles

Bitter Taste Receptor Agonist Denatonium Inhibits Stemness Characteristics in Hematopoietic Stem/Progenitor Cells.

Bone marrow microenvironmental stimuli profoundly impact hematopoietic stem cell fate and biology. As G protein-coupled receptors, the bitter taste receptors (TAS2Rs) are key in transmitting extracellular stimuli into an intracellular response, within the oral cavity but also in extraoral tissues. Their expression in the bone marrow (BM)-derived cells suggests their involvement in sensing the BM microenvironmental fluctuation. In the present study, we demonstrated that umbilical cord blood (UCB)-derived CD34+ cells express fully functional TAS2Rs along with the signal transduction cascade components and their activation by the prototypical agonist, denatonium benzoate, significantly modulated genes involved in stemness maintenance and regulation of cell trafficking. The activation of these specific pathways was confirmed in functional in vitro experiments. Denatonium exposure exerted an antiproliferative effect on UCB-derived CD34+ cells, mainly affecting the most undifferentiated progenitor frequency. It also reduced their clonogenicity and repopulating potential in vitro. In addition, the TAS2R signaling activation impaired the UCB-derived CD34+ cell trafficking, mainly reducing the migration toward the chemoattractant agent CXCL12 and modulating the expression of the adhesion molecules CD62L, CD49d, and CD29. In conclusion, our results in UCB-derived CD34+ cells expand the observation of TAS2R expression in the setting of BM-resident cells and shed light on the role of TAS2Rs in the extrinsic regulation of hematopoietic stem cell functions.

Intracellular complement (complosome) is expressed in hematopoietic stem/progenitor cells (HSPCs) and regulates cell trafficking, metabolism and proliferation in an intracrine Nlrp3 inflammasome-dependent manner

Intracellular complement (complosome) is expressed in hematopoietic stem/progenitor cells (HSPCs) and regulates cell trafficking, metabolism and proliferation in an intracrine Nlrp3 inflammasome-dependent manner

Effect of physical and geometrical stimuli on microvascular dynamics.

The microvasculature is not only a passive organ composed of pipes responsible for the delivery of blood to tissues. It is a highly dynamic organ that actively regulates the passage of nutrients and immune cells to the tissue. More specific, this regulation is bi-directional, where immune cells signal with the blood vessel cells to regulate the recruitment of immune cells to the vasculature, and the subsequent passage of immune cells through the vasculature into the tissue. This regulation of cell trafficking through the vasculature is deregulated in diseases such as atherosclerosis or in cancer, where cancer cells are trafficking through the vasculature in similar manners to immune cells. Besides cell-cell interactions, the dynamics of the vasculature can also be regulated by physical stimuli, such as the properties of the underlying extracellular matrix, the forces the vascular cells exert on each other through cytoskeletal activity, or the geometrical properties of the vascular network. We present mathematical models that elucidate how physical, together with molecular or cellular, stimuli can regulate the dynamic movements of the vasculature, the formations of gaps between vascular cells, and the resulting trafficking of immune or cancer cells through the vasculature. With this model, validated by experiments, we demonstrate that gaps in the vasculature preferentially occur at tricellular junctions, and that cancer cells consequently transmigrate at these locations. We also demonstrate how force distributions change in realistic 3D microvasculature, compared to 2D monolayers, and how this changes gap formation, leakage, and transmigration in 2 versus 3D.

243 PROGNOSTIC IMPACT OF CXCR7 AND CXCL12 EXPRESSION IN PATIENTS WITH ESOPHAGEAL ADENOCARCINOMA

Abstract Chemokines are major regulators of cell trafficking and adhesion. The chemokine CXCL12 and its receptors, CXCR4 and CXCR7, have recently been reported as biomarkers in various cancers, including esophageal squamous cell carcinoma; however, there are few studies of these chemokines in esophageal adenocarcinoma (EAC). In this study, we investigated the relationship between expression of CXCL12, CXCR4 and CXCR7, and prognosis in patients with EAC. Methods This study examined 55 patients with EAC who were treated in Toronto General Hospital from 2001 to 2010. Tissue microarray immunohistochemistry was used to evaluate the expression of CXCL12, CXCR4 and CXCR7. Evaluation of immunohistochemistry was performed by a pathologist without knowledge of patients’ information and scored based on a semiquantitative scoring system. The average score from multiple cancer tissues on the microarray was utilized and patients were divided into high or low expression groups using the median score as a cutoff point. These results were compared with the patients’ clinicopathological features and survival. Results The score of CXCR7 was positively correlated with that of CXCL12 (r = 0.3154). High CXCR7 expression was significantly associated with lymphatic invasion (present vs absent, P = 0.005), higher number of lymph node metastases (pN0–1 vs pN2–3, P = 0.0014) and TNM stage (Stage I-II vs III-IV, P = 0.0168). Patients with high CXCR7 (n = 23) expression was associated with worse overall (OS) and disease-free survival (DFS) (P = 0.0221, 0.0090, respectively), and patients with high CXCL12 (n = 24) tended to have worse OS and DFS (P = 0.1091, 0.1477, respectively). High expression of both CXCR7 and CXCL12 was an independent prognostic factor for DFS on multivariable analysis (HR0.3, 95%CI: 0.1–0.8, P = 0.0115). Conclusion High CXCR7 expression was associated with poor prognosis in patients with EAC, and high expression of CXCR7 and its ligand CXCL12, had a stronger association on prognosis. Further study of this potential biomarker using whole tissue samples and larger sample size is warranted.

Tick saliva protein Evasin-3 modulates chemotaxis by disrupting CXCL8 interactions with glycosaminoglycans and CXCR2

Chemokines are a group of chemotaxis proteins that regulate cell trafficking and play important roles in immune responses and inflammation. Ticks are blood-sucking parasites that secrete numerous immune-modulatory agents in their saliva to evade host immune responses. Evasin-3 is a small salivary protein that belongs to a class of chemokine-binding proteins isolated from the brown dog tick, Rhipicephalus sanguineus. Evasin-3 has been shown to have a high affinity for chemokines CXCL1 and CXCL8 and to diminish inflammation in mice. In the present study, solution NMR spectroscopy was used to investigate the structure of Evasin-3 and its CXCL8–Evasin-3 complex. Evasin-3 is found to disrupt the glycosaminoglycan-binding site of CXCL8 and inhibit the interaction of CXCL8 with CXCR2. Structural data were used to design two novel CXCL8-binding peptides. The linear tEv3 17–56 and cyclic tcEv3 16–56 dPG Evasin-3 variants were chemically synthesized by solid-phase peptide synthesis. The affinity of these newly synthesized variants to CXCL8 was measured by surface plasmon resonance biosensor analysis. The Kd values of tEv3 17–56 and tcEv3 16–56 dPG were 27 and 13 nm, respectively. Both compounds effectively inhibited CXCL8-induced migration of polymorphonuclear neutrophils. The present results suggest utility of synthetic Evasin-3 variants as scaffolds for designing and fine-tuning new chemokine-binding agents that suppress immune responses and inflammation.

Tissue Factor Pathway Inhibitor Enhances Transendothelial Migration of Chronic Lymphocytic Leukemia Cells through Binding to Glypican-3

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. Cell trafficking and homing of CLL cells play a critical role in organ infiltration and contribute to the clinical course of CLL. Interaction between CLL cells and endothelial cells affects gene expression in CLL cells and further regulates cell trafficking. Endothelial cells are the main source of tissue factor (TF) pathway inhibitor (TFPI), which is the primary inhibitor of TF. However, the role of TFPI in leukemic cell trafficking has not been explored before. CXCL12 and its receptor CXCR4 play an important role in CLL cell trafficking and homing. To assess the effect of TFPI on CXCL12-mediated migration of leukemic cells, we investigated transendothelial migration (TEM) of B cells from CLL patients by using CXCL12 as a chemoattractant. We found that full-length recombinant TFPI (rTFPI) increased CXCL12-mediated migration of CLL cells dose-dependently by using MACS flow cytometry to count the migrated cells. Accordingly, Western blot showed the binding of rTFPI to the CLL cells after 24 h treatment. However, the chemokine receptor CXCR4 expression in CLL cells was not increased or even slightly decreased by rTFPI treatment, suggesting that TFPI enhances leukemic cell migration probably through interacting with other factor(s) than overexpressing CXCR4 in CLL cells. Surface expression and intracellular trafficking of TFPI is regulated by glycosylphosphatidylinositol (GPI)-anchored co-receptor(s). Glypican-3, a member of proteoglycan family, binds to the plasma membrane by a GPI anchor and its heparan sulfate (HS)-type glycosaminoglycan chains are considered to mediate the interaction with other cell membrane proteins. To determine whether TFPI interacts with glypican-3, immunofluorescence staining and co-immunoprecipitation were performed in K562 and HL60 cells. Immunofluorescence analysis revealed colocalization of TFPI and glypican-3 on the surface of K562 and HL60 cells. Co-immunoprecipitation confirmed the binding between TFPI and glypican-3 in K562 cells. Moreover, co-localized TFPI and glypican-3 were found in bone marrow biopsies of leukemia patients. We further employed an antibody against HS chains of glypican-3, which inhibits the activity of glypican-3. Western blot showed that rTFPI treatment failed to increase TFPI protein in CLL cells when glypican-3 inhibitor HS20 was applied. In addition, TEM assay showed that HS20 treatment abolished the effect of TFPI on cell migration, which suggests that inhibition of glypican-3 blocks the binding of TFPI to CLL cells and then abolishes the effect of TFPI on cell migration. It indicates that TFPI affects CLL cell migration through binding to glypican-3. Using a factor Xa generation assay, we found that HS20 suppressed TF activity on the surface of HL60 cells, suggesting that the inhibition of glypican-3 impaired the procoagulant activity of leukemic cells. In addition, Western blot and RT-PCR showed that HS20 downregulated TF mRNA and protein expression in CLL cells. Finally, we explored the role of Wnt/β-catenin signaling, which has been shown to regulate tumor progression and metastasis. We found a dose-dependent increase of activated β-catenin in the CLL cells after rTFPI treatment. Inhibition of Wnt/β-catenin signaling by a potent inhibitor IWP4 strongly impaired the migration of CLL cells induced by rTFPI. In this study, we show that TFPI enhances CLL cell migration through binding to glypican-3 and activating Wnt/β-catenin signaling pathway. Our findings suggest a novel molecular mechanism regulating the transmigration of leukemic cells. It may open a new window for therapeutic targeting in the treatment of organ infiltration of CLL patients. DisclosuresNo relevant conflicts of interest to declare.

A Task Force Against Local Inflammation and Cancer: Lymphocyte Trafficking to and Within the Skin.

The skin represents a specialized site for immune surveillance consisting of resident, inflammatory and memory populations of lymphocytes. The entry and retention of T cells, B cells, and ILCs is tightly regulated to facilitate detection of pathogens, inflammation and tumors cells. Loss of individual or multiple populations in the skin may break tolerance or increase susceptibility to tumor growth and spread. Studies have significantly advanced our understanding of the role of skin T cells and ILCs at steady state and in inflammatory settings such as viral challenge, atopy, and autoimmune inflammation. The knowledge raised by these studies can benefit to our understanding of immune cell trafficking in primary melanoma, shedding light on the mechanisms of tumor immune surveillance and to improve immunotherapy. This review will focus on the T cells, B cells, and ILCs of the skin at steady state, in inflammatory context and in melanoma. In particular, we will detail the core chemokine and adhesion molecules that regulate cell trafficking to and within the skin, which may provide therapeutic avenues to promote tumor homing for a team of lymphocytes.

Sphingosine Kinases promote IL-17 expression in human T lymphocytes

Sphingosine 1-phosphate (S1P) has a role in many cellular processes. S1P is involved in cell growth and apoptosis, regulation of cell trafficking, production of cytokines and chemokines. The kinases SphK1 and SphK2 (SphKs) phosphorilate Sphingosine (Sph) to S1P and several phosphatases revert S1P to sphingosine, thus assuring a balanced pool that can be depleted by a Sphingosine lyase in hexadecenal compounds and aldehydes. There are evidences that SphK1 and 2 may per se control cellular processes. Here, we report that Sph kinases regulate IL-17 expression in human T cells. SphKs inhibition impairs the production of IL-17, while their overexpression up-regulates expression of the cytokine through acetylation of IL-17 promoter. SphKs were up-regulated also in PBMCs of patients affected by IL-17 related diseases. Thus, S1P/S1P kinases axis is a mechanism likely to promote IL-17 expression in human T cells, representing a possible therapeutic target in human inflammatory diseases.

Functional and structural consequences of chemokine (C-X-C motif) receptor 4 activation with cognate and non-cognate agonists.

Chemokine (C-X-C motif) receptor 4 (CXCR4) regulates cell trafficking and plays important roles in the immune system. Ubiquitin has recently been identified as an endogenous non-cognate agonist of CXCR4, which activates CXCR4 via interaction sites that are distinct from those of the cognate agonist C-X-C motif chemokine ligand 12 (CXCL12). As compared with CXCL12, chemotactic activities of ubiquitin in primary human cells are poorly characterized. Furthermore, evidence for functional selectivity of CXCR4 agonists is lacking, and structural consequences of ubiquitin binding to CXCR4 are unknown. Here, we show that ubiquitin and CXCL12 have comparable chemotactic activities in normal human peripheral blood mononuclear cells, monocytes, vascular smooth muscle, and endothelial cells. Chemotactic activities of the CXCR4 ligands could be inhibited with the selective CXCR4 antagonist AMD3100 and with a peptide analogue of the second transmembrane domain of CXCR4. In human monocytes, ubiquitin- and CXCL12-induced chemotaxis could be inhibited with pertussis toxin and with inhibitors of phospholipase C, phosphatidylinositol 3 kinase, and extracellular signal-regulated kinase 1/2. Both agonists induced inositol trisphosphate production in vascular smooth muscle cells, which could be inhibited with AMD3100. In β-arrestin recruitment assays, ubiquitin did not sufficiently recruit β-arrestin2 to CXCR4 (EC50>10μM), whereas the EC50 for CXCL12 was 4.6nM (95% confidence interval 3.1-6.1nM). Both agonists induced similar chemical shift changes in the 13C-1H-heteronuclear single quantum correlation (HSQC) spectrum of CXCR4 in membranes, whereas CXCL11 did not significantly alter the 13C-1H-HSQC spectrum of CXCR4. Our findings point towards ubiquitin as a biased agonist of CXCR4.

Dermal γδ T-Cells Can Be Activated by Mitochondrial Damage-Associated Molecular Patterns.

BackgroundGamma delta T-cells have been shown to be important to the early immunoinflammatory response to injury, independent of infection. This unique T-cell population acts to regulate cell trafficking and the release of cytokines and growth factors. We propose this sterile inflammatory response is in part associated with damage associated molecular patterns (DAMPs) generated by major injury, such as burn, and mediated via toll-like receptors (TLRs). It is unknown whether DAMPs can activate resident γδ T-cells that reside in skin.MethodsGamma delta T-cells were isolated from the skin of male C57BL/6 mice by enzymatic digestion. Mitochondrial DAMPs (MTDs) were generated from mitochondria isolated from mouse livers by sonication and centrifugation. Dermal γδ T-cells were incubated with MTDs (0–500 μg/ml) for 24 hr and cells and supernatants were collected for analysis.ResultsMTDs activated dermal γδ T-cells, as evidenced by increased TLR2 and TLR4 expression following in vitro exposure. MTDs also induced the production of inflammatory cytokines (IL-1β, IL-6), and growth factors (PDGF and VEGF) by γδ T-cells.ConclusionsThese findings herein support the concept that MTDs released after tissue/cellular injury are capable of activating dermal γδ T-cells. We propose that the activation of this unique T-cell population is central in the initiation of sterile inflammation and also contributes to the subsequent healing processes.

Abstract 249: Increased Vascular CCRL2 Expression by Disturbed Blood Flow Promotes Atherosclerotic Plaque Formation in ApoE Deficient Mice

Atherosclerosis is a chronic inflammatory disease of the arterial wall elicited by accumulation of LDL and leucocytes in the subendothelium at predilection sites with disturbed laminar flow. Chemokines and their receptors appear to act as critical players in atherosclerosis as they not only direct atherogenic recruitment of leucocytes but also exert cell hemostatic functions by chemokine ligand-receptor axes and their specific or combined contributions. Atypical chemokine (C-C motif) receptor-like 2 (CCRL2) cooperates with its ligand chemerin and leukocyte-expressed chemerin receptor chemokine-like receptor 1 (CMKLR1) to regulate cell trafficking and inflammatory responses,but its role in atherosclerosis is not clear. To investigate whether CCRL2 contributes to the pathomechanism of atherogenesis, we generated CCRL2 -/- mice in hyperlipidemic atherosclerosis-prone ApoE -/- background and found that the atherosclerotic plaque area of the total aorta was significantly reduced compared with CCRL2 +/+ ApoE -/- mice on a high fat diet. The protective effect of CCRL2 deficiency was anatomically isolated primarily to the site of disturbed blood flow (D-flow) in the aortic arch but not in the descending aorta. Endothelial CCRL2 was upregulated in response to D-flow and either CCRL2 or CMKLR1 deletion reduced plaque formation. Further studies showed that CCRL2 co-localized with CMKLR1 and chemerin within the atherosclerotic aorta root. CCRL2 deficiency led to significantly less lipid deposition in aortic root, reduced CMKLR1 + leukocyte rolling on lesional vascular endothelium, diminished macrophage accumulation and foam cell formation, and polarized macrophage to an M2-like phenotype. These results demonstrate that D-flow induction of vascular CCRL2 is required for optimal formation of atherosclerotic plaques via coordinating the accumulation of CMKLR1 + monocytes/macrophages within the vascular wall, and thus identifies CCRL2 as a novel drug target to prevent or treat atherosclerosis. This work was supported by Natural Science Foundation of China (grant 81370373 to L.Z. and 31300781 to C.T.) Key Words: atherosclerosis, CCRL2, chemerin, macrophage

Biased signaling pathways via CXCR3 control the development and function of CD4+ T cell subsets.

Structurally related chemotactic cytokines (chemokines) regulate cell trafficking through interactions with 7-transmembrane domain, G protein-coupled receptors. Biased signaling or functional selectivity is a concept that describes a situation where a 7-transmembrane domain receptor preferentially activates one of several available cellular signaling pathways. It can be divided into 3 distinct cases: ligand bias, receptor bias, and tissue or cell bias. Many studies, including those coming from our lab, have shown that only a limited number of chemokines are key drivers of inflammation. We have referred to them as "driver chemokines." They include the CXCR3 ligands CXCL9 and CXCL10, the CCR2 ligand CCL2, all 3 CCR5 ligands, and the CCR9 ligand CCL25. As for CXCR3, despite the proinflammatory nature of CXCL10 and CXCL9, transgenic mice lacking CXCR3 display an aggravated manifestation of different autoimmune disease, including Type I diabetes and experimental autoimmune encephalomyelitis. Recently, we showed that whereas CXCL9 and CXCL10 induce effector Th1/Th17 cells to promote inflammation, CXCL11, with a relatively higher binding affinity to CXCR3, drives the development of the forkhead box P3-negative IL-10(high) T regulatory 1 cell subset and hence, dampens inflammation. We also showed that CXCL9/CXCL10 activates a different signaling cascade than CXCL11, despite binding to the same receptor, CXCR3, which results in these diverse biologic activities. This provides new evidence for the role of biased signaling in regulating biologic activities, in which CXCL11 induces ligand bias at CXCR3 and receptor-biased signaling via atypical chemokine receptor 3.

F-BAR family proteins, emerging regulators for cell membrane dynamic changes-from structure to human diseases.

Eukaryotic cell membrane dynamics change in curvature during physiological and pathological processes. In the past ten years, a novel protein family, Fes/CIP4 homology-Bin/Amphiphysin/Rvs (F-BAR) domain proteins, has been identified to be the most important coordinators in membrane curvature regulation. The F-BAR domain family is a member of the Bin/Amphiphysin/Rvs (BAR) domain superfamily that is associated with dynamic changes in cell membrane. However, the molecular basis in membrane structure regulation and the biological functions of F-BAR protein are unclear. The pathophysiological role of F-BAR protein is unknown. This review summarizes the current understanding of structure and function in the BAR domain superfamily, classifies F-BAR family proteins into nine subfamilies based on domain structure, and characterizes F-BAR protein structure, domain interaction, and functional relevance. In general, F-BAR protein binds to cell membrane via F-BAR domain association with membrane phospholipids and initiates membrane curvature and scission via Src homology-3 (SH3) domain interaction with its partner proteins. This process causes membrane dynamic changes and leads to seven important cellular biological functions, which include endocytosis, phagocytosis, filopodium, lamellipodium, cytokinesis, adhesion, and podosome formation, via distinct signaling pathways determined by specific domain-binding partners. These cellular functions play important roles in many physiological and pathophysiological processes. We further summarize F-BAR protein expression and mutation changes observed in various diseases and developmental disorders. Considering the structure feature and functional implication of F-BAR proteins, we anticipate that F-BAR proteins modulate physiological and pathophysiological processes via transferring extracellular materials, regulating cell trafficking and mobility, presenting antigens, mediating extracellular matrix degradation, and transmitting signaling for cell proliferation.

Abstract 3605: The inhibitor of NF-ĸB kinase, IKKβ, regulates the stability of GLI1 transcription factor

Abstract Lymphoma microenvironment has a crucial role in lymphoma initiation, progression and drug resistance. Fibroblastic reticular cells (FRCs) participate dynamically in the cytokine microenvironment of the lymph node paracortex as well as the regulation of cell trafficking and access of T-cells into the paracortex. The FRC niche also participates in the cytokine microenvironment of lymphomas providing homing and survival signals including CCL19, CCL21, IL-7, CXCL12 and hedgehog (Hh) ligands. Hh signaling is evolutionary conserved signaling pathway that serves several physiological and development processes mediated by externally secreted Hh ligands. We previously demonstrated that the canonical Hh ligand-PTCH1-SMO-GLI axis is functional and contributes to cell survival, proliferation and enhances chemo tolerance in DLBCL. We further showed that GLI1, Hh signaling transcription factor is aberrantly activated in ∼87% of DLBCL and this activation is in part mediated by sonic Hh ligands secreted by stromal cells. Although the importance of GLI1 in tumor development is well recognized, the molecular mechanisms controlling the transcriptional activity of GLI1 is limited. To identify regulatory components that participate in the transcriptional activity of GLI1, we explored GLI1 putative interacting proteins by liquid chromatography tandem mass spectrometry following immunoprecipitation of endogenous GLI1. We identified the inhibitor of NF-ĸB kinase, IKKβ as a novel GLI1-binding protein. In addition, we found that the kinase activity of IKKβ is critical to recruit and phosphorylate GLI1 in the C-terminal fragment. We further showed that in DLBCL cells stimulated with TNFα, IKKβ phosphorylates GLI1 and restricts the binding between GLI1 with HECT-type E3 ubiquitin ligase (ITCH) resulting in GLI1 stability and increased oncogenic potential. Inhibition of IKKβ-mediated GLI1 phosphorylation restored the binding between GLI1 and ITCH and promoted ubiquitination and degradation of GLI1. Collectively, these results indicate that IKKβ regulates the transcriptional activity of GLI1 by phosphorylating GLI1 and modulating the binding between GLI1 and ITCH. In DLBCL, canonical NF-κB and GLI1 mediated Hh signaling pathways are strongly responsive with tumor microenvironment; our study will help to understand the cross talk between these two pathways which could represents a novel therapeutic approach for DLBCL. Citation Format: Nitin K. Agarwal, Kranthi Kunkalla, Francisco Vega. The inhibitor of NF-ĸB kinase, IKKβ, regulates the stability of GLI1 transcription factor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3605. doi:10.1158/1538-7445.AM2014-3605

Control of the Adaptive Immune Response by Tumor Vasculature

The endothelium is nowadays described as an entire organ that regulates various processes: vascular tone, coagulation, inflammation, and immune cell trafficking, depending on the vascular site and its specific microenvironment as well as on endothelial cell-intrinsic mechanisms like epigenetic changes. In this review, we will focus on the control of the adaptive immune response by the tumor vasculature. In physiological conditions, the endothelium acts as a barrier regulating cell trafficking by specific expression of adhesion molecules enabling adhesion of immune cells on the vessel, and subsequent extravasation. This process is also dependent on chemokine and integrin expression, and on the type of junctions defining the permeability of the endothelium. Endothelial cells can also regulate immune cell activation. In fact, the endothelial layer can constitute immunological synapses due to its close interactions with immune cells, and the delivery of co-stimulatory or co-inhibitory signals. In tumor conditions, the vasculature is characterized by an abnormal vessel structure and permeability, and by a specific phenotype of endothelial cells. All these abnormalities lead to a modulation of intra-tumoral immune responses and contribute to the development of intra-tumoral immunosuppression, which is a major mechanism for promoting the development, progression, and treatment resistance of tumors. The in-depth analysis of these various abnormalities will help defining novel targets for the development of anti-tumoral treatments. Furthermore, eventual changes of the endothelial cell phenotype identified by plasma biomarkers could secondarily be selected to monitor treatment efficacy.

Chemokines and cytokines network in the pathogenesis of the inflammatory skin diseases: atopic dermatitis, psoriasis and skin mastocytosis.

Chemokines are signaling peptides which regulate cell trafficking and provide control of the tissue-specific cell homing. In the skin, chemokines are secreted both by the resident cells such as keratinocytes, melanocytes, fibroblasts, dendritic cells and mast cells, as well as by infiltrated cells – lymphocytes, eosinophils, and monocytes. Chemokines, together with cytokines, participate in induction and maintenance of inflammation in the skin and regulate the composition of the cellular infiltrates. Inflammation within the skin is a feature shared by atopic dermatitis and psoriasis, two of the most common dermatoses. Accumulation of activated mast cells in the affected skin is seen both in atopic dermatitis and in psoriasis. This paper presents a concise overview of the current knowledge on the role chemokines have in pathogenesis of atopic dermatitis, psoriasis, and mastocytosis, a disease caused directly by the accumulation and activation of mast cells in the skin.

Lamins Regulate Cell Trafficking and Lineage Maturation of Hematopoietic Cells

Hematopoietic stem and progenitor cells reside preferentially in adult marrow microenvironments together with nucleated erythroblasts and megakaryocytes, whereas other blood cells readily cross the endothelial barrier into the circulation. Since the nucleus is the largest organelle in blood cells, we hypothesized that [1] cell sorting across microporous barriers is regulated by nuclear deformability as controlled by lamin-A and -B, and [2] lamin levels directly modulate hematopoietic programs. Mass spectrometry-calibrated intracellular flow cytometry indeed reveals a lamin expression map that partitions human blood lineages between marrow and circulating compartments (P = 0.00006). B-type lamins are highly variable and predominate only in CD34+ cells, but migration through micropores and nuclear flexibility in micropipette aspiration both appear limited by lamin-A:B stoichiometry across hematopoietic lineages. Differentiation is also modulated by overexpression or knockdown of lamins as well as retinoic acid addition, which regulates lamin-A transcription. In particular, erythroid differentiation is promoted by high lamin-A and low lamin-B1 expression, whereas megakaryocytes of high ploidy are inhibited by lamin suppression. Lamins thus contribute to both trafficking and differentiation.

Regulation of PKD1-mediated Golgi to cell surface trafficking by Gαq subunits

Heterotrimeric GTP-binding proteins play a key role in cell trafficking regulation. Above all, specific Gβγ subunits have been shown to be a major component of a signal transduction pathway, which also involves phospholipases C (PLC), protein kinases C (PKC) and D (PKD), whose main function is to regulate transport between Golgi and plasma membrane. It was the involvement of PLC which led us to study the role of the other member of this G protein family, the α subunits, in the regulation of membrane fission at the Golgi apparatus. Among constitutive active (QL) variants of different G protein α subunit sub-families, only GαqQL subunits were able to induce Golgi fragmentation, a phenotype that mainly reflects a membrane fission increase at this organelle. This phenotype was not observed with a GαqQL palmitoylation mutant, showing the need for a membrane-bounded subunit. Besides, GαqQL-dependent Golgi fission was blocked by specific PLC and PKC inhibitors, and in the presence of a PKD1-kinase dead variant. In addition, GαqQL was the only α subunit capable of inducing PKD1 phosphorylation. Finally, Vesicular Stomatitis Virus thermosensitive mutant glycoprotein (VSVG tsO45) transport assays have demonstrated that GαqQL acts directly on Golgi membranes to regulate trafficking between this organelle and plasma membrane. All these results indicate Gαq subunits for the first time as a regulator of PKD-mediated intracellular trafficking between Golgi apparatus and plasma membrane, opening new perspectives in the understanding of internal trafficking regulation by external signals through G protein-coupled receptors.

Transcriptional profiling reveals molecular signatures associated with HIV permissiveness in Th1Th17 cells and identifies peroxisome proliferator-activated receptor gamma as an intrinsic negative regulator of viral replication.

BackgroundWe previously demonstrated that primary Th1Th17 cells are highly permissive to HIV-1, whereas Th1 cells are relatively resistant. Molecular mechanisms underlying these differences remain unknown.ResultsExposure to replication competent and single-round VSV-G pseudotyped HIV strains provide evidence that superior HIV replication in Th1Th17 vs. Th1 cells was regulated by mechanisms located at entry and post-entry levels. Genome-wide transcriptional profiling identified transcripts upregulated (n = 264) and downregulated (n = 235) in Th1Th17 vs. Th1 cells (p-value < 0.05; fold change cut-off 1.3). Gene Set Enrichment Analysis revealed pathways enriched in Th1Th17 (nuclear receptors, trafficking, p38/MAPK, NF-κB, p53/Ras, IL-23) vs. Th1 cells (proteasome, interferon α/β). Differentially expressed genes were classified into biological categories using Gene Ontology. Th1Th17 cells expressed typical Th17 markers (IL-17A/F, IL-22, CCL20, RORC, IL-26, IL-23R, CCR6) and transcripts functionally linked to regulating cell trafficking (CEACAM1, MCAM), activation (CD28, CD40LG, TNFSF13B, TNFSF25, PTPN13, MAP3K4, LTB, CTSH), transcription (PPARγ, RUNX1, ATF5, ARNTL), apoptosis (FASLG), and HIV infection (CXCR6, FURIN). Differential expression of CXCR6, PPARγ, ARNTL, PTPN13, MAP3K4, CTSH, SERPINB6, PTK2, and ISG20 was validated by RT-PCR, flow cytometry and/or confocal microscopy. The nuclear receptor PPARγ was preferentially expressed by Th1Th17 cells. PPARγ RNA interference significantly increased HIV replication at levels post-entry and prior HIV-DNA integration. Finally, the activation of PPARγ pathway via the agonist Rosiglitazone induced the nuclear translocation of PPARγ and a robust inhibition of viral replication.ConclusionsThus, transcriptional profiling in Th1Th17 vs. Th1 cells demonstrated that HIV permissiveness is associated with a superior state of cellular activation and limited antiviral properties and identified PPARγ as an intrinsic negative regulator of viral replication. Therefore, triggering PPARγ pathway via non-toxic agonists may contribute to limiting covert HIV replication and disease progression during antiretroviral treatment.

Class I PI3K Isoforms Exert a Differential Role On Survival and Cell Trafficking In Multiple Myeloma

BackgroundThe phosphatidylinositol-3 kinase (PI3K) pathway is a critical regulator of tumor progression, protein translation and cytoskeletal dynamics, collectively required for cell proliferation, survival, adhesion and migration in many malignancies including multiple myeloma (MM). Despite the absence of mutations in the PI3K/Akt genes, many studies have demonstrated that this pathway is constitutively activated in MM cells. In this study, we investigated the role of inhibition of class I PI3K isoforms known as p110α, p110β, p110γ and p110δ in cell trafficking of MM cells using isoform-specific knockdown (KD). We have also evaluated the effect of pan-PI3K inhibitior, NVP-BKM120, on survival, adhesion and migration of MM cells both in vitro and in vivo. MethodsThe baseline expression of class I PI3K isoforms in MM cell lines (MM.1S, OPM1, OPM2, H929, RPMI, INA6, U266, and U266LR7) has been evaluated by immnunobloting. MM tumor cells (MM.1S-GFP+/luc+) were infected with lentivirus mediated shRNA targeting class I PI3K isoforms. RT-qPCR and immunoblotting were performed to show infection efficiency. In vivo tumor growth of isoform specific KDs were assessed by using in vivo bioluminescence (BLI) in SCID mice. Detection of circulating MM-GFP+ cells ex vivo was performed by flow cytometry. Analysis of circulating tumor cells for each isoform-specific KD cells against relative tumor volume was performed by lineer regression using GraphPad software. Survival, adhesion and migration of KD cells were tested by MTT, adhesion and migration in vitro assay, respectively. NVP-BKM120, a pan-PI3K-inhibitor (Novartis, MA) has been tested both in vitro and in vivo. Ex vivo detection of mobilization and tumor growth of MM cells (MM.1S-GFP+/luc+) treated with 1) vehicle; 2) NVP-BKM120 in SCID mice were assessed by using flow cytometry and in vivo BLI. Homing of MM cells to the BM of mice pre-treated with NVP-BKM120 was evaluated by in vivo confocal. Increased concentrations of NVP-BKM120 have been tested on survival, cell cycle and apoptotic pathways in MM cells, by using MTT, PI staining in flow cytometry and immunoblotting, respectively. NVP-BKM120 induced dose-dependent effect on chemotaxis and adhesion of MM.1s to BM stromal cells (BMSCs) and fibronectin were tested by migration and adhesion assays. ResultsPI3K-p110β was highly expressed in all cell lines; while other isoforms were expressed in some of the MM cell lines tested. Of note, MM.1S expressed all isoforms. Mice injected with PI3K isoform specific knockdown MM.1S cells presented with different tumor burdens; p110β and p110δ mice showed significantly slower tumor progression compared to scramble control cell line (P<.05), whereas tumor growth was similar in p110α and p110γ to control mice. We next compared the number of circulating tumor cells (CTCs) at the same tumor burden between groups, which showed only p110β presented with a higher number of CTCs compared to the scramble group (P=0.01). In vitro, we observed reduced adhesion and enhanced migration of KD cells compared to control with no cell survival difference. The effect of pan-inhibition of PI3K with NVP-BKM120 induced MM cell mobilization from the BM to the circulation (Vehicle: 0.002 % vs NVP-BKM120: 0.023%; P<.05). This was supported by the inhibition of homing of MM cells to the BM (84% decrease) in the mice pre-treated with NVP-BKM120 (P<.05). Furthermore, treatment of mice with 50mg/kg of NVP-BKM120 once a day by oral gavage for five weeks significantly decreased the rate of tumor progression in MM compared to the vehicle treated group, as shown by BLI (69% decrease, P<.01). NVP-BKM120 decreased the activation of adhesion-related signaling in MM cells induced by co-culture with stroma, including pFAK, pSrc, pCoffilin and pMLC, as shown by immunoblotting. Moreover, it caused cell cycle arrest, as detected by PI staining and analyzed by flow cytometry. ConclusionThis study suggests that inhibition of Class I PI3K isoforms, particularly p110β and p110δ, can play an important role in the regulation of cell trafficking in MM by disrupting adhesion of MM cells to the BM and inducing mobilization. Thus, pan-PI3K inhibition by NVP-BKM120 is a promising approach, which may enhance therapeutic response and overcome resistance in the treatment of MM. Disclosures:Ghobrial:Onyx: Advisoryboard Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.