Development of FluoAHRL: A Novel Synthetic Fluorescent Compound That Activates AHR and Potentiates Anti-Inflammatory T Regulatory Cells

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.This research was funded by the Ministry of Science, Technological Development and Innovations of the Republic of Serbia No. 451-03-66/2024-03/200007, Serbian Clinical Immunology Fund New Castle, UK. Also, this study has been supported by the European Regional Development Fund of the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation, under the call RESEARCH—CREATE—INNOVATE (project code: TAEDK-06189/Τ2EDK-0326, Acronym: Glioblastoma). Pedro Moura-Alves, Sérgio Marinho and Inês Castro-Almeida were funded by the European Union’s Horizon 2020 research and innovation programme under grant agreement No 951921 (ImmunoHUB). Graphical abstract was created by BioRender.com

Ispitivanje inaktivisane dvovalentne vakcine pripremljene od serotipova 1/2a i 4b Listeria monocytogenes u kontroli listerioze kod ovaca

In this study, the protective effects of two bivalent inactivated vaccines were evaluated. Vaccines were prepared from Listeria monocytogenes, serotypes 1/2a and 4b, as the most frequent in our and surrounding epidemiological areas. Vaccine A consists of whole L. monocytogenes bacteria cells, inactivated with 0.4% formaldehyde and aluminium hydroxide as a carrier. Vaccine B contains 0.1% saponin in addition to ingredients of vaccine A. Evaluations of these vaccines were performed in 60 sheep, divided into four groups (n=10) with a corresponding negative control group (n=5). After 14 days, boosterisation of all animals was performed. In order to evaluate the immune response, blood samples were obtained every 14 days during the next 6 months. Antibody titres were determined by microaglutitation (MAT) and complement fixation tests (CFT). Comparative analyses of antibody titres, induced by vaccines A and B, show that the latter (with saponine) significantly increased the level of antibody titres (p lt 0.01). The levels of immune response were also significantly impacted by the total number of bacteria and vaccine dosage (p lt 0.01). The bivalent vaccine containing 0.1% saponin (vaccine B) in 5.0 mLx 106 cfu/mL (colony-forming units per milliliter) dosage shows a protective effect after challenge with L. monocytogenes. The protective levels of this antibody were 1/80 and 1/16, determined by MAT and CFT, respectively. Antibody titres were significantly higher after boosterisation (p lt 0.01) and protective levels could be detected in the sera of vaccinated animals during the next 6 months. Therefore, it is strongly recommended to perform boosterisation two weeks after the initial vaccination.U ovom radu ispitivano je protektivno dejstvo eksperimentalno inaktivisane dvovalentne vakcine protiv listerioze ovaca.Vakcina je pripremana od sojeva L. monocytogenes 1/2a i 4b koji su najzastupljeniji na našem epizootiološkom području i u zemljama u okruženju. Vakcina A sadrži cele bakterijske ćelije koje su inaktivisane 0,4% formalinom uz dodatak aluminijum hidroksida kao nosača. Vakcina B pored gore navedenih sastojaka sadrži i 0,1% saponina. Ispitivanje ovako pripremljene eksperimentalne inaktivisane bivalentne vakcine protiv listerioze ovaca izvedeno je na 60 ovaca podeljenih u 4 grupe (n=10), pri čemu je svaka grupa imala kontrolnu grupu (n=5). Nakon 14 dana urađena je revakcinacija svih oglednih životinja. Krv je uzorkovana svakih 14 dana, tokom narednih 6 meseci i praćeno je kretanje titra antitela, metodom spore aglutinacije (MAT) i reakcijom vezivanja komplementa (RVK). Uporednim ispitivanjem visine titra antitela kod životinja koje su imunizovane vakcinom bez saponina i vakcinom sa 0,1% saponina ustanovljeno je da saponin značajno podstiče imunski odgovor. Ustanovljeno je da ukupan broj mikrooganizama u vakcini, kao i doza vakcine, utiču na kvalitet imunskog odgovora. Utvrđen je viši titar antitela ako se aplikuje doza vakcine od 5,0 ml × 106 cfu/ml nego kada je aplikovana doza od 2,5 ml × 106 cfu/ml (p lt 0,01). Dvovalentna vakcina pripremljena od inaktivisanih serotipova listerija sa saponinom u dozi od 5,0 ml × 106ml štitila je jagnjad od veštačke infekcije, a protektivni titar iznosio je 1:80 utvrđen metodom mikroaglutinacije, odnosno 1:16 metodom reakcije vezivanja komlementa. Titri antitela nakon revakcinacije su značajno viši nego posle prve vakcinacije (p,01) i mogli su da se otkriju u serumu životinja 6 meseci nakon vakcinacije, zbog čega se preporučuje obavezna revakcinacija 2 nedelje nakon vakcinacije

A study on the inactivated bivalent vaccine prepared from serotypes 1/2a and 4b Listeria monocytogenes for the control of listeriosis in sheep

In this study, the protective effects of two bivalent inactivated vaccines were evaluated. Vaccines were prepared from Listeria monocytogenes, serotypes 1/2a and 4b, as the most frequent in our and surrounding epidemiological areas. Vaccine A consists of whole L. monocytogenes bacteria cells, inactivated with 0.4% formaldehyde and aluminium hydroxide as a carrier. Vaccine B contains 0.1% saponin in addition to ingredients of vaccine A. Evaluations of these vaccines were performed in 60 sheep, divided into four groups (n=10) with a corresponding negative control group (n=5). After 14 days, boosterisation of all animals was performed. In order to evaluate the immune response, blood samples were obtained every 14 days during the next 6 months. Antibody titres were determined by microaglutitation (MAT) and complement fixation tests (CFT). Comparative analyses of antibody titres, induced by vaccines A and B, show that the latter (with saponine) significantly increased the level of antibody titres (p<0.01). The levels of immune response were also significantly impacted by the total number of bacteria and vaccine dosage (p<0.01). The bivalent vaccine containing 0.1% saponin (vaccine B) in 5.0 mLx 106 cfu/mL (colony-forming units per milliliter) dosage shows a protective effect after challenge with L. monocytogenes. The protective levels of this antibody were 1/80 and 1/16, determined by MAT and CFT, respectively. Antibody titres were significantly higher after boosterisation (p<0.01) and protective levels could be detected in the sera of vaccinated animals during the next 6 months. Therefore, it is strongly recommended to perform boosterisation two weeks after the initial vaccination. [Projekat Ministarstva nauke Republike Srbije, br. 31085 i br. 31088