Macrophage Transcriptome Profiling After CD58 and TAGAP Silencing

Human monocyte-derived macrophages were derived as described previously^{38 (link)}. Macrophages from five different volunteers were transfected with CD58 siRNA (L-004538-00-0005), TAGAP siRNA (L-008711-01-0005) or control siRNA (D-001810-10-20) by Dharmafect 4 for 2 days (Thermo Scientific). Total RNA was isolated at 6 hours and 24 hours, and global gene expression was profiled using Illumina Human HT-12 Expression BeadChip^{39 (link)}. Differentially expressed genes by at least 1.25-fold between control and CD58 siRNA cells were subjected to pathway enrichment analysis using GeneNetwork analysis^{40 (link)}. After siRNA transfection, macrophages were exposed to live C. albicans at multiplicity of infection (MOI)= 1 for 24 hours, after which the phagocytosis and fungal outgrowth was determined by microscopy. The role of fungal germination was assessed by using the yeast-locked Hgc1-deficient C. albicans strain (provided by Dr. Bernhard Hube, Jena University). Cytokine concentrations were determined by ELISA.

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Kumar V., Cheng S.C., Johnson M.D., Smeekens S.P., Wojtowicz A., Giamarellos-Bourboulis E., Karjalainen J., Franke L., Withoff S., Plantinga T.S., van de Veerdonk F.L., van der Meer J.W., Joosten L.A., Bochud P.Y., Marchetti O., Perfect J.R., Xavier R., Kullberg B.J., Wijmenga C, & Netea M.G. (2014). Immunochip SNP array identifies novel genetic variants conferring susceptibility to candidemia. Nature communications, 5, 4675.

Publication 2014

Dharmafect 4 Human HT-12 Expression BeadChip

C albicans Cells Cytokine Elisa Gene expression Genes Germination Human Infection Macrophages Microscopy Phagocytosis Sirna Strain Transfection Volunteers Yeast

Corresponding Organization : University of Groningen

Other organizations : Radboud University Medical Center, Radboud University Nijmegen, Duke Medical Center, Duke University Hospital, University Hospital of Lausanne, National and Kapodistrian University of Athens, Broad Institute, Harvard University, Massachusetts Institute of Technology, Max Planck Institute for Molecular Genetics, Campbell University

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Variable analysis

independent variables

SiRNA transfection (CD58 siRNA, TAGAP siRNA, control siRNA)

dependent variables

Global gene expression at 6 hours and 24 hours
Phagocytosis of live C. albicans
Fungal outgrowth
Cytokine concentrations

control variables

Monocyte-derived macrophages from five different volunteers
Exposure to live C. albicans at MOI = 1 for 24 hours
Use of yeast-locked Hgc1-deficient C. albicans strain

controls

Positive control: Not explicitly mentioned
Negative control: Control siRNA

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