The Lipopolysaccharide Mutant Re-LPS Is a Useful Tool for Detecting LPS Contamination in Rheumatoid Synovial Cell Cultures
The Lipopolysaccharide Mutant Re-LPS Is a Useful Tool for Detecting LPS Contamination in Rheumatoid Synovial Cell Cultures
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DOI：
10.1159/000520022
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发表时间：
2021-11
期刊：
影响因子：
5
通讯作者：
H. Kohno;K. Ouhara;S. Mokuda;T. Tokunaga;T. Sugimoto;H. Watanabe;M. Ishitoku;Y. Yoshida;N. Mizuno;Tatsuhiko Ozawa;Masatoshi Kawataka;S. Hirata;H. Kishi;E. Sugiyama
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文献类型：
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作者：
H. Kohno;K. Ouhara;S. Mokuda;T. Tokunaga;T. Sugimoto;H. Watanabe;M. Ishitoku;Y. Yoshida;N. Mizuno;Tatsuhiko Ozawa;Masatoshi Kawataka;S. Hirata;H. Kishi;E. Sugiyama
前言：脂多糖(LPS)对商业蛋白的污染严重阻碍了类风湿滑膜细胞(RSCs)中瓜氨酸纤维蛋白原(Cit-Fb)的研究。方法：取4例风湿性关节炎患者行全膝关节置换术，体外培养RSC，用Cit-Fb刺激，检测细胞因子表达水平。然后，我们评估了多粘菌素-B(PMB)、热灭活和Rough(R)型内毒素突变体，以快速检测内毒素污染。结果：Cit-FB通过Toll样受体诱导RSCs表达CXCL10和IFNB。PMB可抑制20μg/mLCit-Fb诱导的CXCL10基因表达，但不抑制蛋白表达。热灭活不影响内毒素介导的CXCL10或IL-6的诱导，但抑制Cit-FB介导的CXCL10的表达。野生型大肠杆菌脂多糖(WT-LPS)对CXCL10的诱导作用较强，Ra-LPS诱导作用较弱，Rc-和Re-LPS诱导作用较弱。Re-LPS抑制WT-LPS介导的RSCs和外周血单核细胞CXCL10诱导的CXCL10表达呈剂量依赖关系。此外，Re-内毒素可完全抑制Cit-FB介导的CXCL10在RSCs和PBMS中的诱导。结论：与PMB和热处理相比，在常规实验中，Re-LPS是一种更好的快速检测LPS污染的方法。
Introduction: Lipopolysaccharide (LPS) contamination of commercially available proteins has seriously impeded research on citrullinated fibrinogen (cit-Fb) in rheumatoid synovial cells (RSCs). Methods: RSCs obtained from 4 rheumatoid arthritis patients who underwent full knee arthroplasty were cultured, stimulated with cit-Fb, and cytokine expression levels were measured. We then evaluated polymyxin-B (PMB), heat inactivation, and rough (R)-type LPS mutants for rapid detection of LPS contamination. Results: cit-Fb induced expression of CXCL10 and IFNB in RSCs via the toll-like receptor. PMB inhibited cit-Fb-mediated CXCL10 gene expression but not protein expression induced by 20 μg/mL cit-Fb. Heat inactivation did not affect LPS-mediated CXCL10 or IL-6 induction; however, cit-Fb-mediated CXCL10expression was inhibited. Wild-type LPS from Escherichia coli (WT-LPS) strongly induces CXCL10 expression, but induction by Ra-LPS was weak, and induction by Rc- and Re-LPS was minimal. Re-LPS suppression of WT-LPS-mediated CXCL10 induction in RSCs and peripheral blood monocytes (PBMs) was dose dependent. Furthermore, Re-LPS completely suppressed cit-Fb-mediated CXCL10 induction in RSCs and PBMs. Conclusion: To easily identify LPS contamination during routine experiments, our results suggest that Re-LPS is a better tool for rapid detection of LPS contamination compared to PMB and heat treatment.