Background: The previous study showed that mycophenolic acid (MPA) synergizing with lipopolysaccharide (LPS) promoted interleukin (IL)-1β release, but the mechanism is unclear. This study aimed to investigate the mechanism of MPA synergizing with LPS to induce IL-1β release.
Methods: Undiluted human blood cells, THP-1 human myeloid leukemia mononuclear cells (THP-1) cells, or monocytes were stimulated with LPS and treated with or without MPA, and the supernatant IL-1β was detected by enzyme-linked immunosorbent assay. The mRNA levels of IL-1β were detected by real-time quantitative polymerase chain reaction. The intracellular protein levels of nuclear factor kappa B (NF-κB) phospho-p65 (p-p65), precursor interleukin-1β (pro-IL-1β), NOD-like receptor pyrin domain containing-3 (NLRP3), and cysteine aspartic acid-specific protease-1 (caspase-1) p20 in THP-1 cell were measured by Western blot.
Results: The MPA alone failed to induce IL-1β, whereas MPA synergized with LPS to increase IL-1β in a dose-dependent manner (685.00 ± 20.00 pg/ml in LPS + 5 μmol/L MPA group, P = 0.035; 742.00 ± 31.58 pg/ml in LPS + 25 μmol/L MPA group, P = 0.017; 1000.00 ± 65.59 pg/ml in LPS + 75 μmol/L MPA group, P = 0.024; versus 408.00 ± 35.50 pg/ml in LPS group). MPA alone has no effect on the IL-1β mRNA expression, LPS induced the expression of IL-1β mRNA 2761 fold, and LPS + MPA increased the IL-1β expression 3018 fold, which had the same effect with LPS group (P = 0.834). MPA did not affect the intracellular NF-κB p-p65 and pro-IL-1β protein levels but activated NLRP3 inflammasome. Ac-YVAD-cmk blocked the activation of caspase-1 and subsequently attenuated IL-1β secretion (181.00 ± 45.24 pg/ml in LPS + MPA + YVAD group vs. 588.00 ± 41.99 pg/ml in LPS + MPA group, P = 0.014).
Conclusions: Taken together, MPA synergized with LPS to induce IL-1β release via the activation of caspase-1, rather than the enhanced production of pro-IL-1β. These findings suggested that patients immunosuppressed with mycophenolate mofetil may have overly activated caspase-1 during infection, which might contribute to a more sensitive host defense response to invading germs.
霉酚酸协同脂多糖通过活化caspase-1促进白介素-1β分泌摘要背景：我们的既往研究表明MMF的活性代谢产物霉酚酸（MPA）协同LPS可促进白介素-1β分泌，但其机制不明确。本研究拟探讨MPA协同LPS促进白介素-1β的机制。 方法：未稀释的人外周血用地塞米松、霉酚酸（MPA，MMF的活性代谢产物）、硫唑嘌呤或CYC单独使用或联合脂多糖（LPS）培养12小时后，用ELISA方法检测培养上清的白细胞介素（IL）-1β水平。THP-1细胞或单核细胞，MPA单独使用或与LPS联用处理细胞后，用ELISA方法检测培养上清的IL-1β水平。实时荧光定量PCR的方法检测THP-1细胞的IL-1β mRNA水平。 Western blot法检测THP-1细胞中NF-κB p-p65，pro-IL-1β，NLRP3和caspase-1（p20）的胞内蛋白水平。 结果：MPA单独使用不能诱导IL-1β产生，而MPA与LPS协同可增加IL-1β的分泌，并呈剂量依赖性 (685.00±20.00 pg/ml in LPS+5 μmol/L MPA group, P=0.035; 742.00±31.58 pg/ml in LPS+25 μmol/L MPA group, P=0.017; 1000.00±65.59 pg/ml in LPS+75 μmol/L MPA group, P=0.024; vs. 408.00±35.50 pg/ml in LPS group)。 MPA对细胞内IL-1β的mRNA转录水平无影响，LPS可使IL-1β的mRNA转录水平增加2761倍，而LPS+MPA组IL-1β的mRNA转录水平与对照组比较升高3018倍，与LPS组比较无统计学差异 (P=0.834)。MPA对细胞内NF-κB p-p65和pro-IL-1β蛋白水平无影响，但可以激活NLRP3炎性体。Caspase-1的特异性抑制剂Ac-YVAD-cmk可阻断caspase-1的活化并减少IL-1β的分泌，LPS+MPA组IL-1β浓度为588.00±41.99 pg/ml，而LPS+MPA+ YVAD组为181±45.24 pg/ml (P= 0.014)。 结论：MPA协同LPS促进IL-1β产生是通过激活caspase-1使pro-IL-1β转化为IL-1β增多所致，而不是通过增加pro-IL-1β的产生。这些发现表明，用MMF免疫抑制的患者可能在感染期间过度激活caspase-1，可能有助于宿主对入侵的细菌产生更敏感的防御反应。.
Keywords: Autoimmune Diseases; Caspase-1 Host Defense; Interleukin-1β; Lipopolysaccharide Mycophenolic Acid.