Development of FluoAHRL: A Novel Synthetic Fluorescent Compound That Activates AHR and Potentiates Anti-Inflammatory T Regulatory Cells
Natalija Jonić,
Ivan Koprivica,
Christos M. Chatzigiannis,
Antonis D. Tsiailanis,
Stavroula G. Kyrkou,
Eleftherios Paraskevas Tzakos,
Aleksandar Pavić,
Mirjana Dimitrijević,
Andjelina Jovanović,
Milan B. Jovanović,
Sérgio Marinho,
Inês Castro-Almeida,
Vesna Otašević,
Pedro Moura-Alves,
Andreas G. Tzakos,
Ivana Stojanović
Affiliations
Natalija Jonić
Department of Immunology, Institute for Biological Research “Siniša Stanković”—National Institute of the Republic of Serbia, University of Belgrade, 11108 Belgrade, Serbia
Ivan Koprivica
Department of Immunology, Institute for Biological Research “Siniša Stanković”—National Institute of the Republic of Serbia, University of Belgrade, 11108 Belgrade, Serbia
Christos M. Chatzigiannis
Section of Organic Chemistry & Biochemistry, Department of Chemistry, University of Ioannina, 45110 Ioannina, Greece
Antonis D. Tsiailanis
Section of Organic Chemistry & Biochemistry, Department of Chemistry, University of Ioannina, 45110 Ioannina, Greece
Stavroula G. Kyrkou
Section of Organic Chemistry & Biochemistry, Department of Chemistry, University of Ioannina, 45110 Ioannina, Greece
Eleftherios Paraskevas Tzakos
Department of Biology, National and Kapodistrian University of Athens, 15772 Athens, Greece
Aleksandar Pavić
Laboratory for Microbial Molecular Genetics and Ecology, Institute for Molecular Genetics and Genetic Engineering, University of Belgrade, 11000 Belgrade, Serbia
Mirjana Dimitrijević
Department of Immunology, Institute for Biological Research “Siniša Stanković”—National Institute of the Republic of Serbia, University of Belgrade, 11108 Belgrade, Serbia
Andjelina Jovanović
Department of Otorhinolaryngology with Maxillofacial Surgery, Clinical Hospital Center “Zemun”, 11080 Belgrade, Serbia
Milan B. Jovanović
Department of Otorhinolaryngology with Maxillofacial Surgery, Clinical Hospital Center “Zemun”, 11080 Belgrade, Serbia
Sérgio Marinho
Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, 4200-135 Porto, Portugal
Inês Castro-Almeida
Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, 4200-135 Porto, Portugal
Vesna Otašević
Department of Molecular Biology, Institute for Biological Research “Siniša Stanković”—National Institute of the Republic of Serbia, University of Belgrade, 11108 Belgrade, Serbia
Pedro Moura-Alves
Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, 4200-135 Porto, Portugal
Andreas G. Tzakos
Section of Organic Chemistry & Biochemistry, Department of Chemistry, University of Ioannina, 45110 Ioannina, Greece
Ivana Stojanović
Department of Immunology, Institute for Biological Research “Siniša Stanković”—National Institute of the Republic of Serbia, University of Belgrade, 11108 Belgrade, Serbia
Aryl Hydrocarbon Receptor (AHR) ligands, upon binding, induce distinct gene expression profiles orchestrated by the AHR, leading to a spectrum of pro- or anti-inflammatory effects. In this study, we designed, synthesized and evaluated three indole-containing potential AHR ligands (FluoAHRL: AGT-4, AGT-5 and AGT-6). All synthesized compounds were shown to emit fluorescence in the near-infrared. Their AHR agonist activity was first predicted using in silico docking studies, and then confirmed using AHR luciferase reporter cell lines. FluoAHRLs were tested in vitro using mouse peritoneal macrophages and T lymphocytes to assess their immunomodulatory properties. We then focused on AGT-5, as it illustrated the predominant anti-inflammatory effects. Notably, AGT-5 demonstrated the ability to foster anti-inflammatory regulatory T cells (Treg) while suppressing pro-inflammatory T helper (Th)17 cells in vitro. AGT-5 actively induced Treg differentiation from naïve CD4+ cells, and promoted Treg proliferation, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) expression and interleukin-10 (IL-10) production. The increase in IL-10 correlated with an upregulation of Signal Transducer and Activator of Transcription 3 (STAT3) expression. Importantly, the Treg-inducing effect of AGT-5 was also observed in human tonsil cells in vitro. AGT-5 showed no toxicity when applied to zebrafish embryos and was therefore considered safe for animal studies. Following oral administration to C57BL/6 mice, AGT-5 significantly upregulated Treg while downregulating pro-inflammatory Th1 cells in the mesenteric lymph nodes. Due to its fluorescent properties, AGT-5 could be visualized both in vitro (during uptake by macrophages) and ex vivo (within the lamina propria of the small intestine). These findings make AGT-5 a promising candidate for further exploration in the treatment of inflammatory and autoimmune diseases.
