Project description:Glucocorticoids are extensively used to treat inflammatory diseases, however their chronic intake increases the risk of mycobacterial infections. Meanwhile, the effects of glucocorticoids on innate host responses are incompletely understood. Here, we studied the direct effects of glucocorticoids on antimycobacterial host defense in primary human macrophages. We found that glucocorticoids triggered the expression of cathelicidin, an antimicrobial critical for antimycobacterial response, independent of the intracellular vitamin D metabolism. Despite upregulating cathelicidin, glucocorticoids failed to promote macrophage antimycobacterial activity. Gene expression profiles of human macrophages treated with glucocorticoids and/or IFN-gamma, which promotes induction of cathelicidin, as well as antimycobacterial activity, were investigated. Using weighted gene coexpression network analysis (WGCNA), we identified a module of highly connected genes that was strongly inversely correlated with glucocorticoid treatment and associated with IFN-gamma stimulation. This module was linked to the biological functions “autophagy”, “phagosome maturation” and “lytic vacuole/lysosome”, and contained the vacuolar H+-ATPase (v-ATPase) subunit a3, alias TCIRG1, a known antimycobacterial host defense gene, as a top hub gene. We next found that glucocorticoids, in contrast to IFN-gamma, failed to trigger expression and phagolysosome recruitment of TCIRG1, as well as to promote lysosome acidification. Finally, we demonstrated that the tyrosine kinase inhibitor imatinib induces lysosome acidification and antimicrobial activity in glucocorticoid-treated macrophages without reversing the anti-inflammatory effects of glucocorticoids. Taken together, we provide evidence that the induction of cathelicidin by glucocorticoids is not sufficient for macrophage antimicrobial activity, and identify the v-ATPase as a potential target for host-directed therapy in the context of glucocorticoid therapy.

Project description:Glucocorticoids are extensively used to treat inflammatory diseases, however their chronic intake increases the risk of mycobacterial infections. Meanwhile, the effects of glucocorticoids on innate host responses are incompletely understood. Here, we studied the direct effects of glucocorticoids on antimycobacterial host defense in primary human macrophages. We found that glucocorticoids triggered the expression of cathelicidin, an antimicrobial critical for antimycobacterial response, independent of the intracellular vitamin D metabolism. Despite upregulating cathelicidin, glucocorticoids failed to promote macrophage antimycobacterial activity. Gene expression profiles of human macrophages treated with glucocorticoids and/or IFN-gamma, which promotes induction of cathelicidin, as well as antimycobacterial activity, were investigated. Using weighted gene coexpression network analysis (WGCNA), we identified a module of highly connected genes that was strongly inversely correlated with glucocorticoid treatment and associated with IFN-gamma stimulation. This module was linked to the biological functions âautophagyâ, âphagosome maturationâ and âlytic vacuole/lysosomeâ, and contained the vacuolar H+-ATPase (v-ATPase) subunit a3, alias TCIRG1, a known antimycobacterial host defense gene, as a top hub gene. We next found that glucocorticoids, in contrast to IFN-gamma, failed to trigger expression and phagolysosome recruitment of TCIRG1, as well as to promote lysosome acidification. Finally, we demonstrated that the tyrosine kinase inhibitor imatinib induces lysosome acidification and antimicrobial activity in glucocorticoid-treated macrophages without reversing the anti-inflammatory effects of glucocorticoids. Taken together, we provide evidence that the induction of cathelicidin by glucocorticoids is not sufficient for macrophage antimicrobial activity, and identify the v-ATPase as a potential target for host-directed therapy in the context of glucocorticoid therapy. Peripheral blood mononuclear cells (PBMCs) of three healthy human donors were isolated by Ficoll-Paque (GE Healthcare). Monocytes were isolated via CD14+ MACS cell separation (Miltenyi Biotec) according to the manufacturers instructions. Monocyte-derived macrophages (MDMs) were prepared by culturing peripheral blood monocytes in RPMI media containing 10% FCS for four to seven days in the presence of M-CSF (50 ng/ml). Afterwards cells were cultured in fresh media with 10% vitamin D-sufficient human AB serum. Cells were stimulated with media, dexamethasone, interferon-gamma and dexamethasone/interferon-gamma for 20h. Total RNA of was isolated with TRIZOL (Life Technologies) and RNA quality was confirmed using micro capillary electrophoresis (2100 Bioanalyzer, Agilent). 100ng RNA was labeled and hybridized to Sureprint G3 human GE 8x60K whole genome mRNA microarray according to the manufacturerâs specifications. The arrays were scanned (Agilent G2595C scanner), data extracted and processed using the Genespring XII software (Agilent).

Project description:A multi-omic approach in a clinical experimental study identified circulating biomarkers reflecting glucocorticoid exposure. Background: Endogenous glucocorticoids (GC) are mechanistically linked to common diseases and are important as drugs in the treatment of many disorders. There is no marker that can measure and quantify GC action. Our aim was to identify circulating biomarkers of GC action using a clinical experimental study. Methods: In a randomized, crossover, single-blind trial, subjects with primary adrenal insufficiency received intravenous hydrocortisone infusion in a circadian pattern (physiological GC exposure) or isotonic saline (GC withdrawal) over 22 hours. Samples were collected at 7 AM (end of infusion). Integrated multi-omic analysis was used because of the complexity in GC action and the low number of subjects. The transcriptome in peripheral blood mononuclear cells (PBMCs) and adipose tissue, plasma miRNAomic, and serum metabolomics were compared between the interventions. Replication of the plasma miRNA findings was performed in three independent studies. Results: During GC withdrawal, overnight urinary cortisol and cortisone excretion were undetectable. Correlation and hypernetwork analyses identified a transcriptomic profile derived from PBMCs and adipose tissue predictive of GC exposure, and a multi-omic cluster predictive of GC exposure. From the circulating ‘omic data, decreased expression of plasma miR-122-5p was associated with increased GC exposure. This finding was reproduced in three independent studies. Conclusion: We developed a human experimental model for physiological GC exposure and withdrawal. The integrated multi-omic data identified circulating miRNAs and metabolites associated with GC-responsive genes. In independent studies, miR-122-5p was shown to be associated with GC exposure. Background: Endogenous glucocorticoids (GC) are mechanistically linked to common diseases and are important as drugs in the treatment of many disorders. There is no marker that can measure and quantify GC action. Our aim was to identify circulating biomarkers of GC action using a clinical experimental study.

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Expression data from mouse adrenals during recovery after dexamethasone treatment

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