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Ceramide synthase 2 deficiency aggravates AOM-DSS-induced colitis in mice: role of colon barrier integrity

Abstract

Loss of intestinal barrier functions is a hallmark of inflammatory bowel disease like ulcerative colitis. The molecular mechanisms are not well understood, but likely involve dysregulation of membrane composition, fluidity, and permeability, which are all essentially regulated by sphingolipids, including ceramides of different chain length and saturation. Here, we used a loss-of-function model (CerS2+/+ and CerS2−/− mice) to investigate the impact of ceramide synthase 2, a key enzyme in the generation of very long-chain ceramides, in the dextran sodium salt (DSS) evoked model of UC. CerS2−/− mice developed more severe disease than CerS2+/+ mice in acute DSS and chronic AOM/DSS colitis. Deletion of CerS2 strongly reduced very long-chain ceramides (Cer24:0, 24:1) but concomitantly increased long-chain ceramides and sphinganine in plasma and colon tissue. In naive CerS2−/− mice, the expression of tight junction proteins including ZO-1 was almost completely lost in the colon epithelium, leading to increased membrane permeability. This could also be observed in vitro in CerS2 depleted Caco-2 cells. The increase in membrane permeability in CerS2−/− mice did not manifest with apparent clinical symptoms in naive mice, but with slight inflammatory signs such as an increase in monocytes and IL-10. AOM/DSS and DSS treatment alone led to a further deterioration of membrane integrity and to severe clinical symptoms of the disease. This was associated with stronger upregulation of cytokines in CerS2−/− mice and increased infiltration of the colon wall by immune cells, particularly monocytes, CD4+ and Th17+ T-cells, and an increase in tumor burden. In conclusion, CerS2 is crucial for the maintenance of colon barrier function and epithelial integrity. CerS2 knockdown, and associated changes in several sphingolipids such as a drop in very long-chain ceramides/(dh)-ceramides, an increase in long-chain ceramides/(dh)-ceramides, and sphinganine in the colon, may weaken endogenous defense against the endogenous microbiome.

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Acknowledgements

We thank Prof. Anthony Futerman and Dr. Yael Pewzner-Jung for providing us CerS2-wt and -ko breeding mice. Many thanks to Kerstin Birod and Annett Häussler for technical assistance. Furthermore, the authors would like to thank Prof. Simone Fulda for kindly providing viral packaging plasmids. The authors thank Prof. Dr. Michael Parnham for the linguistic revision of the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) [SFB 1039/1, A03, A08, B03, B04, B05, B06, Z01]; DFG project [GR2011/3-2], and the LOEWE Lipid Signaling Forschungszentrum Frankfurt (LiFF).

Author information

SO: data collection, data analysis, and writing; KSch: Melk-analysis; AW: FACS analysis, writing; DT and ST: LC–MSMS analysis; JSch and NF: data analysis and calculation; M-SW: data interpretation; HHR: AOM/DSS mouse model; BB, GG, and IT: writing and figures, SG: study design, writing, data interpretation, data collection, and data analysis.

Correspondence to Sabine Grösch.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplement 1 Immuno-fluorescence staining of CerS2. Colon sections were fixed with 100% ice-cold methanol and permeabilized in PBS containing 0.025% Triton X-100. Subsequent tissue sections were either stained immunohistochemically by DIFF-Quick staining or used for immuno-fluorescence staining. This procedure determines whether the DIFF-Quick sections are comparable to the fluorescence stained section. After blocking, the tissue sections were incubated with anti-CerS2 at 4 °C overnight, followed by a Cy3 labeled anti-rabbit antibody diluted 1:800 for 2 h. Subsequently, tissue sections were stained with DAPI (1:1000). Fluorescent measurements were done with the Keyence BZ-9000 microscope with 20-fold magnification. Representative pictures are shown from CerS2+/+ and CerS2−/− mice with or without AOM and two cycles of DSS treatment. (PPTX 2860 kb)

Supplement 2 Depletion of CerS2 in human Caco-2 cells. CerS2 was depleted in Caco-2 cells by lentiviral transduction of a CerS2-shRNA. As a control, we transduced a non-specific (NC) shRNA. A) mRNA for CerS expressed in Caco-2 cells (CerS1 and Cer3 are only very weakly expressed in Caco-2 cells). CerS2 mRNA was reduced by 80% in CerS2-shRNA treated Caco-2 cells. B) CerS2-protein expression was also significantly reduced in these cells. We show one representative Western-blot of three and the calculation of the relative protein expression (CerS2/GAPDH, CaCo-2 untreated cells were set to 100%) based on findings from three blots. C) Down-regulation of CerS2 in Caco-2 cells led to alterations in various sphingolipid levels. Sphingolipid levels were determined by LC-MS/MS. Changes in sphingolipids are comparable to those observed in the colon of CerS2−/− mice in comparison to CerS2+/+ mice (see Table 2). Statistical analysis was done by unpaired t test, p* < 0.05; p** < 0.01; p*** < 0.001 (PPTX 413 kb)

Supplement 3 MELC analysis of colon sections from CerS2+/+ and CerS2−/− mice with and without AOM and two cycles of DSS treatment. Ten micrometer cryosections of the colon were applied to silane-coated coverslips, fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X100, and blocked with 3% BSA. The sample was placed on the stage of an Leica DM IRE2 and a phase contrast picture was taken. Then, the sample was automatically incubated for 15 min with fluorescence-labeled antibodies [FITC-Ly-6C (Gr1) antibody, FITC-CD11b and FITC-F4/80 antibody] and rinsed with PBS. To delete fluorescence signals, a bleaching step was performed after each antibody incubation step and at least 7-amino actinomycin D (7-AAD) was used to stain nuclei. After the MELC run, the tissue slices were stained with Diff-Quick (Dade Behring) (PPTX 10272 kb)

Supplement Table 1 RT-PCR primer used in this study. (PPTX 41 kb)

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Oertel, S., Scholich, K., Weigert, A. et al. Ceramide synthase 2 deficiency aggravates AOM-DSS-induced colitis in mice: role of colon barrier integrity. Cell. Mol. Life Sci. 74, 3039–3055 (2017). https://doi.org/10.1007/s00018-017-2518-9

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Keywords

  • Sphingosine
  • Occludin
  • Cytokines
  • FITC dextran
  • Colon