Development of new anti-rheumatic drugs

抑制CDK4/6的细胞周期蛋白依赖的激酶抑制物(CDKI)p16和lt；INK4a和gt；在滑膜组织中的表达上调，以及全身应用小分子(Sm)CDK4/6抑制剂抑制类风湿关节炎的动物模型。虽然两者都抑制细胞周期进程，但它们在调节炎症介质产生方面发挥着不同的作用。本研究的这一部分是为了研究p16^&lt；INK4a&gt；表达如何通过简单的CDK4/6激酶抑制来调节巨噬细胞炎性细胞因子的产生。在骨髓巨噬细胞中强制表达p16^-lt；INK4a&gt可抑制脂多糖诱导的IL-6的表达，但不能抑制肿瘤坏死因子α的表达。在RSF中没有观察到这一点。Sm CDK4/6抑制剂和阻断CDK4的shRNA不能抑制IL-6的产生，表明这种抑制不依赖于CDK4/6激酶活性的降低。P16INK4a的表达加速了脂多糖诱导的IRAK1的降解，并抑制了…P38MAPK/AP-1通路较多，而NFκB通路较少。用小发夹RNA直接下调IRAK1也可以诱导AP-1通路的特异性抑制。IRAK-1的加速降解可能是由蛋白酶体降解介导的，因为蛋白酶体抑制剂恢复了p16INK4a表达的BMM中p38MAPK的激活。此外，IRAK1基因过表达可恢复p16INK4a表达的THP-1巨噬细胞系IL-6的产生。最后，用si RNA敲除p16INK4a可促进表达内源性p16INK4a的衰老骨髓细胞产生IL-6。这些结果表明，p16INK4a以CDK4/6非依赖性的方式抑制巨噬细胞产生IL-6。这是由于蛋白酶体介导的IRAK1降解以及随后AP-1途径的抑制。因此，与sm CDK抑制剂不同，p16INK4a可以对滑膜巨噬细胞发挥抗炎作用。髓系细胞上表达的触发受体(TREM)-1是我们正在研究的另一个治疗靶点。它由巨噬细胞和中性粒细胞表达。它的激活增加了Toll样受体参与触发的炎性细胞因子的产生。在小鼠脓毒症模型中，通过给予TREM-1胞外区/Ig Fc区融合蛋白(TREM-1-Ig)阻断TREM-1可延长受感染小鼠的存活时间。这表明，TREM-1阻滞剂通过维持最低限度的炎性细胞因子的产生来抑制病理性炎症，以进行抗微生物防御。我们之前曾报道，TREM-1在类风湿关节的滑膜巨噬细胞上表达，并且TREM-1阻断可以改善类风湿关节炎的动物模型--小鼠胶原(CII)诱导的关节炎(CIA)。然而，由于TREM-1的配体尚不清楚，TREM-1-配体的生理作用以及TREM-1与TREM-1-配体之间的相互作用仍有待阐明。这部分研究是为了确定TREM-1配体分子，以确定其与关节炎的关系。为了寻找表达TREM-1配体的细胞，将不同类型的细胞与TREM-1-Ig孵育。它与小鼠B细胞和A20 B细胞淋巴瘤细胞结合。利用A20细胞cDNA文库进行表达克隆，我们发现了一个编码TREM-1配体的基因。此外，我们还制备了抗TREM-1配体封闭的单抗。给CIA小鼠注射这种抗体可以改善这种疾病。抗TREM-1配体单抗处理对T、B细胞对CII的反应无明显影响。因此，类似于TREM-1-Ig的作用，这种作用似乎是由于减弱了炎症反应，而不是阻止了适应性免疫反应。在未来的研究中，对人类TREM-1配体的鉴定将保证建立一种新的抗风湿疗法，该疗法与严重感染的风险无关。较少

Up-regulated expression of cyclin-dependent kinase inhibitor(CDKI) p16^<INK4a>, which inhibits CDK4/6, in the synovial tissues as well as systemic administration of small molecule(sm) CDK4/6 inhibitors suppress animal models of rheumatoid arthritis. Although both inhibit cell cycle progression, they exert differential effects especially in modulating inflammatory mediator production. This part of the present studies was conducted to investigate how p16^<INK4a> expression modulates inflammatory cytokine production from macrophages in relation to simple CDK4/6 kinase inhibition. Forced expression of p16^<INK4a> in bone marrow-derived macrophages(BMM) suppressed LPS-induced expression of IL-6 but not TNFα. This was not observed in RSF. A sm CDK4/6 inhibitor and shRNA to knock down CDK4 failed to suppress IL-6 production, demonstrating that the inhibition did not depend on decrease in CDK4/6 kinase activity. The p16INK4a expression accelerated LPS-induced IRAK1 degradation, and suppressed … More the p38 MAPK/AP-1 pathway but not the NFκB pathway. Direct down-regulation of IRAK1 with small hairpin RNA also induced specific inhibition of the AP-1 pathway. The accelerated IRAK-1 degradation should be mediated by proteasomal degradation because a proteosome inhibitor restored p38 MAPK activation in p16INK4a-expressing BMM. Also, IRAK1 gene overexpression restored IL-6 production in p16INK4a-expressing THP-1 macrophage cell line. Finally, p16INK4a knock down with si RNA enhanced IL-6 production from senescent BMM that expressed endogenous p16INK4a. These results showed that p16INK4a suppressed IL-6 production from macrophages in a CDK4/6-independent manner. This was due to proteosome-mediated IRAK1 degradation and following suppression of the AP-1 pathway. Thus, unlike sm CDK inhibitors, p16INK4a could exert anti-inflammatory effects on synovial macrophages.Triggering receptor expressed on myeloid cells(TREM)-1 is the other therapeutic target we are pursuing. It is expressed by macrophages and neutrophils. Its activation augments inflammatory cytokine production triggered by Toll-like receptor engagement. In a mouse sepsis model, blockade of TREM-1 by administration of a TREM-1 extracellular domain/Ig Fc domain fusion protein(TREM-1-Ig) prolonged survival of affected mice. This indicated TREM-1 blockade suppressed pathological inflammation with maintaining minimal inflammatory cytokine production for anti-microbial defense. We reported previously that TREM-1 is expressed on synovial macrophages in the rheumatoid joints and that TREM-1 blockade ameliorated mouse collagen(CII)-induced arthritis(CIA), which is an animal model of rheumatoid arthritis. However, since a ligand for TREM-1 was unknown, physiological roles of TREM-1-ligand and interactions between TREM-1 and TREM-1-ligand remained to be clarified. This part of the studies was conducted to identify the TREM-1-ligand molecule for discerning its involvement in arthritis. To search for cells expressing the TREM-1-ligand, various types of cells were incubated with TREM-1-Ig. It bound to mouse B cells and A20 B-cell lymphoma cells. Expression cloning using A20 cell cDNA library led us to identify a gene encoding the TREM-1-ligand. Furthermore, we raised anti-TREM-1-ligand blocking monoclonal antibody(mAb). Administration of this antibody to CIA mice ameliorated the disease. Anti-TREM-1-ligand mAb treatment exerted no apparent effects on T and B cell responses to CII. Thus, in analogous to the effect of TREM-1-Ig, this effect appeared attributable to attenuation of the inflammatory responses rather than prevention of the adaptive immune responses. Identification of the human TREM-1-ligand in the future study will warrant establishment of a new anti-rheumatic therapy that is not associated with a risk of serious infection. Less