Tissue-resident macrophages are heterogeneous as a consequence of anatomical niche-specific functions. Many populations self-renew independently of bone marrow in the adult, but the molecular mechanisms of this are poorly understood. We determined a transcriptional profile for the major self-renewing population of peritoneal macrophages in mice. These cells specifically expressed the transcription factor Gata6. Selective deficiency of Gata6 in myeloid cells caused substantial alterations in the transcriptome of peritoneal macrophages. Gata6 deficiency also resulted in dysregulated peritoneal macrophage proliferative renewal during homeostasis and in response to inflammation, which was associated with delays in the resolution of inflammation. Our investigations reveal that the tissue macrophage phenotype is under discrete tissue-selective transcriptional control and that this is fundamentally linked to the regulation of their proliferation renewal.

Exosomes are nanometer-sized vesicles, secreted by various cell types, present in biological fluids that are particularly rich in membrane proteins. Ex vivo analysis of exosomes may provide biomarker discovery platforms and form non-invasive tools for disease diagnosis and monitoring. These vesicles have never before been studied in the context of bladder cancer, a major malignancy of the urological tract. We present the first proteomics analysis of bladder cancer cell exosomes. Using ultracentrifugation on a sucrose cushion, exosomes were highly purified from cultured HT1376 bladder cancer cells and verified as low in contaminants by Western blotting and flow cytometry of exosome-coated beads. Solubilization in a buffer containing SDS and DTT was essential for achieving proteomics analysis using an LC-MALDI-TOF/TOF MS approach. We report 353 high quality identifications with 72 proteins not previously identified by other human exosome proteomics studies. Overrepresentation analysis to compare this data set with previous exosome proteomics studies (using the ExoCarta database) revealed that the proteome was consistent with that of various exosomes with particular overlap with exosomes of carcinoma origin. Interrogating the Gene Ontology database highlighted a strong association of this proteome with carcinoma of bladder and other sites. The data also highlighted how homology among human leukocyte antigen haplotypes may confound MASCOT designation of major histocompatability complex Class I nomenclature, requiring data from PCR-based human leukocyte antigen haplotyping to clarify anomalous identifications. Validation of 18 MS protein identifications (including basigin, galectin-3, trophoblast glycoprotein (5T4), and others) was performed by a combination of Western blotting, flotation on linear sucrose gradients, and flow cytometry, confirming their exosomal expression. Some were confirmed positive on urinary exosomes from a bladder cancer patient. In summary, the exosome proteomics data set presented is of unrivaled quality. The data will aid in the development of urine exosome-based clinical tools for monitoring disease and will inform follow-up studies into varied aspects of exosome manufacture and function. Molecular & Cellular Proteomics 9:1324 -1338, 2010.

We performed a two-stage genome screen to search for novel risk factors for late-onset Alzheimer disease (AD). The first stage involved genotyping 292 affected sibling pairs using 237 markers spaced at approximately 20 cM intervals throughout the genome. In the second stage, we genotyped 451 affected sibling pairs (ASPs) with an additional 91 markers, in the 16 regions where the multipoint LOD score was greater than 1 in stage I. Ten regions maintained LOD scores in excess of 1 in stage II, on chromosomes 1 (peak B), 5, 6, 9 (peaks A and B), 10, 12, 19, 21, and X. Our strongest evidence for linkage was on chromosome 10, where we obtained a peak multipoint LOD score (MLS) of 3.9. The linked region on chromosome 10 spans approximately 44 cM from D10S1426 (59 cM) to D10S2327 (103 cM). To narrow this region, we tested for linkage disequilibrium with several of the stage II microsatellite markers. Of the seven markers we tested in family-based and case control samples, the only nominally positive association we found was with the 167 bp allele of marker D10S1217 (chi-square=7.11, P=0.045, df=1).

Chronic age-related degenerative disorders, including the formation of chronic leg wounds, may occur due to aging of the stromal tissues and ensuing dysfunctional cellular responses. This study investigated the impact of environmental-driven cellular aging on wound healing by conducting a comprehensive analysis of chronic wound fibroblast (CWF) behavior in comparison with patient-matched healthy skin normal fibroblasts (NF). The dysfunctional wound healing abilities of CWF correlated with a significantly reduced proliferative life span and early onset of senescence compared with NF. However, pair-wise comparisons of telomere dynamics between NF and CWF indicated that the induction of senescence in CWF was telomere-independent. Microarray and functional analysis suggested that CWFs have a decreased ability to withstand oxidative stress, which may explain why these cells prematurely senescence. Microarray analysis revealed lower expression levels of several CXC chemokine genes (CXCL-1, -2, -3, -5, -6, -12) in CWF compared with NF (confirmed by ELISA). Functionally, this was related to impaired neutrophil chemotaxis in response to CWF-conditioned medium. Although the persistence of non-healing wounds is, in part, due to prolonged chronic inflammation and bacterial infection, our investigations show that premature fibroblast aging and an inability to correctly express a stromal address code are also implicated in the disease chronicity.

International audienceLittle is known about the senescent phenotype of human vascular smooth muscle cells (VSMCs) and the potential involvement of senescent VSMCs in age-related vascular disease, such as atherosclerosis. As such, VSMCs were grown and characterised to generate senescent VSMCs needed for microarray analysis (Affymetrix). Comparative analysis of the transcriptome profiles of early (14 CPD) and late (39-42 CPD) passage VSMCs found a total of 327 probesets called as differentially expressed: 149 are up-regulated in senescence and 178 repressed (-value < 0.5%, minimum effect size of at least 2-fold differential regulation, explore data at www.madras.cf.ac.uk/vsmc). Data mining shows a differential regulation of genes at senescence associated with the development of atherosclerosis and vascular calcification. These included genes with roles in inflammation (IL1β, IL8, ICAM1, TNFAP3, ESM1 and CCL2), tissue remodelling (VEGF, VEGFβ, ADM and MMP14) and vascular calcification (MGP, BMP2, SPP1, OPG and DCN). The microarray data for IL1β, IL8 and MGP were validated by either, ELISA, Western blot analysis or RT-PCR. These data thus provide the first evidence for a role of VSMC senescence in the development of vascular calcification and provides further support for the involvement of senescent VSMCs in the progression of atherosclerosis

The thyrotropin receptor (TSHR) is expressed during lineage-specific differentiation (e.g. adipogenesis) and is activated by TSH, thyroid-stimulating antibodies, and gain-of-function mutations (TSHR*). Comparison of gene expression profiles of nonmodified human preadipocytes (n = 4) with the parallel TSHR* population revealed significant up-regulation of 27 genes including hyaluronan (HA) synthases (HAS) 1 and 2. The array data were confirmed by quantitative PCR of HAS1 and HAS2 and enzyme-linked immunosorbent assay measurement of HA; all values were significantly increased (p < 0.03) in TSHR*-expressing preadipocytes (n = 10). Preadipocytes (n = 8) treated with dibutyryl (db)-cAMP display significantly increased HAS1 and HAS2 transcripts, HAS2 protein, and HA production (p < 0.02). HAS1 or HAS2 small interfering RNA treatment of db-cAMP-stimulated preadipocytes (n = 4) produced 80% knockdown in HAS1 or 61% knockdown in HAS2 transcripts (compared with scrambled), respectively; the corresponding HA production was reduced by 49 or 38%. Reporter assays using A293 cells transfected with HAS1 promoter-driven plasmids containing or not containing the proximal CRE and treated with db-cAMP revealed that it is functional. Chromatin immunoprecipitation, using a cAMP-responsive element-binding protein antibody, of db-cAMP-treated preadipocytes (n = 4) yielded products for HAS1 and HAS2 with relative fold increases of 3.3 ± 0.8 and 2.6 ± 0.9, respectively. HA accumulates in adipose/connective tissues of patients with thyroid dysfunction. We investigated the contributions of TSH and thyroid-stimulating antibodies and obtained small (9–24%) but significant (p < 0.02) increases in preadipocyte HA production with both ligands. Similar results were obtained with a TSHR monoclonal antibody lacking biological activity (p < 0.05). We conclude that TSHR activation is implicated in HA production in preadipocytes, which, along with thyroid hormone level variation, explains the HA overproduction in thyroid dysfunction.

The inflammatory activation and recruitment of defined myeloid populations is essential for controlling the bridge between innate and adaptive immunity and shaping the immune response to microbial challenge. However, these cells exhibit significant functional heterogeneity and the inflammatory signals that differentially influence their effector characteristics are poorly characterized. In this study, we defined the phenotype of discrete subsets of effective antigen‐presenting cells (APCs) in the peritoneal cavity during peritonitis. When the functional properties of these cells were compared to inflammatory monocyte‐derived macrophages we noted differential responses to the immune‐modulatory cytokine IL‐10. In contrast to the suppressive actions of IL‐10 on inflammatory macrophages, the recruitment of APCs was relatively refractory and we found no evidence for selective inhibition of APC differentiation. This differential response of myeloid cell subsets to IL‐10 may thus have limited impact on development of potentially tissue‐damaging adaptive immune responses, while restricting the magnitude of the inflammatory response. These findings may have clinical relevance in the context of peritoneal dialysis patients, where recurrent infections are associated with immune‐mediated membrane dysfunction, treatment failure, and increased morbidity.

Guitart et al. performed an in vivo genetic dissection of the Krebs cycle enzyme fumarate hydratase (Fh1) in the hematopoietic system. Their investigations revealed multifaceted functions of Fh1 in the regulation of hematopoietic stem cell biology and leukemic transformation.