GM-CSF Down-Regulates TLR Expression via the Transcription Factor PU.1 in Human Monocytes

<div><p>Toll-like receptors (TLR) are crucial sensors of microbial agents such as bacterial or viral compounds. These receptors constitute key players in the induction of inflammation, e.g. in septic or chronic inflammatory diseases. Colony-stimulating factors (CSFs) such as granulocyte-macrophage-CSF (GM-CSF) or granulocyte-CSF (G-CSF) have been extensively investigated in their capacity to promote myelopoiesis in febrile neutropenia or to overcome immunosuppression in patients suffering from sepsis-associated neutropenia or from monocytic immunoincompetence. We report here that GM-CSF, downregulates TLR1, TLR2 and TLR4 in a time- and dose-dependent fashion in human monocytes. Diminished pathogen recognition receptor expression was accompanied by reduced downstream p38 and extracellular-signal-regulated kinase (ERK) signaling upon lipoteichoic acid (LTA) and lipopolysaccharide (LPS) binding—and accordingly led to impaired proinflammatory cytokine production. Knockdown experiments of the transcription factors PU.1 and VentX showed that GM-CSF driven effects on TLR regulation is entirely PU.1 but not VentX dependent. We further analysed monocyte TLR and CD14 expression upon exposure to the IMID® immunomodulatory drug Pomalidomide (CC-4047), a Thalidomide analogue known to downregulate PU.1. Indeed, Pomalidomide in part reversed the GM-CSF-mediated effects. Our data indicate a critical role of PU.1 in the regulation of TLR1, 2, 4 and of CD14, thus targeting PU.1 ultimately results in TLR modulation. The PU.1 mediated immunomodulatory properties of GM-CSF should be taken into consideration upon usage of GM-CSF in inflammatory or infection-related conditions.</p></div

TLR and CD14 downregulation by GM-CSF is dose-dependent.

<p>Monocytes were treated with increasing doses of GM-CSF from 0.1 to 1000 U/ml for 48 hours and TLR1 (a), TLR2 (b), TLR4 (c) and CD14 (d) mean fluorescence intensities (MFI) were analysed by flow cytometry. Data of four individual experiments were analysed, error bars show means of MFI ± SD.</p

PU.1 knock down reverses CD14 and TLR downregulation in human monocytes.

<p>(a) Silencing of PU.1 results in almost 70% reduction of PU.1 mRNA levels after a time period of 24 hours. (b) Overlay histograms of GM-CSF treated human monocytes GM-CSF-induced TLR downregulation (dark-grey histograms) is abrogated in PU.1 silenced cells (blue histograms). Shaded histograms represent TLR baseline expression in untreated cells. (c) Silencing of VentX, however, did not reverse GM-CSF induced TLR-downregulation in human monocytes. Overlay histograms shown are representative for three individually performed experiments.</p

GM-CSF downregulates TLR1, TLR2, TLR4 and CD 14 in a time-dependent fashion.

<p>(a-d) Monocytes were cultured in presence or absence of 100 U/ml GM-CSF in a time course from 6,12, 24, to 48 h. TLR and CD14 mean fluorescence intensities (MFI) on human monocytes were determined by flow cytometry. Changes of mean fluorescence intensities (MFI) over time were calculated at each time point (MFIGM-CSF−MFI_medium) and compared to baseline expression at the time point 0. Data in Fig 1 a-d represent means ± SD; *** p<0.001. (e-g) Representative flow cytometry dot plots and overlay histograms of GM-CSF treated (red) und untreated (black) CD14⁺ cells are shown for TLR1 (e), TLR2 (f) and TLR 4 (g). (h and i) mRNA transcription of TLR2 and TLR4 genes in monocytes was quantified by multiplex real-time PCR in presence or absence of 100 U/ml GM-CSF in a time course of 2, 4 and 8 hours. TLR mRNA levels were normalized to 18S house keeping gene expression and changes over time were compared to mRNA baseline expression at time point 0 which was set to 1. Data of 3 individually performed experiments show fold change ± SD; * p<0.05.</p

Pomalidomide counters GM-CSF-triggered CD14 and TLR downregulation and partly reverts the GM-CSF-induced effects on cytokine production.

<p>(a-d) Treatment with pomalidomide alone did not change monocyte TLR and CD 14 but revert the GM-CSF-induced downregulation of TLR1 (a), TLR2 (b), TLR4 (c) and CD14 (d). (e and f) Cytokine staining on single cell level by flow cytometry shows that presence of GM-CSF (dark grey histograms) inhibited TNF-α cytokine production in CD14⁺ monocytes upon LPS (e) or LTA (f) stimulation. Additional administration of pomalidomide prior to GM-CSF partly reverted the GM-CSF-induced dampening of proinflammatory cytokine production after LPS or LTA challenge (open black histograms). Shaded histograms show robust TNF-α production in monocytes stimulated with LTA or LPS alone. Histograms shown are from three individually performed experiments.</p

Decreased of downstream signalling protein phosphorylation and intracellular TNF-α production in GM-CSF treated CD14⁺ cells upon TLR ligand induction.

<p>(a) Negatively depleted CD14⁺ cells were stimulated for 10 min with 100 ng LPS. Cells were fixed, permeabilized and stained with phospho-specific mAb to determine p-p38, p-ERK 1/2 and p-p65 activation. Dark-gray histograms show cells treated with medium only while shaded histograms show cells challenged with LPS. GM-CSF treatment prior to TLR engagement (100U/ml, red histograms) resulted in markedly reduced phosphorylation of p38, p65 and ERK1/2. Histograms shown are representative of three individually performed experiments. (b and c) Monocytes were incubated for 48 h in absence or presence of 100 U/ml GM-CSF and afterwards stimulated for 4 h with 10 μg/mL LTA (b) or 100 ng/mL LPS (c), respectively. Cells were double-stained with CD14-FITC, TNF-α-PE or with corresponding isotype antibodies and analyzed by flow cytometry. Dot plots presented are representative from three individually performed experiments.</p