Pattern recognition receptors for fungi include dectin-1 and mannose receptor, and these mediate phagocytosis, as well as production of cytokines, reactive oxygen species, and the lipid mediator leukotriene B4 (LTB4). The influence of G protein-coupled receptor ligands such as LTB4 on fungal pattern recognition receptor expression is unknown. In this study, we investigated the role of LTB4 signaling in dectin-1 expression and responsiveness in macrophages. Genetic and pharmacologic approaches showed that LTB4 production and signaling through its high-affinity G protein-coupled receptor leukotriene B4 receptor 1 (BLT1) direct dectin-1–dependent binding, ingestion, and cytokine production both in vitro and in vivo. Impaired responses to fungal glucans correlated with lower dectin-1 expression in macrophages from leukotriene (LT)- and BLT1-deficent mice than their wild-type counterparts. LTB4 increased the expression of the transcription factor responsible for dectin-1 expression, PU.1, and PU.1 small interfering RNA abolished LTB4-enhanced dectin-1 expression. GM-CSF controls PU.1 expression, and this cytokine was decreased in LT-deficient macrophages. Addition of GM-CSF to LT-deficient cells restored expression of dectin-1 and PU.1, as well as dectin-1 responsiveness. In addition, LTB4 effects on dectin-1, PU.1, and cytokine production were blunted in GM-CSF−/− macrophages. Our results identify LTB4-BLT1 signaling as an unrecognized controller of dectin-1 transcription via GM-CSF and PU.1 that is required for fungi-protective host responses.

Candida albicans is the most common opportunistic fungal pathogen and causes local and systemic disease in immunocompromised patients. Alveolar macrophages (AMs) are pivotal for the clearance of C. albicans from the lung. Activated AMs secrete 5-lipoxygenase-derived leukotrienes (LTs), which in turn enhance phagocytosis and microbicidal activity against a diverse array of pathogens. Our aim was to investigate the role of LTB 4 and LTD 4 in AM antimicrobial functions against C. albicans and the signaling pathways involved. Pharmacologic and genetic inhibition of LT biosynthesis as well as receptor antagonism reduced phagocytosis of C. albicans when compared with untreated or WT controls. Conversely, exogenous LTs of both classes augmented base-line C. albicans phagocytosis by AMs. Although LTB 4 enhanced mainly mannose receptor-dependent fungal ingestion, LTD 4 enhanced mainly dectin-1 receptor-mediated phagocytosis. LT enhancement of yeast ingestion was dependent on protein kinase C-␦ (PKC␦) and PI3K but not PKC␣ and MAPK activation. Both LTs reduced activation of cofilin-1, whereas they enhanced total cellular F-actin; however, LTB 4 accomplished this through the activation of LIM kinases (LIMKs) 1 and 2, whereas LTD 4 did so exclusively via LIMK-2. Finally, both exogenous LTB 4 and LTD 4 enhanced AM fungicidal activity in an NADPH oxidase-dependent manner. Our data identify LTB 4 and LTD 4 as key mediators of innate immunity against C. albicans, which act by both distinct and conserved signaling mechanisms to enhance multiple antimicrobial functions of AMs.

Human pluripotent stem cells (hPSCs) may significantly improve drug development pipeline, serving as an in vitro system for the identification of novel leads, and for testing drug toxicity. Furthermore, these cells may be used to address the issue of differential drug response, a phenomenon greatly influenced by genetic factors. This application depends on the availability of hPSC lines from populations with diverse ancestries. So far, it has been reported that most lines of hPSCs derived worldwide are of European or East Asian ancestries. We have established 23 lines of hPSCs from Brazilian individuals, and we report the analysis of their genomic ancestry. We show that embryo-derived PSCs are mostly of European descent, while induced PSCs derived from participants of a national-wide Brazilian cohort study present high levels of admixed European, African and Native American genomic ancestry. Additionally, we use high density SNP data and estimate local ancestries, particularly those of CYP genes loci. Such information will be of key importance when interpreting variation among cell lines with respect to cellular phenotypes of interest. The availability of genetically admixed lines of hPSCs will be of relevance when setting up future in vitro studies of drug response.Human pluripotent stem cells (hPSCs) are an ideal cell source for the development of cell based assays for drug response. In addition to their extensive proliferation and genetic stability in culture, these human cells can give rise to primary cell types relevant for drug response, including cardiomyocytes, hepatocytes and neurons 1 . Individual differences in drug response can result from the effects of age, sex, disease, ancestry, or drug interactions, but genetic factors play a major role in influencing adverse drug reactions and ineffective therapy 2 . Thus, a collection of genetically diverse lines of hPSCs is required for a broader study of differential drug response in vitro 3 .To date, most lines of hPSCs available are of European or Eastern Asian ancestry 4 , although one hiPSC line from a Native American, and one from an African (Yoruba) have been reported 5 . More recently, one hiPSC line of African American and of Hispanic Latino ancestry each have been described, although the authors do not show the genetic evidence of those ethnicities 6 .The Brazilian population results from 500 years of admixture among the original Native Americans, Europeans (mostly Portuguese), and sub-Saharan Africans, most of which were brought to the country as slaves 7 . Different analyses of genomic ancestry in Brazil have shown that, on average, the urban population has 60% contribution from European, 25% from African, and 15% from Native American populations, although these proportions vary according to the Brazilian geographic region analyzed [7][8][9][10][11] . Therefore, from the genetic point of view the Brazilian population is significantly distinct from the ancestral populations, containing novel genotypes and haplotypes that may impact various phenotyp...

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The ability to reprogram somatic cells into induced pluripotent stem cells (hiPSCs) has led to the generation of large collections of cell lines from thousands of individuals with specific phenotypes, many of which will be shared among different research groups as invaluable tools for biomedical research. As hiPSC-based research involves extensive culture of many cell lines, the issue periodic cell line identification is particularly important to ensure that cell line identity remains accurate. Here we analyzed the different commercially available genotyping methods considering ease of in-house genotyping, cost and informativeness, and applied one of them in our workflow for hiPSC generation. We show that the chosen STR method was able to establish a unique DNA profile for each of the 35 individuals/hiPSC lines at the examined sites, as well as identify two discrepancies resulting from inadvertently exchanged samples. Our results highlight the importance of hiPSC line genotyping by an in-house method that allows periodic cell line identification and demonstrate that STR is a useful approach to supplement less frequent karyotyping and epigenetic evaluations.