LPS那么多，你选对了吗？

 LPS是革兰氏阴性细菌的主要成分，可被Toll样受体4（TLR4）识别以激活先天免疫系统。通过信号级联反应，导致NF-κB的活化和促炎性细胞因子的产生，因此LPS可以应用到免疫细胞的刺激、激活、分化等实验中。但是LPS种类好多，而且并不是所有的LPS都能够激活TLR4受体，LPS-RS作为TLR4的拮抗剂，可以竞争性抑制LPS的活性。

在选订LPS时，您是否有不知道选择哪一种的困惑？在实验效果不好的时候，您是否有怀疑过是不是使用的LPS不正确？希望以下的内容，能够在您选择产品时为您提供帮助。

图1. 不同TLR4受体激动剂的结构

不同的细菌种类表达不同的LPS，其生物活性也有所不同。在某些细菌中，可能不存在O-抗原，表达粗糙型LPS。与光滑型LPS的典型表型相反，O-抗原的缺乏可调节免疫系统细胞识别LPS的过程。 

LPS选择指南

LPS-EB from E. coli 0111:B4

特点：

- E. coli 0111:B4菌株是已知可引起显著胃部疾病的致病性大肠埃希菌血清型

-文献中引用最多的LPS菌株

-在体外和体内都可以发现广泛的应用，包括巨噬细胞分化

应用实例：

-在体外诱导人巨噬细胞(M1)极化¹

-刺激小鼠巨噬细胞表达siglec²

-诱导大西洋鳕鱼和鲑鱼单核细胞的炎症反应³

-在小鼠模型中诱导脓毒性休克和急性肝衰竭⁴

-腹腔注射LPS治疗小鼠低铁血症研究⁵

-诱导诺如病毒感染后人肠上皮细胞的形态学变化⁶

相关应用文献

1.Vogel, D. Y. et al., 2014. Human macrophage polarization in vitro: maturation and activation methods compared. Immunobiology, 219(9), 695-703.

2. Nagala, M. et al., 2018. Expression of Siglec-E alters the proteome of lipopolysaccharide (LPS)-activated macrophages but does not affect LPS-driven cytokine production or toll-like receptor 4 endocytosis. Frontiers in immunology, 8, 1926.

3. Seppola, M. et al., 2015. Ultrapure LPS induces inflammatory and antibacterial responses attenuated in vitro by exogenous sera in Atlantic cod and Atlantic salmon. Fish & shellfish immunology, 44(1), 66-78.

4. Rialdi, A. et al., 2016. Topoisomerase 1 inhibition suppresses inflammatory genes and protects from death by inflammation. Science, 352(6289).

5. Guida, C. et al., 2015. A novel inflammatory pathway mediating rapid hepcidin-independent hypoferremia. Blood, The Journal of the American Society of Hematology, 125(14), 2265-2275.

6. Herbst-Kralovetz, M. M. et al., 2013. Lack of norovirus replication and histo-blood group antigen expression in 3-dimensional intestinal epithelial cells. Emerging infectious diseases, 19(3), 431.

LPS-EK from E. coli K12

特点：

-E. coli K12菌株为典型实验室菌株

-具有结构异质性

-一种主要用于体外试验的强效TLR4激动剂

应用实例：

-刺激骨髓源性巨噬细胞和人外周血单核细胞产生促炎细胞因子⁷

-诱导小鼠脾细胞的B细胞增殖⁸

-刺激小鼠造血和气道上皮细胞用于哮喘研究⁹

-用于HIV感染后刺激树突状细胞和CD4+T细胞¹⁰

-诱导COVID-19患者PBMC产生IL-6、TNF-α和CCL2¹¹

-用于启动THP-1细胞以促进炎症小体活化¹²

相关应用文献：

7. Saz-Leal, P. et al., 2018. Targeting SHIP-1 in myeloid cells enhances trained immunity and boosts response to infection. Cell reports, 25(5), 1118-1126.

8. Kang, S. S. et al., 2018. Staphylococcal LTA antagonizes the B cell-mitogenic potential of LPS. Scientific reports, 8(1), 1-12.

9. McAlees, J. W. et al., 2015. Distinct Tlr4-expressing cell compartments control neutrophilic and eosinophilic airway inflammation. Mucosal immunology, 8(4), 863-873.

10. Rosa, A. et al., 2015. HIV-1 Nef promotes infection by excluding SERINC5 from virion incorporation. Nature, 526(7572), 212-217.

11. Carvelli, J. et al., 2020. Association of COVID-19 inflammation with activation of the C5a–C5aR1 axis. Nature, 1-9.

12. Kroes, M. M. et al., 2019. Activation of human NK cells by Bordetella pertussis requires inflammasome activation in macrophages. Frontiers in Immunology, 10, 2030.

LPS-B5 from E. coli 055:B5

特点：

- E. coli 055：B5菌株是LAL试验中经常用作内毒素标准品的原型内毒素

-高致热原性

-常用于体外细胞活化

应用实例：

-在小鼠巨噬细胞中产生促炎性细胞因子分泌¹³

-诱导小鼠脾脏B细胞分泌IL-6¹⁴

-诱导大鼠骨髓源性MSCs4中IL-6和生长因子的表达¹⁵

-诱导硬骨鱼巨噬细胞中TNFα和IL-1β的表达¹⁶

-研究猪瘟病毒感染后猪组织中TLR4的mRNA表达¹⁷

相关应用文献

13.Krukenberg, K. et al., 2015. Extracellular poly (ADP-ribose) is a pro-inflammatory signal for macrophages. Chemistry & biology, 22(4), 446-452.

14. Kiripolsky, J. et al., 2020. Activation of Myd88-Dependent TLRs Mediates Local and Systemic Inflammation in a Mouse Model of Primary Sjögren's Syndrome. Frontiers in Immunology, 10, 2963.

15. Evaristo-Mendonça, F. et al., 2019. Preconditioning of Rat Bone Marrow-Derived Mesenchymal Stromal Cells with Toll-Like Receptor Agonists. Stem cells international, 2019.

16. Bi, D. et al., 2018. Recognition of lipopolysaccharide and activation of NF-κB by cytosolic sensor NOD1 in teleost fish. Frontiers in immunology, 9, 1413.

17. Cao, Z. et al., 2018. Tissue expression of Toll-like receptors 2, 3, 4 and 7 in swine in response to the Shimen strain of classical swine fever virus. Molecular Medicine Reports, 17(5), 7122-7130

LPS-PG from Porphyromonas gingivalis

特点：

-牙周病机制中的一个重要毒力因子

-具有独特的异质化学结构，不同于传统公认的肠道细菌来源的LPS

-通常与牙周炎相关研究相关的应用

应用实例：

-在小鼠和大鼠牙周炎模型中引起炎症反应^18.19

-研究牙龈卟啉单胞菌-诱导类风湿性关节炎患者树突状细胞的细胞因子应答²⁰

-诱导人牙周膜(PDL)细胞中NF-κB活化²¹

-用于刺激人牙髓细胞(HDPCs)以研究mRNA表达²²

-用于刺激破骨细胞前体细胞以研究破骨细胞基因调控²³

相关应用文献：

18. Park, J. et al., 2017. Fucoidan inhibits LPS-induced inflammation in vitro and during the acute response in vivo. International immunopharmacology, 43, 91-98.

19. Chang, P. C. et al., 2013. Irradiation by light‐emitting diode light as an adjunct to facilitate healing of experimental periodontitis in vivo. Journal of periodontal research, 48(2), 135-143.

20. Santegoets, K. C. et al., 2016. Impaired Porphyromonas gingivalis–induced tumor necrosis factor production by dendritic cells typifies patients with rheumatoid arthritis. Arthritis & Rheumatology, 68(4), 795-804.

21. Gölz, L. et al., 2015. Hypoxia and P. gingivalis synergistically induce HIF-1 and NF-κB activation in PDL cells and periodontal diseases. Mediators of inflammation, 2015.

22. Feng, Z. et al., 2018. METTL 3 regulates alternative splicing of MyD88 upon the lipopolysaccharide‐induced inflammatory response in human dental pulp cells. Journal of cellular and molecular medicine, 22(5), 2558-2568.

23. Chen, Z. et al., 2015. IL-1R/TLR2 through MyD88 divergently modulates osteoclastogenesis through regulation of nuclear factor of activated T cells c1 (NFATc1) and B lymphocyte-induced maturation protein-1 (Blimp1). Journal of Biological Chemistry, 290(50), 30163-30174.

LPS-SM from Salmonella minnesota R595

特点：

-仅由脂质A和3-脱氧-D-甘露-辛酮糖酸组成

-通常用于体外细胞活化

应用实例：

-刺激肉鸡血小板中促炎性细胞因子的产生²⁴

-诱导PBMC分泌IL-6、TNF-α和IL-8²⁵

-刺激腹腔巨噬细胞产生IL-1β²⁶

-启动小鼠骨髓来源的树突状细胞产生IL-1β前体和Caspase1前体²⁷

-诱导大西洋鳕鱼和鲑鱼单核细胞的炎症反应3

相关应用文献

24. Ferdous, F. et al., 2008. Broiler chick thrombocyte response to lipopolysaccharide. Poultry science, 87(1), 61-63.

25. Berthet, J. et al., 2012. Human platelets can discriminate between various bacterial LPS isoforms via TLR4 signaling and differential cytokine secretion. Clinical Immunology, 145(3), 189-200.

26. Brykczynska, U. et al., 2020. Distinct Transcriptional Responses across Tissue-Resident Macrophages to Short-Term and Long-Term Metabolic Challenge. Cell Reports, 30(5), 1627-1643.

27. Ravindran, R. et al., 2016. The amino acid sensor GCN2 controls gut inflammation by inhibiting inflammasome activation. Nature, 531(7595), 523-527.

LPS-RS from Rhodobacter sphaeroides (TLR4 拮抗剂)

特点：

-LPS-RS Standard 是TLR2的强效激动剂，而LPS-RS Ultrapure特异性拮抗TLR4

-在拮抗剂过量100倍时，可能会完全竞争性抑制LPS活性

-可通过LAL测定法检测

应用实例：

-抑制人子宫内膜内皮细胞(HEECs)中TLR-4依赖性细胞因子分泌²⁸

-抑制中耳胆脂瘤干细胞(ME-CSCs)中TLR4依赖性炎症²⁹

-研究LPS诱导的大鼠急性肺损伤的减轻³⁰

-研究小鼠血管痉挛和神经功能损伤的减轻³¹

-抑制TLR4诱导的CD14内吞作用³²

相关应用文献

28. Krikun, G. et al., 2012. Lipopolysaccharide appears to activate human endometrial endothelial cells through TLR‐4‐dependent and TLR‐4‐independent mechanisms. American journal of reproductive immunology, 68(3), 233-237.

29. Schürmann, M. et al., 2020. Stem Cell-Induced Inflammation in Cholesteatoma Is Inhibited by the TLR4 Antagonist LPS-RS. Cells, 9(1), 199.

30. Abdelmageed, M. E. et al., 2016. LPS-RS attenuation of lipopolysaccharide-induced acute lung injury involves NF-κB inhibition. Canadian Journal of Physiology and Pharmacology, 94(2), 140-146.

31. Kawakita, F. et al., 2017. Effects of Toll-like receptor 4 antagonists against cerebral vasospasm after experimental subarachnoid hemorrhage in mice. Molecular neurobiology, 54(8), 6624-6633.

32. Tan, Y. et al., 2015. Mechanisms of Toll-like receptor 4 endocytosis reveal a common immune-evasion strategy used by pathogenic and commensal bacteria. Immunity, 43(5), 909-922.

InvivoGen提供两种纯度的高品质LPS

 市售脂多糖(LPS)的纯度可能存在显著差异。一些混合物中含有额外的细菌成分，例如肽聚糖或脂肽，能够激活TLR2和TLR4受体。InvivoGen提供两种等级的LPS：通过苯酚提取制备的Standard 级别LPS，能够激活TLR2和TLR4，而连续酶处理的Ultrapure 级别的LPS不含脂蛋白，仅激活TLR4。InvivoGen是特异性激活TLR4的Ultrapure LPS的唯一提供者。

图2.激活TLR2受体活性对比。使用不同浓度的其他品牌的LPS-EB和LPS-B5，InvivoGen的Ultrapure的LPS-EB和LPS-B5以及Pam3CSK4刺激HEK-Blue™hTLR2细胞。孵育后，通过读取655 nm的OD值来确定NF-κB诱导的SEAP活性。

图3.激活TLR4受体活性对比。用其他品牌的LPS-EB和InvivoGen的LPS-EB Ultrapure以不同浓度刺激HEK-Blue™hTLR4细胞。孵育后，通过读取655 nm的OD值来确定NF-κB诱导的SEAP活性。

 其实TLR4的激动剂不只有LPS呦，InvivoGen还提供天然的和合成的LPS类似物。后续小编还会继续分享其他TLR4受体的选择指南，欢迎关注。

InvivoGen 1977年成立于法国，是法国第一家生物科技公司。公司成立的初期阶段主要提供细胞培养相关试剂，如抗支原体、细菌、真菌污染的抗生素以及基因筛选抗生素（Blasticidin, Puromycin, Hygromycin B, G418, Zeocin）。随着天然免疫研究热点的兴起，InvivoGen于2000年开始致力于天然免疫相关产品的研发和生产，并成为全世界最优质、最专业的天然免疫相关产品生产商。InvivoGen为天然免疫研究提供一整套研究方案，包括不断更新的模式识别受体（PRRs）激动剂和拮抗剂，PRRs报告基因细胞以及天然免疫信号通路的诱导剂和抑制剂等。