The epigenetic architecture at gene promoters determines cell type-specific LPS tolerance.

Synovial fibroblasts (SF) drive inflammation and joint destruction in chronic arthritis. Here we show that SF possess a distinct type of LPS tolerance compared to macrophages and other types of fibroblasts. In SF and dermal fibroblasts, genes that were non-tolerizable after repeated LPS stimulation included pro-inflammatory cytokines, chemokines and matrix metalloproteinases, whereas anti-viral genes were tolerizable. In macrophages, all measured genes were tolerizable, whereas in gingival and foreskin fibroblasts these genes were non-tolerizable. Repeated stimulation of SF with LPS resulted in loss of activating histone marks only in promoters of tolerizable genes. The epigenetic landscape at promoters of tolerizable genes was similar in unstimulated SF and monocytes, whereas the basal configuration of histone marks profoundly differed in genes that were non-tolerizable in SF only. Our data suggest that the epigenetic configuration at gene promoters regulates cell-specific LPS-induced responses and primes SF to sustain their inflammatory response in chronic arthritis.

Nucleofection: a new, highly efficient transfection method for primary human keratinocytes*.

Transfection is an essential tool for numerous in vitro applications including studies of gene expression, promoter analysis, and intracellular signaling pathways and also for therapeutic strategies such as tissue engineering and gene therapy. However, transfection of primary cells including keratinocytes with common methods such as calcium phosphate, DEAE-dextran, liposome-mediated transfer, electroporation or viral vectors is problematic because of low transfection efficiency and the induction of terminal differentiation. Here we analyzed the use of nucleofection, a new, electroporation-based transfection method that enables the DNA to enter directly the nucleus, for the transfection of keratinocytes. Several different conditions were tested and optimized, resulting in a final transfection efficiency of 56% in primary human epidermal keratinocytes. This efficiency is superior to all non-viral transfection methods reported so far. The number of non-viable keratinocytes after nucleofection was low, varying between 14 and 16%. In contrast to other transfection protocols, nucleofection did not induce terminal differentiation in the transfected keratinocytes. In addition, nucleofection is a fast method, because the results can be analyzed within 7 h. In summary, nucleofection is a fast, easy and highly effective alternative for the transfection of primary human keratinocytes, which offers new opportunities for various research applications.

Osteoclast-independent bone resorption by fibroblast-like cells.

To date, mesenchymal cells have only been associated with bone resorption indirectly, and it has been hypothesized that the degradation of bone is associated exclusively with specific functions of osteoclasts. Here we show, in aseptic prosthesis loosening, that aggressive fibroblasts at the bone surface actively contribute to bone resorption and that this is independent of osteoclasts. In two separate models (a severe combined immunodeficient mouse coimplantation model and a dentin pit formation assay), these cells produce signs of bone resorption that are similar to those in early osteoclastic resorption. In an animal model of aseptic prosthesis loosening (i.e. intracranially self-stimulated rats), it is shown that these fibroblasts acquire their ability to degrade bone early on in their differentiation. Upon stimulation, such fibroblasts readily release acidic components that lower the pH of their pericellular milieu. Through the use of specific inhibitors, pericellular acidification is shown to involve the action of vacuolar type ATPases. Although fibroblasts, as mesenchymal derived cells, are thought to be incapable of resorbing bone, the present study provides the first evidence to challenge this widely held belief. It is demonstrated that fibroblast-like cells, under pathological conditions, may not only enhance but also actively contribute to bone resorption. These cells should therefore be considered novel therapeutic targets in the treatment of bone destructive disorders.