Microarray analysis of human monocyte-derived macrophages stimulated with dexamethasone, interferon-gamma and dexamethasone/interferon-gamma. Homo sapiens

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. Overall design: 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 manufacturer’s specifications. The arrays were scanned (Agilent G2595C scanner), data extracted and processed using the Genespring XII software (Agilent).

糖皮质激素（glucocorticoids）被广泛应用于炎症性疾病的临床治疗，但长期摄入会增加分枝杆菌感染风险。目前，糖皮质激素对宿主先天免疫应答的调控效应尚未完全阐明。本研究旨在探究糖皮质激素对原代人巨噬细胞抗分枝杆菌宿主防御的直接作用。研究发现，糖皮质激素可诱导抗菌肽（cathelicidin）的表达——该抗菌肽在宿主抗分枝杆菌应答中发挥关键作用——且该诱导过程不依赖于细胞内维生素D代谢通路。尽管可上调cathelicidin的表达，糖皮质激素却无法增强巨噬细胞的抗分枝杆菌活性。我们对经糖皮质激素、干扰素-γ（IFN-γ，可诱导cathelicidin表达并增强抗分枝杆菌活性）以及二者联合处理的人巨噬细胞开展了基因表达谱分析。通过加权基因共表达网络分析（WGCNA），我们鉴定出一个高度连通的基因模块：该模块与糖皮质激素处理呈显著负相关，且与IFN-γ刺激密切关联。该模块富集于“自噬”“吞噬体成熟”与“溶解性液泡/溶酶体”等生物学功能，其中包含已知的抗分枝杆菌宿主防御基因——液泡型H+-ATP酶（v-ATPase）亚基a3（又名TCIRG1），其为该模块的核心枢纽基因。进一步研究显示，与IFN-γ不同，糖皮质激素无法诱导TCIRG1的表达及其在吞噬溶酶体的招募，也不能促进溶酶体酸化。最后，我们证实酪氨酸激酶抑制剂伊马替尼（imatinib）可在糖皮质激素处理的巨噬细胞中诱导溶酶体酸化与抗菌活性，且不会逆转糖皮质激素的抗炎作用。综上，本研究证实糖皮质激素诱导的cathelicidin表达不足以赋予巨噬细胞抗菌活性，并鉴定出v-ATP酶可作为糖皮质激素治疗背景下宿主导向治疗的潜在靶点。整体实验设计：采用Ficoll-Paque（GE Healthcare）分离3名健康人类供者的外周血单个核细胞（PBMCs）；依照制造商说明书，通过CD14+磁珠细胞分选（MACS，Miltenyi Biotec）分离单核细胞。将外周血单核细胞置于含10%胎牛血清（FCS）的RPMI培养基中，添加巨噬细胞集落刺激因子（M-CSF，50 ng/ml）培养4至7天，以制备单核细胞来源的巨噬细胞（MDMs）。随后将细胞换用含10%维生素D充足的人AB血清的新鲜培养基培养。以培养基、地塞米松、IFN-γ以及地塞米松/IFN-γ联合处理细胞20小时。采用TRIZOL（Life Technologies）提取总RNA，并通过微毛细管电泳（2100生物分析仪，Agilent）验证RNA质量。取100ng RNA进行标记，依照制造商操作规程与Sureprint G3人全基因组GE 8x60K mRNA微阵列进行杂交。采用Agilent G2595C扫描仪对芯片进行扫描，通过Genespring XII软件（Agilent）提取并处理数据。