Chemokines (CCL3, CCL4, CCL5) inhibit ATP-induced release of IL-1beta by monocytic cells

ATP and chemokines are among the first inflammatory mediators that can enter the circulation via damaged blood vessels at the site of injury, leading to an activation of the host’s immune response. The main function of chemokines is leukocyte mobilization, guiding immune cells towards the injured tissue along a chemotactic concentration gradient. In monocytes, ATP typically triggers inflammasome assembly, a multiprotein complex necessary for the maturation and secretion of IL-1beta. IL-1beta is a potent inflammatory cytokine of innate immunity, essential for pathogen defense. However, excessive IL-1beta may cause life-threatening systemic inflammation. Here, we hypothesize that chemokines control ATP-dependent secretion of monocytic IL-1beta, by engaging a cholinergic signaling pathway. LPS-primed human monocytic U937 cells were treated with chemokines in the presence or absence of nAChR antagonists or iPLA2beta inhibitors and concomitantly stimulated with the P2X7 agonist BzATP. IL-1beta concentration was determined in the cell culture supernatants. Silencing of the chemokine receptor and iPLA2b gene expression was achieved by transfecting cells with the appropriate siRNA. CCL3, CCL4, and CCL5 dose-dependently inhibited BzATP-stimulated release of IL-1beta, whereas CXCL16 was ineffective. The effect of CCL3 was confirmed for primary mononuclear leukocytes. The inhibitory effect of CCL3 was blunted after silencing CCR1 or iPLA2beta gene expression by siRNA and was sensitive to antagonists of nAChRs containing subunits alpha7 and alpha9/alpha10. U937 cells secreted small factors in response to CCL3 that mediated the inhibition of IL-1beta release. We suggest that CCL chemokines inhibit ATP-induced release of IL-1beta from U937 cells by a triple-membrane-passing mechanism involving CCR, iPLA2, release of small mediators, and nAChR subunits alpha7 and alpha9/alpha10. We speculate that whenever chemokines and ATP enter the circulation concomitantly, systemic release of IL-1beta is minimized

The Genetic, Molecular, And Cellular Bases Of Unidentified Primary Immunodeficiencies

The immune system is inseparable from every part of human biology. From cell intrinsic mechanisms of pathogen recognition to multi-cellular interactions over vast ranges of time and space, the immune system is both essential for protection from infection and central to the pathogenesis of many diseases. Thus understanding it has long been a focus of biomedical research. While in vitro molecular, biochemical, and cellular techniques as well as complex genetically modified animal models have been developed, these approaches still only approximate true human disease and in vivo human biology. Primary immunodeficiencies are inborn genetic defects of immunity and present rare opportunities to observe, study, and understand how genetic perturbations impact human immunity directly. I therefore clinically and genetically analyzed three patient families with unidentified primary immunodeficiencies. Using whole exome sequencing coupled with in vitro and in vivo biochemical and cellular assays, I identified two novel genetic etiologies of primary immunodeficiency. I first identified de novo missense mutations in GNAI2, the gene encoding the ubiquitously expressed heterotrimeric G-protein Gαi2, in 2 families with life-threating multi-organ system autoimmunity and immunodeficiency to mucocutaneous infections. Gαi2 is essential for chemokine mediated leukocyte migration as well as regulating development, inflammation, and metabolism in the immune system and beyond. The heterozygous dominant gain-of-function patient proteins impaired chemokine signaling and chemotaxis in addition to augmenting T cell activation by constitutively activating costimulatory pathways and reducing the requirement for T cell costimulation. I also identified homozygous missense mutations in IFIH1, the gene encoding the cytosolic pattern recognition receptor of dsRNA MDA5, in the third family of study. The affected individual presented with recurrent severe respiratory infections with RNA viruses including human rhinovirus, coronaviruses (HKU1, OC43, NL63), influenza virus, and respiratory syncytial virus. The mutant protein lost the ability to bind dsRNA and failed to initiate antiviral interferon-β and pro-inflammatory NF-κB responses. Using gene knockdown and gene editing in immortalized and patient derived cell lines, I demonstrated an essential role for MDA5 in restricting rhinovirus infection in human respiratory epithelium. Thus this work demonstrates the power of human genetics to identify disease causing mutations in rare individuals and reveal how the immune system uses molecules involved in cell migration, activation, and nucleic acid sensing to robustly protect us from virus infections without causing autoimmunity

Growth and apoptosis pathways in human cutaneous melanoma: in vitro and in vivo studies by using biological and proteomics approaches

Purpose. Melanoma is the most aggressive cutaneous cancer without effective treatment. Diagnosis is achieved very often too late and prognosis is poor. Aim of this work is to identify proteomic pathways potentially involved in melanoma aggressiveness. Materials and Methods. We analyzed 5 human metastatic melanoma cell lines and human keratinocyte and melanocyte cell lines as a control. Their proliferation and apoptotic behaviors under serum stimulation and starvation were analyzed in order to identify the most aggressive one. Melanoma cell proteome from the most aggressive cell line (A375), compared to the less aggressive one (SK mel 28), was analyzed by means of two complementary approaches: 1) multiplexed assay to measure the levels of 27 cytokines both in cell extracts and in conditioned media; 2) proteomic study through LC-MS/MS analysis of cell extracts. Data obtained were analyzed using bioinformatic analysis. Results. A375 cells were found to possess the highest growth rate both under serum stimulation and under serum starvation, while SK mel 28 cells under similar conditions were significantly less aggressive, confirmed also by invasion assays. The effect was markedly cell-density dependent, suggesting that cell-cell interaction and/or secretory signals dependent phenomena are important. Proteome analyses indicated that several proteins are differentially expressed and possibly related the aggressiveness. Some of these proteins have been identified as transport and proteasome components. The Bio-Plex analysis of the melanoma cell lines under study indicated that melanoma cells contain significantly (p<0.001) different levels of some inflammatory cytokines and angiogenic growth factors: the most significantly modified factors were IL-6, Il-7, RANTES and VEGF, suggesting a novel interesting viewpoint to explain melanoma cell aggressiveness. Conclusions. The reported results show that transport, proteasome components and an altered cytokine balance may be responsible for human melanoma aggressiveness

Understanding ligand binding, selectivity and functions on the G protein-coupled receptors: A molecular modeling approach

The assessment of target protein molecular structure provides a distinct advantage in the rational drug design process. The increasing number of available G protein-coupled receptor crystal structures has enabled utilization of a varied number of computational approaches for understanding the ligand-receptor interactions, ligand selectivity and even receptor response upon ligand binding. The following dissertation examines the results from three different projects with varied objectives – i) structural modeling of human C-C chemokine receptor type 5 (CCR5) and assessment of the ligand binding pocket of the receptor, ii) assessment of the selectivity profile of naltrexone derivatives on the three opioid receptors (μ-opioid, κ-opioid, δ-opioid) with an aim towards designing selective μ-opioid receptor antagonists, and iii) structural modeling of the ‘active’ state conformation of the κ-opioid receptor in response to agonist binding and determination of a plausible molecular mechanism involved in activation ‘switch’ of the κ-opioid receptor. In absence of a crystal-based molecular structure of CCR5, a homology model of the receptor was built and the ligand binding pocket was validated. On the basis of evaluation of the ligand-receptor interactions on the validated binding pocket, structural and chemical modifications to anibamine, a natural plant product, were proposed to enhance its receptor binding. The selectivity of naltrexone (a universal antagonist) was assessed with respect to the three opioid receptors by employing ligand docking studies and the ‘message-address’ concept. Multiple address sites were identified on the opioid receptors and structural modifications were proposed for the naltrexone derivatives for their enhanced selectivity. In the third project, structural modeling of the active state conformation of the κ-opioid receptor covalently bound to a salvinorin A derivative (agonist) was attempted via molecular dynamics simulations. Although the obtained molecular model lacked the signature ‘agonist-like’ conformations, the result provides a template for such studies in the future

Harnessing ion-binding sites for GPCR pharmacology

Endogenous ions play important roles in the function and pharmacology of G-protein coupled receptors (GPCRs). Historically the evidence for ionic modulation ofGPCRfunction dates to 1973 with studies of opioid receptors, where it was demonstrated that physiologic concentrations of sodium allosterically attenuated agonist binding. This Na+-selective effect was distinct from effects of other monovalent and divalent cations, with the latter usually counteracting sodium’s negative allosteric modulation of binding. Since then, numerous studies documenting the effects of mono- and divalent ions on GPCR function have been published. While ions can act selectively and nonselectively at many sites in different receptors, the discovery of the conserved sodium ion site in class A GPCR structures in 2012 revealed the unique nature of Na+ site, which has emerged as a near-universal site for allosteric modulation of class A GPCR structure and function. In this review, we synthesize and highlight recent advances in the functional, biophysical, and structural characterization of ions bound to GPCRs. Taken together, these findings provide a molecular understanding of the unique roles of Na+ and other ions as GPCR allosteric modulators. Wewill also discuss how this knowledge can be applied to the redesign of receptors and ligand probes for desired functional and pharmacological profiles. © 2019, American Society for Pharmacology and Experimental Therapy. All rights reserved

Targeting tumor microenvironment crosstalk through GPCR receptors and PI3K pathway

[eng] The tumor microenvironment (TME) is gaining momentum due to its contribution to cancer progression and therapy resistance. This TME has a direct crosstalk with tumor cells that involves the activation of different pathways. Follicular lymphoma (FL) is the most common indolent non-Hodgkin lymphoma. Although FL is generally characterized by slow progression and high response rates to therapy, it is still considered incurable, because almost virtually all the patients relapse. FL is probably the NHL with the highest dependence on microenvironment. PI3K is a common denominator transducing the signaling from FL crosstalk with the TME and plays an important role in multiple cellular functions, and also contributes to cancer promoting aspects of the TME, such as angiogenesis and inflammatory cell recruitment. Idelalisib is a first-in-class δ isoform- specific PI3K inhibitor that receive regulatory approval for relapsed CLL, SLL and FL in 2014. Idelalisib blocks PI3K δ which is restricted to leukocytes. BCL2 deregulation is paramount in the pathogenesis of FL, as a consequence of the t(14;18), and therefore it is an attractive target for novel therapeutic approaches. Venetoclax (ABT-199, AbbVie) is a small BCL-2 inhibitors. Even though 85% of FL patients harbor the t(14;18), the results of the first clinical trial with venetoclax were not satisfactory (overall response 38%). From this first study we conclude that Idelalisib modulates key pathways in the germinal center and shapes the FL immune microenvironment by decreasing the recruitment of TFH and Treg to the tumor site leading to less immunesuppresive phenotype. Furthermore Idelalisib induces a moderate cytotoxic effect on FL cells in co-cultures. This co-culture decrease FL dependence on BCL-2 and consequently, venetoclax cytotoxicity, but Idelalisib sensitizes FL co-cultures to venetoclax. In summary, Idelalisib interferes with the crosstalk of FL and its immune microenvironment and potentiates the activity of venetoclax targeting the tumor cells, thus representing a promising combination therapy that may improve FL outcome. Colorectal cancer (CRC) is the third most common cancer in males and the second in females, and the fourth most common cause of cancer-related death worldwide. Patients with advanced and distal metastatic disease (stage IV), the survival rate drops to 10%, which accounts for approximately 18% of cases. The TME in CRC, is a complex structure composed by different type of cells, which are interacting each other’s and secreting a variety of growth factors and other molecules, such as cytokines and chemokines. Tumor development is based on the crosstalk between tumor cells and their surrounding microenvironment, and this crosstalk is mediated by the receptors and its ligand expression in both types of cells. G protein-coupled receptors (GPCRs) are an important family of membrane signaling receptors, which have an important role in cancer growth and development. Originally, GPCRs were considered as monomeric functional entities, nevertheless, in recent years has become evident that GPCRs form dimers and this dimers formation may modify the cellular response. In cancer, CXCR4 (has been studied extensively) plays an important role at different stages of cancer development, and is involved in the metastasis process of tumor cells. The up- regulation of CXCR4 in CRC correlates with a poor prognosis. Another GPCR, CB2 receptor modulates the downstream signaling and it is able to activate a wide range of signaling pathways, including extracellular signal-regulated kinases 1/2 (ERK1/2). In CRC, it has been described an up-regulation of CB2 receptor expression. GPCRs show differential expression in cancer cells and tissues, and they are highly druggable sites. From this second study we concluded that CXCR4 and CB2 expression is increased in primary colon tumor cells and in metastasis cells compared to normal epithelial cells from colon mucosa, and they formed heterodimers in colon tumoral cells and are associated with more aggressive phenotypes. Moreover, a bidirectional cross-antagonism crosstalk is established between these receptors. These heterodimers regulate in vitro CXCL12-induced migration, and in vivo, the simultaneous inhibition of both receptors shows superior anti-tumoral and anti-metastatic activities than the single agent inhibition. In summary, targeting the heterodimerization of CXCR4 and CB2 that are biologically relevant in cancer can be an effective way to reduce proliferation and dissemination in CRC.[spa] El estudio del microambiente tumoral está ganando importancia en las últimas décadas debido a su contribución en la formación y desarrollo del cáncer, además de contribuir en la resistencia de las células tumorales a diferentes terapias. Este microambiente interactúa con las células tumorales y activa diferentes vías. El linfoma folicular (FL), es el linfoma no Hodgkin indolente más común y con mayor dependencia del microambiente tumoral, además es considerado incurable. PI3K desempeña un papel importante en la comunicación con el microambiente, y es importante en múltiples funciones celulares, además de contribuir en la angiogénesis, reclutamiento de células inflamatorias y promover el crecimiento tumoral. Idelalisib es un inhibidor de PI3K (específicamente de la isoforma δ), que se aprobó en 2014 por la FDA. Paralelamente la desregulación de BCL2 es primordial en la patogénesis de FL, como consecuencia de la t (14; 18), presente en un 85% de los pacientes, y por lo tanto es un objetivo atractivo para novedosos enfoques terapéuticos. Venetoclax (ABT-199, AbbVie) es un pequeño inhibidor de BCL2, que mostró unos resultados del primer ensayo clínico no satisfactorios (respuesta global del 38%). De este primer estudio concluimos que Idelalisib interfiere en la comunicación de FL y su microambiente inmune, además potencia la actividad de venetoclax atacando a las células tumorales, lo que representa una terapia de combinación prometedora que puede mejorar el resultado del tratamiento de FL

G-protein coupled receptors activation mechanism: from ligand binding to the transmission of the signal inside the cell

G-protein coupled receptors (GPCRs) are the largest family of pharmaceutical drug targets in the human genome and are modulated by a large variety of en- dogenous and synthetic ligands. GPCRs activation usually depends on agonist binding (except for receptors with basal activity), which stabilizes receptor con- formations and allow the requirement and activation of intracellular transducers. GPCRs are unique receptors and very well studied, since they play an important role in a great number of diseases. They interact with different type of ligands (such as light, peptides, proteins) and different partners in the intracellular part (such as G-proteins or β-arrestins). Based on homology and function GPCRs are divided in five classes: Class A or Rhodopsin, Class B1 or Secretin, Class B2 or Adhesion, Class C or Glutamate, Class F or Frizzled. What is still missing in the state of the art of these receptor, and in particular in Class A, is a global study on different binding cavities with divergent properties, with the aim to discover common binding characteristics, preserved during years of evolution. Gaining more knowledge on common features for ligand recognition shared among all the recep- tors may become crucial to deeply understand the mechanism used to transmit the signal into the cell. In the first step of this thesis we have used all the solved Class A receptors structures to analyze and find, if exist, a common way to transmit the signal inside the cell. We identified and validated ten positions shared between all the binding cavities and always involved in the interaction with ligands. We demonstrated that residues in these positions are conserved and have co-evolved together. In a second step, we used these positions to understand how ligands could be positioned in the binding cavities of three study cases: Muscarinic receptors, Kisspeptin receptors and the GPR3 receptor. We did not have any experimental information a priori. We used homology modeling and docking techniques for the first two cases, adding molecular dynamics simulations in the third case. All the predictions and suggestions from the computational point of view, turned out to be very successful. In particular for the GPR3 receptor we were able to identify and validate by alanine-scanning mutagenesis the role of three functionally relevant residues. The latter were correlated with the constitutive and agonist-stimulated adenylate cyclase activity of GPR3 receptor. Taken together, these results suggest an important role of computational structural biology and pave the way of strong collaborations between computational and experimental researches

Human Immunodeficiency Virus Type 1 Envelope Protein Interaction with Host Cell Coreceptor C-X-C Chemokine Receptor Type 4

Human Immunodeficiency Virus Type 1 (HIV-1) envelope protein is the sole determinant for viral entry and tropism. The ability of HIV-1 to infect susceptible host cells depends on the ability of its envelope protein to engage host cell receptor CD4 and coreceptor C-C Chemokine Receptor Type 5 (CCR5) and/or C-X-C Chemokine Receptor Type 4 (CXCR4). Most naturally occurring infections start with a single CCR5-tropic virion. In approximately 50% of HIV-1 Clade B infected patients, the viral population spontaneous develops the ability to utilize CXCR4 at a late disease stage, and this coreceptor shift corresponds to a poor prognosis for the patients. Clinical application of a CCR5 antagonist drug accelerates this coreceptor shift process. Despite the important implications of HIV-1 tropism on disease pathogenesis, prognosis, and treatment, molecular mechanisms for coreceptor shift and the contributing envelope determinants have not yet been clearly defined due to the dynamic, multimeric, multi-step nature of envelope-coreceptor interaction. In order to better understand the regions on envelope that are important for coreceptor specificity, we studied a panel of HIV-1 envelope amplicon samples from patients who experienced coreceptor shift and treatment failure in a CCR5 antagonist clinical trial. In order to further understand the structure-function relationship of HIV-1 envelope, we utilized both in-depth single clone analysis to fully characterize the functional attributes of individual envelope isolates, and high-throughput deep sequencing analysis to investigate the global envelope variant landscape before and after treatment. In the single clone analysis, we applied both tissue-culture based phenotypic tropism characterization, as well as sequence-based genotypic analysis towards 97 single envelope clones isolated from four samples of two study subjects. Unique quasispecies composition were found post coreceptor shift in two individuals who had different courses of disease. In addition, one subject showed drastic sequence variation between isolates from before and after treatment, and a highly homogeneous viral population post treatment. This suggested a rapid shift to CXCR4-using variants that accounted for failure to respond to CCR5 antagonist treatment. In the deep sequencing analysis, we developed a novel comprehensive approach to sequence a large number of phenotypic validated variants on two next-generation sequencing platforms. This approach offered an unprecedented view of the viral quasispecies landscape in vivo, as well as on the dynamic population change in response to drug selection. Regions of interest that strongly associated with the usage of CXCR4 coreceptor were identified, including previously reported coreceptor specificity sites within and outside V3, the CD4 binding site, and gp41-gp120 interaction site within the same protomer and between neighboring protomers in the envelope trimer spike. These findings have potentially implications in rational design of better coreceptor inhibitors, and development of more accurate predictive algorithms for HIV-1 envelope tropism