Loss of Adenomatous polyposis coli function renders intestinal epithelial cells resistant to the cytokine IL-22

Interleukin-22 (IL-22) is a critical immune defence cytokine that maintains intestinal homeostasis and promotes wound healing and tissue regeneration, which can support the growth of colorectal tumours. Mutations in the adenomatous polyposis coli gene (Apc) are a major driver of familial colorectal cancers (CRCs). How IL-22 contributes to APC-mediated tumorigenesis is poorly understood. To investigate IL-22 signalling in wild-type (WT) and APC-mutant cells, we performed RNA sequencing (RNAseq) of IL-22-treated murine small intestinal epithelial organoids. In WT epithelia, antimicrobial defence and cellular stress response pathways were most strongly induced by IL-22. Surprisingly, although IL-22 activates signal transducer and activator of transcription 3 (STAT3) in APC-mutant cells, STAT3 target genes were not induced. Our analyses revealed that ApcMin/Min cells are resistant to IL-22 due to reduced expression of the IL-22 receptor, and increased expression of inhibitors of STAT3, particularly histone deacetylases (HDACs). We further show that IL-22 increases DNA damage and genomic instability, which can accelerate cellular transition from heterozygosity (ApcMin/+) to homozygosity (ApcMin/Min) to drive tumour formation. Our data reveal an unexpected role for IL-22 in promoting early tumorigenesis while excluding a function for IL-22 in transformed epithelial cells

Novel PDE4 inhibitors derived from Chinese medicine forsythia.

Cyclic adenosine monophosphate (cAMP) is a crucial intracellular second messenger molecule that converts extracellular molecules to intracellular signal transduction pathways generating cell- and stimulus-specific effects. Importantly, specific phosphodiesterase (PDE) subtypes control the amplitude and duration of cAMP-induced physiological processes and are therefore a prominent pharmacological target currently used in a variety of fields. Here we tested the extracts from traditional Chinese medicine, Forsythia suspense seeds, which have been used for more than 2000 years to relieve respiratory symptoms. Using structural-functional analysis we found its major lignin, Forsynthin, acted as an immunosuppressant by inhibiting PDE4 in inflammatory and immune cell. Moreover, several novel, selective small molecule derivatives of Forsythin were tested in vitro and in murine models of viral and bacterial pneumonia, sepsis and cytokine-driven systemic inflammation. Thus, pharmacological targeting of PDE4 may be a promising strategy for immune-related disorders characterized by amplified host inflammatory response

Tested compounds reduce TNF secretion in LPS stimulated mouse RAW264.7 and human PBMCs.

<p><b>A.</b> 5×10⁵ RAW264.7 cells were seeded in 96 wells for 18 h. Cells were primed with compounds at different concentration for 3 h before treated with LPS (1 ng/ml) for additional 8 h. TNF cytokine releases were monitored by ELISA. <b>B.</b> PBMC (0.2 ml at 1×10⁵/ml) were primed with compounds at different concentration for 3 h before treated with LPS (1 ng/ml) for additional 8 h. TNF cytokine releases were monitored by ELISA. % of TNF secretion were calculated and graphed. <b>C.</b> Summary of compound IC₅₀. The data represent <i>n</i> = 3–6 experiments.</p

Tested compounds exhibit high potency and selectivity towards PDE4.

<p>For the PDE activity assay, all test compounds were diluted in DMSO with final concentrations in each assay of 100, 10, 1, 0.1, 0.01, 0.001, 0.0001 µM. For the PDE4 activity assay, 10 mU of purified PDE4 (Millipore) was used per reaction. Compounds 6, 7, 9 and 13 (blue underline) were further tested in PDE3, 5, 7, 10 activity assays in which 25 mU of purified enzyme was used per reaction. Summary of compound IC₅₀ in lower right corner.</p

Forsythin is an inhibitor of PDE4.

<p><b>A–C.</b> The chemical structure of Forsythin extracted from <i>Forsythia suspensa</i> seeds. Forsythin is an o-linked β-D-glucopyranosylated lignin that can be hydrolyzed (Red line). <b>D.</b> Predicted docking site of Forsythin with PDE4. <b>E.</b> Predicted residue electrostatic and van der Waals interactions between PDE4 amino acids and Forsythin. Using ZINCPharmer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115937#pone.0115937-Koes1" target="_blank">[24]</a>, a pharmacophore model was generated (upper left) and used to screen the lead compounds from an 18.3 million purchasable compound library.</p

PDE4 inhibitors reduce H1N1 influenza-induced lung injury.

<p>C57/BL6 mice were challenged with H1N1 (10⁵ pfu/mouse, i.t.) for up to 6 days. For compound treatment, a stock solution (5 mg/ml) was added to the drinking water (containing 2% sucrose) to a final concentration of 30 µg/ml. <b>A.</b> Survival studies of mice administered i.t. with H1N1 (10⁵ pfu/mouse, <i>n</i> = 6 mice/group). Mice were then euthanized using pentobarbital and lungs were lavaged with saline, harvested, and then homogenized. Cell counts and lavage protein were measured (<b>B, C</b>). Lavage cytokine secretions were measured (<b>D, E</b>). Serum samples were also collected and cytokine levels were measured (<b>F, G</b>). <b>H.</b> H&E staining was performed on lung samples. Original magnification, ×60. The data represent <i>n</i> = 4–6 mice/group, *<i>P</i><0.05 versus vehicle.</p

PDE4 inhibitors lessen cytokine storm induced by LPS septic shock.

<p>C57/BL6 mice were administered i.p. nothing (CON), vehicle, 10 ug/kg, 100 ug/kg, 1 mg/kg or 10 mg/kg of compounds 6, 7 or 9. Mice were given LPS (<i>E. coli,</i> 100 µg) 10 min later through an i.p. injection. 2 h later mice were euthanized using pentobarbital and blood was collected for IL-6 and TNF measurements. Shown in panel <b>A–B</b> is the % inhibition of cytokine levels as a function of drug dose. The data represent <i>n</i> = 3 mice/group at each dose. C57/BL6 mice were also pretreated with compound 5, 6, 7 or 9 at 1 mg/kg. Mice were given LPS (<i>E. coli,</i> 100 µg) 18 h later through an i.p. injection. 2 h later the mice were euthanized using pentobarbital and blood was collected for IL-6 and TNF measurements (<b>C–D</b>). The data represent <i>n</i> = 4–6 mice/group, *<i>P</i><0.05 versus vehicle.</p

PDE4 inhibitors ameliorate LPS induced lung injury.

<p>C57/BL6 mice were challenged with LPS <i>(E.coli</i>, 3 mg/kg, i.t.) followed by i.p. administration of vehicle, 1 mg/kg of compound 2, 6, 7 or 9. Mice were then euthanized 18 h later using pentobarbital, and lungs were lavaged with saline, harvested, and then homogenized. Lavage cytokine secretion (<b>A–C</b>), protein concentrations (<b>D</b>) and cell counts (<b>E</b>) were measured. <b>F.</b> H&E staining was performed on lung samples. Original magnification, ×20. The data represent <i>n</i> = 4–6 mice/group, *<i>P</i><0.05 versus vehicle.</p