Crohn's disease patient serum changes protein expression in a human mesenchymal stem cell model in a linear relationship to patients' disease stage and to bone mineral density

Abstract

Background: Crohn's disease (CD) is associated with a higher prevalence of osteoporosis, a complication that is recognized as a significant cause of morbidity. Its pathogenesis is controversial, but the activity of CD is one contributing factor.

Methods: We stimulated SCP-1 cells (mesenchymal stem cell line) under osteogenic conditions with serum from adult patients with CD in the symptomatic phase (SP) and in remission (R) and with control sera. Concentrations of IL-6, IL-1 beta, and TNF alpha in the sera were measured. Patients were classified as normal or osteopenic/osteoporotic based on bone mineral density (BMD) T-score measurements. After 14 days in culture, protein expression and gene ontology (GO) annotation analysis was performed.

Results: Cytokine concentrations (IL-6, IL-1 beta, TNF alpha) varied within sera groups. None of the cytokines were significantly increased in the symptomatic phase compared to remission. Protein analysis revealed 17 proteins regulated by the SP versus R phase sera of disease. A linear relationship between CDAI (Crohn's disease activity index) and normalized protein expression of APOA1 and 2, TTR, CDKAL1 and TUBB6 could be determined. Eleven proteins were found to be differentially regulated comparing osteoporosis-positive and osteoporosis-negative sera. Gene annotation and further analysis identified these genes as part of heme and erythrocyte metabolism, as well as involved in hypoxia and in endocytosis. A significant linear relationship between bone mineral density and normalized protein expression could be determined for proteins FABP3 and TTR.

Conclusion: Our explorative results confirm our hypothesis that factors in serum from patients with CD change the protein expression pattern of human immortalized osteoblast like cells. We suggest, that these short time changes indeed influence factors of bone metabolism.

Keywords: Crohn’s disease; Cytokines; Gene ontology; Osteoporosis; Proteome analysis.

Fig. 1

Protein expression (normalized area) in SCP-1 cells after incubation with CD sera: Sera classified as CD symptomatic phase or remission sera (N = 6; n = 3) N = experimental replicates. n = technical replicates; ^*∼p < 0.05. ^**∼p < 0.01 (unpaired t-test) Scattered dot plot with mean and SD.

Fig. 2

Mean protein expression (normalized area; n = 3) in SCP-1 cells after incubation with CD sera (osteoporosis positive sera (patient O1, O2, O3) are depicted as black symbols; osteoporosis negative sera (C1, C2, C3) displayed as open symbols): Correlation of CDAI as determined during disease phases (symptomatic and remission phase) to mean protein expression of three technical replicates of protein expression (n = 3).

Fig. 3

Protein expression (normalized area) in SCP-1 cells after incubation with CD sera (SP or R) or healthy control sera with (H + C; n = 3) or without addition of cytokines (H; n = 3) CD-Sera were classified as CD symptomatic phase (SP) or remission sera (R) (N = 6; n = 3) N = experimental replicates. n = technical replicates; Scattered dot plot with mean and SD. The first two data sets are identical to Fig. 1 and depicted again for better understanding of the comparative analysis to the healthy controls.

Fig. 4

Protein expression (normalized area) in SCP-1 cells after incubation with CD sera (SP or R) or healthy control sera with (H + C; n = 3) or without addition of cytokines (H; n = 3) CD-Sera were classified as CD symptomatic phase (SP) or remission sera (R) (N = 6; n = 3) N = experimental replicates. n = technical replicates ^*∼p < 0.05, ^**∼p < 0.01, ^***∼p < 0.001 (unpaired t-test or One way Anova (H + cyt vs CD-sera (SP + R)) Scattered dot plot presenting mean and SD.

Fig. 5

Protein expression (normalized area) in SCP-1 cells after incubation with CD patient sera or healthy control sera: Osteoporosis positive sera (Symptomatic phase (black) and remission sera (grey); N = 6; n = 18), osteoporosis negative sera (Symptomatic phase (black) and remission sera (grey); N = 6; n = 18) and healthy control sera (with (open) or without added cytokines (closed symbols) N = 2; n = 6). N = experimental replicates. n = technical replicates; ^*∼p < 0.05, ^**∼p < 0.01 (One-way Anova followed by Bonferroni multiple comparison testing).

Fig. 6

Mean protein expression (normalized area) in SCP-1 cells after incubation with CD patient sera (osteoporosis positive sera (patient O1, O2, O3) are depicted as black symbols; osteoporosis negative sera (C1, C2, C3) are open symbols). Correlation of BMD at femoral neck (g/cm²) as measured during symptomatic and remission phase to mean protein expression of three technical replicates of protein expression (n = 3).