Adipose tissue macrophages in non-rodent mammals: a comparative study

The stromal vascular fraction (SVF) of adipose tissue in rodents and primates contains mesenchymal stem cells and immune cells. SVF cells have complex metabolic, immune and endocrine functions with biomedical impact. However, in other mammals, the amount of data on SVF stem cells is negligible and whether the SVF hosts immune cells is unknown. In this study, we show that the SVF is rich in immune cells, with a dominance of adipose tissue macrophages (ATMs) in cattle (Bos primigenius taurus), domestic goat (Capra aegagrus hircus), domestic sheep (Ovis aries), domestic cat (Felis catus) and domestic dog (Canis familiaris). ATMs of these species are granulated lysosome-rich cells with lamellipodial protrusions and express the lysosome markers acid phosphatase 5 (ACP-5) and Mac-3/Lamp-2. Using ACP-5 and Mac-3/Lamp-2 as markers, we additionally detected ATMs in other species, such as the domestic horse (Equus ferus caballus), wild boar (Sus scrofa) and red fox (Vulpes vulpes). Feline and canine ATMs also express the murine macrophage marker F4/80 antigen. In the lean condition, the alternative macrophage activation marker CD206 is expressed by feline and canine ATMs and arginase-1 by feline ATMs. Obesity is associated with interleukin-6 and interferon gamma expression and with overt tyrosine nitration in both feline and canine ATMs. This resembles the obesity-induced phenotype switch of murine and human ATMs. Thus, we show, for the first time, that the presence of ATMs is a general trait of mammals. The interaction between the adipose cells and SVF immune cells might be evolutionarily conserved among mammals.University of Ul

Cell-based fuzzy metrics enhance High Content Screening (HCS) assay robustness

High-content screening (HCS) allows the exploration of complex cellular phenotypes by automated microscopy and is increasingly being adopted for small interfering RNA genomic screening and phenotypic drug discovery. We introduce a series of cell-based evaluation metrics that have been implemented and validated in a mono-parametric HCS for regulators of the membrane trafficking protein caveolin 1 (CAV1) and have also proved useful for the development of a multiparametric phenotypic HCS for regulators of cytoskeletal reorganization. Imaging metrics evaluate imaging quality such as staining and focus, whereas cell biology metrics are fuzzy logic–based evaluators describing complex biological parameters such as sparseness, confluency, and spreading. The evaluation metrics were implemented in a data-mining pipeline, which first filters out cells that do not pass a quality criterion based on imaging metrics and then uses cell biology metrics to stratify cell samples to allow further analysis of homogeneous cell populations. Use of these metrics significantly improved the robustness of the monoparametric assay tested, as revealed by an increase in Z′ factor, Kolmogorov-Smirnov distance, and strict standard mean difference. Cell biology evaluation metrics were also implemented in a novel supervised learning classification method that combines them with phenotypic features in a statistical model that exceeded conventional classification methods, thus improving multiparametric phenotypic assay sensitivity.The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by Fondo de Investigaciones Sanitarias PS09/01028 to María C. Montoya. Miguel A. del Pozo is supported by the Ministry of Economy and Competitiveness grants SAF2011-25047 and CSD 2009-00016. José R. Dorronsoro received partial support from the MINECO grant TIN201021575-C02-01 and from the UAM-ADIC Chair for Machine Learning. Carlos Alaíz is supported by the FPU-MEC grant AP2008-00167. Alberto Torres is supported by an FPU grant of the Universidad Autónoma de Madrid FPU12/05163. The Centro Nacional de Investigaciones Cardiovasculares is supported by the MINECO and the Pro-CNIC Foundation

ITGB1-dependent upregulation of Caveolin-1 switches TGF beta signalling from tumour-suppressive to oncogenic in prostate cancer

Caveolin-1 (CAV1) is over-expressed in prostate cancer (PCa) and is associated with adverse prognosis, but the molecular mechanisms linking CAV1 expression to disease progression are poorly understood. Extensive gene expression correlation analysis, quantitative multiplex imaging of clinical samples, and analysis of the CAV1-dependent transcriptome, supported that CAV1 re-programmes TGF beta signalling from tumour suppressive to oncogenic (i.e. induction of SLUG, PAI-1 and suppression of CDH1, DSP, CDKN1A). Supporting such a role, CAV1 knockdown led to growth arrest and inhibition of cell invasion in prostate cancer cell lines. Rationalized RNAi screening and high-content microscopy in search for CAV1 upstream regulators revealed integrin beta1 (ITGB1) and integrin associated proteins as CAV1 regulators. Our work suggests TGF beta signalling and beta1 integrins as potential therapeutic targets in PCa over-expressing CAV1, and contributes to better understand the paradoxical dual role of TGF beta in tumour biology.Peer reviewe

Caveolar domain organization and trafficking is regulated by Abl kinases and mDia1

54 páginas, 8 figurasCaveolin-1 (Cav1)/caveolae biology is intimately linked to actin dynamics and adhesion receptors. Caveolar domains are organized in hierarchical levels of complexity from curved or flatten caveolae to large, higher-order caveolar rosettes. We report that stress fibers controlled by Abl kinases and mDia1 determine the level of caveolar domain organization, which conditions the subsequent inward trafficking of caveolar domains induced upon loss of cell adhesion from the extracellular matrix. Abl-deficient cells show decreased content of stress fibers, a smaller stress-fiber co-aligned Cav1 pool and increased clustering of Cav1/caveolae at the cell surface. Defective caveolar linkage to stress fibers prevents the formation of big caveolar rosettes upon loss of cell adhesion, correlating with a lack of inward trafficking. Live imaging of stress fibers and Cav1 showed that the actin-linked Cav1 pool loses its spatial organization in the absence of actin polymerization and is dragged and clustered by depolymerizing filaments. We identify mDia1 as the actin polymerization regulator downstream of Abl kinases that controls the stress fiber-linked Cav1 pool. mDia1 knockdown results in Cav1/caveolae clustering and defective inward trafficking upon loss of cell adhesion. In contrast, cell elongation imposed by the excess of stress fibers induced by active mDia1 flattens caveolae. Furthermore, active mDia1 rescues the actin co-aligned Cav1 pool and Cav1 inward trafficking upon loss of adhesion in Abl-deficient cells. Thus, caveolar domain organization and trafficking are tightly coupled to adhesive and stress fiber regulatory pathwaysWe thank A. M. Pendergast, T. Koleske, J. Wehland, K. Rottner, S. Narumiya, R. Wedlich-Soldner and F. Sánchez-Madrid for valuable reagents. We thank S. Sánchez for excellent technical assistance, S. Calleja, I. Salanueva, C. Patiño, J. Bueno, I. Cotillo and the CNIC Microscopy Unit for technical assistance. We thank S. Bartlett and T. Pellinen for critical reading of the manuscript and R. Parton and A. G. Arroyo for helpful suggestions. AE was supported by the Ramón y Cajal Program (MICINN, Spanish Ministry of Science and Innovation), OM by a predoctoral fellowship from the Instituto de Salud Carlos III (MICINN). MAdP was supported by the MICINN through grants SAF2008-02100, RTICC RD06/0020/1033 and CSD 2009-00016, by EUROHORCS (European Heads Of Research Councils) and the European Science Foundation (ESF) through a EURYI (European Young Investigator) award, and by the EMBO Young Investigator Programme. OL was supported by grants SAF2008-00451, SAF2011-22988 and RD06/0020/1001 (MICINN). The CNIC is supported by the MICINN and the Pro-CNIC Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of this manuscriptPeer reviewe