Characteristic features of in vitro differentiation of human naïve CD4+ T cells to induced regulatory T cells (iTreg) and T helper (Th) 17 cells: Sharing of lineage-specific markers.

In vitro induced regulatory T cells (iTreg) and IL-17 producing T cells (Th17-like cells) can be generated in culture from native CD4+ T cells in peripheral blood by different sets of cytokines. In the presence of transforming growth factor (TGF)-ß plus interleukin (IL)-2, cells differentiate into Treg cells with increased expression of the forkhead box P3 (FOXP3). In the presence of TGF-ß, IL-6, IL-1ß and IL-23, cells differentiate into Th17 cells that produce IL-17A. However, protocols for the generation of human iTreg and Th17 are still controversial. In this study, we characterized the biological features of iTreg and Th17 cells differentiated from peripheral blood naïve CD4+ T cells in vitro using the established protocols. We showed that cells obtained from Treg or Th17 culture conditions shared some phenotypic markers. Cells under Treg conditions had an up-regulated FOXP3 gene and a down-regulated RAR-related orphan receptor C (RORC) gene. Cells derived from the Th17 condition exhibited a down-regulated FOXP3 gene and had significantly higher RORC gene expression than Treg cells. Both resulting cells showed intracellular production of IL-17A and IL-10. Th17 condition-cultured cells exhibited more glycolytic activity and glucose uptake compared to the Treg cells. The findings suggest that cells obtained from established protocols for the differentiation of iTreg and Th17 cells in vitro are possibly in the intermediate stage of differentiation or may be two different types of cells that share a lineage-specific differentiation program.

Identification of Corosolic and Oleanolic Acids as Molecules Antagonizing the Human RORγT Nuclear Receptor Using the Calculated Fingerprints of the Molecular Similarity.

RORγT is a protein product of the RORC gene belonging to the nuclear receptor subfamily of retinoic-acid-receptor-related orphan receptors (RORs). RORγT is preferentially expressed in Th17 lymphocytes and drives their differentiation from naive CD4+ cells and is involved in the regulation of the expression of numerous Th17-specific cytokines, such as IL-17. Because Th17 cells are implicated in the pathology of autoimmune diseases (e.g., psoriasis, inflammatory bowel disease, multiple sclerosis), RORγT, whose activity is regulated by ligands, has been recognized as a drug target in potential therapies against these diseases. The identification of such ligands is time-consuming and usually requires the screening of chemical libraries. Herein, using a Tanimoto similarity search, we found corosolic acid and other pentacyclic tritepenes in the library we previously screened as compounds highly similar to the RORγT inverse agonist ursolic acid. Furthermore, using gene reporter assays and Th17 lymphocytes, we distinguished compounds that exert stronger biological effects (ursolic, corosolic, and oleanolic acid) from those that are ineffective (asiatic and maslinic acids), providing evidence that such combinatorial methodology (in silico and experimental) might help wet screenings to achieve more accurate results, eliminating false negatives.

Evaluation of correlation between transcription factors and IL-17 in oral and cutaneous lichen planus lesions and Leukocytes.

BACKGROUND: Lichen planus (LP) is a chronic autoimmune disease with different clinical subtypes including cutaneous LP (CLP) and oral LP (OLP). We aimed to compare mRNA expression of RORγt and IL-17 in paraffin-embedded blocks of OLP and CLP lesions with normal oral mucosa (NOM), and also its correlation with hematologic parameters. MATERIALS & METHODS: This study included 89 paraffin-embedded blocks contain OLP (44 cases), CLP (45 cases) and NOM from the archive of Mashhad University of Medical Sciences, Mashhad, Iran. The expression of RORγt and IL-17 was evaluated by Real-time RT-PCR method. The result was compared to Leukocyte counts and the other hematological parameters of studied patients. RESULTS: The results of our study showed IL-17 and RORγt expression in OLP lesions were significantly higher than CLP and NOM groups (P = 0.001). Although we found high expression of RORγt and IL-17 in erosive OLP in compared to classic OLP lesion, but this increment was not significant for IL-17 (P = 0.26) and RORγt (P = 0.14). Further, Leukocyte and monocyte counts were substantially high in OLP group in compared to the CLP and NOM groups (P < 0.05). CONCLUSIONS: We concluded that increased expression of RORγt and IL-17 in LP lesions could play role in the pathogenesis of LP. As well, higher expression of RORγt and IL-17 in oral LP more than cutaneous LP might be associated with difference in clinical behavior of the two types of disease and role of these factors in premalignant behavior of OLP lesions.

Effects of crystalline penicillin G sodium on human T-cells differentiation.

BACKGROUND: Although antibiotics are well-known for their anti-bacterial effects, their inaugurated immunomodulatory roles in chronic inflammatory diseases have not elucidated yet. Anecdotal reports support the beneficial effects of parenteral penicillin in arthritis suggesting an immunomodulatory other than antibacterial effects for penicillin. The present study was designed to address the possible effects of penicillin G sodium (PCN-G) on different T-helper cells differentiation. MATERIALS AND METHODS: In this experimental study, peripheral blood mononuclear cells (PBMCs) of 10 healthy donors were isolated using Ficoll density gradient. The stimulated PBMCs by anti-CD3, anti-CD28, and anti-CD69 were cultured in the presence of 120 µg/ml of PCN-G. Foxp3, T-bet, RORγT, GATA3 as well as interferon-gamma (IFN-γ) and interleukin (IL)-17A mRNA in stimulated cells were measured by the real-time polymerase chain reaction. Mann-Whitney U-test was used for determining differences between the medium of gene expression levels of stimulated cell population and unstimulated cells by PCN. Correlations between the related genes were determined using the Spearman test. RESULTS: Based on the results, T-bet gene expression levels were similar in stimulated cells by PCN G after 24 and 48 h while significant reduction was observed after 72 incubation with PCN G (difference = 3; 0.09-0.34; P = 0.031). Meanwhile, treated cells with PCN G expressed decreased levels of IFN-γ (difference = 8.0; 0.49-1.07; P = 0.001) and IL-17A (difference = 2.2; 0.05-0.75; P ≤ 0.05) genes comparing to unstimulated cell by PCN-G. GATA3 genes expression levels downregulated by PCN G after 72 h of incubation by PBMCs (difference = 1.1; 0.77-0.88; P = 0.035). CONCLUSION: Our results confirmed the immunomodulatory role of PCN G by affecting the expression of different cytokines genes in PBMCs.

The Dichotomous Nature of AZ5104 (an EGFR Inhibitor) Towards RORγ and RORγT.

The RORC (RAR related orphan receptor C) gene produces two isoforms by alternative promoter usage: RORγ (nuclear receptor ROR-gamma isoform 1) and RORγT (nuclear receptor ROR-gamma isoform 1). Both proteins have distinct tissue distributions and are involved in several physiological processes, including glucose/lipid metabolism and the development of Th17 lymphocytes. Previously, we developed a stably transfected reporter cell line and used it to screen a library of kinase inhibitors. We found that AZ5104 acts as an RORγ agonist at low micromolar concentrations. Molecular docking analysis showed that this compound occupies the ligand binding domain of the receptor with a significant docking score. However, analysis of the biological activity of this compound in Th17 cells revealed that it downregulates RORγT expression and Th17-related cytokine production via inhibition of SRC-ERK-STAT3 (SRC proto-oncogene - extracellular regulated MAP kinase - signal transducer and activator of transcription 3). We thus identified a compound acting as an agonist of RORγ that, due to the inhibition of downstream elements of EGFR (epidermal growth factor receptor) signaling, exerts different biological activity towards a Th17-specific isoform. Additionally, our results may be relevant in the future for the design of treatments targeting signaling pathways that inhibit Th17-related inflammation in certain autoimmune disorders.