Dose-Dependent Immunomodulation of Human Dendritic Cells by the Probiotic Lactobacillus rhamnosus Lcr35

The response of the immune system to probiotics remains controversial. Some strains modulate the cytokine production of dendritic cells (DCs) in vitro and induce a regulatory response, while others induce conversely a pro-inflammatory response. These strain-dependent effects are thought to be linked to specific interactions between bacteria and pattern recognition receptors. We investigated the effects of a well characterized probiotic strain, Lactobacillus rhamnosus Lcr35, on human monocyte-derived immature DCs, using a wide range of bacterial concentrations (multiplicity of infection, MOI, from 0.01 to 100). DNA microarray and qRT-PCR analysis showed that the probiotic induced a large-scale change in gene expression (nearly 1,700 modulated genes, with 3-fold changes), but only with high doses (MOI, 100). The upregulated genes were mainly involved in immune response and identified a molecular signature of inflammation according to the model of Torri. Flow cytometry analysis also revealed a dose-dependent maturation of the DC membrane phenotype, until DCs reached a semi-mature state, with an upregulation of the membrane expression of CD86, CD83, HLA-DR and TLR4, associated with a down-regulation of DC-SIGN, MR and CD14. Measurement of the DC-secreted cytokines showed that Lcr35 induced a strong dose-dependent increase of the pro-Th1/Th17 cytokine levels (TNFα, IL-1β, IL-12p70, IL-12p40 and IL-23), but only a low increase in IL-10 concentration. The probiotic L. rhamnosus Lcr35 therefore induce a dose-dependent immunomodulation of human DCs leading, at high doses, to the semi-maturation of the cells and to a strong pro-inflammatory effect. These results contribute to a fuller understanding of the mechanism of action of this probiotic, and thus of its potential clinical indications in the treatment of either infectious or IgE-dependent allergic diseases.机体免疫系统对益生菌的应答仍存在争议。部分菌株可在体外调控树突状细胞（dendritic cells, DCs）的细胞因子产生并诱导调节性应答，而另一些菌株则相反，可诱导促炎性应答。此类菌株特异性效应被认为与细菌和模式识别受体（pattern recognition receptors, PRRs）之间的特异性相互作用密切相关。本研究针对一系列梯度浓度的细菌（感染复数multiplicity of infection, MOI范围为0.01至100），探究了经过充分表征的益生菌菌株鼠李糖乳杆菌（Lactobacillus rhamnosus）Lcr35对人单核细胞来源未成熟树突状细胞的影响。DNA微阵列（DNA microarray）与实时定量聚合酶链反应（quantitative real-time polymerase chain reaction, qRT-PCR）分析结果显示，仅在高剂量（MOI=100）条件下，该益生菌才可诱导大规模基因表达改变——近1700个基因出现3倍及以上的表达变化。上调基因主要参与免疫应答过程，且参照Torri等建立的炎症分子特征模型，可鉴定出炎症相关的分子特征。流式细胞术（flow cytometry）分析同样显示，树突状细胞膜表型呈现剂量依赖性成熟过程，最终达到半成熟状态：CD86、CD83、人类白细胞抗原DR（human leukocyte antigen DR, HLA-DR）以及Toll样受体4（Toll-like receptor 4, TLR4）的膜表达上调，同时DC-SIGN（dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin, DC-SIGN）、甘露糖受体（mannose receptor, MR）以及CD14的表达下调。对树突状细胞分泌的细胞因子进行检测后发现，Lcr35可显著诱导促Th1/Th17型细胞因子[肿瘤坏死因子α（tumor necrosis factor α, TNFα）、白细胞介素1β（interleukin 1β, IL-1β）、IL-12p70、IL-12p40以及IL-23]的水平呈剂量依赖性升高，但仅能使IL-10的浓度小幅上升。综上，鼠李糖乳杆菌Lcr35可对人树突状细胞产生剂量依赖性免疫调节作用：高剂量下可诱导细胞进入半成熟状态，并产生强烈的促炎性效应。本研究结果有助于更全面地阐明该益生菌的作用机制，进而明确其在感染性疾病或IgE依赖性过敏性疾病治疗中的潜在临床适应证。