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. These alarmins, if administered together with an antigen result in considerable augmentation of both in vivo cellular and humoral immune responses. We previously identified both alpha and beta types of defensins as alarmins with chemotactic and activating effects on immature dendritic cells (iDCs) as having in vivo immunoadjuvant effects. Some of the beta defensins interact with the CCR6 chemokine receptor, others with CCR2. Defensins by binding to DNA also can activate DC's by triggering TLR-9. The resultant cytokine production has considerable proinflammatory effects. We recently prepared genetically engineered mice deficient in Beta-Defensins 4 and 14 in collaboration with Drs. Teizo Yoshimura and Lino Tessarollo. These mice remained phenotypically normal for 1-2 years. However, when sensitized and challenged with the contact sensitizer, Dinitrochlorobenzene (DNCB), they were markedly deficient in developing cutaneous delayed hypersensitivity responses and antibodies to DNCB. This reinforced our hypothesis that defensins promote 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. To maximize the adjuvant effects of HMGN1, we covalently linked it to a gp100 melanoma tumor antigen. We have immunized mice with gp100 linked to HMGN1 in the form of plasmid DNA using gene gun technology. This succeeded in inducing about 70% of the immunized mice to be resistant to a challenge with B16 melanoma tumor cells. However, therapy of mice with this plasmid DNA, which had been injected with B16 melanoma tumor cells four days previously, failed to inhibit tumor growth. 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 it in conjunction with other antitumor therapies to cure mice with larger tumors. We used R848, a TLR7/8 ligand, together with HMGN1, a TLR4 ligand, because they synergistically stimulated the maturation of dendritic cells and markedly increased their production of IL-12 and TNF. We have been able to show that intratumoral injections of several TLR ligands together with checkpoint inhibitors cures mice with five types 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 liver tumors. In addition, we have even been able to cure 80% of mice bearing B16/F10 melanoma tumors by adding cGAMP, a a ligand of the STING pathway to our TheraVac antitumor protocol. In collaboration with Dr. Mihai Netea's laboratory at Radboud University at the Nijmegen Medical Center in the Netherlands, we investigated the possibility that HMGN1 induces epigenetic effects. Pre-exposure of TLR4 expressing cells to HMGN1 or LPS has desensitizing effects on each of them and inhibits the activating capacity of the second stimulant . Presumably this occurs downstream of their distinct MD2 binding sites. Both HMGN1 and LPS induced Sirtuin-1 expression. The capacity of HMGN1 to induce TLR4 desensitization was associated with histone acetylation via Sirtuin-1. Conversely, both blocking of TLR4 signaling and specific inhibition of Sirtuin-1 reduced HMGN1 induced desensitization. This suggests that targeting Sirtuin-1 may reduce the desensitizing effect of HMGN1 and thus increase its immunostimulatory potency. 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 IFN, which was associated with enhanced expression of the T-bet transcription factor and Th1 polarization favoring cellular antitumor immunity. We are also investigating 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 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 characterized 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 response to a request by 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. Thus this represents the identification of a moiety causally associated with Neurodegenerative Diseases as having potent proinflammatory immunological effects.
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