Anti-inflammatory Effects of Fungal Metabolites in Mouse Intestine as Revealed by In vitro Models

Abstract

Inflammatory bowel diseases (IBD), which include Crohn's disease and ulcerative colitis, are chronic inflammatory disorders that can affect the whole gastrointestinal tract or the colonic mucosal layer. Current therapies aiming to suppress the exaggerated immune response in IBD largely rely on compounds with non-satisfying effects or side-effects. Therefore, new therapeutical options are needed. In the present study, we investigated the anti-inflammatory effects of the fungal metabolites, galiellalactone, and dehydrocurvularin in both an in vitro intestinal inflammation model, as well as in isolated myenteric plexus and enterocyte cells. Administration of a pro-inflammatory cytokine mix through the mesenteric artery of intestinal segments caused an up-regulation of inflammatory marker genes. Treatment of the murine intestinal segments with galiellalactone or dehydrocurvularin by application through the mesenteric artery significantly prevented the expression of pro-inflammatory marker genes on the mRNA and the protein level. Comparable to the results in the perfused intestine model, treatment of primary enteric nervous system (ENS) cells from the murine intestine with the fungal compounds reduced expression of cytokines such as IL-6, TNF-α, IL-1β, and inflammatory enzymes such as COX-2 and iNOS on mRNA and protein levels. Similar anti-inflammatory effects of the fungal metabolites were observed in the human colorectal adenocarcinoma cell line DLD-1 after stimulation with IFN-γ (10 ng/ml), TNF-α (10 ng/ml), and IL-1β (5 ng/ml). Our results show that the mesenterially perfused intestine model provides a reliable tool for the screening of new therapeutics with limited amounts of test compounds. Furthermore, we could characterize the anti-inflammatory effects of two novel active compounds, galiellalactone, and dehydrocurvularin which are interesting candidates for studies with chronic animal models of IBD.

Keywords: ENS; dehydrocurvularin; dexamethasone; galiellalactone; inflammatory bowel disease; intestine; mesenterial perfusion; neuroinflammation.

Figure 1

Scanning electron microscopy images (A,B) of unperfused intestinal tissue (upper row) and after 5 h of perfusion (lower row). Intestinal segments were perfused as described in the Materials and Methods section. Tissue damage is most pronounced at the tips of the mucosal villi (A). Ultrastructural characteristics (microvilli) remain intact (B). Images from the side of a villus are shown in (B).

Figure 2

Multiplex ELISA of tissue lysates (A,B) and qRT-PCR (C) of pro-inflammatory transcripts in perfused, intestinal tissue samples after 5 h of perfusion. During the first hour of perfusion, the indicated concentrations of test compounds were added to the perfusion medium. During the following 4 h, a CM-stimulus was added in addition. Multiplex ELISA: Tissue samples of five (induced, uninduced, dehydrocurvularin treated) or four (galiellalactone and dexamethasone treated) independent experiments were measured twice. The cytokines CXCL10, RANTES, IL-6, Eotaxin, and MCP-1 which are also regulated in adult ENS networks (Figure 3) are shown in (A). Further regulated cytokines are shown in (B). The data are presented in comparison to uninduced control tissue (±SD). The uninduced control tissue corresponds to 100%. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001 treated tissue vs. cytokine induced tissue. qRT-PCR: Induced vs. uninduced samples and treated vs. induced samples are shown. Results were normalized to GAPDH, n = 5. The data are presented as log2 ratios in comparison to cytokine induced tissue (±SD). ^*p < 0.05; ^**p < 0.01; ^***p < 0.001 treated tissue vs. cytokine induced tissue.

Figure 3

Multiplex ELISA of culture supernatants from adult ENS networks (A) and qRT-PCR analysis of pro-inflammatory transcripts in adult ENS networks (B) after treatment with the anti-inflammatory compounds galiellalactone and dehydrocurvularin. Cells were pretreated for 1 h with the indicated concentrations of test compounds prior to stimulation with CM for 24 h. Multiplex ELISA: The data are presented in comparison to untreated control cells (±SD). The untreated control cells correspond to 100%. n = 3 for the test substances galiellalactone and dehydrocurvularin, n = 2 for the positive control dexamethasone. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001 treated cells vs. cytokine induced cells. qRT-PCR: Induced vs. uninduced samples and treated vs. induced samples are shown. Results were normalized to GAPDH, n = 5. The data are presented as log2 ratios in comparison with cytokine induced control cells (±SD). ^*p < 0.05; ^**p < 0.01; ^***p < 0.001 treated tissue vs. cytokine induced cells.

Figure 4

iNOS immunofluorescence staining of adult ENS networks after anti-inflammatory treatment. Cells were pretreated for 1 h with the indicated concentrations of test compounds prior to stimulation with CM for 24 h. iNOS positive cells (red, iNOS) and nuclei (blue, DAPI). Merge image (upper row), iNOS (lower row). Induced, cytokine induced culture; uninduced, uninduced control culture; Deh 10.3 μM, dehydrocurvularin 10.3 μM; Gal 10.3 μM, galiellalactone 10.3 μM; Dex 2.5 μM, dexamethasone 2.5 μM.

Figure 5

Effects of the anti-inflammatory compounds dehydrocurvularin, galiellalactone, and dexamethasone on iNOS expression in the postnatal ENS single cell culture. Cells were pretreated for 1 h with the indicated concentrations of test compounds prior to stimulation with CM for 24 h. (A) Neurons (green, βIII-Tubulin), iNOS positive cells (red, iNOS) and nuclei (blue, DAPI). The upper row shows a merge of all three fluorescence channels, the lower row the iNOS expression. (induced): cytokine induced culture, (uninduced): uninduced control culture, (Deh 2.6 μM): dehydrocurvularin 2.6 μM, (Deh 5.3 μM): dehydrocurvularin 5.3 μM, (Gal 5.1 μM): galiellalactone 5.1 μM, (Gal 10.3 μM): galiellalactone 10.3 μM, (Dex 10.2 μM): dexamethasone 10.2 μM. 5000 cells / coverslip. (B) Results are relative to positive control. The data are presented in comparison to cytokine induced control cells (±SD). ^*p < 0.05; ^**p < 0.01; ^***p < 0.001, treated cells vs. cytokine induced cells, respectively cytokine induced cells vs. uninduced cells. At least 4 independent experiments were performed.

Figure 6

Effect of dehydrocurvularin and galiellalactone on mRNA levels and synthesis of pro-inflammatory genes in CM (TNF-α/IFN-γ/IL-1β) treated DLD-1 cells. (A,C) DLD-1 cells were pretretated with the indicated concentrations of test compounds before cytokine stimulation for 5 h. Total RNA was reversely transcribed and cDNAs measured as described in the Materials and Methods section. Values were expressed as relative mRNA content of CM induced vs. unstimulated cells [(###: p < 0.001 vs. unstimulated cells) and CM stimulated and compound treated cells vs. untreated, stimulated cells, corrected for gapdh as reference determined in the same sample in parallel. Data are shown as mean values ± SD of three independent experiments (^***p < 0.001; ^*p < 0.05 vs. stimulated cells; ns: not significant vs. stimulated cells)]. (B,D) Effect of galiellalactone and dehydrocurvularin on CM induced synthesis of pro-inflammatory proteins. DLD-1 cells were pretreated for 1 h with the indicated concentrations of test compounds prior to stimulation with CM for 16 h. The production of pro-inflammatory proteins in cell supernatants was determined with the human Cytokine Array (A) (R&D systems) according to the manufacturer's instructions. The analysis of two biological replicates (each one with two technical replicates) with ImageJ is shown. The data (means ± SD) represent relative protein amounts of significantly (>2-fold) regulated inflammation-related proteins (^***p < 0.001; ^**p < 0.01; ^*p < 0.05 vs. stimulated cells; ns: not significant vs. stimulated cells).