益生菌(prbiotics),也称直接饲喂微生物(direct-fed microbial,DFM),是指一类自然存在的有益于宿主的活微生物[1]。它们可以促进机体肠道微生态平衡、增强机体免疫功能,最终达到促生长和提高抗病力等益生作用。由于其具有效果显著、安全性好等诸多优点,已成为替代饲用抗生素的首选[2, 3]。
益生菌主要是通过非免疫调节(改善肠道微生态平衡和肠道形态结构)[4, 5, 6]和免疫调节(增强肠道免疫、活化全身免疫系统及减轻炎症反应)[7]2种途径改善动物肠道健康,从而促进动物生长。益生菌口服进入肠道黏附在固有层和淋巴集结后,被肠道内相关淋巴组织的巨噬细胞和树突状细胞识别,从而刺激细胞因子的分泌和共刺激分子的表达,产生相关的免疫反应[8]。因此,对肠黏膜免疫功能的影响和调节是其发挥免疫作用的关键。本文主要综述益生菌对巨噬细胞的免疫调节作用及其可能的机理。
巨噬细胞来源于血液单核细胞,可非特异性吞噬细菌、异物等,参与天然抗感染、抗肿瘤等过程,是天然免疫系统的重要组成部分。由于其本身的生物学特点,可表达多种与抗原摄取相关的表面分子,包括Fc段受体(FcR)、补体受体、Toll样受体(Toll-like receptor,TLR)等,也可表达大量的主要组织相容性复合体(MHC)Ⅰ类、Ⅱ类分子以及CD80、CD86、CD40等表面共刺激分子,能在细胞内加工处理外源抗原,形成的抗原肽-MHCⅡ类分子复合物在细胞表面表达并提呈给T细胞[9]。巨噬细胞在免疫过程中的具体作用为:1)免疫起始阶段,巨噬细胞可作为抗原递呈细胞摄取处理抗原并递呈给辅助性T细胞(helper T cell,简称Th细胞),启动特异性免疫应答;2)免疫应答阶段,活化的巨噬细胞趋化到病灶周围,更有效地吞噬细菌、杀伤靶细胞。同时,分泌多种活性物质,如溶菌酶、补体、细胞因子等,发挥相应的生物学功能[10, 11]。
巨噬细胞对损伤和感染产生迅速的反应,活化后的巨噬细胞分为2个不同的类型:M1和M2型。Ⅰ型炎症因子和微生物的代谢产物活化的巨噬细胞称为M1型巨噬细胞,具有杀灭微生物及促炎作用。M2型巨噬细胞根据其被活化的蛋白质不同而分成3个亚群:1)M2a,由白细胞介素(IL)-4或IL-13诱导;2)M2b,由免疫复合体和TLR或IL-1R的激动剂诱导;3)M2c,由IL-10和糖皮质激素诱导[12]。M2型巨噬细胞具有很强的免疫调节和组织修复功能,而杀灭微生物的功能很弱。因此,活化的巨噬细胞可以是促炎的也可以是抗炎的。M1和M2型代表巨噬细胞的2个极端和简化的功能状态,实际上巨噬细胞的活化是一个复杂的连续的功能状态[9]。
益生菌进入消化道后被肠黏膜内特有的M细胞摄取,进而递呈给巨噬细胞,益生菌表面的多种微生物相关分子模型(MAMP)与巨噬细胞的表面模式识别受体(PRR)相互作用[13, 14, 15, 16],刺激它们产生下游效应器细胞因子,调节免疫相关反应。
活化的巨噬细胞可以产生氧气派生的中间体、一氧化氮(NO)、一氧化氮合成酶(iNOS)、细胞因子、抗菌肽和其他防御分子[20]。益生菌可激活巨噬细胞分泌以上活性蛋白的能力,增强免疫功能。
一些益生菌能够刺激巨噬细胞产生生理性炎症反应,提高机体的免疫力。研究表明,鼠李糖乳杆菌可诱导巨噬细胞J774分泌NO[21],青春型双歧杆菌和两歧双歧杆菌可激活巨噬细胞产生IL-1和IL-6[22],几乎所有的干酪乳杆菌都能诱导巨噬细胞产生IL-12[23],纳豆枯草芽孢杆菌和乳酸乳球菌能够诱导巨噬细胞产生NO和细胞因子[24, 25],屎肠球菌能够诱导巨噬细胞系产生超氧阴离子(O-2)、肿瘤坏死因子-α(TNF-α)、干扰素-γ(INF-γ)、IL-6和IL-10[26]。乳酸菌和双歧杆菌均能诱导巨噬细胞产生NO和炎症因子如IL-6、干扰素-α(INF-α),增强机体的抗病毒能力[27]。
而有些益生菌能够通过调节巨噬细胞抗炎细胞因子的分泌来调节炎症反应,避免组织损伤。例如,短乳杆菌G-101能够促进脂多糖(LPS)诱导的巨噬细胞IL-10的分泌,抑制TNF-α、IL-1β和IL-6的产生[28],干酪乳杆菌代田株能够降低LPS诱导的巨噬细胞系RAW264.7对IL-6的分泌量[29],瑞士乳杆菌Bc-10能够抑制巨噬细胞产生白三烯B4,以上效果均能减轻炎症反应[30]。
某些益生菌在调节巨噬细胞极化方面起着重要的作用。解淀粉芽孢杆菌能够促进M1型巨噬细胞极化,并增强其吞噬功能[31],短乳杆菌G-101可以诱导巨噬细胞由M1型向M2型转化[28]。M1型和M2型巨噬细胞的平衡也能反映Th1和Th2的平衡[32],所以巨噬细胞可能是决定Th1或Th2型免疫应答的重要因素。益生菌与巨噬细胞的相互作用会有助于调节免疫应答的强度和类型[33],例如养乐多代田菌(L. casei Shirota)可以通过调节巨噬细胞分泌IL-12来促进Th1细胞的分化[8],乳酸菌可通过活化巨噬细胞来调节Th1/Th2平衡,并用于治疗因Th2引起的过敏反应[34]。
细菌都具有MAMP,它们可以是细胞表面分子LPS、鞭毛蛋白、脂磷壁酸(LTA)或者肽聚糖,也可以是细菌的DNA。在肠黏膜表层下存在大量的巨噬细胞,其细胞表面有PRR[37]。益生菌的MAMP与巨噬细胞的PRR相互作用产生信号级联,产生免疫反应。
TLR是PRR中的一种重要受体,能区分自身与非自身抗原,并能有效协调信号通路下游分子及各种炎症因子之间的作用。益生菌的细胞壁成分如脂蛋白、脂磷壁酸、脂阿拉伯甘露聚糖等可被巨噬细胞的TLR识别[25, 38]。同时,由于细菌的成分较为复杂,一种益生菌可同时能通过多种TLR介导发挥作用,所以无法预测特定益生菌产生的免疫反应,如细胞因子的分泌[25]。
益生菌如罗伊氏乳杆菌、乳酸菌、干酪乳杆菌分泌的因子能抑制NF-κB抑制蛋白α(IκBα)磷酸化和降解,抑制NF-κB信号通路[35, 39]。另一些益生菌如乳酸杆菌YIT 9029和发酵乳杆菌YIT 0159能够激活NF-κB信号通路,分泌TNF-α[38]。
益生菌同样可通过增强或减弱c-Jun氨基末端激酶(JNK)、p38和细胞外信号调节激酶1/2(ERK1/2)磷酸化作用,调节肠巨噬细胞的MAPK信号通路。干酪乳杆菌的分泌物能降低ERK1/2的磷酸化,抑制MAPK信号通路[35],Iyer等[40]报道,罗伊乳杆菌ATCC PTA 6475的分泌物通过加强JNK和p38磷酸化,抑制ERK1/2磷酸化,调节肠巨噬细胞MAPK信号通路。也有报道表明,p38 MAPK信号通路是益生菌调控细胞因子信号转导抑制因子3(SOCS3)基因表达的关键环节[41]。
益生菌除了调节巨噬细胞NF-κB和MAPK信号通路外,还调节其他信号通路。Lin等[42]报道,一些特殊的乳酸菌如罗伊乳杆菌ATCCFTA 6475能抑制激活蛋白-1(AP-1)的产生,从而降低由LPS诱导的肠巨噬细胞的肿瘤坏死因子(TNF)分泌量。Kim等[43]报道,鼠李糖乳杆菌GR-1可以通过激活Janu激酶2/信号转导子和转录激活子3(JAK2/STAT3)抑制肠巨噬细胞JNK的激活。
NF-κB和MAPK是益生菌调节肠巨噬细胞功能的关键信号通路。此外,益生菌也可通过AP-l和Janu激酶/信号转导子和转录激活子(JAK/STAT)等信号通路发挥作用,这是其调控肠巨噬细胞的一种特异方式。
巨噬细胞是一类重要的免疫细胞,在非特异性免疫和特异性免疫中起着重要的作用。益生菌能够通过NF-κB和MAPK等信号通路调节巨噬细胞,产生相应的免疫反应。目前,益生菌及其组分对巨噬细胞免疫功能的影响及其作用机理已成为研究热点。益生菌对巨噬细胞免疫功能的影响具有菌株特异性,因此对不同菌株及其组分的免疫功能需作系统研究,以找出其规律性。
巨噬细胞的极化也是近些年的研究热点,但有关益生菌对巨噬细胞极化的影响及其机理的研究较少,有待于进一步的研究。另外,目前有关益生菌对巨噬细胞的影响大多为体外试验结果,不能代表动物体内的真实情况。在动物体内益生菌要通过M细胞与巨噬细胞发生作用,期间益生菌的结构和成分是否发生变化并不清楚,所以下一步的研究重点应是益生菌在动物体内的具体作用效果和机制。
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