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Loss of DDX3X compromises the immune response of macrophages to Listeria monocytogenes.

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posted on 2018-11-26, 20:22 authored by Daniel Szappanos, Roland Tschismarov, Thomas Perlot, Sandra Westermayer, Katrin Fischer, Ekaterini Platanitis, Fabian Kallinger, Maria Novatchkova, Caroline Lassnig, Mathias Müller, Veronika Sexl, Keiryn L. Bennett, Michelle Foong-Sobis, Josef M. Penninger, Thomas Decker

A Bone marrow-derived macrophages (BMDMs) of Ddx3xfl/y CreERT2 male and Ddx3xfl/fl CreERT2 female mice were infected with L. monocytogenes (strain LO28) with a multiplicity of infection (MOI) of 10. Colony-forming units (CFU) were determined 1, 2, 4 or 8 h after infection by plating serial dilutions of lysed cells on brain–heart infusion (BHI) agar plates. The last two columns in the right panel represent cells pretreated with IFNγ over night. n = 6, analyzed in 2 independent experiments, for male BMDMs (left panel). n = 12, analyzed in 4 independent experiments, for female BMDMs (right panel). Data are represented as mean values +/- standard deviations (SD). Statistical analysis was performed using the unpaired, two-tailed Student's t-test. * P<0.05. B BMDM of mice with the indicated genotypes were left untreated or infected with L. monocytogenes at MOI 10 for 4 hours as described below. RNA isolated from the infected cells was isolated and subjected to RNA-Seq. Left panel: heat map of log 2-fold gene expression changes between Listeria-treated and untreated samples for genes differentially expressed upon treatment in WT samples of either sex (padj <0.01, abs(logFC)>1; DeSeq2 v1.18.1). Genes assigned to the GO category GO:0045087 innate immune response are indicated on the left. From left to right, genes from female knock-out, female wild-type, male knock-out and male wild-type cells (as indicated below the lanes) are compared. Upper right panel: functional enrichment analysis of the differentially expressed genes using DAVID v6.8; the top 10-enriched GO terms ranked by p-value for the category GOTERM_BP_3 are shown. Lower right panel: Venn diagram of gene sets differentially expressed upon Lm infection in the four experimental conditions displayed in the heat map (DESeq2 v 1.18.1; absolute log2 fold change > 1; FDR < 0.01). Solid blue: Lm-infected versus uninfected in macrophages representing a male wildtype genotype (DDX3Xfl/y); light blue: Lm-infected versus uninfected in macrophages representing a male DDX3X-deficient genotype (DDX3X-/y); Solid red: Lm-infected versus uninfected in macrophages representing a female wildtype genotype (DDX3Xfl/fl); light red: Lm-infected versus uninfected in macrophages representing a female DDX3X-deficient genotype (DDX3X-/-). C Bone marrow-derived macrophages (BMDMs) of Ddx3xfl/y CreERT2 male and Ddx3xfl/fl CreERT2 female mice with or without 4_OHT-mediated DDX3X deletion, were infected with L. monocytogenes (strain LO28) with a multiplicity of infection (MOI) of 10. After 4 h of infection total RNA was isolated and analyzed by qPCR for expression of the indicated genes. Upper panels show results from female cells, lower panels show results from male cells, normalized to their respective controls (male and female macrophages without 4-OHT treatment = 100%). Bars represent mean values +/- standard deviations (SD) of at least three independent experiments. Statistical significance was calculated using the paired, two-tailed Student's t-test. *P<0.05, **P<0.01, ***P<0.005. D Primers flanking intron sequences were chosen to analyze the primary unprocessed transcripts in untreated or 4-OHT treated BMDMs of Ddx3xfl/fl CreERT2 female mice. Cells were infected with L. monocytogenes (strain LO28) with a multiplicity of infection (MOI) of 10. After 2 and 4 h of infection total RNA was isolated and analyzed by qPCR for expression of the indicated primary transcripts. Bars represent mean values +/- standard deviations (SD) of at least three independent experiments, normalized to male and female controls (macrophages without 4-OHT treatment = 100%). Statistical significance was calculated using the paired, two-tailed Student's t-test. *P<0.05, **P<0.01, ***P<0.005.

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