Macrophage polarization by apoptotic cancer cells - a RNAi high-throughput screen and validation of interleukin 10 regulation

Tumor-associated macrophages (TAM) are a major supportive component within neoplasms and by their plasticity promote all phases of tumor development. Mechanisms of macrophage (M Phi) attraction and differentiation to a tumor-promoting phenotype, defined among others by distinct cytokine patterns such as pronounced immunosuppressive interleukin 10 (IL-10) production, are largely unknown. However, a high apoptosis index within tumors and strong M Phi infiltration correlate with poor prognosis. Thus, I aimed at identifying signaling pathways contributing to generation of TAM-like M Phi by using supernatant of apoptotic cancer cells (ACM) as stimulus. To distinguish novel factors involved in generating TAM-like M Phi, I used an adenoviral RNAi-based approach. The primary read-out was production of IL-10. However, mediators modulating IL-10 were re-validated for their impact on regulation of the cytokines IL-6, IL-8 and IL-12. Following assay development, optimization and down-scaling to a 384-well format, primary human M Phi were transduced with 8495 constructs of the adenoviral shRNA SilenceSelect® library of Galapagos BV, followed by activation to a TAM-like phenotype using ACM. I identified 96 genes involved in IL-10 production in response to ACM and observed a pronounced cluster of 22 targets regulating IL-10 and IL-6. Principal validation of five targets of the IL-10/IL-6 cluster was performed using siRNA or pharmacological inhibitors. Among those, IL-4 receptor-alpha and cannabinoid receptor 2 were confirmed as regulators of IL-10 and IL-6 secretion. One protein identified in the screen, the nerve growth factor (NGF) receptor TRKA was chosen for in-depth validation, based on its involvement in IL-10, IL-6 and IL-12 secretion from ACM-stimulated human M Phi. TRKA possesses a cardinal role in neuronal development, but compelling evidence emerges suggesting participation of TRKA in cancer development. First experiments using pharmacological inhibitors principally confirmed the involvement of TRKA in IL-10 secretion by ACM-stimulated M Phi and revealed PI3K/AKT and to a lesser extend MAPK p38 as important signaling molecules downstream of TRKA activation. Signaling through TRKA required the presence of its ligand NGF, as indicated by NGF neutralization experiments. NGF was not induced by or present in ACM, but was constitutively secreted by M Phi. Interestingly, M Phi responded to authentic NGF with neither AKT and p38 phosphorylation nor IL-10 production. TRKA is well known to be transactivated by other receptors and in neurons its cellular localization is decisive for its function. Inhibitors of common transactivation partners did not influence IL-10 production by human M Phi. Rather, ACM-treatment provoked pronounced translocation of TRKA to the plasma membrane within 10 minutes as observed by immunofluorescence staining. Consequently, I was intrigued to clarify mechanisms of TRKA trafficking in response to ACM. The bioactive lipid sphingosine-1-phosphate (S1P) has been previously identified as important apoptotic cell-derived mediator involved in TAM-like M Phi polarization. Indeed, I observed S1P and src kinase involvement in ACM-mediated IL-10 induction. Furthermore, inhibition of S1P receptor (S1PR) signaling or src kinase activity prevented TRKA translocation, whereas a TRKA inhibitor or anti-NGF did not block TRKA trafficking to the plasma membrane in response to ACM. Thus, autocrine secreted NGF activated TRKA to promote IL-10 secretion, which required previous S1PR/src-dependent translocation of TRKA to the plasma membrane. Following the detailed analysis of IL-10 regulation, I was interested whether other TAM phenotype markers were influenced by ACM and whether their expression was regulated through TRKA-dependent signaling. Five of six markers were up-regulated on mRNA level by ACM, and secretion of IL-6, IL-8 and TNF-alpha was triggered. S1PR-signaling was essential for induction of all but one marker, whereas TRKA signaling was only required for cytokine secretion. Interestingly, none of the investigated TAM markers was regulated identically to IL-10, emphasizing a tight and exclusive regulation machinery of this potent immunosuppressive cytokine. Finally, I aimed to validate the in vitro findings in human ACM-stimulated M Phi. Therefore, I isolated murine TAM as well as other major mononuclear phagocyte populations from primary oncogene-induced breast cancer tissue. Indeed, TRKA-dependent signaling was required for spontaneous cytokine production selectively by primary murine TAM. Besides IL-10, the TRKA pathway was decisive for secretion of IL-6, TNF-alpha and monocyte chemotactic protein-1, indicating its relevance in cancer-associated inflammation. In summary, my findings highlight a fine-tuned regulatory system of S1P-dependent TRKA trafficking and autocrine NGF signaling in TAM biology. Both factors, S1P as well as NGF, might be interesting targets for future cancer therapy.Tumor-assoziierte Makrophagen (TAM) begünstigen verschiedene Phasen der Tumorentwicklung. Bisher ist das Wissen über die Mechanismen der Rekrutierung und Differenzierung von Makrophagen (M Phi) zu TAM, die sich unter anderem durch eine usgeprägte Produktion von immunsuppressivem Interleukin 10 (IL-10) auszeichnen, unvollkommen. Eine Korrelation zwischen dem Ausmaß der M Phi-Infiltration sowie der Zelltotrate innerhalb von Tumoren und einer schlechten Prognose für Krebspatienten wurde berichtet. Auf Grundlage dessen, untersuchte ich in der vorliegenden Arbeit Signalwege, die zur Entstehung von TAM-ähnlichen M Phi führen. Dazu stimulierte ich humane primäre M Phi mit Überständen von apoptotischen Tumorzellen (ACM). Zuerst führte ich ein RNAi-basiertes adenovirales Hochdurchsatzverfahren (HTS) zur Identifizierung unbekannter Faktoren, die zur Entstehung von TAM-ähnlichen M Phi beitragen, durch. Als primären Endpunkt analysierte ich zunächst die Produktion von IL-10. Die identifizierten IL-10-regulierenden Faktoren wurden im nächsten Schritt auf ihre Beteiligung an der Synthese von weiteren tumorfördernden (IL-6, IL-8) und tumorinhibierenden (IL-12) Zytokinen getestet. M Phi wurden mit 8495 adenovirale shRNAs der SilenceSelect® Bibliothek der Firma Galapagos BV transduziert und mit ACM zu TAM-ähnlichen M Phi polarisiert. 96 Gene, die eine Rolle in der ACM-vermittelten IL-10 Produktion spielen, wurden identifiziert. Davon zeigten 22 Gene eine bisher nicht beobachtete Koregulation von IL-10 und IL-6 auf. Fünf Kandidaten aus dieser Gruppe wurden mit Hilfe von Inhibitoren oder siRNA validiert und eine Beteiligung des IL-4 Rezeptors alpha sowie des Cannabinoid Rezeptors 2 an der IL-10 und IL-6 Regulation konnte bestätigt werden. In darauffolgenden Versuchen verifizierte ich im Detail die Beteiligung des nerve growth factor (NGF) Rezeptors TRKA an der Ausbildung TAM-ähnlicher M Phi nach ACM-Behandlung. Die Ausschaltung des Gens im HTS hatte zu einer verminderten Sekretion von IL-10 und IL-6 sowie einer Zunahme von IL-12 geführt. TRKA ist essentiell an der Entwicklung des Nervensystems beteiligt und neuere Berichte weisen auf seine Beteiligung in der Tumorbiologie hin. Pharmakologische Inhibitoren bestätigten die Notwendigkeit von TRKA an der ACM-vermittelten IL-10 Produktion, sowie die TRKA-abhängige Aktivierung der Signalmoleküle PI3K/AKT und, wenn auch weniger ausgeprägt, MAPK p38. Die Neutralisation von NGF demonstrierte, dass von M Phi konstitutiv autokrin sezerniertes NGF für die IL-10 Sekretion benötigt wurde. Synthetisches NGF zeigte jedoch keinen Effekt auf die AKT und p38 Phosphorylierung oder auf die Produktion von IL-10. Interessanterweise legten Immunfluoreszenzaufnahmen von ACM-stimulierten M Phi eine deutliche Zunahme von TRKA an der Plasmamembran nach 10-minütiger Simulation offen, die durch Inhibition von TRKA oder Neutralisation von NGF nicht beeinflusst wurde. Dieses Phänomen war Anlass für weiterführende Studien. Sphingosine-1-Phosphat (S1P) wurde im Vorfeld als wichtiger Mediator in der ACMvermittelten M Phi Polarisierung identifiziert und ich konnte eine Abhängigkeit der IL-10 Produktion von S1P und Src Kinasen zeigen. Des Weiteren wurde die Anreicherung von TRKA an der Plasmamembran durch Inhibitoren von Src sowie S1P Rezeptoren verhindert. Diese Daten indizieren eine komplexe Regulation der ACM-vermittelten IL-10 Produktion in primären M Phi. S1P, generiert von apoptotischen Krebszellen, vermittelt S1P Rezeptor- und Src Kinasen-abhängig die Translokalisation von TRKA an die Zelloberfläche von M Phi. Der Rezeptor wird daraufhin von autokrin sezerniertem NGF aktiviert und initiiert darauffolgend die Produktion von IL-10. In einem weiteren Schritt untersuchte ich zusätzliche biologische Marker, die eine Rolle bei der Funktion von TAM spielen. ACM bewirkte eine starke Sekretion von IL-6, IL-8 und TNF-alpha, sowie eine Hochregulation weiterer Marker auf mRNA-Ebene. Die S1P Rezeptoraktivität war für nahezu alle Marker essentiell. Die Zytokinsekretion war ebenfalls abhängig von TRKA, jedoch wurde keiner der untersuchten Faktoren analog zu IL-10 reguliert. Diese Befunde unterstreichen die fein abgestimmte und exklusive Regulation dieses hochpotenten immunsuppressiven Zytokins. Um meine in vitro Daten zu bestärken, isolierte ich TAM und weitere mononukleare Phagozytenpopulationen aus primärem Onkogen-induzierten Brustkrebsgewebe der Maus und untersuchte die spontane Zytokinproduktion dieser Zellen. Auch in diesem Modell zeigte sich eine, auf TAM beschränkte, TRKA-abhängige Produktion von IL-10, IL-6, TNF-alpha und monocyte chemotactic protein-1, und die Rolle von TRKA im Tumor-assoziierten Entzündungsgeschehen wurde bekräftigt. Zusammenfassend konnte ich Mechanismen der S1P- und NGF-abhängigen TRKA Signaltransduktion und deren Funktion im Bezug auf Makrophagen im Tumorgeschehen identifizieren. Sowohl S1P als auch NGF stellen somit interessante Zielmoleküle in der künftigen Krebstherapie dar

Dialogic science-policy networks for water security governance in the arid Americas

Addressing wicked problems challenging water security requires participation from multiple stakeholders, often with conflicting visions, complicating the attainment of water-security goals and heightening the need for integrative and effective science-policy interfaces. Sustained multi-stakeholder dialogues within science-policy networks can improve adaptive governance and water system resilience. This paper describes what we define as “dialogic science-policy networks,” or interactions -- both in structural and procedural terms -- between scientists and policy-makers that are: 1) interdisciplinary, 2) international (here, inter-American), 3) cross-sectoral, 4) open, 5) continual and iterative in the long-term, and 6) flexible. By fostering these types of interactions, dialogic networks achieve what we call the 4-I criteria for effective science-policy dialogues: inclusivity, involvement, interaction, and influence. Here we present several water-security research and action projects where some of these attributes may be present. Among these, a more comprehensive form of a dialogic network was intentionally created via AQUASEC, a virtual center and network initially fostered by a series of grants from the Inter-American Institute for Global Change Research. Subsequently, AQUASEC has significantly expanded to other regions through direct linkages and additional program support for the International Water Security Network, supported by Lloyd's Register Foundation and other sources. This paper highlights major scientific and policy achievements of a notable suite of science-policy networks, shared practices, methods, and knowledge integrating science and policy, as well as the main barriers overcome in network development. An important gap that remains for future research is the assessment and evaluation of dialogic science-policy networks' long-term outcomes.Fil: Lutz Ley, America N.. El Colegio de Sonora; MéxicoFil: Scott, Christopher A.. University of Arizona; Estados UnidosFil: Wilder, Margaret. University of Arizona; Estados UnidosFil: Varady, Robert G.. University of Arizona; Estados UnidosFil: Ocampo Melgar, Anahi. Universidad de Chile.; ChileFil: Lara Valencia, Francisco. Arizona State University; Estados UnidosFil: Zuniga Teran, Adriana. University of Arizona; Estados UnidosFil: Buechler, Stephanie. University of Arizona; Estados UnidosFil: Díaz Caravantes, Rolando. El Colegio de Sonora; MéxicoFil: Ribeiro Neto, Alfredo. El Colegio de Sonora; MéxicoFil: Pineda Pablos, Nicólas. El Colegio de Sonora; MéxicoFil: Martin, Facundo Damian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Ciencias Humanas, Sociales y Ambientales; Argentin

Saturated Fats Compared With Unsaturated Fats and Sources of Carbohydrates in Relation to Risk of Coronary Heart Disease A Prospective Cohort Study

AbstractBackgroundThe associations between dietary saturated fats and the risk of coronary heart disease (CHD) remain controversial, but few studies have compared saturated with unsaturated fats and sources of carbohydrates in relation to CHD risk.ObjectivesThis study sought to investigate associations of saturated fats compared with unsaturated fats and different sources of carbohydrates in relation to CHD risk.MethodsWe followed 84,628 women (Nurses’ Health Study, 1980 to 2010), and 42,908 men (Health Professionals Follow-up Study, 1986 to 2010) who were free of diabetes, cardiovascular disease, and cancer at baseline. Diet was assessed by a semiquantitative food frequency questionnaire every 4 years.ResultsDuring 24 to 30 years of follow-up, we documented 7,667 incident cases of CHD. Higher intakes of polyunsaturated fatty acids (PUFAs) and carbohydrates from whole grains were significantly associated with a lower risk of CHD comparing the highest with lowest quintile for PUFAs (hazard ratio [HR]: 0.80, 95% confidence interval [CI]: 0.73 to 0.88; p trend <0.0001) and for carbohydrates from whole grains (HR: 0.90, 95% CI: 0.83 to 0.98; p trend = 0.003). In contrast, carbohydrates from refined starches/added sugars were positively associated with a risk of CHD (HR: 1.10, 95% CI: 1.00 to 1.21; p trend = 0.04). Replacing 5% of energy intake from saturated fats with equivalent energy intake from PUFAs, monounsaturated fatty acids, or carbohydrates from whole grains was associated with a 25%, 15%, and 9% lower risk of CHD, respectively (PUFAs, HR: 0.75, 95% CI: 0.67 to 0.84; p < 0.0001; monounsaturated fatty acids, HR: 0.85, 95% CI: 0.74 to 0.97; p = 0.02; carbohydrates from whole grains, HR: 0.91, 95% CI: 0.85 to 0.98; p = 0.01). Replacing saturated fats with carbohydrates from refined starches/added sugars was not significantly associated with CHD risk (p > 0.10).ConclusionsOur findings indicate that unsaturated fats, especially PUFAs, and/or high-quality carbohydrates can be used to replace saturated fats to reduce CHD risk

Enhanced efficacy and increased long-term toxicity of CNS-directed, AAV-based combination therapy for Krabbe disease

Infantile globoid cell leukodystrophy (GLD, Krabbe disease) is a demyelinating disease caused by the deficiency of the lysosomal enzyme galactosylceramidase (GALC) and the progressive accumulation of the toxic metabolite psychosine. We showed previously that central nervous system (CNS)-directed, adeno-associated virus (AAV)2/5-mediated gene therapy synergized with bone marrow transplantation and substrate reduction therapy (SRT) to greatly increase therapeutic efficacy in the murine model of Krabbe disease (Twitcher). However, motor deficits remained largely refractory to treatment. In the current study, we replaced AAV2/5 with an AAV2/9 vector. This single change significantly improved several endpoints primarily associated with motor function. However, nearly all (14/16) of the combination-treated Twitcher mice and all (19/19) of the combination-treated wild-type mice developed hepatocellular carcinoma (HCC). 10 out of 10 tumors analyzed had AAV integrations within the Rian locus. Several animals had additional integrations within or near genes that regulate cell growth or death, are known or potential tumor suppressors, or are associated with poor prognosis in human HCC. Finally, the substrate reduction drug L-cycloserine significantly decreased the level of the pro-apoptotic ceramide 18:0. These data demonstrate the value of AAV-based combination therapy for Krabbe disease. However, they also suggest that other therapies or co-morbidities must be taken into account before AAV-mediated gene therapy is considered for human therapeutic trials

Reducing bias in auditory duration reproduction by integrating the reproduced signal

Duration estimation is known to be far from veridical and to differ for sensory estimates and motor reproduction. To investigate how these differential estimates are integrated for estimating or reproducing a duration and to examine sensorimotor biases in duration comparison and reproduction tasks, we compared estimation biases and variances among three different duration estimation tasks: perceptual comparison, motor reproduction, and auditory reproduction (i.e. a combined perceptual-motor task). We found consistent overestimation in both motor and perceptual-motor auditory reproduction tasks, and the least overestimation in the comparison task. More interestingly, compared to pure motor reproduction, the overestimation bias was reduced in the auditory reproduction task, due to the additional reproduced auditory signal. We further manipulated the signal-to-noise ratio (SNR) in the feedback/comparison tones to examine the changes in estimation biases and variances. Considering perceptual and motor biases as two independent components, we applied the reliability-based model, which successfully predicted the biases in auditory reproduction. Our findings thus provide behavioral evidence of how the brain combines motor and perceptual information together to reduce duration estimation biases and improve estimation reliability

Next-generation sequencing identifies the natural killer cell microRNA transcriptome

Natural killer (NK) cells are innate lymphocytes important for early host defense against infectious pathogens and surveillance against malignant transformation. Resting murine NK cells regulate the translation of effector molecule mRNAs (e.g., granzyme B, GzmB) through unclear molecular mechanisms. MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate the translation of their mRNA targets, and are therefore candidates for mediating this control process. While the expression and importance of miRNAs in T and B lymphocytes have been established, little is known about miRNAs in NK cells. Here, we used two next-generation sequencing (NGS) platforms to define the miRNA transcriptomes of resting and cytokine-activated primary murine NK cells, with confirmation by quantitative real-time PCR (qRT-PCR) and microarrays. We delineate a bioinformatics analysis pipeline that identified 302 known and 21 novel mature miRNAs from sequences obtained from NK cell small RNA libraries. These miRNAs are expressed over a broad range and exhibit isomiR complexity, and a subset is differentially expressed following cytokine activation. Using these miRNA NGS data, miR-223 was identified as a mature miRNA present in resting NK cells with decreased expression following cytokine activation. Furthermore, we demonstrate that miR-223 specifically targets the 3′ untranslated region of murine GzmB in vitro, indicating that this miRNA may contribute to control of GzmB translation in resting NK cells. Thus, the sequenced NK cell miRNA transcriptome provides a valuable framework for further elucidation of miRNA expression and function in NK cell biology

Birth weight and later life adherence to unhealthy lifestyles in predicting type 2 diabetes: prospective cohort study

Objectives To prospectively assess the joint association of birth weight and established lifestyle risk factors in adulthood with incident type 2 diabetes and to quantitatively decompose the attributing effects to birth weight only, to adulthood lifestyle only, and to their interaction. Design: Prospective cohort study. Setting: Health Professionals Follow-up Study (1986-2010), Nurses’ Health Study (1980-2010), and Nurses’ Health Study II (1991-2011). Participants: 149 794 men and women without diabetes, cardiovascular disease, or cancer at baseline. Main outcome measure Incident cases of type 2 diabetes, identified through self report and validated by a supplementary questionnaire. Unhealthy lifestyle was defined on the basis of body mass index, smoking, physical activity, alcohol consumption, and the alternate healthy eating index. Results: During 20-30 years of follow-up, 11 709 new cases of type 2 diabetes were documented. The multivariate adjusted relative risk of type 2 diabetes was 1.45 (95% confidence interval 1.32 to 1.59) per kg lower birth weight and 2.10 (1.71 to 2.58) per unhealthy lifestyle factor. The relative risk of type 2 diabetes associated with a combination of per kg lower birth weight and per unhealthy lifestyle factor was 2.86 (2.26 to 3.63), which was more than the addition of the risk associated with each individual factor, indicating a significant interaction on an additive scale (P for interaction<0.001). The attributable proportions of joint effect were 22% (95% confidence interval 18.3% to 26.4%) to lower birth weight alone, 59% (57.1% to 61.5%) to unhealthy lifestyle alone, and 18% (13.9% to 21.3%) to their interaction. Conclusion: Most cases of type 2 diabetes could be prevented by the adoption of a healthier lifestyle, but simultaneous improvement of both prenatal and postnatal factors could further prevent additional cases

Divergent responses of viral and bacterial communities in the gut microbiome to dietary disturbances in mice

To improve our understanding of the stability of mammalian intestinal communities, we characterized the responses of both bacterial and viral communities in murine fecal samples to dietary changes between high- and low-fat (LF) diets. Targeted DNA extraction methods for bacteria, virus-like particles and induced prophages were used to generate bacterial and viral metagenomes as well as 16S ribosomal RNA amplicons. Gut microbiome communities from two cohorts of C57BL/6 mice were characterized in a 6-week diet perturbation study in response to high fiber, LF and high-refined sugar, milkfat (MF) diets. The resulting metagenomes from induced bacterial prophages and extracellular viruses showed significant overlap, supporting a largely temperate viral lifestyle within these gut microbiomes. The resistance of baseline communities to dietary disturbances was evaluated, and we observed contrasting responses of baseline LF and MF bacterial and viral communities. In contrast to baseline LF viral communities and bacterial communities in both diet treatments, baseline MF viral communities were sensitive to dietary disturbances as reflected in their non-recovery during the washout period. The contrasting responses of bacterial and viral communities suggest that these communities can respond to perturbations independently of each other and highlight the potentially unique role of viruses in gut health

Impact of the Resident Microbiota on the Nutritional Phenotype of Drosophila melanogaster

Background: Animals are chronically infected by benign and beneficial microorganisms that generally promote animal health through their effects on the nutrition, immune function and other physiological systems of the host. Insight into the host-microbial interactions can be obtained by comparing the traits of animals experimentally deprived of their microbiota and untreated animals. Drosophila melanogaster is an experimentally tractable system to study host-microbial interactions. Methodology/Principal Findings: The nutritional significance of the microbiota was investigated in D. melanogaster bearing unmanipulated microbiota, demonstrated by 454 sequencing of 16S rRNA amplicons to be dominated by the a-proteobacterium Acetobacter, and experimentally deprived of the microbiota by egg dechorionation (conventional and axenic flies, respectively). In axenic flies, larval development rate was depressed with no effect on adult size relative to conventional flies, indicating that the microbiota promotes larval growth rates. Female fecundity did not differ significantly between conventional and axenic flies, but axenic flies had significantly reduced metabolic rate and altered carbohydrate allocation, including elevated glucose levels. Conclusions/Significance: We have shown that elimination of the resident microbiota extends larval development and perturbs energy homeostasis and carbohydrate allocation patterns of of D. melanogaster. Our results indicate that th

American Gut: an Open Platform for Citizen Science Microbiome Research

McDonald D, Hyde E, Debelius JW, et al. American Gut: an Open Platform for Citizen Science Microbiome Research. mSystems. 2018;3(3):e00031-18