A forskolin-mediated increase in cAMP promotes T helper cell differentiation into the Th1 and Th2 subsets rather than into the Th17 subset.

The adenylyl cyclase (AC) signaling pathway is suggested to be a key regulator of immune system functions. However, specific effects of cyclic adenosine monophosphate (cAMP) on T helper (Th) cell differentiation and functions are unclear. The involvement of cAMP in the Th cell differentiation program, in particular the development of Th1, Th2, and Th17 subsets, was evaluated employing forskolin (FSK), a labdane diterpene well known as an AC activator. FSK mediated an elevation in Th1-specific markers reinforcing the Th1 cell phenotype. The Th2 differentiation was supported by FSK, though cell metabolism was negatively affected. In contrast, the Th17 immunophenotype was severely suppressed leading to the highly specific upregulation of CXCL13. The causality between FSK-elicited cAMP production and the observed reinforcement of Th2 differentiation was established by using AC inhibitor 2',5'-dideoxyadenosine, which reverted the FSK effects. Overall, an FSK-mediated cAMP increase affects Th1, Th2 and Th17 differentiation and can contribute to the identification of novel therapeutic targets for the treatment of Th cell-related pathological processes.

Convergent Assembly of the Tricyclic Labdane Core Enables Synthesis of Diverse Forskolin-like Molecules.

We report a new synthetic strategy for the flexible preparation of forskolin-like molecules. The approach is different from the previously published works and employs a convergent assembly of the tricyclic labdane-type core from pre-functionalized cyclic building blocks. Stereoselective Michael addition enabled the fragment coupling with excellent control over three newly created contiguous stereocenters, all-carbon quaternary centers included. Silyl enol ether-promoted ring-opening metathesis paired with ring closure were the other key steps enabling concise assembly of the tricyclic core. Late-stage functionalization sequences transformed the tricyclic intermediates into a set of different forskolin-like molecules. The modular nature of the synthetic scheme described herein has the potential to become a general platform for the preparation of analogs of forskolin and other complex tricyclic labdanes.

Pseurotin D inhibits delayed type IV hypersensitivity response.

Pseurotins, secondary metabolites of fungi, represent a group of bioactive natural products with newly recognized biological activities, including the modulation of specific immune response. However, the type of immune response affected by pseurotins and the mechanistic details underlying these effects are still not understood. Thus, the aim of the current study was to examine the effects of pseurotin D on delayed-type IV hypersensitivity (DTH) reaction induced by chicken ovalbumin in vivo and to examine the effects of pseurotin D on major types of leukocytes responsible for DTH development in vitro. Pseurotin D significantly decreased paw swelling, the major symptom of DTH, as well as the DTH-related production of pro-inflammatory cytokine IL-1ß, IL-4, IL-6, IFN-γ and anti-inflammatory cytokine IL-10 in paws tissue, spleen enlargement, and DTH-related changes in leukocyte counts in peripheral blood. In vitro, pseurotin D mediated a decrease in the proliferation and differentiation of both Th1 and Th2 cells, as was concluded on the basis of the inhibition of the gene expressions of Gata3 and Tbx21 and the production of effector cytokines IFN-γ and IL-13 in vitro. Further, pseurotin D significantly inhibited the activation and differentiation of B cells, as was documented by the significant inhibition of B cell proliferation, CD138 expression, and IgE production. In conclusion, the results show the potential of pseurotin D to inhibit DTH reaction, this phenomenon involving the inhibition of the activation and differentiation of both T cells and B cells.

Single-cell RNA sequencing analysis of T helper cell differentiation and heterogeneity.

Single-cell transcriptomics has emerged as a powerful tool to investigate cells' biological landscape and focus on the expression profile of individual cells. Major advantage of this approach is an analysis of highly complex and heterogeneous cell populations, such as a specific subpopulation of T helper cells that are known to differentiate into distinct subpopulations. The need for distinguishing the specific expression profile is even more important considering the T cell plasticity. However, importantly, the universal pipelines for single-cell analysis are usually not sufficient for every cell type. Here, the aims are to analyze the diversity of T cell phenotypes employing classical in vitro cytokine-mediated differentiation of human T cells isolated from human peripheral blood by single-cell transcriptomic approach with support of labelled antibodies and a comprehensive bioinformatics analysis using combination of Seurat, Nebulosa, GGplot and others. The results showed high expression similarities between Th1 and Th17 phenotype and very distinct Th2 expression profile. In a case of Th2 highly specific marker genes SPINT2, TRIB3 and CST7 were expressed. Overall, our results demonstrate how donor difference, Th plasticity and cell cycle influence the expression profiles of distinct T cell populations. The results could help to better understand the importance of each step of the analysis when working with T cell single-cell data and observe the results in a more practical way by using our analyzed datasets.

Pseurotin D Inhibits the Activation of Human Lymphocytes.

BACKGROUND: Pseurotins, a family of secondary metabolites of different fungi characterized by an unusual spirocyclic furanone-lactam core, are suggested to have different biological activities including the modulation of immune response. PURPOSE: Complex characterization of the effects of pseurotin D on human lymphocyte activation in order to understand the potential of pseurotin to modulate immune response in humans. METHODS: CD4+ and CD8+ T cells and CD19+ B cells isolated from human blood were activated by various activators simultaneously with pseurotin D treatment. The effects of pseurotin were tested on the basis of changes in cell viability, apoptosis, activation of signal transducers and activators of transcription (STAT) signaling pathways, production of tumor necrosis factor (TNF)-α by T cells, expression of activation markers CD69 and CD25 on T cells and Human Leukocyte Antigen-DR isotype (HLA-DR) on B cells, and the differentiation markers CD20, CD27, CD38, and immunoglobulin (Ig) D on B cells. RESULTS: Pseurotin D significantly inhibited the activation of both CD4+ and CD8+ human T cells complemented by the inhibition of TNF-α production without significant acute toxic effects. The Pseurotin D-mediated inhibition of T-cell activation was accompanied by the induction of the apoptosis of T cells. This corresponded with the inhibited phosphorylation of STAT3 and STAT5. In human B cells, pseurotin D did not significantly inhibit their activation; however, it affected their differentiation. CONCLUSIONS: Our results advance the current mechanistic understanding of the pseurotin-induced inhibition of lymphocytes and suggest pseurotins as new attractive chemotypes for future research in the context of immune-modulatory drugs.