Knockout collections are invaluable tools for studying model organisms such as yeast. However, there are no large-scale knockout collections of human cells. Using gene-trap mutagenesis in near-haploid human cells, we established a platform to generate and isolate individual 'gene-trapped cells' and used it to prepare a collection of human cell lines carrying single gene-trap insertions. In most cases, the insertion can be reversed. This growing library covers 3,396 genes, one-third of the expressed genome, is DNA-barcoded and allows systematic screens for a wide variety of cellular phenotypes. We examined cellular responses to TNF-α, TGF-β, IFN-γ and TNF-related apoptosis-inducing ligand (TRAIL), to illustrate the value of this unique collection of isogenic human cell lines.

Sulfur bath therapy represents the oldest form of treatment for patients with different types of rheumatic disorders. However, scientific reports about the beneficial effects of this form of therapy are controversial, rare and of poor scientific quality. Also, little is known about the role and underlying molecular mechanisms of H2S. Therefore, this topic encouraged us to investigate the influence of H2S on fibroblasts isolated from the synovial membrane of RA (rheumatoid arthritis) patients. FLSs (fibroblast-like synoviocytes) were treated with different concentrations of an exogenous H2S donor (NaHS). At defined time points, secretion of IL-6 was quantified by ELISA. Activation/deactivation of MAPKs (mitogen-activated protein kinases), p38 and p44/42 MAPK (ERK1/2) were confirmed by Western blot experiments. FLSs constitutively express and secrete large quantities of IL-6 and IL-8. Data provided prove that, in FLSs, constitutive as well as IL-1beta-induced expression of IL-6 is transiently and partially down-regulated by the short treatment of cells with low concentrations of NaHS. Another key finding is that H2S deactivates p44/42 MAPK (ERK1/2). Long-term exposure of FLSs to H2S provides stimulatory effects, leading to reinforced activation of p38 MAPK and ERK1/2 accompanied by upregulation of IL-6 expression. Presented data seem of importance for studying (patho-) physiological functions of H2S and also for re-evaluating sulfur spa therapy as one of the oldest forms of therapy for rheumatic disorders.

Mitogen-activated protein kinases (MAPKs) play a central role in inflammatory processes, and their blockage represents pharmacological approaches in the treatment of autoimmune diseases like rheumatoid arthritis (RA). Alternatively, H(2)S has long been used in sulphur bath therapy for patients suffering from different types of rheumatic disorders, but reports about the beneficial effects of this form of therapy are controversial, rare and of poor scientific quality. The human chondrocyte cell line C-28/I2 was treated with two different MAPK inhibitors (SB203580 and U0126) or with various concentrations of the H(2)S donor Natrium hydrogen sulphide (NaHS). Thereafter, the secretion of IL-6 and IL-8 was quantified by enzyme-linked immunosorbent assays (ELISAs). The impact of NaHS on the regulation of p38 and ERK1/2 MAPK was confirmed by Western blot experiments. Furthermore, IL-6 and IL-8 expression was quantified by real-time polymerase chain reaction (RT-PCR) and ELISAs from cells which were exposed to SB203580, U0126 and NaHS and stimulated by IL-1β. The C-28/I2 cells constitutively expressed large quantities of IL-6 and IL-8. The data provided prove that in these cells, constitutive as well as IL-1β-induced IL-6 and IL-8 expression was partially and transiently blocked by the treatment of cells with both MAPK inhibitors and NaHS. Presented data seem to be important in evaluating the beneficial functions of MAPK inhibitors and H(2)S in immune-pathophysiological processes.

Balneotherapy employing sulphurous thermal water is still applied to patients suffering from diseases of musculoskeletal system like osteoarthritis (OA) but evidence for its clinical effectiveness is scarce. Since the gasotransmitter hydrogen sulphide (H2S) seems to affect cells involved in degenerative joint diseases, it was the objective of this study to investigate the effects of exogenous H2S on fibroblast-like synoviocytes (FLS), which are key players in OA pathogenesis being capable of producing pro-inflammatory cytokines and matrix degrading enzymes. To address this issue primary FLS derived from OA patients were stimulated with IL-1β and treated with the H2S donor NaHS. Cellular responses were analysed by ELISA, quantitative real-time PCR, phospho-MAPkinase array and Western blotting. Treatment-induced effects on cellular structure and synovial architecture were investigated in three-dimensional extracellular matrix micromasses. NaHS treatment reduced both spontaneous and IL-1β-induced secretion of IL-6, IL-8 and RANTES in different experimental settings. In addition, NaHS treatment reduced the expression of matrix metallo-proteinases MMP-2 and MMP-14. IL-1β induced the phosphorylation of several MAPkinases. NaHS treatment partially reduced IL-1β-induced activation of several MAPK whereas it increased phosphorylation of pro-survival factor Akt1/2. When cultured in spherical micromasses, FLS intentionally established a synovial lining layer-like structure; stimulation with IL-1β altered the architecture of micromasses leading to hyperplasia of the lining layer which was completely inhibited by concomitant exposure to NaHS. These data suggest that H2S partially antagonizes IL-1β stimulation via selective manipulation of the MAPkinase and the PI3K/Akt pathways which may encourage development of novel drugs for treatment of OA.