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. 2012 Nov;44(11):1839-46.
doi: 10.1016/j.biocel.2012.06.016. Epub 2012 Jun 19.

Lipidomic profiling in Crohn's disease: abnormalities in phosphatidylinositols, with preservation of ceramide, phosphatidylcholine and phosphatidylserine composition

Gavin W Sewell  1 , Yusuf A Hannun, Xianlin Han, Grielof Koster, Jacek Bielawski, Victoria Goss, Philip J Smith, Farooq Z Rahman, Roser Vega, Stuart L Bloom, Ann P Walker, Anthony D Postle, Anthony W Segal
Affiliations

Lipidomic profiling in Crohn's disease: abnormalities in phosphatidylinositols, with preservation of ceramide, phosphatidylcholine and phosphatidylserine composition

Gavin W Sewell et al. Int J Biochem Cell Biol. 2012 Nov.

Abstract

Crohn's disease is a chronic inflammatory condition largely affecting the terminal ileum and large bowel. A contributing cause is the failure of an adequate acute inflammatory response as a result of impaired secretion of pro-inflammatory cytokines by macrophages. This defective secretion arises from aberrant vesicle trafficking, misdirecting the cytokines to lysosomal degradation. Aberrant intestinal permeability is also well-established in Crohn's disease. Both the disordered vesicle trafficking and increased bowel permeability could result from abnormal lipid composition. We thus measured the sphingo- and phospholipid composition of macrophages, using mass spectrometry and stable isotope labelling approaches. Stimulation of macrophages with heat-killed Escherichia coli resulted in three main changes; a significant reduction in the amount of individual ceramide species, an altered composition of phosphatidylcholine, and an increased rate of phosphatidylcholine synthesis in macrophages. These changes were observed in macrophages from both healthy control individuals and patients with Crohn's disease. The only difference detected between control and Crohn's disease macrophages was a reduced proportion of newly-synthesised phosphatidylinositol 16:0/18:1 over a defined time period. Shotgun lipidomics analysis of macroscopically non-inflamed ileal biopsies showed a significant decrease in this same lipid species with overall preservation of sphingolipid, phospholipid and cholesterol composition.

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Figures

Supplementary Fig. 1
Supplementary Fig. 1
Ceramide and sphingoid bases in HC and CD macrophages after HkEc stimulation. Alterations in the amount of (A) C16:0, (B) C24:0, (C) C24:1 ceramides and (D) dihydrosphingosine (dhSph) are shown for HC (left panel) and CD (right panel) macrophages. * represents p < 0.05, ** p < 0.01 and *** p < 0.001.
Supplementary Fig. 2
Supplementary Fig. 2
Ceramide and sphingoid base composition of HkEc-stimulated HC and CD macrophages. (A) Amounts of individual ceramide species expressed as pmol/mg protein. (B) Amounts of sphingosine (Sph) and dihydrosphingosine (dhSph). (C) Total amount of ceramide. No significant differences were found between HC (n = 7) and CD (n = 12) macrophages. Results are presented as mean + SEM.
Supplementary Fig. 3
Supplementary Fig. 3
ESI-MS analysis of endogenous and newly synthesised PC. (A) Precursor scan of m/z 184+, indicating endogenous PC species. (B) Precursor scan of m/z 193+ showing newly synthesised PC species 9 m/z units higher than the endogenous species.
Supplementary Fig. 4
Supplementary Fig. 4
Incorporation of methyl-D9-choline into HC and CD macrophage PC. Total lipid was extracted and analysed by ESI-MS as precursor scans of m/z 184+ and m/z 193+. The fractional incorporation of methyl-D9-choline as a % of the total PC was determined.
Supplementary Fig. 5
Supplementary Fig. 5
ESI-MS analysis of endogenous and newly synthesised PS. (A) Neutral loss scan of m/z 87 endogenous PS species. (B) Neutral loss scan of m/z 90, showing newly synthesised PS species 3 m/z units higher than the endogenous species.
Supplementary Fig. 6
Supplementary Fig. 6
Incorporation of serine-D3 into HC and CD macrophage PS. Results are shown for unstimulated (HC n = 7, CD n = 9) and HkEc-stimulated (HC n = 6, CD n = 5) macrophages. Total lipid was extracted and analysed by ESI-MS as neutral loss scans of m/z 87- and m/z 90-. The fractional incorporation of serine-D3 as a percentage of the total PS was determined.
Supplementary Fig. 7
Supplementary Fig. 7
ESI-MS analysis of endogenous and newly synthesised PI species in macrophages. (A) Precursor scan of m/z 241-, indicating endogenous PI species. (B) Precursor scan of m/z 247-, indicating newly synthesised PI species.
Supplementary Fig. 8
Supplementary Fig. 8
Incorporation of myo-D6-inositol into HC and CD macrophage PI. Results are shown for unstimulated (HC n = 7, CD n = 9) and HkEc-stimulated macrophages (HC n = 6, CD n = 5). No significant differences were identified.
Fig. 1
Fig. 1
Ceramide and sphingoid base composition of CD macrophages is unaltered. The amounts of individual ceramide species were quantified in HC (n = 7) and CD (n = 8) macrophages by HPLC-MS. (A and B) Pie charts depicting the proportion of various ceramides in HC and CD macrophages respectively, expressed as a percentage of the total amount of ceramide detected. (C) The amounts of individual ceramide species (in pmol/mg protein) in unstimulated HC and CD macrophages. No significant differences were identified between HC and CD. (D) The amounts of sphingosine (Sph) and dihydrosphingosine (dhSph) in HC and CD macrophages. (E) Total amounts of ceramide in HC and CD macrophages. Results are expressed as mean + SEM.
Fig. 2
Fig. 2
Composition of endogenous and newly synthesised PC in HC and CD macrophages, in the presence and absence of HkEc. Composition of (A) endogenous PC, consisting of various carbon chain length fatty acid species, and (B) newly synthesised (D9) PC in unstimulated (HC n = 10, CD n = 13) and HkEc-stimulated (HC n = 7, CD n = 8) macrophages, expressed as a molar percentage of total PC. Results are mean + SEM. * indicates p < 0.05.
Fig. 3
Fig. 3
Composition of endogenous and newly synthesised PS in HC and CD macrophages, in the presence and absence of HkEc. Composition of (A) endogenous PS and (B) synthesised (D3) PS, in unstimulated (HC n = 7, CD n = 9) and HkEc-stimulated (HC n = 6, CD n = 5) macrophages. Results are expressed as a molar percentage of total PS and are mean + SEM. No statistically significant differences were identified between HC and CD.
Fig. 4
Fig. 4
Composition of endogenous and newly synthesised PI in HC and CD macrophages, in the presence and absence of HkEc. Composition of (A) endogenous PI and (B) newly synthesised PI in unstimulated (HC n = 7, CD n = 9) and HkEc-stimulated (HC n = 6, CD n = 5) macrophages. Results are expressed as a molar percentage of the total PI and are mean + SEM. * indicates p < 0.05.
Fig. 5
Fig. 5
Shotgun lipidomics analysis of ileal biopsies. (A) Total phospholipid (PC, PE, CL, PG, PA, PS), sphingolipid (sphingomyelin, SM and ceramide, Cer) and cholesterol (Chol) content did not differ between HC (n = 5) and CD (n = 5) patients. (B) Molar percentage composition of phosphatidylinositol (PI) species. (C) Reduced molar percentage of PI 16:0/18:1 in CD biopsies compared to HC. ** represents p < 0.01.
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