弓形虫基因型Chinese 1不同毒力的分离株诱导巨噬细胞偏移应答的研究
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摘要
背景刚地弓形虫是一种机会致病性的专性有核细胞内寄生原虫，猫科动物为其终宿主；中间宿主为几乎所有的温血动物，包括人类。全球约1/3的人口受到感染。食入含有弓形虫包囊的生肉或被卵囊污染的食物均可发生感染。免疫功能正常的个体感染后通常表现为无明显临床症状的隐性感染状态；但在免疫机能低下的患者，弓形虫感染可能导致危及生命的严重后果，宫内感染弓形虫可致胎儿发生严重缺陷。
感染的急性期，速殖子迅速增殖，诱导Th1型免疫反应，产生促炎症性细胞因子IL-6，IFN-γ和TNF-α等。作为抗弓形虫的第一道防线，巨噬细胞是最易受到感染的细胞，而且可以提供弓形虫繁殖的场所，也是弓形虫急性感染全身播散的主要运载细胞。已知巨噬细胞能够在周围微环境的影响下发生表型的变化，以发挥特定的功能。活化的巨噬细胞可分成两类：经典活化的巨噬细胞（M1）和旁路活化的巨噬细胞(M2)。在炎症细菌分子如脂多糖（LPS）和IFN-γ的作用下，巨噬细胞活化为M1，促进Th1型免疫应答，通过触发呼吸爆发、释放反应氧和反应氮等中间产物如一氧化氮(NO)等，发挥抗微生物的功能，杀死胞内的寄生虫；Th2型细胞因子IL-4和IL-13则活化M2，后者通常具有高水平的清道夫受体、甘露糖受体（CD206）和半乳糖受体，且可合成精氨酸酶，生成的多胺类物质有利于虫体的增殖，此外，M2还可以通过分泌IL-10等参与负向免疫调控，以及组织修复。
根据传统的生物学分类，弓形虫属（Toxoplasma）下只有一个物种，即刚地弓形虫（T.gondii，Tg）。然而，近期对弓形虫遗传多态性的研究发现，世界各地的Tg分类株具有丰富的基因型，而不同基因型的弓形虫感染会活化不同的巨噬细胞亚群，其原因在于Tg调节巨噬细胞活化的毒力因子的多态性，并调节不同的信号通路，最终影响弓形虫建立慢性感染。已经明确棒状体蛋白激酶ROP16和致密颗粒蛋白GRA15都是毒力决定因子。Ⅰ型和Ⅲ型弓形虫的ROP16能够组成性地磷酸化STAT6，使巨噬细胞极化为M2；而Ⅱ型弓形虫GRA15则活化NF-κb，诱导促炎症性的M1基因表达。
大多数从北美和欧洲的人和动物体内分离到的弓形虫虫株都属于三个基因型：Ⅰ型、Ⅱ型和Ⅲ型，三型之间的遗传差异不到1%。无论感染小鼠的遗传背景如何，Ⅰ型弓形虫都是强毒株，可致感染小鼠100%的死亡率。而Ⅱ型和Ⅲ型弓形虫的毒力则与宿主的遗传背景有关，表现为弱毒性，半数致死率分别约是102和105。近些年有文献报道，在不同地域的弓形虫存在着不同于上述三种基因型的非典型谱系（atypical lineages）。例如：12型是北美洲野生动物的最常见的基因型，Africa1和Africa3是非洲的主要弓形虫型别。我们的前期研究发现，Chinese1基因型（ToxoDB#9）是中国流行的优势基因型。有趣的是，尽管同属于Chinese1型，TgCtwh3和TgCtwh6分离株对于小鼠的毒力却显著不同。TgCtwh3是强毒株，可致感染小鼠死亡；而TgCtwh6则是弱毒株，能够使小鼠建立慢性感染，并在小鼠体内形成包囊。
目的比较同一基因型的TgCtwh3和TgCtwh6虫株感染后宿主的免疫应答特点，观察活化巨噬细胞的表型变化，研究两种分离株在诱导活化巨噬细胞的偏移及调节信号通路方面的差异，初步探讨我国优势基因型Chinese1弓形虫的毒力相关的致病机制。
方法（1）传代、纯化TgCtwh6速殖子：昆明小鼠腹腔接种含TgCtwh6包囊的小鼠脑组织匀浆，7-8天后获取含TgCtwh6速殖子的腹水；接种于单层小鼠成纤维细胞（L929）中培养。当大多数L929细胞自然裂解时，刮起细胞、收集裂解液，用27G针头的注射器反复抽吸，离心，收集速殖子，连续细胞传代约4周后即可获得较纯的TgCtwh6速殖子。TgCtwh3速殖子在L929中进行连续细胞传代，约4天即可获得较纯的TgCtwh3速殖子。调整TgCtwh3和TgCtwh6速殖子浓度，以用于体内或体外实验。（2）6-8周龄的BALB/C小鼠腹腔接种1×105TgCtwh3或1×106TgCtwh6速殖子，未感染小鼠为对照组。感染后2-6天麻醉后断颈处死小鼠，冲洗腹腔获得腹腔冲洗细胞，置12孔板中（1×106/孔），贴壁培养12h后分别收集上清液和细胞（分别称为Wh3-Mφ和Wh6-Mφ）。流式细胞术CBA试剂检测上清液中细胞因子IL-6,IFN-γ, TNF, IL-4和IL-10浓度，Griess法检测NO，按文献步骤检测尿素浓度。（3）予骨髓来源的巨噬细胞(BMMφ)或RAW264.7细胞rMuIFN-γ和LPS或rMu IL-13和IL-4刺激24h，或不予刺激，按弓形虫：细胞为2:1的比例分别感染TgCtwh3或TgCtwh6速殖子，继续培养24h。收集上清液，检测尿素浓度；瑞氏-吉姆萨染色计纳虫泡内的速殖子数，评价速殖子在M1/M2细胞中的增殖情况。（4）收集未感染小鼠的Mφ、Wh3-Mφ和Wh6-Mφ，与相应抗体或同型对照抗体4℃避光反应20min后，FACS缓冲液洗3次，2%多聚甲醛固定，应用流式细胞技术检测细胞表面的共刺激分子CD80、CD86和MHC-Ⅱ及M1/M2相关的标记PD-L1、PD-L2和CD206。（5）TRIzol裂解正常小鼠Mφ、Wh3-Mφ和Wh6-Mφ、感染TgCtwh3或TgCtwh6的BMMφ细胞，提取总RNA，按试剂盒说明合成cDNA。采用荧光定量PCR技术对M1/M2或Th1/Th2相关标记物进行mRNA水平检测。（6）BMMφ感染TgCtwh3或TgCtwh6速殖子（按弓形虫：细胞为2:1的比例），24h后收集细胞，加入磷酸化酶/蛋白酶抑制剂，加入裂解缓冲液提取总蛋白，或按试剂盒说明提取核蛋白。蛋白印迹分析M1/M2和信号通路相关蛋白。（7）TgCtwh3或TgCtwh6速殖子感染爬片生长的HeLa细胞（按弓形虫：细胞为2:1的比例），感染后不同时间终止。固定、透膜处理爬片，山羊血清封闭后与抗-NF-κB p65和抗-T.gondii p30-FITC抗体孵育，4℃避光过夜。PBS洗后加二抗tetramethylrhodamine goat-anti-rabbit IgG (H+L)室温反应1h，Hoechst染色5分钟后荧光倒置显微镜观察NF-κB p65表达。
结果⑴Wh3-Mφ和Wh6-Mφ的细胞因子表达谱存在差异。两种速殖子均可诱导Th1型免疫反应。与未感染的正常小鼠的Mφ相比，Wh6-Mφ产生的IL-6，IL-10，IL-12p40，IFN-γ和TNF水平显著升高；而Wh3-Mφ上述细胞因子仅中度升高，且IL-10水平和正常Mφ无差异（p＞0.05）。实时定量RT-PCR显示，Wh6-Mφ的IL-10，IL-12p40，IFN-γ和TNF-α的mRNA水平也升高。尽管Wh3-Mφ的IL-4在蛋白水平未能检测到，但IL-4mRNA水平升高。⑵TgCtwh3和TgCtwh6均下调Mφ表面共刺激分子的表达。与正常小鼠的Mφ相比，Wh3-Mφ和Wh6-Mφ的F4/80的荧光强度（MFI）均显著减弱，从正常的900.6±144.33分别下降到40.51±6.22和38.86±5.69（P值均＜0.001）。Wh3-Mφ和Wh6-Mφ均不能上调MHC-Ⅱ分子的表达，且CD80的MFI在Wh3-Mφ和Wh6-Mφ的表达均低于正常Mφ。Wh3-Mφ表达CD86的水平与正常Mφ相似，MFI分别为41.3±4.60和42.0±4.43（p＞0.05），但Wh6-Mφ的CD86水平低于正常组，MFI为29.54±6.12（p＜0.01）。⑶Wh3-Mφ上调PD-L2和CD206的表达，而Wh6-Mφ中等强度上调PD-L1和PD-L2的表达。Wh3-Mφ的PD-L2和CD206的表达显著升高，MFI分别从正常组的5.97±2.96和10.28±3.02上升到112.78±19.91和105.32±25.59（P值均＜0.001）。Wh6-Mφ的PD-L1中度升高，从正常组的27.86±4.88上升到139.83±20.82（p＜0.01）。⑷Wh6-Mφ产生高浓度的NO，Wh3-Mφ上调Ym1和Arg-1表达。与Wh3-Mφ产生的低NO相比（是正常Mφ NO的2倍），Wh6-Mφ诱导产生高水平的NO（是正常Mφ NO的14倍）；反之，Wh3-Mφ诱导产生高水平的尿素，为1.33±0.37mM (与正常组0.91±0.27mM相比，p＜0.05）。体外实验表明，BMMφ或RAW264.7感染TgCtwh3时产生高水平的精氨酸酶活性。实时定量RT-PCR和蛋白印迹结果显示：感染TgCtwh3的BMMφ上调Arg-1和Ym1mRNA和蛋白表达，而TgCtwh6可诱导iNOS mRNA和蛋白水平的升高。⑸M2细胞中高水平的精氨酸酶与弓形虫增殖有关。IL-4和IL-13预刺激24h的RAW264.7细胞感染TgCtwh3后产生的尿素水平最高，并促进速殖子在细胞内的增殖。⑹细胞免疫荧光显示，TgCtwh6感染HeLa细胞后，NF-κB p65活化进入细胞核；而TgCtwh3感染后NF-κBp65仍在细胞浆内表达。蛋白印迹结果显示，TgCtwh3感染诱导STAT6的try641磷酸化；而TgCtwh6感染诱导IκBα的磷酸化和细胞核内NF-κB p65的表达。
结论⑴同一基因型Chinese1不同毒力的分离株TgCtwh3和TgCtwh6均诱导Th1型免疫应答，但感染的巨噬细胞分泌细胞因子的水平和种类存在差异；⑵Wh3-Mφ和Wh6-Mφ均不能上调表达CD80、CD86和MHC-Ⅱ共刺激分子；⑶TgCtwh3和TgCtWh6分离株通过不同的途径诱导巨噬细胞的偏移：强毒株TgCtwh3活化p-STAT6，诱导M2偏移；而成囊株TgCtwh6通过NF-κB p65的活化，诱导M1偏移。
Background Toxoplasma gondii is an intracellular parasite capable of infecting a broadspectrum of hosts including up to30%of the world’s human population. Infectionoccurs when individual ingest undercooked meat containing cysts of the parasite orconsume food contaminated with oocysts. Although most infections are mild in healthyadults, life-threatening consequences may develop in immunocompromised patients andin utero infection can lead to major defects in the fetus.
During acute infection, tachyzoite, the rapidly replicative form of the parasiteelicits an extremely strong type1immune response, characterized by proinflammatorycytokine production such as IL-6, IFN-γ and TNF-α. Macrophages can provide a nichepermissive for parasite replication and are the most abundant cell type infected byToxoplasma. It has been clarified that macrophages can be phenotypically polarized bythe micro-environment to mount specific functional programs. Polarized macrophageshave been classified in two main groups: classically activated macrophages (or M1)whose typical activating stimuli are IFN-γ and LPS, and alternatively activatedmacrophages (or M2) induced by exposure to IL-4and IL-13. M1exhibits antimicrobialfunctions against intracellular pathogens which is conducted by the production ofreactive oxygen and nitrogen intermediates such as NO and promote strong Th1 responses, while M2is accompanied by diminished proinflammatory cytokine secretionand shares high expression of scavenger, mannose (CD206) and galactose receptors.
Recent work on T. gondii polymorphisms show that mouse macrophages infectedwith type I and III strains are polarized toward an M2activation state through activationof STAT6, while type II infected macrophages are M1-like cells elicited by activationof NF-kB. Type I and type III rhoptry kinase ROP16, inferred as a virulencedeterminant, can constitutively activate STAT6, and type II dense granule proteinGRA15is responsible for strain-specific NF-kB activation.
The majority of T. gondii strains isolated from humans and animals in NorthAmerica and Europe have been grouped into three predominant clonal lineages (types I,II and III) that differ genetically less than1%. Regardless of the genetic background ofmice, type I strains are highly virulent with an LD100=1, whereas type II or type IIIstrains associate with host genetic background and display lower virulence with anLD50≈102and≈105, respectively. Recently there were several studies revealingthat a few major clonal lineages of T.gondii dominate in different geographical regions.For example, the type12lineage is most common in wildlife in North America, and theAfrica1and3are among the major types in Africa. Our previous study showed that thegenotype Chinese1(known as ToxoDB#9) is dominantly prevalent in China.Interestingly, although belonging to the same genotype Chinese1, isolates of TgCtwh3and TgCtwh6have remarkably different virulence to mice, in which TgCtwh3is highlyvirulent and cause lethal infection prior to encystation, whereas TgCtwh6is mildlyvirulent and able to establish latent infection.
Objective To compare the difference in the acute immune response of macrophagesinfected with TgCtwh3or TgCtwh6tachyzoites in vitro and in vivo; to observe thephenotypical changes on activated macrophages; to evaluate the activation state ofmacrophages and determine modulation of the signaling pathway elicited by TgCtwh3 or TgCtwh6; to briefly explore the mechanism of human toxoplasmosis caused byisolates of Chinese1genotype.
Methods (1) Purification and cell passages of TgCtwh3and TgCtwh6tachyzoites.Tachyzoites of mouse-virulent TgCtwh3isolates were maintained in vitro by serialpassages in L929fibroblasts monolayers cultured in RPMI1640. The TgCtwh6tachyzoites were initially obtained by inoculating brain homogenate containingTgCtwh6cysts from infected mice and then cultured with L929fibroblasts. For initialpassages, fibroblast monolayers were detached by scraping, and cells were forcedthrough a27-gauge needle to release the intracellular parasites. About4weeks laterTgCtwh6were maintained by continual cell passages.(2) For in vivo infection,1×105tachyzoites of TgCtwh3or1×106tachyzoites of TgCtwh6were inoculated i.p. into six-to eight-week old BALB/C mice. At2-6d postinfection mice were euthanized andperitoneal exudates cells (PECs) were harvested by washing of the peritoneal cavitywith10ml of ice-cold D-Hanks solution. PECs were cultured in12-well plates (1×106cells per well) in RPMI1640, and three hours later nonadherent cells were washed offand the remaining adherent Mφ (termed Wh3-Mφ and Wh6-Mφ, respectively) were leftuntreated12h. Supernatants were collected for cytokine measurement, NO productionand arginase activity. The simultaneous detection of IL-6, IFN-γ, TNF, IL-4and IL-10was performed by Cytometric Bead Array Cytokine Kit, and IL-12p40was detected byMouse IL-12/IL-23p40Flex Set according to manufacturer’s instructions. Samples wereacquired on a cytometer and data were analyzed using FCAP Array Software. NOproduction was assessed by nitrite accumulation using the Griess Reagent System andarginase activity was measured according to previously published protocols.(3) For invitro infection assays, maintained in6-well plates (2×106cells per well), BMMφ andRAW264.7cells were pre-stimulated for24h with rMuIFN-γ plus LPS, or withrMuIL-13plus IL-4, or left unstimulated, then infected with freshly lysed T. gondii tachyzoites at a parasite to host cell ratio of2:1. Cells were incubated for an additional24h. Supernatants were collected for detection of arginase activity.(4) After beingwashed twice with PBS containing3%FCS, Wh3-Mφ and Wh6-Mφ were labeled withthe antibodies of interest and appropriate isotypes at the appropriate dilution for20minat4°C in the dark. The antibodies include FITC-conjugated anti-F4/80,PE-Cy5-conjugated anti-MHC Ⅱ, PE-conjugated PD-L2, PE-conjugated PD-L1,PE-Cy5-conjugated anti-CD80, APC-conjugated anti-CD86as well as Alexa Fluor647-conjugated anti-CD206. Noninfected macrophages were performed in parallel.Cells were washed three times in FACS buffer and fixed in2%paraformaldehydebefore FACS analysis.(5) By resuspension in TRIzol reagent RNA was prepared fromWh3-Mφ or Wh6-Mφ after being cultured for12h, or TgCwh3or TgCwh6-infectedBMMφ infected at24h. Total RNA was extracted and cDNA was synthesized using theRevertAid First Strand cDNA Synthesis Kit. The mRNA levels of Th1/Th2cytokineand M1/M2markers were detected by real-time RT-PCR.(6) BMMφ grown in a6-wellplate were infected with TgCtwh3and TgCtwh6parasites respectively (2:1ratio ofparasites to Mφ) for24h. After being washed with ice-cold PBS, in the presence ofProtease/Phosphatase Inhibitor Cocktail infected cells were lysed by addition of lysisbuffer, or nuclear proteins were extracted according to manufacturer’s instructions.Total cell lysates were subjected to SDS-PAGE and immunoblotting using several Abs,including anti-iNOS, anti-Ym1, anti-arginase1, anti-Stat6, anti-pY641-Stat6, anti-IκBα,anti-p-IκBα, anti-T.gondii SAG1. Nuclear proteins were subjected to SDS-PAGE fordetecting anti-NF-κB p65.(7) TgCtwh3or TgCtWh6parasites were allowed to invadecells on coverslips and incubated for different time points. Then coverslips were fixed,permeabilized and blocked in goat serum. To determine the nuclear or cytoplasmicNF-κB p65expression, HeLa cells were simultaneously incubated with anti-NF-κB p65along with anti-T.gondii p30-FITC overnight at4°C. Coverslips were incubated for1hat room temperature with tetramethylrhodamine goat-anti-rabbit IgG (H+L), and then incubated with Hoechst dye for5min for DNA visualization. Cell preparations wereexamined by inverted fluorescence microscope.
Results (1) Both TgCtwh3and TgCtwh6triggered Th1immune response with distinctcytokines profile. Compared with non-infected Mφ, remarkedly elevated IL-6, IL-10,IL-12p40, IFN-γ and TNF were found in Wh6-Mφ, whereas modest increases of thesecytokines were displayed in Wh3-Mφ except for IL-10. In concert with protein levels,Wh6-Mφ also showed elevated mRNA expression of IL-10, IL-12p40, IFN-γ andTNF-α. Unexpectedly, although IL-4concentration was below the level of detectionincreased transcription of IL-4mRNAs was exhibited on Wh3-Mφ.(2) TgCtwh3andTgCtwh6down-regulated CD80, CD86and MHC Ⅱ molecules expression onmacrophages. Our results indicated that both Wh3-Mφ and Wh6-Mφ expressed lowerF4/80, with MFI being40.51±6.22and38.86±5.69respectively, than uninfectedmacrophages with900.6±144.33(both p＜0.001). Additionally, compared withuninfected controls neither Wh3-Mφ nor Wh6-Mφ could up-regulate MHC class IImolecule expression, and both CD80expression in Wh3-Mφ and Wh6-Mφ was less asshown by MFI. Besides, Wh3-Mφ expressed the levels of CD86similar to uninfectedcontrols with MFI being41.3±4.60vs42.0±4.43(p＞0.05), contrary to lower levelsof CD86in Wh6-Mφ with MFI being29.54±6.12(p＜0.01).(3) Wh3-Mφ up-regulatedPD-L2and CD206expression whereas Wh6-Mφ modestly expressed PD-L1. Comparedwith uninfected Mφ TgCtwh3strongly promoted PD-L2expression and stained positivefor CD206, with MFI being112.78±19.91vs5.97±2.96(p＜0.001) and105.32±25.59vs10.28±3.02(p＜0.001) respectively. On the other hand, relative to residentperitoneal Mφ with low PD-L1and no PD-L2expression, Wh6-Mφ modestlyup-regulated the expression of PD-L1and PD-L2with MFI being139.83±20.82vs27.86±4.88(p＜0.001) and37.48±10.34vs5.97±2.96(p＜0.01) respectively.(4)Wh6-Mφ generated high levels of NO whereas Wh3-Mφ up-regulated Ym1and argniase expression. Our result showed that in contrast to low level of NO in Wh3-Mφ(2-fold higher than uninfected Mφ), less virulent TgCtwh6isolates induced robustproduction of NO,14-fold higher than uninfected Mφ. At the same time TgCtwh3infection elicited high arginase activity in Mφ as measured by the production of ureawhen compared with TgCtwh6. Additionally, in vivo experiment also suggested thatTgCtWh6triggered enhanced expression of iNOS on transcript and protein levels,whereas TgCtwh3up-regulated not only argniase activity, but also Ym1and argnise-1mRNA and protein expressions.(5) Highly expression of arginase in M2associatedwith parasites proliferation. To evaluate the effects of M1or M2on parasites,RAW264.7cells, pretreated either with IL-4plus IL-13or with IFN-γ plus LPS for24h,were infected with TgCtwh3and TgCtwh6respectively, and assayed for arginaseactivity and parasites growth. In general, arginase activity was all enhanced in IL-4andIL-13-treated cells, especially in those co-infected with TgCtwh3. Moreover, theproliferation of TgCtwh3in IL-4and IL-13-treated cells was also obvious by countingthe number of parasites per vacuole.(6) By immunofluorescence we found that manyHeLa cells infected with TgCtwh6showed high levels of NF-κB p65in their nucleus,whereas no translocation or only low levels of NF-κB p65to the nucleus induced byTgCtwh3. Furthermore, the results from Western blotting revealed that TgCtwh3induced phospho-STAT6whereas TgCtWh6led to the phosphorylation of IκBα andactivation and nuclear translocation of NF-κBp65.
Conclusions (1) Although sharing the Chinese1genotype, different virulent TgCtwh3and TgCtWh6isolates elicited Th1immune response in distinct extent with variantkinds of cytokines.(2) Both TgCtwh3and TgCtWh6tachyzoites down-regulated CD80,CD86and MHCⅡ molecules expression on macrophages.(3) TgCtwh3and TgCtWh6isolates induced macrophage-biased responses in different pathways. Virulent TgCtwh3 whereas cyst-forming isolate of TgCtwh6activated NF-κB pathway and promoteinfected macrophages similar to M1phenotype.
感染的急性期，速殖子迅速增殖，诱导Th1型免疫反应，产生促炎症性细胞因子IL-6，IFN-γ和TNF-α等。作为抗弓形虫的第一道防线，巨噬细胞是最易受到感染的细胞，而且可以提供弓形虫繁殖的场所，也是弓形虫急性感染全身播散的主要运载细胞。已知巨噬细胞能够在周围微环境的影响下发生表型的变化，以发挥特定的功能。活化的巨噬细胞可分成两类：经典活化的巨噬细胞（M1）和旁路活化的巨噬细胞(M2)。在炎症细菌分子如脂多糖（LPS）和IFN-γ的作用下，巨噬细胞活化为M1，促进Th1型免疫应答，通过触发呼吸爆发、释放反应氧和反应氮等中间产物如一氧化氮(NO)等，发挥抗微生物的功能，杀死胞内的寄生虫；Th2型细胞因子IL-4和IL-13则活化M2，后者通常具有高水平的清道夫受体、甘露糖受体（CD206）和半乳糖受体，且可合成精氨酸酶，生成的多胺类物质有利于虫体的增殖，此外，M2还可以通过分泌IL-10等参与负向免疫调控，以及组织修复。
根据传统的生物学分类，弓形虫属（Toxoplasma）下只有一个物种，即刚地弓形虫（T.gondii，Tg）。然而，近期对弓形虫遗传多态性的研究发现，世界各地的Tg分类株具有丰富的基因型，而不同基因型的弓形虫感染会活化不同的巨噬细胞亚群，其原因在于Tg调节巨噬细胞活化的毒力因子的多态性，并调节不同的信号通路，最终影响弓形虫建立慢性感染。已经明确棒状体蛋白激酶ROP16和致密颗粒蛋白GRA15都是毒力决定因子。Ⅰ型和Ⅲ型弓形虫的ROP16能够组成性地磷酸化STAT6，使巨噬细胞极化为M2；而Ⅱ型弓形虫GRA15则活化NF-κb，诱导促炎症性的M1基因表达。
大多数从北美和欧洲的人和动物体内分离到的弓形虫虫株都属于三个基因型：Ⅰ型、Ⅱ型和Ⅲ型，三型之间的遗传差异不到1%。无论感染小鼠的遗传背景如何，Ⅰ型弓形虫都是强毒株，可致感染小鼠100%的死亡率。而Ⅱ型和Ⅲ型弓形虫的毒力则与宿主的遗传背景有关，表现为弱毒性，半数致死率分别约是102和105。近些年有文献报道，在不同地域的弓形虫存在着不同于上述三种基因型的非典型谱系（atypical lineages）。例如：12型是北美洲野生动物的最常见的基因型，Africa1和Africa3是非洲的主要弓形虫型别。我们的前期研究发现，Chinese1基因型（ToxoDB#9）是中国流行的优势基因型。有趣的是，尽管同属于Chinese1型，TgCtwh3和TgCtwh6分离株对于小鼠的毒力却显著不同。TgCtwh3是强毒株，可致感染小鼠死亡；而TgCtwh6则是弱毒株，能够使小鼠建立慢性感染，并在小鼠体内形成包囊。
目的比较同一基因型的TgCtwh3和TgCtwh6虫株感染后宿主的免疫应答特点，观察活化巨噬细胞的表型变化，研究两种分离株在诱导活化巨噬细胞的偏移及调节信号通路方面的差异，初步探讨我国优势基因型Chinese1弓形虫的毒力相关的致病机制。
方法（1）传代、纯化TgCtwh6速殖子：昆明小鼠腹腔接种含TgCtwh6包囊的小鼠脑组织匀浆，7-8天后获取含TgCtwh6速殖子的腹水；接种于单层小鼠成纤维细胞（L929）中培养。当大多数L929细胞自然裂解时，刮起细胞、收集裂解液，用27G针头的注射器反复抽吸，离心，收集速殖子，连续细胞传代约4周后即可获得较纯的TgCtwh6速殖子。TgCtwh3速殖子在L929中进行连续细胞传代，约4天即可获得较纯的TgCtwh3速殖子。调整TgCtwh3和TgCtwh6速殖子浓度，以用于体内或体外实验。（2）6-8周龄的BALB/C小鼠腹腔接种1×105TgCtwh3或1×106TgCtwh6速殖子，未感染小鼠为对照组。感染后2-6天麻醉后断颈处死小鼠，冲洗腹腔获得腹腔冲洗细胞，置12孔板中（1×106/孔），贴壁培养12h后分别收集上清液和细胞（分别称为Wh3-Mφ和Wh6-Mφ）。流式细胞术CBA试剂检测上清液中细胞因子IL-6,IFN-γ, TNF, IL-4和IL-10浓度，Griess法检测NO，按文献步骤检测尿素浓度。（3）予骨髓来源的巨噬细胞(BMMφ)或RAW264.7细胞rMuIFN-γ和LPS或rMu IL-13和IL-4刺激24h，或不予刺激，按弓形虫：细胞为2:1的比例分别感染TgCtwh3或TgCtwh6速殖子，继续培养24h。收集上清液，检测尿素浓度；瑞氏-吉姆萨染色计纳虫泡内的速殖子数，评价速殖子在M1/M2细胞中的增殖情况。（4）收集未感染小鼠的Mφ、Wh3-Mφ和Wh6-Mφ，与相应抗体或同型对照抗体4℃避光反应20min后，FACS缓冲液洗3次，2%多聚甲醛固定，应用流式细胞技术检测细胞表面的共刺激分子CD80、CD86和MHC-Ⅱ及M1/M2相关的标记PD-L1、PD-L2和CD206。（5）TRIzol裂解正常小鼠Mφ、Wh3-Mφ和Wh6-Mφ、感染TgCtwh3或TgCtwh6的BMMφ细胞，提取总RNA，按试剂盒说明合成cDNA。采用荧光定量PCR技术对M1/M2或Th1/Th2相关标记物进行mRNA水平检测。（6）BMMφ感染TgCtwh3或TgCtwh6速殖子（按弓形虫：细胞为2:1的比例），24h后收集细胞，加入磷酸化酶/蛋白酶抑制剂，加入裂解缓冲液提取总蛋白，或按试剂盒说明提取核蛋白。蛋白印迹分析M1/M2和信号通路相关蛋白。（7）TgCtwh3或TgCtwh6速殖子感染爬片生长的HeLa细胞（按弓形虫：细胞为2:1的比例），感染后不同时间终止。固定、透膜处理爬片，山羊血清封闭后与抗-NF-κB p65和抗-T.gondii p30-FITC抗体孵育，4℃避光过夜。PBS洗后加二抗tetramethylrhodamine goat-anti-rabbit IgG (H+L)室温反应1h，Hoechst染色5分钟后荧光倒置显微镜观察NF-κB p65表达。
结果⑴Wh3-Mφ和Wh6-Mφ的细胞因子表达谱存在差异。两种速殖子均可诱导Th1型免疫反应。与未感染的正常小鼠的Mφ相比，Wh6-Mφ产生的IL-6，IL-10，IL-12p40，IFN-γ和TNF水平显著升高；而Wh3-Mφ上述细胞因子仅中度升高，且IL-10水平和正常Mφ无差异（p＞0.05）。实时定量RT-PCR显示，Wh6-Mφ的IL-10，IL-12p40，IFN-γ和TNF-α的mRNA水平也升高。尽管Wh3-Mφ的IL-4在蛋白水平未能检测到，但IL-4mRNA水平升高。⑵TgCtwh3和TgCtwh6均下调Mφ表面共刺激分子的表达。与正常小鼠的Mφ相比，Wh3-Mφ和Wh6-Mφ的F4/80的荧光强度（MFI）均显著减弱，从正常的900.6±144.33分别下降到40.51±6.22和38.86±5.69（P值均＜0.001）。Wh3-Mφ和Wh6-Mφ均不能上调MHC-Ⅱ分子的表达，且CD80的MFI在Wh3-Mφ和Wh6-Mφ的表达均低于正常Mφ。Wh3-Mφ表达CD86的水平与正常Mφ相似，MFI分别为41.3±4.60和42.0±4.43（p＞0.05），但Wh6-Mφ的CD86水平低于正常组，MFI为29.54±6.12（p＜0.01）。⑶Wh3-Mφ上调PD-L2和CD206的表达，而Wh6-Mφ中等强度上调PD-L1和PD-L2的表达。Wh3-Mφ的PD-L2和CD206的表达显著升高，MFI分别从正常组的5.97±2.96和10.28±3.02上升到112.78±19.91和105.32±25.59（P值均＜0.001）。Wh6-Mφ的PD-L1中度升高，从正常组的27.86±4.88上升到139.83±20.82（p＜0.01）。⑷Wh6-Mφ产生高浓度的NO，Wh3-Mφ上调Ym1和Arg-1表达。与Wh3-Mφ产生的低NO相比（是正常Mφ NO的2倍），Wh6-Mφ诱导产生高水平的NO（是正常Mφ NO的14倍）；反之，Wh3-Mφ诱导产生高水平的尿素，为1.33±0.37mM (与正常组0.91±0.27mM相比，p＜0.05）。体外实验表明，BMMφ或RAW264.7感染TgCtwh3时产生高水平的精氨酸酶活性。实时定量RT-PCR和蛋白印迹结果显示：感染TgCtwh3的BMMφ上调Arg-1和Ym1mRNA和蛋白表达，而TgCtwh6可诱导iNOS mRNA和蛋白水平的升高。⑸M2细胞中高水平的精氨酸酶与弓形虫增殖有关。IL-4和IL-13预刺激24h的RAW264.7细胞感染TgCtwh3后产生的尿素水平最高，并促进速殖子在细胞内的增殖。⑹细胞免疫荧光显示，TgCtwh6感染HeLa细胞后，NF-κB p65活化进入细胞核；而TgCtwh3感染后NF-κBp65仍在细胞浆内表达。蛋白印迹结果显示，TgCtwh3感染诱导STAT6的try641磷酸化；而TgCtwh6感染诱导IκBα的磷酸化和细胞核内NF-κB p65的表达。
结论⑴同一基因型Chinese1不同毒力的分离株TgCtwh3和TgCtwh6均诱导Th1型免疫应答，但感染的巨噬细胞分泌细胞因子的水平和种类存在差异；⑵Wh3-Mφ和Wh6-Mφ均不能上调表达CD80、CD86和MHC-Ⅱ共刺激分子；⑶TgCtwh3和TgCtWh6分离株通过不同的途径诱导巨噬细胞的偏移：强毒株TgCtwh3活化p-STAT6，诱导M2偏移；而成囊株TgCtwh6通过NF-κB p65的活化，诱导M1偏移。
Background Toxoplasma gondii is an intracellular parasite capable of infecting a broadspectrum of hosts including up to30%of the world’s human population. Infectionoccurs when individual ingest undercooked meat containing cysts of the parasite orconsume food contaminated with oocysts. Although most infections are mild in healthyadults, life-threatening consequences may develop in immunocompromised patients andin utero infection can lead to major defects in the fetus.
During acute infection, tachyzoite, the rapidly replicative form of the parasiteelicits an extremely strong type1immune response, characterized by proinflammatorycytokine production such as IL-6, IFN-γ and TNF-α. Macrophages can provide a nichepermissive for parasite replication and are the most abundant cell type infected byToxoplasma. It has been clarified that macrophages can be phenotypically polarized bythe micro-environment to mount specific functional programs. Polarized macrophageshave been classified in two main groups: classically activated macrophages (or M1)whose typical activating stimuli are IFN-γ and LPS, and alternatively activatedmacrophages (or M2) induced by exposure to IL-4and IL-13. M1exhibits antimicrobialfunctions against intracellular pathogens which is conducted by the production ofreactive oxygen and nitrogen intermediates such as NO and promote strong Th1 responses, while M2is accompanied by diminished proinflammatory cytokine secretionand shares high expression of scavenger, mannose (CD206) and galactose receptors.
Recent work on T. gondii polymorphisms show that mouse macrophages infectedwith type I and III strains are polarized toward an M2activation state through activationof STAT6, while type II infected macrophages are M1-like cells elicited by activationof NF-kB. Type I and type III rhoptry kinase ROP16, inferred as a virulencedeterminant, can constitutively activate STAT6, and type II dense granule proteinGRA15is responsible for strain-specific NF-kB activation.
The majority of T. gondii strains isolated from humans and animals in NorthAmerica and Europe have been grouped into three predominant clonal lineages (types I,II and III) that differ genetically less than1%. Regardless of the genetic background ofmice, type I strains are highly virulent with an LD100=1, whereas type II or type IIIstrains associate with host genetic background and display lower virulence with anLD50≈102and≈105, respectively. Recently there were several studies revealingthat a few major clonal lineages of T.gondii dominate in different geographical regions.For example, the type12lineage is most common in wildlife in North America, and theAfrica1and3are among the major types in Africa. Our previous study showed that thegenotype Chinese1(known as ToxoDB#9) is dominantly prevalent in China.Interestingly, although belonging to the same genotype Chinese1, isolates of TgCtwh3and TgCtwh6have remarkably different virulence to mice, in which TgCtwh3is highlyvirulent and cause lethal infection prior to encystation, whereas TgCtwh6is mildlyvirulent and able to establish latent infection.
Objective To compare the difference in the acute immune response of macrophagesinfected with TgCtwh3or TgCtwh6tachyzoites in vitro and in vivo; to observe thephenotypical changes on activated macrophages; to evaluate the activation state ofmacrophages and determine modulation of the signaling pathway elicited by TgCtwh3 or TgCtwh6; to briefly explore the mechanism of human toxoplasmosis caused byisolates of Chinese1genotype.
Methods (1) Purification and cell passages of TgCtwh3and TgCtwh6tachyzoites.Tachyzoites of mouse-virulent TgCtwh3isolates were maintained in vitro by serialpassages in L929fibroblasts monolayers cultured in RPMI1640. The TgCtwh6tachyzoites were initially obtained by inoculating brain homogenate containingTgCtwh6cysts from infected mice and then cultured with L929fibroblasts. For initialpassages, fibroblast monolayers were detached by scraping, and cells were forcedthrough a27-gauge needle to release the intracellular parasites. About4weeks laterTgCtwh6were maintained by continual cell passages.(2) For in vivo infection,1×105tachyzoites of TgCtwh3or1×106tachyzoites of TgCtwh6were inoculated i.p. into six-to eight-week old BALB/C mice. At2-6d postinfection mice were euthanized andperitoneal exudates cells (PECs) were harvested by washing of the peritoneal cavitywith10ml of ice-cold D-Hanks solution. PECs were cultured in12-well plates (1×106cells per well) in RPMI1640, and three hours later nonadherent cells were washed offand the remaining adherent Mφ (termed Wh3-Mφ and Wh6-Mφ, respectively) were leftuntreated12h. Supernatants were collected for cytokine measurement, NO productionand arginase activity. The simultaneous detection of IL-6, IFN-γ, TNF, IL-4and IL-10was performed by Cytometric Bead Array Cytokine Kit, and IL-12p40was detected byMouse IL-12/IL-23p40Flex Set according to manufacturer’s instructions. Samples wereacquired on a cytometer and data were analyzed using FCAP Array Software. NOproduction was assessed by nitrite accumulation using the Griess Reagent System andarginase activity was measured according to previously published protocols.(3) For invitro infection assays, maintained in6-well plates (2×106cells per well), BMMφ andRAW264.7cells were pre-stimulated for24h with rMuIFN-γ plus LPS, or withrMuIL-13plus IL-4, or left unstimulated, then infected with freshly lysed T. gondii tachyzoites at a parasite to host cell ratio of2:1. Cells were incubated for an additional24h. Supernatants were collected for detection of arginase activity.(4) After beingwashed twice with PBS containing3%FCS, Wh3-Mφ and Wh6-Mφ were labeled withthe antibodies of interest and appropriate isotypes at the appropriate dilution for20minat4°C in the dark. The antibodies include FITC-conjugated anti-F4/80,PE-Cy5-conjugated anti-MHC Ⅱ, PE-conjugated PD-L2, PE-conjugated PD-L1,PE-Cy5-conjugated anti-CD80, APC-conjugated anti-CD86as well as Alexa Fluor647-conjugated anti-CD206. Noninfected macrophages were performed in parallel.Cells were washed three times in FACS buffer and fixed in2%paraformaldehydebefore FACS analysis.(5) By resuspension in TRIzol reagent RNA was prepared fromWh3-Mφ or Wh6-Mφ after being cultured for12h, or TgCwh3or TgCwh6-infectedBMMφ infected at24h. Total RNA was extracted and cDNA was synthesized using theRevertAid First Strand cDNA Synthesis Kit. The mRNA levels of Th1/Th2cytokineand M1/M2markers were detected by real-time RT-PCR.(6) BMMφ grown in a6-wellplate were infected with TgCtwh3and TgCtwh6parasites respectively (2:1ratio ofparasites to Mφ) for24h. After being washed with ice-cold PBS, in the presence ofProtease/Phosphatase Inhibitor Cocktail infected cells were lysed by addition of lysisbuffer, or nuclear proteins were extracted according to manufacturer’s instructions.Total cell lysates were subjected to SDS-PAGE and immunoblotting using several Abs,including anti-iNOS, anti-Ym1, anti-arginase1, anti-Stat6, anti-pY641-Stat6, anti-IκBα,anti-p-IκBα, anti-T.gondii SAG1. Nuclear proteins were subjected to SDS-PAGE fordetecting anti-NF-κB p65.(7) TgCtwh3or TgCtWh6parasites were allowed to invadecells on coverslips and incubated for different time points. Then coverslips were fixed,permeabilized and blocked in goat serum. To determine the nuclear or cytoplasmicNF-κB p65expression, HeLa cells were simultaneously incubated with anti-NF-κB p65along with anti-T.gondii p30-FITC overnight at4°C. Coverslips were incubated for1hat room temperature with tetramethylrhodamine goat-anti-rabbit IgG (H+L), and then incubated with Hoechst dye for5min for DNA visualization. Cell preparations wereexamined by inverted fluorescence microscope.
Results (1) Both TgCtwh3and TgCtwh6triggered Th1immune response with distinctcytokines profile. Compared with non-infected Mφ, remarkedly elevated IL-6, IL-10,IL-12p40, IFN-γ and TNF were found in Wh6-Mφ, whereas modest increases of thesecytokines were displayed in Wh3-Mφ except for IL-10. In concert with protein levels,Wh6-Mφ also showed elevated mRNA expression of IL-10, IL-12p40, IFN-γ andTNF-α. Unexpectedly, although IL-4concentration was below the level of detectionincreased transcription of IL-4mRNAs was exhibited on Wh3-Mφ.(2) TgCtwh3andTgCtwh6down-regulated CD80, CD86and MHC Ⅱ molecules expression onmacrophages. Our results indicated that both Wh3-Mφ and Wh6-Mφ expressed lowerF4/80, with MFI being40.51±6.22and38.86±5.69respectively, than uninfectedmacrophages with900.6±144.33(both p＜0.001). Additionally, compared withuninfected controls neither Wh3-Mφ nor Wh6-Mφ could up-regulate MHC class IImolecule expression, and both CD80expression in Wh3-Mφ and Wh6-Mφ was less asshown by MFI. Besides, Wh3-Mφ expressed the levels of CD86similar to uninfectedcontrols with MFI being41.3±4.60vs42.0±4.43(p＞0.05), contrary to lower levelsof CD86in Wh6-Mφ with MFI being29.54±6.12(p＜0.01).(3) Wh3-Mφ up-regulatedPD-L2and CD206expression whereas Wh6-Mφ modestly expressed PD-L1. Comparedwith uninfected Mφ TgCtwh3strongly promoted PD-L2expression and stained positivefor CD206, with MFI being112.78±19.91vs5.97±2.96(p＜0.001) and105.32±25.59vs10.28±3.02(p＜0.001) respectively. On the other hand, relative to residentperitoneal Mφ with low PD-L1and no PD-L2expression, Wh6-Mφ modestlyup-regulated the expression of PD-L1and PD-L2with MFI being139.83±20.82vs27.86±4.88(p＜0.001) and37.48±10.34vs5.97±2.96(p＜0.01) respectively.(4)Wh6-Mφ generated high levels of NO whereas Wh3-Mφ up-regulated Ym1and argniase expression. Our result showed that in contrast to low level of NO in Wh3-Mφ(2-fold higher than uninfected Mφ), less virulent TgCtwh6isolates induced robustproduction of NO,14-fold higher than uninfected Mφ. At the same time TgCtwh3infection elicited high arginase activity in Mφ as measured by the production of ureawhen compared with TgCtwh6. Additionally, in vivo experiment also suggested thatTgCtWh6triggered enhanced expression of iNOS on transcript and protein levels,whereas TgCtwh3up-regulated not only argniase activity, but also Ym1and argnise-1mRNA and protein expressions.(5) Highly expression of arginase in M2associatedwith parasites proliferation. To evaluate the effects of M1or M2on parasites,RAW264.7cells, pretreated either with IL-4plus IL-13or with IFN-γ plus LPS for24h,were infected with TgCtwh3and TgCtwh6respectively, and assayed for arginaseactivity and parasites growth. In general, arginase activity was all enhanced in IL-4andIL-13-treated cells, especially in those co-infected with TgCtwh3. Moreover, theproliferation of TgCtwh3in IL-4and IL-13-treated cells was also obvious by countingthe number of parasites per vacuole.(6) By immunofluorescence we found that manyHeLa cells infected with TgCtwh6showed high levels of NF-κB p65in their nucleus,whereas no translocation or only low levels of NF-κB p65to the nucleus induced byTgCtwh3. Furthermore, the results from Western blotting revealed that TgCtwh3induced phospho-STAT6whereas TgCtWh6led to the phosphorylation of IκBα andactivation and nuclear translocation of NF-κBp65.
Conclusions (1) Although sharing the Chinese1genotype, different virulent TgCtwh3and TgCtWh6isolates elicited Th1immune response in distinct extent with variantkinds of cytokines.(2) Both TgCtwh3and TgCtWh6tachyzoites down-regulated CD80,CD86and MHCⅡ molecules expression on macrophages.(3) TgCtwh3and TgCtWh6isolates induced macrophage-biased responses in different pathways. Virulent TgCtwh3 whereas cyst-forming isolate of TgCtwh6activated NF-κB pathway and promoteinfected macrophages similar to M1phenotype.
引文
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3. Mylonas KJ, Nair MG, Prieto-Lafuente L, Paape D,Allen JE. Alternatively activatedmacrophages elicited by helminth infection can be reprogrammed to enable microbial killing. JImmunol,2009.182(5):3084-94.
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5. Namangala B, De Baetselier P, Noel W, Brys L,Beschin A. Alternative versus classicalmacrophage activation during experimental African trypanosomosis. J Leukoc Biol,2001.69(3):387-96.
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8. Loke P, Nair MG, Parkinson J, Guiliano D, Blaxter M,Allen JE. IL-4dependentalternatively-activated macrophages have a distinctive in vivo gene expression phenotype. BMCImmunol,2002.3:7.
9. Raes G, De Baetselier P, Noel W, Beschin A, Brombacher F,Hassanzadeh Gh G. Differentialexpression of FIZZ1and Ym1in alternatively versus classically activated macrophages. JLeukoc Biol,2002.71(4):597-602.
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