Studies of Receptor Interactions and Effects of Alarmins

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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.