Laboratório de Imunologia Celular (LIM-17) - Faculdade de Medicina FMUSP, Universidade de Sao Paulo, Sao Paulo, SP, Brazil.
Laboratório de Lípides (LIM-10) - Faculdade de Medicina FMUSP, Universidade de Sao Paulo, Sao Paulo, SP, Brazil.
Adv Rheumatol. 2019 Jul 24;59(1):30. doi: 10.1186/s42358-019-0072-x.
Adaptive immune cells, including CD4CD69 and CD4CD25FoxP3 regulatory T (Treg) cells, are important for maintaining immunological tolerance. In human systemic lupus erythematosus (SLE), CD4CD25FoxP3 Treg cells are reduced, whereas CD69 expression is increased, resulting in a homeostatic immune imbalance that may intensify autoreactive T cell activity. To analyze the mechanisms implicated in autotolerance failure, we evaluated CD4CD69 and CD4CD25FoxP3 T cells and interleukin profiles in a pristane-induced SLE experimental model.
For lupus induction, 26 female Balb/c mice received a single intraperitoneal 0.5 ml dose of pristane, and 16 mice received the same dose of saline. Blood and spleen samples were collected from euthanized mice 90 and 120 days after pristane or saline inoculation. Mononuclear cells from peripheral blood (PBMC), peritoneal lavage (PL) and splenocytes were obtained by erythrocyte lysis and cryopreserved for further evaluation by flow cytometry using the GuavaEasyCyte TM HT. After thawing, cells were washed and stained with monoclonal antibodies against CD3, CD4, CD8, CD25, CD28, CD69, FoxP3, CD14 and Ly6C (BD Pharmingen TM). Interleukins were quantified using Multiplex® MAP. The Mann-Whitney test and the Pearson coefficient were used for statistical analysis, and p < 0.05 considered significant.
Compared with the controls, SLE-induced animals presented increased numbers of CD4CD69 T cells in the blood on T90 and T120 (p = 0.022 and p = 0.008) and in the spleen on T120 (p = 0.049), but there were decreased numbers in the PL (p = 0.049) on T120. The percentage of Treg was lower in blood (p < 0.005 and p < 0.012) on T90 and T120, in spleen (p = 0.043) on T120 and in PL (p = 0.001) on T90. Increased numbers of CD4 + CD69+ T cells in the PL were positively associated with high IL-2 (p = 0.486) and IFN-γ (p = 0.017) levels, whereas reduced Treg cells in the blood were negatively correlated with TNFα levels (p = 0.043) and positively correlated with TGFβ1 (p = 0.038).
Increased numbers of CD4CD69 T cells and reduced numbers of CD4CD25FoxP3 Treg cells with an altered interleukin profile suggests loss of autotolerance in pristane-induced lupus mice, which is similar to human lupus. Therefore, this model is useful in evaluating mechanisms of cellular activation, peripheral tolerance and homeostatic immune imbalance involved in human SLE.
适应性免疫细胞，包括 CD4CD69 和 CD4CD25FoxP3 调节性 T（Treg）细胞，对于维持免疫耐受至关重要。在人类系统性红斑狼疮（SLE）中，CD4CD25FoxP3 Treg 细胞减少，而 CD69 的表达增加，导致免疫稳态失衡，可能加剧自身反应性 T 细胞的活性。为了分析与自身耐受失败相关的机制，我们在苍耳烷诱导的 SLE 实验模型中评估了 CD4CD69 和 CD4CD25FoxP3 T 细胞和白细胞介素谱。
为了诱导狼疮，26 只雌性 Balb/c 小鼠接受单次腹腔内 0.5ml 剂量的苍耳烷，16 只小鼠接受相同剂量的生理盐水。在苍耳烷或生理盐水接种后 90 和 120 天，处死小鼠并采集血液和脾脏样本。外周血（PBMC）、腹腔灌洗液（PL）和脾细胞通过红细胞裂解获得，并通过 GuavaEasyCyte TM HT 进行流式细胞术评估，然后冷冻保存。解冻后，用针对 CD3、CD4、CD8、CD25、CD28、CD69、FoxP3、CD14 和 Ly6C（BD Pharmingen TM）的单克隆抗体进行染色。使用 Multiplex®MAP 定量白细胞介素。使用曼-惠特尼检验和皮尔逊系数进行统计学分析，p<0.05 认为有统计学意义。
与对照组相比，SLE 诱导的动物在 T90 和 T120 时血液中 CD4CD69 T 细胞数量增加（p=0.022 和 p=0.008），在 T120 时脾脏中 CD4CD69 T 细胞数量增加（p=0.049），但在 T120 时 PL 中 CD4CD69 T 细胞数量减少（p=0.049）。T90 和 T120 时血液中 Treg 的百分比降低（p<0.005 和 p<0.012），T120 时脾脏中 Treg 的百分比降低（p=0.043），T90 时 PL 中 Treg 的百分比降低（p=0.001）。PL 中 CD4+CD69+T 细胞数量增加与高 IL-2（p=0.486）和 IFN-γ（p=0.017）水平呈正相关，而血液中 Treg 细胞减少与 TNFα 水平呈负相关（p=0.043），与 TGFβ1 水平呈正相关（p=0.038）。
苍耳烷诱导狼疮小鼠中 CD4CD69 T 细胞数量增加和 CD4CD25FoxP3 Treg 细胞数量减少，白细胞介素谱发生改变，提示自身耐受丧失，与人类狼疮相似。因此，该模型可用于评估人类 SLE 中涉及细胞活化、外周耐受和免疫稳态失衡的机制。
