Open Access Open Badges Research article

Novel application of multi-stimuli network inference to synovial fibroblasts of rheumatoid arthritis patients

Peter Kupfer1*, René Huber25, Michael Weber1, Sebastian Vlaic1, Thomas Häupl3, Dirk Koczan4, Reinhard Guthke1 and Raimund W Kinne5

Author Affiliations

1 Leibnitz Institute for Natural Product Research and Infection Biology - Hans-Knöll-Institute, Beutenbergstr. 11a, 07745 Jena, Germany

2 Institute of Clinical Chemistry, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany

3 Department of Rheumatology and Clinical Immunology, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany

4 Institute of Immunology, University of Rostock, Schillingallee 68, 18057 Rostock, Germany

5 Experimental Rheumatology Unit, Department of Orthopedics, Jena University Hospital, Waldkrankenhaus, Rudolf Elle, Klosterlausnitzer Str. 81, 07607 Eisenberg, Germany

For all author emails, please log on.

BMC Medical Genomics 2014, 7:40  doi:10.1186/1755-8794-7-40

Published: 3 July 2014



Network inference of gene expression data is an important challenge in systems biology. Novel algorithms may provide more detailed gene regulatory networks (GRN) for complex, chronic inflammatory diseases such as rheumatoid arthritis (RA), in which activated synovial fibroblasts (SFBs) play a major role. Since the detailed mechanisms underlying this activation are still unclear, simultaneous investigation of multi-stimuli activation of SFBs offers the possibility to elucidate the regulatory effects of multiple mediators and to gain new insights into disease pathogenesis.


A GRN was therefore inferred from RA-SFBs treated with 4 different stimuli (IL-1 β, TNF- α, TGF- β, and PDGF-D). Data from time series microarray experiments (0, 1, 2, 4, 12 h; Affymetrix HG-U133 Plus 2.0) were batch-corrected applying ‘ComBat’, analyzed for differentially expressed genes over time with ‘Limma’, and used for the inference of a robust GRN with NetGenerator V2.0, a heuristic ordinary differential equation-based method with soft integration of prior knowledge.


Using all genes differentially expressed over time in RA-SFBs for any stimulus, and selecting the genes belonging to the most significant gene ontology (GO) term, i.e., ‘cartilage development’, a dynamic, robust, moderately complex multi-stimuli GRN was generated with 24 genes and 57 edges in total, 31 of which were gene-to-gene edges. Prior literature-based knowledge derived from Pathway Studio or manual searches was reflected in the final network by 25/57 confirmed edges (44%). The model contained known network motifs crucial for dynamic cellular behavior, e.g., cross-talk among pathways, positive feed-back loops, and positive feed-forward motifs (including suppression of the transcriptional repressor OSR2 by all 4 stimuli.


A multi-stimuli GRN highly concordant with literature data was successfully generated by network inference from the gene expression of stimulated RA-SFBs. The GRN showed high reliability, since 10 predicted edges were independently validated by literature findings post network inference. The selected GO term ‘cartilage development’ contained a number of differentiation markers, growth factors, and transcription factors with potential relevance for RA. Finally, the model provided new insight into the response of RA-SFBs to multiple stimuli implicated in the pathogenesis of RA, in particular to the ‘novel’ potent growth factor PDGF-D.

Network modeling; Reverse engineering; Rheumatoid arthritis; Synovial fibroblasts; Cytokines; Growth factors; Cartilage development; Multi-stimuli modeling