Inhibition of Francisella tularensis LVS infection of macrophages results in a reduced inflammatory response: evaluation of a therapeutic strategy for intracellular bacteria
Inhibition of Francisella tularensis LVS infection of macrophages results in a reduced inflammatory response: evaluation of a therapeutic strategy for intracellular bacteria
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DOI：
10.1111/j.1574-695x.2011.00817.x
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发表时间：
2011-08-01
影响因子：
--
通讯作者：
Atkins, Helen S.
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文献类型：
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作者：
D'Elia, Riccardo;Jenner, Dominic C.;Atkins, Helen S.
土拉热弗朗西丝菌是一种细胞内病原体，能够侵入几种不同的细胞类型，特别是巨噬细胞，最常见的是通过吞噬作用。使用异硫氰酸荧光素标记的抗F，开发了流式细胞术测定细菌摄取。土拉热菌脂多糖抗体与细胞表面标志物的抗体结合，以确定细菌阳性的特定细胞表型。在巨噬细胞系和肺匀浆测定中评价了几种吞噬抑制剂，以确定F。土拉热菌LVS菌株的遗传变异。我们的数据显示，细胞松弛素B、LY 294002、渥曼青霉素、诺考达唑、MG 132和XVA 143抑制剂在这些测定中使LVS摄取减少> 50%，而没有显著的细胞毒性作用。此外，当存在抑制性化合物时，在感染LVS的肺组织上清液中发现炎性细胞因子单核细胞趋化蛋白-1、白细胞介素-6和肿瘤坏死因子-α的减少。类似地，在对LV S感染的小鼠施用渥曼青霉素后，细菌摄取发生改变，炎性细胞因子反应减少。虽然单独的渥曼青霉素治疗与提高LV感染小鼠的存活率无关，但这些抑制剂可能在联合治疗方法中或针对使用吞噬机制进入其最佳生态位的其他细胞内病原体具有实用性。
Francisella tularensis is an intracellular pathogen and is able to invade several different cell types, in particular macrophages, most commonly through phagocytosis. A flow cytometric assay was developed to measure bacterial uptake, using a fluorescein isothiocyanate-labelled anti-F. tularensis lipopolysaccharide antibody in conjunction with antibodies to cell surface markers, in order to determine the specific cell phenotypes that were positive for the bacteria. Several phagocytic inhibitors were evaluated in macrophage cell lines and a lung homogenate assay to determine whether the uptake of F. tularensis strain LVS could be altered. Our data show that cytochalasin B, LY294002, wortmannin, nocodazole, MG132 and XVA143 inhibitors reduced LVS uptake by > 50% in these assays without having significant cytotoxic effects. Furthermore, a reduction in the inflammatory cytokines monocyte chemoattractant protein-1, interleukin-6 and tumour necrosis factor-alpha was found in the supernatant of lung tissue infected with LVS when the inhibitory compounds were present. Similarly, there was an alteration in bacterial uptake and a reduction in the inflammatory cytokine response following the administration of wortmannin to LVS-infected mice. Although wortmannin treatment alone did not correlate with the enhanced survival of LVS-infected mice, these inhibitors may have utility in combination therapeutic approaches or against other intracellular pathogens that use phagocytic mechanisms to enter their optimal niche.