Studies of Receptor Interactions and Effects of Alarmins

警报器的特点是对表达GiPCR的细胞具有体外趋化或体内募集活性，同时具有与其他受体相互作用的能力，导致未成熟的树突状细胞（iDC）活化为能够与T淋巴细胞相互作用的成熟抗原提呈。这使得DC能够将它们吞噬的抗原呈递到T淋巴细胞，并产生适应性免疫反应。因此，体外对Dc的刺激作用可以用来预测警报素促进体内免疫反应的能力。因此，如果这些警报与抗原一起使用，会导致体内细胞和体液免疫反应的显著增强。我们之前的研究表明，HMGN-1敲除小鼠对肿瘤（EG-7或EL-4）的抗性降低。相反，当转染过表达HMGN1的肿瘤细胞（EG-7或EL-4）时，正常小鼠的生长速度明显降低。这些观察结果表明，HMGN1能够增强肿瘤免疫。因此，我们将重组HMGN1蛋白直接注射到小鼠CT26结肠肿瘤中，以接近佐剂和抗原。这种治疗性疫苗试验确实减缓了肿瘤的生长，延长了小鼠的生存时间，但没有治愈任何小鼠。因此，我们通过联合使用免疫治疗抗肿瘤试剂来治疗具有较大肿瘤的小鼠，从而提高了肿瘤疫苗的效力。我们筛选了所有TLR配体，以确定与HMGN1配合最好的TLR4配体。这导致了TLR7/8配体R848的鉴定，该配体能够最大限度地与TLR4协同刺激树突状细胞的成熟，从而显着增加其IL-12和TNF的产生。我们已经能够证明，在肿瘤内注射这两种TLR配体以及各种检查点抑制剂可以治愈患有五种不同肿瘤的小鼠，并导致它们随后对这些肿瘤的再挑战产生抵抗力。由HMGN1、R848 （Resiquimod）、检查点抑制剂（如抗PDL-1或抗CTLA4抗体）或低剂量环环酰胺组成的免疫治疗剂联合使用，成功治愈了小鼠结肠（CT26）、肾脏（RENCA）、胸腺瘤（EG7）、肺（Lewis lung）和肝脏（Hepa1-6）的大（1cm直径）肿瘤。我们将这种抗肿瘤药物的组合称为“TheraVac”。我们还开发了一种将HMGN1和R848滴注在金纳米颗粒上的方法，成功地治愈了位于其侧翼的大结肠和肝细胞系衍生的肿瘤。我们还用TheraVac治疗了小鼠的遗传性自发性肿瘤，并成功地减缓了它们的生长，显著延长了它们的寿命。此外，通过在我们的TheraVac抗肿瘤方案中添加STING途径的配体cGAMP，我们已经能够治愈70%携带耐药B16/F10黑色素瘤肿瘤的小鼠。我们还研究了HMGN1和R848协同刺激作用的机制。众所周知，刺激细胞表面TLR4激活MyD88和TRIF依赖通路，从而激活MAP激酶、NFKB和I型ifn的产生。虽然R848对TLR7/8的刺激发生在细胞内的核内体中，不刺激TRIF通路，但它也激活MyD88通路、MAP激酶、NFKB和IRF7，导致I型IFN的产生。我们发现，HMGN1和R848同时刺激导致NFKB、MAPK、IRF3和IRF7转录因子协同激活，介导促炎细胞因子和I型ifn的协同产生，并显著上调指示活化DC成熟的表型标记。细胞因子如IL-12的协同产生增强了IFNgamma的产生，这与T-bet转录因子和Th1极化的增强表达有关，有利于细胞抗肿瘤免疫。此外，我们使用RNAseq技术研究了HMGN1和R848单独和联合使用的效果。这项研究已经确定了由这些配体激活的独特基因，并且通过将这些TLR配体一起给予其中一种TLR配体，可以显著增加许多促炎基因的表达。在过去的十年里，我们与中国北京广安门医院的林教授和她的同事合作，测试了许多中药，以检测任何具有免疫刺激作用的中药。我们最终确定，纯化和化学合成的中药隐丹参酮（Cryptotanshinone， CT）不仅对肿瘤细胞具有细胞毒作用，而且还能在体外激活树突状细胞成熟并产生细胞因子（如IL-12和TNF）。我们还能够证明，当与检查点抑制剂一起在瘤内或静脉注射时，CT具有治愈大（直径1厘米）结肠肿瘤的能力。我们最近也能够证明CT需要表达MyD88通路和TLR7受体来激活树突状细胞的成熟，这表明其作用机制与R848相似。在与Michael Zasloff博士的合作中，我们发现α -突触核蛋白（α -s）是中性粒细胞和单核细胞的有效趋化蛋白。这一结果有助于理解在以急性中性粒细胞和慢性单核炎症细胞浸润为特征的多种肠壁炎症患者的活检标本中，人类肠神经系统中α - s的高表达。随后，我们也证明了α - s也是树突状细胞的有效激活剂，因此具有警报蛋白的功能。这是特别有趣的，因为α - s突变与帕金森病的发展有关。缺失α - s基因的小鼠存在细胞和体液免疫应答缺陷，表明α - s具有重要的免疫功能。因此，我们的研究结果表明，与神经退行性疾病有因果关系的α -s具有强大的促炎免疫作用。

Alarmins are characterized by having in vitro chemotactic or in vivo recruitment activity for cells expressing GiPCR, together with the capacity to interact with other receptors resulting in the activation of immature dendritic cells (iDC) into mature antigen-presenting capable of interacting with T lymphocytes.This enables the DC to present antigens they have phagocytized and processed to T lymphocytes and results in adaptive immune responses. Consequently, the in vitro stimulant effects on Dc can be used to predict the capacity of an alarmin to promote immune responses in vivo. Thus, these alarmins, if administered together with an antigen result in considerable augmentation of both in vivo cellular and humoral immune responses. We previously showed that HMGN-1 knockout mice exhibit reduced resistance to tumor (EG-7 or EL-4) challenge. Conversely, tumor cells (EG-7 or EL-4) when transfected to overexpress HMGN1 showed a marked reduction in the rate of growth in normal mice. These observations indicated that HMGN1 is capable of augmenting tumor immunity. . We therefore injected a recombinant HMGN1 protein directly intratumorally into CT26 colon tumors in mice to proximate the adjuvant and antigen. This therapeutic vaccine trial did slow the tumor growth and prolonged the survival of mice, but did not cure any of the mice. We therefore improved the potency of the tumor vaccine by employing combinations of immunotherapeutic antitumor reagents to cure mice with larger tumors .We screened all the TLR ligands to identify which cooperated best with HMGN1 our TLR4 ligand. This led to the identification of R848, a TLR7/8 ligand as capable of maximally synergizing with TLR4 in stimulating the maturation of dendritic cells to markedly increase their production of IL-12 and TNF. We have been able to show that intratumoral injections of these two TLR ligands together with various checkpoint inhibitors can cure mice with five different of tumors and results in their subsequent resistance to re-challenge with these tumors. The combination of immunotherapeutic agents consisting of HMGN1, R848 (Resiquimod), a checkpoint inhibitor such as anti PDL-1 or anti CTLA4 antibody or a low dose of cytoxan successfully cured large (1cm diam.) tumors of the colon (CT26), kidney (RENCA), thymoma (EG7) lung (Lewis Lung) and liver (Hepa1-6) in mice. We have termed this combination of antitumor therapeutics, "TheraVac". We have also developed a means of delivering the HMGN1 and R848 on gold nanoparticles intravenously with success in curing large colon and hepatic cell line derived tumors located in their flanks. We have also treated genetically derived spontaneous tumors of mice with TheraVac and succeeded in slowing their growth and prolonging their life span significantly. In addition, we have been able to cure 70% of mice bearing resistant B16/F10 melanoma tumors by adding cGAMP, a a ligand of the STING pathway to our TheraVac antitumor protocol. We also investigated the mechanisms accounting for the synergistic stimulatory effects of HMGN1 and R848. It is well known that stimulation of cell surface TLR4 activates MyD88 and TRIF dependent pathways resulting in the activation of MAP kinases, NFKB and the production of type I IFNs . Although stimulation of TLR7/8 by R848 occurs intracellularly in the endosome and does not stimulate the TRIF pathway, it also activates the MyD88 pathway, MAP kinases, NFKB and IRF7 resulting in type I IFN production. We showed that simultaneous stimulation by HMGN1 and R848 resulted in synergistic activation of NFKB, MAPK, IRF3 and IRF7 transcription factors mediating the synergistic production of proinflammatory cytokines and type I IFNs as well as markedly upregulating the phenotypic markers indicative of maturation of activated DC's. Synergistic production of cytokines such as IL-12 enhanced the production of IFNgamma, which was associated with enhanced expression of the T-bet transcription factor and Th1 polarization favoring cellular antitumor immunity. Furthermore, we investigated the effects if HMGN1 and R848 alone and in combination using RNAseq technology. This has identified unique genes activated by these ligands and marked increases in the expression of many proinflammatory genes stimulated by just one of the TLR ligands by giving these TLR ligands together. In collaboration with Professor Lin and her colleagues at the Traditional Chinese Medicine (TCM) Guang'anmen Hospital, Beijing, China we have tested a number of TCM's over the past decade in an effort to detect any with immunostimulant effects. We finally determined that a purified and chemically synthesized TCM known as Cryptotanshinone (CT) not only had cytotoxic effects on tumor cells, but also activated dendritic cells in vitro to mature and produce cytokines (e.g. IL-12 and TNF). We also were able to show that CT had the capacity to cure mice with large (1 cm diam.) colon tumors, when injected either intratumorally or intravenously together with a checkpoint inhibitor. We were recently also able to show that CT required expression of the MyD88 pathway and TLR7 receptor to activate maturation of Dendritic Cells suggesting its mechanism of action is like that of R848. In collaboration with Dr. Michael Zasloff, we showed that alpha-synuclein (alpha-S) was a potent chemotactic protein for neutrophils and monocytes. This result helped to understand the high expression of alpha-S within the human enteric nervous system in biopsy specimens from patients with a variety of inflammatory conditions of the intestinal wall characterized by infiltrations of acute neutrophil and chronic mononuclear inflammatory cells. Subsequently we also demonstrated that alpha-S was also a potent activator of dendritic cells and therefore functioned as an alarmin. This is of particular interest since mutations of alpha-S are associated with the development of familiar Parkinson's Disease. Mice with deletion of the Alpha-S genes have deficient cellular and humoral immune responses, indicative of the important immunological function of Alpha-S. Thus, our findings show Alpha-s, which is causally associated with Neurodegenerative Diseases as having potent proinflammatory immunological effects.